The organ of Corti sensory progenitor cell competence is controlled by SoxC transcription factors

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Content Copyright 2021 Xizi Wang

The Organ of Corti
Sensory Progenitor Cell Competence
is Controlled by SoxC Transcription Factors

by
Xizi Wang

Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(DEVELOPMENT, STEM CELL AND REGENERATIVE MEDICINE)

December 2021
ii

Dedication
To my parents, Mr. WANG Junfeng and Mrs. YANG Ying
For their endless love and support

iii

Acknowledgements
In the past 27 years of my life, the most correct decision I’ve made is to pursue
my Ph.D. study at USC. USC gives me this chance to know many great people that I
can learn from, thank you all for making me who I am today.
First and foremost, I want to thank my mentor Professor Neil Segil, for always
listening, guiding, encouraging, and advising, with wisdom and humor. Dr. Segil has
always been supportive during my study and gave me a lot of precious advice not only
scientifically but also in life. There are many things he taught me, one is how to think
scientifically and critically. Every time we talk about science, he has his way to lead me
to think deeper about the questions and how to solve the problem in a better way. In the
meantime, he shares with me his knowledge on those questions, making the
conversations fruitful and beneficial. Besides the scientific part, he always encourages
me and helps me build confidence. I am always a shy person and lack confidence. After
Dr. Segil noticed this, he first encouraged me to be a teaching assistant where I can
communicate with different people to improve. Whenever I feel frustrated or confusing,
he can notice that very soon and talk with me to help solve the problems I face. Thanks
for always encouraging me to try new stuff, if it’s not for his support, I won’t find myself
love bioinformatics, which is a career I would like to pursue in the future. There are too
many things I learned from Dr. Segil, though cannot state all here, I will keep all of those
in mind moving forward. Thanks for shaping me into who I am now, my appreciation is
beyond expression.
I would also like to thank all my lab members, especially Dr. Ksenia Gnedeva,
Dr. Litao Tao, Juan Llamas, and Welly Makmura. I’m super thankful for Dr. Ksenia
iv

Gnedeva who helped a lot in guiding my thesis project. Thanks for always being
available to answer my questions and discuss the details of the project. This project
would not have been the same without her instructions. Also, I need to thank Dr. Litao
Tao for leading me to the field of bioinformatics, and always being patient with all my
questions. Our collaboration on the HiC assay development project is fantastic and
without him, I won’t gain that much knowledge of the next-generation sequencing
techniques. I’m super grateful for having the chance to learn from Juan Llamas. He
taught me a lot of dissection and culturing skills starting from my rotation in the Segil
lab. Juan is not only a great teacher of experimental skills; his optimistic and cheerful
attitude always makes the lab happy. Additionally, I must say thanks to Welly Makmura,
who’s been helping a lot with the genotyping stuff. Without him, the lab won’t be as
organized and efficient. I would also like to thank Talon Trecek, we are classmates and
joined the Segil lab at the same time, we share information all the time and support
each other a lot. Thanks to all my lab members Dr. Robert Rainey, Dr. Haoze Yu,
Francis James, John Duc Ngyuen, thanks for giving me such a wonderful lab
experience.
Additionally, I would like to thank all my great committee members: Dr. Robert
Maxson, Dr. Gage Crump, and Dr. Min Yu, as well as Dr. Andy Groves, for their
continuous support and all the great suggestions. My first rotation was done in Dr. Gage
Crump’s lab, thanks for giving me such a wonderful experience and expanded my
knowledge in the zebrafish development field. I appreciated all the things I learned in
the Crump lab. A special thanks to Dr. Min Yu, for giving me this chance to study at
USC. She was one of the interviewers during the process of application. I still remember
v

the questions and details we talked about during that interview. It is my pleasure to have
these great scientists guide my Ph.D. study.
Finally, I would like to thank my family and friends. To my parents, thank you for
your love and support all the time. To Xi Chen, thank you for being someone I can rely
on no matter what. To all my friends, I am lucky to have you in my life, may our
friendship last forever.

vi

TABLE OF CONTENTS
Dedication ................................................................................................................................... ii
Acknowledgements ................................................................................................................... iii
List of Tables ............................................................................................................................ viii
List of Figures ............................................................................................................................ ix
Abstract ..................................................................................................................................... xii
Chapter 1. Introduction .............................................................................................................. 1
1.1 The establishment of progenitor cell competence during embryogenesis ......................... 1
1.2 The development of the inner ear ...................................................................................... 4
1.3 Hearing loss and failure of hair cell regeneration .............................................................. 8
Chapter 2. The competence establishment in the sensory progenitor cells ...................... 17
2.1 Sensory progenitors in the organ of Corti acquire competence to differentiate as hair cells
and supporting cells upon transition to the post-mitotic state. ............................................... 18
2.2 Progenitor cell competence for sensory differentiation is associated with emergence of
newly established accessible chromatin regions. .................................................................. 20
1.3 The competent chromatin state established in the postmitotic progenitor cells is required
for Atoh1-mediated hair cell differentiation. ........................................................................... 23
Chapter 3. The pivotal role of SoxC transcription factors involved in the sensory
progenitor cell competence establishment process. ............................................................ 27
3.1 SoxC transcription factors are essential for initiation of sensory differentiation during the
development of organ of Corti. .............................................................................................. 27
3.2 SoxC transcription factors promote hair cell differentiation by regulating key sensory
lineage genes. ....................................................................................................................... 33
vii

3.3 SoxC transcription factors regulate chromatin accessibility to promote hair cell
differentiation in the E13.5 organ of Corti. ............................................................................. 37
Chapter 4. SoxC transcription factors are sufficient to trigger new hair cell generation in
the utricles and the organ of Corti in vitro and in vivo. ........................................................ 41
Chapter 5. Conclusion and Discussion .................................................................................. 46
5.1 The role of SoxC transcription factors in neuronal systems ............................................ 46
5.2 “Sox after Sox” theory ...................................................................................................... 48
5.3 Cell cycle exit is not interrupted in SoxC mutants ........................................................... 48
5.4 SoxC transcription factors regulate Atoh1 expression ..................................................... 49
5.5 The potential of SoxC transcription factors in regeneration ............................................. 50
Chapter 6. Assay development ............................................................................................... 51
6.1 Development of the in vitro enhancer reporter assay ...................................................... 51
6.2 Identification of Gjb2 enhancers using the reporter assay .............................................. 54
6.3 Development of a de novo HiC method for low cell number input ................................... 58
Chapter 6. Experimental methods ........................................................................................... 59
Appendix II ................................................................................................................................ 73
Appendix III ............................................................................................................................... 78
References .............................................................................................................................. 102

viii

List of Tables
Table 1. Sox transcription factor family subgroups and their functional domains…….13
Table 2. Putative enhancers validated through the enhancer reporter assay…………52

ix

List of Figures
Chapter1:
Figure 1.1. Schematic of the development of mouse inner ear………………………..…..5
Figure 1.2. Early development of the cochlea duct. …………………………………..…....6
Figure 1.3. Notch inhibition leads to supporting cell transdifferentiation through the
epigenetic de-repression of the hair cell-specific gene regulatory network……….…….10

Chapter2:
Figure 2.1. Sensory progenitors in the organ of Corti acquire competence to differentiate
as hair cells and supporting cells upon transition to the post-mitotic state…….….…….19
Figure 2.2. Progenitor cell competence for sensory differentiation is associated with
emergence of newly established accessible chromatin regions……………………….…21
Figure 2.3. High infection efficiency in the dissociated organ of Corti cultures…………23
Figure 2.4. The competent chromatin state established in the postmitotic progenitor
cells is required for Atoh1-mediated hair cell differentiation………………………………24

Chapter3:
Figure 3.1. SoxC transcription factors are essential for initiation of sensory
differentiation during the development of organ of Corti …….……………………………28
Figure 3.2. Overexpression of SoxC genes in the prosensory progenitors
promotes hair cell formation………………………………………………………………….29
Figure 3.3. Representative low magnification immunofluorescent images demonstrate
that overexpression of Sox2 gene fails to induce sensory differentiation……………….30
x

Figure 3.4. Loss of SoxC transcription factors does not affect hair cell survival organ of
Corti……………………………………………………………………………………………..31
Figure 3.5. Atoh1 overexpression in absence of SoxC genes failed to induce hair cell
formation………………………………………………………………………………………..32
Figure 3.6. Single cell RNA-seq indicates that SoxC transcription factors promote hair
cell differentiation by regulating key sensory lineage genes……………………………...34
Figure 3.7. Single cell RNA-seq analysis show the regulons of key sensory lineage
genes are changed upon SoxC deletion…………………………………………………….36
Figure 3.8. SoxC transcription factors control chromatin accessibility to promote hair cell
differentiation in the E13.5 organ of Corti………………………………………………...…38

Chapter4:
Figure 4.1. SoxC transcription factors are sufficient to trigger new hair cell generation in
the utricles and the organ of Corti in vitro and in vivo…………………………………..…42
Figure 4.2. In vivo injection of Anc80 Adeno-associated virus through the lateral
ventricle infects the nervous system as well as the inner ear…………………………….43

Chapter6:
Figure 6.1. Schematic of the enhancer reporter assay…………………………………....50
Figure 6.2. Enhancer reporter assay demonstrated 100% hair cell specificity of a
previous established Atoh1 enhancer and a novel Rasd2 enhancer at P1…………..…51
Figure 6.3. Summary of the identified deletions upstream of Gjb2 gene………………..53
xi

Figure 6.4. UCSC genome browser view of the ATAC-seq peaks detected within the
95.4kb common interval………………………………………………………………………54
Figure 6.5. two candidate enhancers are predicted to regulate Gjb2 expression by
snATAC-seq and histone modification ChIP-seq data….…………………………………55
Figure 6.6. Enhancer reporter assay demonstrates the activity of Enh4 in supporting
cells……………………………………………………………………………………………..56
Figure 6.7. Schematic of the modified Tn5 protein and the transposition and enrichment
steps…………………………………………………………………………………………….57

xii

Abstract
How progenitor cells acquire the ability to respond to the inductive stimuli to
differentiate towards defined cell lineages during embryogenesis remains a major
obscure question in developmental biology. The Organ of Corti – the auditory sensory
organ – represents a unique system to study the mechanisms governing competence
establishment, where cellular differentiation and terminal mitosis are uncoupled. In the
organ of Corti, upregulation of a single transcription factor, Atoh1, represents an
inductive stimulus both necessary and sufficient for sensory lineage specification.
Through in vitro cultures, we showed that the competence to respond to Atoh1 is
established in the organ of Corti progenitor cells prior to the initiation of sensory
differentiation following the cell cycle exit. By analyzing chromatin accessibility and
transcriptome of actively dividing (E12.0) and post-mitotic (E13.5) sensory progenitors,
we demonstrated that the transition to the competent state is rapid and is associated
with extensive chromatin remodeling controlled by the SoxC transcription factors.
Conditional loss of the two members of SoxC family, Sox4 and Sox11, does not affect
progenitor cell specification or the timing of cell cycle exit, but blocks sensory lineage
differentiation. Mechanistically, we demonstrated that SoxC binds to and regulates the
accessibility of the regulatory elements, many of which become targets of Atoh1 in the
differentiating sensory cells at later developmental stages. Further, transcriptomic
analysis at a single cell resolution reveals that SoxC also controls the expression of
other key sensory lineage genes. Consistently, overexpression of SoxC in the
progenitor cells prior to the establishment of competence for differentiation or after the
developmental window of plasticity enhances sensory differentiation both in vitro and in
xiii

vivo. Our findings demonstrate the pivotal role of SoxC as competence factors and may
facilitate the studies on repair and regeneration of the inner ear.

1

Chapter 1. Introduction
1.1 The establishment of progenitor cell competence during
embryogenesis
During the early development of mammalian tissues, a number of complex
structures start from a small pool of progenitor cells that will further give rise to diverse
cell types to achieve distinct functions. These progenitor cells gradually gain the
competence for differentiation as part of a developmental continuum between self-
renewing and committed states, where they are responsive to a specific intrinsic or
extrinsic cue. This transition of competence is highly regulated to ensure the progenitor
cells differentiate as distinct cell types in a spatiotemporal manner.
The notion of competence was first demonstrated through a series of studies
using isochronic and heterochronic transplantation assays (Desai & McConnell, 2000;
Frantz & McConnell, 1996; McConnell, 1988). These pioneering works show that when
the young cortical progenitors isolated from ferrets are transplanted into an older
embryo at the later-stage of cortical development, they are capable of differentiating as
superficial layer II-IV neurons, even though normally they would give rise to the early-
born deep layer V and VI neurons first in the original environment. In contrast, later
stage cortical progenitors that produce the late-born superficial layer II-IV neurons
cannot generate early-born deep layer neurons when transplanted into an earlier
embryo even if these progenitor cells undergo multiple rounds of cell division. These
results demonstrated that progenitor cells acquire the competence to specify into
distinct cell types at specific time windows and this process is not governed by cell
2

proliferation and division. The extrinsic molecular cues driving the specification of
progenitor cells have been studied extensively (reviewed by Miller and Gauthier 2007;
Barnabé-Heider et al. 2005; Koblar et al. 1998), however, little is known about the
molecular mechanisms underlying the competence establishment intrinsically.
During the development of the mammalian ventral hindbrain, neural progenitor
cells give rise to distinct types of neurons sequentially. A specific group of neural
progenitors expressing Nkx2.2 rapidly produces motor neurons between embryonic day
9.5 (E9.5) and E11.5 (Briscoe et al., 1999). Thereafter, these progenitor cells switch fate
to generating serotonergic neurons. This temporal regulation of the competence switch
in the neural progenitors relies on the homeodomain transcription factor Phox2b
(Pattyn, 2003). Phox2b is highly expressed in the progenitor cells during the active
production of motor neurons, but its expression decreases significantly at the time when
serotonergic neurons are generated. In Phox2b knockout animals, the premature
serotonergic neurons are solely generated by the progenitor cells with the motor
neurons being skipped out. While extended activation of Phox2b expression promotes
motor neuron fate and suppresses the production of serotonergic neurons.
In addition, another type of progenitor competence switch -- the temporal
transition from neurogenesis to gliogenesis – has been observed in multiple areas
during the development of the central nervous system (CNS). Active proliferating neural
progenitor/stem cells (NPCs) undergo a series of differentiation processes to produce
neurons and glial cells including astrocytes and oligodendrocytes (Gage, 2000). In the
context of the embryonic cortex, a pool of progenitor cells transforms into radial glial
cells (RGCs) and acquire the competence to generate neurons. At a later stage, these
3

progenitor cells terminate neurogenesis and switch to a gliogenesis-competent state
(Götz & Huttner, 2005). This timed transition of progenitor competence is shown to be
regulated by multiple transcription factors intrinsically. The proneural basic helix-loop-
helix transcription factors, Neurogenin1, Neurogenin2, and Ascl1, play essential roles in
the transition from progenitor proliferation to neurogenesis: These proneural factors
initiate and promote neurogenesis, each of them is involved in the terminal
differentiation into distinct neural subtypes (Ross et al., 2003). Inhibition of these
proneural transcription factors results in gliogenesis. Reciprocally, Sox9 as a glial fate
transcription factor is indispensable for progenitors to acquire competence for
gliogenesis (Stolt, 2003). Additionally, it has been shown that the nuclear receptors
Nr2f1 and Nr2f2 are required for NPCs to acquire gliogenic competence (Naka et al.,
2008). They are co-expressed transiently in the ventricular zone during the limited time
window of progenitor competence switch, and ablation of Nr2f1 and Nr2f2 in the
developing mouse brain results in extended neurogenesis at the cost of gliogenesis.
Similarly, the differentiation of major retinal cell types requires retinal progenitor
cells to first acquire the competence for fate specification (Marquardt & Gruss, 2002).
During retinogenesis, retinal progenitor cells (RPCs) located at the inner layer of the
optic cup produce all seven major types of retinal cells: Retinal ganglion cells and the
horizontal cells are generated first starting at E11, followed by the differentiation of
cones, amacrine cells, rods, and bipolar cells, and muller glia cells at later stages
(Young, 1985). Few studies were conducted to understand the competence
establishment during retinogenesis. Ikaros, a zinc-finger transcription factor, expressing
in the early progenitor cells can trigger early-born retinal cell fates when being
4

ectopically expressed in later stage progenitor cells that are Ikaros-negative. The
production of retinal ganglion cells, horizontal cells, and amacrine cells is compromised
upon Ikaros deletion. This piece of evidence suggests that Ikaros is indispensable for
establishing the early RPC competence to give rise to early-born retinal cells.
In all systems above, this competence establishment process is precisely
controlled to ensure the proper lineage development in a specific time window. Yet, how
the responsiveness to differentiation cues is acquired remains poorly understood, partly
because differentiation closely follows the cell cycle exit, and proliferating, post-mitotic,
and committed progenitor states to co-exist in most tissues. Understanding the
molecular control of competence and commitment is needed to provide insights on
generating specific cell types for tissue repair and remains one of the key questions in
developmental biology.
The organ of Corti, the auditory sensory epithelium in the inner ear, represents a
unique system to study the molecular underprints of competence establishment as the
processes of sensory differentiation and terminal mitosis are spatially and temporally
uncoupled in this system.

1.2 The development of the inner ear
The mammalian inner ear originates from the otic placode, a thickened patch of
the cranial ectoderm that locates alongside the hindbrain, at around embryonic day 8.5
(Groves & Fekete, 2012; Noramly & Grainger, 2002). Following the induction of the otic
fate by the intrinsic and extrinsic stimuli located in the otic placode region, invagination
of the placode leads to the formation of the otic vesicle at E9.5 (M. W. Kelley, 2006b).
5

Soon after the formation of otic vesicles, the inner ear undergoes a series of cell
patterning processes and morphological changes to give rise to the sensory organs as
development continues. The developing inner ear contains five vestibular organs –
consist of three semi-circular canals including three cristae, the utricle and saccule, and
the auditory organ – the cochlea (Alsina et al., 2009; Bok et al., 2007; Groves & Fekete,
2012) (Figure 1.1).
The development of a functional auditory system depends on the differentiation
and maturation of the hair cells and supporting cells, both of which come from the same
pool of progenitor cells in the sensory epithelium of the cochlear duct – the organ of
Corti (D M Fekete et al., 1998). The organ of Corti originates from a Sox2-positive
domain established in the cochlear duct, where cells undergo active mitosis until
embryonic day 12.0 (E12.0) (Kiernan et al., 2005; Lee, 2006; Matei et al., 2005; Ruben,
1967). A narrow strip of cells within the broader prosensory domain then start to exit the
cell cycle in a wave, spreading from the apex towards the base of the cochlea, and by
Figure 1.1. Schematic of the development of mouse inner ear. During the time window
from embryonic day 9.5 (E9.5) to E17.5, the inner ear undergoes dramatic
morphological changes, from a small patch of otocyst to an organ contains six in total
sensory regions. Adapted from (M W Kelly, 2006).
6

E13.5 most of the organ of Corti progenitors transition to a post-mitotic state where they
express a high level of cyclin-dependent kinase inhibitor Cdkn1b (p27kip1) (P Chen &
Segil, 1999; Lee, 2006) (Figure 1.2). After proliferation ceases, the wave of sensory
differentiation is initiated by the upregulated expression of the basic helix-loop-helix
transcription factor Atoh1, in an opposing base-to-apex gradient and results, under the
influence of notch-mediated lateral inhibition, in the stereotyped mosaic of the hair cells
and supporting cells – the two major cell types in the organ of Corti (Ping Chen et al.,
2002; Groves et al., 2013b; Lee, 2006). The construction of a functional auditory system
depends on this strict regulation of the timing for cell cycle exit and differentiation. In the
p27Kip1-deficient animals, where the
cell cycle exit and sensory
differentiation become coupled in the
organ of Corti, similarly to the rest of
the sensory organs and the CNS,
patterning defects arise leading to
profound hearing deficits (P Chen &
Segil, 1999; Lowenheim et al., 1999;
Oesterle et al., 2011).
Upregulation of Atoh1 in the progenitor cells at the mid-base of the cochlear duct
at E14.5 is the very first sign of hair cell differentiation (Bermingham et al., 1999; Ping
Chen et al., 2002). In the process of lateral inhibition through Notch signaling, this basic
helix-loop-helix transcription factor is down-regulated in the cells directly adjacent to the
forming sensory receptors, which acquire the fate of supporting cells (Donna M. Fekete
E12.0
E13.5
Sox2 p27
kip1
Figure 1.2. Early development of the cochlea
during E12.0 to E13.5. Cochlea duct
elongates during E12.0 to E13.5 while the
progenitor cells located in the sensory region
transit from active-proliferating stage to a
post-mitotic state.
7

et al., 1998). One row of inner hair cells starts to form first upon Atoh1 induction at
E14.5, while three rows of outer hair cells are produced later at E15.5. The inner hair
cells are the true sensory receptors that convert the sound waves amplified by the outer
hair cells into electrical signals. 95% of the auditory nerve fibers project to the auditory
brainstem and the auditory cortex arise from the inner hair cell population. While the
outer hair cells govern the sensitivity of hearing. They are capable of amplifying the low
amplitude sound waves received from the external ear and transmitted by the middle
ear. Supporting cells also contain multiple subgroups: Border cells, inner phalangeal
cells, pillar cells, Deiters’ cells, and Hensen’s cells (Wan et al., 2013). These subsets of
supporting cells differ both in their function and gene expression pattern. For instance,
pillar cells are required to maintain the tunnel of Corti structure and exclusively express
the growth factor receptors FGFR3 (Toshinori Hayashi et al., 2007); The prospero
homeobox protein PROX1 expression is only detected in outer pillar cells and Deiters’
cells (Bermingham-McDonogh et al., 2006); Glial fibrillary acid protein GFAP is
restricted to the inner border, inner phalangeal and Deiters’ cells (Rio et al., 2002; Smeti
et al., 2011). Notably, several of the specific markers for supporting cells are expressed
in the glial cells (Choi & Kim, 1984; Cid et al., 2010), indicating the similar functions of
glial cells in the nervous system and supporting cells in the auditory system.
These glial-like cells of the inner ear serve as hair cell precursor populations in
non-mammals where they replenish lost sensory receptors either directly through
transdifferentiation or after undergoing mitosis (Adler & Raphael, 1996; Duncan et al.,
2006; Roberson et al., 2004). During embryonic and neonatal development in
mammals, supporting cells retain the capacity for sensory differentiation (Bermingham
8

et al., 1999; Gao et al., 2016; Izumikawa et al., 2005; Kelly et al., 2012; Liu et al., 2012;
Shou et al., 2003; (Doetzlhofer et al., 2009). Upon overexpression of the master
regulator Atoh1 or inhibition of the Notch signaling, supporting cells at the postnatal 1
(P1) stage is capable of transdifferentiating into hair cells. However, this plasticity is
gradually lost after birth rendering the organ of Corti incapable of hair cell regeneration
by postnatal day 6 (P6) (B. C. Cox et al., 2014). These altogether indicate that
progenitor cells in the organ of Corti undergo the process where they develop the
competency that allows hair cell and supporting cell formation first; translate this
competency into differentiation upon upregulation of Atoh1, the master regulatory for the
hair cell lineage; then lose this competency during maturation. Because hair cells are
limited in number and can be easily damaged by noise or ototoxic drugs (Bramhall et
al., 2014; B. C. Cox et al., 2014; M. Kelley et al., 1995), the inability of supporting cells
to give rise to new sensory receptors leads to irreversible hearing loss and balance
disorders.

