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Figure 1. Observation of coeloms and skeletogenic mesenchyme cells in the starfish Patiria pectinifera. Bipinnaria larvae at 48 h postfertilization (hpf) possess two types of coeloms: a posterior enterocoel (PE, arrowhead in a) and two bilateral coelomic pouches (arrows in a). After the onset of feeding, the bilateral coelomic pouches extend posteriorly along the lateral walls of stomach, and the left coelomic pouch and PE fuse (b; the left coeloms are shown in blue encircled by dotted lines in b’). At 5 days postfertilization (dpf), the bilateral coeloms fuse around the pharynx (arrowhead in c), and a mesenchyme cell population appears in the dorsal posterior region (dotted circle in c; d, enlarged image). By 7 dpf, adult skeletal rudiments first emerge on the dorsal left side (e; arrowheads in f). Scale bars: 50 μm.
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Figure 2. Expression of skeletogenesis-related genes in starfish larvae. Expression patterns of vegf (a1–a3), vegfr (b1–b3), ets1/2 (c1–c3), erg (d1–d3), alx1 (e1–e3), ca1 (f1–f3), and clect (g1–g3) were examined at 3 dpf (a1–g1, b1’–d1’), 5 dpf (a2–g2, f2’, g2’), and 7 dpf (a3–g3) by whole-mount in situ hybridization (WMISH). The numbers shown in the lower right corner of each image indicate the number of larvae showing a positive WMISH signal among the larvae examined. Arrowheads indicate the regions or cells expressing each gene. Enlarged images are shown in (b1’–d1’, f2’, g2’). See details in the text. Scale bars: 50 μm.
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Figure 3. Observations of skeleton formation in the experimental starfish larvae. Morphology and skeleton formation were observed in control larvae (a1–a3), nonfeeding larvae (b1–b5), rapamycin (rapa)-treated larvae (c1–c5), and axitinib (axi)-treated larvae (d1–d5) at 7 dpf. (a1–d1, a2–d2) Living larvae. In the panel a2, the arrowhead indicates an adult skeletal rudiment and the dotted circle indicates aggregated mesenchyme cells. (a3–d3) Fluorescence images of larvae examined by immunohistochemistry using the mesenchyme-specific marker MC5. The green signal shows MC5 expression, whereas the red signal shows Chaetoceros calcitrans in the stomach or autofluorescence. At 7 pdf, the ratio of larvae with adult skeletons (b4–d4) and that of larvae with aggregated mesenchyme cells in the posterior dorsal region (b5–d5) were evaluated. Feeding larvae and DMSO-treated larvae were used as controls for nonfeeding larvae and inhibitor-treated larvae, respectively. Scale bars: 50 μm. M: mol/L.
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Figure 4. Expression of skeletogenesis-related genes in the experimental starfish larvae. The expression of vegf (a1–d1), vegfr (a2–d2), ets1/2 (a3–d3), erg (a4–d4), alx1 (a5–d5), ca1 (a6–d6), and clect (a7–d7) was examined by WMISH in the control (feeding and DMSO-treated larvae) and inhibitor-treated larvae. The numbers in the lower corner of each image indicate the number of larvae showing a positive WMISH signal/the total number of larvae examined. In the control larvae, the number in the lower left corner is for feeding larvae, and that in the lower right corner is for DMSO-treated larvae. Scale bar: 50 μm. M: mol/L.
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Figure 5. Morphology and gene expression in TALEN-mediated vegfr knockout starfish larvae. Adult skeleton formation and the expression of alx1, ca1, and clect were examined in control (a–f) and vegfr knockout larvae (g–l). In the panel b, arrowheads indicate adult skeletal rudiments. In panels b and h, the number in the lower right corner shows the number of larvae with skeleton rudiments/the total number of larvae examined. In the WMISH images (c–f, i–l), the number in the lower right corner shows the number of larvae with a positive WMISH signal/the total number of larvae examined. Scale bars: 50 μm.
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Figure 6. Expression of phbA and phbB in the starfish. The expression of phbA and phbB was examined by WMISH (a–h) and quantitative PCR (qPCR; i and j). (a–h) The number in the lower right corner shows the number of embryos or larvae showing a positive WMISH signal/the total number examined. Asterisks indicate nonspecific signals for phb genes (see “Methods”). (i, j) The X-axis shows the time after fertilization, and the Y-axis shows the mRNA amounts of phbA (i) and phbB (j) relative to that of EF1α. Data are shown as the mean ± standard deviation. Scale bars: 50 μm.
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Figure 7. Treatment with rapamycin or axitinib in embryos of the sea urchin Hemicentrotus pulcherrimus. Morphology, the formation of larval skeletons, and gene expression were observed in the control (a1–a3, e1–e6), rapamycin (rapa)-treated (b1–b3, f1–f6) and axitinib (axi)-treated larvae (c1–c3, g1–g6). (a–c) Living embryo or larva at the early gastrula (eGs; 25 hpf) and pluteus (50 hpf) stages. For the observation of larval skeletons, the larvae were pressed (a3–c3). In panels of a3 and b3, arrowheads indicate larval skeletons. (d) The ratio of larvae with skeletons was evaluated. The total numbers of larvae examined are shown at the bottom. The expression of alx1, ets1, vegf and vegfr was examined in the control embryos treated with DMSO (e1–e6), rapamycin-treated embryos (f1–f6) and axitinib-treated embryos (g1–g6) at the hatched blastula (hBl; 16 hpf) and gastrula (Gs; 36 hpf) stages. The numbers in the lower right corner show the number of embryos showing a positive WMISH signal/the total number of embryos examined. All WMISH images except for that in panel f1 show typical expression patterns, whereas panel f1 shows an atypical expanded expression pattern (5 of 15 embryos). Scale bars: 50 μm. M: mol/L.
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Figure 8. Illustration of skeletogenesis-related regions in starfish larvae (a) and comparison of gene regulatory network (GRN) models (b). (a) Our data suggest that adult skeletogenic cells are derived from cells included in the posterior coelom, and Vegf ligand emitted from the posterior ectoderm binds and activates the receptor Vegfr in dorsal mesenchyme cell lineage, which leads to the formation of adult skeletal rudiments on the dorsal left side of starfish larvae. (b) GRN models for adult skeletogenesis in starfish (left) or larval skeletogenesis in cidaroids (center) and euechinoids (right). The regulatory relationships shown by dotted lines were indicated by gene expression analyses. The cidaroid and euechinoid models are based on previous studies27,29,45,46.
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