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First published online 7 February 2007
doi: 10.1242/dev.02805

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The Snail repressor is required for PMC ingression in the sea urchin embryo

DCMB group, Biology Department, Duke University, Durham, NC 27708, USA.

View larger version (73K): [in this window] [in a new window]   Fig. 1. Sequence comparisons of Lytechinus Snail and related Snail family proteins. (A) The zinc finger region of Lytechinus Snail compared with related proteins in other organisms. Positions of zinc-finger II-V are shown and the conserved cysteines and histidines are indicated with asterisks. (B) The SNAG domain of Lytechinus Snail fits the consensus and is identical to those of several other snail family members, including Acropora Snail and mouse Slug and Scratch. (C) Rooted neighbor-joining tree showing the relationship of Lytechinus Snail with other snail family proteins (1000 bootstraps, values indicated on nodes). Mouse and Drosophila Scratch served as outgroups.

View larger version (63K): [in this window] [in a new window]   Fig. 2. WMISH showing the dynamic pattern of Lvsnail mRNA expression during sea urchin development. (A) No staining is detected at the 16-cell stage. (B) At late-hatched blastula, Lvsnail staining appears in the vegetal region. (C) In a mesenchyme blastula, undergoing ingression, Lvsnail mRNA is detected in the ingressing PMCs (arrow). (D) In early gastrula, Lvsnail mRNA expression disappears from PMCs (arrow), and is expressed instead in SMCs. (E) Late gastrula, Lvsnail mRNA continues to be expressed in SMCs. (F,G) Lateral and vegetal (vv) views, respectively, of Prism stage embryos, showing Lvsnail mRNA in ventrolateral PMC clusters (arrow in F). (H) Early pluteus. Lvsnail mRNA is detected in the PMCs at the tips of the larval arms (arrow).

View larger version (48K): [in this window] [in a new window]   Fig. 3. PMC ingression is blocked by SnaMASO injection. (A-C) Control embryos show normal PMC ingression (arrow), and normal gastrulation. (D-G) Embryos of the same age as A-C injected with SnaMASO. Compared with the control, SnaMASO-injected embryos (Sna morphants) show no PMC ingression (D), even at gastrula stages (E-G). (G) Invagination is also delayed, though occurs normally later. MB, mesenchyme blastula; EG, early gastrula; LG, late gastrula.

View larger version (73K): [in this window] [in a new window]   Fig. 4. Chimeric embryos demonstrate Snail is required in micromeres for ingression. (A) Control embryo. (B) SnaMASO-injected embryo showing no PMC ingression. (C,C') A SnaMASO-containing vegetal half (red) was combined with a control animal half (green). Resulting embryos lack PMCs. (D,D') A SnaMASO-containing animal half (red) was combined with a control vegetal half (green). Resulting embryos develop PMCs as in sibling controls. (G) A schematic diagram of the experimental designs of C and D. (E,E') Single SnaMASO-containing micromere (red) transplanted onto a control host embryo lacking one micromere (green). The SnaMASO micromere failed to ingress (arrow in E'), whereas all other control micromeres ingressed and migrated normally (arrowheads). (F,F') The reciprocal experiment to that in E. One normal micromere (green) ingresses into the blastocoele (arrow in F') when transplanted to a SnaMASO-injected host embryo lacking one micromere (red). (H) The schematic diagram of the experimental designs of E and F. See text for details.

View larger version (37K): [in this window] [in a new window]   Fig. 5. Effects of SnaMASO on PMC specification and differentiation. (A-D) In situ hybridization with Lvalx1 and Lvets1 probes. Control mesenchyme blastula embryos show strong expression of Lvalx1 and Lvets1 in PMCs (A,C). Expression of alx1 and ets1 are not affected by SnaMASO injection (B,D). (E,F) Immunostaining with mAb 1d5, shows the presence of 1d5 in control PMCs (E), but little to no expression of 1d5 in Sna morphants (F). (G) QPCR analysis of the expression of PMC differentiation genes, Lvsm30, Lvsm50 and Lvmsp130, in Sna morphants at the HB and MB stage relative to controls. Data are shown as net CT ± s.e.m.