1.3 Hearing loss and failure of hair cell regeneration
Hearing is one of the most important senses which allows us to communicate.
However, mammals can easily lose hearing permanently as a result of the cumulative
death of the sensory receptor hair cells and cause sensorineural hearing loss.
Sensorineural hearing loss, a worldwide common disease that affects over 16% of
adults in the United States (NICDC, 2021), is irreversible due to the failure of hair cell
regeneration (Chardin & Romand, 1995; Rubel et al., 1994). Along with hearing loss,
patients also bear isolation or depression (C.-M. Li et al., 2014; Sun et al., 2016; Tseng
9

et al., 2016). Additionally, there are hundreds of key sensory genes have been identified
that would lead to hearing loss (Shearer et al., 1993), including Gjb2, Myo7a, Cdh23,
etc (El-Amraoui & Petit, 2005; Estivill et al., 1998; Green, 1999; Well et al., 1995; Wilch
et al., 2010; Zelante, 1997). Despite the existing prosthetics such as a cochlear
implant, which only provides limited recovery, hair cell regeneration in the inner ear
induced by genetic tools represents promising approaches for defeating sensorineural
hearing loss. Understanding the molecular basis at early developmental processes of
hair cell generation will provide insights on tissue repair to make this attractive treatment
a reality.
Although hair cell regeneration is blocked in adult mammals, non-mammalian
vertebrates such as chicken, birds, and zebrafish have been shown to regenerate hair
cells robustly after damage through rapid cell cycle re-entry of the surrounding
supporting cells (Lush & Piotrowski, 2014; Sliwińska-Kowalska et al., 1999). These
remaining supporting cells either undergo asymmetrical division to produce hair cells
and supporting cells at the same time, or generate new hair cells by direct
transdifferentiation at the cost of supporting cells (Corwin & Cotanche, 1988; Rubel et
al., 1994; Ryals & Rubel, 1988). Forced regeneration can be achieved through ectopic
overexpression of the “master regulator” Atoh1 or inhibition of Notch signaling pathways
(Figure 1.3A and B) in the embryonic or perinatal supporting cells in the cochlea, which
generate hair cell-like cells. This transient potential for regeneration is rapidly lost by
early postnatal stages (Basch et al., 2016; Bramhall et al., 2014; Brandon C Cox et al.,
2012; Doetzlhofer et al., 2004, 2009; Groves et al., 2013a; Michael C Kelly et al., 2012;
Liu et al., 2012). A recently published work from our lab suggests that the loss of
10

supporting cell transdifferentiation potential is due to the epigenetic barrier established
during maturation (Tao et al., 2021) (Figure 1.3C). Through the analysis of the
transcriptomic and epigenetic
landscapes of hair cells and
supporting cells using RNA-seq
ATAC-seq and Cut&Run ChIP-seq,
Tao et al. identified the gene
regulatory networks that are essential for hair cell fate. By looking at how the hair cell
gene regulatory networks behave epigenetically in the supporting cells, they found that
the regulatory elements for hair cell genes remain “poised” – according to the histone
Figure 1.3. Notch inhibition leads to
supporting cell transdifferentiation
through the epigenetic de-repression
of the hair cell-specific gene
regulatory network. A. Whole mount
organ of Corti cultured in vitro for 48
hours treated with DMSO as control
or DAPT at P1, supporting cells
transdifferentiated into hair cell-like
cells. Adopted from (Doetzlhofer et
al., 2009). B. Organ of Corti isolated
from P1 or P6 were treated with
DMSO or DAPT for 72 hours. P6
supporting cells do not respond to
Notch inhibition. Adopted from (Tao
et al. 2021). C. Enhancer
decommissioning model blocks
mature supporting cell
transdifferentiation. Hair cell gene
enhancers are “poised” and “primed”
at early stage supporting cells but
are decommissioned after one week
of birth, leading to the failure of
regeneration in mature animals.
Adopted from (Tao et al. 2021).
B
A
C
P1
P6
DMSO DAPT (72h)
DMSO DAPT (48h)
P1
P1(primed) P6(decommissioned)
H3k4me1
11

modification marker H3k4me3 and H3k4me1 for promoters and enhancers – at early
stage supporting cells that are capable of transdifferentiation, while most of these
enhancers lose the priming state and become silenced at a later stage where they bear
the repressive histone marker H3k27me3 and undergo rapid histone deacetylation.
These “decommissioned” enhancers are not capable of initiating hair cell gene
expression, leading to the failure of regeneration in the mature organ of Corti. Upon
Notch inhibition, where supporting cells are forced to transdifferentiate into hair cells,
these “poised” regulatory elements transit to an “active” state to promote the hair cell
fate.

1.4 Transcription factors involved in early development
Despite the clear temporal regulation in the organ of Corti during the early
development, the underlying molecular mechanisms of acquisition and loss of
competence for sensory differentiation remain poorly understood. Eya1, Six1, and Sox2
were all proposed to initiate hair cell differentiation (Ahmed et al., 2012; Kempfle et al.,
2016). Recent studies have shown that Six1 together with Eya1, its transcriptional co-
activator, are necessary for the induction of Atoh1 expression and are sufficient to
promote hair cell fate in the greater epithelial ridge (GER) (Ahmed et al., 2012b).
Despite the compelling evidence for direct binding to the 3’ enhancer to induce Atoh1
expression, sensory receptor specification was still observed when Six1 deletion was
induced prior to the onset of hair cell differentiation in the organ of Corti (Zhang et al.,
2017). Both Eya1 and Six1 are activated at the otic vesicle stage and are maintained at
12

the high levels of expression at P6, when the organ of Corti loses its regenerative
capacity (Cox et al., 2014).
Similarly, Sox2 labels supporting cell population throughout the postnatal period,
and despite clear evidence for Sox2 being both necessary and sufficient to establish the
prosensory domain in the cochlear duct, its role in hair cell induction is less defined
(Kiernan et al., 2005; Lee, 2006; Matei et al., 2005; Ruben, 1967). While Atoh1
overexpression alone is sufficient to induce hair cell formation in the non-sensory
regions in the cochlea, Sox2 co-expression mitigates this inductive effect (Dabdoub et
al., 2008). This antagonistic relationship between bHLH and Sox2 transcription factors
also exists in the nervous system (Bylund et al., 2003). Additionally, haploinsufficiency
for Sox2 was shown to promote hair cell differentiation (Atkinson et al., 2018).
Furthermore, Sox2 expression is associated with stem cell and progenitor populations in
the developing mammalian embryo, including CNS and the retina, where the
transcription factor maintains an undifferentiated state (Arnold et al., 2011; Graham et
al., 2003).

1.5 The role of Sox transcription factors during embryogenesis
The Sox family of transcription factors play versatile roles including progenitor
cell fate determination and subtype specification during development, with most of them
serve as transcriptional activators. All twenty Sox transcription factors share a high-
mobility-group (HMG) domain (more than 80% amino acid similarity) that can
specifically bind to DNA (Sinclair et al., 1990) (Table 1). According to the differences in
biochemical properties, these Sox proteins are separated into 8 distinct groups, Sox A
13

to H (Wegner, 2010). Despite that all Sox transcription factors share a high level of
sequence identity, each group has characteristic biological functions under a distinct
biological context due to the difference in binding affinity, co-factors, or post-
translational modifications (Wegner, 2010).

SOX Proteins Functional Domains (base pair)
SOXB1 SOX1 HMG + SoxB Homology + TAD (391)
SOX2 HMG + SoxB Homology + TAD (317)
SOX3 HMG + SoxB Homology + TAD (446)
SOXB2 SOX14 HMG + SoxB Homology +TRD (240)
SOX21 HMG + SoxB Homology +TRD (276)
SOXC SOX4 HMG + TAD (474)
SOX11 HMG + TAD (441)
SOX12 HMG + TAD (315)
SOXD SOX5 CC + HMG (763)
SOX6 CC + HMG (828)
SOX13 CC + HMG (622)
SOXE SOX8 DIM + HMG + TAM + TAD (446)
SOX9 DIM + HMG + TAM + TAD (509)
SOX10 DIM + HMG + TAM + TAD (466)
SOXF SOX7 HMG + TAD (388)
SOX17 HMG + TAD (414)
SOX18 HMG + TAD (384)
SOXG SOX15 HMG + TAD (233)
SOXH SOX30 HMG (753)
Extensive studies have been conducted to reveal the distinct functions of each
Sox transcription factors. Members of the SoxB2 group are involved in the development
of neuronal systems as repressors (Uchikawa et al., 1999) to regulate neuronal cell fate
specification and cell proliferation. SoxD subgroup of transcription factors is actively
expressed during spermatogenesis and chondrogenesis (Connor et al., 1995; Lefebvre,
1998). Despite that each of the Sox genes functions diversely under different biological
environments, Sox transcription factors can act together to regulate one developmental
Table 1. Sox transcription factor family subgroups and their functional domains. HMG,
High-mobility group domain; TAD, Transactivation domain; TRD, Transrepression
domain; CC, Coiled-coil domain; DIM, Dimerization domain.
14

process. The “SOX after SOX” regulation of the neurogenesis represents one of these
processes (Bergsland et al., 2011; Wegner, 2011), where the genome-wide
characterization of Sox2, Sox3, and Sox11 transcription factors’ targetomes using ChIP-
seq demonstrated a sequential binding of these proteins on the neuronal gene
enhancers to precisely control the neurogenesis. During this process, SoxB1
transcription factors are responsible for maintaining the neuronal precursor fate through
activation of the progenitor cell-specific genes. Sequentially, the SoxC transcription
factors replace SoxB1 to make the progenitor cells competent for neuron differentiation
through robustly upregulating lineage-specific genes and shutting down the proliferation
genes.
Besides Sox2, the most well-studied transcription factor in the Sox family, the
SoxC group of transcription factors also plays an essential role in cell-type specification
and fate determination in the nervous system, heart, and sensory organs. Sox4, Sox11,
and Sox12 in the SoxC subgroup share a high level of homology. However, Sox4 and
Sox11 overall are higher in expression level and binding affinity than Sox12.
Additionally, Sox12 knock-out animals are grossly normal without significant defects,
whereas Sox4 or Sox11 mutants die during embryogenesis (Dy et al., 2008; Hoser et
al., 2008; Schilham et al., 1996). During early neurogenesis, Sox4 and Sox11
expression is rapidly increased during the transition from active proliferating progenitor
cells to committed neuronal precursors (Haslinger et al., 2009a; Mu et al., 2012a).
Homozygote knock out of Sox4 and Sox11 in the adult hippocampus inhibited neuron
differentiation (Mu et al., 2012b), while overexpression of Sox4 gene or Sox11 is
sufficient to promote neurogenesis and upregulate expression of neuronal subtype-
15

specific genes in progenitor cells from a variety of tissues including the spinal cord,
hippocampus, and retina (Bergsland et al., 2006; Haslinger et al., 2009b; Jiang et al.,
2013; Mu et al., 2012b; Usui et al., 2013).
Beyond transcriptional regulation, SoxC transcription factors have been reported
to remodel the chromatin configurations and epigenetic markers (Koumangoye et al.,
2015; Smith et al., 2016b; Usui et al., 2013). During the reprogramming process from
fibroblast to neurons, a group of regulatory elements that are indispensable for this
process relies on Sox4 expression, and Sox4 knockdown results in significant loss of
the accessibility of these chromatin regions.

1.6 The organ of Corti progenitor competence is controlled by SoxC
transcription factors
Understanding the molecular control of the commitment of progenitor cells to hair
cell fate is necessary to uncover the failure of regeneration in this system. By utilizing
unbiased genome-wide analyses, in this study, we characterized transcriptomic and
epigenetic changes that are associated with acquisition and loss of competence for
sensory differentiation in the inner ear. Utilizing RNA sequencing (RNA-seq) and assay
for transposase-accessible chromatin (ATAC-seq), we identified genomic loci
accessible in progenitors and supporting cells capable of hair cell generation and
pinpointed few candidate transcription factors that may be required for maintaining the
accessibility of these elements. We further focused on the Sox4 transcription factor of
the SoxC family, one of such candidates transiently expressed in the organ of Corti
during the window of plasticity. Using single-cell RNA-seq, we demonstrated that
16

conditional loss of Sox4 and its close homolog, Sox11, led to the failure of initiation of
hair cell generation in the organ of Corti without affecting the timing of the cell cycle exit
or cellular identities within the cochlear duct. Overexpression of SoxC genes prior to the
onset of differentiation promoted hair cell fate in the prosensory progenitor cells in vitro.
Importantly, re-introducing the factor to the postnatal inner ear extended the window of
plasticity and boosted the rates of transdifferentiation after hair cell loss in vivo.
Together our data suggest that SoxC transcription factors activate the expression of key
sensory-lineage genes and set up permissive chromatin states in progenitors and
nascent supporting cells, rendering them competent to respond to sensory
differentiation cues.

17

Chapter 2. The competence establishment in the sensory
progenitor cells
All vertebrates use sensory organs containing mechanosensitive hair cells to
perceive sound and motion. These sensory receptors are limited in number and cannot
be regenerated after damage, thus leading to permanent hearing loss and balance
disorders (Bohne & Harding, 2000). During embryonic development, cells that give rise
to the organ of Corti in the mammalian inner ear first exit the cell cycle to form a well-
defined prosensory domain, and subsequently initiate a process of differentiation that
gives rise to a stereotyped mosaic of hair cells and supporting cells. The mechanisms
governing the competence switch within the forming prosensory domain remain poorly
understood, yet understanding the transition from proliferative progenitor to competent
precursor is important for a full understanding of the failure of regeneration in this
system.
Here, we observed the competence switch of progenitor cells during the E12.0 to
E13.5 development window through explantation and in vitro culture assays. Transgenic
mice expressing either Sox2-GFP or p27kip1-GFP reporters provided a means to
FACS- purify proliferating vs. post-mitotic progenitor cells from the developing (E12.0 -
E13.5) organ of Corti, respectively. Using RNA- and ATAC- sequencing, we observe
that a group of potential gene regulatory elements associated with hair cell fate-
commitment and inner ear morphogenesis emerge during the transition to post-mitotic
progenitor cells.
18

2.1 Sensory progenitors in the organ of Corti acquire competence to
differentiate as hair cells and supporting cells upon transition to the
post-mitotic state.
During auditory organ development, Sox2-positive progenitor cells in the
prosensory domain of the cochlear duct rapidly exit the cell cycle between E12.0 and
E14.5 (Bermingham et al., 1999) and differentiate into hair cells and supporting cells to
form the auditory sensory organ – the organ of Corti (M. W. Kelley, 2006a) (Figure
2.1A). It has been previously shown that p27Kip1-positive progenitor cells retain the
capacity for sensory differentiation in vitro either in dissociated cultures (Doetzlhofer et
al., 2004) or in whole-organ explants (Haque et al., 2015). However, hair cell
differentiation is disrupted when cochlear cultures are initiated prior to p27Kip1
upregulation (Montcouquiol & Kelley, 2003).
To confirm these observations, we utilized three-dimensional organotypic
cultures that preserve innate anatomy and surrounding tissues of the inner ear sensory
organs (Gnedeva et al., 2018). Cochlear ducts were explanted from reporter mice
expressing GFP under the control of a 3’ enhancer for Atoh1 (Helms et al., 2000).
Because Atoh1 is a key regulator of sensory receptor cell fate in the inner ear
(Bermingham et al., 1999), GFP activation indicates hair cell formation. Cochlear ducts
were explanted prior to or after initiation of the wave of progenitor cell-cycle exit at
E12.0 or E13.5 accordingly, and cultured for 3 days or 2 days before sensory
differentiation was assessed (Figure 2.1B). The sensory differentiation was not initiated
in E12.0 cochlear explants, where only 0.01% of Sox2-positive progenitor cells have
activated Atoh1-GFP expression (n=6; Figure 2.1B, C). In contrast, a robust reporter
19

activation was observed in the E13.5 organ of Corti explants, where 47% of progenitor
cells phenotypically converted to the sensory receptors – over a 40-fold increase
compared to E12.0 explants (n=3; p=0.01704). As during normal development, newly
forming hair cells were arranged in rows along the cochlea duct (Figure 2.1B). These
data suggest that the competence for sensory differentiation is acquired by the
prosensory progenitor cells after they transition to the post-mitotic state at E13.5.
A D
B
Non-competent Comepetent
E12.0
Explant
D0 D3
Harvest organs to
assess differentiation
D1
Atoh1-nGFP
E13.5
Explant
p27kip1
312 677
E12.0 3D culture
0.008%
47%
E13.5 3D culture
Atoh1
+
cells
Sox2
+
cells
5 628
C
E12.0 E13.5 E14.5
Sox2
E12.0 + 3d E13.5 + 2d
100µm
Atoh1:GFP Sox2 Atoh1-nGFP
Atoh1
Sox2
Differentiation
Figure 2.1. Sensory progenitors in the organ of Corti acquire competence to
differentiate as hair cells and supporting cells upon transition to the post-mitotic state. A.
Diagram demonstrates early embryonic development of the organ of Corti. At E12.0,
Sox2-positive proliferating progenitors (yellow) start to exit cell cycle from the apex of
the organ of Corti. By E13.5, most progenitors become post-mitotic and express high
levels of p27
kip1
(cyan). At E14.5, Atoh1-positive hair cells (HCs, green) and the
surrounding Sox2-positive supporting cells (SCs, red) are formed. Scale bars = 100 μm.
B. The diagram shows the experimental design for in vitro 3D cochlea culture
establishment and analysis demonstrated in panel C. C. Representative
immunofluorescent images of the whole cochleae isolated from E12.0 and E13.5 Atoh1-
nGFP transgenic reporter animals and harvested for characterization after 3-day or 2-
day in culture respectively. Atoh1-nGFP-positive hair cells (green) and Sox2-positive
cells (red) are labeled. Scale bars = 50 μm. D. Quantitative analysis of the cultures
demonstrated in panel C shows activation of Atoh1-nGFP reporter in E12.0 explants
(0.008%; p < 0.5; n=6) and E13.5 explants (47%: p < 0.5; n=3).

