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

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

<div><p>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 <i>barx1</i>, as well as <i>in vivo</i> 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 <i>barx1</i> expression in the dorsal arches, with mutation of <i>barx1</i> rescuing some aspects of dorsal skeletal patterning in <i>jag1b</i> mutants. We also identified <i>prrx1a</i> and <i>prrx1b</i> as negative Edn1 and positive Bmp targets that function in parallel to Jagged-Notch signaling to restrict the formation of dorsal <i>barx1</i>+ pre-cartilage condensations. Simultaneous loss of <i>jag1b</i> and <i>prrx1a/b</i> better rescued lower facial defects of <i>edn1</i> 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 <i>edn1</i> 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.</p></div

<i>prrx1a</i> and <i>prrx1b</i> are repressed by Edn1 and activated by Bmp4 signaling.

<p>(A-F) Two-color fluorescent <i>in situs</i> of 36 hpf wild-type embryos show that, relative to all arch NCCs (<i>dlx2a</i>, green), <i>prrx1a</i> and <i>prrx1b</i> (magenta) are expressed in dorsal arch NCCs and mesenchyme surrounding the ear (white arrow), as well as in a more limited ventral arch domain (white arrowhead). <i>prrx1a</i> and <i>prrx1b</i> are upregulated in ventral arch NCCs (white open arrowhead) of <i>edn1</i> mutants and nearly lost upon overexpression of Edn1 in <i>hsp70I</i>:<i>Gal4</i>; <i>UAS</i>:<i>Edn1</i> embryos subjected to a 20–24 hpf heat-shock treatment. (G, H) <i>prrx1a/b</i> and <i>barx1</i> are expressed complementarily in the arches of wild types. (I, J) Overexpression of Bmp4 in <i>hsp70I</i>:<i>Gal4</i>; <i>UAS</i>:<i>Bmp4</i> embryos heat-shocked from 20–24 hpf resulted in broad upregulation of <i>prrx1a/b</i> throughout the arches, with <i>barx1</i> restricted to domains showing lower <i>prrx1a/b</i> expression. (K) <i>prrx1b</i> overlaps only slightly with <i>jag1b</i> expression at the dorsal-posterior tips of the first and second arches (yellow arrows). (L) Schematic depicting the expression patterns of <i>prrx1a/b</i> (magenta), <i>barx1</i> (green), and <i>jag1b</i> (blue). Scale bar = 20 μm.</p

Regulation of <i>barx1</i>+ condensations by Edn1 and Notch.

<p>(A) At 36 hpf, the intermediate <i>sox9a</i> domain (green) only partially overlaps with zones of <i>barx1</i> expression (magenta) at the ventral and dorsal poles of each arch. The oral ectoderm (oe) and first pharyngeal pouch (p1) are shown for reference. (B, C) <i>jag1b</i> (green) and <i>barx1</i> (magenta) are anti-correlated in dorsal NCCs at 36 and 48 hpf. (D-J) <i>barx1</i> expression at 36 hpf in the first and second arches of wild-type controls, mutants, and overexpression embryos. Open arrowheads show the loss of ventral <i>barx1</i> in <i>edn1</i> mutants (E) and its restoration in 5/6 <i>jag1b</i>; <i>edn1</i> mutants (J). The blue arrow in E indicates weak upregulation of <i>barx1</i> in the intermediate domain of <i>edn1</i> mutants. Upregulation of <i>barx1</i> in the dorsal first arch (white arrowhead) and dorsal second arch (white arrow) is seen in Edn1-overexpressing embryos (F), <i>jag1b</i> mutants (G), <i>notch2</i>; <i>notch3</i> mutants (H), and <i>jag1b</i>; <i>edn1</i> mutants (J). Dotted lines in (I) show the arches of NICD-overexpression embryos in which <i>barx1</i> is nearly absent. (K, L) Ectopic <i>barx1</i> persists in <i>jag1b</i> mutants at least until 48 hpf, but no ectopic expression of <i>sox9a</i> is observed. (M, N) Representative <i>barx1</i> expression patterns and skeletal preparations in embryos treated with the Notch inhibitor DBZ (10 μM) starting at the indicated time points. Earlier exposure to the DBZ inhibitor correlated with stronger ectopic <i>barx1</i> expression (M) and more severe and penetrant Notch-type skeletal phenotypes (N). Fractions indicate the number of embryos in each treatment that exhibited unambiguous ectopic <i>barx1</i> expression in the dorsal first arch (arrowheads in M) or showed posterior Pq malformations (arrowheads in N. DBZ treatment also caused systemic effects, including spinal curvature and cardiac edema, which reduced bone mineralization and led to a general reduction in the size of the craniofacial skeleton. Scale bars in B, C, J, L, M = 20 μm; scale bar in N = 100 μm. </p

Combined loss of <i>prrx1a</i> and <i>prrx1b</i> results in ectopic dorsal cartilage.

