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Exploring the function of distal nephron enhancers in zebrafish

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Exploring the function of distal nephron enhancers in zebrafish

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Exploring the function of distal nephron
Enhancers in zebrafish

by
Tianming Zhou

A Thesis Presented to the
FACULTY OF THE USC KECK SCHOOL OF MEDICINE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(STEM CELL AND REGENERATIVE MEDICINE)

December 2023

Copyright 2023 Tianming Zhou
ii
ACKNOWLEDGEMENTS
This thesis would not have been possible without the support of many people.
First of all, I would like to express my sincere gratitude to my advisor, Dr. Nils Lindströ m from the Eli and
Edythe Broad CIRM Center for Regenerative Medicine and Stem Cell Research at USC, who read my
revisions and helped me understand what I needed to do with patience and kindness. He not only taught me
how to think scientifically, but also provided me with many valuable suggestions for my future life. This
experience will definitely become an asset in my future.
I would also like to thank my committee members, Dr. Francesca Mariani and Dr. Unmesh Jadhav, both
from Eli and Edythe Broad CIRM Center for Regenerative Medicine and Stem Cell, who continued
offering guidance and support in this year. I am gratefully indebted to them for their valuable comments on
the thesis.
I would also like to acknowledge in Peter Fabian, Kelsey Elliott and Mathi Thiruppathy who carefully
guided me how to conduct related experiments with zebrafish. Without their help, my project would not be
able to go any further. Also my lab mates Connor-James Fausto, MaryAnne Achieng, Jack Schnell, for their
guidance and comments on my experiments, as well as for their encouragement and comfort.
Finally, I would like to thank my parents, not only for their financial support in the past two years, but also
for their complete understanding of my personal choices for future development. This achievement would
not have been possible without them. I’m really lucky to have your help.

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TABLE OF CONTENTS

ACKNOWLEDGEMENTS ............................................................................................................ ii
List of Tables .................................................................................................................................. v
List of Figures ................................................................................................................................ vi
ABSTRACT ................................................................................................................................. viii
CHAPTER 1 INTRODUCTION .................................................................................................... 1
1.1 Clinical Significance of Understanding Nephron Gene Regulati on in Model
Organisms ............................................................................................................................... 1
1.2 Biological Process and Related Molecules of the Kidney ................................................ 1
1.2.1 Nephron Development in Mammals ...................................................................... 1
1.2.2 Nephron Development in Zebrafish....................................................................... 4
1.3 Enhancer Function and Regulation of POU3F3 Expression During
Nephrogenesis ......................................................................................................................... 7
1.4 Choice of zebrafish as a model organism ....................................................................... 10
1.4.1 Current models in studying enhancers ................................................................. 10
1.4.2 The advantages of zebrafish models in this project ............................................. 11
1.5 Analysis of the advantages and disadvantages of this project ........................................ 13
1.5.1 Goals of the Study ................................................................................................ 13
1.5.2 Shortcomings of the Study ................................................................................... 13
CHAPTER 2 METHODS ............................................................................................................. 15

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2.1 Using snATAC-seq data from mouse and human nephrogenesis to identify
candidate enhancers of POU3F3 .......................................................................................... 15
2.2 Analysis of candidate regions list and order primers ...................................................... 16
2.3 Primers based on the chosen regions .............................................................................. 16
2.4 Polymerase chain reaction (PCR) Profiling .................................................................... 17
2.4.1 Template DNA ..................................................................................................... 17
2.4.2 Perform PCR ........................................................................................................ 19
2.5 In-Fusion Cloning ........................................................................................................... 22
2.6 Fish experiments ............................................................................................................. 26
2.6.1 Oocyte microinject the plasmid DNA.................................................................. 26
2.6.2 Check the injection animals with confocal .......................................................... 28
CHAPTER 3 RESULTS ............................................................................................................... 30
3.1 Identification of the putative enhancers using snATAC-seq. ......................................... 30
3.2 PCR results of the candidate enhancers .......................................................................... 32
3.3 Preparation and testing of plasmid .................................................................................. 32
3.4 Injected animals .............................................................................................................. 35
CHAPTER 4 DISCUSSION ......................................................................................................... 38
4.1 Problems encountered during the experiments and solutions ......................................... 38
4.2 Conclusion and Future Perspectives ............................................................................... 39
REFERENCES ............................................................................................................................. 45

v
List of Tables

Table 1. Details of five candidate regions .................................................................................... 17
Table 2. Forward or reverse candidate enhancer primers from successful PCR with
overhang list .................................................................................................................................. 17
Table 3. Q5 High-Fidelity 2X master mix PCR ........................................................................... 20
Table 4. Recommended In-Fusion Reactions for Purified Fragments(adapted from
In-Fusion kit) ................................................................................................................................ 25
Table 5. In-Fusion cloning reaction .............................................................................................. 25
Table 6. zebrafish oocyte inject reaction ...................................................................................... 28

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List of Figures

Figure 1. Cells gradually aggregate to nephrons and form putative precursor domains
(adapted from Schnell et al., 2022). ........................................................................................ 3
Figure 2. Early pronephros development and gene expression analysis in renal
progenitor regions. .................................................................................................................. 6
Figure 3. Mesonephros Development: Gradual Addition of Nephrons to the
Mesonephros. .......................................................................................................................... 7
Figure 4. Regulation of gene expression patterns by enhancers. The enhancer-
promoter chromosomal loop (mediated by CTCF and cohesin) ............................................. 9
Figure 5. Workflow of In-Fusion Cloning. Cited from the In-Fusion® HD Cloning
Kit User Manual Protocol of In-Fusion cloning adapted from In-Fusion® HD
Cloning Kit User Manual. ..................................................................................................... 24
Figure 6. Media with growth (left) and without growth (right). Tubes with bacterial
growth are cloudy, while controls should be clear (cited from Addgene, 2019). ................. 27
Figure 7. Successful injected embryos. After injection, stable red spherical
droplets were observed, which did not dissipate (cited from Rosen et al., 2009). ............... 30
Figure 8. Top candidate enhancer regions selected. ............................................................. 32
Figure 9. Representative PCR results. .................................................................................. 33
Figure 10. The map of origional plasmid, eda_E1B:mCherry Tol2 plasmid
(Crump lab -Fabian et al., 2022). .......................................................................................... 34
Figure 11. Double-digestion of circular vectors. .................................................................. 35
Figure 12. In-Fusion cloning LB plates after overnight incubation at 37° C. ....................... 36

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Figure 14. Transgenic fish generated by oocyte injections. .................................................. 37
Figure 15. Failed In-Fusion clone LB plates. ....................................................................... 39

viii
ABSTRACT
The kidney is a structurally complex organ system affected by many congenital anomalies. About 3% of
babies are born with renal or genitourinary abnormalities. These developmental defects are associated with
multiple genes, many of which have unknown functions in the renal organogenesis program (Schnell et al.,
2022). The distal nephron plays a crucial role in regulating electrolyte and fluid balance in vertebrates. The
transcription factor POU3F3 is implicated in the development and function of the mammalian distal
nephron; however, its regulation remains unclear. This study aimed to investigate the function of putative
POU3F3 nephron enhancers using the zebrafish pronephros as model.
To address this question, I performed cross-species comparisons across all cell-types in the developing
mouse and human nephron, identifying putative cis-regulatory regions matching the known expression
dynamics of Pou3f3 and POU3F3 using single-nuclear assay for transposase-accessible chromatin
(ATAC)- seq data. I found regions of DNA surrounding Pou3f3 and POU3F3 that correspond to their
respective expression profiles. The majority of these are deeply conserved while others are present in mouse
or human but not the other. A series of potential enhancers of POU3F3 were then assayed by generating
enhancer reporter constructs and tested by generating transgenic zebrafish lines. Positive controls enhancers
with known function were used to successfully drive reporter expression, 5/7enhancers tested failed to
express in the distal zebrafish pronephros, the purpose of this project to analyze POU3F3 via enhancer
reporter constructs failed.