20

2.2 Progenitor cell competence for sensory differentiation is
associated with the emergence of newly established accessible
chromatin regions.
We have recently demonstrated that a rapid switch to the post-mitotic state in the
organ of Corti progenitor cells is associated with repression of a self-renewal gene
network that is accompanied by changes in chromatin accessibility (Gnedeva et al.,
2020). We noted, however, that 13,352 regions gained accessibility upon transition to
the post-mitotic state at E13.5. We separated these putative regulatory elements into
proximal and distal regulatory elements based on their distance to the transcription start
sites (TSS) (Figure 2.2A). The regulatory elements within 2kb upstream or downstream
of the TSS were considered proximal promoters (1,104 ATAC peaks), the remaining
regions were considered distal regulatory regions (12,248 ATAC peaks). To better
characterize the distal regulatory elements, we performed CUT&RUN ChIP-seq (Skene
& Henikoff, 2017) to analyze the status of the well-known enhancer marker H3K4me1
(Rada-Iglesias et al., 2011). We identified 9,811 enhancers that are newly opened at
E13.5. These promoters and enhancers were emerged prior to the onset of sensory
differentiation and were maintained in the developing hair cells and supporting cells at
E17.5, but lost in the P6 supporting cells incapable of sensory differentiation (Figure
2.2A). Because the accessibility of these genomic regions coincided with the window of
plasticity, we hypothesized that gain in their accessibility may represent an
establishment of competence for sensory differentiation. The GREAT analysis
demonstrated that genes involved in cell fate commitment (GO: 0045165, -logPvalue =
21

6.91) and cell differentiation (GO: 0030154, -logPvalue =4.14) were associated with
these accessible genomic regions (Figure 2.2B).
Transcriptomics data revealed that expression of the genes involved in cell fate
commitment was significantly upregulated during E12.0 to E13.5 transition (Figure 2.2C;
22

n=85; p=0.000257), including many established sensory lineage specification genes,
such as Isl1, Eya1, and Fgf20 (T. Hayashi et al., 2008; Radde-Gallwitz et al., 2004; Zou
et al., 2008). The genes associated with the epithelial cell differentiation, however,
represented hair cell (HC) and supporting cell (SC) specific genes that were not yet
upregulated at E13.5 (Figure 2.2D; n=14; p=0.0012(HC) and 0.0024(SC)). Examples of
these genes included important sensory cell fate regulators such as Atoh1, Myo6, Hes5,
and Prox1 (Figure 2.2E) (Bermingham-McDonogh et al., 2006; Bermingham, 1999;
Hertzano et al., 2008; Azel Zine et al., 2001), strongly suggesting that the chromatin
state, permissive for sensory differentiation, is established in the E13.5 progenitor cells
prior to sensory receptor specification.
Figure 2.2. Progenitor cell competence for sensory differentiation is associated with
emergence of newly established accessible chromatin regions. A. Heatmap
demonstrates that most of the ATAC-seq accessible genomic regions newly emerged
during the transition from E12.0 to E13.5 (1,104 promoters and 9,811 enhancers)
remain open in E17.5 hair cells (HC) and supporting cells (SC) but lose their
accessibility in the postnatal supporting cells (P6 SC). Scale of each sample column is
±3kb from the centered ATAC-seq peaks. B. Gene ontology (GO) analysis showing the
top eight most-enriched biological process terms for E13.5 newly appeared peaks (in A)
is generated using GREAT. C. Violin plot shows that expression (Log10(FPKM+1)) of
the genes in the cell fate commitment GO term is significantly upregulated (p = 9.1e-15;
Wilcoxon signed-rank test) in E13.5 progenitors (Prog) compared to E12.0.
D. Violin plot shows that the expression (Log10(FPKM+1)) of hair cell-specific and
supporting cell-specific genes in the inner ear morphogenesis GO term is unchanged at
E13.5 (p = 0.26; Wilcoxon signed-rank test) but is significantly upregulated upon
differentiation (p = 2.4e-4 for E17.5 hair cells (HC); p = 1.2e-4 for E17.5 supporting cells
(SC); Wilcoxon signed-rank test). E. Integrative Genomics Viewer (IGV) tracks show
ATAC-seq profiles of representative genomic loci of hair cell-specific (Atoh1, Myo6) and
supporting cell-specific genes (Hes5, Prox1) at E12.0 and E13.5. Their putative
enhancers, highlighted in grey boxes, gain accessibility at E13.5. F. The expression
(FPKM) of the hair cell- and supporting cell-specific genes in panel E in the progenitor
cells (Prog), hair cells (HC) and supporting cells (SC) is shown.

23

1.3 The competent chromatin state established in the postmitotic
progenitor cells is required for Atoh1-mediated hair cell
differentiation.
Because inner ear sensory lineage specification is controlled by Atoh1, we tested
the ability of this transcription factor to promote sensory differentiation at E12.0 and
E13.5. The dissociated organ of Corti cells isolated from the Atoh1-GFP reporter mice
were infected with adenoviral vectors carrying either the control RFP (Ad-RFP) or RFP
and Atoh1 (Ad-Atoh1-RFP) constructs. We showed high (90-100%) infection efficiency
for the viral vectors used (Figure 2.3). The GFP-reporter activation was analyzed after 3
days in culture. Consistent with the previous observations (Doetzlhofer et al., 2004), no
reporter activity was detected at E12.0 and limited activation was observed at E13.5 in
the RFP control groups (Fig 2.4A). The number of GFP+ hair cells increased
dramatically in the Ad-Atoh1-RFP infected E13.5 cultures, where we observed over 8-
fold increase compared to the RFP control. In addition, hair cells and the surrounding
supporting cells induced in the E13.5 cultures were organized in polarized rosettes in
which the actin-rich apical cell surfaces formed a small lumen. By stark contrast, only a
Figure 2.3. High infection
efficiency in the dissociated
organ of Corti cultures.
Immunofluorescence analysis
of the dissociated organ of
Corti cells isolated from E12.0
Atoh1-nGFP animals infected
with Ad-RFP or Ad-Atoh1-RFP
viral vectors, demonstrates
the adenoviral transduction
efficiency, widespread RFP
expression (red) is observed.
Scale bar = 50 μm.
24

A
B C
E12.0 Prog E13.5 Prog
ATAC-seq accessible regions
E17.5 SC P6 SC
Atoh1 Cut&Run
3,163 Atoh1 targets
E17.5 HC
±3kb
E17.5 HC
0
50
10
20
E12.0 E13.5
Ad-RFP Ad-Atoh1
***
E12.0 E13.5
30
40
Percentage of hair cells
p = 1.5e-5
Ad-RFP Control Ad-Atoh1-RFP
E12.0
Atoh1 Sox2
E13.5 E12.0 E13.5
Atoh1 Sox2 Phal Atoh1 Sox2
***
p = 2.9e-6
***
p = 9.5e-5
25

sparse reporter activation was seen in Atoh1 overexpression group at E12.0,
over 3-fold decrease compared to E13.5 (p=1.5e-5) (Fig 2.4B). Additionally, these newly
forming GFP-positive hair cells were not polarized to form sensory rosettes resembling
RFP-control conditions.
Because Atoh1 was proposed to have no pioneer factor activity (Kim et al. 2014;
Yu and Tao et al., 2021), we hypothesized that its insufficiency to induce sensory
differentiation at E12.0 was due to the non-permissive progenitor chromatin state. To
test this hypothesis, we assessed whether any of the E13.5 newly emerging putative
regulatory elements represented future targets of Atoh1. By comparing the accessibility
of Atoh1 targetome identified in the differentiating sensory receptors (Yu and Tao et al.,
2021; Tao et al., 2021) in E12.0 and E13.5 progenitor cells, we demonstrated that
approximately one fifth (3,163 out of 13,352) of the E13.5 newly emerged ATAC peaks
represented putative regulatory elements occupied by Atoh1 in the E17.5 hair cells
(Figure 2.4C). Consistent with their role in the maintenance of the permissive state, the
Figure 2.4. The competent chromatin state established in the postmitotic progenitor
cells is required for Atoh1-mediated hair cell differentiation. A. Representative
immunofluorescent images show the dissociated progenitor cells isolated at E12.0 and
E13.5 from the Atoh1-nGFP reporter mice, infected with Ad-RFP control or Ad-Atoh1-
RFP virus, and maintained in culture for 3 days. Note that only in E13.5 cultures Atoh1
overexpression results in formation of the sensory rosettes with a small lumen formed
by the actin-rich (Phalloidin, white) apical surfaces of the polarized Atoh1-nGFP-positive
hair cells (green) and surrounding Sox2-positive supporting cells (blue). Panels of low
magnification (left column, scale bar = 50 μm) and high magnifications (right column,
scale bar = 50 μm) are shown. B. Bar graphs show the quantitative analysis of the
cultures demonstrated in panel A. A significant increase of the percentage of Atoh1-
nGFP-positive hair cells is observed in the E13.5 Atoh1-overexpression cultures
compared to other conditions (n=3 for each condition; p = 9.5e-5, 2.9e-6, 1.5e-5 for
comparison with E12.0 Ad-RFP, E13.5 Ad-RFP, E12.0 Ad-Atoh1 conditions
respectively; Welch t-test). C. Heatmap demonstrates that 3,163 genomic loci bound by
Atoh1 in E17.5 hair cells (Atoh1 C&R, blue) gain accessibility at E13.5 progenitors
(Prog), remain open in E17.5 hair cells (HC) and supporting cells (SC), and lose
accessibility in P6 supporting cells (ATAC-seq, orange).

26

same regions were also accessible in E17.5 supporting cells, capable of direct
conversion into the sensory receptors, but were closed by P6 when the capacity for
trans-differentiation is lost (Cox et al., 2014).

27

Chapter 3. The pivotal role of SoxC transcription factors involved
in the sensory progenitor cell competence establishment process.

3.1 SoxC transcription factors are essential for the initiation of
sensory differentiation during the development of the organ of Corti.
To understand what transcription factors may set up the E13.5 chromatin state,
permissive for sensory differentiation, we performed motif enrichment analysis using
HOMER software (Heinz et al., 2010). Sox transcription factor binding motifs were the
most significantly enriched in E13.5 unique accessible chromatin regions (Figure 3.1A).
Sox family of transcription factors contains many well-known regulators of cell fate
commitment and specification (reviewed in Bowles et al., 2000). To investigate which
family members may be involved in the establishment of competence for sensory
differentiation in the inner ear, we assessed the expression pattern of Sox genes in the
organ of Corti during development. We reasoned that the expression of the transcription
factors, necessary for the establishment of competence for sensory differentiation at
E13.5, may be upregulated at this stage.
Among all the Sox genes present in the cochlea, the expression pattern of Sox4
correlated best with the temporal dynamics of acquisition and loss of the competence
for sensory differentiation (Figure 3.1B). While, consistent with their potential inductive
role, expression of Sox2, Sox4, Sox10, and Sox13 was significantly upregulated in
E13.5 progenitors as compared to E12.0 (Sox2: fold=5, p<0.001; Sox4: fold=32,
p<0.001; Sox10: fold=5, p<0.001; Sox13: fold=5, p<0.001). Only the expression of the
28

Sox4 gene, the SoxC family member, was also significantly downregulated in maturing
supporting cells (Figure 3.1B), as these cells gradually lost the chromatin structure,
permissive for differentiation towards the hair cell lineage (Figure 2.2A).
To test if SoxC transcription factors can promote sensory differentiation, we
overexpressed either Sox4 or Sox11, the two closely related members of the family, in
the dissociated E13.5 progenitor cells (Figure 3.2A), not capable of differentiating
towards hair cells spontaneously (Doetzlhofer et al., 2004). Isolated from the Atoh1
reporter mice, the progenitor cells were infected with adenovirus containing RFP, Sox2,
Sox4, or Sox11 gene, and hair cell formation was analyzed 3 days later (Figure 3.2A).
In the control RFP condition, cells grew as a monolayer and almost no Atoh1-GFP
Figure 3.1. SoxC transcription factors are essential for initiation of sensory
differentiation during the development of organ of Corti. A. HOMER motif analysis
shows top four most enriched DNA binding motifs in the newly emerged accessible
chromatin regions at E13.5. B. Gene expression (FPKM) of Sox4, Sox11, and Atoh1 in
the progenitors (Prog) and supporting cell (SC) at different developmental stages of the
organ of Corti is demonstrated (n=2 for Progs, n=3 for SCs). Sox4 expression is
upregulated in E13.5 progenitors prior to the onset of Atoh1 expression. C.
Representative immunofluorescent images show the whole cochlea isolated from the
WT and SoxC KO littermate embryos at E14.5. Ki67-positive (magenta) proliferating
cells and the p27
kip1
-positive (green) post-mitotic progenitor cells (left panels) are
labeled. Note that progenitor cell cycle exit is not affected in the SoxC KO organs.
Sox2-positive supporting cells (magenta) and one row of Pou4f3-positive inner hair cells
(green) are detected in the WT but not in the SoxC KO organs. Cell nuclei are labeled
with DAPI (blue). Scale bar = 100 μm.

29

reporter activation was detected. Similarly, Sox2 overexpression failed to promote hair
cell formation (Supplementary figure 3.3). In stark contrast, overexpression of either the
Sox4 or Sox11 resulted in robust Atoh1-GFP reporter activation (Figure 3.2B and C).
Additionally, as Atoh1-overexpression, the newly formed hair cells were polarized and
organized as sensory rosettes. Quantification further confirmed that overexpression of
either Sox4 or Sox11 gene strongly promoted sensory differentiation resulting in a
Figure 3.2. Overexpression of SoxC genes in the prosensory progenitors
promotes hair cell formation. A. Representative immunofluorescent images demonstrate
that overexpression of SoxC genes promotes sensory differentiation. Many Atoh1-
nGFP-positive hair cells (green) are detected in the Sox4 and Sox11 overexpressing
cultures where these sensory receptors and surrounding Sox2-positive supporting cells
(blue) are organized into the sensory rosettes. Actin is labeled in white (phalloidin).
Scale bar = 50 μm. B. Bar graph shows quantitative analysis of the cultures
demonstrated in panel D. A significant increase in the number of Atoh1-nGFP-positive
hair cell is observed in Sox4 and Sox11 overexpression conditions compared to the RFP
control (n=3 for each condition; p = 0.0003665 for Ad-Sox4 condition; p = 0.0009526 for
Ad-Sox11 condition; Welch t-test). C. Bar graph shows quantitative analysis of the
cultures demonstrated in panel D. A significant increase in the number of Sox2-positive
supporting cells is observed in Sox4 and Sox11 overexpression conditions compared to
the RFP control (n=3 for each condition; p = 0.001838 for Ad-Sox4 condition; p =
0.008096 for Ad-Sox11 condition; Welch t-test).

A
B
C
30

significant increase in the number of hair calls compared to RFP controls (n=3,
p=0.0003665 for Sox 4; n=3, p=0.0009526 for Sox 11; Figure 3.2B and C).
Although we previously showed that by birth hair cells formation is disrupted in
the conditional Sox4 and Sox11 knockout animals (Gnedeva and Hudspeth, 2015),
mechanistically the role of these transcription factors remained undefined in the inner
ear. To test if these genes are necessary for the establishment of competence for
sensory differentiation, we assessed whether initiation of hair cell differentiation was
affected in the mutants. By E14.5, the p27kip1-positive post-mitotic prosensory domain
Figure 3.3. Representative low magnification immunofluorescent images demonstrate
that overexpression of Sox2 gene fails to induce sensory differentiation. While
overexpression of SoxC genes promotes hair cell differentiation. Many Atoh1-nGFP-
positive hair cells (green) are detected in the Sox4 and Sox11 overexpressing cultures
and are organized into the sensory rosettes. Actin is labeled in white (phalloidin). Scale
bar = 100 μm.
31

surrounded by proliferating Ki67-positive cells was established in the wildtype cochlea
(Figure 3.1C). The cells in this domain also expressed Sox2, and a single row of
Pou4f3-positive inner hair cells was detected in the mid-base region of the cochlea,
signifying the initiation of sensory differentiation. However, although p27Kip1- and Sox2-
positive prosensory domain was established in the knockouts, progenitor cells failed to
differentiate towards the sensory lineage in absence of Sox4 and Sox11 expression. To
exclude the possibility that hair cell survival rather than differentiation was affected, we
assessed for activated Caspase-3 labeling. This analysis revealed no difference in the
rate of apoptosis in the cochlear sensory epithelia between the wildtype and the
knockout organs (Figure 3.4). Together, these results suggest that Sox4 and Sox11
transcription factors are both necessary and sufficient for sensory cell fate initiation in
the organ of Corti progenitor cells. Interestingly, it also revealed that sensory cell fate
commitment is established independent of the cell cycle exit, as loss of Sox4 and Sox11
did not affect the timing of p27Kip1 upregulation.
Figure 3.4. Loss of SoxC transcription factors does not affect hair cell survival organ of
Corti. Representative immunofluorescent images show the whole cochlea isolated from
the WT and SoxC KO littermate embryos at E14.5. Sox2-positive supporting cells
(magenta) and one row of Pou4f3-positive inner hair cells (green) are detected in the
WT but not in the SoxC KO organs. Note that no dying cells were detected by activated
caspase 3 staining (white) in both WT and SoxC KO organs. Cell nuclei are labeled with
DAPI (blue). Scale bar = 50 μm
32

The failure of sensory fate induction upon loss of SoxC indicates two non-
mutually exclusive possibilities. The first one is that SoxC directly controls the onset of
Atoh1 upregulation. The second one is that these transcription factors may be a part of
the network necessary to establish the state permissive for sensory differentiation both
epigenetically and transcriptionally. To distinguish between these two possibilities, we
overexpressed Atoh1 in the progenitor cells isolated from the SoxC double knockout
cochlea at E14.5 and analyzed for sensory differentiation after 3 days in culture (Figure
3.5A). In the control Ad-RFP condition, the hair cell induction rate in the knockout
progenitor cells was comparable to that in the non-permissive E12.0 wildtype (p=0.61)
and significantly lower than in E13.5 competent wildtype progenitors (p=0.002). Atoh1
overexpression in absence of SoxC genes failed to induce hair cell formation, as only
2.6% of Sox2-positive transduced cells upregulated Brn3C – the marker of the sensory
A B
Figure 3.5. Atoh1 overexpression in absence of SoxC genes failed to induce hair cell
formation. A. Representative immunofluorescent images show the Pou4f3-positive hair
cells (green) and Sox2-positive supporting cells (blue) in the dissociated SoxC KO
organs after 3 days in culture. Note that Atoh1 fails to induce Pou4f3-positive hair cells.
Actin is labeled in white (Phalloidin). Scale bar = 50 μm. B. Histogram shows quantitative
analysis of the cultures demonstrated in panel I and Fig. S3. The percentage of Pou4f3-
positive hair cells in the Sox2-positive cells is significantly decreased in the SoxC KO
cultures transduced with Ad-RFP or Ad-Atoh1 virus compared to the WT cultures (n=3; p
= 0.004037 for Ad-Sox4 condition; p = 1.486e-5 for Ad-Sox11 condition; Welch t-test).

33

receptors. Strikingly, this rate of differentiation was significantly reduced compared to
the E12.0 wildtype progenitor cells (p=0.00087) and over 12-fold lower than that in the
E13.5 wildtype progenitor cells, where 31.6% progenitor cells converted to the sensory
receptor upon Atoh1 upregulation (p=2.696e-6) (Figure 3.5B). This evidence clearly
demonstrated that SoxC transcription factors are necessary for the establishment of
competence for sensory differentiation and allow progenitor cells to differentiate as hair
cells in response to Atoh1 induction.

3.2 SoxC transcription factors promote hair cell differentiation by
regulating key sensory lineage genes.
To gain a mechanistic understanding of how SoxC transcription factors establish
the competence for sensory differentiation, we performed a single-cell RNA-sequencing
analysis of SoxC-deficient cochlear duct epithelia at E13.5, prior to the onset of hair cell
differentiation. For a comparative analysis between wildtype and knockout organs, we
integrated the two datasets through the canonical correlation analysis (CCA) algorithm
(Figure 3.6A). Cells were visualized by Uniform Manifold Approximation and Projection
(UMAP) which was performed using the top fifteen principal components. After
excluding non-epithelial cell types (e.g., blood and mesenchyme), the same 11 clusters
were identified in both wildtype and knockout datasets, suggesting that loss of Sox4 and
Sox11 did not affect the overall cellular composition of the cochlear duct (Figure 3.6A).
Established markers for known cochlear cell populations were used to assign the
identity to each cluster. Post-mitotic sensory progenitor cells grouped distinctly as
cluster 2 (Figure 3.6B) in wildtype and knockout organ, characterized by the overlapped
34

35

Cdkn1b (P Chen & Segil, 1999), Sox2 (Kiernan et al., 2005), and Hey2 (S. Li et al.,
2008) expression (Figure 3.6C). In addition, other cell populations such as Otx2-positive
roof domain of the cochlea (Morsli et al., 1999), Bmp4-positive outer sulcus (Morsli et
al., 1998), as well as Sox2 and Jag1-positive Kölliker's organ (Dabdoub et al., 2008; A
Zine et al., 2000) were identified in both WT and KO cochlear ducts.
To investigate the prosensory role of SoxC transcription factors, we assessed the gene
expression changes associated with the loss of Sox4 and Sox11 in the postmitotic
progenitor cells. Suggesting that SoxC function as transcriptional activators, two-thirds
of the most significantly differentially expressed genes were downregulated in the
knockouts (Log2(Fold change) >1.5, p-value <0.01; Figure 3.6D). These most
significantly upregulated and downregulated genes are enriched for hair cell lineage
Figure 3.6. Single cell RNA-seq indicates that SoxC transcription factors promote hair
cell differentiation by regulating key sensory lineage genes. A. UMAP plots of 7,365
cells collected from WT cochlea duct epithelium and 5,693 cells collected from SoxC
KO cochlea duct epithelium at E13.5 generated by integration using Seurat Canonical
Correlation Analysis (CCA) are shown. Note that cells group together based on shared
cell type regardless of the genotype, and the same eleven clusters are identified in the
WT and SoxC KO organs. B. UMAP visualization of WT and SoxC KO cochlea cell
populations at E13.5 split based on the genotype. C. UMAP feature plots demonstrate
transcript levels for cell markers specific to the known cochlear cell types. The organ of
Corti progenitor cell population is identified based on the Cdkn1b, Sox2, and Hey2
expression. The roof domain of the cochlear duct is identified by high level of Otx2
expression. D. Heatmap visualizes the 109 genes most significantly differentially
expressed in SoxC KO compared to control progenitor cells (Log2(Fold change) > 1.5, p
< 0.001). Key sensory lineage genes, including Isl1, Sox9 and Eya1, are highlighted.
High expression level is shown in yellow, low expression level is depicted in purple. E.
Gene ontology analysis performed using DAVID shows top six biological process terms
enriched in the genes most significantly downregulated in SoxC KO progenitor cells
compared to control. F. Violin plots show the average expression (Average FPKM) of
the representative genes significantly downregulated in SoxC KO progenitor cells
compared to control. Accompanying immunofluorescent images of the sections through
the E14.5 cochlear ducts validate a decrease in the protein levels for the same genes.
Sox2-positive progenitor cells (red) are counterstained in each section for the
investigated protein (Ebf1, Sox9, Isl1; green). The organ of Corti is highlighted in white
dashed box in the WT and SoxC KO. Scale bar = 50 μm.

