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Nr5a2 balances lineage decisions to promote diverse connective tissue fates in the jaw
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Nr5a2 balances lineage decisions to promote diverse connective tissue fates in the jaw
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Content
Nr5a2 Balances Lineage Decisions
to Promote Diverse Connective Tissue Fates in the Jaw
By
Hung-Jhen Chen
A 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 CELLS, AND REGENERATIVE MEDICINE
May 2023
Copyright 2023 Hung-Jhen Chen
ii
Acknowledgements
Hung-Jhen Chen and J. Gage Crump conceived of experiments. Hung-Jhen Chen conducted
zebrafish and mouse experiments and multi-omic data analysis. Lindsey Barske, Jared C.
Talbot, and Christian Jimenez initiated the categorization of zebrafish phenotypes. Hung-Jhen
Chen, Olivia M. Dinwoodie, Ryan R. Roberts, Amy E. Merrill, Abigail S. Tucker, and D'Juan T.
Farmer categorized mouse phenotypes. Hung-Jhen Chen, Ryan R. Roberts, and D'Juan T.
Farmer performed the single-nuclei multiome. J. Gage Crump, Amy E. Merrill, and Abigail S.
Tucker obtained funding and oversaw the project. Portions of this work have been submitted for
publication.
I thank Jenna Galloway for Tg(scxa:mCherry)
fb301
, the Broad CIRM Center Flow Cytometry
Facility for FACS, the CHLA Center for Personalized Medicine Molecular Genomics Core for
NGS sequencing, Megan Matsutani and Olivia Ruffins for fish care, and Samantha Brugmann
for sharing the protocol of colorimetric whole-mount in situ for mouse embryos.
iii
TABLE OF CONTENTS
Acknowledgements ....................................................................................................................................... ii
List of Tables ............................................................................................................................................... iv
List of Figures .............................................................................................................................................. v
Abstract ........................................................................................................................................................ vi
Graphical Abstract ...................................................................................................................................... vii
Introduction .................................................................................................................................................. 1
Chapter 1. Conserved expression of Nr5a2 within the aboral domain of the zebrafish
and mouse mandibular arches ..................................................................................................................... 4
Chapter 2. Requirement for nr5a2 in zebrafish lower jaw development ...................................................... 6
Chapter 3. Nr5a2 functions cell-autonomously in zebrafish CNCCs to specify tenocytes
at the expense of chondrocytes ................................................................................................................... 8
Chapter 4. Nr5a2 misexpression mildly suppresses chondrogenesis and disrupts tendon formation
in the zebrafish lower jaw ........................................................................................................................... 10
Chapter 5. Requirement for Nr5a2 in mouse NCCs for lower jaw and middle ear development ............... 11
Chapter 6. Single-cell multi-omics analysis of mandibular CNCC defects in nr5a2 mutants ..................... 13
Chapter 7. Identification of direct targets of Nr5a2 for diverse connective tissue fates ............................. 15
Discussion .................................................................................................................................................. 17
Figures ....................................................................................................................................................... 21
Tables ........................................................................................................................................................ 42
References ................................................................................................................................................. 47
Appendices ................................................................................................................................................ 52
Appendix A. Experimental model and subject details ............................................................................ 52
Appendix B. Method details ................................................................................................................... 54
Appendix C. Quantification and statistical analysis ................................................................................ 67
iv
List of Tables
Table 1. Differentially accessible chromatin regions in nr5a2 mutant mandibular
mesenchyme associated with differentially expressed genes and Nr5a2 motifs………………42
Table 2. Key resources table………………………………………………………………...………43
v
List of Figures
Graphical abstract……………………………………………………………………………………….vii
Figure 1. Nr5a2 expression within the developing zebrafish and mouse face……………………21
Figure 2. Detailed facial expression of nr5a2
mGFP
in zebrafish and Nr5a2 in mouse…………….23
Figure 3. Requirement of nr5a2 for zebrafish lower jaw development……………….……………25
Figure 4. Characterization of nr5a2 alleles and cell proliferation assays…………………………27
Figure 5. Nr5a2 functions cell-autonomously in zebrafish CNCCs to promote tenocyte
at the expense of chondrocyte fate……………………………………………………………………29
Figure 6. Nr5a2 functions in CNCCs and is sufficient to suppress chondrogenesis
and disrupt tendon formation in the zebrafish lower jaw……………………………………………30
Figure 7. CNCC requirement for Nr5a2 in mouse mandibular arch development……………….31
Figure 8. Detailed phenotypic analysis of mouse conditional Nr5a2 mutants……………………33
Figure 9. Single-cell analysis of gene expression and chromatin changes in nr5a2 mutants…..35
Figure 10. Single-cell analysis of marker gene expression in entire nr5a2 mutant
and control datasets…………………………………………………………………………………….36
Figure 11. Nr5a2 regulates jaw-specific enhancer accessibility and gene expression for
perichondrium and tendon genes…………………..…………………………………………………38
Figure 12. Analysis of Nr5a2-regulated enhancers, upregulated genes in nr5a2 mutants,
and normal axis patterning in nr5a2 mutants………………………………………………………...40
vi
Abstract
Organ development involves sustained production of diverse cell types with spatiotemporal
precision. This is especially true in the vertebrate jaw, where neural-crest-derived multipotent
progenitors produce not only the skeletal tissues but also the later-forming tendons and salivary
glands. Here we identify the pluripotency factor Nr5a2 as essential for balancing cell fate
decisions to generate the full repertoire of neural-crest-derived fates in the jaw. In zebrafish and
mice, we observe transient expression of Nr5a2 in a subset of mandibular post-migratory
neural-crest-derived cells. In zebrafish nr5a2 mutants, nr5a2-expressing cells generate excess
cartilage, resulting in an expanded lower jaw skeleton and loss of later forming tendons. In mice,
neural crest-specific Nr5a2 loss results in analogous skeletal and tendon defects in the jaw and
middle ear, as well as salivary gland loss. Single-cell profiling in mutants reveals that Nr5a2,
distinct from its roles in pluripotency, promotes jaw-specific chromatin accessibility and gene
expression essential for tendon and gland fates. Thus, repurposing of Nr5a2 within post-
migratory neural-crest-derived cells restricts skeletal and promotes connective tissue fates to
generate the full repertoire of derivatives required for proper jaw and middle ear function.
vii
Graphical Abstract
1
Introduction
The gnathostome jaw is a remarkable evolutionary innovation that facilitated predation, feeding,
and communication. In addition to the jaw skeleton (bone, cartilage, and teeth), multiple soft
connective tissue components are required for its function. For example, tendons connect the
jaw skeleton to musculature, ligaments stabilize the jaw joint, and salivary glands lubricate the
oral cavity. Remarkably, the jaw skeleton, tendons, ligaments, and salivary gland mesenchyme
all arise from cranial neural crest-derived cells (CNCCs) of the mandibular arch (Chai et al.,
2000; Chen and Galloway, 2014; Crump et al., 2006; Platt, 1893; Schilling and Kimmel, 1994).
CNCCs closer to the future oral cavity gives rise to Meckel’s cartilage, intramembranous regions
of the dentary bone, and teeth, while CNCCs located in the more posterior (i.e. aboral) domain
of the mandibular arch contribute to jaw tendons and salivary gland mesenchyme. In mammals,
the more proximal domains of the mandibular arch also give rise to skeletal and connective
tissue elements of the middle ear (Anthwal and Thompson, 2016). The mechanisms that induce
skeletal versus non-skeletal connective tissue fates from mandibular arch CNCCs remain
unclear.
Transcription factors for skeletal and connective tissue fates in the jaw and middle ear, as well
as the rest of the body, have been identified. Sox9 and Runx2 are essential for the specification
of cartilage (Bi et al., 1999; Yan et al., 2005) and bone (Ducy et al., 1997), respectively, and
Scleraxis (Scx) is important for tendon and ligament formation (Kague et al., 2019; Murchison et
al., 2007). Sox9 is also regionally activated in distal mandibular epithelium, in part by a local
mesenchymal source of Fgf10, to induce salivary gland development (Chatzeli et al., 2017).
However, we have an incomplete understanding of how the expression of lineage-specific
transcription factors, such as Sox9 and Scx, is restricted to precise spatiotemporal
mesenchymal domains. While the expression of these transcription factors is likely controlled by
2
region-specific enhancers, the upstream factors controlling their region-specific expression have
yet to be identified.
CNCCs contribute to a wide diversity of cell types (Bronner and LeDouarin, 2012). It remains
debated, however, if their multilineage capacity is endowed during specification or acquired after
their migration into the head (reviewed in (Fabian and Crump, 2022)). Whereas experiments in
frog suggested that CNCCs might retain pluripotency from blastula stages (Buitrago-Delgado et
al., 2015), later studies in mouse support a reacquisition of multipotency at CNCC specification
stages due to induction of pluripotency factors Oct4 and Sox2 (Zalc et al., 2021). Our recent
single-cell genomic analysis of CNCC differentiation in zebrafish shows that the majority of
enhancers required for CNCC lineage capacity gain accessibility after CNCCs migrate into the
jaw-forming mandibular arch (Fabian et al., 2022). This suggests that opening of lineage-
specific enhancers, and therefore access to transcription factors, plays a major role in
determining the range of potential fates for CNCCs in the developing face, yet few factors that
regulate CNCC enhancer accessibility have been identified.
Through single-cell profiling of zebrafish CNCCs, we recently identified nr5a2 as a highly
specific marker of the aboral domain of the mandibular arch of zebrafish (Fabian et al., 2022).
Nr5a2 is a member of the nuclear receptor transcription factor family and is considered orphan
as a physiological ligand remains unknown. Nr5a2 functions to maintain Oct4 expression in
mouse embryonic stem cells (Gu et al., 2005) and can replace Oct4 in reprogramming to
induced pluripotent cells (Heng et al., 2010). Reflective of this key role in pluripotency, loss of
Nr5a2 in mouse results in lethality at blastula stages, around embryonic (E) day 6.5 (Gu et al.,
2005; Pare et al., 2004). Nr5a2 also has roles in development of the liver (Pare et al., 2004),
pancreas (Hale et al., 2014), and nervous system (Stergiopoulos and Politis, 2016). Whereas
zebrafish nr5a2 mutants have liver and pancreas defects, effects on CNCC differentiation and
3
jaw development were not examined (Nissim et al., 2016). Here we found that Nr5a2 plays a
conserved role in the aboral mandibular domain of zebrafish and mouse embryos where it
promotes tendon and salivary gland fates at the expense of skeletal fates. In particular, we
found that Nr5a2 establishes accessibility of enhancers required for maintaining undifferentiated
progenitors while priming them for later tendon and gland differentiation. These targets of Nr5a2
in CNCCs are distinct from those described in embryonic stem cells (Heng et al., 2010). Nr5a2
therefore promotes diverse connective tissue fates at the expense of skeletal fates in a
restricted subset of postmigratory mandibular CNCCs through binding to genomic targets
distinct from those important for pluripotency.
4
Chapter 1. Conserved expression of Nr5a2 within the aboral
domain of the zebrafish and mouse mandibular arches
In single-cell transcriptome and chromatin accessibility analysis of zebrafish CNCCs, we
previously identified nr5a2 expression and the Nr5a2 DNA-binding motif as being highly
enriched in the aboral domain of the mandibular arch (i.e. opposite to the oral epithelium) at 1.5
days post-fertilization (dpf) (Fabian et al., 2022). In situ hybridization for nr5a2 mRNA revealed
highly specific expression in dlx2a+ mesenchyme of the mandibular arch at 1.5 dpf, posterior to
the sox9a+ lower jaw Meckel’s cartilage at 2 and 3 dpf (Fig. 1A-C). Previously published in situ
patterns indicate a lack of CNCC expression at stages earlier than 1.5 dpf (Bertrand et al.,
2007), which agrees with bulk RNA sequencing studies of migratory CNCCs in zebrafish
(Askary et al., 2017). We also generated a nr5a2:membrane-GFP knock-in reporter line
(nr5a2
mGFP
) by inserting membrane-GFP in the 5’ untranslated region of the nr5a2 locus via
homologous recombination. Consistent with endogenous expression, nr5a2
mGFP
labeled the
aboral mandibular domain at 2 and 3 dpf, with mesenchymal nr5a2
mGFP
expression partially co-
localizing with the tenocyte marker scxa:mCherry but not the cartilage maker col2a1a:mCherry
(Fig. 1D, 2A, 4A). We also observed weak nr5a2:mGFP expression in mesenchyme adjacent to
cartilage in the neurocranium (Fig. 2B). Sequential live imaging of individuals revealed that
nr5a2
mGFP
expression precedes that of scxa:mCherry, with nr5a2
mGFP
being downregulated in
scxa:mCherry+ tenocytes as they mature (Fig. 2C). Thus, nr5a2 expression is transiently
upregulated in postmigratory mandibular CNCCs that are precursors for tendon but not cartilage
cells.
To understand the regulatory logic on local expression of nr5a2, I visited our previously
published CNCC snATACseq data of 2 dpf facial CNCCs (Fabian et al., 2022) and found two
accessible genome regions at nr5a2 locus showed enriched accessibility in the nr5a2-active
5
domain opposite to the oral domain (pitx1-active). When combined with a minimal promoter in
transgenic assays, nr5a2-p1 drove GFP expression corelated to the mesenchymal nr5a2 and
nr5a2:mGFP expressions posterior to the Meckel’s cartilage and not overlapping with tendon
marker scxa:mCherry at 3 dpf. Whereas nr5a2-p2 drove GFP expression overlapped with
scxa:mCherry at intermandibular tendon (Fig. 1F). The partial correlation of nr5a2 enhancer
activity and nr5a2 expression suggested that local expression of nr5a2 in the mandibular arch is
achieved by a set of celebrative enhancers.
We next examined whether aboral mandibular expression of Nr5a2 is conserved in mouse.
Examination of mouse mandibular arch single-cell transcriptome data at E10.5 (Yuan et al.,
2020) revealed expression of Nr5a2 in a putative aboral domain complementary to an oral
domain marked by Pitx1 expression (Fig. 2F), as in zebrafish (Fabian et al., 2022). In situ
hybridization at E11.5 and E12.5 revealed mesenchymal expression of Nr5a2 at the posterior
boundary of the mandibular arch and anterior boundary of the hyoid arch, similar to the reported
aboral expression domain of Gsc (Yuan et al., 2020). As in zebrafish, mandibular Nr5a2
expression was observed just posterior to developing Sox9+ Meckel’s cartilage in mouse (Fig.