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CHAPTER 1 INTRODUCTION
1.1 Clinical Significance of Understanding Nephron Gene Regulati on in Model Organisms
Studying the development and function of the kidney is critical to understanding human health, as kidney
disease has a major impact on patient outcomes. Kidney disease, including chronic kidney disease, renal
hypertension, and electrolyte imbalances, affects over 900 million people worldwide (Chukwuka Elendu et
al., 2023). These conditions can lead to organ failure, requiring costly and invasive treatments such as
dialysis or kidney transplants (Marquez et al. , 2011).
There are several challenges to studying kidney development. Although human fetal tissue provides
valuable insights into developmental processes, access is limited due to ethical concerns and modifications
and perturbation experiments are not very feasible. These limitations hinder our ability to comprehensively
study the molecular mechanisms of kidney development and identify potential therapeutic targets.
1.2 Biological Process and Related Molecules of the Kidney
1.2.1 Nephron Development in Mammals
The distal nephron, comprising the distal convoluted tubule (DCT) and connecting tubule (CNT), plays a
crucial role in regulating electrolyte balance and acid-base homeostasis in the kidney (McCormick &
Ellison, 2014). The process of distal nephron domain formation is a complex and tightly regulated
developmental event, involving intricate cellular interactions and molecular signaling pathways (see Fig.1).
The progenitor cells, localized in the cap mesenchyme, undergo a series of morphogenetic events and
molecular signaling cascades to give rise to the various segments of the nephron, including the distal
nephron.
The initial steps of distal nephron domain formation involve the reciprocal signaling between the cap
mesenchyme and the ureteric bud. The ureteric bud, an outgrowth from the Wolffian duct, interacts with
the cap mesenchyme to induce nephron formation. This interaction is mediated by signaling molecules such

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as Wnts (Carroll et al. , 2005), BMPs (bone morphogenetic proteins) (Oxburgh et al., 2005), and FGFs
(fibroblast growth factors) (Barak et al., 2012).
As nephron development progresses, specific molecular markers are expressed in the distal nephron domain.
For example, the transcription factor Pax8 is expressed in the early distal nephron progenitors and is
essential for the differentiation of these cells into mature distal nephron segments (Narlis et al., 2007). Other
markers, such as Jagged1 and Jagged2, are expressed in the developing DCT and CNT and are involved in
the Notch signaling pathway, which plays a crucial role in segment specification (Schnell et al., 2022).
During distal nephron domain formation, intricate molecular signaling cascades are activated to establish
segment-specific cell types and functions. Specific molecular markers begin to be expressed in the distal
nephron domain. Among them, the POU transcription factor Brn1 (now named Pou3f3) gene plays a key
role in the formation and function of the distal convoluted tubule in the mammalian kidney. Nakai et al.
(2003) found that the development of the loop of Henle (HL) and the distal convoluted tubule and macula
densa was severely affected when Pou3f3 is knocked-out, suggesting that Pou3f3 is essential for both the
development and function of the distal nephron of the kidney. Of note, while functional, the distal segments
did not differentiate normally, structurally only an epithelial tubule remains (Nakai et al., 2003). Lindströ m
et al., (2018) localized transcription factors in human and mouse kidney vesicles and S-shaped nephrons
using single-channel immunofluorescence and observed POU3F3 in human and mouse distal nephron cell
nuclei, demonstrated Pou3f3/POU3F3 expression is deeply conserved across species.
The process of distal nephron domain formation is also influenced by extrinsic factors, including retinoic
acid signaling. Retinoic acid signaling is known to regulate the expression of Hox genes, which are crucial
for segment identity and patterning (Szatmari et al., 2010).
In summary, the process of distal nephron domain formation during kidney development involves
reciprocal signaling between the ureteric bud and the developing nephron, activation of specific molecular
markers, and the coordinated activity of signaling pathways and transcription factors, previous studies have

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already provided valuable insights into these processes and serve as important references for understanding
the mechanisms driving distal nephron domain formation.

Figure 1. Cells gradually aggregate to nephrons and form putative precursor domains (adapted from Schnell
et al., 2022). Cells gradually aggregate into nephrons and form putative precursor domains. a. Pretubular aggregates.
Pretubular aggregates have strong cellular associations with nephron progenitor cell (NPC) populations. WNT9B is
thought to signal recruitment from the ureteric epithelium to NPCs and neonatal nephrons. b. In renal vesicles, NPCs
are continuously recruited to proximal vesicles and form dissociated distally and medially area. β-catenin is active in
the gradient. A slight curvature at the proximal end of the vesicle indicates the site of glomerular cleft formation.
MAFB, a transcription factor that regulates podocyte development, was first detected at this stage. c. Late alveoli
elongate as they transform into comma-shaped nephrons. Glomerular fissures are forming and small indentations
appear distally. At this point the distal and medial cell populations separate and the parietal epithelium is slightly
flattened. d. Prominent glomerular fissures and distal gyri are clearly visible when late comma-shaped nephrons
transform into S-shaped nephrons. Cell types evident at this stage include emerging connecting tubules, putative

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precursors of distal tubules, and the loop of Henle (medial segment) containing putative proximal tubules. e, f. S-
shaped nephrons continue to undergo elongation, morphogenesis, and gene expression profiles segregating into
distinct populations of putative precursors. g. Proposed anatomy of the nephron at the capillary loop stage and the
association of the renal corpuscle with the distal segment of the renal tubule forming plaque compaction. h. Mature
medullary (left) and cortical (right) nephrons (Schnell et al., 2022).
1.2.2 Nephron Development in Zebrafish
Early development of the zebrafish embryo includes rapid blastomere division and activation of genomic
transcription at the mid-blastocyst transition. Within 4 to 10 hours after fertilization, the single-layer
blastocyst embryo begins ectoderm motility to form a three-layer gastrulation embryo with ectoderm,
mesoderm, and endoderm (Kimmel et al., 1995). The zebrafish pair of pronephros originates from
intermediate mesoderm (Pfeffer et al., 1998). The bilateral band of cells of the intermediate mesoderm is
located anatomically between the paraxial and lateral plate mesoderm. It produces erythroid cells,
endothelial cells, and pronephric cells (Davidson and Zon 2004).
Early glomerular development in zebrafish roughly occurs at the 3-somite (11hpf) stage, as observed by
detecting a glomerular marker: a population of cells expressing Wilms tumor suppressor 1a (wt1a). By 48
hpf, the fused midline glomeruli form the pronephros and gradually connect with the tubules through the
neck and then gradually extend into the cloaca (Drummond et al., 1998). The anterior tubule is divided into
four segments along the anterior-posterior axis: proximal convoluted tubule (PCT), proximal straight tubule
(PST), distal early tubule (DE), and distal late segment (DL) and can be visualized by tracing the respective
solute carriers it develops (see Fig.2A). Gene expression in progenitor regions of the zebrafish kidney is
actually a complex nested pattern that is both complex and dynamic (see Fig 2B).
After approximately 2 weeks, the mesonephros begin to develop and nephrons are added to the developed
pronephric tubules from individual mesenchymal cells associated with the prenephric tubules (Fig 3A), at
which point the larva is about 4-5 mm long. The production of new nephrons can be observed by tracking
the expression of several developmental genes. For example, single mesenchymal cells produced green
fluorescent protein (GFP) in a transgenic line under the control of the lhx1a promoter (Choe et al., 2021).

5
When the larval length reaches 5 mm (approximately 13 dpf), the renal tubules begin to elongate, and the
location of the mesonephros is dorsal to the adult body cavity (Fig 3B).
All of the above indicate that the zebrafish kidney development process is very rapid and occurs within a
few days (Gerlach & Wingert, 2012).

A

B
Figure 2. Early pronephros development and gene expression analysis in renal progenitor regions.

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A. Early pronephros development in zebrafish. The view of the whole embryo on the left is a lateral view. A schematic
overview of the spatial arrangement of the pronephros at the stage indicated is shown. The dorsal view of the
complementary stage is shown on the right. 6-ss stage embryo highlighted by arrowhead distal tubule migrating
towards the caudal part of the cloaca. Arrowheads in 24 hpf embryos show subsequent midline migration of the P/PEC
lineage. Arrowheads in 48 hpf embryos indicate collective anterior migration of pronephric cells starting at 29 hpf.
P/PEC - podocytes and parietal epithelial cells. PCT - proximal convoluted tubule. PST proximal straight tubule. DE
distal early tubule. DL distal late tubule/duct hybrid segment. MCC multi-ciliated cell (adapted from Naylor et al.,
2017).
B. Gene expressor analysis in renal progenitor regions. At the fifth somite stage, the rostral region contained a subset
of wt1a+ cells, which included podocyte progenitors. By the 15th somite stage, pou3f3a, pou3f3b expression was
detected and underpinned the concept of central domain identity supporting this time point. By the 28th somite stage,
segments have become contiguous domains defined by the expression of specific transcription factors (adapted from
Gerlach & Wingert, 2012).

Figure 3. Mesonephros Development: Gradual Addition of Nephrons to the Mesonephros.
A. Mesonephrons (pink) gradually increase over time, forming a dense collection of nephrons that penetrate deeply
into a pair of pronephrons (blue) (panel is from Gerlach & Wingert, 2012).
B. Schematic diagram of an adult fish mesonephros. Following a conserved pattern of blood filters, tubules, and ducts,
the tubules comprise multiple segments that typically include proximal and distal regions and possibly intermediate
segments.
B
A

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1.3 Enhancer Function and Regulation of POU3F3 Expression During Nephrogenesis
Enhancers, one of the non-coding DNA elements, regulate complex gene expression programs in specific
time and space (Long et al., 2016). These regulatory elements play an important role in controlling the
activity of gene promoters to ensure the normal development and function of specific tissues and cell types
((Fig.4) (Carullo & Day, 2019).
In zebrafish embryos, H3K4me3 and H3K27me3 marks are present at the promoters of active and inactive
genes in the absence of sequence-specific transcriptional activators or RNAPII (Lindeman et al., 2011),
suggesting that histone modifications precede gene activation . In addition to this, Fulco et al. (2016) studied
enhancer activation by using small molecules to disrupt chromatin loops, aiming to understand the
mechanism of enhancer activation. They observed that disruption led to activation of the enhancer-promoter
interaction and increased expression of the genes it regulates. They demonstrate that disruption of these
loops activates enhancer-promoter interactions and increases specifically regulated gene expression.
But how do enhancer sequences acquire specific patterns that precede cell fate decisions?
At its most basic level, enhancers are usually short regions of DNA (50-1500 bp), which can be located
upstream or downstream of the gene they regulate, and activates the transcription of one or more genes by
recruiting activators (transcription factors) to drive transcription (Pennacchio et al., 2013). Multiple studies
have found that enhancers are often located thousands or even tens of thousands of base pairs away from
gene transcriptional start sites, so DNA-wrapping, bringing the enhancer closer to the promoter, has been
suggested as a mechanism (Panigrahi & O’Malley, 2021) .
Active enhancer regulatory regions of DNA are able to interact with the promoter DNA regions of their
target genes by forming chromosomal loops (Carullo & Day, 2019). This initiates messenger RNA (mRNA)
synthesis by binding of RNA polymerase II (RNAP II) to the promoter at the gene's transcription start site
(Krivega & Dean, 2012). Specificity is ensured by transcription factors binding to DNA sequence motifs.
Transcription factors bind enhancers and act on promoters driving RNA polymerase mediated transcription.
Mediators (complexes of approximately 26 proteins in an interacting structure) transmit regulatory signals

