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Foxc1 establishes enhancer accessibility for craniofacial cartilage differentiation

ABSTRACT: The specification of cartilage requires Sox9, a transcription factor with broad roles for organogenesis outside the skeletal system. How Sox9 gains selective access to cartilage-specific cis-regulatory regions during skeletal development had remained unclear. By analyzing chromatin accessibility during the differentiation of neural crest cells into chondrocytes of the zebrafish head, we find that cartilage-associated chromatin accessibility is dynamically established. Cartilage-associated regions that become accessible after neural crest migration are co-enriched for Sox9 and Fox transcription factor binding motifs. In zebrafish lacking Foxc1 paralogs, we find a global decrease in chromatin accessibility in chondrocytes, consistent with a later loss of dorsal facial cartilages. Zebrafish transgenesis assays confirm that many of these Foxc1-dependent elements function as enhancers with region- and stage-specific activity in facial cartilages. We propose that Foxc1-dependent chromatin accessibility helps directs the versatile Sox9 protein to a chondrogenic program in the face.

ORGANISM(S): Danio rerio

PROVIDER: GSE157575 | GEO | 2020/12/31

REPOSITORIES: GEO

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Project description:Background: Human pluripotent stem cells (hPSCs) give rise to chondrogenic activity in culture. Such activity was found in mesodermal and neural crest like progeny of hPSCs, and readily expanded as SOX9+ msenchymal cells in a medium containing FGF2. However, once expanded, the type of chondrocytes they perfer to form is growth plate-ike transient chondrocytes that are destined to form bone. However, we have recently developed a method to generate GDF5+ mesenchymal cells from such SOX9+ cells which ehibit activity to preferentially form articular-like permanent chondrocytes. The aim of the present prospective follow-up study was to reveal the cell-type and developmental origin of the GDF5+ cells as well as the SOX9+ cells. Methods: Paraxial mesoderm was generated from human PSCs, purified by FACS, and expanded to generate SOX9+ chondroprogenitors. Then, the SOX9+ cells were further differentiated into joint progenitor-like GDF5+ chondroprogenitors. These two chondroprogenitors were subjcted to bulk RNA-seq. Results: Based on the transcriptomic data, we examined differentially expressed genes, cell-type deconvolution analysis, and cell-type correlation analysis. The results suggested that GDF5+ cells might contain a teno/ligamento-genic potential (e.g., SCX+ cells), while SOX9+ cells were related to (neural crest-derived) ectomesenchymal chondroprogenitors. The followup biological analyses indicated as follows. The fast-growing SOX9+ cells formed cartilage that expressed chondrocyte hypertrophy markers and was readily mineralized after ectopic transplantation. In contrast, the slowly-growing GDF5+ cells were derived from SOX9+ cells. They formed cartilage that tended to express low to no chondrocyte hypertrophy markers, while expressing PRG4, a marker of embryonic articular chondrocytes, and to remain largely unmineralized in vivo. Conclusions: Our results imply that GDF5+ cells specified to generate permanent-like cartilage are likely to emerge in association with the commitment of SOX9+ cells to the tendon/ligament lineage.

Project description:Distinct shaping of the upper versus lower facial skeleton is essential for function of the vertebrate jaw and middle ear, yet the cellular mechanisms by which this occurs have remained unclear. Here, we show that Endothelin1 (Edn1) signaling accelerates mesenchymal condensation and subsequent cartilage formation in the lower face through antagonism of Jagged-Notch signaling and Prrx1 transcription factors. A genomic analysis of facial skeletal precursors in mutants and overexpression embryos reveals that Jagged-Notch signaling represses genes that are strongly induced as pharyngeal arch neural crest-derived cells begin skeletal differentiation. In wild types, initial Jagged-Notch repression dorsally ensures that barx1+ condensations and cartilage differentiation occur first in ventral-intermediate zones of the pharyngeal arches. Reduced Jagged-Notch signaling results in an expansion of pre-cartilage condensations in the upper face, with loss of barx1 partially restoring dorsal cartilage shapes in jag1b mutants. Further, by studying new mutants for zebrafish prrx1a and prrx1b, we find that Prrx1 genes function in parallel to Jagged-Notch signaling to restrict the formation of dorsal barx1+ pre-cartilage condensations. Consistently, combined losses of jag1b and prrx1a/b robustly rescue ventral barx1+ condensations and lower facial cartilage development in edn1 mutants. Together, our work suggests that Edn1 works through parallel inhibition of Jagged-Notch and Prrx1 pathways to promote an earlier and more extensive establishment of cartilage condensations in the lower face. We performed RNAseq on FACS-sorted neural crest-derived pharyngeal arch cells (fli1a:GFP; sox10:DsRed double positive) from wild-type embryos at 3 different stages (20, 28, and 36 hours post fertilization) and embryos with altered levels of Edn1 and Notch signaling (edn1 mutants and hsp70I:Gal4; UAS:Edn1 transgenics; jag1b mutants, dibenzazepine-treated embryos, and hsp70I:Gal4; UAS:NICD transgenics. We also sequenced RNA from heat-shocked UAS:Edn1+ and hsp70I:Gal4+ transgenics and jag1b+/+ controls.

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Foxc1 establishes enhancer accessibility for craniofacial cartilage differentiation

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