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zTollip contributes to the canonical Wnt pathway in embryonic development. (A-E) Embryos overexpressing ztollip (B) or injected with either ztollip morpholinos (MOs) (C), or β-catenin2 MO (D), or both (E) were analyzed for their morphological phenotype at 48 hpf. Typical phenotypes are shown. Lateral views with head to the left. Injection of ztollip mRNA led to reduction of posterior structures (arrow in B) compared with wild-type, control embryos (A). Knockdown of ztollip resulted in tail curvature (arrow in C) and pericardial oedema (asterisk in representative embryo in inset of C). Injection of β-catenin2 MO caused morphological defects similar to those found in embryos overexpressing ztollip (compare B and D). Tail curvature and pericardial oedema in embryos overexpressing ztollip could be rescued by injection of 2 ng of β-catenin2 MO (compare B and E). (F-R) In situ hybridization using different mRNA probes. Embryos injected with ztollip mRNA showed expanded expression of both the dorsal mesendodermal marker goosecoid (gsc) at 6 hpf (shield stage) (brackets in G) and the dorsal marker notail (ntl) at tail bud stage (brackets in I) compared to uninjected embryos (F and H, respectively). Expression of the cardiac marker cmlc2 (J, K) indicated the failure of the heart to form the loop in ztollip morphants (asterisk in J) compared to wild-type embryos (asterisk in K) at 48 hpf. Expression of fgf8 in the tail was reduced or absent in embryos overexpressing ztollip (arrow in M), while embryos injected with ztollip MOs showed an increase in fgf8 expression in the brain and in the tail (arrows in O) compared to uninjected, control embryos (L) at 24 hpf. Embryos injected with 8 ng β-catenin2 MO exhibited reduced fgf8 expression (N). Blocking endocytosis by injecting embryos with 4 ng rab5a MO or by expressing the Dnm2-K44A displayed no clear effect on fgf8 expression (arrows in P and Q). ztollip overexpression together with the Dnm2-K44A mutant showed a Tollip-like phenotype on the expression of fgf8 (arrow in R).
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