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from enhancer DNA-binding transcription factors to promoters (Koch et al., 2011). Mediators can
coordinate enhancer signaling to the transcriptional machinery by interacting with enhancer-binding
transcription factors and Pol II, and serve as hubs for long-range enhancer transcriptional regulation.
Another mediator component, TBP-associated factor 3 (TAF3), directly interacts with CCCTC-binding
factor (CTCF) and is recruited to distal sites in ESCs shared by CTCF and cohesin (Liu et al., 2011). These
interactions are summarized in (Fig.4) from Carullo and Day, (2019).

Figure 4. Regulation of gene expression patterns by enhancers. The enhancer-promoter chromosomal loop (mediated
by CTCF and cohesin) allows distal enhancer elements to physically interact with and activate gene promoters. These
interactions increase the binding of transcription factors, chromatin modifiers, and mediator complexes at gene
promoters to recruit RNA polymerase II (RNAPII). The work of enhancers is characterized by increased DNA
sequence conservation, open chromatin, transcription factor binding motifs, characteristic histone modifications, DNA
hypomethylation, and bidirectional transcription to generate enhancer RNA (eRNA) (adapted from Carullo & Day,
2019).

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Enhancers interact with gene promoters and regulators to modulate gene expression, showcasing their
functional diversity and impact on gene regulation (Visel et al., 2009). Enhancer evolution contributes to
phenotypic innovation and drives cell type and tissue diversification (Villar et al., 2015). Comparative
epigenomics studies highlight enhancer function and their dynamic nature, influencing gene expression
regulation across different species (Gross et al., 2018). In contrast to variations in protein-coding sequences,
the importance of non-coding DNA variation in human disease has been poorly explored. Many recent
publications and studies have shown that non-coding variants are important risk factors for common
diseases, but the mechanisms by which they cause disease remain largely unknown. Enhancers, a major
class of functional non-coding DNA, may be involved in many developmental and disease-related processes
(Visel et al., 2009).
Enhancers also play a vital role in developmental biology and disease, controlling gene expression during
embryonic development and tissue differentiation, and their dysfunction contributes to developmental
disorders and disease pathogenesis (Kvon and Kazmar, 2015). Dysregulation of enhancers can have
profound effects on kidney development and function, possibly leading to kidney disease. Therefore,
understanding the regulatory mechanisms of enhancers and their effects on gene expression is crucial for
elucidating the molecular basis of kidney development and identifying potential therapeutic targets.
The activation of Pou3f3/POU3F3 in kidney development involves intricate regulatory mechanisms that
ensure its timely and spatially controlled expression. Several studies have shed light on the factors and
processes contributing to the activation:
1. Transcription Factor Networks: Pou3f3/POU3F3 expression is regulated by interactions with specific
transcription factors. The transcription factor HNF-1β (Hepatocyte Nuclear Factor 1 Beta) is known to play
a role in kidney development and differentiation (Heliot et al., 2013). The interaction between HNF1B and
Irx1/Irx2 transcription factors is discussed in the same paper by Heliot et al. (2013). Irx2 plays a role in
defining segmental identity along the nephron. It has been associated with the development of distal
nephron segments, which include the distal convoluted tubules (DCT) and connecting tubules (CNT) where

10
Pou3f3 is expressed. The coordinated expression of both Irx2 and Pou3f3 in these segments suggests a
functional relationship between these two transcription factors.
2. Cognate Promoters: The activation of Pou3f3 can be influenced by the properties of its cognate
promoter. Understanding the regulatory elements within the promoter region is crucial (Ong and Corces,
2012).
3. Tissue-Specific Enhancers: Enhancer elements play a vital role in controlling tissue-specific gene
expression (Visel et al., 2009).
4. Signaling Pathways: Signaling pathways such as Wnt and TGF-β are known to influence kidney
development. These pathways can impact the expression of transcription factors like Pou3f3 (Arnold et al.,
2020).
5. Epigenetic Modifications: Epigenetic modifications, such as histone modifications and DNA
methylation, can impact the accessibility of the Pou3f3 promoter and enhancer regions (Lindeman et al.,
2011).
Overall, understanding the regulatory network and molecular mechanisms underlying distal nephron
domain formation, including the role of enhancers and transcription factors such as POU3F3, is critical to
unraveling the complex processes that drive kidney development and function.
1.4 Choice of zebrafish as a model organism
1.4.1 Current models in studying enhancers
Many enhancers have been tested with success in mice and other models, such as iPSCs. Although enhancer
testing in these models has now yielded valuable insights, there are still respective limitations.
Cell models: Cellular models including iPSCs, provide simplified representations of in vivo systems. They
may not fully capture the complexity of tissue-specific enhancer activity and the interactions of multiple
cell types present in native tissues (Brooks et al., 2022). Enhancers can also exhibit context-specific activity,
meaning that their function may vary depending on cell type or developmental stage. Testing enhancers in

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specific cell models may not fully represent their activity in other cellular contexts. Fulco et al., 2016 found
that while cell models allow the identification of some enhancer-promoter properties, they missed a large
number of functional enhancers. In addition, epigenetic modifications can affect enhancer activity. Cell
models may exhibit epigenetic differences compared to their in vivo counterparts, potentially affecting
enhancer function and regulation (Ong & Corces, 2012). Finally, experimental techniques used to test
enhancers in cell models have limitations. These include the potential for off-target effects of perturbation
methods such as short palindromic repeat (CRISPR)-associated system (Cas)-mediated genome (CRISPR-
Cas), the difficulty of precisely defining enhancer boundaries, and the challenge of distinguishing causality.
(Subramanian et al., 2019).
Mouse models: First, mice have limitations in capturing the full complexity of enhancer function due to
evolutionary divergence. Thus, differences in genome structure and gene regulation between mice and
humans may not fully reproduce enhancer regulation observed in human tissue complexity and diversity
(Visel et al., 2009). Second, genetic background and environmental factors can influence the phenotypic
outcome of mouse enhancer perturbations, and studying mouse enhancer function is often complicated by
redundancy, pleiotropy, and context-specific effects, which make generalizing results across different
contexts challenging (Ong and Corces, 2012). Furthermore, the scale and efficiency of manipulating and
testing enhancers in mice can be labor-intensive and time-consuming, limiting the scope and throughput of
functional studies. Fulco et al., 2016 describe how their traditional method of generating transgenic mice
to study enhancer-promoter junctions is time-consuming.
1.4.2 The advantages of zebrafish models in this project
The zebrafish pronephros is a valuable model for studying mammalian nephrogenesis due to following
reasons:
Conserved Developmental Pathways: Thermes et al. (2002) demonstrate that the zebrafish pronephros
shares developmental pathways with the mammalian metanephros, indicating evolutionary conservation of
kidney development mechanisms.

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Segmentation and Patterning: Lindeman et al. (2011) describe the presence of similar gene expression
patterns in early kidney development of zebrafish and mammals, including conserved expression of genes
involved in patterning and differentiation.
Functional Analogy: Wingert and Davidson (2008) explain how zebrafish nephrogenesis involves similar
molecular and cellular processes as mammalian kidney development, such as the differentiation of renal
progenitors.
Gene Expression Profiles: The work by Wingert et al. (2007) in PLoS Genetics discusses the role of cdx
genes and retinoic acid in positioning and segmenting the zebrafish pronephros, which highlights the
conserved signaling pathways involved in kidney development.
Genetic Conservation: Naylor et al. (2017) suggest that key genes involved in zebrafish kidney
development, such as zp2e, are conserved and have counterparts in mammalian kidneys, reinforcing the
relevance of the zebrafish model.
Conservation of regulatory elements: The shared enhancer activity profiles observed in zebrafish and
human nephron development suggest the existence of conserved gene regulatory networks (Gehrig et al.,
2009).
High degree of genetic tractability: The zebrafish genome has been fully sequenced, and researchers have
access to a wealth of genetic tools and resources. This genetic tractability enables the manipulation of
specific genes and regulatory elements to gain insight into their function and contribution to organ
development (Lawson & Weinstein, 2002). For example, the zebrafish is an ideal model organism to study
the development of specific organs such as the heart because it is suitable for both loss-of-function and
gain-of-function assays (Stainier, 2001). Zebrafish embryos also exhibit several features that make them
suitable for studying kidney development. Zebrafish embryos are optically transparent, allowing real-time
observation of organogenesis and cellular processes. This transparency facilitates direct observation of
nephron development, allowing researchers to analyze the formation and morphogenesis of distal nephron
fragments.