1E, 2D,E). In more lateral regions, we observed Nr5a2 expression between the first pharyngeal
pouch and first pharyngeal cleft, adjacent to but outside of the nascent middle ear cartilage
domains (Fig. 2D,E). At E14.5, Nr5a2 expression was observed in mesenchyme of the palate
and flanking the basisphenoid-forming region of the skull base, in mesenchyme surrounding the
malleus, incus, and external auditory meatus, and in salivary gland mesenchyme (Fig. 2G)
(Visel et al., 2004), with single-cell transcriptome profiles of mouse submandibular glands
(Hauser et al., 2020) further confirming salivary gland mesenchyme expression that decreased
from E12 to E16 (Fig. 2H). Expression of Nr5a2 in the aboral mandibular arch is therefore
conserved from fish to mammals.
6
Chapter 2. Requirement for nr5a2 in zebrafish lower jaw
development
Given the restricted mandibular expression of nr5a2, we investigated its requirement in
zebrafish jaw development. We first examined homozygous nr5a2
oz3/oz3
mutant zebrafish, in
which an early frameshift mutation results in premature truncation of the protein before the
DNA-binding and ligand-binding domains (Nissim et al., 2016) (Fig. 4A). Mutant fish displayed
an open mouth, and skeletal staining revealed an enlarged Meckel’s cartilage of the lower jaw,
but no other craniofacial skeletal defects, at 6 and10 dpf (Fig. 3A, 4B). Repeated live imaging
revealed increased numbers of col2a1a:h2az2a-mCherry-2A-EGFP-CAAX+ Meckel’s
chondrocytes in nr5a2 mutants versus wild-type sibling controls from 2.5 dpf, the earliest stage
at which labeled chondrocytes appear, through 7 dpf (Fig. 3B,C). The numbers of proliferating
Meckel’s chondrocytes, as assessed by anti-phospho-Histone-H3 staining, was unchanged at
2.5 and 3 dpf (Fig. 4C). Enlargement of Meckel’s cartilage in mutants is therefore likely due to
increased specification and not increased proliferative expansion of chondrocytes.
We next examined whether increased numbers of Meckel’s chondrocytes are accompanied by
concomitant loss of other CNCC derivatives. Meckel’s cartilage is connected to lower jaw
muscles by CNCC-derived scxa:mCherry+ tenocytes at 2.5 and 3 dpf, with these condensing at
intermandibular tendons by 4 dpf (Fig. 1A, 3D). In nr5a2 mutants, we observed variable
disorganization and loss of scxa:mCherry+ lower jaw tenocytes, which was accompanied by
disorganization of the lower jaw muscles (Fig. 3D,E, 4D). The interopercular–mandibular
ligament, which stabilizes the jaw joint, was also missing in 12/13 mutants at 6 dpf, yet hyoid
arch-derived tendons were largely unaffected (Fig. 3D, 4E). The lower-jaw-specific expansion of
cartilage, loss of tendon and ligament, and disorganization of muscle precisely correlates with
7
the highly localized expression of nr5a2 in the aboral mandibular arch from which these
structures derive.
8
Chapter 3. Nr5a2 functions cell-autonomously in zebrafish
CNCCs to specify tenocytes at the expense of chondrocytes
To investigate the cellular basis of jaw tenocyte loss in nr5a2 mutant zebrafish, we performed
short-term lineage tracing with nr5a2
mGFP
. Homozygous nr5a2
mGFP/mGFP
fish displayed normal
lower jaw cartilage and muscle and were adult viable; we did observe subtle jaw muscle defects
in nr5a2
mGFP/oz3
but not nr5a2
+/oz3
fish, suggesting that the nr5a2
mGFP
allele is a weak hypomorph
(Fig. 4B,D). To create a severe loss-of-function allele in cis to the mGFP insertion, we injected
CRISPR-Cas9 reagents into nr5a2
mGFP
fish and obtained progeny with an in-frame deletion of
three amino acids (nr5a2
mGFP-DBD-del
) that removes a critical zinc finger motif-forming cysteine
residue in the DNA-binding domain (Fig. 4A). We observed similar enlargement of Meckel’s
cartilage and muscle disorganization in nr5a2
mGFP-DBD-del/mGFP-DBD-del
and nr5a2
oz3/oz3
fish,
indicating that disruption of the DNA-binding domain generates a severe loss-of-function allele
(Fig. 4B,D). The similarity of cartilage defects in the mGFP-DBD-del and early frameshift oz3
alleles also argues against genetic compensation, i.e. transcriptional adaptation in response to
nonsense-mediated decay, in the oz3 allele (Rossi et al., 2015).
In nr5a2
mGFP-DBD-del/+
controls, mGFP+ cells contributed to scxa:mCherry+ jaw tenocytes but only
rarely to Meckel’s chondrocytes at 3 dpf, which we confirmed by lack of co-expression of the
cartilage marker col2a1a:mCherry-NTR+ at 2.5 dpf. In contrast in nr5a2
mGFP-DBD-del/mGFP-DBD-del
mutants, many mGFP+ cells contributed to Meckel’s cartilage and co-expressed
col2a1a:mCherry-NTR, with a concomitant decrease in contribution to scxa:mCherry+ jaw
tenocytes (Fig. 5A,B). These observations indicate that nr5a2-expressing cells shift from a
tenocyte to chondrocyte fate in the absence of Nr5a2.
9
To further test the sufficiency of nr5a2 in CNCCs for jaw patterning, we performed unilateral
transplantation of wild-type BFP+ CNCC precursors into nr5a2 mutants (Fig. 5C). In contrast to
contralateral mutant sides, 4/4 sides with mandibular contribution of wild-type BFP+ CNCCs
displayed rescue of Meckel’s cartilage morphology based on col2a1a:h2az2a-mCherry-2A-
EGFP-CAAX expression and Alcian Blue staining. We also observed rescue of lower jaw
muscle organization in transplanted sides, with wild-type CNCCs forming cells with tenocyte
morphology at the junction of muscle and cartilage. In two examples, wild-type CNCCs rescued
Meckel’s cartilage morphology without contributing to chondrocytes, indicating a non-cell-
autonomous role of Nr5a2 in patterning Meckel’s cartilage (Fig. 5C, 6A). Thus, Nr5a2 functions
to suppress chondrocyte differentiation in cells adjacent to the developing Meckel’s cartilage.
10
Chapter 4. Nr5a2 misexpression mildly suppresses
chondrogenesis and disrupts tendon formation in the
zebrafish lower jaw
To test whether Nr5a2 is sufficient to repress lower jaw chondrogenesis, we generated a
UAS:nr5a2 transgenic line and crossed it to a CNCC mesenchyme-specific fli1a:Gal4VP16
driver (Xu et al., 2018), resulting in nr5a2 expression throughout CNCC mesenchyme.
Compared to single-positive fli1:Gal4VP16 and UAS:nr5a2 controls, Meckel’s cartilage of
fli1a:Gal4VP16; UAS:nr5a2 fish had an abnormal pointed morphology and a 12-16% reduction
in chondrocyte number, as assessed by col2a1a:EGFP and nuclear DAPI staining (Fig. 6B).
Misexpression of nr5a2 also resulted in ectopic scxa:mCherry+ tenocytes and disorganized
lower jaw muscle fibers, with occasional fibers connecting to ectopic tenocytes; the number of
scxa:mCherry+ tenocytes in the normal lower jaw attachment region, however, were unchanged
(Fig. 6C). While tenocyte defects could reflect increased specification and/or disrupted migration
and morphogenesis, our data show that Nr5a2 misexpression only mildly suppresses cartilage
differentiation in the lower jaw.
11
Chapter 5. Requirement for Nr5a2 in mouse NCCs for lower
jaw and middle ear development
Given the conserved expression of Nr5a2 in mouse, we examined whether this reflects a similar
requirement for development of mandibular arch-derived structures. As homozygous loss of
Nr5a2 results in lethality at epiblast stages (Pare et al., 2004), we utilized the Wnt1-Cre driver
and a Nr5a2-flox allele to delete both copies of Nr5a2 in NCCs (Nr5a2
NCC
). Nr5a2
NCC
animals
died shortly after birth due to unidentified causes, with air bubbles apparent in their intestines
(Fig. 8A). Whereas overall head length was similar between newborn controls and Nr5a2
NCC
animals, staining for cartilage and bone revealed abnormalities in skeletal structures derived
from the mandibular arch (Fig. 7A, 8F). While Meckel’s cartilage was largely unaffected at E14.5
(Fig. 8B), at birth the malleus cartilage, which derives from the proximal portion of Meckel’s, was
enlarged. We also observed shortening and thickening of the angular process of the mandibular
bone, as well as the tympanic ring and gonial bones of the middle ear. In the pterygoid plate of
the cranial base, mutants displayed abnormal ossification at lateral edges and truncated medial
bones (Fig. 8C) consistent with Nr5a2 expression in this region at E14.5 (Fig. 2G).
We next used Scx-GFP to examine the effects of NCC-specific Nr5a2 deletion on tendon
development. In Nr5a2
NCC
; Scx-GFP newborn mice, the tendons connecting to the angular
process of the lower jaw were reduced and dysmorphic (Fig. 7B, 8D,F). Histology revealed
dysmorphology of the connective tissue at the muscle insertion site, and detachment of muscle
fibers that was confirmed by MF20 staining (Fig. 7B; 8E). The tensor tympani tendon connecting
to the malleus was similarly dysmorphic, in some cases with ectopic insertion into the wall of the
middle ear cavity (Fig. 7C). In contrast, deletion of Nr5a2 from limb mesenchyme using Prrx1-
Cre had no effect on the bones or Scx-GFP+ tendons of the forelimb and hindlimb (Fig. 8G,H),
showing specificity of skeletal and tendon defects to the jaw and middle ear.
12
In addition to tendon defects, Nr5a2
NCC
animals had a complete absence of submandibular and
sublingual salivary glands and their connecting ducts (Fig. 7D), consistent with these arising
from the Nr5a2-expressing mandibular domain. The parotid and lacrimal glands arising from the
mid-oral and eye field, respectively, were unaffected (Fig. 8I). These findings reveal a highly
region-specific requirement for Nr5a2 in development of tendons and glands from the
mandibular arch.
13
Chapter 6. Single-cell multi-omics analysis of mandibular
CNCC defects in nr5a2 mutants
We next sought to understand the mechanistic basis by which Nr5a2 regulates cell fate
decisions in the jaw. Given its role in transcriptional regulation, we performed integrated single-
nuclei analysis (10X Genomics Multiome) of mRNA expression (snRNAseq) and assay for
transposase accessible chromatin (snATACseq) in nr5a2-expressing cells sorted from control
(nr5a2
mGFP-DBD-del/+
) and mutant (nr5a2
mGFP-DBD-del/oz3
)
fish heads at 2.5 dpf (Fig. 9A). After filtering
for nuclei with high quality reads (see Appendix B. Method details), we recovered 4,304 control
and 6,589 mutant cells with a median of 11,505 and 6,546 ATAC fragments and 1,411 and 821
genes per nucleus, respectively. UMAP clustering of integrated RNAseq and ATACseq data
from control and mutant heads using Seurat (see Appendix B. Method details), combined with
analysis of known cell type markers, revealed major clusters of neurons and mesenchyme,
consistent with expression of nr5a2
mGFP
in both these populations (Fig. 10A,B). We also
observed clusters of epithelial, vascular, and blood cells, likely representing contaminating cells.
We then extracted and combined control and mutant mesenchyme clusters based on co-
expression of fli1a and prrx1a, which resolved into 14 clusters upon sub-clustering (Fig. 10C).
For detailed analysis, we further extracted a cartilage cluster (acana+), a tendon cluster
(tnmd+), and four mandibular arch mesenchyme clusters (based on co-expression of dlx2a,
dlx5a, and hand2) (Fig. 9B, 10C). The six remaining dlx2a-; dlx5a- mesenchyme clusters, which
express lower levels of nr5a2 (Fig. 10C), may represent the neurocranium mesenchyme that we
found also weakly expresses nr5a2
mGFP
(Fig. 2B).
Differential analysis of snATACseq data by chromVAR revealed that the top DNA-binding motif
underrepresented in mutant versus control mandibular mesenchyme accessible regions was
Nr5a2, followed by Egr1 (and versions of these) (Fig. 9C). Feature plots of mandibular
14
mesenchyme clusters confirmed that, while expression of nr5a2 persists in mutants, the Nr5a2
binding motif was nearly completely absent in mutant accessible chromatin regions (Fig. 9C,
10C). Whereas 559 regions displayed reduced accessibility in mutants, with 47% containing
Nr5a2 motifs, only 34 regions had increased accessibility, with none containing Nr5a2 motifs (>
1.19 fold change, p < 0.01; Fig. 9D). We also identified 204 genes that were downregulated in
mutants, versus only 80 that were upregulated (> 1.19 fold change, p < 0.01; Fig. 9D). Gene
Ontogeny analysis of the 204 downregulated genes revealed enrichment for biological
processes that included cell adhesion, extracellular matrix organization, negative regulation of
transcription, neural crest cell migration, cartilage development, and embryonic viscerocranium
morphogenesis. Although our data do not support upregulated genes being directly regulated by
Nr5a2, we observed increased expression of igfbp5b and ogna in the mutant perichondrium
(Fig. 11A). In contrast, expression of genes involved in regionalization of the mandibular arch
mesenchyme along the dorsoventral (hand2) and oral-aboral (pitx1, gsc, msx1a) axes were
unaffected in nr5a2 mutants at 1.5 dpf (Fig. 12B), suggesting that Nr5a2 functions largely
downstream of initial regional patterning factors. These findings are consistent with a primary
requirement for Nr5a2 to initiate or maintain open chromatin in mandibular mesenchyme to
activate gene expression.
