Douglas WB and Ettensohn CA (2026), BMP signaling regulates dorsal skeletal growth ...

ECB-ART-54958

Development 2026 Apr 15;15316:. doi: 10.1242/dev.205344.

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BMP signaling regulates dorsal skeletal growth in the sea urchin embryo.

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The development of the elaborate, calcified endoskeleton of sea urchin embryos is a model for understanding the dynamic nature of developmental gene regulatory networks and the control of biomineralization. While several signaling pathways have been shown to regulate gene expression and biomineral formation by sea urchin skeletogenic cells, important gaps in our understanding remain. Here, we focused on signals that regulate skeletogenesis along the dorsal-ventral axis of the late-stage embryo. We used a specific inhibitor of Type I BMP receptors, K02288, to show that BMP signaling regulates skeletal growth selectively in the dorsal region. K02288 treatment led to dorsal skeletal defects and inhibited the expression of genes typically expressed specifically in the dorsal skeletogenic cells, including biomineralization genes. Using RNA sequencing, we identified genes that were uniquely downstream of either the BMP or a ventral signaling pathway (the VEGF pathway) at late developmental stages and genes downstream of both pathways. Our findings establish BMP signaling as a key pathway regulating dorsal skeleton formation and show that BMP signaling functions in concert with VEGF signaling to define the dorsal-ventral axis of the skeleton.

