Fig. 2.

LMO4 is required for neural crest formation. (A) Whole mount in situ hybridization of embryos co-injected in one animal micromere at the eight-cell stage with LMO4 morpholino and β-gal as a lineage tracer. Embryos were examined at mid-neurula stage (St17) with neural crest markers Slug, Snail, Sox8, Twist, Sox10, FoxD3. * indicates side of injection which is also denoted by red gal staining. (B) Effects of LMO4 depletion can be rescued with a morpholino resistant form of LMO4 (LMO4 M1). Whole mount in situ hybridization injected probed for neural crest marker Slug. (C) Whole mount in situ of stage 13 LMO4 depleted embryos probed for neural plate marker Sox3, epidermal marker Epk, and neural plate border markers Opl, and Pax3. (D) In situs of LMO4 MO injected embryos probed for mesoderm marker MyoD. Normal expression of mesodermal markers indicates that effects of LMO4 depletion on the neural crest are not a consequence of mesodermal defects. (E) Animal cap assay demonstrating that mesoderm independent induction of neural crest by Wnt/Slug is blocked by LMO4 depletion. (F) Western blot analysis validating the knockdown of LMO4 immunoblotting with antibodies against the tagged protein. Actin is used as a loading control. (G) Western blot of in vitro translated (IVT) LMO4 proteins demonstrating that the mutant form is resistant to translation blocking morpholino.