15
Chapter 7. Identification of direct targets of Nr5a2 for diverse
connective tissue fates
To identify potential Nr5a2 direct targets, we queried chromatin regions that lost chromatin
accessibility in mutants, contained predicted Nr5a2 binding sites, and were located within 500
kb of a gene with decreased expression in mutants. The top five genes included tnksa
(genomically linked to fgf10a), foxp2, and cdh6 (Fig. 9D; Table 1). We found fgf10a, foxp2, and
cdh6 to be co-expressed with nr5a2 in the mesenchyme and perichondrium posterior to the
developing lower jaw cartilage at 2.5 dpf, with foxp2 and fgf10a labelling largely distinct regions
within a broader nr5a2+; cdh6+ domain (Fig. 11A-C, 12C). Perichondrium is a tissue
surrounding the cartilage that is thought to house progenitors for tendon, cartilage, bone, and
other connective tissues (Colnot et al., 2004). In nr5a2 mutants, fgf10a, foxp2, and cdh6
expression was specifically reduced in the mesenchyme of the lower jaw, including the
perichondrium associated with Meckel’s cartilage (Fig. 11A-C). In Fgf10
-/-
mice, all the salivary
glands are lost (Entesarian et al., 2005), similar to the loss of the mandibular glands observed in
Nr5a2
NCC
mice. In keeping with this result, Fgf10 expression was selectively lost in the
mandibular domain of Nr5a2
NCC
mice at E12.5 (Fig 11D).
We next focused on chromatin regions with reduced accessibility in mutants (> 1.19 fold
change, p < 0.01) that contained predicted Nr5a2 binding sites. An unbiased analysis of the
most highly significant regions revealed regions near fgf10a (#3, p = 1.95E-15), cdh6 (#6, p =
7.19E-14), foxp2 (#10, p = 1.66E-12), and scxa (#43, p = 1.39E-08) (Table 1). Genome views
confirmed reduced accessibility of these regions in mutants, and analysis of previously
published snATACseq data of 1.5 dpf facial CNCCs (Fabian et al., 2022) revealed enriched
accessibility of all three regions in the aboral mandibular domain (Fig. 11, 12F). Moreover,
analysis of published chromatin accessibility data at NCC migration stages (Trinh et al., 2017)
16
revealed that these regions gain accessibility only after migration into the arches (Fig. 12F).
When combined with a minimal promoter in transgenic assays, all four regions drove highly
specific expression of GFP in the mesenchyme posterior to Meckel’s cartilage, partially
overlapping with scxa:mCherry expression in jaw tendons and ligaments (Fig. 11, 12D,E). In
addition, GFP expression driven by all four regions was completely lost in mutants at 3 and 6
dpf, with the exception of a small amount of residual GFP expression driven by the scxa region
(Fig. 11, 12D). These findings are consistent with Nr5a2 directly binding jaw-specific enhancers
for fgf10a, cdh6, foxp2, and scxa to activate their expression.
To identify direct target genome regions of Nr5a2, I created a nr5a2-HA allele with 3X HA tag in-
frame inserted upstream of the translation stop site (Fig. 4A) that allows me to perform Cut &
Run-seq (alternative ChIPseq accommodating low cell number input) with anti-HA antibody due
to lack of Nr5a2 antibody in zebrafish. However, with two attempts of Cut & Run-seq using the
EpiCypher CUTANA™ ChIC/CUT&RUN Kit pipeline with two anti-HA antibodies validated by
EpiCypher or the Henikoff Lab, I was not able to recover Nr5a2-targeted genome regions. The
low recovery of genome regions was possibly resulted from the insufficient cell numbers as
input of the pipeline when ten-times more of the cell numbers were recommended.
17
Discussion
Here we uncovered a local and transient redeployment of the pluripotency factor Nr5a2 to
preserve CNCC-derived progenitors for alternative non-skeletal fates in the vertebrate jaw and
middle ear. Despite substantial differences in the types of structures arising from the aboral
domain of the zebrafish and mouse mandibular arch, we found that Nr5a2 has a common role in
repressing skeletogenesis and promoting tendon, gland, and potentially other mesenchymal
derivatives in this domain. Transplantation rescue in zebrafish and conditional deletion
experiments in mouse revealed that Nr5a2 functions cell-autonomously in CNCCs for jaw and
middle ear development.
In fish, nr5a2 was required for the accessibility of jaw-specific enhancers linked to uncommitted
mesenchyme and tendon development. These enhancers were enriched for Nr5a2 binding
sites, suggesting they are direct Nr5a2 targets. It also seems likely that Nr5a2 functions to open
rather than simply maintain accessibility of these enhancers, as nr5a2 CNCC expression is not
observed before 1.5 dpf in zebrafish (Bertrand et al., 2007) and the Nr5a2 target enhancers
studied here do not gain accessibility until after NCC migration (Trinh et al., 2017). These
findings are consistent with the recently reported pioneer factor activity of Nr5a2 during zygotic
genome activation in mice (Gassler et al., 2022), although additional experiments will be needed
to confirm this in CNCCs.
Our data support a highly localized role of Nr5a2 in the jaw to limit skeletal fate commitment
while priming chromatin competency for connective tissue fates such as tendons and glands.
The types of mandibular derivatives affected in zebrafish and mice lacking Nr5a2 reflect
substantial evolutionary transitions, in particular the striking transformation of the fish jaw joint
into the mammalian middle ear (Gould, 1990). Whereas in zebrafish nr5a2 mutants the entire
18
Meckel’s cartilage was substantially enlarged, in Nr5a2
NCC
mice only the proximal portion of
Meckel’s that forms the malleus cartilage of the middle ear was enlarged. In both species, the
tendons and ligaments normally associated with the affected jaw and middle ear structures were
missing or dysmorphic. Nr5a2 therefore functions similarly in fish and mice to repress
chondrogenesis and promote tendon and ligament development, yet the requirement of Nr5a2
has shifted from the entire lower jaw of fish to a more localized region of the jaw joint and middle
ear of mice.
In addition to enlargement of the malleus cartilage, Nr5a2
NCC
mice displayed shortening and
thickening of several bones, including the angular process of the mandibular bone, and the
tympanic and gonial bones. Whether this also reflects an expansion of the initial skeletogenic
domain, which in turn depletes progenitors required for sustained growth, remains to be
determined. Nr5a2
NCC
mice also had specific loss of the mandible-associated salivary glands
(submandibular and sublingual), yet the lacrimal and parotid glands that form in different regions
of the head were unaffected. Interestingly, despite fish lacking salivary glands, a conserved jaw-
specific role of Nr5a2 in fgf10a/Fgf10 regulation could explain the selective loss of mandibular
salivary glands in Nr5a2
NCC
mice. We also uncovered other predicted Nr5a2 target genes that
we did not validate here, such as itga8 that is required in mandibular mesenchyme to promote
continued morphogenesis of endodermal pouch epithelia (Talbot et al., 2016). Nr5a2 may
therefore promote additional connective tissue fates in the mandibular arch beyond tendon,
ligament, and salivary gland mesenchyme.
Several of the direct target genes of Nr5a2, including foxp2 and cdh6, are co-expressed with
nr5a2 in the lower jaw perichondrium, a tissue known to contain progenitors for diverse
connective tissue fates (Colnot et al., 2004). Members of the Foxp1/2/4 family are also
expressed in the perichondrium of mouse where they inhibit osteogenic differentiation (Zhao et
19
al., 2015). As Foxp2 has been shown to repress gene expression in the nervous system by
closing chromatin (Hickey et al., 2019), one possibility is that Nr5a2 indirectly represses
skeletogenesis through promoting jaw-specific expression of foxp2. Interestingly, deletion of
both Scleraxis genes in zebrafish appears to result in a similar expansion of Meckel’s cartilage
as seen in nr5a2 mutants (Kague et al., 2019). It is therefore also possible that Nr5a2 inhibits
lower jaw cartilage formation through jaw-specific regulation of scxa.
Our data argue against redeployment of Nr5a2 in the mandibular arch reflecting re-utilization of
the Nr5a2-regulated pluripotency network. In embryonic stem cells, Nr5a2 regulates the
expression of Oct4 and other genes (Gu et al., 2005; Pare et al., 2004), in part by binding as a
complex with the pluripotency factors Sox2 and Klf4 (Heng et al., 2010). Although a recent study
has shown that Oct4 and Sox2 have important roles in early neural crest formation (Hovland et
al., 2022), we had previously shown that zebrafish homologs of oct4, klf4, and most other
pluripotency genes are not expressed in postmigratory CNCCs (Fabian et al., 2022). Sox2 and
Klf4 motifs are also not strongly enriched in Nr5a2-dependent chromatin regions in CNCCs.
Instead, we found that Nr5a2 is repurposed to regulate a largely distinct set of genes in
mandibular CNCCs important for perichondrium biology and formation of tendons, ligaments,
and glands. Along with previous studies showing that pluripotency factors Sox2 and Oct4 bind
targets in neural progenitors (Peterson et al., 2012) and neural crest cells (Hovland et al., 2022)
distinct from those in embryonic stem cells, our findings highlight the context-dependent activity
of transcription factors associated with pluripotency at later stages of development.
The role of nuclear receptor transcription factors in balancing cell fate decisions in postmigratory
CNCCs may be a general theme. Nr2f (COUP-TF) family nuclear receptors have been shown to
restrict progenitors from committing to cartilage fates in the upper jaw, thus facilitating later
dermal bone formation (Barske et al., 2018). A key difference is that Nr2f factors are expressed
20
during initial formation of CNCC mesenchyme and persist in specific jaw regions, whereas
nr5a2 expression initiates only after migration of CNCCs to the mandibular arch. In the future, it
will be interesting to examine what makes this class of transcription factor particularly well suited
for regulating CNCC potential in diverse contexts.
More experiments will be needed to understand how nr5a2 function is specific to the lower jaw
and middle ear. I have shown that nr5a2 promotes jaw-specific enhancers of downstream
genes. One reason for specificity in function could be due to its restricted expression to the
aboral domain of mandible. Current data show that misexpression of nr5a2 affects first arch
derivatives but the effect on back arch derivatives need to be further examined. Un-
affected back arch derivatives would suggest that nr5a2 requires jaw-specific co-activators for
its local function. To understand how nr5a2 expression is restricted to the lower jaw,
experiments examining nr5a2 expression and nr5a2 enhancers in animals with loss- or gain-of-
function in a variety of signaling pathways will be needed, as well as defining the regulatory
elements that drive nr5a2 expression from the current single-cell data with in-vivo validations.
Nr5a2-target genes, like foxp2, cdh6 and fgf10a, exhibit partially overlapped but distinct
expression domains in the jaw suggesting mesenchyme diversity in nr5a2+ mesenchyme. In my
nr5a2:mGFP-DBD-del+ single-cell data, multiple Nr5a2-dependent mesenchyme clusters were
identified. Interestingly, despite fish lacking salivary glands, fish have conserved Nr5a2-
dependent fgf10a expression in the aboral domain of mandible. The contributions of these
mesenchyme populations to the face requires further characterization with lineage-tracing tools.
21
Figures
Figure 1. Nr5a2 expression within the developing zebrafish and mouse face
(A) Diagrams of zebrafish heads showing pharyngeal arch CNCCs (grey, numbered), facial
cartilages (magenta), and tendons and ligaments (pink). Image at right is a ventral view of the
lower jaw in transgenic animals at 6 dpf, with cartilage in blue (col2a1a:GFP), tendon and
ligament in magenta (scxa:mCherry), and muscle in white (phalloidin). Ceratohyal cartilage
(Ch), Hyosymplectic cartilage (Hs), Intermandibular tendon (Imt), Interopercular–mandibular
ligament (Ioml), Mandibulohyoid junction tendon (Mhj), Meckel’s cartilage (M), Palatoquadrate
cartilage (Pq).
(B,C) In situ hybridizations show expression of nr5a2 (green) in mandibular (arrow) and hyoid
(arrowhead) arch CNCCs (dlx2a+) at 1.5 dpf, and posterior to developing sox9a+ M and Pq jaw
cartilages at 2 and 3 dpf (lateral views in B and ventral view in C).
22
(D) Ventral views of the jaw at 3 dpf show nr5a2:membrane-GFP (mGFP) expression posterior
to col2a1a:mCherry-NTR+ M and Pq chondrocytes and partially overlapping with the tenocyte
and ligamentocyte marker scxa:mCherry at the Imt, Ioml, and Mhj.
(E) Diagram shows ventral view of jaw region in E11.5 mouse embryo with dashed lines
indicating sagittal sections shown below (numbered). Overviews of sagittal sections with DAPI
staining only (1, 2 - white). RNAscope in situ hybridizations of boxed regions show Nr5a2
expression at the posterior boundary of the mandibular (1st) arch and anterior boundary of the
hyoid (2nd) arch, posterior to Sox9+ Meckel’s cartilage and anterior to Hyoid (H) cartilage.
Similar staining was observed in multiple sections. Scale bars = 50 μm (A-D); 500 μm (E).
(F) Two accessible genome regions at nr5a2 locus drove GFP expressions correlated to
partially nr5a2 and nr5a2:mGFP expression domains. Feature plots of 2 dpf CNCC snATAC
data (Fabian et al., 2022) highlighting their accessibility in the nr5a2+ domain opposite to oral
(pitx1+) domain.
23
Figure 2. Detailed facial expression of nr5a2
mGFP
in zebrafish and Nr5a2 in mouse
(A) At 2 dpf, confocal image in ventral view shows that nr5a2
mGFP
(green) labels mesenchyme
posterior to Meckel’s (M) and palatoquadrate (Pq) cartilages (magenta).
24
(B) At 2.5 dpf, confocal image of the neurocranium shows weak nr5a2:mGFP expression in
mesenchyme (arrows) adjacent to the ethmoid plate (Ep) and trabecula (Tr) cartilages
(col2a1a:GFP+, grey). Ch, ceratohyal cartilage.
(C) Repeated live imaging shows the emergence of lower jaw scxa:mCherry+ tenocytes (boxed
regions, arrows) from a broader domain of nr5a2:mGFP+ mesenchyme at intermandibular (Imt)
and mandibulohyoid junction (Mhj) tendons in wild type (nr5a2
mGPF/+
), with nr5a2:mGFP being
downregulated as scxa:mCherry+ jaw tenocytes mature. Lower magnification images are
confocal projections and insets are digital sections. Scale bars = 50 μm (A-C).
(D and E) Diagrams show ventral views of the jaw region in mouse embryos at E11.5 and
E12.5. Numbers and dashed lines indicate locations of sagittal sections below, with lower
magnification views stained with DAPI only to label nuclei. RNAscope in situ hybridizations of
boxed regions show Nr5a2 expression (green), with some sections also showing Sox9
expression (magenta) to highlight chondrogenic condensations. Expression of Nr5a2 is seen in
the aboral-proximal domain of the mandibular (1st) arch, extending into the hyoid (2nd) arch,
and between the first pharyngeal cleft (c1) and first pharyngeal pouch (p1) in mesenchyme
associated with the middle ear-forming region. Expression is also seen in the cranial base
(yellow arrow). Similar expression was observed in multiple sections. Incus (In), malleus (Ma),
stapes (St). Scale bars = 500 μm (D, E).
