NEURODEVELOPMENT. Shared regulatory programs suggest retention of blastula-stage potential in neural crest cells

Abstract

Neural crest cells, which are specific to vertebrates, arise in the ectoderm but can generate cell types that are typically categorized as mesodermal. This broad developmental potential persists past the time when most ectoderm-derived cells become lineage-restricted. The ability of neural crest to contribute mesodermal derivatives to the bauplan has raised questions about how this apparent gain in potential is achieved. Here, we describe shared molecular underpinnings of potency in neural crest and blastula cells. We show that in Xenopus, key neural crest regulatory factors are also expressed in blastula animal pole cells and promote pluripotency in both cell types. We suggest that neural crest cells may have evolved as a consequence of a subset of blastula cells retaining activity of the regulatory network underlying pluripotency.

Figure 1

Neural crest cells and pluripotent blastula cells share a common regulatory circuitry. (A-B) In situ hybridization of wildtype blastula (stage 9) Xenopus embryos examining expression of genes associated with pluripotency (A) or neural crest formation (B). Scale bars, 250 µM. (C) qRT-PCR of wildtype ectodermal explants examining relative expression of pluripotency genes and neural crest genes over developmental time.

Figure 2

Neural crest regulatory factors are required for the expression of blastula pluripotency factors. (A-B) In situ hybridization of embryos injected with ΔSnail mRNA (A) or Sox5 MO (B). Embryos were collected at blastula stages (stage 9) and examined for expression of genes associated with pluripotency/neural crest formation. Asterisk denotes injected side with β-gal staining (red) serving as a lineage tracer. Scale bars, 250 µM.

Figure 3

Neural crest regulatory factors are required for pluripotency of blastula cells. (A-D) Nieuwkoop recombinant assay examining expression of Brachyury (A,C) and MyoD (B,D) after depleting Snail1 (A,B) or Sox5 function (C,D). Recombinants were harvested at gastrulation stages for Brachyury expression (stage 11.5) or early neurula stages (stage 13/14) for MyoD expression. (E-H) Ectodermal explant assay examining expression of MyoD (E,G) and Endodermin (F,H). Explants were injected with ΔSnail mRNA (E,F) or Sox5 MO (G,H) and cultured with or without activin until early neurula stages for MyoD expression (stage 13/14) and midgastrula stages (stage 11.5) for Endodermin expression. Scale bars, 250 µM.

Figure 4

Establishing a neural crest state prevents loss of potency in blastula-derived cells. (A-D) Ectodermal explant assay examining expression of MyoD (A,C) or Endodermin (B,D) in embryos that were injected with Pax3-GR/Zic1-GR mRNA (A,B) or Snail2/Wnt8 mRNA (C,D). Explants were treated with activin at either stage 8 or 12 and cultured until late neurula stages (stage 18). Scale bars, 250 µM.

Figure 5

Neural crest cells possess the capacity for endoderm formation. (A) Schematic representation of neural plate border/neural crest isolation. Neural folds are dissected at early neurula stages (stage 14/15) and cultured with or without activin until late neurula stages (stage 18). (B-E) In situ hybridization examining expression of MyoD (B), Endodermin (C), Brachyury (D), and Sox17 (E) in neural plate border/neural crest tissue treated with or without activin. Scale bars, 250 µM.

Figure 6

(A) Historic model of neural crest induction in which neural crest cells were believed to gain potential relative to predecessor cells. (B) Proposed model for the generation of neural crest cells via retention of pluripotency characteristics of earlier blastula cells.