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Fig. 5. Early neural crest marker expression is impaired in RhoV-depleted embryos. (A) Comparison of RhoVa and RhoVb mRNA initiator regions. Shown in red are the nucleotides that differ between the two sequences. RhoV-MO was designed to target the sequence underlined in black. (B) Western blot analysis of lysates prepared from control embryos (Uninjected) or embryos expressing the RhoV-GFP fusion alone (no MO) or co-injected with 20 ng per embryo of control MO (Ctrl-MO), RhoV-MO or RhoU-MO. Arrow indicates the position of the RhoV-GFP fusion. Only RhoV-MO inhibited RhoV-GFP translation. (C) Western Blot analysis of lysates prepared from control embryos (Uninjected), or embryos expressing the RhoU-GFP fusion alone (no MO) or co-injected with 20 ng per embryo of control MO (Ctrl-MO), RhoV-MO or RhoU-MO. Arrow indicates the position of the RhoV-GFP fusion. RhoV-MO does not inhibit RhoU-GFP translation. (D) Xbra and MyoD in situ hybridization analyses. Embryos were injected in one cell of two-cell stage embryo with 20 ng of RhoV-MO or control MO. β-galactosidase mRNA was co-injected to identify the injected side (red staining). Embryos were harvested at gastrula (a–d) and neurula stages (e, f) and analyzed respectively for Xbra and MyoD stainings. Shown are vegetal (a, c) or dorso-vegetal (b, d) or lateral (e, f) views. Control non-injected side (e) and RhoV-MO-injected side (f) of the same embryo. (E) In situ hybridization of early neurula stage embryos injected in one cell of two cell-stage embryos with 20 ng of control MO (Ctrl-MO) or RhoV-MO. Dorsal views, anterior to the top. RhoV depletion inhibits or strongly reduces the expression of the early neural crest markers Sox9 (b), Slug (d), Sox10 (e) and Twist (f) but has little effect on Snail expression (g). Note that depletion of RhoV also reduces the expression of Sox9 in the otic placodes (b). Injection of Ctrl-MO has no effect on Sox9 (a) or Slug (c). (F) Western Blot analysis of lysates from control embryos (Uninjected) or embryos expressing a RhoV-GFP fusion alone (no MO) or in combination with 20 ng of control MO (Ctrl-MO) or RhoV-MO, and embryos expressing a GFP-RhoV fusion alone (no MO) or in combination with 20 ng of RhoV-MO. Arrow indicates the position of GFP-RhoV and RhoV-GFP. GFP-RhoV mRNA is insensitive to RhoV-MO. (G) The loss of Sox9 expression upon RhoV depletion is rescued by injection of mRNA (100 pg) encoding GFP-RhoV or the same amount of its closely relative GFP-RhoU. The translation of GFP-Rho fusion proteins is insensitive to RhoV-MO (see panels C and F). Note that expression of RhoB is not able to restore Sox9 expression. In all cases, the injected side is oriented to the right (red staining is from the lineage tracer β-Gal). |