(F) Published single-cell transcriptome data of E10.5 mandible (Yuan et al., 2020) show cells
with Nr5a2 transcripts overlapping with transcripts for an aboral marker (Gsc) and largely non-
overlapping with an oral marker (Pitx1).
(G) Diagram shows ventral view of the jaw region in E14.5 mouse embryo and locations of
sagittal sections from the GenePaint database (Visel et al., 2004). Magnified regions of boxed
areas show Nr5a2 mesenchymal expression in (1) salivary gland (SG) mesenchyme
underneath the tongue (T) and in the cranial base (yellow arrow), (2) mid palate (red arrow), and
(3) mesenchyme associated with the malleus (Ma) and incus (In) of the middle ear and external
auditory meatus (EAM).
(H) Published single-cell transcriptome datasets of E12, E14, and E16 submandibular salivary
glands (Hauser et al., 2020) show specific expression of Nr5a2 in the gland mesenchyme
cluster. Expression progressively diminishes from E12 to E16.
25
Figure 3. Requirement of nr5a2 for zebrafish lower jaw development
(A) Heads of 10 dpf zebrafish and flat-mount dissections of the first and second arch skeleton of
6 dpf zebrafish stained with Alcian Blue (cartilage) and Alizarin Red (bone). Arrows denote
thickening of Meckel’s cartilage (M) in mutants. Branchiostegal ray bone (Br), Ceratohyal
cartilage (Ch), Entopterygoid bone (En), Hyosymplectic cartilage (Hs), Opercle bone (Op),
Palatoquadrate cartilage (Pq).
(B) Repeat imaging of individual animals shows progressive thickening of Meckel’s cartilage
(outlined) in mutant versus control col2a1a:h2az2a-mCherry-2A-EGFP-CAAX+ fish. H2az2a-
mCherry labels nuclei. EGFP-CAAX channel is not shown.
(C) Quantification of Meckel’s chondrocyte number. Difference of wild type and mutant
chondrocyte numbers was compared by Wilcoxon rank-sum test at each stage. Error bars
represent standard error of the mean. * = p < 0.05; *** = p < 0.0001.
(D) Repeated imaging of individuals shows thickening of the col2a1a:GFP+ Meckel’s cartilage
and loss of the scxa:mCherry+ intermandibular tendon (Imt, white arrow), interopercular–
mandibular ligament (Ioml, yellow arrow), and mandibulohyoid junction tendon (Mhj, arrowhead)
in 4/4 mutants compared to 0/3 wild types. Note that the hyoid tendons (far right at 4 dpf) are
26
unaffected in mutants, as is the midline scxa:mCherry expression at 2.5 and 3 dpf that does not
contribute to tendons by 4 dpf in wild types.
(E) Phalloidin staining (white) shows that the jaw muscles connected to col2a1a:GFP+ Meckel’s
cartilage are highly disorganized in 5/5 mutants compared to 0/8 wild types. Scale bars = 50
μm.
27
Figure 4. Characterization of nr5a2 alleles and cell proliferation assays
(A) Schematic of nr5a2 alleles. nr5a2
oz3
is a frameshift mutation resulting in premature
truncation of the DNA binding domain (DBD). GFP-CAAX was inserted at the translation start
site to generate nr5a2
mGFP
. In-frame deletion of a critical zinc finger motif-forming cysteine
residue in the DBD generated nr5a2
mGFP-DBD-del
. 3X-HA tag was inserted at the translation stop
site to generate nr5a2
HA
.
28
(B) Alcian Blue staining shows Meckel’s cartilages (M) of wild type and various nr5a2 mutant
allele combinations; the ethmoid plate cartilage is out of focus. Hypomorphic allele nr5a2
mGFP
over oz3 displays a slight thickening of Meckel’s cartilage, compared to striking enlargement of
Meckel’s cartilage in various combinations of nr5a2
oz3
and nr5a2
mGFP-DBD-del
alleles.
(C) Confocal sections of Meckel’s cartilages in wild type and nr5a2 mutants are shown in ventral
views. Anti-phospho-Histone-H3 staining (red, pHH3) shows proliferating chondrocytes labeled
by H2az2a-mCherry (white, col2a1a:h2az2a-mCherry-2A-EGFP-CAAX+, EGFP-CAAX channel
not shown). Yellow arrows in insets of boxed regions denote rare pHH3+ chondrocytes.
Quantification to the right shows no changes in proliferation of Meckel’s chondrocytes in nr5a2
mutants at 2.5 and 3 dpf (p = 0.8879 and 0.5797 by Wilcoxon rank-sum test). Error bars
represent standard error of the mean.
(D) Phalloidin staining of lower jaw muscle fibers (white) in ventral view shows mild muscle
defects in nr5a2
mGFP
over oz3, no defect in nr5a2
mGFP-DBD-del
over nr5a2
HA
and severe muscle
disorganization in various combinations of nr5a2
oz3
and nr5a2
mGFP-DBD-del
alleles.
(E) In 12/13 nr5a2 mutants (5 nr5a2
oz3/oz3
and 8 nr5a2
mGFP-DBD-del/oz3
), ventral views of
scxa:mCherry (magenta) with Phalloidin staining of muscle (white) reveal loss of the
interopercular–mandibular ligament (Ioml, arrowheads in control) and mandibulohyoid junction
tendon (Mhj). Note the midline hyoid tendons are unaffected in mutants. Scale bars = 50 μm.
29
Figure 5. Nr5a2 functions cell-autonomously in zebrafish CNCCs to promote tenocyte at
the expense of chondrocyte fate
(A) In representative confocal sections at 3 dpf, nr5a2:mGFP-DBD-del+ cells contributed to
12+/-3.9 Meckel’s (M) chondrocytes (arrowheads) across 10 mutants (nr5a2
mGFP-DBD-del/mGFP-DBD-
del
) versus 0.4+/-0.8 chondrocytes across 12 controls (nr5a2
mGFP-DBD-del/+
) (p = 4.76E-05,
Wilcoxon rank-sum test). Reciprocally, nr5a2:mGFP-DBD-del+ cells contributed to 1.3+/- 0.7
scxa:mCherry+ intermandibular tendon (Imt) cells (arrows) in mutants versus 9.6+/-2.0 in
controls (p = 7.2E-05, Wilcoxon rank-sum test). Mhj, mandibulohyoid junction.
(B) At 2.5 dpf, nr5a2:mGFP-DBD-del+ cells contributed to 6.1+/-3.5% col2a1a:mCherry-NTR+
Meckel’s chondrocytes (arrowhead, inset) across 6 mutants versus 1.2+/-1.7% chondrocytes
across 10 controls (p = 0.0198, Wilcoxon rank-sum test). Lower magnification images are
confocal projections and insets are digital sections. Ch, ceratohyal cartilage.
(C) Schematic shows shield-stage transplantation of BFP+ (green, actb2:LOXP-BFP-LOXP-
DsRed) wild-type ectoderm cells into the CNCC precursor domain of nr5a2 mutant hosts. In 4/4
mutants, unilateral contribution of wild-type CNCCs rescued morphology of Meckel’s cartilage
(magenta, col2a1a:h2az2a-mCherry-2A-EGFP-CAAX+ in fluorescent images; Alcian Blue+ at
right) and muscle organization (white, Phalloidin, in middle panel). Inset shows magnified digital
section in which wild-type CNCCs non-cell-autonomously rescued Meckel’s cartilage. Scale
bars = 50 μm. Pq, palatoquadrate cartilage.
30
Figure 6. Nr5a2 functions in CNCCs and is sufficient to suppress chondrogenesis and
disrupt tendon formation in the zebrafish lower jaw
(A) Additional example of shield-stage transplantation of wild-type ectoderm cells into the CNCC
precursor domain of nr5a2 mutant host. Shown in ventral view, contribution of BFP+ wild-type
CNCCs (actb2:LOXP-BFP-LOXP-DsRed, green) to the left side of the jaw (top) rescued
morphology of Meckel’s cartilage (M, magenta, col2a1a:h2az2a-mCherry-2A-EGFP-CAAX+ in
fluorescent images, h2az2a-mCherry channel is not shown; Alcian Blue+ at right) only on the
transplanted side. Organization of muscles (Phalloidin, white) was also rescued only on the
transplanted side, with muscles appearing to connect to wild-type BFP+ tenocytes. Note that
BFP signal is decreased after the fixation step required for Phalloidin staining. Ceratohyal
cartilage (Ch), palatoquadrate cartilage (Pq).
(B) Ventral views of Meckel’s cartilages labeled by col2a1a:GFP. CNCC-specific misexpression
of nr5a2 in fli1a:Gal4VP16; UAS:nr5a2 animals results in a more pointed morphology of
Meckel’s cartilages. Quantification shows a mild reduction in Meckel’s chondrocyte number in
fli1a:Gal4VP16; UAS:nr5a2 animals versus single-positive fli1a:Gal4VP16 or UAS:nr5a2
controls.
(C) Ventral views show the jaw muscles labeled by Phalloidin (white) and the intermandibular
tendon (Imt), interopercular–mandibular ligament (Ioml), and mandibulohyoid junction tendon
(Mhj) labeled by scxa:mCherry (magenta). CNCC-specific misexpression of nr5a2 in
fli1a:Gal4VP16; UAS:nr5a2 animals results in disorganized Imt and Mhj tendons and associated
muscles; arrow in example below shows ectopic scxa:mCherry+ cells connecting to a stray
muscle fiber. Quantification reveals no changes in Imt tenocyte number in fli1a:Gal4VP16;
UAS:nr5a2 animals versus single-positive fli1a:Gal4VP16 or UAS:nr5a2 controls. DAPI staining
(not shown) was performed to identify individual chondrocyte or tenocyte nuclei. Statistical
difference of chondrocyte or tenocyte numbers across genotypes was compared by Tukey's
range test. ns, not significant. Error bars represent standard error of the mean. Scale bars = 50
μm.
31
Figure 7. CNCC requirement for Nr5a2 in mouse mandibular arch development
(A) Newborn (P0) skulls of control (Nr5a2-f/f) and Nr5a2
NCC
(Wnt1-Cre; Nr5a2-f/f) mice stained
with Alcian Blue (cartilage) and Alizarin Red (bone). Dashed boxes designate magnified regions
shown at right. Mutants display an enlarged malleus cartilage (Ma), and shorter and thicker
lower jaw angular process (Ang), tympanic bone (Ty), and gonial bone (G). Consistent
phenotypes were seen across 12 wild-type (Nr5a2-f/+, Nr5a2-f/f or Wnt1-Cre; Nr5a2-f/+) and 6
Nr5a2
NCC
heads. Condylar process (Con), coronoid process (Cor), incus (In), body of Meckel’s
cartilage (M), stapes (St).
(B) Angular processes are labeled for tendon (Scx-GFP, magenta) and bone (Alizarin Red,
green) in whole-mount imaging, and trichrome staining in sagittal sections. Diagrams depict the
dysmorphic tendons (arrow) and detached muscle (yellow arrowhead) in 4/4 Nr5a2
NCC
mice.
(C) Dissected middle ears and accompanying diagrams show dysmorphology of the Scx-GFP+
(magenta) tensor tympanic tendon (Ttt, which connects the malleus (blue) to the tensor
tympanic muscle (grey)) in Nr5a2
NCC
mice. Tendon defects were seen in 6/6 middle ears from
Nr5a2
NCC
mice, with abnormal connections to the middle ear wall (yellow) in three ears. The
abnormal gonial and tympanic bones are visualized by Alizarin Red staining (green).
32
(D) Trichrome staining of sagittal sections and freshly dissected tissue show absence of the
submandibular gland (SMG), sublingual gland (SLG), and salivary gland duct (asterisk) in 4/4
Nr5a2
NCC
mice. The tongue (T) appears unaffected. Scale bars = 1 mm (A, D); 500 μm (B, C).
33
34
Figure 8. Detailed phenotypic analysis of mouse conditional Nr5a2 mutants
(A) Death at birth of Nr5a2
NCC
animals (Wnt1-Cre; Nr5a2-f/f) correlates with numerous air
bubbles (arrows) in their stomach and intestines (freshly dissected). N = 4 each for Nr5a2
NCC
mutants and wild types (Nr5a2-f/+, Nr5a2-f/f or Wnt1-Cre; Nr5a2-f/+).
(B) Flat-mount dissections of E14.5 Meckel’s cartilages (M, Alcian Blue staining) show no
difference in 4/4 Nr5a2
NCC
mutants versus 10 littermate controls. Incus (In), malleus (Ma).
(C) Alcian Blue (cartilage) and Alizarin Red (bone) staining of newborns show that, in the
pterygoid plate of the cranial base, the lateral edges are enlarged (black arrow) and the medial
ridges are shorter (red arrow) in 6/6 Nr5a2
NCC
mutants versus 12 littermate controls.
(D) Whole-mount fluorescent images of bone (Alizarin Red, pseudo-colored green) and facial
tendons and ligaments (Scx-GFP+, pseudo-colored magenta) show dysmorphic lateral jaw
tendons (arrows) associated with mandibular bone (Mand) in 4/4 Nr5a2
NCC
mutants versus 3
littermate controls. Dashed lines indicate locations of coronal sections in (E).
(E) Coronal sections of the proximal lower jaw at E16.5 reveal dysmorphic muscle fibers
(MF20+, green, arrows) in the lower jaw of Nr5a2
NCC
mutants versus Nr5a2-f/+ controls.
Tendons are labeled by Scx-GFP (magenta) and chondrocytes by anti-Sox9 staining (blue).
Similar staining was seen in multiple sections. Angular process (Ang), condyle process (Con).
(F) Measurements of facial skeleton and tendon parameters for the indicated genotypes at P0.
Significance was calculated with a Tukey's range test. Error bars represent standard error of the
mean. Nonsignificant p values (p > 0.05) are not shown.
(G) Newborn forelimb and hindlimb skeletons stained with Alcian Blue (cartilage) and Alizarin
Red (bone) are unaffected in 10 Prrx1-Cre; Nr5a2-f/f mutant limbs versus 38 sibling controls
(Nr5a2-f/+, Nr5a2-f/f, or Prrx1-Cre; Nr5a2-f/+) limbs.