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Relatively fast generation times in zebrafish: Zebrafish can produce hundreds of offspring in a short
period of time, facilitating large-scale genetic screens and experiments (Iwanami et al., 2016). This rapid
generation time allows the study of gene function and the identification of new regulatory elements
affecting kidney development.
In conclusion, the choice of zebrafish as my model organism for this project was well-reasoned based on
the advantages zebrafish offer for studying kidney development. Genetic tractability, optical transparency,
mammalian conservation, fast generation time, and experimental maneuverability make zebrafish an ideal
system for studying the function of distal nephron enhancers and their role in kidney development.
1.5 Analysis of the advantages and disadvantages of this project
1.5.1 Goals of the Study
The primary goal of this research project is to explore the function of distal nephron enhancers, particularly
those regulating the expression of POU3F3, in zebrafish kidney development. By investigating these
enhancers, the study aims to uncover their role in the formation and maturation of the distal nephron
segments, shedding light on the molecular mechanisms underlying distal nephron development.
The importance of studying enhancer elements in understanding the precise gene regulatory networks
governing kidney development. By elucidating the function of distal nephron enhancers, this study
contributes to the broader goal of deciphering the regulatory landscape of kidney development.
1.5.2 Shortcomings of the Study
It is important to acknowledge the limitations and potential shortcomings of this study. One limitation is
that zebrafish, while sharing many similarities with mammals in terms of kidney structure and development,
may also have subtle differences. These differences could affect the interpretation and generalizability of
the findings to human kidney development. Peterson et al., 2000 discuss the need for caution when
extrapolating zebrafish research findings to human biology.
Another potential limitation is the inherent complexity of studying enhancer function in vivo. The precise
identification and characterization of enhancers and their target genes require sophisticated molecular

14
techniques and thorough validation. Visel et al. (2013) highlight the challenges and complexities associated
with studying enhancer function in mammalian systems.
Furthermore, the study may face challenges in specifically manipulating and studying the distal nephron
enhancers and their impact on kidney development. Enhancers are often cell-type-specific and context-
dependent, requiring precise experimental strategies for their isolation and functional characterization.
Levine et al. (2014) provide insights into the complexities of enhancer biology and the challenges associated
with dissecting their functions.
Despite these potential shortcomings, this study contributes to our understanding of enhancer-mediated
gene regulation in kidney development, providing valuable insights into the role of distal nephron enhancers,
particularly POU3F3, in zebrafish. The findings serve as a foundation for further investigations and
potentially contribute to future therapeutic strategies for kidney-related disorders.

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CHAPTER 2 METHODS
2.1 Using snATAC-seq data from mouse and human nephrogenesis to identify candidate
enhancers of POU3F3
A brief introduction of how the single-nuclear assay for transposase-accessible chromatin with sequencing
(snATAC-seq) results were generated.
Cell Calling and Clustering: Use computational methods to identify individual cells or nuclei in the
snATAC-seq data. snATAC-seq begins with the isolation of single cells from the tissue or organism of
interest. The cells are then subjected to lysis and transposition, where a Tn5 transposase is used to
simultaneously fragment the DNA and add sequencing adapters. This step allows the transposase to insert
the sequencing adapters into regions of open chromatin, resulting in the preferential fragmentation of
accessible regions. Following transposition, the DNA fragments with sequencing adapters are purified,
amplified, and prepared for sequencing. Unique molecular identifiers (UMIs) are incorporated during the
amplification step to distinguish between genuine biological signal and potential amplification artifacts.
The resulting libraries are then sequenced using high-throughput sequencing platforms. After sequencing,
the data undergoes quality control and preprocessing steps, including read alignment to a reference genome
and removal of duplicates. Single-cell barcodes and UMIs are used to assign reads to individual cells,
enabling downstream analysis at the single-cell level. The snATAC-seq data can then be analyzed to
identify cell types, infer cell states, and explore the chromatin accessibility landscape of individual cells.
Various computational tools and algorithms are available for analyzing snATAC-seq data, including peak
calling, clustering, and visualization methods (Buenrostro et al., 2015; Cusanovich et al., 2015).
Data Acquisition and Preprocessing: snATAC-seq data from mouse and human nephrogenesis was
obtained from from Sunghyun Kim’s database. To expand the understanding of the conserved and unique
features of kidney organogenesis in humans and mice, and detail their respective cellular diversity, genome
organization, and gene activity, Kim et al., (2023) performed single-cell (mouse) and Single-nucleus
(human) RNA-seq (sc/snRNA-seq) analysis and single-nuclei ATAC-seq (snATAC-seq) analysis and close

16
inspection of gene expression and epigenetic maps. Detailed data were obtained and uploaded to the UCSC
Genome Browser.

Table 1. Details of five candidate regions
Name Region Length
POU3F3_candidate enhancer_1 chr2:104,842,583-104,843,466 884
POU3F3_candidate enhancer_2 chr2:104,844,842-104,845,495 654
POU3F3_candidate enhancer_3 chr2:104,862,098-104,862,805 708
POU3F3_candidate enhancer_4 chr2:104,866,994-104,868,001 1008
POU3F3_candidate enhancer_5 chr2:104,901,099-104,902,196 1098

2.2 Analysis of candidate regions list and order primers
The protocol I used was as follows - After selecting the region that meets the requirements, I entered "DNA"
in the toolbar view to obtain the specific sequence. Then I used the primer blast website
(https://www.ncbi.nlm.nih.gov/tools/primer-blast/) to design primers, and adjust the potential product size
according to the sequence length. Then I chose the ideal primers and ordered them. Primers can be viewed
using Snapgeneviewer. The specific method is to add an extra length, such as 50bp, to the beginning and
end of the primer when obtaining the DNA in the UCSC browser, to obtain a long sequence with an extra
length, and to highlight the ideal band on this band part, that is, the original sequence. Then copy and paste
the forward and reverse primers you picked in the website and see if they completely cover the part you
want to study.
2.3 Primers based on the chosen regions

Table 2. Forward or reverse candidate enhancer primers from successful PCR with overhang list
Primers Tm
(℃, 50mM NaCl)
Product
Size (bp) Name Sequence
POU3F3_C1_F
GTATAGAAAAGTTGcccgggTGTAGAGAA
CCGAGGAGGGG
62 982

17
Primers Tm
(℃, 50mM NaCl)
Product
Size (bp) Name Sequence
POU3F3_C1_R
GTACAAACTTGgatatcCCCCTCCCAACCC
CTTAGTA
62.8
POU3F3_C2_F
GTATAGAAAAGTTGcccgggTTAACCAGG
CACCGACCTGA
61
731
POU3F3_C2_R
GTACAAACTTGgatatcAGGTACAAGGAGT
GAGGTGGA
62
POU3F3_C3_F
GTATAGAAAAGTTGcccgggCCCAAAAGC
CAAAGAGAAAGCA
61.1
784
POU3F3_C3_R
GTACAAACTTGgatatcCTGGCCCAGGATC
ACTTTTAC
62
POU3F3_C4_F
GTATAGAAAAGTTGcccgggCCAGCCCAG
AATACACTTCACT
61
1029
POU3F3_C4_R
GTACAAACTTGgatatcTTGTTTACGTGCTT
GGCAGC
63
POU3F3_C5_F
GTATAGAAAAGTTGcccgggTGCAAAATG
ATCTGCTTAGTTCTGT
61
1117
POU3F3_C5_R
GTACAAACTTGgatatcATCCCCCTTCTGAGT
GTCCC
62

2.4 Polymerase chain reaction (PCR) Profiling
PCR is a molecular cloning technique, invented by Mullis in 1983, based on the ability of DNA polymerase
to synthesize a new DNA strand complementary to the provided template strand, since DNA polymerase
can only add nucleotides to pre-existing 3' In the -OH group, primers are therefore required to add the first
nucleonucleotide to a specific region of the template sequence desired for amplification. This results in
hundreds of millions or even billions of copies of a particular sequence of interest at the end of the PCR
reaction, with no change in the amount of DNA that is not of interest (Kadri, 2019).
I assembled these three components to run this experiment: a DNA template (template DNA containing the
sequence of interest), primers(which have been designed in advance), and a DNA polymerase (provided in
the kit).
2.4.1 Template DNA
I used the QIAamp DNA Micro Kit to purify human pluripotent stem cells (iPSCs) and get genomic DNA.

18
Protocol adapted from QIAamp DNA Micro kit
Required instruments and consumables: QIAamp DNA Micro kit, centrifuge, 1.5 ml microcentrifuge tube
(which have been autoclaved), micropipette gun and tips in 10ul, 200ul and 1000ul.

