Oestrogen shuts the door on SOX9

Oestrogen exerts a robust yet imperfectly understood effect on sexual development in vertebrate embryos. New work by Pask and colleagues in BMC Biology indicates that it may interfere with male development by preventing nuclear localization of SOX9, a master regulator of the testis differentiation pathway

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

Jagged-Notch signaling represses genes strongly induced during pharyngeal arch differentiation.

<p>(A) 36 hpf <i>fli1a</i>:<i>EGFP</i>; <i>sox10</i>:<i>DsRed</i> embryo showing colocalization of GFP and DsRed in the arches (yellow), with DsRed-only cells (red) in the ear and GFP-only cells (green) in the vasculature (and macrophages). Scale bar = 20 μm. (B) Double-positive embryos were dissociated and subjected to FACS to isolate GFP/DsRed+ arch cells (yellow) for RNA sequencing. Wild-type embryos were profiled at three stages, and Notch and Edn1 loss- and gain-of-function (LOF, GOF) models at 36 hpf only. (C-D) Scatterplots depicting changes in expression (RPKM) among the genes on each list from 20 to 28 hpf (C) and 28 to 36 hpf (D). Genes to the left of the diagonal were upregulated, while genes to the right were downregulated. For the ‘total’ arch NCC-enriched gene charts, black points indicate genes with fold-changes ≥ 1.5, and grey points indicate genes with fold changes ≤ 1.5. In the other charts, black points correspond to genes uniquely present on a given list, whereas purple points indicate genes oppositely regulated by Notch and Edn1 (dark purple: Notch-activated/Edn1-inhibited; light purple: Notch-inhibited/Edn1-activated). The grey blocks in D reflect the filtering threshold (RPKM ≥ 3 at 36 hpf in wild types) used to generate the total list of arch NCC-enriched genes. (E) Venn diagrams display the numbers of genes on each list, with genes shared between the Notch-activated/Edn1-inhibited or Notch-inhibited/Edn1-activated lists indicated in dark and light purple, respectively. (F) Summary of gene expression changes (RPKM) in wild types between 20 and 28 hpf (left chart) and 28 and 36 hpf (right chart), for genes classified as activated (red) or inhibited (blue) by Notch or Edn1 signaling. The data are shown as the median fold-change value for all genes on a given list (black central line), with flanking second and third quartiles as the lower and upper boxes, respectively, and the 5^th and 95^th percentiles indicated by the whiskers. For the lists in which the 95^th percentile value exceeded the upper boundary of the chart, the value is indicated at the top of the upper whisker. Compared with the total list of arch NCC-enriched genes, genes inhibited by Notch signaling and those that were both inhibited by Notch and activated by Edn1 (light purple) showed a significantly larger increase in expression from 20 to 28 hpf (p < 0.001). Genes inhibited by Edn1 also increased slightly but significantly from 20–28 and 28–36 hpf relative to the total list. The gray dashed line indicates a fold-change value of 1 (no change in expression) between the two time points.</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

Partially overlapping functions of Prrx1a/b and Jagged-Notch signaling in dorsal cartilage development.

<p>(A-F) The expression of <i>prrx1a</i> and <i>prrx1b</i> (magenta) is largely normal in <i>jag1b</i> mutants (B, D) but is upregulated in ventral arch NCCs (white arrows) and reduced in dorsal NCCs upon forced Notch activation in <i>hsp70I</i>:<i>Gal4</i>; <i>UAS</i>:<i>NICD</i> embryos subjected to a 20–24 hpf heat-shock treatment. <i>dlx2a</i> expression (green) marks all arch NCCs. (G-I) <i>edn1</i> mutants display a loss of ventral <i>barx1</i> expression (green) and gain of <i>prrx1b</i> (magenta) (white arrowhead) (H). In <i>jag1b</i>; <i>edn1</i> mutants, there is partial recovery of ventral <i>barx1</i> expression in the second arch (white open arrowhead), which corresponds to regions where the ectopic expression of <i>prrx1b</i> is restored to control levels. (J, K) In 7/10 <i>prrx1a</i>; <i>prrx1b</i> mutants, <i>jag1b</i> expression is partially reduced in the dorsal second arch (yellow arrow). (L-O) Dissections of facial cartilage and bone derived from the first two arches show additive phenotypes in <i>jag1b</i>; <i>prrx1a</i>; <i>prrx1b</i> triple mutants. Similar to <i>prrx1a</i>; <i>prrx1b</i> double mutants, triple mutants display ectopic cartilage connecting Pq to the otic cartilage (black arrows). However, similar to <i>jag1b</i> single mutants, <i>jag1b</i>; <i>prrx1a</i>; <i>prrx1b</i> triple mutants also display irregularities in the main body of Pq (black arrowheads). Scale bar in K = 20 μm; scale bar in O = 100 μm.</p

Reduced growth of Hm cartilage in <i>jag1b</i> mutants.

<p>(A-F) <i>kikGR</i> RNA was injected into control <i>fli1a</i>:<i>EGFP</i> or <i>jag1b</i>; <i>fli1a</i>:<i>EGFP</i> embryos, and kikGR protein was photoconverted in small groups of GFP+ arch NCCs using a UV laser at 36 hpf. The same larvae were then reimaged at 6 dpf to assess contributions of kikGR-converted cells (magenta) to cartilage. In both controls (A-C) and <i>jag1b</i> mutants (D-F), photoconverted cells from the anterior, center, and posterior regions of the dorsal second arch contributed to the anterior Hm (A,D), posterior Hm (B,E) and posterior edge of the Hm and opercle bone (C,F). Relative to controls, labeled NCCs from <i>jag1b</i> mutants contributed to qualitatively smaller domains of cartilage by 6 dpf. Reproducible differences were seen in each mutant or control examined, with <i>n</i> numbers listed in each panel. (G) Summary of these fate maps showing contribution to anterior Hm (red), posterior Hm (dark blue), and opercle bone (light blue). Scale bars = 20 μm.</p