36

related gene ontology terms including neuronal differentiation (GO:0030182, P = -
log105.34) and sensory perception of sound (GO:0007605, P = -log105.21), indicating
Figure 3.7. Single cell RNA-seq analysis show the regulons of key sensory lineage
genes are changed upon SoxC deletion. A. Quality control data of the single cell RNA-
seq datasets collected for E13.5 wildtype organ of Corti and the E13.5 SoxC knockout.
Bubble plot and violin plot together show the differentially expressed known cell
population features in each dataset. B. UMAP plots for E13.5 WT and KO datasets
generated separately after filtering cells based on the quality control parameters.
Feature plots show distinct marker expression in each cluster. C. SCENIC analysis on
WT and KO datasets reveal the downstream regulons of key sensory lineage genes are
altered upon deletion of SoxC transcription factors.
A
B
C
37

their roles in hair cell differentiation in the inner ear (Figure 3.6E). These genes included
many crucial regulators of sensory lineage specification, such as Isl1 (Radde-Gallwitz et
al., 2004), Eya1 (Zou et al., 2008), Sox9, and Fgf20. Amongst potential targets of SoxC,
we have also identified genes with no established functions in the inner, but shown to
be essential for neuronal lineage specification, such as Ebf1 (Garel et al., 1999) and
Sall3 (Kuroda et al., 2019). We confirmed the downregulation of the key sensory lineage
factors in the knockout using immunohistochemistry (Figure 3.6F), asserting the role of
SoxC transcription factors as crucial regulators of hair cell fate initiation.

3.3 SoxC transcription factors regulate chromatin accessibility to
promote hair cell differentiation in the E13.5 organ of Corti.
To test if SoxC transcription factors establish the chromatin state, permissive for
sensory differentiation, we profiled chromatin accessibility changes in the organ of Corti
upon ablation of Sox4 and Sox11 genes. This analysis demonstrated that the
accessibility of 10% of wildtype genomic regions was dependent on SoxC and was lost
in the knockouts (Cluster I in Figure 3.8A). The GREAT analysis demonstrated that
genes associated with regulation of timing of cell differentiation and auditory receptor
cell fate commitment were associated with these SoxC-dependent regions. Strikingly,
the peak that lost accessibility in the knockouts overlapped with the genomic regions
newly emerged at E13.5, representing over 60% of these regions (Figure 3.8B). In
contrast, over 70% of genomic regions that gained accessibility in the knockouts
(Cluster II in Figure 3.8A) overlapped with the E12.0-specific regions that closed at
E13.5. These data suggested that upon SoxC ablation, the chromatin state of the
38

39

progenitor cells resemble that at the E12.0 stage, when cells are not yet capable of
spontaneous differentiation towards the sensory lineage (Figure 3.8C). Notably, around
20% (515 out of 2,659) SoxC-dependent E13.5 newly emerged peaks represented
Atoh1 targets (Figure 3.8D). This potentially explains the observation that sensory
differentiation was not initiated after the loss of SoxC even when Atoh1 was present
(Figure 3.5A).
To assess if SoxC transcription factors directly control the accessibility of the
competency-related regulatory elements, we profiled the Sox4 targetome in the E13.5
progenitor cells using CUT&RUN (Figure 3.8E). This analysis demonstrated that while
none of the chromatin regions accessible in the knockouts (Clusters II and III)
overlapped with Sox4-occupied peaks, 21% of genomic regions, that lost accessibility in
the knockout (Cluster I), were direct targets of Sox4 in the wildtype progenitors. To
Figure 3.8. SoxC transcription factors control chromatin accessibility to promote hair cell differentiation in
the E13.5 organ of Corti. A. Heatmap shows accessible genomic regions identified by ATAC-seq in the
WT and SoxC KO cochlea at E13.5. Half of the regulatory elements lose (5,303; SoxC-dependent
regions, Cluster I) or gain (39,611; SoxC-dependent regions, Cluster II) accessibility in the SoxC KO
cochlea (SoxC-dependent regions). Common peaks are labeled as SoxC-independent regions (45,242;
Cluster III). B. The accessibility of SoxC-dependent chromatin regions is analyzed in the E12.0 and E13.5
purified progenitors (Prog). Out of 5,303 peaks in Cluster I, 4,656 were identified in either E12.0 or E13.5
progenitors. Note that over half of these regions overlap with the chromatin regions that are newly
emerged at E13.5 compare to E12.0. Out of 39,611 peaks in Cluster II, 4,368 were identified in either
E12.0 or E13.5 progenitors. Note that over half of these regions overlap with the chromatin regions that
lose accessibility at E13.5 compare to E12.0. C. GREAT GO term analysis shows the top 10 biological
process terms most-enriched in the genes associated with Cluster I and Cluster II – SoxC-dependent
regions that lose and gain accessibility, respectively. D. Heatmap demonstrates that out of 2,659 SoxC-
dependent regions that are newly emerged at E13.5 compare to E12.0 progenitor cells (ATAC-seq,
orange), 19% represent future targets of Atoh1 in E17.5 hair cells (Atoh1 Cut&Run, blue). E. Heatmap
demonstrates that out of 2,659 SoxC-dependent regions (Cluster I) that are newly emerged at E13.5
compare to E12.0 progenitor cells, 22% are direct targets of Sox4 transcription factor (Sox4 Cut&Run,
blue) in E13.5 progenitors (Prog). F. Violin plots show the expression (log10(FPKM)) changes of genes
associated with SoxC-dependent regions (577 direct targets and 2,082 indirect targets demonstrated in
panel E) and SoxC-independent regions (45,242 peaks demonstrated in panel A). Gene expression
(FPKM) profiles were obtained from WT and SoxC KO single cell RNA-seq datasets. Genes associated
with SoxC-dependent regions are significantly downregulated in SoxC KO progenitor cells compared to
control (p = 0.0025 for direct targets, p = 0.01214 for indirect targets; Wilcoxon signed rank test). Genes
associated with SoxC-independent regulatory elements are unchanged (p = 0.4558; Wilcoxon signed rank
test). G. HOMER motif analysis shows top four most enrichen DNA binding motifs on SoxC-dependent
peaks lost their accessibility (Cluster I).

A. Integrative Genomics Viewer (IGV) tracks show ATAC-seq profiles (orange) and Sox4 Cut&Run
profile (blue) of representative genomic loci of significantly downregulated key sensory lineage genes
(Atoh1, Eya1 and Bcl11a) in SoxC KO progenitor cells compared to WT. Their putative enhancers,
highlighted in grey boxes, gain accessibility in E13.5 progenitors (Prog, yellow), lose their accessibility
upon SoxC ablation (KO, yellow), and are bound by Sox4 transcription factor (Sox4 Cut&Run, blue).

40

determine if the chromatin accessibility changes altered gene expression, we then
assessed the expression changes of the closest genes assigned to SoxC-dependent
genomic regions (Cluster I) after ablating SoxC genes based on our single-cell RNA
sequencing data (Figure 3.8F). These SoxC-dependent peaks associated genes, both
directly and indirectly regulated by Sox4, are significantly down-regulated after the loss
of SoxC (p=0.0025; p=0.01214), while the expression of genes associated with SoxC-
independent genomic regions remains unchanged (p=0.4558). HOMER analysis
revealed that, in addition to Sox, GATA and Isl1 motifs were specifically enriched in the
SoxC-dependent peaks lost in the knockouts, suggesting these factors may also be
involved in setting up the permissive chromatin state (Figure 3.8G). By correlating the
chromatin accessibility, Sox4 CUT&RUN, and gene expression changes seen in the
SoxC knockouts, we have identified a number of confident targets of Sox4. These
included transcription factors previously shown to control hair cell fate initiation,
including Atoh1, Bcl11a, and Isl1 (Kuwajima et al., 2017) (Figure 3.8H). These data
together demonstrated that SoxC transcription factors establish competence for sensory
differentiation by directly binding to and opening the putative sensory regulatory
elements, as well as by upregulating the key sensory lineage genes.

41

Chapter 4. SoxC transcription factors are sufficient to trigger new
hair cell generation in the utricles and the organ of Corti in vitro
and in vivo.
The organ of Corti maturation is associated with a reduction in transdifferentiation
potential of supporting cells in mammals and is culminated in the loss of capacity for
hair cell regeneration by P6 (B. C. Cox et al., 2014). Concomitantly, the expression of
Sox4 and Sox11 genes is reduced (Fig 3.1B). To test if SoxC transcription factors can
re-establish competence for sensory differentiation, we overexpressed Sox4 or Sox11 in
the mature supporting cells. Lfng
GFP
transgenic mice were used to purify supporting
cells via FACS from P7 cochlea and transfected with adenoviruses carrying RFP
control, Sox4 or Sox11 genes (Figure 4.1A). As demonstrated by Myo7a-positivity,
SoxC overexpression significantly increased the number of hair cells formed in the
cultures compared to control (9-fold; n=3; p=0.006056; Figure 4.1A and B). We also
noted the presence of GFP-positive neuron-like cells in the Sox11 overexpression
condition.
Because these results suggested that SoxC overexpression enhances
supporting cell transdifferentiation potential in culture, we then tested whether Sox4 or
Sox11 overexpression may trigger hair cell generation in vivo after injury (Figure 4.1C).
By crossing the Lfng
CreERT2
mice (Semerci et al., 2017), tdTomato reporter mice
(Madisen et al., 2010), and the Pou4f3
DTR/+
mice (J. S. Golub et al., 2012), we
generated transgenic animals in which supporting cells can be permanently labeled with
42

tdTomato prior to induction of hair cell damage with diphtheria toxin. After tamoxifen
administration at P3, diphtheria toxin was injected to ablate hair cells a day later. At P5,
43

B
D
C
Lfng
CreERT2/Tdt
; Pou4f3
DTR/+
P3: Tamoxifen Injection
Permanently label SCs
P4: DT Injection
Specifically kill HCs
P5: Virus Injection
Reintroducing SoxC
P15
Collect ears
E
A
MyoVIIa MyoVIIa Lfng:GFP
Ad-RFP Ad-Sox4 Ad-Sox11
RFP/Sox4/11
Utricle Cochlea
0
Anc80-GFP
Anc80-Sox4
Anc80-Sox11
10
20
30
40
50
Percentage of SC transdifferentiation
Merge MyoVIIa Anc80-Sox4 Lfng
Ad-RFP
Ad-Sox4
Ad-Sox11
0
40
80
120
Numbers of hair cells/supporting cells
HC
SC
*
p=0.0103
**
p=0.0081
*
p=0.02
*
p=0.05
Figure 4.1. SoxC transcription factors are sufficient to trigger new hair cell generation in the utricles and
the organ of Corti in vitro and in vivo. A. Representative immunofluorescent images show the Lfng-GFP-
positive supporting cells (green) FACS purified from the reporter mice, infected with Ad-RFP control or
Ad-Sox4 or Ad-Sox11 virus, and maintained in culture for 7 days. Many supporting cells transdifferentiate
into MyoVIIa-positive hair cells (red) in the Sox4 and Sox11 overexpressing cultures. Scale bar = 50 μm.
B. Bar graph shows quantitative analysis of the cultures demonstrated in panel A. A significant increase
in number of hair cells is observed in Sox4 and Sox11 overexpressing conditions compared to the RFP
control (n=3 for each condition; p = 0.0103 for Ad-Sox4 condition; p = 0.0081 for Ad-Sox11 condition;
Welch t-test). C. The diagram shows the experimental design for the analyses of the ability of SoxC
transcription factors to promote sensory differentiation in vivo. Tamoxifen is administrated at postnatal
day 3 (P3) to permanently label supporting cells (SCs) in the Lfng
CreERT2/Tdt
; Pou4f3
DTR/+
animals.
Diphtheria toxin (DT) is injected at P4 to specifically ablate hair cells (HCs). Anc80-AAV viruses are then
injected through the posterior semicircular canal at P5 to re-introduce the expression of Sox4, Sox11, or
RFP-control. Sensory organs are collected at P15 for analysis. D. Representative immunofluorescent
images show the P15 whole-mount utricles and cochlea isolated from the animals in which Sox4
expression was re-introduced (green). Lfng-positive supporting cells and their derivatives are labeled in
white and hair cells are stained with MyoVIIa in red. White arrows indicate a number of triple-labeled
supporting cells transdifferentiated to sensory receptors upon Sox4 overexpression. Scale bars = 50 μm.
E. Bar graph shows quantitative analysis of the percentage of supporting cells transdifferentiated to the
sensory receptors in the utricles. The rate of transdifferentiation is significantly increased in the Sox4 and
Sox11 overexpression conditions compare to the GFP control (n=3 for each condition; p = 0.05 for Ad-
Sox4 conditions and p = 0.02 for Ad-Sox11 conditions; Welch t-test).

44

we introduced RFP control, Sox4, or Sox11 overexpression vectors using a recently
published synthetic adeno-associated virus, Anc80 (Landegger et al., 2017). The
cerebrospinal fluid rout, via lateral ventricle injections, was used as we described
previously (Gnedeva et al., 2020) (Figure 4.2). After 10 days, utricles and the organ of
Corti are dissected out for analysis (Figure 4.1C). By assessing the percent of
transduced supporting cells that had converted to Myo7a-positive hair cells, we first
determined a spontaneous rate of transdifferentiation in GFP controls to be 6%.
Introduction of either Sox4 or Sox11 expression dramatically busted the proportion of
transdifferentiating supporting cells over 7-fold to 42% (n=3; p=0.04432; Figure 4.1D
and E). Although the rate of supporting cell to hair cell conversion was greatly reduced
Figure 4.2. In vivo injection
of Anc80 Adeno-associated
virus through the lateral
ventricle infects the nervous
system as well as the inner
ear. A. Schematic of the
injection method achieve at
the lateral ventricle.
Adopted from (Gnedeva et
al., 2020) B.
Immunofluorescence
analysis of the whole mount
organs that are infected by
the Anc80-CMV-GFP virus
injected through this new
method at P3. The utricle
and the organ of Corti are
isolated 48 hours post
infection. Cells locate in the
utricle and the organ of
Corti are effectively
infected, including hair cells
(Myo7a, blue).
Perilymph
Endolymph
CSF
Cochlea
Vestibule
5ul
Cochlear
aqueduct
A
B
Myo7a Anc80-GFP Merge
Utricle Cochlea
45

in the cochlea, the newly formed hair cells were only detected in the cochlea in Sox4
and Sox11 overexpression conditions (Figure 4.1D).

46

Chapter 5. Conclusion and Discussion
Understanding the molecular underprints required for the establishment of
competence for differentiation in progenitor cells is crucial for manipulating tissue repair.
Through transcriptomic and epigenomic analysis, we uncovered a de novo molecular
mechanism of how SoxC transcription factors control the competence establishment
through chromatin remodeling, and consequent sensory lineage-specific gene
expression. We demonstrated that the window of plasticity is acquired by establishing
sensory differentiation competence related chromatin structure during E12.0 to E13.5
transition and is gradually lost by postnatal day 6. Sox4 and Sox11 of SoxC family
transcription factors promote progenitor cell progression to sensory lineage
competence. Elimination of these activators disrupts sensory progenitor cell
differentiation towards hair cells and supporting cells without interrupting the cell cycle
exit. Contrarily, sox4/11 overexpression is sufficient to promote hair cell fate during both
early development and postnatally. Our study builds a model of the mechanisms
governing sensory fate commitment and provides insights for a better understanding of
the failure of regeneration in this system.
5.1 The role of SoxC transcription factors in neuronal systems
Consistent with our result, SoxC transcription factors are shown to be critical for
neuronal differentiation in other systems (Haslinger et al., 2009b; Kuwajima et al., 2017;
Mu et al., 2012b). Ablation of Sox4 and Sox11 in chick spinal cord and mouse
hippocampus leads to failure of neuronal differentiation, while overexpression of
Sox4/Sox11 transcription factor promotes neurogenesis by targeting neuron-specific
genes such as Tuj1 and Map2 (Bergsland et al., 2006; Mu et al., 2012; Tanaka et al.,
47

2004; Bergsland et al., 2006). Similarly, SoxC genes are necessary and sufficient to
promote retinal ganglion cell differentiation and expression of lineage-specific genes like
Isl1 (Kuwajima et al., 2017).
Beyond transcriptional regulation of gene expression, SoxC transcription factors
co-operate with chromatin remodelers to alter chromatin states (Kavyanifar et al., 2018;
Smith et al., 2016; Tiwari et al., 2013). Sox4 has been shown to recruit SWI/SNF
subunits to remodel chromatin structure during human MRC-5 fibroblast to neuron
reprogramming. Sox4 knockdown abolishes the reprogramming event due to loss of
around 60% accessible chromatin regions. Additionally, ChIP-seq results reveal that
Sox4 induces the active histone marker, H3K27 acetylation, on the regulatory elements
of key neuronal lineage genes to promote neuronal differentiation (Smith et al., 2016).
As a master regulator for EMT (Epithelial-Mesenchymal Transformation), Sox4
upregulates the expression of the epigenetic modifier, Ezh2, a functional enzymatic
component within the Polycomb Repressive Complex 2 (PRC2) and co-operates with
Ezh2 to establish repressive chromatin structure by introducing tri-methylation of lysine
27 on histone H3 (H3K27me3)(Tiwari et al., 2013). Consistently, our single-cell RNA-
seq and ATAC-seq experiments revealed that upon SoxC deletion at E13.5, Ezh2
expression is largely down-regulated, and the chromatin accessibility remains in the
state resembling that of E12.0 prosensory cells. Whether SoxC transcription factors
cooperate with chromatin remodelers to shift histone modifications, in particular the
active histone mark H3K27ac and the repressive mark H3K27me3, requires further
characterization.
48

5.2 “Sox after Sox” theory
Transcription factors in the Sox family are well-known regulators of the molecular
cascades during neuronal development, stem cell maintenance, cell fate specification
and commitment, and differentiation (Sarkar & Hochedlinger, 2013). In the “Sox after
Sox” model it has been proposed that the SoxB1 and SoxC family of transcription
factors sequentially bind to a shared set of regulatory elements to generate neural
precursors and neurons (Wegner, 2011). We show that the expression of Sox4 is
initiated at E12.5, while the Sox2 (SoxB1) transcription factor is expressed from the
initiation of otic lineage (Gu et al., 2016). This “Sox after Sox” model could be a
conserved process in the context of the organ of Corti development: Sox2 involves in
the early establishment of the prosensory domain and Sox4 binds on competence-
related regulatory regions prior to the initiation of the differentiation process, this
sequential binding of Sox factors leads to the selection and fine adjustment of sensory
cell fate.

5.3 Cell cycle exit is not interrupted in SoxC mutants
During the organ of Corti development, the differentiation wave proceeds in a
basal-to-apical gradient along with the prosensory domain following cell cycle exit from
the apex to base (Ping Chen et al., 2002). Despite the fact that cells locate in the apex
exit cell cycle first and differentiate last, it has been reported that post-mitotic apical
progenitors are capable of giving rise to hair cells immediately following the cell cycle
exit, when repressive Shh signaling is conditionally inactivated in culture (Bok et al.,
2013). This implicated that the sensory fate commitment is tightly coupled with cell cycle
49

exit: loss of inhibitory stimuli could release the differentiation program in the competent
progenitors. However, from our observation, although progenitor cells lose the
competence for sensory differentiation upon SoxC deletion, they still exit the cell cycle
and are trapped in this post-mitotic state from which they are not able to initiate the
differentiation process, even in the presence of the strong differentiation stimuli – the
master regulator Atoh1. This evidence demonstrates that sensory cell fate commitment
is not directly dependent on the cell cycle, suggesting that SoxC-dependent chromatin
remodeling in the postmitotic progenitor cells may be a common mechanism for the
competence establishment of neuronal lineages.

5.4 SoxC transcription factors regulate Atoh1 expression
The master regulator Atoh1 is required for the selection and differentiation of
sensory hair cell fate (Ping Chen et al., 2002). What transcription factors initiate Atoh1
expression at the onset of differentiation remains unknown. According to our RNA-seq
data, the expression pattern of SoxC correlates with the temporal dynamics of Atoh1
up-regulation. We did not detect Atoh1 expression after the loss of SoxC and
overexpression of Sox4/Sox11 in dissociated cultures promotes Atoh1 expression.
These demonstrate that SoxC transcription factors are upstream of Atoh1 and regulate
its expression. However, we didn’t detect Sox4 binding on the pre-established
autoregulatory 3’ enhancer of Atoh1 according to our ChIP-seq data. This indicates two
possibilities: one, SoxC transcription factors set up chromatin structure and regulate the
expression of other transcription factors that directly initiate Atoh1 expression; Or Sox4
regulates unidentified putative enhancers of Atoh1 that are essential for initiating its
50

expression. Evidence from transgenic animals shows that the GFP reporter expression
driven by the 3’ autoregulatory enhancer lags the endogenous Atoh1 expression in the
prosensory domain, suggesting the initial upregulation of Atoh1 relies on other
regulatory elements. We noted several other putative enhancers, and one of these sites
located around 90kb downstream of the Atoh1 transcription start site is occupied by the
Sox4 transcription factor according to our ChIP-seq results. It would be interesting to
test if Sox4 binding on this putative enhancer regulates early Atoh1 upregulation, prior
to the initiation of the autoregulatory loop.