(H) Limb tendons and ligaments (Scx-GFP, magenta) and muscles (Phalloidin, white) appear
normal in 4/4 Prrx1-Cre; Nr5a2-f/f mutant limbs versus 8 control littermates at P3.
(I) Trichrome staining of coronal sections show that parotid and lacrimal glands (arrowheads)
are still present in Nr5a2
NCC
mutants. N = 4 each. Scale bars = 200 μm (B, E, I), 1 mm in all
others.
35
Figure 9. Single-cell analysis of gene expression and chromatin changes in nr5a2
mutants
(A) Image of 2.5 dpf nr5a2:GFP+ cells and scheme for jaw dissection (boxed region), FACS,
and Multiome analysis. UMAPs show distribution of mandibular mesenchyme from controls and
mutants. Scale bar = 50 μm.
(B) UMAPs show distribution of tendon, cartilage, and mandibular mesenchyme (Mes1-4)
clusters from controls and mutants, and feature plots show expression of tendon (tnmd) and
cartilage (acana) markers.
(C) Feature plots of the Nr5a2 binding motif (predicted by chromVAR) show its absence in
mutant accessible chromatin. Top underrepresented motifs in mutants fall into two classes:
Nr5a/Esrr and Egr1/Klf9.
(D) Schematic of regions with reduced or increased accessibility in mutants (> 1.19-fold change,
p value < 0.01). Peaks were filtered based on predicted Nr5a2 motifs. We then intersected
peaks with genes within 500 kb showing down or up regulation (> 1.19-fold change, p value <
0.01) to identify potential Nr5a2 target genes and their enhancers.
36
Figure 10. Single-cell analysis of marker gene expression in entire nr5a2 mutant and
control datasets
(A, B) Individual UMAPs and clustering of the integrated single-cell chromatin accessibility and
transcriptome datasets of control (A, nr5a2:mGFP-DBD-del/+) and mutant (B, nr5a2:mGFP-
DBD-del/oz3). Feature plots show major marker genes used to identify subsets of mesenchyme
37
(fli1a+ and prrx1a+), epithelium (epcam+), neurons (elavl3+ or neurod1+), endothelium (kdrl+),
and blood cells (hbbe2+).
(C) Re-clustering of mesenchyme (fli1a+ and prrx1a+ in A and B above) from combined control
and mutant datasets separated into 14 clusters. Feature plots show major marker genes used to
identify the mandibular mesenchyme (dlx+ and hand2+), tendon (tnmd+), and cartilage
(acana+) clusters used for detailed analysis.
38
Figure 11. Nr5a2 regulates jaw-specific enhancer accessibility and gene expression for
perichondrium and tendon genes
(A-C) For each gene, feature plots in top left show downregulation of transcripts in mutant
(nr5a2:mGFP-DBD-del/oz3) versus control (nr5a2:mGFP-DBD-del/+) mandibular mesenchyme.
Middle left shows genomic tracks of chromatin accessibility, with the Nr5a2 motif-harboring
regions with decreased accessibility shown in grey. Bottom left shows feature plots of 1.5 dpf
CNCC snATAC data (Fabian et al., 2022) highlighting accessibility of the shaded regions in the
aboral mandibular domain. Top right shows fluorescent in situ hybridizations of the mandibular
and hyoid arches in ventral views, with sox9a expression labeling chondrocytes (A, C) or DAPI
labeling all nuclei (B). White arrowheads denote loss of perichondrium expression in mutants,
and yellow arrowheads loss of midline fgf10a expression. Bottom right shows transgenic lines in
which GFP is driven by the highlighted genomic regions. For each line, GFP expression is
observed posterior to Meckel’s (M) cartilage and partially overlapping with scxa:mCherry in 5/5
wild types and completely lost in 5/5 mutants. Intermandibular tendon (Imt), interopercular–
mandibular ligament (Ioml), mandibulohyoid junction tendon (Mhj).
(D) Oral view of in situ hybridizations for Fgf10 in dissected mandibular arches from control
(Nr5a2-f/+) and mutant (Wnt1-Cre; Nr5a2-f/f) mouse embryos at E12.5. Fgf10 expression is
selectively lost in mandibular domains (arrows). T, tongue.
(E) Top left shows genome tracks of chromatin accessibility at the scxa locus, with the region
containing Nr5a2 motifs and having decreased accessibility in mutants highlighted in grey. At
39
top right, feature plot from 1.5 dpf CNCC snATAC data shows accessibility of this region in the
aboral mandibular domain. At bottom, transgenic line in which GFP is driven by the highlighted
genomic region shows expression in the scxa:mCherry+ Ioml ligament in 5/5 wild types and loss
of Ioml expression in 5/5 nr5a2
oz3/oz3
mutants. Scale bars = 50 μm (A-C, E); 500 μm (D). See
Supp. Fig. 6 for additional examples of transgenic line expression and expanded genomic views
of open chromatin.
40
41
Figure 12. Analysis of Nr5a2-regulated enhancers, upregulated genes in nr5a2 mutants,
and normal axis patterning in nr5a2 mutants
(A) Feature plots show increased igfbp5b and ogna transcripts in mutant snRNAseq datasets of
mandibular mesenchyme. Ventral views of fluorescent in situ hybridizations show increased
expression of igfbp5b and ogna posterior to Meckel’s (M) cartilage (arrowheads) in mutants
(nr5a2:mGFP-DBD-del/oz3) compared to controls (nr5a2:mGFP-DBD-del/+). DAPI staining
labels all nuclei for igfbp5b in situ, and sox9a expression highlights Meckel’s cartilage for ogna
in situ.
(B) Lateral facial views of in situ hybridizations show that markers of the aboral (gsc and
msx1a), oral (pitx1), and ventral (hand2) domains are largely unaffected in nr5a2 mutants
(arrows denote jaw expression domains). Anti-GFP staining for sox10:mGFP highlights arch
CNCCs in white.
(C) RNAscope in situ hybridizations at 2.5 dpf show Meckel’s cartilage and surrounding
mesenchyme, with sox9a expression labeling chondrocytes in blue (top panels) and DAPI
labeling all nuclei in white. Top, white arrowheads denote co-localization of nr5a2 with foxp2,
cdh6, and fgf10a expression within the perichondrium, and yellow arrowhead denotes co-
localization of nr5a2 with midline fgf10a expression. Bottom, white arrowheads denote co-
localization of cdh6 with foxp2 and fgf10a in the perichondrium; fgf10a and foxp2 expression are
largely non-overlapping.
(D) Earlier transgenic activity at 3 dpf for the same individuals shown in Figure 6. foxp2-p1,
cdh6-p1, and fgf10a-p1 lines display GFP expression posterior to Meckel’s cartilage, yet only
few scxa-p2:GFP+ cells are seen at this stage. For each transgenic line, GFP expression was
absent in nr5a2
oz3/oz3
mutants. Tendons and ligaments are labeled by scxa:mCherry in magenta.
Intermandibular tendon (Imt), interopercular–mandibular ligament (Ioml), mandibulohyoid
junction tendon (Mhj).
(E) Independently isolated transgenic lines for each enhancer tested in Figure 6 show
consistent GFP expression posterior to Meckel’s cartilage. Scale bars = 50 μm.
(F) Genome views of the foxp2, cdh6, fgf10a, and scxa loci for the snATACseq datasets of
controls and mutants at 2.5 dpf show the highly specific reduction of chromatin accessibility in
the regions highlighted in grey (tested in D and E). Tested enhancer regions had little to no
accessibility in migratory CNCCs at 18 hours post-fertilization (hpf) (Trinh et al., 2017). Note that
the 18 hpf ATACseq tracks and the snATACseq tracks are not at the same scale. Omitted
regions display minimal accessibility.
42
Tables
Table 1. Differentially accessible chromatin regions in nr5a2 mutant mandibular
mesenchyme associated with differentially expressed genes and Nr5a2 motifs
Downregulated In mutant
Peak (tested) p value avg_log2FC
Associated genes (downregulated
genes, fgf10a, scxa)
Nr5a2_
motif
#
21-19692350-19693259 1.95E-15 0.553275366 fgf10a,tnksa 1
2-28158978-28159848 7.19E-14 0.513019837 CR925777.1,cdh6 1
8-43428555-43429447 2.72E-13 0.428184234 ncor2 1
8-43489206-43489722 4.02E-13 0.451525229 ccdc92,ncor2 1
4-6133307-6133884* 1.66E-12 0.433689405 CR356233.1,ppp1r3aa,foxp2 1
13-5424873-5425735 7.77E-11 0.34377373 meis1b 1
11-32624835-32625608 2.87E-10 0.44992116 pcdh17 1
6-24479944-24480641 3.71E-10 0.394336895 si:ch73-389b16.2,tgfbr3 1
8-30284831-30285657 5.23E-09 0.378906631 aopep 1
16-28695735-28696499 1.09E-08 0.402149263 itga8,fam171a1,abcb4,zgc:171704 1
19-3044102-3044993 1.39E-08 0.345148445 fam126a,si:ch211-251g8.5, scxa, bop1,
hsf1,si:ch211-133n4.4
1
3-28818384-28819219 1.80E-08 0.37348766 rbfox1 1
21-23738263-23739107 3.52E-08 0.429820298 ncam1a,cadm1a 1
4-6133885-6134115* 3.62E-08 0.354684355 tmem63a,CR356233.1,MDFIC,foxp2 1
11-27856973-27857773 1.51E-07 0.326422759 ptpdc1a,cstf1,ephb2b 1
16-28484397-28485253 1.77E-07 0.322700889 itga8,zgc:171704 1
11-27866129-27866962 3.97E-07 0.320575038 cstf1,ephb2b 1
15-30741175-30742012 5.94E-07 0.312261952 msi2b 1
16-35036351-35037253 6.44E-07 0.27055534 ptprub 1
7-68307237-68307879 9.44E-07 0.3645936 zfhx3 1
11-27673533-27674353 3.00E-06 0.390176085 fbln2,ptpdc1a,barx1,fam120a,lactbl1b,
ephb2b
1
12-29688831-29689677 5.29E-06 0.371425058 si:ch211-214e3.5, BX072570.1,
nrg3b,atrnl1b,ablim1b
1
12-44337589-44338443 1.92E-05 0.344154095 dock1 1
13-40087901-40088745 2.39E-05 0.320144687 hpse2 1
7-56751125-56751952 6.13E-05 0.27899442 myo9aa 1
16-35758783-35759687 6.33E-05 0.258762143 ptprub,sh3d21,eva1ba 1
16-40861775-40862644 0.000127475 0.297005277 si:dkey-242e21.3, ndufaf6,
BX324007.2,si:dkey-22o22.2
1
9-41948595-41949463 0.000220836 0.297160563 col18a1a 1
17-51565965-51566836 0.000263298 0.301494474 pxdn 1
15-30787749-30788572 0.000331413 0.259045369 nos2b,msi2b 1
14-20490721-20491556 0.000426445 0.264492109 aff2 1
1-6010650-6011459 0.00059351 0.268367723 fn1b,nrp2a 1
16-9624173-9625042 0.000894352 0.257545828 enpp2 1
11-5057505-5058377 0.00110577 0.254356021 ptprga 1
7-68355795-68356664 0.001227104 0.261945284 CU467042.1,zfhx3 1
23-26977059-26977852 0.001644965 0.259511489 si:dkey-205h13.1,hspg2 1
16-9877591-9878499 0.002601055 0.280135439 adcy2a,enpp2,ncalda 1
Upregulated in mutant
Peak p value avg_log2FC Associated genes (upregulated genes)
Nr5a2_
motif
#
11-35664934-35665629 8.29E-07 0.282584177 sema3fb 0
25-7844197-7845074 9.95E-07 0.255412659 prdm11,dgkzb,mdkb,hal 0
* Two adjacent regions were cloned and tested together
#
Nr5a2_motif; 1 = Nr5a2 motif, 0 = no Nr5a2 motif
43
Table 2. Key resources table
REAGENT or RESOURCE SOURCE IDENTIFIER
Antibodies
rabbit-anti-phospho-histone H3
(Ser10)
Santa Cruz Biotechnology sc-8656-R
chicken-anti-mCherry Novus Biologicals NBP2-25158
chicken-anti-GFP Abcam ab13970
mouse-anti-MF-20 Developmental Studies Hybridoma Bank MF 20
rabbit-anti-Sox9 Novus Biologicals NBP1-85551
Alexa Fluor 488 anti-chicken ThermoFisher A-11039
Alexa Fluor 568 anti-chicken ThermoFisher A-11041
Alexa Fluor 568 anti-rabbit ThermoFisher A11011
Alexa Fluor 647 anti-rabbit ThermoFisher A-31573
Alexa Fluor 647 anti-mouse ThermoFisher 21242
rabbit-anti-HA-tag (C29F4) Cell Signaling 3724T
rabbit HA Tag CUTANA™
CUT&RUN Antibody
EpiCypher SKU:13-2010
Critical Commercial Assays
RNAscope Multiplex Fluorescent
Reagent Kit v2 Assay
Advanced Cell Diagnostics Protocol: 323100-USM,