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	Fig. 1. Smad1/5/8 activation is progressively restricted to dorsal PMCs. (A-G′) S. purpuratus embryos were fixed and immunostained with anti-pSmad1/5/8 at the indicated times, from mesenchyme blastula to late pluteus stage. Embryos are oriented with dorsal sides on the left and ventral sides on the right. Enriched pSmad1/5/8 signal in dorsal PMCs is marked (white arrowheads). Diminishing pSmad1/5/8 in more ventral PMCs of the syncytium is also marked (F, blue arrowheads). A representative 5 dpf pluteus is shown at multiple focal planes to highlight the persistence of pSmad1/5/8 enrichment in the dorsal PMCs (G). pSmad1/5/8 is also strongly enriched in the coelomic pouches of pluteus stage embryos (white arrows) (G′). D, dorsal; V, ventral.
	Fig. 2. K02288 treatment phenocopies BMP pathway knockdowns and inhibits Smad1/5/8 phosphorylation. (A-C′) Morphology of sibling L. variegatus embryos treated continuously from fertilization with DMSO (control) or 0.5 μM K02288. Morphologies were quantified at 24 hpf. Embryos were more sensitive to the drug at earlier stages and so a lower concentration of K02288 was used when treating immediately after fertilization. Images sharing the same letter show the same embryo under different optical conditions (DIC versus polarized light). (D,E) Effects of K02288 on the enrichment of pSmad1/5/8 in the DS (white arrowhead) was analyzed and pooled from three biological replicates (separate mating pairs). D and E are oriented with dorsal sides on the left and ventral sides on the right. Numbers at the bottom of images indicate the number of embryos exhibiting the morphology shown. D, dorsal; V, ventral.
	Fig. 3. Inhibition of the BMP signaling pathway during Phase 2 of skeletogenesis selectively affects body rod and recurrent rod development. (A) Timeline of treatment and embryo analysis. (B-C′) Representative images of DMSO-treated (34 embryos measured) and K02288-treated (32 embryos measured) embryos, showing the five skeletal elements that were measured: anterolateral rods (AL), recurrent rods (RR), post-oral rods (PO), ventral-transverse rods (VT) and body rods (BR). (D,D′) Representative K02288-treated embryo with a shortened body rod and a hooked scheitel (22/32) (white arrowheads). (E,E′) Representative K02288-treated embryo with perturbed recurrent rods that failed to bend and lack distal segments (27/32) (black arrowhead). (F) Average lengths for each skeletal element were compared between DMSO-treated and K02288-treated embryos by Student's t-test. (G) Proportional average length of each element in K02288-treated embryos compared to DMSO-treated embryos. Images sharing the same letter show the same embryo at different focal planes or under different optical conditions (DIC versus polarized light). Numbers at the bottom of images indicate the number of embryos exhibiting the morphology shown or the total number of embryos examined. n.s., not significant.
	Fig. 4. Components of the BMP signaling pathway are expressed in the DS and the distal RR-PMCs. Fixed L. variegatus embryos from various stages were stained by HCR in situ hybridization. White arrowheads mark the DS when expression is present. Black arrowheads mark RR-PMCs when expression is present. The embryos show strong autofluorescence in the gut at later stages, which is exacerbated by the body being compressed on the slide. Some panels show the same embryo stained for multiple mRNAs and there are three such groups of images: C,G; L,L′,P,P′; M,M′,Q,Q′. Images of late gastrula embryos show lateral views. Prism and later stage embryos are oriented with the blastopore against the slide/coverslip such that the anterior-posterior axis is perpendicular to the image. Prism and later stage images show high magnification views of the dorsal scheitel (C-E,G,H,I,K,L,M,O,P,Q) or the recurrent rod PMCs (E′,H′,I′,L′,M′,P′,Q′). At least 30 embryos of each stage for each gene of interest were observed, and representative images showing expression patterns consistent with >90% of observed embryos are shown.
	Fig. 5. The expression of mRNAs enriched in the DS is dependent upon BMP signaling. (A-O′) L. variegatus embryos were treated with DMSO, 1 µM K02288 or 75 nM axitinib continuously starting after the formation of the tri-radiate spicule rudiments. Embryos were fixed at the late pluteus stage (2 dpf) and the expression of various genes of interest was analyzed by HCR in situ hybridization (magenta). sm37 and vegfr-Ig10 were used as counterstains to label the entire PMC syncytium (cyan). The number of DMSO-treated embryos with gene expression in the DS (marked with white arrowheads) is shown (A′,D′,G′,J′,M′). Representative images of K02288-treated embryos completely lacking detectable gene expression in the DS and the fraction of these embryos are shown (B′,E′,H′,K′). Representative images of axitinib-treated embryos with DS expression and the fraction of these embryos are shown (C′,F′,I′,L′,O′). In all cases (40/40), enriched expression of sm29 in the DS is qualitatively reduced in K02288-treated embryos compared to control embryos and distal RR-PMC expression of sm29 appears visually the same as other PMCs within the syncytium of K02288-treated embryos (N′). C′ shows the number of embryos with both DS expression and ectopic expression in ventral PMCs of the syncytium (yellow arrowheads). Images sharing the same letter(s) show the same embryo stained for different mRNAs.
	Fig. 6. The expression of mRNAs enriched in the RR-PMCs of the syncytium is dependent upon BMP signaling. (A-O′) L. variegatus embryos were treated, collected and stained as previously described in Fig. 5. The number of DMSO-treated embryos with gene expression in the RR-PMCs (marked with outline arrowheads) is shown (A′,D′,G′,J′,M′). Representative images of K02288-treated embryos completely lacking detectable gene expression in the RR-PMCs and the fraction of these embryos are shown (B′,E′,H′,K′). Representative images of axitinib-treated embryos with RR-PMC expression and the fraction of these embryos are shown (C′,F′,I′,L′,O′). In all cases (40/40), enriched expression of sm29 in the distal most RR-PMCs is qualitatively reduced in K02288-treated embryos compared to control embryos and distal RR-PMC expression of sm29 appears visually the same as other PMCs within the syncytium of K02288-treated embryos (N′). Distal anterolateral PMC positions showing lack of wild-type sm29 expression are marked (red arrowheads) (M,M′). Images sharing the same letter(s) show the same embryo stained for different mRNAs. Additionally, several images show the same embryos as corresponding images in Fig. 5 at different focal planes or of the same focal plane to draw attention to the recurrent rods instead. These groups of images are as follows: B,B′ and Fig. 5B,B′ ; E,E′ and Fig. 5E,E′; F,F′ and Fig. 5F,F′; I,I′ and Fig. 5I,I′; and N,N′ and Fig. 5N,N′.
	Fig. 7. Venn diagram showing the proportion of curated PMC-enriched mRNAs with expression sensitive to K02288, axitinib or both drugs. Approximately 20% (74/367) of the curated list of PMC-enriched mRNAs was affected by the drug treatments. The list of enriched mRNAs is based on 321 mRNAs identified as PMC enriched at late stages of development in previous single-cell RNA-seq work (Massri et al., 2021) and 46 manually curated genes based on known expression patterns (Solek et al., 2013; Valencia, 2018).
	Fig. 8. Provisional model of signaling pathway contributions to dorsal/ventral skeletal growth. BMP ligands (orange) are expressed by the RR-PMCs and dorsal (scheitel) PMCs. Autocrine BMP signaling acts to maintain expression of BMP ligands in these cells (1). Scheitel PMCs export BMP ligands to the overlying dorsal ectoderm, activating SMAD1/5/8 in this tissue (2). BMP signaling may weakly contribute to ventral skeletal growth via a gradient of BMP ligands originating from the dorsal skeletal elements that signals to alk1/2-expressing cells at the growing tips of the arms (3). VEGF3 ligand is exported from the ectoderm overlying the arm tips and VEGF signaling (magenta) is essential for elongation of the ventral skeletal elements but not the dorsal elements (4). VEGF signaling maintains vegfr-Ig10 expression in the PMCs at the tips of the post-oral and anterolateral rods (5).