Mutations in lrr6l do not affect general pronephric patterning or cilia structure. (A-D) Expression of pronephric specification genes at eight somites is not affected in sea mutants (B,D) compared with wild type (A,C). (A,B) wt1 is correctly expressed in the intermediate mesoderm from the first somite to the beginning of the fourth somite. (C,D) pax2.1 is correctly expressed in the intermediate mesoderm posterior to the third somite. In A-D, RNA in situ hybridization for α-tropomyosin was used to visualize the somites. (E,F) Expression of pax2.1 in the neck segments at 48 hpf is unaffected in sea mutants (F) compared with siblings (E). (G,H) Acetylated tubulin immunofluorescence of KV at six somites in sea mutant (H) and sibling (G) shows that cilia are not affected in sea mutants. (I-K) In wild-type embryos at 72 hpf (I), wt1 is expressed in the fused glomerulus in the midline. In sea mutants, defects in wt1 expression were seen, including separation of the glomeruli (J) and drastic expansions of the wt1 domain (K). These effects are probably caused by the expansion of the glomerular region. (L,M) Acetylated tubulin staining in sibling (L) and sea mutants (M) at 27 hpf indicate that cilia formation in the pronephros is unaffected by mutations in sea. All above images are from seafa20rmutants, but similar results were obtained for seatg238a. (N-Q) TEM analysis of pronephric cilia show no alterations in axoneme structure in seafa20r mutants (O,P) compared with siblings (N). Basal body localization at the apical membrane is also unaffected in sea mutants (Q; arrow indicates basal body). Anterior is towards the top in A-K. L and M are lateral views of the pronephric tubules; anterior is towards the left.
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