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Fig. 1. Endogenous grem1 expression pattern, and effects of knockdown and overexpression on the neural crest. (A–C) Stage 36 grem1 expression pattern. (A) Whole-mount stained embryo showing strong expression in the kidney primordia (k), posterior roofplate (rp) and tailbud (tb), and neural crest (nc). (B-C) Transverse sections from an embryo like the one shown, with approximate anteroposterior locations marked by dashed lines in A. (B) In the most anterior region, grem1 is expressed in neural crest cells ventral to the developing eye fields (e). (C) In the pharyngeal region, expression is seen in neural crest cells flanking the neural tube (nt) and invading the tissue around the pharyngeal pouches (pp). (D–E) Effect of grem1 overexpression on the neural crest. (D) Cranial neural crest (nc) around stage 18 is marked by the expression of twist1, flanking the anterior neural plate. (E) Overexpression by injection of Xenopus grem1 RNA causes a substantial expansion (arrowhead) of the neural crest (n = 12/19). (F–G) Effect of grem1 knockdown. (F) At stage 21 twist1 marks the neural crest as it begins to surround the eye fields (e) and to migrate in two more posterior streams. It is unaffected by unilateral injection of a standard control morpholino (n = 30/30). (G) Knockdown of grem1 by injection of a translation-blocking morpholino severely impairs neural crest formation (asterisk, n = 80/93). (H) Injection of murine Grem1 RNA is also able to expand the domain of twist1 expression (arrowhead), indicating functional conservation between the mouse and Xenopus orthologues (n = 59/74). (I–K) Rescue of neural crest formation with murine Grem1. (J) Bilateral grem1 knockdown depletes the neural crest from both sides (asterisks). (K) Unilateral rescue using murine Grem1 RNA, mismatched with the morpholino, which only targets the Xenopus orthologue, is able to restore neural crest development (arrowhead, n = 14/20). (L–Q) Effect of gain and loss of grem1 on other neural crest markers. (L) Control neural crest expresses sox10 (n = 25/25). (M) Unilateral grem1 knockdown abolishes expression on the injected side (asterisk, n = 19/24). (N) Unilateral overexpression expands the domain of sox10 (arrowhead, n = 21/30). (O) Control neural crest also expresses foxd3. (P) Knockdown similarly abolishes expression (asterisk, n = 24/24). (Q) Likewise, the domain is expanded by overexpression (arrowhead, n = 12/15). Embryo in A is shown in side view with anterior towards the left, sections in B-C are transverse with dorsal towards the top, and embryos in D-Q are shown in frontal view. |