5.5 The potential of SoxC transcription factors in regeneration
Previous studies concluded that re-introducing Atoh1 alone in the adult
mammalian cochlea is not sufficient to trigger hair cell regeneration (M. C. Kelly et al.,
2012; Liu et al., 2012). We attribute this to a decrease in accessibility of the
competence-related genomic regions in adult supporting cells due to downregulation of
SoxC transcription factors. By reintroducing SoxC genes, we were able to boost the
transdifferentiation rate in the inner ear postnatally. Further experiments on co-
expressing Atoh1 with SoxC factors may achieve a better regeneration rate. This finding
is consistent with the result that Sox4 is one of the very first responding genes during
hair cell regeneration in zebrafish after hair cell loss (unpublished data from Piotrowski
lab). Additionally, during the reprogramming process of induced hair cells from mouse
embryonic fibroblasts, SoxC genes are significantly upregulated (Menendez et al.,
2020). Together with our result that SoxC factors bind and regulate the competence-
51

related genomic regions embryonically, these demonstrate that SoxC expression is
required to promote plasticity in mature supporting cells after hair cell damage.
Chapter 6. Assay development
6.1 Development of the in vitro enhancer reporter assay
Understanding the Cis-regulatory control of gene expression is essential for
constructing the gene regulatory networks of different developmental processes.
Enhancers, as the major regulators of gene expression, are normally located in the non-
coding genomic regions including introns (Levine, 2010). They are usually under 1kb in
size and contains multiple transcription factor binding sites, this allows enhancers to
function as hubs integrating all the transcription factors for the target genes.
Identification of the enhancers is the fundamental step for later characterization
of its activity, target genes and functions. In the lab, we build an enhancer testing
pipeline to characterize the specificity of the bioinformatic analysis selected putative
regulatory elements for hair cells and supporting cells (Figure 6.1) (Protocol attached in
Figure 6.1. Schematic of the enhancer reporter assay. A. Dissociated organ of Corti
cells are infected with lentivirus containing the enhancer-reporter. 3 days post
transduction, immunostaining of cell type specific marker shows the specificity of the
enhancers. B. Sequence map of the enhancer-reporter construct.

A
B
52

Appendix I). Our assay starts from the dissociated organ of Corti, since currently, we
don’t have the means to infect the intact Organ of Corti utilizing lentivirus. Then the
dissociated cells are infected with lentivirus consisting of two parts: one is the innate
control, where a PGK promoter-driven mRFP sequence is inserted in the pLKO
backbone; the second part, constructed in cis to avoid read through, is a minimal β-
globin promoter-driven eGFP sequence, in front of this cassette, putative enhancers are
cloned and inserted (Figure 6.1). Three days post virus infection, the cell culture is
collected and stained using hair cell or supporting cell-specific marker to assess the
specificity of the predicted enhancer. Ideally, a hair cell-specific enhancer should be
triple positive: RFP expression confirms the viral infection, GFP expression suggests
the activity of this enhancer, and hair cell marker (Myo6) staining indicates the specific
cell type this enhancer is activated. Therefore, the specificity of a given enhancer is
revealed by the percentage of triple-positive cells among all infected RFP+ cells.
Atoh1 enhancer
H2B:RFP Enh-GFP Myo6 Merge
Rasd2 enhancer
Figure 6.2. Enhancer reporter assay demonstrated 100% hair cell specificity of a
previously established Atoh1 enhancer and a novel Rasd2 enhancer at P1. Arrows point
at infected (RFP, red) hair cells (Myo6, cyan) that show enhancer activity (GFP, green).
DAPI shown in blue.
53

To validate if our assay is functioning properly, we cloned a previously mapped
Atoh1 3’ enhancer into our viral vector as a positive control for this testing
system(Helms et al., 2000). Results show that among all the 255 hair cells analyzed,
the reporter activity is only seen in the infected hair cells. This suggests a 100%
specificity of the Atoh1 3’ enhancer and is consistent with the previous characterization
of this enhancer using transgenic animals (Helms et al., 2000) (Figure 6.2). In addition,
we tested another newly identified Rasd2 enhancer predicted by our bioinformatics
data: this enhancer is open in hair cells and is active indicating by the histone marker
H3K27ac (Figure 6.2). The testing result using the reporter assay confirmed the
specificity of this enhancer in hair cells. Here I attached a list of tested putative
enhancers and their activity (Table 2) (All putative enhancer sequences and designed
primers are attached in Appendix III).
ENHANCERS ACTIVITY
HAIR CELL
ENHANCERS
Atoh1 3’ Hair cell specific
Atoh1_75kb downstream No activity
Atoh1_95kb downstream No activity
Rasd2 Hair cell specific
Pou4f3_proximal
Activation restricted to ~60% hair
cells: no activation in other cell types
but not all hair cells show activation.
Pou4f3_middle No activity
Pou4f3_distal No activity
SUPPORTING
CELL
ENHANCERS
Gjb2_1 No activity
Gjb2_2 No activity
Gjb2_4
Supporting cell specific (6% hair cell
misexpression)
Id1 up Supporting cell specific
Id1 down
Show activation in ~20% supporting
cells
Id2 up No activity
Id2 down No activity
POU4F3
DEPENDENT
Gfi1
Show activation, but not specific to
hair cell or supporting cell
Table 2. Putative enhancers validated through the enhancer reporter assay.
54

GENE
ENHANCERS
Dll1 No activity
Ptprq Not tested
Dnaic2 Not tested
Ccer2 Not tested
Ttc34 Not tested
Xylt1 Not tested
Otof_1 No activity
Otof_2 No activity
Cep851 Not tested
Neurod1 Not tested

6.2 Identification of Gjb2 enhancers using the reporter assay
Most of the hereditary hearing loss cases are non-syndromic hearing loss, which
usually occurs as a consequence of sensory cell destruction in the inner ear. Among all
the autosomal recessive non-syndromic hearing loss cases, although over 200 genes
identified to be associated with hearing loss so far (Shearer et al., 1993), over 50% of
patients bearing mutations on a single protein – gap junction protein beta 2 (GJB2), also
known as connexin 26 (Cx26) (Snoeckx et al., 2005). GJB2 gene encodes the subunit
of the gap junction protein that helps regulate the recycling of potassium and maintain
the electric potential difference between endolymph and perilymph in the cochlea
(Kikuchi et al., 1995). Mutations in the Gjb2 gene are the predominant cause of
autosomal recessive hearing loss (DFNB), which make up 50% of autosomal recessive
non-syndromic sensorineural hearing loss (SNHL). The most common mutation cause
DFNB1, which is 70% of the cases, is a nonsense mutation (35delG). This 35delG
Figure 6.3. Summary of the identified deletions upstream of the Gjb2 gene. Schematic
of the large deletions upstream of the Gjb2 gene coding region. The red arrow indicates
the 95.4kb common interval.
55

mutation will induce a premature stop of GJB2 translation (Zelante et al. 1997).
Normally, individuals who have DFNB1 are either homozygous or compound
heterozygous for GJB2, or compound heterozygous for one GJB2 plus one of the large
deletions upstream of GJB2 (Figure 6.3). These large deletions without touching the
coding region of the Gjb2 gene resulted in decreased expression of GJB2 and GJB6,
indicating the presence of cis-regulatory elements within the deleted genomic regions.
Summarizing all the deletions discovered by the previous genetic studies, there’s a
95.4kb common critical interval locates at 100kb upstream of Gjb2 coding region.
To identify potential cis-regulatory elements within this 95.4kb common interval,
first, we analyzed the chromatin accessibility at different developmental time points
using ATAC-seq (Figure 6.4). Results show there are 5 genomic regions (Enh1-5,
proximal to distal) that are highly opened in the developing P1 supporting cells, 2 of
these are also opened in the E13.5 embryonic progenitor cells and the mature P6
supporting cells. The conservation track reveals that these putative regulatory elements
Enh1 Enh5 Enh4 Enh3 Enh2
E13.5 Prog
P1 SC
P6 SC
Conservation
Figure 6.4. UCSC genome browser view of the ATAC-seq peaks detected within the
95.4kb common interval. Putative enhancers are emphasized by blue box and are
named Enh1-5, Enh1 is the most proximal region to Gjb2 gene coding region.
56

are conserved in vertebrates. After identification of these 5 potential regulatory
elements, we collected single cell nuclei ATAC-seq sample to predict if there are any
DNA interactions between these sites and the Gjb2 promoter (Figure 6.5). The analysis
was done using two packages: Seurat Signac and CICERO (Adorf et al., 2018; Pliner et
al., 2018). To correlate the clusters identified by snATAC data, we projected snATAC
onto scRNA Data collected at the same developmental stage to help identify cell
populations within the organ of Corti using a calculated gene activity matrix. Using this
canonical correlation analysis, we identified the progenitor cells population in the
snATAC-seq UMAP plot by the canonical markers highly expressed in the RNA profile
(Figure 6.5A). Then, we analyzed co-accessible peaks in the progenitor cell population
scRNA snATAC A
B
C
CICERO predicted 3D interactions
using snATAC-seq data
ATAC
H3k4me1
H3k4me3
H3k27ac
Gjb2 Gjb6 Cryl1
Figure 6.5. two
candidate enhancers are
predicted to regulate
Gjb2 expression by
snATAC-seq and
histone modification
ChIP-seq data.
A. Integrated analysis of
scRNA and scATAC on
E13.5 organ of Corti.
Cluster 0 represents the
progenitor population.
B. Cis-regulatory DNA
interactions predicted by
CICERO. C. Epigenetic
markers demonstrate
the active state of the
predicted enhancers.
ATAC shows the open
chromatin regions,
H3k4me1 and H3k4me3
labels enhancers and
promoters respectively.
H3k27ac marks active
cis-regulatory elements.
57

from scATAC-seq data using the Cicero package to predict their potential interactions
between the open chromatin regions at the Gjb2 locus. The result shows that Enh2 and
Enh4 are predicted to have 3D looping interaction with the Gjb2 promoter to regulate its
expression (Figure 6.5B). Epigenetic analysis on the histone modification markers
further confirmed that Enh2 and Enh4 are active enhancer candidates for Gjb2
expression regulation. We investigated the behavior of the active enhancer markers:
H3k4me1 and H3k27ac at these two putative enhancers. H3K4me1 and H3k27ac
Cut&Run ChIP-seq results together with the promoter histone marker H3K4me3
demonstrate the active state of these two genomic regions (Figure 6.5C).
We then validated the activity of Enh2 and Enh4 using our established enhancer
testing reporter assay. Enh2 and Enh4 sequences were cloned into the enhancer-
reporter viral vector and
infected the dissociated organ
of Corti culture. Analysis after
three days in culture showed
that Enh2 failed to drive GFP
expression in the infected cells while Enh4 drives GFP expression specifically in
supporting cells. Among 325 GFP
+
RFP
+
cells analyzed, 79% are ProxI labeled
supporting cells, while 6% of them are Myo6 labeled hair cells (Fig. 6.6). This result
demonstrates that Enh4 potentially serves as an enhancer to regulate the expression of
Gjb2. Here, we identified this enhancer as a supporting cell enhancer, further
characterization of how this enhancer regulates Gjb2 expression still requires
Figure 6.6. Enhancer reporter assay demonstrates the
activity of Enh4 in supporting cells.
58

exploration through other approaches such as generation of transgenic animals or use
CRISPR technology to study the loss-of-function effect.

6.3 Development of a de novo HiC method for low cell number input
Enhancer validation is just the first step to understand the cis-regulatory
networks, the capture of the three-dimensional (3D) chromatin structures is required to
explain the cis-regulatory controls. Although a lot of 3D chromatin capture methods
emerged rapidly in the past decades, the requirement of millions of cells as input makes
those techniques unreachable to characterize the chromatin structure in the inner ear,
where the number of hair cells and supporting cells are extremely low. We developed a
de novo HiC technique in the lab for low cell number input taking advantage of the Tn5
protein (Protocol attached in Appendix II). With modified Tn5 instead of the restriction
enzymes in the conventional methods, we speculate the background ratio will decrease
a lot, therefore low input could still get sufficient results (Figure 6.7).

ME sequence
OE_17A/18G
P7 adapter
P5 adapter
Linker DNA
Biotin
Modified Tn5
A B
Figure 6.7. Schematic of the modified Tn5 protein and the transposition and enrichment
steps.
59

Chapter 6. Experimental methods

Animal care and strains
All experiments were conducted according to the policies of the Institutional
Animal Care and Use Committees of the Keck School of Medicine at the University of
Southern California.
P27
Kip1
-GFP mice were previously described in our laboratory (Lee, 2006).
Tg(Atoh1-GFP)1Jejo mice were obtained from Dr. Jane Johnson at the University of
Texas Southwestern (Lumpkin et al., 2003). Sox2
GFP
mice were obtained from the
Jackson laboratory (Arnold et al., 2011). Pax2-Cre mice (Bhattaram et al., 2010;
Ohyama & Groves, 2004) were provided by Dr. Groves, Baylor College of Medicine. To
create conditional double knockout mice, Sox4
fl/fl
Sox11
fl/fl
Sox12
−/−
mice were bred to
Pax2-Cre mice. F1 was then bred back with Sox4
fl/fl
Sox11
fl/fl
Sox12
−/−
animals to
generate homozygous double knockouts of Sox4 and Sox11. Lfng-
Cre
ERt2
::Rosa26tdTomato (Tg(Lfng-cre/ERT2)) transgenic mice were bred with
Pou4f3
DTR
mice that were obtained from Dr. Jennifer Stone, University of Washington
(Justin S Golub et al., 2012; Semerci et al., 2017). These transgenic mice were used for
lineage-tracing of supporting cells after hair cell damage.

Primary cochlear and hair cell cultures
Cochlea were dissected at E12.0 and E13.5 from Atoh1-GFP mice. Isolated
organs were placed in 500 ul collagen culture containing 100ul collagen I gel solution
(Sigma), 0.9M sodium bicarbonate (Sigma) 70 ul, 0.34M sodium hydroxide (Sigma) 133
60

ul and 1M 4(2hydroxyethyl)-1 piperazineethanesulfonic acid (HEPES) 40 ul in 160 ul
10x PBS. After embedding the organs, the wells were flooded with 100 ul hair cell
medium consist of DMEM-F12 medium plus N2 (100x) and B27 (100x), 20mg/L
epidermal growth factor, 20mg/L fibroblast growth factor,15mM HEPES and Insulin
Transferrin-sodium Selenite (ITS). ROCK Inhibitor (Y-27632) is added into the medium
for the first 24hr for better survival.
The cochlea duct was dissected, and the epithelium portion was isolated using
Dispase (Gibco) and collagenase I (Gibco) at 0.25% concentration for 10 min at E12.0
and E13.5 from Atoh1-GFP mice. The cells were dissociated to single cell suspension
and plated onto Matrigel coated 96-well tissue culture plates or cover slips. Cells were
infected with Adenovirus Type 5 (Ad5) containing the full-length coding sequence of
RFP or Sox4-IRES-RFP or Sox11-IRES-RFP or Atoh1-IRES-RFP. The medium
containing virus was replaced after 24hr with fresh hair cell medium.

Immunohistochemistry
For whole-mount organ staining, the epithelium of the cochlea ducts was
obtained as described above and fixed for 30min in 4% paraformaldehyde. Organs were
blocked using the normal donkey serum blocking buffer consist of 20 mM Tris-Buffered
Saline (10x TBS; Bio-Rad), 0.1% Tween-20 (Sigma-Aldrich) and 5% normal donkey
serum (Sigma-Aldrich) around 24hr at room temperature. The primary antibodies were
diluted in the blocking buffer and incubated overnight at 4 ˚C. Primary antibodies used
include goat anti-Sox2 (Invitrogen), rabbit anti-Myo7A (Proteus Bioscience), chicken
anti-GFP (Abcam), rabbit anti-GFP (Torrey Pines Biolabs), guinea pig anti-Sox4 (Thein
61

et al., 2010). Samples were washed with 20 mM TBS supplemented with 0.1% Tween
20 (Sigma-Aldrich) (TBST) for 3 times, each wash for 5 min. Secondary antibodies
conjugated to Alexa Fluor dyes (Life Technologies) were diluted in TBST for 2hr at room
temperature or at 4˚C overnight. 3 μM DAPI (Sigma-Aldrich) were used for nuclei
labeling.
For cryo-sections, the whole inner ears were dissected and fixed using 4%
paraformaldehyde overnight at 4˚C. Then transfer organs to 30% sucrose in PBS until
tissues sank. Organs were then embedded in Tissue-Tek O.C.T. (Sakura) and snap
freeze on dry ice. Cryo-sections at 12 μm were obtained using cryostats and sit at room
temperature for 24hr. If necessary, antigen retrieval was performed in citrate acid buffer
for 5 min at 95˚C. Sections were then blocked using the previously described blocking
buffer at room temperature for 1hr to overnight. primary antibodies including mouse
anti-Isl1 (Invitrogen), rabbit anti-Ebf1 (Millipore), mouse anti-Bcl11a (Abcam), goat anti-
Sox2 (Invitrogen) in blocking solution were applied overnight at 4 ˚C. Secondary
antibodies (Life Technologies) were diluted in TBST for 2hr at room temperature or 4 ˚C
overnight after washing 3 times using TBST. 3 μM DAPI (Sigma-Aldrich) were used for
nuclei labeling.

Molecular cloning of viral plasmids and virus production
Full-length coding sequence for Sox4, Sox11 transcription factors were cloned in
frame with T2A-GFP and then each cloned under the control of a cytomegalovirus
promoter into adeno-associated virus vectors using Gibson assembly (NEB). The
pAnc80L65AAP viral capsid (Landegger et al., 2017; Addgene plasmid 92307) was
62

used to create adeno-associated virus. HEK293T cells were transfected with both
vectors to package the adeno-associated virus. Virus were then purified by CsCl-
gradient centrifugation followed by dialysis (Viral Vector Core Facility, Sanford Burnham
Prebys Medical Discovery Institute). To achieve transduction of the inner ear sensory
organs, each animal was injected at P5 into the lateral ventricle with 5 ul of virus at a
titer of 1012 PFU/mL (Gnedeva et al., 2020).

RNA sequencing
RNA was isolated from collected tissue using Quick-RNA MicroPrep kit (Zymo
Research). QIAseq FX Single Cell RNA Library Kit (Qiagen) was used for library
preparation from the total RNA and the quality of the library was evaluated by
Bioanalyzer (Quick Biology Inc.). We collected two biological replicates for both wildtype
and SoxC genes double knockouts at E13.5. For sequencing, at least 20 million 150
base paired-end reads were sequenced for each replicate. Raw fastq files were mapped
to GRCm38/mm10 genome assembly using STAR at default setting. Gene expression
matrix was generated using RSEM and differentially expressed protein coding genes
were detected by DESeq2 (Log2FoldChange > 2, FDR<0.05).

Single cell RNA sequencing and analysis
The epithelium of the cochlea duct were collected as described above from
E13.5 wildtype and SoxC conditional double knockout animals. Single-cell suspensions
were obtained after trypsin treatment for 10min at 37 ˚C. The standard protocol for the
63

10X single cell kit (V3.0) was followed and each sample was loaded onto separate
lanes on the same chip. Samples were then sequenced using Illumina NextSeq kit.
Sequencing data from 10x runs was aligned and quantified using the CellRanger
software using default parameters. Sequencing results for both reached 50% saturation.
Quality controls and data analysis were done using Seurat package generated by Satija
lab (Butler et al., 2018; Stuart et al., 2019) and Scenic package (Aibar et al., 2017).

ATAC sequencing
Progenitor cells from different developmental stages were purified by FACS into
cold PBS and centrifuged 600g for 5 min. Cell pellet were resuspended with Lysis buffer
containing 10mM TrisHCl (pH=8.0), 5mM MgCl2, 10% DMF, 0.2% NP40 in ddH2O.
Transposition buffer consists of 10 mM Tris-HCL (pH=8.0), 5 mM MgCl2, 10% DMF,
0.2% NP40, and Tn5 transposes was then added and incubate at 37°C for 20 min.
Amplification and library construction was then performed after collecting DNA.
Five thousand FACS purified progenitor cells were used for each of three biological
replicates sequenced for E12.0 and E13.5 organ of Corti. For Sox4 and Sox11 double
knockout experiments, the whole epithelium was collected and processed similarly as
progenitor cells. At least 50 million paired-end reads were sequenced for each sample.
Encode pipelines were adapted for analysis for ATAC-seq data.

CUT&RUN ChIP-seq
The CUT&RUN method for in situ chromatin immunoprecipitation was described
previously (Skene & Henikoff, 2017) and was used to profile Sox4 occupancy of the
64

chromatin in E13.5 progenitors. Protein A-MNase fusion protein was obtained from Dr.
Henikoff’s laboratory at Fred Hutchinson Cancer Research Center. Guinea pig anti-
Sox4 (Thein et al., 2010) antibody were used. CUT&RUN libraries were constructed
using Accel-NGS 2S plus DNA prep kits with single index and MIDs (Swift Bioscience).
At least 20 million paired-end reads were sequenced for each sample.
Encode pipelines were adapted for analysis of CUT&RUN data. Reads from the
raw fastq files were aligned to GRCm38/mm10 genome assembly using STAR package
(Dobin et al., 2013). PCR duplicates were detected, and peaks were called by Model-
based analysis of ChIP-Seq (MACS2) with FDR< 0.01 and the dynamic lambda (--
nolambda) option for individual replicates. For each sample, IDR peaks, overlap peaks
and pooled peaks were identified between the biological replicates. BamCoverage files
were generated directly from BAM files after sorting. Bigwig files were generated with
deepTools (Ramírez et al., 2016). IGV (Robinson et al., 2011) was used to visualize the
genomic loci from Bigwig files. Heatmaps were generated with deepTools based on
normalized Bigwig signal files. HOMER (Heinz et al., 2010) suite was used for
identifying transcription factor motif enrichment of subsets of the picked genomic
regions.