1) Transfer the culture medium (within 100ul) containing the cells to a 1.5 ml microcentrifuge tube.
2) Add Buffer ATL to a final volume of 100 µ l.
3) Add 10 µ l proteinase K.
4) Add 100 µ l Buffer AL, cap and pulse vortex for 15 seconds.
5) Incubate at 56° C for 10 minutes. Gently shake the tube every two minutes to increase DNA yield.
6) Centrifuge the 1.5 ml tube briefly to get the droplet inside the cap.
7) Add 50 µ l of ethanol (96–100%), cap and mix well by pulse vortexing for 15 seconds. Incubate for 3
minutes at room temperature (15–25° C).
8) Centrifuge the 1.5 ml tube briefly to get the droplet inside the cap.
9) Carefully transfer the entire lysate from step 8 to the center of the QIAamp MinElute® tube without
wetting the rim. Cover and centrifuge at 6000 x g (8000 rpm) for 1 min. Then place the QIAamp MinElute
column into a clean 2 ml collection tube and discard the collection tube containing the flow-through. If the
lysate does not pass completely through the membrane after centrifugation, centrifuge again at a higher
speed until the QIAamp MinElute column is empty.
10) Carefully open the QIAamp MinElute column and add 500 µ l Buffer AW1 to the center of the tube
without wetting the edges. Cap and centrifuge at 6000 x g (8000 rpm) for 1 min. Place the QIAamp
MinElute column into a clean 2 ml collection tube and discard the collection tube containing the flow-
through.
11) Carefully open the QIAamp MinElute column and add 500 µ l Buffer AW2 to the center of the tube
without wetting the edges. Cap and centrifuge at 6000 x g (8000 rpm) for 1 min. Place the QIAamp
MinElute column into a clean 2 ml collection tube and discard the collection tube containing the flow-
through.

19
12) Then centrifuge at 20,000x g (14,000 rpm) for 3 minutes to completely remove excess liquid in the
membrane.
13) Place the QIAamp MinElute column into a new clean 1.5 ml microcentrifuge tube and discard the
collection tube. Then carefully add 20ul Buffer AE or distilled water to the center of the membrane.
Incubate at room temperature for 1-3 minutes to increase yield.
14) Finally, centrifuge at 20,000 x g (14,000 rpm) for 1 minute to obtain the final gDNA solution. Store at
-20° C.
2.4.2 Perform PCR
I followed the steps below (Table 3) to run the reactions and store the PCR tubes at 4℃ (use within 2 days)
or -20℃(long-term storage).

Table 3. Q5 High-Fidelity 2X master mix PCR
Component 25 ul Reaction 50 ul Reaction Final Concentration
Q5 2X master mix 12.5 μl 25 μl 1X
10 uM Forward Primer 1.25 μl 2.5 μl 0.5 uM
10 uM Reverse Primer 1.25 μl 2.5 μl 0.5 uM
Template DNA 50-100 ng 50-100 ng ＜1000 ng
Nuclease-Free Water To 25 μl to 50 μl
Notes: Because the ordered primer is 100 uM, it needs to be diluted to 10 uM with nuclease-free water in
advance. Be very precise when adding solutions to the PCR set, as the reaction is very sensitive.
Protocol of making the 0.8% Agarose Gel
Required instruments and consumables: agarose powder, microwave, comb, gel cassette, autoclaved 200
mL volumetric flask, 1xTAE buffer, tissue.
15) Add 0.8g Agarose to 100ml 1xTAE buffer into the bottle.
16) Microwave until you see bubbles (1min).

20
17) Take out of the bottle with tissue since the bottle will be super hot, and shake lightly then continue
microwaving (30s) until all particles are dissolved.
18) Add 3.5 μl of Ethidium bromide (EtBr) to the liquid after you could hold the bottle for a few seconds
(15 mins).
19) Pour the liquid gel to the container with a proper size comb, leaving approximately ⅓ of the comb
exposed.
*Run out 10 of the PCR product on a 2% agarose gel to check for amplification. Keep the majority of the
PCR product, and purify it using a spin-column kit. Spec to determine concentration.

Protocol of gel electrophoresis
Gel electrophoresis is a standard laboratory procedure used to separate DNA by size (eg, base pair length)
for visualization and purification. Electrophoresis uses an electric field to move negatively charged DNA
through an agarose gel matrix towards a positive electrode. Shorter DNA fragments pass through the gel
faster than longer fragments, so they can be used to determine the approximate length of a DNA fragment
(Addgene, 2018. Addgene: Protocol - How to Run an Agarose Gel). Etbr, a non-radioactive label used to
stain DNA, intercalates between base pairs and binds to DNA, allowing DNA banding patterns to be
visualized when illuminated with a UV light source (Sigmon & Larcom, 1996).
Required instruments and consumables: gel, gel electrophoresis machine, 1xTAE buffer, PCR products,
1kb ladder, ddH₂O, 6X Loading Dye, micropipette gun and tips in 10ul and 200ul.

1) Add ladder at the first hole (and the last one in option) of the gel and then add the first group of PCR
products next to the ladder hole and skip one hole between every sample.
2) Make ladder tube: Add 4 μl of dH₂O, 1 μl ladder and 1 μl of 6X Loading Dye (purple lids for PCR
products, no DMSO).

21
3) Make PCR product tubes: add 6x Loading Dye (white lids) and mix lightly. Amount running on the gel
divide by six.
4) Fill the gel cassette with 1xTAE until the gel is covered.
5) Add an extra 7 μl of EtBr to the solution at the bottom of the gel, since EtBr is positively charged and
runs in the opposite direction to DNA, before starting the run to help better observe the results.
6) Carefully load the samples into the wells of the gel, no more than 35 μl for every well, preventing
interference from the addition of air bubbles. Place the very top of the pipette tip into the buffer above the
well, and using the other hand to support the forearm portion of the arm holding the pipette, push out the
sample slowly and steadily.
7) Close the lid of the gel cassette. Check the poles! Black is the negative pole, red is the positive pole.
DNA is negatively charged and will flow to the positive pole. Always runs to red.
8) Start the machine by running at 120V for 30 min, then check (the lower the voltage, the clearer the band
but the longer it takes, like 80V for 1h).
9) Carefully take out the gel! And check the results under Ultraviolet (UV).

Protocol adapted from QIAquick Gel Extraction Kit
Required instruments and consumables: centrifuge, QIAquick gel extraction kit, blade, 2 ml
microcentrifuge tube, micropipette gun and tips in 200 μl and 1000 μl.
Notes: All centrifuge speeds at 13000 rpm. Since both UV light and temperature can denature DNA, cut
the gel as soon as possible. Subsequent heating to dissolve the gel also speeds up the process by shaking
the tube every minute.

1) Excise the DNA fragment(should be clean bands at the correct size) from the agarose gel under UV with
a clean, sharp blade as soon as possible. And place the excised product in a clean labeled tube.
2) Weigh the mass of gel in the tube. Add 3 volumes of Buffer QG to 1 volume of gel (approximately 100
µ l is required for a 100 mg gel).

22
3) The tubes are then incubated at 50° C for 10 minutes (or until the gel pieces are completely dissolved).
Vortex the tube every minute to help dissolve the gel. After the gel pieces have completely dissolved, check
that the color of the mixture is yellow (similar to Buffer QG with undissolved agarose).
4) Add 1 gel volume of isopropanol to the sample and pipette 4-6 times to mix.
5) Place the QIAquick spin column into the provided 2 ml collection tube and centrifuge for 1 min. For
sample volumes >800 µ l, repeat the above steps until all the sample has passed through the column. Discard
the flow-through and put the QIAquick column back in the same tube.
6) If the DNA will subsequently be used for sequencing, in vitro transcription, or microinjection, add 500
μl Buffer QG to the QIAquick column and centrifuge for 1 min. Discard the flow-through and put the
QIAquick column back in the same tube.
7) Add 750 μl Buffer PE to the QIAquick column, let stand for 2-5 minutes, and centrifuge for 1 minute to
remove residual washing buffer.
8) Discard the flow-through and place the QIAquick column into a clean 1.5 ml microcentrifuge tube.
9) To elute DNA, add 20 μl water to the center of the QIAquick membrane, let the column sit for 4-6
minutes (increased yield) and centrifuge for 1 minute.
2.5 In-Fusion Cloning
We chose In-Fusion cloning as it is simple and time-saving by just combining the PCR product and the In-
Fusion cloning vector in the provided reaction buffer, waiting 5 minutes, then transforming into an E. coli
strain. The pipeline is shown in Figure 5.

23

Figure 5. Workflow of In-Fusion Cloning. Cited from the In-Fusion® HD Cloning Kit User Manual Protocol of In-
Fusion cloning adapted from In-Fusion® HD Cloning Kit User Manual.

Typically, good cloning efficiencies are obtained using 50–200 ng of vector and inserting each vector,
regardless of their length. More is not better because too much DNA can be toxic to competent cells. Table
4 lists the recommended volume, cited from In-Fusion® HD Cloning Kit User Manual. (Positive control:
provided in the kit. Negative control: known amount of linearized vector.)

Table 4. Recommended In-Fusion Reactions for Purified Fragments(adapted from In-Fusion kit)
Component Cloning Rxn Negative Control Rxn Positive Control Rxn
Purified PCR products 10-200 ng* - 2 μl of 2 kb control insert
Linearized vector 50-200 ng** 1 μl 1 μl of 2 kb control insert

24
Component Cloning Rxn Negative Control Rxn Positive Control Rxn
5X In-Fusion HD enzyme premix 2 μl 2 μl 2 μl
Deionized water To 10 μl To 10 μl To 10 μl
* ＜0.5 kb: 10-50 ng, 0.5 to 10 kb: 50-100 ng, ＞10 kb: 50-200 ng
** ＜10 kb: 50-100 ng, ＞10 kb: 50-200 ng

According to the original manual, I tested the In-Fusion cloning reaction set up as shown in Table 5, and
followed the subsequent steps to test. I found that this worked well in my hands, because first the dna size
met the requirement, and then in order to add an equal amount to the Linearized vector to combine with it,
you only need to add 1 μl Purified PCR fragment (too much dna is toxic to competent cells). At the same
time, because I used PCR instrument instead of the incubator for cultivation, the binding efficiency can be
improved and the enzyme can be saved because of its expensive price.

