Sequential signaling crosstalk regulates endomesoderm segregation in sea urchin embryos

Abstract

The segregation of embryonic endomesoderm into separate endoderm and mesoderm fates is not well understood in deuterostomes. Using sea urchin embryos, we showed that Notch signaling initiates segregation of the endomesoderm precursor field by inhibiting expression of a key endoderm transcription factor in presumptive mesoderm. The regulatory circuit activated by this transcription factor subsequently maintains transcription of a canonical Wnt (cWnt) ligand only in endoderm precursors. This cWnt ligand reinforces the endoderm state, amplifying the distinction between emerging endoderm and mesoderm. Before gastrulation, Notch-dependent nuclear export of an essential β-catenin transcriptional coactivator from mesoderm renders it refractory to cWnt signals, insulating it against an endoderm fate. Thus, we report that endomesoderm segregation is a progressive process, requiring a succession of regulatory interactions between cWnt and Notch signaling.

Figure 1. Notch inhibits a Hox11/13b-dependent regulatory circuit in mesoderm

(A) Diagram of endomesoderm blastomeres: Large (red) micromere progeny are adjacent to veg₂ cells in which early cWnt activates endoderm and mesoderm specification (hatched light green) in early blastulae. By HB stage, veg₂ descendants form outer (presumptive endoderm, yellow) and inner (presumptive mesoderm, green) rings. Large micromere progeny (red) ingress by MB stage. (B to Q) Endodermal brachyury (bra), foxa and blimp1b transcripts are downregulated in Hox11/13b morphants (MASO). (B, F, N) hox11/13b mRNA is upregulated in Hox11/13b morphants, because Hox11/13b represses its own transcription (11). (K to M) Ectopic expression of brachyury (bra), foxa and blimp1b mRNA occurs in inner veg₂ cells of Notch morphants. hox11/13b mRNA concentration is low through auto-repression by ectopic Hox11/13b protein (11) (J). (O to Q) In Notch+Hox11/13b double morphants, ectopic expression of Hox11/13b target genes in inner veg₂ cells (arrows) is significantly lower (Q). Scale bar=20 µm.

Figure 2. Through the Hox11/13b circuit, Notch indirectly restricts wnt1 to endoderm, reinforcing its differentiation

(A to D) In Notch morphants, ectopic wnt1 mRNA accumulates in inner veg₂ descendants (arrows). (E) cWnt signaling activity (TOPFLASH-see Methods) is strongly reduced in Wnt1 (MASO or MASO2), Hox11/13b or Brachyury morphants [error bars indicate ±SD from the mean (n=3)]. (F to H) wnt1 mRNA accumulation in control MB endoderm and (I to K) its ectopic expression in inner veg₂ cells (arrows) of Notch morphants requires Hox11/13b and Brachyury. (L to S) Endodermal MB expression of hox11/13b, brachyury (bra), foxa and blimp1b mRNA is reduced in Wnt1 morphants.

Figure 3. Notch down regulates nuclear TCF in mesoderm

(A to C) Nuclear TCF clears from mesoderm (brackets; SoxB1-negative; see fig. S9 for lineage identification using SoxB1) in control embryos. (D to F) Without Notch or (G to I) NLK, nuclear TCF persists in inner veg₂ descendants (brackets). (J) Summary of endomesoderm segregation steps. (Step 1) Notch inhibits the Hox11/13b regulatory circuit in mesoderm (green box) by HB stage to initiate segregation. The Hox11/13b circuit stays active in endoderm (yellow). (Step 2) Consequently, by MB stage, Hox11/13b- and Brachyury-dependent expression of wnt1 is restricted to endoderm, where it reinforces cWnt signaling and endoderm fate. (Step 3) By pregastrula stage, a Notch-NLK pathway clears nuclear TCF from mesoderm, making it unresponsive to cWnt. The endoderm retains nuclear TCF and cWnt sensitivity.