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First published online 16 May 2007
doi: 10.1242/dev.005108

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Localized VEGF signaling from ectoderm to mesenchyme cells controls morphogenesis of the sea urchin embryo skeleton

Developmental Biology Unit, University Pierre et Marie Curie (Paris 6) and CNRS, Observatoire Océanologique, 06230 Villefranche-sur-Mer, France.

View larger version (74K): [in this window] [in a new window]   Fig. 1. The primary mesenchyme lineage of the sea urchin embryo and the expression pattern of VEGFR and VEGF during development. (A) Development of the primary mesenchyme lineage (red). The founder cells form at the 32-cell stage. At the mesenchyme blastula stage, the 32 descendants ingress into the blastocoel and become positioned along the ectoderm wall following a stereotypical pattern that includes two symmetrical ventrolateral clusters in which spiculogenesis begins by the formation of a tri-radiate rudiment. (B) Northern blot with total RNA isolated from embryos at the indicated stages. (C) Spatial distribution of VEGFR and VEGF transcripts detected by whole-mount in situ hybridization (WMISH). Embryos at the indicated stages were hybridized with sense (not shown) and antisense probes. Lateral views with the animal pole at the top, except for those marked (vv), which are ventral views with the oral side to the left. E, unfertilized egg; 16, 16-cell stage; 32, 32-cell stage; 60, 60-cell stage; EB, early blastula; B, blastula; SB, swimming blastula; MB, mesenchyme blastula; EG, early gastrula; G, gastrula; , prism; P, pluteus.

View larger version (108K): [in this window] [in a new window]   Fig. 2. Expression pattern of VEGFR and VEGF during perturbed development. (A) Perturbation along the AV axis. Sea urchin embryos were treated with 30 mM LiCl. Detection of VEGF and VEGFR transcripts by WMISH (as in Fig. 1) on gastrula-stage embryos and DIC images of 48-hour-old embryos. Lateral views with the animal pole at the top. (B) Perturbation along the OA axis. Embryos were treated with 0.5 mM NiCl2. WMISH of early gastrulae-stage embryos and DIC images of 48-hour-old embryos. Lateral views with the animal pole at the top, or ventral views with the oral side to the left when OA polarity is present (vv).

View larger version (93K): [in this window] [in a new window]   Fig. 3. Overexpression of VEGF perturbs skeletogenesis. Sea urchin embryos overexpressing VEGF following microinjection of VEGF mRNA into the egg. (A) WMISH with a Ske-T probe at the gastrula stage. Vegetal view. (B-D) DIC images of pluteus-stage embryos. Arrow, supernumerary elements; circle, abnormal branching. (E-J) WMISH with probes for (E,H) MSP130 at prism stage, (F,I) SM30 and (G,J) MSP130 at pluteus stage.

View larger version (84K): [in this window] [in a new window]   Fig. 4. VEGF or VEGFR loss-of-function blocks PMC spatial patterning and skeleton formation in sea urchin. Embryonic treatment (control or injected with Mo-VEGF or Mo-VEGFR) is indicated on the left of each row. In each column is shown morphology at 24 and 48 hours, expression of Ske-T at the gastrula stage and expression of MSP130 at the pluteus stage. vv, vegetal view.

View larger version (55K): [in this window] [in a new window]   Fig. 5. VEGF/VEGFR signaling controls expression of PMC-specific genes. SM30, SM50 and VEGFR transcripts detected by WMISH at the indicated stages in control sea urchin embryos and those injected with Mo-VEGF or Mo-VEGFR. MB, mesenchyme blastula; EG, early gastrula; LG, late gastrula; EP, early pluteus.

View larger version (61K): [in this window] [in a new window]   Fig. 6. Rescue of spicule formation by expression of VEGF mRNA in sea urchin embryos injected with Mo-VEGF. (A) Co-injection of Mo-VEGF and VEGF mRNA into unfertilized eggs. (B-D) Phenotypes of 72-hour-old embryos treated as in A. (E) Localized ectopic expression of VEGF obtained by microinjection of Mo-VEGF into the egg, and VEGF mRNA together with a fluorescent lineage marker into one blastomere at the 8-cell stage. (F-K) 72-hour-old embryos treated as in E were split into two batches for (F-H) DIC and fluorescence microscopy, with the DIC/fluorescence signal superimposed to show spicule formation, or (I-K) immunolocalization of lineage marker (light red) and WMISH with a SM30 probe (brown); dispersed spherical red `dots' are microinjection artifacts.

View larger version (82K): [in this window] [in a new window]   Fig. 7. VEGF induces spicule formation by PMCs. DIC and dark field images of 72-hour-old sea urchin embryos injected at the egg stage with pmar1 mRNA or co-injected with pmar1 and VEGF mRNAs.

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