Table 5. In-Fusion cloning reaction
1 μl 5X In-Fusion HD enzyme premix
1 μl Linearized vector
1 μl Purified PCR fragment
2 μl dH₂O
5 μl Total volume

1) I used the In-Fusion cloning settings in Dr. Crump’s laboratory PCR machine. I incubated the configured
reaction for 1 hour, and then placed it on ice. If you cannot transform cells immediately, store the cloning
reaction at –20° C until you are ready.
2) Thaw Stellar Competent Cells, Escherichia coli (E. coli), on ice before use. After thawing, mix gently
to ensure even distribution, then pipette 25 µ l of competent cells into 14 ml falcon tubes. Do not vortex.
3) Add 1 µ l of In-Fusion Reaction Mix to the competent cells.
4) Place the tubes on ice for 30 minutes. Put SOC medium at 37° C.

25
5) Pleace the tubes in water bath at 45° C for 30 seconds.
6) Place tubes on ice for 1-2 minutes to recover.
7) Add SOC medium to a final volume of 500 µ l.
8) Incubate at the shaking incubator at 37° C for 1 hour.
9) Pipette 100 µ l of each transformation reaction onto separate LB plates containing 100 µ g/ml ampicillin
with clear labels.
10) Centrifuge the remainder of each transformation reaction at 6,000 rpm for 5 minutes. Discard the
supernatant and resuspend each pellet in 100 µ l fresh SOC medium. Plate each sample on separate LB
plates containing 100 µ g/ml ampicillin with clear labels.
11) Incubate all plates overnight at 37° C.
12) The next day, pick individual isolates from each assay plate. Then follow the next experiment to grow
the products.

Protocol use Liquid Bacterial Culture to grow the colonies products
1. Prepare liquid LB. For example, to make 400 mL of LB, weigh out the following into a 500 mL glass
bottle. And autoclave the solution using protocol ‘liquid 15’ in the machine. Loosely cap the bottle (do not
fully cap or the bottle may explode!) and cover the entire top of the bottle loosely with aluminum foil.
Autoclaved LB can be stored at room temperature.
2. When the culture medium is ready (it takes about 1h to completely cool down), add liquid LB to 14 ml
falcon tubes, and add appropriate antibiotics to the correct concentration. I used 100 µ g/mL ampicillin, so
I added 4 µ l ampicillin to 2 mL liquid LB and pipetted 4-6 times to mix.
3. Using a 10 µ l sterile pipette tip, gently scrape a free-standing colony from the LB agar plate that is not
attached to other colonies.
4. Drop the tip or toothpick into the liquid LB + antibiotic and swirl.
5. Put the tips into the liquid LB + antibiotic and close the lid.

26
6. Incubate the bacterial culture for 12-18 hours in a shaking incubator at 37° C.
7. After incubation, check for growth, which is characterized by a cloudy haze in the media (Fig.6)
(Addgene, 2019. Addgene: Protocol - How to Inoculate a Bacterial Culture).
8. Finally, use QIAmini prep kit to purify the plasmid DNA.

Figure 6. Media with growth (left) and without growth (right). Tubes with bacterial growth are cloudy, while
controls should be clear (cited from Addgene, 2019).

2.6 Fish experiments
2.6.1 Oocyte microinject the plasmid DNA
Part 1: Preparing for the injection
1. One day before the experiment, use Dr. Crump's laboratory needle preparation machine to prepare
injection needles using glass capillary tubes with an outer diameter of 1 mm. Store in needle holder.
2. Production and Collection of Zebrafish Eggs. The night before the injection, the fish were placed in a
culture tank with partitions. In order to increase the total egg production, I set the fish according to the ratio
of two females and three males. (Zebrafish will only spawn in the morning, sometimes they will not spawn
due to various factors such as age, so it is recommended to divide into several groups at a time).

27
Part 2: The day of the injection
3. Follow table 6 to prepare the inject reaction before 9 am, since the tubes of Tol2 are stored at -80℃.

Table 6. zebrafish oocyte inject reaction
6 μl DNA (50 ng/μl)
2 μl Loading dye
1.7 μl dH₂O
0.3 μl Tol2 enzyme
10 μl Total volume

4. After 9am, when the lights come on in the room, pull down a few tank dividers and place the fish in new
plastic strips and allow the fish about 15 minutes of undisturbed mating time. The optimal injection stage
is the one-cell stage, i.e. within 45 min after mating, which will then progress to the two-cell stage (Kimmel
et al., 1995), which may lead to undesirable outcomes where the target cells may not carry the injected gene.
5. Wait for the process to assemble the needle. Use a micropipette to add 1 µ l of plasmid solution to the
needle, and load it onto the injection device lever after waiting until the liquid has completely drained from
the bottom to the needle tip.
6. Slightly turn the valve to open the air source, the normal air source should be at 3kpa-4kpa air pressure.
7. Open the microinjector. I used the handheld joystick (fixed rods are also available) to bring the tip into
the field of view of the microscope, off stage height, and focus on the thinnest area of the tip. Use a pair of
sharp pliers to pinch off the needle at one point, allowing each depressing of the valve needle to produce a
consistent bead size.
8. After the eggs are collected, return the fish to the original breeding tank to re-segregate the sexes. Eggs
were collected in the spawning cages with a strainer by pouring the water with the eggs directly into the
sink. Then, while tapping the strainer, rinse the eggs hanging on the strainer with water into a clean plate.

28
Part 3: Injection
9. Take a new clean plate, use a pipette to absorb the fish eggs and transfer the fish eggs to the plate by
adding as little liquid as possible, and then absorb the excess liquid as much as possible to dry the fish eggs,
so that the fish eggs will be placed next to each other for easy injection.
10. Lower the needle towards the egg post and use the other hand to hold the plate in place.
11. Pierce the surface of the eggshell and enter the egg yolk smoothly, while observing whether the yolk
sac is crushed or torn. The injection material is injected into the egg yolk and it is easy to see if the injection
was successful. A successful injection will have a steady orange-red (from loading dye) spot (Fig.7). The
unsuccessful ones will dissipate. Avoid injecting air bubbles or stretching the yolk, as both are fatal to the
embryo.
12. After the injection is completed, add culture medium to the plate.
13. At the end of the day, transfer the surviving well-developed embryos to a new plate and replace the
medium while recording the number of injected embryos. Just change the culture medium in the plate again
the next morning (the normal culture temperature is 28° C, but we can put the embryos at 22° C to delay the
development, which can be delayed by about double the time).

Figure 7. Successful injected embryos. After injection, stable red spherical droplets were observed, which did not
dissipate (cited from Rosen et al., 2009).
2.6.2 Check the injection animals with confocal

29
Users should complete the confocal training with instruction from people at Dr. Crump’s lab. And keep the
positive injected fish for the second generation.

30
CHAPTER 3 RESULTS
3.1 Identification of the putative enhancers using snATAC-seq.
I followed these steps to get the ideal enhancers:
1. Get the authorization from Sunghyun Kim to view the database. Access the UCSC Genome Browser:
Visit the UCSC Genome Browser website (https://genome.ucsc.edu/) and navigate to the section that allows
we to access and download data.
2. Select the Relevant Tracks: Within the dataset, there may be multiple tracks corresponding to different
aspects of the data, such as raw reads, processed reads, and annotations. Select the tracks that contain the
snATAC-seq data we're interested in.
3. Choose the Organism: Ensure that we are selecting tracks for both mouse and human since we want data
from both species.
4. Download the Data: Most likely, the UCSC Genome Browser will provide options to download the data
in various formats.
5. Differential Accessibility Analysis: Perform differential accessibility analysis to identify genomic
regions that exhibit differential chromatin accessibility between relevant cell clusters. Focus on clusters or
cell types known to be involved in POU3F3 expression during nephrogenesis.
6. Enhancer Identification: Annotate the differentially accessible regions with known genomic features
using available databases and tools. This step helps identify potential enhancers. Prioritize regions that
exhibit characteristics of enhancers, such as enrichment for histone modifications associated with active
enhancers or known transcription factor binding motifs.
7. Cross-Species Analysis: Using Lift-over, a tool at UCSC genome browser, to compare the identified
enhancer candidates between mouse and human datasets. Look for conserved regions that exhibit similar
chromatin accessibility patterns and potential regulatory activity across species. Prioritize enhancer
candidates that show conservation and overlap between the mouse and human datasets.
Then the five candidate regions are selected (Fig.8B).

