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

Wnt-Dependent Epithelial Transitions Drive Pharyngeal Pouch Formation

The pharyngeal pouches, which form by budding of the foregut endoderm, are essential for segmentation of the vertebrate face. To date, the cellular mechanism and segmental nature of such budding have remained elusive. Here, we find that Wnt11r and Wnt4a from the head mesoderm and ectoderm, respectively, play distinct roles in the segmental formation of pouches in zebrafish. Time-lapse microscopy, combined with mutant and tissue-specific transgenic experiments, reveal requirements of Wnt signaling in two phases of endodermal epithelial transitions. Initially, Wnt11r and Rac1 destabilize the endodermal epithelium to promote the lateral movement of pouch-forming cells. Next, Wnt4a and Cdc42 signaling induce the rearrangement of maturing pouch cells into bilayers through junctional localization of the Alcama immunoglobulin-domain protein, which functions to restabilize adherens junctions. We propose that this dynamic control of epithelial morphology by Wnt signaling may be a common theme for the budding of organ anlagen from the endoderm

Identification of the skeletal progenitor cells forming osteophytes in osteoarthritis.

OBJECTIVES: Osteophytes are highly prevalent in osteoarthritis (OA) and are associated with pain and functional disability. These pathological outgrowths of cartilage and bone typically form at the junction of articular cartilage, periosteum and synovium. The aim of this study was to identify the cells forming osteophytes in OA. METHODS: Fluorescent genetic cell-labelling and tracing mouse models were induced with tamoxifen to switch on reporter expression, as appropriate, followed by surgery to induce destabilisation of the medial meniscus. Contributions of fluorescently labelled cells to osteophytes after 2 or 8 weeks, and their molecular identity, were analysed by histology, immunofluorescence staining and RNA in situ hybridisation. Pdgfrα-H2BGFP mice and Pdgfrα-CreER mice crossed with multicolour Confetti reporter mice were used for identification and clonal tracing of mesenchymal progenitors. Mice carrying Col2-CreER, Nes-CreER, LepR-Cre, Grem1-CreER, Gdf5-Cre, Sox9-CreER or Prg4-CreER were crossed with tdTomato reporter mice to lineage-trace chondrocytes and stem/progenitor cell subpopulations. RESULTS: Articular chondrocytes, or skeletal stem cells identified by Nes, LepR or Grem1 expression, did not give rise to osteophytes. Instead, osteophytes derived from Pdgfrα-expressing stem/progenitor cells in periosteum and synovium that are descendants from the Gdf5-expressing embryonic joint interzone. Further, we show that Sox9-expressing progenitors in periosteum supplied hybrid skeletal cells to the early osteophyte, while Prg4-expressing progenitors from synovial lining contributed to cartilage capping the osteophyte, but not to bone. CONCLUSION: Our findings reveal distinct periosteal and synovial skeletal progenitors that cooperate to form osteophytes in OA. These cell populations could be targeted in disease modification for treatment of OA

Histone H3.3 beyond cancer: Germline mutations in Histone 3 Family 3A and 3B cause a previously unidentified neurodegenerative disorder in 46 patients

Although somatic mutations in Histone 3.3 (H3.3) are well-studied drivers of oncogenesis, the role of germline mutations remains unreported. We analyze 46 patients bearing de novo germline mutations in histone 3 family 3A (H3F3A) or H3F3B with progressive neurologic dysfunction and congenital anomalies without malignancies. Molecular modeling of all 37 variants demonstrated clear disruptions in interactions with DNA, other histones, and histone chaperone proteins. Patient histone posttranslational modifications (PTMs) analysis revealed notably aberrant local PTM patterns distinct from the somatic lysine mutations that cause global PTM dysregulation. RNA sequencing on patient cells demonstrated up-regulated gene expression related to mitosis and cell division, and cellular assays confirmed an increased proliferative capacity. A zebrafish model showed craniofacial anomalies and a defect in Foxd3-derived glia. These data suggest that the mechanism of germline mutations are distinct from cancer-associated somatic histone mutations but may converge on control of cell proliferation

Bmps and Id2a Act Upstream of Twist1 To Restrict Ectomesenchyme Potential of the Cranial Neural Crest