65

Appendix I
Enhancer Testing Assay Protocol
I. Enhancer Selection
Pick enhancers using ATAC-seq and H3K27ac ChIP-seq data:
ATAC peak in distal regions
H3K27ac within 250bp
+/-5kb from TSS of highly expressed genes

II. Molecular Cloning
1. PCR predicted enhancers from mouse genome/other vectors
Design primers ~20bp containing KpnI and EcoRI cutting sites
5’- TAAGCAGGTACCNNN…NNN (KpnI)
5’- TAAGCAGAATTCNNN…NNN (EcoRI)
PCR using Phusion:
Component 50 µl rxn Final Concentration
5x Phusion HF Buffer 10 µl 1x
10mM dNTPs 1 µl 200 µlM
Forward Primer 10uM 2.5 µl 0.5 µlM
Reverse Primer 10uM 2.5 µl 0.5 µlM
Template DNA X µl ~100ng
Phusion DNA
polymerase
0.5 µl 0.02 U/ µl
66

H2O Add to 50
µl

Cycling:
Cycle step Temp. Time Cycles
Initial
Denaturation
98C 30s 1
Denaturation 98C 10s
30 Annealing ~58C 30s
Extension 72C 20s/kb
Final Extension 72C 10min 1
4C hold
Purify the PCR product by Gel Extraction using the Zymo kit
2. Restriction Enzyme digestion
Use KpnI and EcoRI to cut both vector and PCR products
10x
CutSmart
2 µl
DNA 2 µg
KpnI 1 µl
EcoRI 1 µl
H2O Up to 20 µl
37℃digest for 1-2h
67

Run on the agarose gel and cut out the desired bands, then gel recovery using
Zymo Kit.
(Optional) Add Alkaline phosphatase to remove phosphate groups.
3. Ligation
Calculate the exact Vector and Insert volume using NEBiocalculator
http://nebiocalculator.neb.com/#!/ligation
10x Ligation Buffer 1 µl
T4 DNA Ligase 1 µl
Vector X µl
Insert X µl
H2O Up to 10 µl
Mix by pipetting and leave at room temperature for 2h or 4C overnight.

4. Transformation
a. Thaw a tube of NEB 5-alpha Competent E. coli on ice for 10 minutes
b. Add 5µl ligation product to 10µl cell mixture, flick tube 4-5 times to mix cells and
DNA
c. Place mixture on ice for 30 minutes
d. Heat shock at 42C for 30s
e. Place back on ice for 5 minutes
f. Add 500µl SOC into mixture
Sequence map of the pLKO construct. H2B-mRFP express in infected cells, regulatory region linked
with eGFP indicates the activity of regulatory elements.
68

g. Place at 37C for 60 minutes in shaking incubator
h. Warm the Amp plates at 37C incubator
i. Spin at 600rpm for 4 minutes, aspirate supernatant (leave around 50µl) and
resuspend to spread onto the selection plate and incubate at 37C.
5. Mini-prep and sequencing
Pick colonies from plate and culture overnight at 37C, Mini-prep then send to
sequencing to verify the sequence of enhancers cloned into plasmids.
Midi-prep if correct for virus making.

III. Virus Packaging
1. Transfection
a. Seed 7x10
5
293FT cells in 10cm tissue culture dish coated with 0.1% Gelatin.
b. After 24 hrs. When cells are 80-85% confluent, start transfection.
c. Dilute Lipofectamine reagent in Opti-MEM medium
Opti-MEM Medium 500
µl
Lipofectamine 2000
Reagent
10 µl

d. Dilute DNA in Opti-MEM medium
pLKO-enhancer 8ug
PAX2 5ug
VSVG 3ug
69

e. Add diluted DNA to diluted Lipofectamine 2000 at 1:1 ratio, incubate for 10-20
minutes at room temperature.
f. Add DNA-lipid complex to cells incubate overnight.

2. Virus Collection
a. RFP expression could be observed after overnight incubation
b. Replace Opti-MEM with fresh medium (DMEM with 10% FBS), incubate for
another 48h
c. Harvest virus-containing supernatants in culture dish after 24h and 48h, replace
with fresh medium after collection. Keep all viral media at 4C until all collections
are done.
d. Filter the viral media using 0.45uM low protein-binding filter (use only acetate or
polyethersulfone(PES) filters, no nitrocellulose filters).
e. Add Lenti-X concentrator reagent to concentrate virus:
Combine 1 volume of Lenti-X concentrator with 3 volumes of supernatant. Mix by
gentle Inversion.
f. Incubate mixture at 4C overnight or for 24h.
g. Centrifuge sample at 1,500 x g for 45 minutes at 4C.
h. Remove supernatant and gently resuspend the pellet in 100 µl DMEM or 0.2%
BSA in PBS.
i. Store virus at -80C in single –use aliquots.

70

IV. Infection
1. Dissociation of Organ of Corti
a. Transfer organ of Corti in 100 µl of clean PBS into 1.5ml centrifuge tube
b. Add 100 µl of 0.25% trypsin for 10 minutes at 37C water bath
c. Quench the enzymatic reaction with 200 µl MEF media containing 10% serum at
room temperature
d. Triturate gently for 3 minutes to ensure a single-cell suspension
e. Spin 1.5ml tube at 1500 RPM for 5 minutes
f. Discard supernatant
g. Resuspend cells in media sufficient for 100 µl of HCM base + ROCK inhibitor +
EGF + FGF per organ to reach 1000 cells/ 1 µl media (referred as cell
suspension)
2. Infection of Dissociated Organ of Corti
a. Determine amount of cells to be incorporated per well and adjust accordingly for
following steps
b. Add HCM base + ROCK inhibitor + EGF + FGF (referred as HCM) per well to be
infected into 15ml tube, totaling 100 µl per well after cell suspension and virus
have been added
c. Add 1 µl polybrene for every 1000 µl of final media that will be used in the
infection
note: polybrene should be added to HCM secondarily and in drop-wise fashion
d. Wait for 10 minutes
71

e. Separate the necessary amount of polybrene + HCM mix for each virus-type that
will be used to infect cells
f. Add 1-10 µl of full-strength virus (1 µl of virus is sufficient to infect 10,000
HEK293 cells) or adjust accordingly as infectivity decreases ~50% with each
subsequent freeze-thaw cycle (referred as virus + PB mix)
g. Gently mix and wait for 10 minutes
h. Add precalculated amount of cell suspension to virus + PB mix
i. Allow infection overnight in incubator at 37C
j. Remove virus mix the next day and gently replace with 100 µl of fresh HCM base
+ ROCK inhibitor + EGF + FGF
k. Incubate at 37C for 4 days, signals start to show at 48h post infection, strongest
signal could be observed at 96h post infection.

V. Immuno Staining
1. Fix cells using 4% formaldehyde diluted in 1x PBS for 10 minutes at room
temperature
2. Wash 3 times in PBS for 5 min each
3. Block wells in blocking buffer for 1h
4. Aspirate blocking solution and apply diluted primary antibody
For Hair cell: Myosin-VI; rabbit polyclonal antibody (Product # 25-6791) 1:500
Foe supporting cell: ProxI mouse monoclonal antibody (Product # PCRP-
PROX1-1A6) 1:1000
5. Incubate 2h at room temperature overnight at 4C
72

6. Wash with PBS for 3 times
7. Aspirate PBS and apply diluted secondary antibody
Alexa Fluor 647 will be applied at 1:1000 for 2h at room temperature
8. Wash with PBS for 3 times
9. Aspirate PBS and stain with hoechst at 1:1000 for 10 minutes
10. Image plate or store at 4C

HCM base: DMEM/F12 + N2 1%+ B27 2%+ Penicillin 1%
HCM+EGF+FGF+ROCK inhibitor: EGF 2.5ng/ml; FGF 5ng/ml; ROCKi 10µM/ml.
Formaldehyde: Paraformaldehyde 20% solution, Methanol-free, EM grade 15713,
use fresh and store opened vials at 4°C in dark. Dilute 1 in 5 in 1X PBS to make a
4% formaldehyde solution.
Blocking Buffer: (1X PBS/10% goat serum/0.2% Triton™ X-100): To prepare 10
ml, add 1 ml serum and mix well. While stirring, add 20 µl Triton™ X-100.
Dilute antibodies in diluted blocking buffer (1:5)

73

Appendix II
HiC protocol for low cell number
I. Prepare Cells:
1. Collect 10,00~50,000 cells in PBS+10%FBS.
2. Spin at 600G for 5 min, remove supernatant.
3. Resuspend cells with 0.5mL PBS.
4. Spin at 600G for 5min, remove supernatant.

II. Lyse cells:
1. Resuspend cells into 20μL lysis buffer:
Lysis Buffer:

2. Incubate on ice for 15min.

III. Tn5-C transposition:
1. Add 30μL transposition reaction mix into the tube:
10mM TrisHCL (pH=8.0) Stock:100mM 2 μL
5mM MgCl2 Stock:50mM 2 μL
10% DMF Stock:100% 2 μL
0.2% NP-40 Stock:1% 4 μL
ddH2O 10 μL
20 μL
74

Reaction Buffer

2. Incubate at 37°C for 1 hour.
3. Add 300μL ERC (Qiagen enzymatic reaction cleanup buffer) to stop reaction.

IV. DNA extraction:
1. Transfer (Tn5+ERC) solution to QIAGEN MinElute column.
2. Spin at 17,900G for 1min, discard the flow through.
3. Add 700μL Buffer PE, spin at 17,900G for 1 min, and discard the flow through.
4. Spin the empty column at 17,900G for 2min.
5. Place the column into 1.5mL Eppendorf tube, add 14μL H
2
O, incubate for 1 min,
spin at 17,900G for 1min.

V. Gap repairing:
1. Add 2μL 10X T4 ligation buffer and 2μL dNTP (0.33mM), mix by pipetting.
2. Add 1μL T4 DNA polymerase, mix by pipetting, and incubate at room
temperature for 60min.
10mM TrisHCL (pH=8.0) Stock:100mM 3 μL
5mM MgCl2 Stock:50mM 3 μL
10% DMF Stock:100% 3 μL
Tn5 (Tetramer) 0.5 μL
ddH2O 20.5 μL
30 μL
75

3. Add 1μL T4 DNA ligase, mix by pipetting, and incubate at room temperature for
60min.
4. Add 300μL ERC to stop reaction, and purify DNA fragments using MinElute
column as step 8~12
5. Elute fragments into 20μL H
2
O.

VI. Chimeric fragment enrichment:
1. Prepare 10uL of MyOne C1 Dynabeads for each sample by washing twice with
1mL of 1x B&W-T buffer.
2. Resuspend beads in 20 uL of 2 x B&W buffer, and mixed with elute from step 18.
3. Mix on end-to-end rotator at 10rpm for 60 minutes at room temperature
4. Place mixture of magnetic stand to separate supernatant (can be used as
negative control for Tn5-C, or for ATACseq) and beads (Tn5-C chimeric
fragments)
5. Wash beads twice with 0.5mL 1x B&W-T buffer, and then suspend beads with
20μL H
2
O.

VII. Library construction:
1. PCR amplification
PCR mix:
DNA(in beads blurry) 20 μL
2 x NEB NEXT mastermix 25 μL
Primer Ad1 2.5 μL
76

Primer barcode 2.5 μL
50 μL

PCR program:
95 °C 2 min
95 °C 10s
63 °C 30s
72 °C 1 min
Repeat 2-4 for 12-14 cycles
72 °C 5 min
4 °C hold

2. Put tubes on magnetic stand, wait for ~2 min, transfer liquid to a new tube.
3. PCR production purification using SPRIselect beads:
a. Add 40μL SPRIselect beads into 50μL PCR reaction mix, and mix by
pipetting.
b. Incubate at room temperature for 5 min, then place on magnetic stand.
c. Remove solutions, and wash beads with fresh 80% ethanol twice.
d. Short spin with mini centrifuge.
e. Place the tube back on a magnetic stand to remove remaining ethanol.
f. Resuspend beads with 20μL Low EDTA TE, wait for 1 min at room
temperature.
g. Place the tube back on a magnetic stand and save the solution.
77

4. Library QC by Tapestation or Bioanalyzer.

2x B&W Buffer:
Reagents Amount Final Concentration
1M Tris (pH=8.0) 0.5 ml 10mM
5M NaCl 20 ml 2M
0.5M EDTA 100 μL 1mM
H2O Bring to 50 ml

1X B&W Buffer:
Reagents Amount Final Concentration
2x B&W Buffer 2 ml 1x
Tween-20 (10%) 20 μL 0.05%
H2O 1.98 ml

78

Appendix III
Enhancer list
1. Hair cell enhancers

Gbs Atoh1 st1 Size 604bp
F: AGAATGGGTTAAATCCTTGGAAG -
cttttatgcccagcccatcgAGAATGGGTTAAATCCTTGGAAG
R: TGGTTCACTGCTTCAGGTGCCT -
gtcattggtcttaaaggtacTGGTTCACTGCTTCAGGTG
AGAATGGGTTAAATCCTTGGAAGcaggggagaggcaggggaggagagaagtcggaggagtataaa
gaaaaggacaggaaccaagaagcgtgggggtggtttgccgtaatgtgagtgtttcttaattagagaacggttgacaatag
agggtctggcagaggctcctggccgcggtgcggagcgtctggagcggagcacgcgctgtcagctggtgagcgcactct
cctttcaggcagctccccggggagctgtgcggccacatttaacaccatcatcacccctccccggcctcctcaacctcggcc
tcctcctcgtcgacagccttccttggccccccaccagcagagctcacagtagcgagcgtctctcgccgtctcccgcactcg
gccggggcccctctcctcccccagctgcgcagcgggagccgccactgcccactgcacctcccagcaaccagcccagc
acgcaaagaagctgcgcaaagttaaagccaagcaatgccaaggggaggggaagctggaggcggcagaggcggcg
gcgggagagctgtggggttgttcaggaaaagttgggctgggggctgAGGCACCTGAAGCAGTGAACCA

Gbs Atoh1 st2 Size 553bp
F: TGAAAGGCAGGGTGGATAATGT -
cttttatgcccagcccatcgTGAAAGGCAGGGTGGATAATG
R: TGGTTTACAATTTCAAACAGTTCC -
gtcattggtcttaaaggtacTGGTTTACAATTTCAAACAGTTCC
79

TGAAAGGCAGGGTGGATAATGTgacacgcgctttactttcagcagacttgttaatatcagctctgaatttttca
tgacggctttggtaacctgtagcctggagttgaagctctgggggtttccaccacaaagaggtggatcagctgagctctcactt
cccatctggcactgggtgacagggacagtcagggggactgcaggaaacctgaagaaggagaaagctgatgtgggac
acccattgtcgtgcataaaaggctaaacatgtgctgttgtgtggatatatctttgagacacttgtttatcactggcttcagaatat
agttagtcaatgtgtttaactttatagtcacaagaacagctgccgctcgtgtaagtaataccctcatacacagtggcatcctga
ttcgccaaaccagattcaggacctctcctcagtgactatattatgctgcctggcatgcttcttatttacccacagaggcaggca
tacacgtgcaggcacacacacggacacacaccgtacagtttcGGAACTGTTTGAAATTGTAAACCA

Gbs Bdnf st1 Size 726bp Ordered 02/16/2017
F: ATCCCAACTCTGCGCAGGTGGA -
cttttatgcccagcccatcgATCCCAACTCTGCGCAGG
R: CCCTAGCAACTGTATTAGGCAGC -
gtcattggtcttaaaggtacCCCTAGCAACTGTATTAGGCAG
ATCCCAACTCTGCGCAGGTGGAttcataggcgaagcgaggatattgtggaaattcagaaggaaaagata
aaaaacaggcgctaggatcagatgacggtgataggctgctcggcacacaaagggagcgtagggcagggtttacggag
caagcctgcagcgaatggggcacagattgttccgagatccagtcgttttctcagtcagatctacgcgaagggaggggag
gggaggggcgggcaggggagcgtggcgggaggggctgagcttgggggcggggggatttctgatcagtctgatgcaatt
ccaagcgtgctgcaaaggaactccaaggcgcccgcatcaccatcgccacccacccttcccagatggtgctgttttaaata
cggatctgcagggctgaacgcagaactgggagatttattgcaaaatcccgggaggggcgggggggggtggtgtgcgg
aacggggaatggaggagcagaatttaaaggtgcaacgcttgctttttccaatcaggcggcaaccggccggaattattatttt
tttctttctgtctgcttgtctctggattctaattcaccaagaaagaggtgtaaatattgtgacattttgaggcagcttgatggatgg
gaaagaaatcatctgtcactctaaattgcagagttccctctccccgcgccatcccttgctagcgaatactcgctGCTGCC
TAATACAGTTGCTAGGG
80

Gbs Bdnf st2 new Size 727bp Ordered 02/16/2017
F: ATCCCAACTCTGCGCAGG - cttttatgcccagcccatcgATCCCAACTCTGCGCAGG
R: GCCCTAGCAACTGTATTAGGCA -
gtcattggtcttaaaggtacGCCCTAGCAACTGTATTAGGC
ATCCCAACTCTGCGCAGGtggattcataggcgaagcgaggatattgtggaaattcagaaggaaaagataa
aaaacaggcgctaggatcagatgacggtgataggctgctcggcacacaaagggagcgtagggcagggtttacggagc
aagcctgcagcgaatggggcacagattgttccgagatccagtcgttttctcagtcagatctacgcgaagggaggggagg
ggaggggcgggcaggggagcgtggcgggaggggctgagcttgggggcggggggatttctgatcagtctgatgcaattc
caagcgtgctgcaaaggaactccaaggcgcccgcatcaccatcgccacccacccttcccagatggtgctgttttaaatac
ggatctgcagggctgaacgcagaactgggagatttattgcaaaatcccgggaggggcgggggggggtggtgtgcgga
acggggaatggaggagcagaatttaaaggtgcaacgcttgctttttccaatcaggcggcaaccggccggaattattatttttt
tctttctgtctgcttgtctctggattctaattcaccaagaaagaggtgtaaatattgtgacattttgaggcagcttgatggatggg
aaagaaatcatctgtcactctaaattgcagagttccctctccccgcgccatcccttgctagcgaatactcgctgcTGCCT
AATACAGTTGCTAGGGC

Gbs Dll1 Size 418bp Ordered 02/16/2017
F: GGCTGCTCCAGAGGAGTCC - cttttatgcccagcccatcgGGCTGCTCCAGAGGAGTC
R: AGCCTGAACCAACATTAGAGGTT -
gtcattggtcttaaaggtacAGCCTGAACCAACATTAGAGG
GGCTGCTCCAGAGGAGTCCcggagagcgcggccgtggaagctttcgaacgcgccgtgcttccccgccgc
ccccgcctccctttctgtgagcatctgccggggagaacagatgtgggcacctgcggtgaccggctgcgtgcgttccatccc
attcacaggcattcagacgtggccacaatgagcggcctttgtgtgcacagagtcactaaatatattaatctgtactccccag
81

agcaccgggctgtttaatttttcactagcaataacatctgatctaatccttaaatcggctcccaaagccttccctccagcccac
cccgcccctccccccgaaaaacgtgtgaagttattcaactttccacctgttgccggctgcactgggcgtgggttattgcAA
CCTCTAATGTTGGTTCAGGCT

Gbs Dll3 Size 464bp Ordered 02/16/2017
F: TTCCCCCTCCGCAAGACAAA -
cttttatgcccagcccatcgTTCCCCCTCCGCAAGACAAAG
R: TTTCCCCTATAATTATTAGC -
gtcattggtcttaaaggtacTTTCCCCTATAATTATTAGCCTCTAAGCAC
TTCCCCCTCCGCAAGACAAAgccaccctcccaaaccccgcatcgcggacgcaccagacgccagggcg
aggagctcggcctcaagagggggctcccagctgtatgtaaatgccgccatctgccgggcggccgagcccctcccccttc
gggccgcagctgggctgcccgtttggcctccggaaggtgtgaggagcaaatgggcaggaaattccgtcttcacatacag
ggacccgctcccccaccccagaagaactggggggggggacatcccgggcccttcagaaaccctttagtattccaggac
ctcagcaggaaaagccaattaggagcagatgtcccagcccctctggtcataattgctccctgccaggcatggggcaaaa
cccttgagctggggtaacttcttttttttcccccccaaagtaaaaaactttagtgcttagagGCTAATAATTATAGG
GGAAA

Gbs Fgf8 Size 696bp Ordered 02/16/2017
F: GAGGGCGAGCGGCCCCAGAC -
cttttatgcccagcccatcgGAGGGCGAGCGGCCCCAG
R: AAGGTAGGCAGAAGGCCACAG -
gtcattggtcttaaaggtacAAGGTAGGCAGAAGGCCACAGGG
82

GAGGGCGAGCGGCCCCAGACagcaggtgggcaccccagcagatggcgaggtgggcaggagccgca
gccccaccccctgcctgggcacccgctctctaatccctcacaactcgcagcccacccggcaacttcctgtcctcccaggc
agcagtgacacatggttgtctacggaggggctgggccccaacgcagaaagacagagagaggggcccagagcctggc
ctgctctggcctcctgcccaccagtctggccacccgtctccccacctgcccctctcacccccccaggacttcggttccctggc
cccagggcagccaccctatgggggaggggaggcctgggagaagatgctaagaccccagccccagaagcccagga
agtggggagctcgaggctggagaggagccgtgactaatggagccatttcagagctcggccgaggggctgggcctgcg
gcttactgaacattccaaatgctggtaccatgtaattggacataatagcaaagcaatttggcgatttgttaaaaagatatatg
gaatttaccaaccaccaccccccagccccttccctagccttcattcttcctccttcgccttttatagttttcatctctcaaacagctt
ccttctccctcactcctgcacccaatcgtgaggtaaccccaagatggccCTGTGGCCTTCTGCCTACCTT