31

Figure 8. Top candidate enhancer regions selected.
A. UMAP of nephrogenic and ureteric lineages were subsetted and progenitors were identified and later development
tracked. The numbers of the clusters match the lineage numbers in B.
B. Five candidate regions were chosen to be injected in the project indicated in the screenshot from UCSC browser.
Since I wanted to study pro-nephrons, I mainly focused on lineages with peaks in NPCs (lineages in green box in
A
B

32
panel b) and distal lineages (lineages in pink box in panel b), rather than lineages in podocytes or proximal tubules. I
then select the blue bar as a candidate. Kim et al. Reanalyzed data in 2023.
3.2 PCR results of the candidate enhancers
After the template DNA was extracted from iPSCs donated by volunteers according to the steps of method
2.4.1, the target DNA region was amplified by PCR using the primers designed in Table 2. The results are
shown in the Fig 9.

Figure 9. Representative PCR results. I performed PCR and checked the results for candidate enhancers of POU3F3
by electrophoresis. 1 kb lader, blue arrows points at 500 bp of the ladder. I ran the gel under 120V for 45 mins. Size
of the candidate regions, C1: 982 bp, C2: 731 bp, C3: 784 bp, C4: 1029 bp, C5: 1117 bp.

3.3 Preparation and testing of plasmid

C7

C4
C5 C3 C4

33

Figure 10. The map of origional plasmid, eda_E1B:mCherry Tol2 plasmid (Crump lab -Fabian et al., 2022).
E1b is the minimal promoter in upstream of mCherry. Tol2AB2 is the destination vector. Bcrystalline:CFP, which
serves as an internal control for our injections. pA, polyadenylation sequence---The poly-A tail makes the RNA
molecule more stable and prevents its degradation. Additionally, the poly-A tail allows the mature messenger RNA
molecule to be exported from the nucleus and translated into a protein by ribosomes in the cytoplasm

First, use EcoRV and SmaI to remove the original 'eda' fragment to obtain a linearized vector (Fig.11) Then
connect the PCR product of the previous enhancer and the linearized vector through the In-fusion cloning
kit to obtain the plasmad carrying the target gene(Fig.12). After the plasmid is injected into a zebrafish
embryo, it undergoes a process called transgenesis, which involves the integration of foreign DNA into the
developing zebrafish's genome. The process works as follows:
1. Integration: The microinjected plasmid DNA contains a specific Tol2 transposon system that allows the
plasmid to integrate into a specific site in the zebrafish genome, thereby enabling stable inheritance of the
transgene in offspring.
2. Promoter activity: The E1B promoter contained in the plasmid is responsible for driving the expression
of mCherry fluorescent protein.

34
3. Fluorescent protein expression: As the zebrafish embryo develops, the integrated plasmid will replicate
with the host genome in each cell division. Potentially results in the production of mCherry fluorescent
protein in cells where the pou3f3_E1B promoter is active.
4. Observation and analysis: Researchers can observe the fluorescence emitted by mCherry protein under
a fluorescence microscope to be able to track the expression pattern of the pou3f3_E1B promoter and thus
the specific tissue or cell in which the promoter is active.

Figure 11. Double-digestion of circular vectors. EcoRV and SmaI double digested the original plasmid overnight
at room temperature.Electrophoresis was carried out under 120V for 2 hours. The top bright band is the linearized
vector, the bottom band is the original inset.

35

Figure 12. In-Fusion cloning LB plates after overnight incubation at 37° C. Examples of In-Fusion cloning LB
plates after overnight incubation at 37° C. The leftmost is the positive control, the first line from left to right is
POU3F3_candidate enhancer 1_GFP_Tol2, POU3F3_candidate enhancer 1_mcherry_Tol2 and the second line is
POU3F3_candidate enhancer 2_GFP_Tol2, POU3F3_candidate enhancer 2_mcherry_Tol2
3.4 Injected animals
I first found the earliest expression of pou3f3 in zebrafish, also used this as a positive control in the injection
groups (see in Fig 13). Then I performed injections by following steps of method 2.6. The resulting fish are
shown in Fig 14.The expression of pou3f3_mCherry in the pronephric region was not observed in the
injected group.

36

Figure 13. Profiling the expression of pou3f3 in zebrafish nephrogenesis. The earliest stage of pou3f3 expression
in the zebrafish nephron. Two growth stages after crossing cdh17:mCherry (Hukriede lab) with pou3f3b:nlsGFP
(Crump lab). The image on the left was taken at 10.5 hours and the image on the right was taken at 22 hours. pou3f3
is not only expressed in the kidney, but also expressed in the nerves, but zebrafish cdh17 is expressed exclusively in
the pronephros during embryogenesis and in the mesonephros during larval development and adulthood, so used it as
a double marker to confirm the expression of pou3f3 in the pronephros.

Figure 14. Transgenic fish generated by oocyte injections. Groups of oocyte injection: a) positive control injected
with original plasmid (col9a3_p1_E1B_mCherry_Tol2). Since the plasmid has Bcrystalline:CFP, which serves as an
internal control for our injections (we also call this eye:CFP, or blue eyes). b) Experimental groups that injected with
a b 1 b 2

37
POU3F3_mcherry_Tol2 into wildtype(Tuebingen), and two injected fish with positive muscle cells were selected.
Results shown represent all 5 injection results.

38
CHAPTER 4 DISCUSSION
4.1 Problems encountered during the experiments and solutions
During the course of this project, I encountered some challenges, one of the main ones was the failure of
the cloning process, as shown in Figure 15. However, through experimentation and refinement, I have
made some important discoveries and modifications to the culture protocol.

Figure 15. Failed In-Fusion clone LB plates. Examples of In-Fusion cloning LB plates after overnight incubation at
37° C. The rightmost is the positive control, from In-Fusion® HD Cloning Kit. The first line from left to right is
POU3F3_candidate enhancer 1_mcherry_Tol2, POU3F3_candidate enhancer 1_GFP_Tol2 and the second line is
POU3F3_candidate enhancer 2_GFP_Tol2, POU3F3_candidate enhancer 2_mcherry_Tol2

First, I found that using a PCR device during the incubation of an In-Fusion reaction significantly improved
efficiency. This improvement increases the success rate of the cloning process. Also, I learned that it is
critical to digest circular vectors into linearized vectors using restriction enzymes (specifically EcoRV and

39
SmaI). It is essential to include controls such as no-cut and single-cut groups and ensure sufficient run time
(120V for 2 hours) during gel electrophoresis. This step is necessary because the size difference between
the linearized vector and the circular vector is not significant, and running the gel for a sufficient time
prevents incomplete digestion and helps distinguish the circular vector from the desired linearized vector.
transformation carrier. Failure to establish a control group and run the gel long enough in the unsuccessful
group may be due to the presence of large numbers of round vectors in the gel extraction product.
In addition, I learned the importance of doing each experiment carefully and paying attention to details. For
example, when loading PCR samples onto gels, precision and accuracy are critical due to the sensitivity of
the reactions. When preparing the extraction gel, I realized that the concentration should not be too high or
too thick. This precaution is necessary because a hot plate will be used later to dissolve the gel, and high
temperatures can adversely affect the DNA.
Also, it is valuable to keep detailed experimental records and seek input from others for discussion. Through
these discussions, I gained valuable insights and suggestions to improve my techniques and experimental
procedures, which ultimately allowed me to gain a better understanding of areas where I could improve my
skills.
Overall, this project has taught me the importance of paying attention to details, carefully optimizing
protocols, and constantly learning from successes and failures.
4.2 Conclusion and Future Perspectives
The primary issue encountered in the current study pertains to the misplacement of fluorescent expression
within zebrafish specimens. Following the initial transgenic injection, a significant proportion of first-
generation zebrafish exhibited mCherry fluorescence predominantly in the muscle and skin cells. Insight
from individuals within Dr. Crump's laboratory suggests that such observations are not uncommon, with
successful injections yielding second-generation zebrafish that display more desirable patterns of

40
fluorescent protein expression along with increased positional accuracy. However, the underlying reasons
supporting these claims remain uncertain, prompting further inquiry into the basis of their assertions.
Differences between species may also be one of the reasons why experiments did not achieve the expected
results. Sperber et al. (2008) showed that even closely related species may exhibit differences in gene
function and regulation. Kos-Echepare et al. (2018) showed that although the pou3f3 gene is highly
conserved across vertebrate species, its precise expression pattern and function in the kidney may vary
across species. This cross-species variation presents challenges when trying to extrapolate findings directly
from one species to another. In this specific context, the authors note that although pou3f3 expression has
been extensively studied in mammals, including mice and humans, its expression and function appear to be
different in Xenopus kidneys. This finding underscores the importance of species-specific studies for an
accurate understanding of the role of genes in development. And Quillion et al. (2017) showed that while
zebrafish are a valuable model for studying vertebrate development, it is important to recognize species-
specific differences in gene regulatory elements, including enhancers, between zebrafish and humans.
These differences may affect the functional characteristics of human gene enhancers in zebrafish.
The observation of GFP fluorescence in muscle and skin in the first-generation injected zebrafish can be
attributed to several factors, including the characteristics of the transgenic construct, the efficiency of
injection, and the developmental stage of the embryos. The explanation provided by the people in Professor
Gage Crump's laboratory regarding the occurrence of this phenomenon and the subsequent generation of
fish with ideal positions and stable expression of the fluorescent protein is likely based on their experience
and empirical evidence.
1. Design of transgene constructs: The design of transgene constructs plays a crucial role in determining
the tissue-specific expression of fluorescent proteins. If the transgenic construct contains a promoter that is
active in multiple tissues, including muscle and skin, it may lead to non-specific expression patterns in the
first generation injected zebrafish. However, the construct may also contain other regulatory elements that
promote specific expression in the desired target tissue (eg kidney).