<div><p>Cranial neural crest cells (CNCCs) have the remarkable capacity to generate both the non-ectomesenchyme derivatives of the peripheral nervous system and the ectomesenchyme precursors of the vertebrate head skeleton, yet how these divergent lineages are specified is not well understood. Whereas studies in mouse have indicated that the Twist1 transcription factor is important for ectomesenchyme development, its role and regulation during CNCC lineage decisions have remained unclear. Here we show that two Twist1 genes play an essential role in promoting ectomesenchyme at the expense of non-ectomesenchyme gene expression in zebrafish. Twist1 does so by promoting Fgf signaling, as well as potentially directly activating <em>fli1a</em> expression through a conserved ectomesenchyme-specific enhancer. We also show that Id2a restricts Twist1 activity to the ectomesenchyme lineage, with Bmp activity preferentially inducing <em>id2a</em> expression in non-ectomesenchyme precursors. We therefore propose that the ventral migration of CNCCs away from a source of Bmps in the dorsal ectoderm promotes ectomesenchyme development by relieving Id2a-dependent repression of Twist1 function. Together our model shows how the integration of Bmp inhibition at its origin and Fgf activation along its migratory route would confer temporal and spatial specificity to the generation of ectomesenchyme from the neural crest.</p> </div

Fgf signaling depends on Twist1 and regulates a subset of ectomesenchyme gene expression.

<p>(A,B) In situs at 18 hpf show that expression of the Fgf target gene <i>pea3</i> is reduced in the arches (numbered) of <i>twist1a/1b</i>-MO embryos (n = 8/8) compared to un-injected controls (n = 0/12). (C–L) <i>sox10</i>:Gal4VP16; <i>UAS</i>:dnFgfr1a embryos show ectopic arch expression of <i>sox10</i> at 18 hpf (n = 8/8) versus controls (n = 0/8), reduction of <i>dlx2a</i> at 24 hpf (n = 3/3) versus controls (n = 0/3), but no change in <i>fli1a</i> at 24 hpf (n = 0/7) versus controls (n = 0/5). <i>twist1a</i> expression was unchanged at 24 hpf in dnFgfr1a embryos (n = 10) compared to controls (n = 7), as was <i>twist1b</i> expression in dnFgfr1a embryos (n = 8) compared to controls (n = 8). Black arrows indicate the second arch, white arrowheads the ear, and red arrowheads the vasculature. Scale bar = 50 µm.</p

Misexpression of Bmp4 in migrating CNCCs inhibits ectomesenchyme formation.

<p>(A–D) Whole mount in situs at 18 hpf show ectopic expression of <i>sox10</i> in the arches (numbered) of <i>sox10</i>:Gal4VP16; <i>UAS</i>:Bmp4; <i>UAS</i>:mKR embryos (n = 8/8) compared to <i>sox10</i>:Gal4VP16; <i>UAS</i>:mKR controls (n = 0/8) and reductions of <i>dlx2a</i> in <i>sox10</i>:Gal4VP16; <i>UAS</i>:Bmp4; <i>UAS</i>:mKR embryos (n = 4/4) compared to <i>sox10</i>:Gal4VP16; <i>UAS</i>:mKR controls (n = 0/4). Arrows indicate the second arch and white arrowheads the developing ear. (E–H) Double fluorescent in situs for <i>mKR</i> (red) and <i>fli1a</i> or <i>dlx2a</i> (green) at 24 hpf show reduction of <i>fli1a</i> arch expression in <i>sox10</i>:Gal4VP16; <i>UAS</i>:Bmp4; <i>UAS</i>:mKR embryos (n = 5/5) compared to <i>sox10</i>:Gal4VP16; <i>UAS</i>:mKR controls (n = 0/9) and reduction of <i>dlx2a</i> arch expression in <i>sox10</i>:Gal4VP16; <i>UAS</i>:Bmp4; <i>UAS</i>:mKR embryos (n = 4/4) compared to <i>sox10</i>:Gal4VP16; <i>UAS</i>:mKR controls (n = 0/3). The mKR transgene was included as a lineage tracer for CNCC-derived cells that migrated into the arches. (I,J) Skeletal staining at 5 dpf shows severe loss of craniofacial skeleton in <i>sox10</i>:Gal4VP16; <i>UAS</i>:Bmp4; <i>UAS</i>:mKR embryos (n = 7/7) compared to <i>sox10</i>:Gal4VP16; <i>UAS</i>:mKR controls (n = 0/9). (K,L) In situs for <i>foxd3</i> at 48 hpf reveal largely normal patterns of glia in <i>sox10</i>:Gal4VP16; <i>UAS</i>:Bmp4; <i>UAS</i>:mKR embryos (n = 8) and <i>sox10</i>:Gal4VP16; <i>UAS</i>:mKR controls (n = 11). (M–P) In situs at 28 hpf show normal <i>dct</i>-positive melanophore precursors in <i>sox10</i>:Gal4VP16; <i>UAS</i>:Bmp4; <i>UAS</i>:mKR embryos (n = 12) and <i>sox10</i>:Gal4VP16; <i>UAS</i>:mKR controls (n = 15) and normal <i>xdh</i>-positive xanthophore precursors in <i>sox10</i>:Gal4VP16; <i>UAS</i>:Bmp4; <i>UAS</i>:mKR embryos (n = 10) and <i>sox10</i>:Gal4VP16; <i>UAS</i>:mKR controls (n = 10). Scale bars = 50 µm.</p