Gbs Fhod3 Size 594bp Ordered 02/16/2017
F: CTTACAAGGTTAAAATCATGGT -
cttttatgcccagcccatcgCTTACAAGGTTAAAATCATGGTC
R: TAGTCCCAACCCAGTGTCAGCAC-
gtcattggtcttaaaggtacTAGTCCCAACCCAGTGTC
CTTACAAGGTTAAAATCATGGTctattacttttatagtaggtctggctataaagatgttaaatttttaccaagtat
caaattcaaggaaaatgttgtgaaataacattggatgcattcaaaggaattttaaaataattataatttgccgtcttctccttggt
cagagggtactttggaatcttactttgagctgtgcaggcaaaaggaattattccacttttctctatttggctttgctctgttgttgact
attcctcttgagttcaagtggaggcctcctggacagctggtggccagggcagctgtagtgacccaactctagtgtgcccctc
ccaaggatggagctcccagtttagctgttgggtttgtacataaaatgccacagcacagcagtctgagctcttgctttctgtttaa
ataaaaaggcagatgcttcacacatgttttcatgaatttatataaaccatggaaggaaagaatgctgatattcaataggaac
cctgtagcaacttgttacttgtaatttttaggtttactgtaagagcaaatgcagaaagcaaacctgcatctGTGCTGACA
CTGGGTTGGGACTA
83

Gbs Gfi1 Size 602bp Ordered 03/13/2017
F: TTGAACAAACAGGCACACAAC - cttttatgcccagcccatcgTTGAACAAACAGGCACAC
R: TGTATCTTGATATCCCCAGTTCAG -
gtcattggtcttaaaggtacTGTATCTTGATATCCCCAG
TTGAACAAACAGGCACACAACaaagtgacttgggtcaaatcactatcaccacccagttttcaagggcaatg
gcagtcaagaagtcgccggggagtgatactcttctatccctttaattaaggcggggcttttaaaactcaattgttccatccgtgt
ctcaaatcaatcagatgaaattcaccttttacgtggcctgaatcctgatagctcagcacacttgataggtaatttaatcacgtct
ggtgtagatgaacgcaaactcaaattagcgctgaaagagttggtcttctcagagatagctgtgcttttccacttttatgcaaga
gctgagcttcaccaactggctagctggaggtgttattaaaaatttagtgaaagaaacaaaggtgtgctctggtgtctcttgtta
ccctttctactgttactccagcagacaaaaggcatagagcctgcctcatgaataatgtgacatggaatattcacaatctattttt
aatggagtcaagtaataacattaagagagatatttgcaatcttcaaatttcacacggtaacaggcacagtaatttgttttgatt
CTGAACTGGGGATATCAAGATACA

Gbs Myo6 Size 397bp Ordered 03/13/2017
F: ATGCATGAGACTTTATTCCAGTA -
cttttatgcccagcccatcgATGCATGAGACTTTATTCC
R: TAGCCACATTTAAACTTAAATA -
gtcattggtcttaaaggtacTAGCCACATTTAAACTTAAATATG
ATGCATGAGACTTTATTCCAGTAtcacctaactgtgtagtcctttggtgactggagataaattaccccactgt
agttctcctgttataaagatggggctgttcattatttttgaacgaagaaaacacatggcttctttgacacttactagatcaaatga
cagtcttaatctattatatcactatcagtttgcctaattgtacttacttaattctgactaaattacatttagtctctgccatctgtccaa
84

gtagtgacagctgggagatagaacttatttattctattccaaaagagtatacttgccatcttctagttatatgtagttttaagacttt
tctccctgattttttcaaaatcaactaaataaagaacaTATTTAAGTTTAAATGTGGCTA

Gbs Pou4f3 st1n Size 239bp
F: GGCCTCTGGCCCCTGAAT - cttttatgcccagcccatcgGGCCTCTGGCCCCTGAAT
R: CGCCCGAACTCAAGATGCCATCG -
gtcattggtcttaaaggtacCGCCCGAACTCAAGATGC
GGCCTCTGGCCCCTGAATccctctgggcgtctcggatcctctgagccactttgcagacaatgcaaggcagcg
gccaacagacgagtcccgcagcagctgcgcaggcggcgagtacatgtgagaagtttgtgaaaggcgaccagagaaa
gggagaggaaggcaagtcaggcggctgcgaacacctggccaggcagccccgccagctgcctcgctgcgCGATG
GCATCTTGAGTTCGGGCG

Gbs Rasd2 Size 470bp
F: AGGAAATGTTAGCCTGACCA - cttttatgcccagcccatcgAGGAAATGTTAGCCTGAC
R: TAGGAGGAGAAATCTTGCAG - gtcattggtcttaaaggtacTAGGAGGAGAAATCTTGC
AGGAAATGTTAGCCTGACCAggtggaaagctcacccctgctcgccatctgctgaagaagggtcacgtaca
tgccacctggctttctgcggggagatagcatctggaaggcagagaaagctcctctgggtggggagaagtttcactgggca
ccatcatgatttattcagcagcgtcagacacctgcaccctggggccagcgccggccttggtggttctccaggattcgttctgg
gtgcacacacctgtctcttcctacacccggggaatggggggtgggggacagaggccagtccagaggcagcagcctctc
ccacccttagcctgggtagggggaggaagcagtcctggccagagccccagactcacagcggatgtggccagctcacct
ggtggcaagagggagccagaaacgtccttggggccctgagtcccagtgccaggactgttccCTGCAAGATTTC
TCCTCCTA

85

Gbs Rtn4rl2 Size 543bp Ordered 03/13/2017
F: TTGGAGAAGGGCATCATGTACAG -
cttttatgcccagcccatcgTTGGAGAAGGGCATCATG
R: TTCCCAAGGCCAAGACTGCACCT -
gtcattggtcttaaaggtacTTCCCAAGGCCAAGACTG
TTGGAGAAGGGCATCATGTACAGaggtgaaaggagatgctcagttttcagaacatctgacaagtctatct
ggaagcattaaaaattcatatagaaacctgaccccagctggaccttgggcctgccacctggtctatccctggcctcctgga
ctctcagagtctgttgggaaatttcccttccacagtgtcctgagagtcccaagtctgtatttcattggagaggagttcctctcgg
attagggatcgaaaattgggagcccgcagctagggaaacagggctgagaaccgtcccccccctactctgggtgtaaga
accttagaggcttaagggcttataaaccttatcatctggtgtcctagtcctctccccaggcggttagatgctcattctctgttcta
gacgactgaagcctggctgtgcttcccaaggtggccagtcgggggcagccgtctgcttggagggagccagagcctgag
cctctgtctgctgctttgaagaacccctgtatatggtcagagAGGTGCAGTCTTGGCCTTGGGAA

2. Supporting cell enhancers

Gbs Jag1B Size 1768bp Ordered 03/13/2017
F: CAGCACTCTTCCCAGGTTGACAA -
cttttatgcccagcccatcgCAGCACTCTTCCCAGGTTG
R: TGATGTCGGTCAGCAACTTCTGAG -
gtcattggtcttaaaggtacTGATGTCGGTCAGCAACTTC
CAGCACTCTTCCCAGGTTGACAAatgactcctttcacctggcagggacgcactggagtcaacatgcatgc
aggcagctcaaagttgagatcaggaaatgccacctcactatatgatatgagagtatttagcatatagtttcccaaaactttcc
agaccctgaccctagtgcatgaggaaacacacacacacacacacacacacacacacacacacacaaagaatttattc
86

taagctaacctaaccaggtatttgtaccgagggggtattgctaaggttacactttgggccctggtgaggcagtctctaggga
ggcccccttttcctttttagaacccctttgaaaactctgtctatgtgagaccatcctggctcccacaagtatcgggtaactctag
gaaacaggagggggtagatgcaaagtgactctgcaagacaaagaagtcaacctcacttgggcggtggagttgggggc
agatcccagggaagaaagtacaaagggagaaacaaccgcacacccatgggttatatttattgatgaacttatctgttaata
agatctaacattccaagaataatcagagtctactaactaaacaaattccctggatactggcaagatgccagctgttaaact
gaaggcaccttttatcttccaagcttatctcagaattccacctccccacccccagccaaacctaagctattagcaaattccct
ccttttctaaaattttccttcgtaatttacagtaaggtcagtctcacaaaacaccagcgtttaaatgaagcttctgctatcttggca
ctgaagaaatgtactcaaggcaggtggggtagacaaggaacatgaactaggttatgaatccctgctttgctgtgtcagaa
accaagccataggaaatagaagcgtgtatagattaaataataccgtgctgtgccaccagaagcacactgctgccaaga
gaagagaaagcacctataagataaaggaaaaagagaaaaagggagggtaaaagggagtttcattcataactcaagg
ccagcttagcgctctcagagtgacacagcaggtcatctctgaatataaaaatatcttgtgatagaccctgccttggaattcag
ccgaaatatgtttcatactcaaagcagagtaggattccatttcgttcactgataactgtgggaaaatgctcagaaaagcaag
caaaaccgggcaagccagggcacatacccagccctctcctgttagtggccacaggtccaaattcattgctgttgttactattt
tattagcgaaagttataccttatttactgcctctttaagtttcttaggacttcctgcaaattacaggactcttccctaagagcagca
agaaattatatttctctactgatttgaggtaagagcacagtcaaaatgaggccagatgtgtggctgattctgtataattaagcc
cttcatttgctggtcacgttgaggcccacgtcctccacttcaacagtattgttgtaattaatcctctgattaaatgcattgatacca
ggagttttgacagacttgcacaagcgctaaacacataatacccacaaaccccactgcaagggaaggtgccgggactga
gctggcatggcagtactaactcagtttaatactcaaagaggaaagcatccttaggtcttgcacaagaacttggacgacca
agcagtctaccaggctcatcctcgccagaactgggaaaacCTCAGAAGTTGCTGACCGACATCA

Gbs Hey1A Size 1824bp Ordered 03/13/2017
F: AATATCCTGGCAGGTTTCCTCAG -
cttttatgcccagcccatcgAATATCCTGGCAGGTTTC
87

R: GCAATCTCTGCTCTTCTAAAAGA -
gtcattggtcttaaaggtacGCAATCTCTGCTCTTCTAAAAG
AATATCCTGGCAGGTTTCCTCAGgcccaggcattgtacaaaactccagaagctgctcagtctgagaagc
ggagcgctatgcagaaagcccccggcatcgtcacaacatgggacctttagtgggcagttgtttgaggtcattttctcatcagt
agcagcaggctcaaaatggatgtgcaatgcaaaccaggagactagggagcaccgagcagcctcaggggttgttttctg
cctggttcaagagaaactgccgtagaaatttttgccgccagcctgttgttgagtgacaggaagagaaaaaaggatcatttg
aaggctccctgattcaaatggcgtatacagaatgccaggccctggctgccctggagaaagccactctggtgccagcttag
ggaacaaatgtgttcccactgtctcctggaaaggctttccccacccctgctgagggggtggatttcaaagcagctgacttctt
gccctttttaataggactgcaagtgttcccattttaattctaaaactttctcccaattttccactcctccaaggcctcccaagaaa
agctggtattctgggtcactgaaatggcgtcaggttattaagcagagaaaggggattagaggaacagttcctagcagcag
cagcgaacacagttacggtattgagagcttgcgttgcatgttaataaaatgctataaaacatcaaaccctaaaggtagcag
tgaaagccacgtaagtcctttccggagtttgccttctgcagctgtggatctaaaagttccctgcctttcctgtactttcataatcac
gcttttcctcctttagagagacacgcagcctcactcccaaaccatggtgtgttttaaatgctgaccaccccatctttccctggca
ccatggagcgtttcagtctaaaaccctagggtctgcgaattctggccagtgcgctggggagaggtagtagagtaattaagtt
agctgttagctgctgtgagcggacttcggtatttcatcgcagctgcctgcctctgtctccccagccgccctcccacatcttccta
tgagaccggctgtgtgtgtgaaggaggtgctgtttcccacagctctgatctaattcagctgaaattcaatctccgtgtgaccaa
cctcagccagcgctctcccacccaccgccggcctcccctctgcaggctgtgtgtgctgcccatttacaccatcaatcccaca
gcctcccctccaccagcccctgggtatagacttacggtgtcagcgggccagccttgggggctcgcctgctgcccggacgc
tcacccctcatccccagtactccgaggtgagacactctatacctgccactcggtggcctgaatggggcctgtgagtcatcgc
aggagaaagggcagtggggccagctgaggctgagaacccaaaacagatgtcagagacatcaggccacgttttccatg
gatcttcagtgcccagaaagatttatgacctcttttaacccccttttaacgtgggttgaactgggcttgtcttataggggggaaa
caagtctggcagtcattcagaaagtcaccagagcgacagggcctgccctgggtctgtggacccttgcttcctttctttgtcatc
cctctcttaagctccatcccttagcctctgtgtttggtggggacattccatcacagactcatcagagctctccatcccttgacag
88

cccaaagctgcttgtggctttgaccctaaatggtattcaatttgggtttgcatcttcatactgaacaaggaaataataagtaTC
TTTTAGAAGAGCAGAGATTGC

Gbs Hey2A Size 774bp Ordered 03/13/2017
F: CAGACAGTTGCATGAAAGGAAAG -
cttttatgcccagcccatcgCAGACAGTTGCATGAAAG
R: GTTCTGAAGGATCCAGATCAGGT -
gtcattggtcttaaaggtacGTTCTGAAGGATCCAGATC
CAGACAGTTGCATGAAAGGAAAGgccctgatttttatgacaagcaagtagaagcaaacccacagaaca
atctatccatttgggtataaatggaaaatctagtgccctcctgccttcttgtcctttcacttccattttcagttttcattttcacacccc
acagaagctgatgctgtgccctatgcactgactcctatcccacaacaaaaagacttccccaactgaataggcatggttgcc
atggcagctagggggtcataaactcctgagctgtgggtcctggcacaattccacgcattatctgcatcctggtgcaaatcag
tccctttatctaattagtagccttacaaatagccacctgccccaaagcttaaccattaaagatgcaatattggctgaatagctt
gagaccagaggtgaggatatcagaaaactgaaagctgatctgagtgaggccctatctctcctggctactgacacctgtga
gccctagcccacccacataccaggagttcagaaggagaccaagaacaaatcacttctgtcttcttttcgtgactcaccttgg
gtgtcttctgatgcactggcctcccagaagctgggcctgctgacctctgtctaaaggacctggttcatcattcctgcttagatgc
ccaacccttccctgcctgagaacttcctccctaagaagtccagccctgtctctctctctctagaacttcatcaactgccctggc
ctgttgtagttcactACCTGATCTGGATCCTTCAGAAC

Gbs Lfng Size 1648bp Ordered 03/13/2017

89

F: GTCAGCTGCCAGCCAGCGGCTGAG -
cttttatgcccagcccatcgGTCAGCTGCCAGCCAGCG
R: ACCCCCTGGCCCACTCTGCCTG -
gtcattggtcttaaaggtacACCCCCTGGCCCACTCTG
GTCAGCTGCCAGCCAGCGGCTGAGgaccagccctgcaatctgagtgggggcctgtgggaggatgcag
ctgaggggagtggcgaagctgaagaaactctcgagaaaggtgggaggggccggggcagtgtcagggccgctgtgca
catccgtgcaggtcgtgcactgactggcagcacacatctgagatccagcccgtgctccgctcatcgggttgtgaagaagc
agtacctctttccaagcagtggcccaggcagtaagcggagcatctggcaggcggggtccacacttccccaagaccctgt
gctggctgtggctgtatgtgcacacttctacatgacttgcagggtgcccaaacggcccggaggaggccgggaccccagc
ccccaggccagggctcccgtgagtagggagacgcatcactctctggccccggaggctgtgcttcccaggccatcacaat
ggccacacaacaggggccctcagggacggaacctaatcttgagctattgaagacctgaaagccgtggcccctggcccg
agccagcagacgcgcctgccggccccaccagagacacctgatccaccggtcgccccgccacaaagggggacactgt
ccccccagggccgcccgccccaccaacagagacaaggaggaaaactgggtcgctgagatagagtgacagccatgg
caggtggggatgagacaggagggaggggcggccaccactgggcacagcccagcctctggggatgccccaggcctg
aagatctgcccgaggggggctcagagccccgaggagggctcaagtgactgccggtttgcagggagtggggtggaggc
tggagtggggggcgcagctgagaccagaaagaaacagctaggaccccacctgaggctggagacacagccaggaca
ggggactctccacccagagagacgccataggccaggttgttggggacaggcctggactggtccccaggccccaccctg
ctgaggcaggccagccagctgcccaccccttggcctgggcatgtccagcttttgctcagtttcccagcctggcagggctttc
cgaggggttgcagggagaagcaggcctggccaaaagaccgccatgcctgaaggttcaccttcaccccctggcaccac
caaagaccccgcccaggggaagtcttggagcctcagcgcctcagaactttgggacttcctggcatatgggaggcacccg
gaggtgtgagcctgaggggttcactttcaagggtggctggagacacctgctgctggccatgtgggcctgggaggccttctg
gggcctccatcttcccatctgtgaaatgggacttggacctagatgctcttcaaggtcccttcttccaagtcttctggcaagagc
caagtccaggaagctcagctccattgcgtcggcatctggactgagtactctgtgggtgccgggctgtgctgagggctggac
90

gggacccaggaacagacaacaggtagagtggaggagggagcagcgaggagacttcccacaggggtggagacgct
gagcactccaggcagacacagaggggcggctggagtgtggggatcctgggcctggcagggcctggggggttcggCA
GGCAGAGTGGGCCAGGGGGT

Gbs Mreg Size 312bp Ordered 03/13/2017
F: CACCTTTGGAACCTCTATGTATCC -
cttttatgcccagcccatcgCACCTTTGGAACCTCTATG
R: ACCTTTTGAGAGTAAAAGCAGCTT -
gtcattggtcttaaaggtacACCTTTTGAGAGTAAAAGC
CACCTTTGGAACCTCTATGTATCCcagaccacgaagggctctggggcctgtgtcaacagaatttagtgc
aaaggcctgggggtctgggcctgaaaacccctgggaggacagatggcgggaaacagaccataagtcagaaggttcc
ctttctctccctcccccatatcatgagttctaaaaggctctaaagaaagtgagagtggttgtgtttcccctggccaccacctgct
gatgataaatattttattaacatcaggcatttgagaggtggctctgatattacacactAAGCTGCTTTTACTCTCA
AAAGGT

Gbs Myo7A Size 407bp Ordered 03/13/2017
F: GCAGCCCCCACTCTCAGGGCCT -
cttttatgcccagcccatcgGCAGCCCCCACTCTCAGG
R: GGTGCTGGGATTAGAACCCAGGA -
gtcattggtcttaaaggtacGGTGCTGGGATTAGAACCCAG
GCAGCCCCCACTCTCAGGGCCTgggtggaggagaagcgggaggggacctttgggattcggtgacgca
gttacgggacaccatctgacatgcaacatgacccaggtgaaactcaagcctgcagacagagtccctggtccccaccctg
ggggctctgggcaggttagcaggcccgtctatgctgccacccagcctgtgcctccccttctgtggctgcaccctcatagtag
91

gttcctagagggtcctagaactggagcctctggaatgatattgcaggccttgggttctacggcccaagggccaaaaatgtg
attagcagaatctgagctccaaagtcgccgctgagataatgctgacaggggaaactcatgcatagaagacagaggTC
CTGGGTTCTAATCCCAGCACC

Gbs Id1 Up Size 764bp Ordered 01/31/2017
F: AGCTGGAAAGAGAACTCAGGCC -
cttttatgcccagcccatcgAGCTGGAAAGAGAACTCAGG
R: AAAGCGGGCGCTCCTTTACATCT -
gtcattggtcttaaaggtacAAAGCGGGCGCTCCTTTAC
AGCTGGAAAGAGAACTCAGGCCtttttccccacgctggaaggggtagctggggcggagaggtgggggg
agcggtgaagaaaccccaagcggcccaagctgtgggtctgggttgggagactcgcaggtgtggggcggggaggtaag
gtgacccttgctcagcgaccgcgcccgcaagaaacgcattcccaggcctcccgcccggggtctgcaggtgacgggctg
gggggagcacgggaactagctagaccagtttgtcgtctccatggcgaccgcccgcgcggcgccagcctgacagtccgt
ccgggttttatgaatgggtgacgtcacaggcctggcgtctaacggtctgagccgctggttcagacgctgacacagaccgg
cccgggaagggaggggggagactgtagctccgcagctgccgcgccgtgggagggagaccctgctctgaggtctttgag
aagaaaatttaaaaagcagccaaaaatgggaaaaaacattaaaaaatcacgaactgttgcaggttcaggaaatttttgc
aaggagctgcaaattcaaggtggaatcgaatgcagcctcactccactgcgctctatctagttcacttcccagccacccagc
cccaaacttactagactttcccgaattaattgctcccacccgggagggatctgggtaggccctccgggtctcaggaacacg
aacagcaacattatttaggaattgagaaagcccaggggagcAGATGTAAAGGAGCGCCCGCTTT