Technical note: MK 50-016
Chromium Next GEM Single Cell
Multiome ATAC + Gene Expression
10X Genomics Protocol: CG000338
CUTANA™ ChIC/CUT&RUN Kit EpiCypher SKU:14-1048
Deposited Data
Raw sequencing fastq files and Cell
Ranger ARC v2.0.0-processed
Multiome data in this paper
Gene Expression Omnibus (GEO) GSE210251
Published scRNAseq of
submandibular salivary gland
Gene Expression Omnibus (GEO)
(Hauser et al., 2020)
GSE150327
Published scRNA-Seq of mouse
mandible
FaceBase (Yuan et al., 2020) 1-DTK2
Published ATAC-Seq of zebrafish
NCC
Gene Expression Omnibus (GEO)
(Trinh et al., 2017)
GSE89670
Experimental Models: Organisms/Strains
Mouse: Nr5a2-flox: Nr5a2
tm1Sakl
The Jackson Laboratory JAX 024054
Mouse: Wnt1-Cre: Wnt1-Cre1 Danielian et al., 1998 N/A
Mouse: Prrx1-Cre: Tg(Prrx1-Cre)1Cjt The Jackson Laboratory JAX 005584
Mouse: Scx-GFP: Tg(Scx-GFP)1Stzr Pryce et al., 2007 N/A
Zebrafish: nr5a2
oz3
: nr5a2
oz3
Nissim et al., 2016 oz3
Zebrafish: sox10:dsRed:
Tg(sox10:DsRedExpress)
Our lab el10
Zebrafish: fli1a:GAL4: Tg(fli1:GAL4-
VP16,myl7:EGFP)
Our lab el360
Zebrafish: sox10:mGFP:
Tg(sox10:EGFP-CAAX)
Our lab el375
Zebrafish: col2a1a:GFP:
TgBAC(col2a1a:EGFP)
Our lab el483
44
Zebrafish: col2a1a:mCherry:
TgBAC(col2a1a:mCherry-NTR)
Our lab el599
Zebrafish: col2a1a:h2az2a-mCherry-
2A-EGFP-CAAX:
Tg(col2a1a:h2az2a-mCherry-2A-
EGFP-CAAX)
Our lab el690
Zebrafish: scxa:mCherry:
Tg(scxa:mCherry)
Chen et al., 2020 fb301
Zebrafish: actb2:LOXP-BFP-LOXP-
DsRed: Tg(actb2:LOXP-BFP-LOXP-
DsRed)
Kobayashi et al., 2014 sd27
Zebrafish: nr5a2
mGFP
:
Tg(nr5a2:GFP-CAAX)
This paper el874
Zebrafish: nr5a2
mGFP-DBD-del
:
Tg(nr5a2:GFP-CAAX-DBD-del)
This paper el875
Zebrafish: UAS-nr5a2: Tg(UAS-
nr5a2,cryaa:Cerulean)
This paper el877
Zebrafish: foxp2-p1:GFP:
Tg(foxp2_p1-Mmu.E1b:GFP,
cryaa:Cerulean)
This paper el887, el888, el889
Zebrafish: cdh6-p1:GFP:
Tg(cdh6_p1-Mmu.E1b:GFP,
cryaa:Cerulean)
This paper el895, el896, el897
Zebrafish: fgf10a-p1:GFP:
Tg(fgf10a_p1-Mmu.E1b:GFP,
cryaa:Cerulean)
This paper el890, el891, el892
Zebrafish: scxa-p2:GFP: Tg(scxa_p2-
Mmu.E1b:GFP, cryaa:Cerulean)
This paper el900, el901
Zebrafish: nr5a2-p1:GFP:
Tg(nr5a2_p1-Mmu.E1b:GFP,
cryaa:Cerulean)
This paper el879, el880
Zebrafish: nr5a2-p2:GFP:
Tg(nr5a2_p2-Mmu.E1b:GFP,
cryaa:Cerulean)
This paper el881, el882, el883
Zebrafish: nr5a2
HA
: Tg(nr5a2-3XHA) This paper el886
Oligonucleotides: RNAscope probe
cdh6 Advanced Cell Diagnostics 1225191-C4
fgf10a Advanced Cell Diagnostics 1090101-C1, 1090101-C4
foxp2 Advanced Cell Diagnostics 1181381-C1
nr5a2 Advanced Cell Diagnostics 578901-C2
ogna Advanced Cell Diagnostics 848241-C4
sox9a Advanced Cell Diagnostics 543491-C3
Nr5a2 Advanced Cell Diagnostics 543571
Sox9 Advanced Cell Diagnostics 401051-C3
Software and Algorithms
Cell Ranger ARC v2.0.0 10X Genomics https://support.10xgenomics.co
m/single-cell-multiome-atac-
gex/software/pipelines/latest/w
hat-is-cell-ranger-arc
Seurat v4 Hao et al., 2021 https://satijalab.org/seurat/
Signac v1.6 Stuart et al., 2021 https://satijalab.org/signac/
45
chromVAR Schep et al., 2017 https://greenleaflab.github.io/ch
romVAR/
LinkPeaks Ma et al., 2020 N/A
DAVID Bioinformatics Resources Huang et al., 2009; Sherman et al., 2022 https://david.ncifcrf.gov/
ImageJ Schneider et al., 2012 https://imagej.nih.gov/ij/
Other
Alexa Fluor 647 Phalloidin ThermoFisher A22287
Oligonucleotides
SEQUENCES SOURCE
guideRNA targeting sequences:
nr5a2 exon1: generating nr5a2
mGFP
5’-GGGACTCGCTCGATCGCATG-3’,
5’-GGTGCATGGATGAGAGTGC-3’
This paper
guideRNA targeting sequences:
nr5a2 DBD: generating nr5a2
mGFP-
DBD-del
5’-CTACTCCTACGACGAAGACT-3’,
5’-CTCATCCAAGTCTTCGTCGT-3’,
5’-AGGTGTCCGGATATCACTAT-3’,
5’-CAGCAACCCATAGTGATATC-3’,
5’-GCTGACCTGTGAGAGCTGTA-3’
This paper
guideRNA targeting sequences:
nr5a2-3’UTR: generating nr5a2
HA
5’-CGCCAAACGTGCCTGAGAAC-3’,
5’-GGCCAGTTCTCAGGCACGTT-3’
This paper
nr5a2-3XHA sequences: nr5a2-
3’UTR: generating nr5a2
HA
5’-GTGCCCTGTAATAATCTTCTTATAGAGA
TGCTGCACGCCAAACGTGCCTACCCATA
CGATGTTCCAGATTACGCTGGCTACCCCT
ACGATGTACCCGATTACGCAGGCTATCCT
TATGATGTACCTGACTACGCTTGAGAACT
GGCCGTGTCTCCAAACACTCATCCCGCG
CACACACACTCT-3’
This paper
in situ hybridization probe: cdh6 Forward:
5′-GCGGAAAAGATGAGGACTTG-3′
Reverse:
5′-CATCCACATCCTCGACACTG-3′
Liu et al., 2006
in situ hybridization probe: igfbp5b Forward:
5′-GCTGGGTACATTTCTGACGG-3′
Reverse:
5′-TGGATGTTACCGCCACTGTA-3′
Askary et al., 2017
in situ hybridization probe: nr5a2 Forward:
5′-ATGGGGAACAGGGGCATATG-3′
Reverse:
5′-AGGGGTCGGGATACTCTGAT-3′
Fabian et al., 2022
in situ hybridization probe: pitx1 Forward:
5′-CCCGAAGAAGAAGAAGCAGC-3′
Reverse:
5′-TATGCTCGTCTCTGCTCCAG-3′
Askary et al., 2017
in situ hybridization probe: sox9a Forward:
5′-CCGATGAACGCGTTTATGGTGT-3′
Reverse:
5′-TTTTCGGGGTGGTGGGAGGAG-3′
Yan et al., 2002
in situ hybridization probe: Fgf10 Forward:
5′-GGGAGGAAGTGAGCAGAGGTG-3′
Reverse:
5′-GGGAGGAAGTGAGCAGAGGTG-3′
Visel et al., 2004, GenePaint
Set ID MH359
Primers to amplify nr5a2 left
homology arm: generating nr5a2
mGFP
Forward:
5’-TGCCAGCTCTTTTCGCTATGT-3’
Reverse:
5’-TCTGCTGAGCCGATGATGAG-3’
This paper
46
Primers to amplify nr5a2 right
homology arm: generating nr5a2
mGFP
Forward:
5’-TCATCCATGCACTGTTGGACA-3’
Reverse:
5’-ACAGACACATGTACAGTAAAGAGGA-3’
This paper
Primers to amplify nr5a2 coding
sequence: generating UAS-nr5a2
Forward:
5’-ATGCTGCCTAAAGTCGAGTC-3’
Reverse:
5’-TCAGGCACGTTTGGCGTGCA-3’
This paper
Primers to amplify foxp2-p1 Forward:
5’-ACTTCTCGCAGGGAGGCCAG-3’
Reverse:
5’-CGACTCCCTTTGAGTTACAG-3’
This paper
Primers to amplify cdh6-p1 Forward:
5’-GTGTCTCATTTGCACCACAC-3’
Reverse:
5’-TGTTGTTTTCAGTCACATTCTG-3’
This paper
Primers to amplify fgf10a-p1 Forward:
5’-GTGCTAGTTCGGAGAAACCC-3’
Reverse:
5’-GTGATGGAATGAGAGTGCAC-3’
This paper
Primers to amplify scxa-p2 Forward:
5’-GTGTGTGTGTGTGTGTGTATGGC-3’
Reverse:
5’-GCGGCCTCCTTCACCCCCGT-3’
This paper
gBlock to clone nr5a2-p1 gBlock of chr22:22609540-22610198
(AATTTTGTGGAAGAAACAGC……CTATCA
AGAATCCATAGAATG)
This paper
gBlock to clone nr5a2-p2 gBlock of chr22:22767424-22767888
(CAAAATTGTCATATTATTAAG……CCCAA
GCCACAGCGCAGGCCA)
This paper
Primers to genotype nr5a2
oz3
Forward:
5’-ACGAACCTCATAACACATGACAGCCA-3’
Reverse:
5’-AGCTCTCACAGGTCAGCAACCCATA-3’
Nissim et al., 2016
Primers to genotype
nr5a2
mGFP-DBD-del
Forward:
5’-ACTCCTACGACGAAGACTTGGA-3’
Reverse:
5’-GTCCACAATTGTTTTTCTCCCT-3’
This paper
Primers to genotype
nr5a2
HA
Forward:
5’-GGGAGACGTGCCCTGTAATAATC-3’
Reverse:
5’-CCAACCCATTCAGCTTTTCAGAT-3’
This paper
47
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52
Appendices
Appendix A. Experimental model and subject details
All experiments on zebrafish (Danio rerio) and mouse (Mus musculus) were approved by the
Institutional Animal Care and Use Committee of the University of Southern California (IACUC
protocols #20771 and #21151). Embryos and animals were humanely euthanized for
experiments.
Zebrafish
Zebrafish are raised in vivarium under standard conditions at 28.5°C with health and water
conditions monitored daily. Published mutant and transgenic lines include: nr5a2
oz3
(Nissim et
al., 2016); Tg(sox10:DsRedExpress)
el10
; Tg(fli1:GAL4-VP16,myl7:EGFP)
el360
; Tg(sox10:EGFP-
CAAX)
el375
; TgBAC(col2a1a:EGFP)
el483
; TgBAC(col2a1a:mCherry-NTR)
el559
;
Tg(col2a1a:h2az2a-mCherry-2A-EGFP-CAAX)
el690
; Tg(scxa:mCherry)
fb301
(Chen et al., 2020);
and Tg(actb2:LOXP-BFP-LOXP-DsRed)
sd27
(ubiquitous BFP reporter) (Kobayashi et al., 2014).
Transgenic lines generated in this study include Tg(nr5a2:GFP-CAAX)
el874
(mGFP);
Tg(nr5a2:GFP-CAAX-DBD-del)
el875
(mGFP-DBD-del); Tg(UAS-nr5a2,cryaa:Cerulean)
el877
;
Tg(foxp2_p1-Mmu.E1b:GFP, cryaa:Cerulean)
el887; el888; el889
; Tg(cdh6_p1-Mmu.E1b:GFP,
cryaa:Cerulean)
el895; el896; el897
; Tg(fgf10a_p1-Mmu.E1b:GFP, cryaa:Cerulean)
el890; el891; el892
;
Tg(scxa_p2-Mmu.E1b:GFP, cryaa:Cerulean)
el900; el901
; Tg(nr5a2_p1-Mmu.E1b:GFP,
cryaa:Cerulean)
el879; el880
; Tg(nr5a2_p2-Mmu.E1b:GFP, cryaa:Cerulean)
el881; el882,el883
; and
Tg(nr5a2-3XHA)
el886
(HA). All transgenic lines generated in this study are on a Tubingen
background. Developmental stages and numbers of embryos used are described for each
experiment. As zebrafish develop gender differences after the stages studied here, the gender
of embryos was undetermined. For oz3 allele genotyping, genomic DNA was amplified by PCR
and digested by restriction enzyme BsrI. Wild type yields cut DNA products, while oz3 yields
53
uncut product. For the nr5a2:mGFP-DBD-del allele, genotyping was based on a reduction in
band size due to the 9 bp deletion. For the nr5a2-HA allele, genotyping was based on an
increase in band size due to the 87 bp 3X-HA insertion.
Mice
Mice are raised in vivarium under standard conditions with health conditions monitored daily. All
mice are in the C57BL/6 background. Alleles utilized in this study are Nr5a2-flox (Nr5a2
tm1Sakl
,
JAX 024054, The Jackson Laboratory), Wnt1-Cre1 (Danielian et al., 1998), Prrx1-Cre (Tg(Prrx1-
Cre)1Cjt, JAX 005584, The Jackson Laboratory), and Scx-GFP (Tg(Scx-GFP)1Stzr) (Pryce et
al., 2007). Developmental stages and numbers of embryos used are described for each
experiment. Both male and female embryos were used. To conditionally delete both Nr5a2
alleles in mutants, Nr5a2-flox/flox was bred onto Wnt1-Cre or Prrx1-Cre drivers and their
siblings were used as controls.
54
Appendix B. Method details
Zebrafish transgenesis
To generate nr5a2:mGFP, GFP-CAAX sequences were inserted just upstream of the translation
start site (ATG) of nr5a2 by injecting a mix of Cas9 protein (final concentration of 800 ng/μl,
Pnabio CP02), two guide RNAs (see Table 2 for detail sequences, final concentration of 200
ng/μl), and linearized dsDNA (final concentration of 20 ng/μl) of GFP-CAAX flanked by a left
homology arm of 1020 bp and right homology arm of 1196 bp centered around the translation
start site. One founder line was identified with mGFP expression recapitulating endogenous
nr5a2 expression. To generate the nr5a2:mGFP-DBD-del allele, five guide RNAs targeting the
DNA binding domain (DBD) of nr5a2 were injected with Cas9 protein into nr5a2:mGFP
embryos. The deletion allele was identified by the smaller band size following PCR
amplification, and sequencing revealed an in-frame 9 bp deletion that removes a critical zinc
finger motif-forming cysteine residue in the DNA-binding domain. To generate nr5a2-HA, 3X-HA
tag sequences were inserted just upstream of the translation stop site (TGA) of nr5a2 targeted
by two guide RNAs (final concentration of 200 ng/μl) and linearized dsDNA (final concentration
of 0.4 ng/μl) of 3X-HA tag flanked by a left homology arm and right homology arm of 48 bp
centered around the translation stop site. The insertion allele was identified by the larger band
size following PCR amplification, and sequences were confirmed through sequencing.