Twist1 genes are required for ectomesenchyme specification in zebrafish.

<p>(A–D) Whole mount in situ hybridizations of <i>sox10</i> expression at 18 hpf show ectopic expression in the arches (numbered and second arch indicated by black arrow) of <i>twist1a/1b</i>-MO (n = 16/16) and <i>sox10</i>:Gal4VP16; <i>UAS</i>:dnTwist1b (n = 4/4) embryos compared to un-injected (n = 0/14) and <i>sox10</i>:Gal4VP16 only (n = 0/9) controls. White arrowheads indicate otic expression. (E–H) Whole mount in situs at 24 hpf show reduction of <i>fli1a</i> expression in the arch ectomesenchyme (numbered) of <i>twist1a/1b</i>-MO (n = 12/12) and <i>sox10</i>:Gal4VP16; <i>UAS</i>:dnTwist1b (n = 5/5) embryos compared to un-injected (n = 0/13) and <i>sox10</i>:Gal4VP16 only (n = 0/8) controls. Insets in E and F highlight arch ectomesenchyme which is reduced in <i>twist1a/1b</i>-MO embryos. Vascular expression of <i>fli1a</i> (red arrowheads) is unaffected. (I–L) Whole mount in situs at 18 hpf show a slight reduction of <i>dlx2a</i> in <i>sox10</i>:Gal4VP16; <i>UAS</i>:dnTwist1b embryos (n = 4/4) but not un-injected (n = 0/6), <i>twist1a/1b</i>-MO (n = 0/8), and <i>sox10</i>:Gal4VP16 only (n = 0/6) embryos. (M-P) Skeletal staining at 5 dpf shows severe loss of CNCC-derived head skeleton in <i>twist1a/1b</i>-MO embryos (n = 21/21) and primarily jaw reductions in <i>sox10</i>:Gal4VP16; <i>UAS</i>:dnTwist1b embryos (n = 9/9) compared to no defects in un-injected (n = 0/24) and <i>sox10</i>:Gal4VP16 only (n = 0/16) controls. Whereas only small remnants remain of the CNCC-derived skeleton (arrows), the mesoderm-derived otic capsule cartilage (arrowheads) and posterior neurocranium are less affected in <i>twist1a/1b</i>-MO embryos. (Q–T) In situs for <i>dct</i> expression at 28 hpf show normal melanophore precursors in un-injected (n = 14), <i>twist1a/1b</i>-MO (n = 12), <i>sox10</i>:Gal4VP16 only (n = 8), and <i>sox10</i>:Gal4VP16; <i>UAS</i>:dnTwist1b (n = 8) embryos. (U–X) In situs for <i>xdh</i> expression at 28 hpf show normal xanthophore precursors in un-injected (n = 7), <i>twist1a/1b</i>-MO (n = 5), <i>sox10</i>:Gal4VP16 only (n = 9), and <i>sox10</i>:Gal4VP16; <i>UAS</i>:dnTwist1b (n = 8) embryos. (Y-AB) In situs for <i>foxd3</i> expression at 48 hpf reveal largely normal patterns of glia in un-injected (n = 9), <i>twist1a/1b</i>-MO (n = 8), <i>sox10</i>:Gal4VP16 only (n = 9), and <i>sox10</i>:Gal4VP16; <i>UAS</i>:dnTwist1b (n = 8) embryos. Scale bar = 50 µm.</p