Gbs Id1 Down Size 973bp Ordered 01/31/2017

92

F: ATCGAAGAACCCAGTACTTGCAGG -
cttttatgcccagcccatcgATCGAAGAACCCAGTACTTG
R: GATAGCACTGGCCTCGTTAACAA -
gtcattggtcttaaaggtacGATAGCACTGGCCTCGTTAAC
ATCGAAGAACCCAGTACTTGCAGGcgaattatgttctttgctacctcctccccaccggctgctgtattacagt
actaacctcacggaatgtagtacagaataaatgaaataaagtaagtgtgtaaaacgtttagcacagtgcttggcacagaa
tgtttcatagttataaattattattgtcgcctgtgagtccttggagtgtcctcggaggcacacgtgtgcacgcacgcactctcac
gtgtgggcccccgcctgcaagcccacccacggggtctgagcgggttccccctcgcgtactccgacatgcagggcctttttc
caggggaggaaggggctgcggcgggggcctgggccacctcccccgccattcaggcacactctggattccacgtttccct
ctgttcccctttggcccagacccagcccatctggaggccggctccgcggcggcctgggagcgtttccatcagctgggccc
gaggaatgcggagctatttaacctgagcatccccaggtgtacggaggcgcctggctgtctggggcccgcagcctttggca
cagccgcgctagacaaacagcgcgccgcccccgccctgcccctccgcccccagctgcagccgggcctgggaaccgg
cgggcgccggcggcttgcataagaggctccgggcctcgcagagggaggggggacagaggccaccggtgtgtgtgcat
gtaacacacaaaaccctgctcctttggaaatcattaactccttaacccctcctggacaggacgaagccaggagctggggt
ggaagatgccgagaaacccgaggaccaagtttcttgggcactggactctgcctttccggggctgtactgttaattttatttaa
gaaatacagagagcttattgtgtgctgggcaccatgctaaactctttacaatatcaattcatttaatcgtcagaacttGTTA
ACGAGGCCAGTGCTATC

Gbs Id2 Up Size 855bp Ordered 01/31/2017
F: TTTTGCAAAAGGAAGATTTCTTTC -
cttttatgcccagcccatcgTTTTGCAAAAGGAAGATTTCTTTCTTCTCCCTCTCC
R: GCGGGCGGTGGGAAGGTGTGCAGG -
gtcattggtcttaaaggtacGCGGGCGGTGGGAAGGTG
93

TTTTGCAAAAGGAAGATTTCTTTCttctccctctcccggcttttttccttcgtcctctcctccgcccctcacccgc
ccacccccccccccccccgagcccttttcttttcttctcggtcctctttccttccccaaatcactcgaaacttaagacgtgcagc
gtctgcaggtgcgcgcgggccggcggccgggcctgggcgagcctgggactgccgggcctcgcaggcattgatcagctg
ggcgcgcgcgctgagtgacggcgcggttgccatggcagccgcctgagcggcgccgcgaggacaaggctgcagggcg
gcgtgaatgggcggcgtcacgcgcctggcgccagagagtctgctccggggctccggctccggccccgccgcggcctgg
cccgcgcgccgccgccgccgccaccgccgccgccaattcatcacctgtcagggatcactcgcggggtcacgggcgga
atggacacagctgtgagaacaaaacccgggaaagaaaggcgaacgctccccattgcgccagatgtgcagggaggac
cttgagacgcgccccccgcccccgcaggctcctcccgccccccggcgcatcacgcgggggtggcaaaggcggcctgg
ccagcgcggagctcccggcccggagctgcttctgattaccgcgaggggcccggacgcgagagccgccgcggggcctg
ccctagaggcggagtgagtgagtcctggagccaggctgggggaccttacgggccggtctgtcgctgccacggggtggg
ggcgtcccggtggggagtcgaggggaaccgcaccaacacccaccagcggaagcCCTGCACACCTTCCCA
CCGCCCGC

Gbs Id2 Up (NEW) Size 1283bp Ordered
F: TTCAAAGGTATTTTTAGGAATCC -
cttttatgcccagcccatcgTTCAAAGGTATTTTTAGGAATCC
R: TGATTTAAACGAGGCCTAAG -
gtcattggtcttaaaggtacTGATTTAAACGAGGCCTAAG
TTCAAAGGTATTTTTAGGAATCCaggatcaaagccattcggcgagaaaccgaaataatccaaaggagt
aaataaatctcgccatgtgctaagacagtgaccttctccccatccacccttccctcgcgccagccccaccctgaacgcctc
ggttgcaaaaacaaaataaagcaaaaccttttgctttggcaacctctaagccacattatgcctccttttgcaaaaggaagat
94

ttctttcttctccctctcccggcttttttccttcgtcctctcctccgcccctcacccgcccacccccccccccccccgagcccttttct
tttcttctcggtcctctttccttccccaaatcactcgaaacttaagacgtgcagcgtctgcaggtgcgcgcgggccggcggcc
gggcctgggcgagcctgggactgccgggcctcgcaggcattgatcagctgggcgcgcgcgctgagtgacggcgcggtt
gccatggcagccgcctgagcggcgccgcgaggacaaggctgcagggcggcgtgaatgggcggcgtcacgcgcctgg
cgccagagagtctgctccggggctccggctccggccccgccgcggcctggcccgcgcgccgccgccgccgccaccgc
cgccgccaattcatcacctgtcagggatcactcgcggggtcacgggcggaatggacacagctgtgagaacaaaacccg
ggaaagaaaggcgaacgctccccattgcgccagatgtgcagggaggaccttgagacgcgccccccgcccccgcagg
ctcctcccgccccccggcgcatcacgcgggggtggcaaaggcggcctggccagcgcggagctcccggcccggagct
gcttctgattaccgcgaggggcccggacgcgagagccgccgcggggcctgccctagaggcggagtgagtgagtcctg
gagccaggctgggggaccttacgggccggtctgtcgctgccacggggtgggggcgtcccggtggggagtcgagggga
accgcaccaacacccaccagcggaagccctgcacaccttcccaccgcccgcggatctagccctccctctagctggggc
cgtttgaactttggaggcctcaaatgcaaacccacccaagccaccaattgacagattttgacttgcctttaaacccacttgga
aaatgctccgacacgattttcccccacgaaagCTTAGGCCTCGTTTAAATCA

Gbs Id2 Down Size 1110bp Ordered 01/31/2017
F: ACACCCGAGGGTTACTGTTTGCAC -
cttttatgcccagcccatcgACACCCGAGGGTTACTGTTTG
R: TGCTCCTCGGGTTCCCAGTGCCC -
gtcattggtcttaaaggtacTGCTCCTCGGGTTCCCAG
ACACCCGAGGGTTACTGTTTGCACagggaactaagctgtcacctggccccgggctcctgaggcctcctt
ggcttcgcctgcgtccacacctgtagcccccccgccacaccccacccacctccgggcctccggagacggggctgggcc
gggcgcattttcccggacttttcgatcgccctcagccttgactgccgccctggactggaaggaagaggcagcgggcagaa
gcctttcccagccaaagaccgcgggtggccacgtgctcaagactggtctgcgttttcagtcccggggcgcgaggtgggct
95

gggaggctctggaaggcgggtcccaggtggacgcgaaccgggggaggaagggatccgggcggggcagcagctca
cctggggcggaacaggtgttcgggcggcgccaccgaggagtccgcgcgtctccccgccctgacccccgatcgctcccc
gcgggcgctggcgctggaggagggcgggggccgtggagccgcggccgcccggatcgccctgcggacccgcgccgg
ggactgggctctgccggggccagcgcggggcggtcgtgtggttgatcatcatacccggaggaagccctgagggccgta
ggggcgtcttccccgtcgcaggctacccgcggacctggcccccaggtccccgccacctgtcgccagccgcggccccgg
ccgacctctgcgcggcggggagggaagcgcgctctggtcccaacccgacccccgcctcgcctccgggagaggcgca
gacgccggccgcacgctcgccccccagccacggggacccgcgcgtagtccgctcggacggccctggggagcgtccc
cggacggaagcagtgcgggcgggactgtggggcggcccgggccctttcgagcccctgccgggcaccgctggcgacgt
tggccccgggacccctggagccgcagccgaggcgcagcagcccctctcggaggcgacttcccgcggcgccagccgc
gctgcccgactccggccgtggagcccgggcgccacctgctggccgacacgaggacggcggctctcgcgcggggcctg
gtgggcctggggcccGGGCACTGGGAACCCGAGGAGCA

Hey1A LT Size 896bp
F: ACCATTACCTAACACTAGTA
R: TAACTTCTCTTGACCAGTCT
ACCATTACCTAACACTAGTAggtagttgtgcatacgtaggaaaccactaaaattgatccaatcacatatttta
agtgagtgaattgaatatgaattgtatctcaataaagctggaggttttaaagtcatcggtgtcctgtactggaaagtaaagctg
agtttccattagcctccatccagggtgaccttgacataggagaacactgagtgtcaagtgcctagagttgctttcagacagct
gatgggctcccaggacagaggccctggtgagcagtgcccaggttaggggctactgggttgagtctagttccacaggata
gggacagggcagtacccctcctctgcttagcacaatgagctggtctctttgtcatcaccaaagcagagtcactaaaccttca
ggacactgacctgcctcaaggatatttggtaacagttgttcttctctggctctgatcaacactgacctcctgctgctggatcgtg
caagagctctggccgtcacaagcctagactgagggaagaccctctgcagggacaccgccctggaggcccctggttctg
96

cgcagagaggatgggggaggggaggtggaactgtggcgcggccggggttcccacgctcctattgttggcgactttgtttc
caattcccatcacgtgtgctaattagttggagctgctggctggctggaaggcgtgtggcttgcctgaaagcagaaagatcg
cttcctgttgggagtcgggcaggcaggactctagctcagaaggggttagacaagaatgatgacaaaggtccctgcaagc
agccgctaaaagctgaggtgacccgaccagtggaagagggaaggggggaggaggcgggggggggagtgggggA
GACTGGTCAAGAGAAGTTA

Notch3B LT Size 1399bp
F: ATAGTCCTGAGTAGTGGGAC
R: AGTTCGTTCATCAGTGTGTT
ATAGTCCTGAGTAGTGGGACaggggtgccacacgcctttaatcccagcacttgggaggcagaggcaggc
gggtttctgagttcgaggccagcctggtctatagagtgagttccagaacagccagggctacacagagaaaccctgtctcg
aaaaaatctaagtcctgagtgctagaaaataaaaactaaaagacgacttaagtttgtaacctttctacggacagaggtga
ggagggctaaggaaggggactggagtagtcaaacaatcctggctatgcaactatgcaatgacacaacgtacatcaccc
ctacctcaaccctcctctcctctaccctacccttccggctcccagcgaggatcccctagctcagtcttgtctcaaacaccaag
ggcagaactcaaagcaggtgactcatttcttagcgtcaccctacaatacccccaccccgaattccgttcctagataaacgt
gtcgaagctcaaccctgtcgaagctcaaccctgcaagggtgtggtggacattcgaaccttggaggtgatgctctcaacccc
ctacccagtcctgcaaggcggcgacgatgacacgccaggagggggcgctgcgtgcagagcactggggaccacgggt
atcatcgccaagctgggcgcggcaccctccctgcgccgcgggcgggttcccacgctctgcacctcgcgcccccgccga
ccggtccggccggttccagccccggacccgcctcgccgccttagctttaggggagacgtagcggagcgggagggggg
cgggggagcagcttccaaggagggaacggactagcaagacagacaaggaccaagcgacggagaactccgaagcc
ttctcccggtcggaagactggggagaagtgggggagaaagtgtcattggggagggggcggtcactcctaaggagaca
gcaggatttgagaagcctgaaagagtgggttgggatgagaagaacataggctgaggtcccccccaaatctctattctgga
gccttaagcgagctccactccgcgcgatccgggctcccggaggccgcgtgagccggtgattcacctgtcgaggggcgg
97

ggcagcctcaggcgtattcacctgtcggcagctgtccaggcgtgtagttctactgcgcaggcgccccgccccacgcctca
gcctcccttccaagcgactgcttccaagtaaggaccctcccaaccctcagcctcaggatgcccctcggccagtgaccccc
aataaagctaaatgagattccagccattgaattttaaaggtttttgtttttctatatcggaagtcatcctggccccacctagcttg
aggctgatccactggctagcccacacagtattgaggaaatgAACACACTGATGAACGAACT

Hes1A LT Size 926bp
F: AATTCCAAGAGAGGAATGAATGGG
R: GGAATTTTTCTCCATTATATCAGCTGGCAT
AATTCCAAGAGAGGAATGAATGGGctagagggagacaagcatccaaaagacaccttgctccgaaaa
ccctggctgtgaaggcatttggggggacggtaagggcatgtttagcgtgtggggtttagtatctccccaacctcatgagcag
ttttgactcccctaaacatacagagttcgagcgggaattccggaagacattagagtaacacatcctctttaccttgttccctcct
tttttcaatcactaaattttgtcttggcctatatctgttcaaaatatttttcaaatgaacttattatacaaagtagttatattgcatgca
gcaagaacaataaaaaccaaaggcctggccacaaaagaaatagactagactaaaactaagcaaagcccagagga
aagagttagcaaagggttaaaatccttttgattgacgttgtagcctccggtgccccgggctcaggcgcgcgccattggccg
ccagaccttgtgcctagcggccaatgggggggcgcagtccacgagcggtgccgcgtgtctcttcctcccattggctgaaa
gttactgtgggaaagaaagtttgggaagtttcacacgagccgttcgcgtgcagtcccagatatatatagaggccgccagg
gcctgcggatcacacaggatctggagctggtgctgataacagcggaatcccctgtctacctctctccttggtcctggaatagt
gctaccgatcactaagtagccctaagacataataaaccttcaactgctcagtagtttttcttatgaaagtcaagtaaaagga
cgtaagcaaaaaaaaattattttttttttgcgtgaaggattccaaaaataaaattctctggggactgagaagaaaaaaaaa
aaaaaaacgaaaATGCCAGCTGATATAATGGAGAAAAATTCC

Jag1A LT Size 680bp
98

F: AGAAGGTGTGAGGCACTCTGAGAGCA
R: ACTCTATTCCTTAACTTCTAGAAC
AGAAGGTGTGAGGCACTCTGAGAGCAgtgggcaaaggcaaaaaaatggatcaactctctttcctgaa
cagatcaaaactgtccgagtgaatcaggttttccaaaggcaaccaggctctggtgataaaggaggaactgtgtatctcact
gggagggagaacgaacgtgttacaatagagttgaccaccatcagcagagtacaccttcactacctctccacacacccca
caagccccagccacttctctgcaggcctgaaaagcaacctgcccagagaacagagccatctttcaaaacggacaggg
caggtcctgactgcggggggctctgaaattcggcttcaatgggaggtttgtggtgagcagtgtgggaaactcaggctgacg
tggggctcacctgcccactgagtgaaatttaaggttggtctttagagcccagcaccaccctttacagctttatctttctgatgag
agattaaatgctcacttaaagtttaaatggacttttaaaaagtcactactttgatcctttggcagtctacaaggttttaaagttgta
aaaccattaagatctctatccagagataaactatgtccctttctgcaaggactgaataaactgggagtctattctggctactta
atatttcagttgaaaGTTCTAGAAGTTAAGGAATAGAGT

Tthy1A LT Size 1222bp
F: TTATGTCCTGGAGCACTCTCA
R: AACTGGGGCATGAGTCTGCA
TTATGTCCTGGAGCACTCTCAccccagtctgggctgtgggccactcttcctcattgtaggaagaagagggtg
tgtgtttgggagtatatgggggttgggcgccctatagccaagattttttttctcatcaggctctccagaatgggggtggatggg
ggtgggatgttgaaaaaggcccagtgggggtgaggcacagccccagccccctcagagcctaggaacaagaggagctt
tgtagggtcctgaacaatggggcaagggcagtaggctcccatccaagggatgaggtggggcctgtgtagcccaaggct
gggaactgtgagggcatcaggctctcgccccctccagtgtggagtacagactaatggaggaaagtgggtccccatattca
agctccacaatggagtctgtgtgttggctgggcagagacagggcccagctcttggcctggcctctccagccctccctccca
cattctctcagtctcttccagctggctggatgcctggatccctgggcatcataggggaaggcttagagctctgggcacaatgt
99

cttttacaaaagaatagggtctagggcatttaggaggaaggggtaaggaacacctgggctagaagagacagcacttgg
cctaatgattcctaagacaaggctgtaaatgctgaatgtctttgcaaaggatgtgggagttgtttgtgagctgatcactattag
gactgattgataacctttaatccagggctagctaggcaaaaaccagggttccactctggggaggtgtgagttaaatctggg
ggctctgtcttttccaccactccacccaccattagcatcacaatgacgtctctccagcagggggagtgaggtcttcaggttac
cttggcaactggtggaggcaaacctcgtgctgttctttttcccacggaggatgaaccaatagggtggatgcagagatcaga
taggcaaatgaagaagctgggaggatgggtatgtgtgtagaggcatggtgacaccctccagggtctgagactggttggta
tttcttcttcctttcccttcagtgccggcattggcattggtttctatggtaacagcgagaccagcgatggggtgtcccagctcagc
tcagcactgctgcacgccaaccacacgctcagcaccatcgatgacgtggtgagagagccagccactgaccagacTG
CAGACTCATGCCCCAGTT
3. Pou4f3 dependent gene enhancers
Gfi1: chr5:107,712,900-107,713,600
Gfi1_F cttttatgcccagcccatcgCAATAACTCAAAAGTGCAG
Gfi1_R gtcattggtcttaaaggtacCTCTGTATCCCTCAGTTTAG

Dll1: chr17:15,381,437-15,382,097
Dll1_F cttttatgcccagcccatcgCTAGGGTCTGAGCTATGC
Dll1_R gtcattggtcttaaaggtacTCTGGAAGCCTGTTCAATAATTATTG

Ptprq: chr10:107,724,603-107,726,069
Ptprq_F cttttatgcccagcccatcgTCATCCATCTTCTGGGAAG
Ptprq_R gtcattggtcttaaaggtacTTGATTAACTTTTCCCCTATC

Dnaic2: chr11:114,741,183-114,742,008
100

Dnaic2_F cttttatgcccagcccatcgCACAACGCAGTTGGTAAG
Dnaic2_R gtcattggtcttaaaggtacCTTTGCCAACAGAGGGGTAAAG

Ccer2: chr7:28,747,469-28,748,130
AK_Ccer2_F cttttatgcccagcccatcgCATTCCATCTACAACCAG
AK_Ccer2_R gtcattggtcttaaaggtacCTGGATAAGTATGTGCTAC

Ttc34: chr4:154,833,012-154,834,124
Ttc34_F cttttatgcccagcccatcgGGAATCTTCAACTGCAATC
Ttc34_R gtcattggtcttaaaggtacCTCCTCTCCTCTCCTCTC

Xylt1: chr7:117,392,695-117,393,979
Xylt1_F cttttatgcccagcccatcgGGCTTAAGGTAGACACTG
Xylt1_R gtcattggtcttaaaggtacCAAGTGAGGGAGGAAGAG

Otof: chr5:30,407,580-30,408,360
Otof_F1 cttttatgcccagcccatcgCTCTGAGAGCTGCAAGGTC
Otof_R1 gtcattggtcttaaaggtacTCCCCCAACCCCAAGTAG

Otof: chr5:30,424,650-30,425,200
Otof_F2 cttttatgcccagcccatcgGAAGCACACCCATTAGAAAG
Otof_R2 gtcattggtcttaaaggtacAGTAAGGTGCTTGGAGCTAG

101

Cep85l: chr10:53,377,162-53,378,390
Cep851_F cttttatgcccagcccatcgAGTTTTAGAACACCCAGAATTAAC
Cep851_R gtcattggtcttaaaggtacCTCTATGTGTGTGTGTATATAATG

Neurod1: chr2:79,450,496-79,451,383
Neurod1_F cttttatgcccagcccatcgTTTTCTTGGTCCAAAGCTATC
Neurod1_R gtcattggtcttaaaggtacATTAAAGCTGGTCCCAAC

102

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Asset Metadata

Creator Wang, Xizi (author)

Core Title The organ of Corti sensory progenitor cell competence is controlled by SoxC transcription factors

Contributor Electronically uploaded by the author (provenance)

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Development, Stem Cells and Regenerative Medicine

Degree Conferral Date 2021-12

Publication Date 09/28/2023

Defense Date 08/31/2021

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag cochlea,competence,epigenetics,inner ear,OAI-PMH Harvest,SoxC transcription factors

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Language English

Advisor Maxson, Robert (committee chair), Crump, Gage (committee member), Segil, Neil (committee member), Yu, Min (committee member)

Creator Email wangxizi1018@gmail.com,xiziw@usc.edu

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Abstract (if available)

Abstract How progenitor cells acquire the ability to respond to the inductive stimuli to differentiate towards defined cell lineages during embryogenesis remains a major obscure question in developmental biology. The Organ of Cortiㅡthe auditory sensory organㅡrepresents a unique system to study the mechanisms governing competence establishment, where cellular differentiation and terminal mitosis are uncoupled. In the organ of Corti, up-regulation of a single transcription factor, Atoh1, represents an inductive stimulus both necessary and sufficient for sensory lineage specification. Through in vitro cultures, we showed that the competence to respond to Atoh1 is established in the organ of Corti progenitor cells prior to the initiation of sensory differentiation following the cell cycle exit. By analyzing chromatin accessibility and transcriptome of actively dividing (E12.0) and post-mitotic (E13.5) sensory progenitors, we demonstrated that the transition to the competent state is rapid and is associated with extensive chromatin remodeling controlled by the SoxC transcription factors. Conditional loss of the two members of SoxC family, Sox4 and Sox11, does not affect progenitor cell specification or the timing of cell cycle exit, but blocks sensory lineage differentiation. Mechanistically, we demonstrated that SoxC binds to and regulates the accessibility of the regulatory elements, many of which become targets of Atoh1 in the differentiating sensory cells at later developmental stages. Further, transcriptomic analysis at a single cell resolution reveals that SoxC also controls the expression of other key sensory lineage genes. Consistently, overexpression of SoxC in the progenitor cells prior to the establishment of competence for differentiation or after the developmental window of plasticity enhances sensory differentiation both in vitro and in vivo. Our findings demonstrate the pivotal role of SoxC as competence factors and may facilitate the studies on repair and regeneration of the inner ear.