To generate zebrafish transgenic lines, donor plasmids (final concentration of 20 ng/μl) were co-
injected with Tol2 transposase RNA (final concentration of 30 ng/μl) into one-cell-stage
embryos. To make UAS:nr5a2 plasmid, the nr5a2 coding sequence was first amplified from
cDNA made from Tubingen mRNA, and then combined with 10X UAS regulatory sequences
and polyA entry vectors into a destination plasmid flanked by Tol2 transposase sequences
(Tol2kit) and containing the co-selection marker cryaa:Cerulean (“eye CFP”) using Gateway
cloning (Thermofisher BP Clonase II 11789020, LR Clonase II plus 12538120). Three
55
independent UAS:nr5a2 alleles were tested and gave similar phenotypes when combined with
fli1a:Gal4VP16. To test putative enhancers of nr5a2, foxp2, cdh6, fgf10a, and scxa, accessible
chromatin regions were cloned with dsDNA gBlocks (nr5a2) or from Tubingen genomic DNA
(foxp2, cdh6, fgf10a, and scxa) into a plasmid containing Mmu.E1b as a minimal promoter,
GFP, and cryaa:Cerulean as a co-selection marker, flanked by Tol2 transposase sequences,
using In-Fusion cloning (Takara Bio 638910). Two to three independent founder lines were
identified for each construct, and consistent GFP expression patterns were observed in at least
three embryos of each allele (see Fig. 6 and Supp. Fig. 6D).
mRNA in situ hybridization
Zebrafish embryos were fixed in 4% PFA/1X PBS at 4
o
C overnight and dehydrated through
MeOH series (25%, 50%, 75% MeOH in PBSTw (0.25% Tween-20 in 1X PBS), 100% MeOH
twice) 5 min each and stored in 100% MeOH at -80
o
C overnight or longer.
In situ probes in this study include digoxigenin-labeled cdh6 (Liu et al., 2006), gsc (Schulte-
Merker et al., 1994), igfbp5b (Askary et al., 2017), msx1a (Akimenko et al., 1995), nr5a2
(Fabian et al., 2022), pitx1 (Askary et al., 2017), and Fgf10 (Visel et al., 2004) (GenePaint Set
ID MH359); dinitrophenol-labeled dlx2a (Akimenko et al., 1994), hand2 (Angelo et al., 2000),
and sox9a (Yan et al., 2002); RNAscope probes of cdh6, fgf10a, foxp2, nr5a2, ogna, sox9a,
Nr5a2, and Sox9 were synthesized by Advanced Cell Diagnostics.For whole-mount detection of
cdh6, dlx2a, gsc, hand2, igfbp5b, msx1a, nr5a2, pitx1, and sox9a by fluorescent in situ, the full
protocol is available online (https://wiki.zfin.org/display/prot/Triple+Fluorescent+In+Situ) with
minor modifications: 1.5 dpf embryos were permeabilized with gentle shaking for 30 min and 2.5
dpf embryos for 1 hr with 1.25 μg/ml protease K. We used 1 ng/μl of each probe, 1:500 of anti-
DIG-peroxidase (Roche 11207733910), 1:200 of anti-DNP-peroxidase (Akoya NEL747A001KT),
1:30 of Try-Cy3 (Akoya NEL704A001KT), and 1:30 of Try-Cy5 (Akoya NEL705A001KT).
Counterstain of nuclei by DAPI staining was performed at 4
o
C overnight.
56
Whole-mount detection of cdh6, fgf10a, foxp2, nr5a2, ogna, and sox9a in zebrafish and
detection of Nr5a2 and Sox9 on mouse sections were performed by RNAscope Multiplex
Fluorescent Reagent Kit v2 Assay per manufacturer’s instructions (Advanced Cell Diagnostics,
protocol 323100-USM, and technical note MK 50-016) with optimization. RNAscope on
zebrafish embryos was performed in tubes (six 2.5 dpf embryos/ml). Target Retrieval was
performed for 20 min at 100
o
C. We permeabilized 2.5 dpf embryos with Protease Plus for 15
min at 40
o
C. RNAscope on mouse embryos was performed on paraformaldehyde-fixed paraffin-
embedded sections with sample preparation, paraffin embedding, and sectioning.
For Fgf10 in situs, mandibular arches of E12.5 mouse embryos were dissected and fixed in 4%
PFA/1X PBS at 4
o
C overnight. Colorimetric whole-mount detection of Fgf10 was performed as
described (Elliott et al., 2018). In brief, embryos were dehydrated through MeOH series and
stored at -20
o
C overnight, rehydrated through MeOH series to PBSTx (0.2% Triton-X in 1X
PBS) before permeabilization by 20 μg/ml of protease K for 30 min with gentle shaking at RT
(room temperature). Secondary fixation in 4% PFA/0.1% glutaraldehyde/1X PBS for 30 min was
followed by two washes of PBSTx for 10 min at RT and prehyb incubation for 2 hr at 65
o
C. 1
ng/μl of probe was used for hybridization for 48 hr followed by 2X SSC and 0.2% SSC washes
three each for 20 min at 65
o
C and two KTBT washes for 10 min each at RT. Blocking and anti-
DIG-AP Fab incubation (1:2000, Roche 11093274910) were performed in 20% sheep
serum/KTBT at 4
o
C overnight. We then washed five times with KTBT for 1 hr and twice with
NTMT for 15 min before color development in 10 μl NBT/BCIP premix stock (Roche
11681451001) per ml of NTMT in the dark. When hybridization signal was optimal, we stopped
the reaction with KTBT followed by PBS washes. Similar expression patterns were observed in
at least three zebrafish embryos of wild type and mutant unless described in the text and
panels. Numbers of mouse embryos examined are described in the figure legends.
57
Sectioning
For paraffin sections, we fixed E11.5 or E12.5 whole embryos or dissected newborn mouse
heads in 4% PFA/1X PBS at 4
o
C overnight, followed by decalcification in 0.5 M EDTA (pH 8)
(for newborn) and dehydration though EtOH series (1X PBS, two 50% EtOH, two 70% EtOH for
1 hr each, 95% EtOH overnight, 95% EtOH, three 100% EtOH for 1 hr each at RT in the second
day, and then 100% EtOH overnight at 4
o
C). Before embedding with paraffin, samples were
infused with Hemo-De (xylene substitute, Electron Microscopy Sciences 23412-01) and paraffin
through a series of 50% EtOH/50% Hemo-De, four Hemo-De at RT. Sections were then placed
in the vacuum incubator at 65
o
C and put through a series of 50% Hemo-De/50% paraffin and
paraffin three times for 30 min each. After embedding in paraffin, paraffin blocks were left
overnight at RT to harden. For mouse embryos, 5 μm thick sections were cut on a Thermo
Scientific Shandon Finesse ME+ Microtome. For newborn mouse heads, 8 μm thick sections
were cut with a Leica Microtome RM2245. For cryo-sectioning, E16.5 mouse heads were fixed
in 4% PFA/1X PBS followed with 1X PBS wash at 4
o
C for 30 min each, and were then infused
with sucrose (10% sucrose overnight and 30% sucrose until sinking at 4
o
C) and embedded in
OCT (with a quick wash of OCT) (Sakura 4583). Cryo-blocks were frozen on dry ice and stored
in -80
o
C. For cryo-sections, 10 μm thick sections were cut on a Leica Cryostat CM3050S.
Trichrome staining
Staining was performed via standard protocols. Frontal and sagittal paraffin sections were first
de-paraffinized and rehydrated through an EtOH series (100%, 90%, 70%, and 50%) to water.
The sections were stained with Alcian Blue (1% Alcian Blue 8GX (BDH) in 3% acetic acid, pH
2.5), Ehrlich’s hematoxylin (Solmedia), and differentiated in 2.5% phosphomolybdic acid (Acros
Organic), with two rinses of water between each solution. Finally, the sections were stained with
58
Sirius red (0.1% Direct Red 80 in saturated aqueous solution of picric acid, Sigma) followed by
two 1% Acetic Acid washes prior to mounting and imaging.
Skeletal staining
For skeletal staining, zebrafish embryos were fixed in 2% PFA/1X PBS for 1 hr and washed with
100 mM Tris (pH 7.5)/10 mM MgCl2 for 10 min at RT. We then incubated embryos with Alcian
stain (0.04% Alcian Blue (Anatech 862), 100 mM Tris (pH 7.5), 10 mM MgCl2, 80% EtOH) for
cartilage staining overnight followed by de-staining in EtOH gradient (80%, 50%, 25% EtOH in
100 mM Tris (pH 7.5)/10 mM MgCl2) for 5 min each. Embryos were then incubated in 3% H2O2
/0.5% KOH with open lids under a light source until pigments bleached. To stain bone, we then
washed in 25% glycerol/0.1% KOH for 10 min before adding Alizarin stain (0.01% Alizarin Red
S (Sigma A5533), 25% glycerol, 100 mM Tris (pH 7.5)) for 30 min. Embryos were de-stained in
50% glycerol/0.1% KOH twice before whole-mount imaging. Zebrafish facial skeletons were
dissected with fine insect pins and mounted on microscope slides for flat-mount imaging.
For whole-mount skeletal staining of E14.5 embryos and newborn mice, we skinned newborn
animals and removed soft tissues before fixation in 95% EtOH for five days with occasional
changes of fresh EtOH at RT. We then added Alcian stain (0.015% Alcian Blue (Alcian Blue
8GX (Sigma A3157), 75% EtOH, 20% Glacial acetic acid) for one day and de-stained in 95%
EtOH for one day, replacing solution once. Soft tissues were cleared with 0.5% KOH for five
days before addition of Alizarin stain (0.002% Alizarin Red S (Sigma A5533), 0.5% KOH) for
four days. We then de-stained in 1% KOH for one day before transfer into a glycerol gradient
(25%, 50%, 75% glycerol / 0.5% KOH and 100% glycerol) one day each for imaging and
storage. E14.5 Meckel’s cartilages were dissected with fine insect pins and mounted on
microscope slides for flat-mount imaging.
59
Freshly dissected, un-fixed Scx-GFP+ mouse heads were stained in Alizarin stain and washed
in PBS for 30 min each. Reported phenotypes were observed in at least three zebrafish or
mouse embryos unless described otherwise.
Immunostaining
For whole-mount immunostaining, zebrafish embryos were fixed in 4% PFA/1X PBS at 4
o
C
overnight and washed by PBS and ddH2O each for 5 min. Permeabilization of embryos was
performed in cold acetone at -20
o
C for 10 min, followed by washing in ddH2O for 5 min,
rehydrating with PBS for 5 min, blocking for 3 hr at RT, a brief wash, and incubation with rabbit
anti-phospho-histone H3 (Ser10) (1:200, Santa Cruz Biotechnology), chicken anti-mCherry
(1:200, Novus Biologicals NBP2-25158), or chicken anti-GFP (1:200, Abcam ab13970) in 2%
goat serum / PBDTx (1% BSA, 1% DMSO, 0.1% Triton-X in 1X PBS, pH 7.3) at 4
o
C overnight.
We then washed three times for 20 min each with PBDTx before incubating with 1:300
secondary antibodies Alexa Fluor 647 anti-rabbit (ThermoFisher A-31573), Alexa Fluor 568 anti-
chicken (ThermoFisher A-11041), or Alexa Fluor 488 anti-chicken (ThermoFisher A-11039), as
well as DAPI, in 2% goat serum / PBDTx at 4
o
C overnight. Embryos were washed twice with
PBSTx (0.1% Triton-X in 1X PBS) before imaging.
For staining of mouse cryo-sections, slides were rinsed with ddH2O, air dried at RT, and fixed in
4% PFA/1X PBS at 4
o
C for 10 min. Tissues were permeabilized in PBSTx for 10 min and
washed twice with PBSTw (0.05% Tween-20 in 1X PBS). We then blocked for 1h at RT before
incubating with mouse anti-MF-20 (1:300, Developmental Studies Hybridoma Bank MF 20),
rabbit anti-Sox9 (1:800, Novus Biologicals NBP1-85551), or chicken anti-GFP (1:200, Abcam
ab13970) at 4
o
C overnight. This was followed with incubation of 1:300 secondary antibodies
Alexa Fluor 647 anti-mouse (ThermoFischer A-21242), Alexa Fluor 568 anti-rabbit
(ThermoFischer A11011), or Alexa Fluor 488 anti-chicken (ThermoFisher A-11039) in 10%
60
FBS/1% BSA/5% goat serum in PBSTw at RT for 1 hr. Slides were rinsed twice with ddH2O
before mounting and imaging. Numbers of zebrafish and mouse embryos examined are
described for each experiment in the figure panels or legends.
Muscle staining
To stain muscle, zebrafish embryos were fixed in 4% PFA/1X PBS for 1 hr at RT (or at 4
o
C
overnight), and Scx-GFP+ mouse limbs were left un-fixed. Both samples were permeabilized
with 0.5% Triton-X in PBS for at least 1 hr followed by phalloidin staining (1:100 in 1X PBS,
Alexa Fluor 647 Phalloidin, ThermoFisher A22287) at 4
o
C overnight. Samples were rinsed
several times with PBS before imaging. Phenotypes were observed in at least three zebrafish
and mouse embryos unless described otherwise.
Transplantation
For CNCC transplants, BFP+ naïve ectoderm from shield-stage actb2:LOXP-BFP-LOXP-DsRed
donors was transplanted into CNCC-forming regions of shield-stage col2a1a:h2az2a-mCherry-
2A-EGFP-CAAX hosts. Transplantation was performed unilaterally with one side as control.
Embryos were held in place with 2% methylcellulose in Ringer’s solution (116 mM NaCl, 2.6 mM
KCl, 5 mM HEPES, pH 7.0) on glass slides flooded with Ringer’s solution. A fine glass needle
was used to move small clumps of cells from donors to hosts. Animals were recovered in
embryo medium with 50 U/ml Penicillin and 0.05 mg/ml Streptomycin, selected by contribution
of BFP+ cells to pharyngeal arches on one side of the embryo at 1.5 dpf, and imaged at 6 dpf
on a confocal microscope before fixation for phalloidin staining of muscle. The same embryos
then underwent skeletal staining as described above.
