Nuclear receptor Nr5a2 promotes diverse connective tissue fates in the jaw - PubMed (original) (raw)

Nuclear receptor Nr5a2 promotes diverse connective tissue fates in the jaw

Hung-Jhen Chen et al. Dev Cell. 2023.

Abstract

Organ development involves the sustained production of diverse cell types with spatiotemporal precision. In the vertebrate jaw, neural-crest-derived progenitors produce not only skeletal tissues but also later-forming tendons and salivary glands. Here we identify the pluripotency factor Nr5a2 as essential for cell-fate decisions in the jaw. In zebrafish and mice, we observe transient expression of Nr5a2 in a subset of mandibular postmigratory neural-crest-derived cells. In zebrafish nr5a2 mutants, nr5a2-expressing cells that would normally form tendons generate excess jaw cartilage. 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 shows that Nr5a2, distinct from its roles in pluripotency, promotes jaw-specific chromatin accessibility and gene expression that is essential for tendon and gland fates. Thus, repurposing of Nr5a2 promotes connective tissue fates to generate the full repertoire of derivatives required for jaw and middle ear function.

Keywords: Nr5a2; craniofacial development; jaw; middle ear; multipotency; neural crest; salivary gland; tendon; zebrafish.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

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). (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). See also Figure S1.

Figure 2.. 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 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. See also Figure S2.

Figure 3.. 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, h2az2a-mCherry channel is not shown; 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. Note that BFP signal is decreased after the fixation step required for Phalloidin staining. Pq, palatoquadrate cartilage. Scale bars = 50 μm. See also Figure S3.

Figure 4.. 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). (D) Trichrome staining of coronal sections shows absence of salivary gland mesenchyme (Mes, red outline) and epithelial invagination (E) at E12.5 in 2/2 Nr5a2 NCC mice. Sagittal sections at birth (P0) show absence of the submandibular gland (SMG), sublingual gland (SLG), and salivary gland duct (asterisk) in 4/4 Nr5a2 NCC newborn (P0) mice. The tongue (T) is unaffected. Scale bars = 500 μm. See also Figure S4.

Figure 5.. Single-cell analysis of gene expression and chromatin changes in nr5a2 mutants

(A) Image of 2.5 dpf _nr5a2:mGFP_+ 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. See also Figure S5.

Figure 6.. 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 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 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/oz mutants. Scale bars = 50 μm (A-C, E); 500 μm (D). See also Figure S6 for additional examples of transgenic line expression and expanded genomic views of open chromatin.

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Nuclear receptor Nr5a2 promotes diverse connective tissue fates in the jaw - PubMed (original) (raw)