<p>(A-F) <i>prrx1a</i>; <i>prrx1b</i> mutants develop ectopic cartilage, both from the dorsal-medial surface of Pq (black arrow) and connecting Pq to the otic cartilage (magenta arrowheads), as well as fusions of Hm to the otic cartilage (yellow arrows). The entopterygoid dermal bone (black arrowhead) that normally forms along the dorsal-medial surface of the Pq is also lost. (G, H) In <i>prrx1a</i>; <i>prrx1b</i> mutants at 36 hpf, <i>barx1</i> (magenta) is ectopically upregulated in the dorsal first arch (white arrowhead), along the dorsal border of the second arch (white bracket, compare with G), and in the posterior dorsal second arch (white arrow). (I, J) By 48 hpf in <i>prrx1a</i>; <i>prrx1b</i> mutants, ectopic <i>barx1</i> expression is no longer evident in the posterior first arch (open white arrowhead), and the second arch <i>barx1</i>+ domain is slightly larger than the sibling control (white arrow). <i>sox9a</i> expression (green) is largely normal at this stage in <i>prrx1a</i>; <i>prrx1b</i> mutants. Scale bars in B and F = 100 μm; scale bars in H and J = 20 μm.</p

Accelerated cartilage differentiation in ventral-intermediate arch NCCs.

<p>(A) Schematic of pharyngeal arch patterning in zebrafish. At early patterning stages (~28 hpf), the first two pharyngeal arches (pa1, pa2) are divided into distinct dorsal (blue) and ventral/intermediate (green stripe) domains, with the latter resolving into intermediate (light green) and ventral (dark green) domains by 36 hpf. Notch activity governs the dorsal domain, Edn1 the intermediate domain, and Bmp signaling the ventral domain. The anterior maxillary domain (grey) is not significantly influenced by any of these pathways. The facial cartilages of the larval skeleton (5 dpf) are color-coded based on their arch origins. Hm, hyomandibula; Pq, palatoquadrate; M, Meckel’s; Sy, symplectic; Ch, ceratohyal. (B) <i>barx1</i> (green) is upregulated ventrally (≤ 26 hpf, white open arrowhead) well before dorsal second arch expression can be detected (~32 hpf, white arrowhead). NCCs express the <i>sox10</i>:<i>GFP</i> transgene (blue). Shown are maximum intensity projections of confocal z-stacks of single-color <i>in situs</i> co-stained with a GFP antibody. The orientation of the dorsal (D)-ventral (V) axis is indicated. (C) Stills from a time-lapse movie (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005967#pgen.1005967.s016" target="_blank">S1 Movie</a>) show the emergence of facial cartilages (<i>sox10</i>:<i>DsRed</i>+, magenta) from <i>fli1a</i>:<i>EGFP</i>+ ectomesenchyme (green). <i>sox10</i>:<i>DsRed</i>+ chondrocytes appear in a stereotyped sequence within the facial cartilages, with cells of the intermediate Sy and Pq cartilages detectable first at 56 hpf, followed by the ventral M and Ch cartilages at 60 hpf and the dorsal Hm at 65 hpf. (D) The same sequence of cartilage differentiation is seen slightly earlier in stills from a time-lapse movie of <i>col2a1a</i>_<i>BAC</i>:GFP fish (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005967#pgen.1005967.s017" target="_blank">S2 Movie</a>). The time-lapses in B and C were performed with a 20x objective using 0.5x digital magnification. Et, ethmoid cartilage. (E) Color-coded schematic of the sequence of chondrocyte differentiation in the facial skeleton. The orientations of the D-V and anterior (A)-posterior (P) axes are indicated. Scale bar in B = 20 μm; scale bars in C, D = 100 μm.</p