Nav3 isoforms, phylogenetic analysis and expression. (A) Northern blot analysis of whole embryo RNA demonstrated that two nav3 isoforms are expressed during zebrafish development: a longer nav3a (8 kb) and a shorter nav3b isoform (7.3 kb). (B) 52 RACE PCR confirmed the expression of nav3a and nav3b in zebrafish embryos at 22 hpf. Nav3a and nav3b have a divergent first exon, indicating transcriptional regulation by independent alternative promoters. (C) Nav3a, in contrast to Nav3b, contains a calponin-homology domain and an LKK-actin binding domain. Both share a SH3 and ATPase domains of the AAA type. (D) Whole-mount in situ hybridization for nav3a in developing zebrafish embryos. Weak nav3a expression could be detected beginning at 16 hpf. At 22 hpf a maximum expression is detectable in intestinal endoderm and in addition in the posterior endoderm along the trunk (white arrowheads). The intestinal and hepatic endoderm expression remains up to 40 hpf (white arrowheads) but is downregulated after completion of liver budding (white arrowhead, 72 hpf). Beginning at 26 hpf up to 72 hpf an additional expression domain could be observed in the developing pharyngeal arches (red arrowheads), parts of the eye (black arrowheads) and at 72 hpf in the hindbrain and the heart (blue arrowheads). (E) Confocal stacks of sox17:gfp embryos between 22 and 26 hpf demonstrating that the foregut endoderm becomes more compact during this period. (F) Nav3a mRNA expression is restricted to the foregut endoderm. Nav3a was detected by in situ hybridization in sox17:gfp embryos at the indicated time points. Endodermal GFP protein was stained by immunofluorescence in the same embryo. E and F show ventral view.
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