61
Imaging
Images of live and fixed zebrafish, as well as sections after in situ hybridization,
immunostaining, or phalloidin staining were captured on a Zeiss LSM800 confocal microscope
using ZEN software. Skeletal staining, freshly dissected mouse tissues (salivary glands,
stomach, and intestines), and mouse whole-mount in situs were imaged using a Leica S8APO
stereo microscope. Images of flat-mount zebrafish skeletons were captured with a Leica
DM2500 compound microscope. Fluorescence images of freshly dissected mouse skeletons
after Alizarin Red staining were taken by a Leica MZ16F fluorescence microscope. Trichrome
stains of mouse histological sections were imaged by a Nikon Eclipse 80i microscope.
Single nuclei isolation
Zebrafish embryos were screened for nr5a2:mGFP-DBD-del+ and scxa:mCherry+ with a
fluorescence dissecting microscope and fin clips were collected for PCR genotyping of the
mutant oz3 allele before 2 dpf. Control and mutant heads were decapitated between the eye
and ear after anesthesia at 2.5 dpf. Separately for controls and mutants, 160 dissected heads
(twenty heads pre tube) were washed twice with fresh and iced Ringer’s solution followed by
dissociation at 28.5 °C for 40 min by mechanical and enzymatic digestion – nutating and
pipetting every 5 min in pre-warmed protease solution (0.25% trypsin, 1 mM EDTA (pH 8.0), and
20 mg/mL Collagenase D (from stock of 400 mg/mL Collagenase D in HBSS) in PBS) until full
dissociation. The reaction was stopped with 6X stop solution (6 mM CaCl2 and 30% fetal bovine
serum (FBS) in PBS). Dissociated cells were collected by centrifugation for 5 min (2000 rpm at
4°C) and washed by suspension solution (1% FBS, 0.8mM CaCl2, 50 U/ml Penicillin, 0.05
mg/ml Streptomycin in Leibovitz Medium) twice and filtered by cell strainer before sorting. Live
cells were fluorescence-activated cell sorted (FACS) to isolate 50,000 GFP+ cells that excluded
the cytoplasmic stain Zombie Violet (control) or nuclear stain DAPI (mutant) into 0.04% BSA/
PBS at 4°C. Nuclei isolation was performed per manufacturer’s instructions (10X Genomics,
62
protocol CG000169, low cell input protocol) with optimization for zebrafish mesenchyme. FACS
cells were pelleted for 15 min (300 rcf at 4
o
C) and incubated with lysis buffer for 100 s on ice.
Isolated nuclei were washed by Wash buffer and Nuclei buffer and checked for nuclear integrity
with a fluorescence confocal microscope with DAPI staining before use in library construction.
Multi-omic library construction, sequencing, and alignment
Multi-omic libraries of snATACseq and snRNAseq from the same barcoded single nuclei were
constructed per manufacturer’s instructions (10X Genomics, Chromium Next GEM Single Cell
Multiome ATAC + Gene Expression, protocol CG000338). In brief, to capture accessible
chromatin and transcripts from the same cells, accessible chromatin regions from isolated nuclei
were first tagged by Tn5 transposase. Tagged chromatin and polyadenylated mRNA from the
same nuclei were pulled down and barcoded with the same sequences within isolated GEMs to
achieve single-nuclei separation. Chromatin fragments were further indexed with sample-
specific i7 indexes and linked with Illumina P5 and P7 sequences. Reverse transcription was
performed in GEMs to synthesize RNA:DNA hybrids from polyadenylated mRNA. cDNAs were
further synthesized, fragmented, and indexed with sample-specific i7 and i5 indexes and linked
with Illumina P5 and P7 sequences. Quality control of libraries was assessed with the 4200
TapeStation system and Qubit dsDNA HS assay kit. Libraries were sequenced on Illumina
HiSeq (control) or NextSeq (mutant) platforms. For sequencing snATACseq libraries, both
Read1 and Read2 were extended to 60 cycles, and for sequencing snRNAseq libraries, Read2
was extended to 102 cycles for longer coverage. Sequencing reads were aligned to customized
genome (built with GRCz11.fa, GRCz11.104.gtf, and JASPAR2020.pfm, with addition of GFP-
CAAX and mCherry gene information; the same genome build is used for analysis by
Seurat/Signac) and peak calling was performed by Cell Ranger ARC v2.0.0 per manufacturer’s
instructions to generate peak-by-cell and gene-by-cell count matrices. Raw sequencing fastq
63
files and Cell Ranger ARC v2.0.0-processed Multiome data can be accessed at GEO
GSE210251.
Multi-omics data analysis
Multii-omics data were processed by Seurat v4 (Hao et al., 2021) and Signac v1.6 (Stuart et al.,
2021) packages in Rstudio following the standard workflow with optimization. To first identify the
mesenchyme populations of individual libraries, Cell Ranger ARC-output count matrices in H5
format of individual libraries were used to create Seurat objects by “CreateSeuratObject” and
“CreateChromatinAssay” functions. Quality control was performed by setting the thresholds of
accessible region counts (nCount_ATAC) < 50000, transcript counts (nCount_RNA) < 7500,
ratio of mononucleosome to nucleosome-free region (nucleosome_signal) < 2, and enrichment
at TSS (TSS.enrichment) > 0.5. In order to compare accessible chromatin regions (peaks)
between control and mutant, peaks-called were combined by “disjoin” function that splits peaks
into overlapping and non-overlapping regions between the control and mutant libraries and
filtered by new peak widths (peakwidths < 10000 & > 20). The disjoined-peak list was used to
recalculate peak-by-cell count matrices by “CreateFragmentObject”, “FeatureMatrix”, and
“CreateChromatinAssay” functions. For further analysis of ATAC data (peak-by-cell count
matrices), 95% of most common peaks were selected by “FindTopFeatures (min.cutoff = 5)”
function. Normalization and linear dimension reduction by LSI were performed by “RunTFIDF”,
and “RunSVD” functions. RNA data (gene-by-cell count matrices) were normalized and scaled,
and the top 3000 most common genes were selected by “SCTransform” function for dimension
reduction by PCA (“RunPCA” function). To cluster cells with ATAC and RNA information in
combination, “FindMultiModalNeighbors” (2:20 dimensions of LSI and 1:25 dimensions of PCA
were used, with the first dimension of LSI excluded due to its high correlation with sequencing
depth), “RunUMAP” (nn.name = “weighted.nn”), and “FindClusters” (resolution = 0.8,
graph.name=”wsnn”, algorithm = 3) functions. Identities of clusters were identified by
64
differentially enriched genes calculated by “FindAllMarkers” (test.use = “wilcox” (Wilcoxon Rank
Sum test), logfc.threshold = 0.25 (log2 Fold Change), return.thresh = 0.01 (p value)) function.
Clusters 0, 2, 5, 8, 20 of control library and clusters 2, 4,10,18 of mutant library were identified
as mesenchyme populations for the high expression of fli1a, prrx1a, and other mesenchyme
genes.
“Merge” function was used to combine count matrices of control and mutant mesenchyme
clusters. Merged ATAC data were processed by “FindTopFeatures” (min.cutoff = 10) and LSI
linear dimension reduction, and merged RNA data were processed and dimensionally reduced
by PCA as described above. 2:30 dimensions of LSI and 1:30 dimensions of PCA were used to
cluster cells with both ATAC and RNA information as described above. After filtering for artificial
clusters with over- and under-accessible profiles, 14 sub-clusters of mesenchyme were
identified and differentially enriched genes were calculated by “FindAllMarkers” (test.use =
“wilcox”, logfc.threshold = 0.25, return.thresh = 0.01, min.pct = 0.25). To calculate motif
enrichments of each cell by chromVAR (Schep et al., 2017), “AddMotifs” was first used to
calculate motif enrichments of each accessible chromatin region (peak) followed by
“RunChromVAR” to calculate motif activity in each mesenchyme cell. To identify accessibility of
chromatin regions correlated with expression of genes whose TSS was located within 500 kb of
the chromatin regions, “LinkPeaks” (method =”spearman”, min.cells = 5, pvalue_cutoff = 0.1,
score_cutoff = 0.02) (Ma et al., 2020) was performed on merged mesenchyme.
For detailed analysis of mandibular arch clusters, we further extracted a cartilage cluster
(acana+, cluster 11), a tendon cluster (tnmd+, cluster 2), and four mandibular arch mesenchyme
(Mes1-4) clusters (dlx2a+; dlx5a+; hand2+; Hox-negative, clusters 8, 4, 7, and 1, respectively).
UMAP visualization of mandibular arch clusters was generated by “FindMultiModalNeighbors”
and “RunUMAP” using the same parameters as mesenchyme analysis above. To investigate
65
the difference of mutant mandibular arch clusters compared to control, differential analysis was
performed. Differential motif enrichments of mutant and control mandibular mesenchyme,
excluding cartilage, were calculated by “FindMarkers” (test.use = "wilcox", mean.fxn =
rowMeans, fc.name = "avg_diff", logfc.threshold = 0.25 (calculating average difference)) using
“chromvar” matrix, differential accessible chromatin regions by “FindAllMarkers” (test.use = ‘LR’
(logistic regression), logfc.threshold = 0.25, return.thresh = 0.01, min.pct = 0.05, latent.vars =
‘nCount_ATAC’) on “ATAC” matrix, and differential gene expressions by “FindAllMarkers”
(test.use = “wilcox”, logfc.threshold = 0.25, return.thresh = 0.01, min.pct = 0.25) on “SCT”
matrix. Gene Ontology analysis (GO) of downregulated genes in mutants was performed using
DAVID Bioinformatics Resources (Huang et al., 2009; Sherman et al., 2022). To identify
potential Nr5a2 direct targets, regions with less or more accessibility in mutants were divided
into two groups: those that contain predicted Nr5a2 binding motifs and those that did not. Genes
whose expression correlated with the accessibility of the Nr5a2 motif regions (predicted by
“LinkPeaks” above) were intersected with genes with decreased or increased expression in
mutants. To investigate the accessibility of enhancers in the published 1.5 dpf CNCC
snATACseq data (Fabian et al., 2022), the most overlapping chromatin regions (peaks) were
identified and their activities were plotted for visualization.
Cut & Run Sequencing library construction, sequencing and analysis
Whole cells (50,000 cells) were dissociated from 2.5 dpf heads of nr5a2
mGFP-DBD-del/HA
animals
(160 animals) and FACS-sorted for live GFP+ cells that excluded the cytoplasmic stain Zombie
Violet following the details described above. Nr5a2 Cut & Run-seq libraries were constructed
per manufacturer’s instructions (EpiCypher, CUTANA™ ChIC/CUT&RUN Kit, SKU:14-1048)
with anti-HA antibodies (rabbit-anti-HA-tag (C29F4), Cell Signaling 3724T or rabbit HA Tag
CUTANA™ CUT&RUN Antibody, EpiCypher SKU:13-2010). Libraries were sequenced on
66
Illumina NextSeq platforms with both Read1 and Read2 extended to 60 cycles. Sequencing
reads were aligned to GRCz11 genome and analyzed per manufacturer’s instructions.
67
Appendix C. Quantification and statistical analysis
Quantification of Meckel’s chondrocyte numbers across time was performed by counting nuclei
labeled by the col2a1a:h2az2a-mCherry-2A-EGFP-CAAX transgene in serial confocal sections
with ImageJ software. Quantification of pHH3+ nuclei labeled by the col2a1a:h2az2a-mCherry-
2A-EGFP-CAAX transgene in wild-type and mutant Meckel’s was counted in serial confocal
sections. Wilcoxon rank-sum test was performed to compare the difference of wild-type and
mutant chondrocyte numbers at each stage. Numbers of nr5a2:mGFP-DBD-del+ tenocytes and
chondrocytes were counted by membrane-GFP+ cell outlines and chondrocyte numbers by
col2a1a:mCherry-NTR labeling in serial confocal sections. Wilcoxon rank-sum test was
performed to compare the difference of GFP+ tenocyte numbers and GFP+ chondrocyte
proportion to total chondrocyte numbers in wild types and mutants. Quantification of Meckel’s
chondrocyte and tenocyte numbers in fixed embryos of fli1a:Gal4VP16; UAS:nr5a2 and single-
transgene animals were counted with nuclei labeled by DAPI within cells marked by
chondrocyte or tenocyte transgenes across serial confocal sections. Measurements of skeleton
and tendon length in newborn mice were measured by ImageJ software. Differences across
genotypes were calculated by a Tukey's range test to account for multiple comparisons.
Abstract (if available)
Abstract
Organ development involves sustained production of diverse cell types with spatiotemporal precision. This is especially true in the vertebrate jaw, where neural-crest-derived multipotent progenitors produce not only the skeletal tissues but also the later-forming tendons and salivary glands. Here we identify the pluripotency factor Nr5a2 as essential for balancing cell fate decisions to generate the full repertoire of neural-crest-derived fates in the jaw. In zebrafish and mice, we observe transient expression of Nr5a2 in a subset of mandibular post-migratory neural-crest-derived cells. In zebrafish nr5a2 mutants, nr5a2-expressing cells generate excess cartilage, resulting in an expanded lower jaw skeleton and loss of later forming tendons. In mice, neural crest-specific Nr5a2 loss results in analogous skeletal and tendon defects in the jaw and middle ear, as well as salivary gland loss. Single-cell profiling in mutants reveals that Nr5a2, distinct from its roles in pluripotency, promotes jaw-specific chromatin accessibility and gene expression essential for tendon and gland fates. Thus, repurposing of Nr5a2 within post-migratory neural-crest-derived cells restricts skeletal and promotes connective tissue fates to generate the full repertoire of derivatives required for proper jaw and middle ear function.
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Asset Metadata
Creator
Chen, Hung-Jhen
(author)
Core Title
Nr5a2 balances lineage decisions to promote diverse connective tissue fates in the jaw
School
Keck School of Medicine
Degree
Doctor of Philosophy
Degree Program
Development, Stem Cells and Regenerative Medicine
Degree Conferral Date
2023-05
Publication Date
02/01/2023
Defense Date
12/08/2022
Publisher
University of Southern California
(original),
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(digital)
Tag
connective tissues,craniofacial,jaw skeleton,middle ear,neural crest cells,Nr5a2,OAI-PMH Harvest,salivary gland,tendon,zebrafish
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Electronically uploaded by the author
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Merrill, Amy (
committee chair
), Crump, Gage (
committee member
), Mariani, Francesca (
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)
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Tags
connective tissues
craniofacial
jaw skeleton
middle ear
neural crest cells
Nr5a2
salivary gland
tendon
zebrafish