Fox proteins are modular competency factors for facial cartilage and tooth specification.

Facial form depends on the precise positioning of cartilage, bone, and tooth fields in the embryonic pharyngeal arches. How complex signaling information is integrated to specify these cell types remains a mystery. We find that modular expression of Forkhead domain transcription factors (Fox proteins) in the zebrafish face arises through integration of Hh, Fgf, Bmp, Edn1 and Jagged-Notch pathways. Whereas loss of C-class Fox proteins results in reduced upper facial cartilages, loss of F-class Fox proteins results in distal jaw truncations and absent midline cartilages and teeth. We show that Fox proteins are required for Sox9a to promote chondrogenic gene expression. Fox proteins are sufficient in neural crest-derived cells for cartilage development, and neural crest-specific misexpression of Fox proteins expands the cartilage domain but inhibits bone. These results support a modular role for Fox proteins in establishing the competency of progenitors to form cartilage and teeth in the face.

Genome-wide analysis of facial skeletal regionalization in zebrafish.

Patterning of the facial skeleton involves the precise deployment of thousands of genes in distinct regions of the pharyngeal arches. Despite the significance for craniofacial development, how genetic programs drive this regionalization remains incompletely understood. Here we use combinatorial labeling of zebrafish cranial neural crest-derived cells (CNCCs) to define global gene expression along the dorsoventral axis of the developing arches. Intersection of region-specific transcriptomes with expression changes in response to signaling perturbations demonstrates complex roles for Endothelin 1 (Edn1) signaling in the intermediate joint-forming region, yet a surprisingly minor role in ventralmost regions. Analysis of co-variance across multiple sequencing experiments further reveals clusters of co-regulated genes, with in situ hybridization confirming the domain-specific expression of novel genes. We then created loss-of-function alleles for 12 genes and uncovered antagonistic functions of two new Edn1 targets, follistatin a (fsta) and emx2, in regulating cartilaginous joints in the hyoid arch. Our unbiased discovery and functional analysis of genes with regional expression in zebrafish arch CNCCs reveals complex regulation by Edn1 and points to novel candidates for craniofacial disorders.

Fat4-Dchs1 signalling controls cell proliferation in developing vertebrae.

The protocadherins Fat4 and Dchs1 act as a receptor-ligand pair to regulate many developmental processes in mice and humans, including development of the vertebrae. Based on conservation of function between Drosophila and mammals, Fat4-Dchs1 signalling has been proposed to regulate planar cell polarity (PCP) and activity of the Hippo effectors Yap and Taz, which regulate cell proliferation, survival and differentiation. There is strong evidence for Fat regulation of PCP in mammals but the link with the Hippo pathway is unclear. In Fat4(-/-) and Dchs1(-/-) mice, many vertebrae are split along the midline and fused across the anterior-posterior axis, suggesting that these defects might arise due to altered cell polarity and/or changes in cell proliferation/differentiation. We show that the somite and sclerotome are specified appropriately, the transcriptional network that drives early chondrogenesis is intact, and that cell polarity within the sclerotome is unperturbed. We find that the key defect in Fat4 and Dchs1 mutant mice is decreased proliferation in the early sclerotome. This results in fewer chondrogenic cells within the developing vertebral body, which fail to condense appropriately along the midline. Analysis of Fat4;Yap and Fat4;Taz double mutants, and expression of their transcriptional target Ctgf, indicates that Fat4-Dchs1 regulates vertebral development independently of Yap and Taz. Thus, we have identified a new pathway crucial for the development of the vertebrae and our data indicate that novel mechanisms of Fat4-Dchs1 signalling have evolved to control cell proliferation within the developing vertebrae.

Competition between Jagged-Notch and Endothelin1 Signaling Selectively Restricts Cartilage Formation in the Zebrafish Upper Face.

The intricate shaping of the facial skeleton is essential for function of the vertebrate jaw and middle ear. While much has been learned about the signaling pathways and transcription factors that control facial patterning, the downstream cellular mechanisms dictating skeletal shapes have remained unclear. Here we present genetic evidence in zebrafish that three major signaling pathways - Jagged-Notch, Endothelin1 (Edn1), and Bmp - regulate the pattern of facial cartilage and bone formation by controlling the timing of cartilage differentiation along the dorsoventral axis of the pharyngeal arches. A genomic analysis of purified facial skeletal precursors in mutant and overexpression embryos revealed a core set of differentiation genes that were commonly repressed by Jagged-Notch and induced by Edn1. Further analysis of the pre-cartilage condensation gene barx1, as well as in vivo imaging of cartilage differentiation, revealed that cartilage forms first in regions of high Edn1 and low Jagged-Notch activity. Consistent with a role of Jagged-Notch signaling in restricting cartilage differentiation, loss of Notch pathway components resulted in expanded barx1 expression in the dorsal arches, with mutation of barx1 rescuing some aspects of dorsal skeletal patterning in jag1b mutants. We also identified prrx1a and prrx1b as negative Edn1 and positive Bmp targets that function in parallel to Jagged-Notch signaling to restrict the formation of dorsal barx1+ pre-cartilage condensations. Simultaneous loss of jag1b and prrx1a/b better rescued lower facial defects of edn1 mutants than loss of either pathway alone, showing that combined overactivation of Jagged-Notch and Bmp/Prrx1 pathways contribute to the absence of cartilage differentiation in the edn1 mutant lower face. These findings support a model in which Notch-mediated restriction of cartilage differentiation, particularly in the second pharyngeal arch, helps to establish a distinct skeletal pattern in the upper face.

Analysis of sphingosine-1-phosphate signaling mutants reveals endodermal requirements for the growth but not dorsoventral patterning of jaw skeletal precursors.

Development of the head skeleton involves reciprocal interactions between cranial neural crest cells (CNCCs) and the surrounding pharyngeal endoderm and ectoderm. Whereas elegant experiments in avians have shown a prominent role for the endoderm in facial skeleton development, the relative functions of the endoderm in growth versus regional identity of skeletal precursors have remained unclear. Here we describe novel craniofacial defects in zebrafish harboring mutations in the Sphingosine-1-phospate (S1P) type 2 receptor (s1pr2) or the S1P transporter Spinster 2 (spns2), and we show that S1P signaling functions in the endoderm for the proper growth and positioning of the jaw skeleton. Surprisingly, analysis of s1pr2 and spns2 mutants, as well as sox32 mutants that completely lack endoderm, reveals that the dorsal-ventral (DV) patterning of jaw skeletal precursors is largely unaffected even in the absence of endoderm. Instead, we observe reductions in the ectodermal expression of Fibroblast growth factor 8a (Fgf8a), and transgenic misexpression of Shha restores fgf8a expression and partially rescues the growth and differentiation of jaw skeletal precursors. Hence, we propose that the S1P-dependent anterior foregut endoderm functions primarily through Shh to regulate the growth but not DV patterning of zebrafish jaw precursors.