41
2. Injection efficiency: The success of the injection, including the precise delivery of the transgene construct,
may vary between individual zebrafish embryos. In some cases, the injected constructs may not be evenly
distributed, or may not integrate into the genome of every cell. This results in a mosaic expression pattern,
where the fluorescent protein is expressed in some cells but not others. This mosaic expression can lead to
the observation of fluorescence in unexpected tissues such as muscle and skin. A study by Thermes et al.
(2002) demonstrated a mosaic expression pattern resulting from transgene injection in zebrafish embryos.
The authors used a transgenic construct containing a green fluorescent protein (GFP) reporter gene and
injected it into zebrafish embryos. They observed mosaic expression of GFP in different tissues, including
muscle and skin, indicating uneven distribution and integration of the transgenic construct.
3. Developmental stage: The developmental stage of the zebrafish embryo at the time of injection affects
the expression pattern of the transgenic construct. During zebrafish embryogenesis, different tissues and
organs develop at different rates. If the injection is performed at an early stage, before the specific tissue of
interest is fully differentiated, it may result in non-specific or ectopic expression of the fluorescent protein.
Taken together, these studies support the notion that efficient delivery and uniform integration of transgenic
constructs in zebrafish embryos can be challenging, resulting in mosaic expression patterns. This mosaic
expression leads to the observation of fluorescence in unexpected tissues such as muscle and skin.
The second-generation of injected zebrafish often exhibits improved expression patterns and stability of
fluorescent proteins can be attributed to several factors as follow:
1. Germline Integration: Successful integration of the transgene into the germline cells during the first
generation allows for its transmission to subsequent generations. This ensures stable and consistent
expression patterns (Amsterdam et al., 1999).
2. Mosaicism Reduction: In the first generation, mosaic zebrafish may exhibit varied expression due to
incomplete transgene integration in different cells. During the second generation, the transgene is more
likely to be present in all cells, resulting in a more uniform and stable expression pattern (Amsterdam et al.,
1999).

42
3. Dilution of Mosaic Expression: Mosaic expression can occur due to random integration of the transgene.
In the second generation, when the transgene is inherited by a higher number of cells, the variation in
expression levels among cells may become less prominent, leading to improved overall expression
(Amsterdam et al., 1999).
4. Enhanced Promoter Activity: Transgene integration in the germline may allow for better utilization of
endogenous regulatory elements, leading to more consistent and robust promoter activity (Parinov et al.,
2004).
5. Selective Advancement: The presence of a transgene that confers a survival advantage to the developing
zebrafish may lead to a biased representation of transgene-bearing individuals in the second generation,
resulting in enhanced expression (Parinov et al., 2004).
6. Transgenerational Epigenetic Effects: Epigenetic modifications acquired in the first generation may
influence transgene expression in subsequent generations. These modifications can stabilize and enhance
expression patterns (Lindeman et al., 2011).
7. Natural Selection: Over generations, individuals with unfavorable transgene integration may be selected
against due to fitness disadvantages, leading to an enrichment of individuals with improved expression
patterns (Lindeman et al., 2011).
Together, these studies support the notion that the presence of germline-transmitted transgenes in progeny
zebrafish contributes to improved expression patterns and stability of fluorescent proteins. Transgene
inheritance through germline integration ensures more consistent and tissue-specific expression.
However, it is important to acknowledge that individual experimental setups and specific transgenic
constructs may exhibit different results. Factors such as construct design, integration site, and genetic
background can affect transgene stability and expression patterns. Therefore, careful evaluation and
optimization of injection protocols, construct design, and developmental stage are critical to achieve the
desired tissue-specific expression in zebrafish models.

43
I tried to combine multiple candidate regions of POU3F3 to design combined DNA fragments, but the plan
was rejected by the companies due to large variation in GC content.
Then, designing a cognate promoter for POU3F3/pou3f3 may be important in subsequent enhancer testing
projects. A cognate promoter or core promoter refers to the native or endogenous promoter of a gene
associated with an enhancer region (F Quillien et al. ,2017). The reason is that homologous promoters have
the following characteristics: 1. Enhancer promoter specificity: homologous promoters contain regulatory
elements specifically recognized by enhancers to ensure precise control of gene expression. This specificity
is critical for accurately studying the functional interactions between enhancers and promoters. 2. Tissue-
specific expression: Cognate promoters often exhibit tissue-specific expression patterns, which can provide
valuable insights into enhancer activity in specific cell types or developmental stages. The use of
homologous promoters allows the study of enhancer function in a context that closely mimics natural gene
expression patterns. 3. Differential regulation: Homologous promoters may respond differently to enhancer
activity compared to heterologous or synthetic promoters. Understanding the nuances of enhancer-promoter
interactions in natural environments can reveal the complex regulatory mechanisms that control gene
expression (Ong and Corces, 2012).
Identifying the homologous promoter of the POU3F3/pou3f3 gene involves several steps, the following is
my own pipeline summarizing the process: 1. Gene annotation and sequence analysis: first retrieve the
genomic sequence of the pou3f3 gene from a reliable database (such as NCBI or Ensembl) . Analyze gene
structure and identify transcription start site (TSS) regions. 2. In silico promoter prediction: Computational
tools and algorithms were used to predict potential promoter regions upstream of the POU3F3/pou3f3 gene.
These tools typically consider specific DNA sequence motifs and transcription factor binding sites
associated with promoter activity. 3. Comparative genomics: Comparative genomic analysis was performed
to identify conserved regions upstream of POU3F3/pou3f3 in different species. Conserved non-coding
sequences often indicate potential regulatory regions, including cognate promoters. 4. Functional analysis:
Perform functional analysis of the putative promoter region, such as mutagenesis studies or deletion

44
analysis, to identify essential regulatory elements. These experiments helped to narrow down the specific
sequences responsible for driving the expression of the POU3F3/pou3f3 gene. 5. Promoter reporter gene
detection: first clone the putative promoter determined by the above steps into the reporter vector proved
to work well by Dr. Crump's lab, and inject it into suitable fish embryos. Measure reporter gene activity. If
it is good, it can be combined with the plasmid vector designed by yourself, and tested by dividing into
three groups, the existing basal promoter and enhancer; cognate promoter; cognate promoter combined with
enhancer.
If the expression of fluorescence in pronephros is successfully observed, subsequent bulk RNA-seq can be
performed to analyze the specific results. The identification regions of the genome that physically interact
with regulatory proteins and may serve as cognate promoters.
To overcome these limitations, alternative model systems are essential. By utilizing model organisms,
researchers can accelerate discovery, gain mechanistic insights, and develop new therapeutic strategies for
kidney disease.

45
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Abstract (if available)

Abstract The kidney is a structurally complex organ system affected by many congenital anomalies. About 3% of babies are born with renal or genitourinary abnormalities. These developmental defects are associated with multiple genes, many of which have unknown functions in the renal organogenesis program (Schnell et al., 2022). The distal nephron plays a crucial role in regulating electrolyte and fluid balance in vertebrates. The transcription factor POU3F3 is implicated in the development and function of the mammalian distal nephron; however, its regulation remains unclear. This study aimed to investigate the function of putative POU3F3 nephron enhancers using the zebrafish pronephros as model.

To address this question, I performed cross-species comparisons across all cell-types in the developing mouse and human nephron, identifying putative cis-regulatory regions matching the known expression dynamics of Pou3f3 and POU3F3 using single-nuclear assay for transposase-accessible chromatin (ATAC)- seq data. I found regions of DNA surrounding Pou3f3 and POU3F3 that correspond to their respective expression profiles. The majority of these are deeply conserved while others are present in mouse or human but not the other. A series of potential enhancers of POU3F3 were then assayed by generating enhancer reporter constructs and tested by generating transgenic zebrafish lines. Positive controls enhancers with known function were used to successfully drive reporter expression, 5/7enhancers tested failed to express in the distal zebrafish pronephros, the purpose of this project to analyze POU3F3 via enhancer reporter constructs failed.

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Asset Metadata

Creator Zhou, Tianming (author)

Core Title Exploring the function of distal nephron enhancers in zebrafish

School Keck School of Medicine

Degree Master of Science

Degree Program Stem Cell Biology and Regenerative Medicine

Degree Conferral Date 2023-12

Publication Date 09/11/2023

Defense Date 08/23/2023

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

Tag enhancer,kidney,nephron,OAI-PMH Harvest,zebrafish

Format theses (aat)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Nils, Lindstrom (committee chair), Jadhav, Unmesh (committee member), Mariani, Francesca (committee member)

Creator Email tzhou239@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC113304705

Unique identifier UC113304705

Identifier etd-ZhouTianmi-12338.pdf (filename)

Legacy Identifier etd-ZhouTianmi-12338

Document Type Thesis

Format theses (aat)

Rights Zhou, Tianming

Internet Media Type application/pdf

Type texts

Source 20230911-usctheses-batch-1093 (batch), University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the author, as the original true and official version of the work, but does not grant the reader permission to use the work if the desired use is covered by copyright. It is the author, as rights holder, who must provide use permission if such use is covered by copyright.

Repository Name University of Southern California Digital Library

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