View larger version (115K): [in this window] [in a new window]   Fig. 6. Upstream regulators of LvSnail. (A,B) WMISH shows a significant reduction of Lvsnail mRNA expression in Alx1 morphants (B), compared with control embryos (A). (C,D) Effect of U0126 treatment on Lvsnail mRNA expression. The U0126-treated embryos show no significant changes in Snail expression (D) compared with controls (C).

View larger version (75K): [in this window] [in a new window]   Fig. 7. LvSnail functions downstream of LvAlx1 in PMCs. (A) Control embryos. (B) Embryos injected with AlxMASO show no PMC ingression. (C) Excess mesenchyme cells form in embryos overexpressing LvSnail. (D) Co-injection of LvSnail rescues the formation of mesenchyme cells in the absence of LvAlx1. (E) Diagram of experiment in F,G. vv, vegetal view. (F-F'') Chimeric embryos generated by combining one control micromere (green) and one AlxMASO-containing micromere (red) with a control 16-cell stage embryo in which two micromeres were removed. Brightfield (F), fluorescent (F') and merged images (F'') show that the AlxMASO-containing micromere progeny do not ingress, unlike the control micromere progeny in the same embryos. (G-G'') LvSnail can rescue the effects of AlxMASO in PMC ingression. The micromere co-injected with Lvsnail mRNA and AlxMASO (green) ingresses into the blastocoele, whereas the AlxMASO-injected micromere (red) fails to ingress. Brightfield (G), fluorescent (G'), and merged (G'') images are shown.

View larger version (74K): [in this window] [in a new window]   Fig. 8. LvSnail downregulates G-cadherin expression and positively regulates its endocytosis. (A) Control embryos. (B) Embryos with ectopically expressed Pmar1 produce extra PMCs and mesenchyme extrusions. (C) Co-injection of SnaMASO rescues the Pmar1 ectopic-expression phenotype. Most co-injected embryos fail to undergo PMC ingression. (D) QPCR analysis of Pmar1- and Pmar1/SnaMASO-injected embryos. The expression of Lvsnail, Lvalx1, and Lvmsp130 is upregulated in Pmar1-injected embryos, whereas G-cadherin is significantly downregulated. The co-injection of SnaMASO rescues the reduction of cadherin expression in Pmar1-overexpressed embryos. Data are shown as net CT±s.e.m. (E) Surface view of an embryo injected at the one- and two-cell stage as illustrated in H. The SnaMASO-injected half is shown in red. The apical localization of cadherin can be clearly observed in adherens junctions (CadTM-GFP; adherens junctions are present on the red side also but obscured in the dual image by the red dye). (F) The internal view of the same embryo as shown in E; the intracellular punctate GFP signals indicate endocytosed cadherins (arrowheads), which do not overlap with SnaMASO-injected cells (with rhodamine-dextran). (G) Another two-cell injection embryo; the punctate GFP signals (arrowheads) can be seen in PMCs. (H) Schematic of the experimental design in E-G.

View larger version (17K): [in this window] [in a new window]   Fig. 9. Model of a Snail-dependent pathway regulating PMC ingression in the PMC-micromere GRN. Pmar1 de-repression system initiates the entire PMC specification program, and activates Alx1, Ets1 and other PMC regulatory genes (light blue oval), which in turn regulate skeletogenic differentiation genes (dark blue box). Alx1 regulates Snail (and other unknown EMT genes, denoted as X), and Snail represses Cadherin to attenuate the cell-cell adhesion, which allows PMCs to ingress into the blastocoele. Ets1 also regulates PMC ingression via unidentified EMT genes (denoted as Y). A subnetwork of EMT genes (light blue square box) regulates the EMT process of PMCs. The developmental stages shown on the left correspond to the chronological sequences of PMC developmental processes shown on the right.