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Fig. 4. Cep4l is required for neurogenesis. (A) Cep4l morpholino design and cep4l constructs. (B) Cep4l MO blocks translation of cep4l mRNA. Immunoblotting against HA in embryo lysates with the indicated constructs and MO doses. ☐-catenin was used as a loading control. ((C)–(T)) Cep4l depletion inhibits endogenous neurogenesis. ((C) and (D)) Phenotypes of controls and embryos injected with 40 ng Cep4l MO. Lateral views, anterior to the left. ((E)–(F)) Tubb2b expression in Cep4l MO-injected embryos. Dorsal (top) and lateral (bottom) views, anterior to the left. ((G)–(L)) Dorsal and ventral Cep4l depletion differentially affects neurogenesis. (G) Diagram of lineage targeting strategy. Tubb2b ((H), (I), (K)–(M)) or islet1 ((J) and (N)) expression in controls ((H), (I) and (J)), and in embryos injected with 8 ng Cep4l MO into the right dorsal animal (DA) (K) or ventral animal (VA) ((L), (M) and (N)) cells. Arrow in (K) marks depleted brain and placode neurons. Brackets in ((M) and (N)) mark depleted sensory neurons, small arrows mark unaffected areas. ((O)–(V)) cep4l rescue injections. Embryos were injected at two cells with 50 pg β-gal mRNA alone ((O) and (S)) or with a sub-phenotypic dose (125 pg) of cep4l ((R) and (V)), and subsequently at eight cells with 8 ng Cep4l MO into the right VA blastomere ((P), (T), (Q) and (U)). Bracket in ((P) and (T)) marks depleted sensory neurons. Panels ((S)–(V)) are closeup images of those above. Arrowheads in ((Q) and (U)) marks rescued sensory neurons. m.n., motorneurons, s.n., sensory neurons. |
