Both overexpression and knockdown of rab23 alter asymmetric gene expression in zebrafish. (A) Dorsal views of spaw and lefty1 expression at 16 to 18-somite stage (top) and lefty1 and lefty2 expression at the 22 to 24-somite stage (bottom) in the different phenotypic classes quantified in B, C, and D. spaw expression is normally observed in the left LPM, lefty1 in the midline, and lefty2 in the left cardiac mesoderm. Injection of rab23 mRNA or rab23 MOs produced embryos with left, right, bilateral, or no gene expression in the LPM. Control embryos included those injected with phenol red (p. red) or with a control morpholino. (B) Quantification of the spaw expression pattern at the 16 to 18-somite stage. Both mRNA injected embryos and MO injected embryos showed randomized spaw expression in the LPM. The majority showed bilateral expression. (C) Quantification of spaw expression patterns at the 9 to 11-somite stage when spaw is just beginning to be expressed in the LPM. A much smaller fraction of rab23 MO injected embryos induced spaw expression at this stage compared to controls, whereas the rab23 mRNA injected fish showed a highly significant increase in the fraction of spaw-expressing embryos. (D) Quantification of lefty2 expression in the cardiac LPM at the 22 to 24-somite stage. The vast majority of mRNA-injected embryos expressed lefty2 on the left side of the heart, whereas most rab23-MO injected fish failed to express lefty2. In all assays, embryos injected with the control morpholino did not differ from those injected with phenol red alone. Statistical significance: ^NNNP<10⁶, ^NNp<10³, and ^Np<0.05 Fisherós exact test.

Rab23 functions in KV to regulate left-right patterning in zebrafish embryos. (A and B) Expression of fluorescein-tagged rab23-atgMO in the dorsal forerunner cells (DFC) at tailbud stage. Embryos were injected with the MO at midblastula stages to target specifically the DFC that form KV later in development. KV morphology appeared normal in the DFC^rab23MO morphants at the 8-somite stage, as indicated by the arrow in (C). (D) Quantification of spaw expression in the DFC^rab23MO morphants at the 16 to 18-somite stage shows that 40% displayed bilateral spaw expression in the LPM. (E) Quantification of lefty2 expression shows that about half of the DFC^rab23MO expressed lefty2 normally on the left, while the remaining 50% showed bilateral, right-sided, or no lefty2 expression in the heart. Differences in morphant versus control patterns in D and E were highly significant (P<10⁶ Fisherós exact test).

Knockdown of Rab23 produces defects in visceral organ asymmetries.

Ventral views of the heart (A-C), and dorsal views of the liver and pancreas (D-F) at 48 hpf. Probes for cmlc2, fkd2, and ins were used to visualize the heart, liver, and pancreas, respectively. Embryos either displayed the wild-type arrangement of organs (situs solitus, shown in A,D), complete reversal (situs inversus, shown in B, E), or a random arrangement (heterotaxia, shown in C, F). V, ventricle; A, atrium; L, liver; P, pancreas. (G) Quantification of visceral organ laterality. Injection of rab23 mRNA had little effect on organ laterality, whereas injection of rab23-MO had a more severe effect. Nearly two-thirds of the rab23-atgMO injected embryos displayed heterotaxia or situs inversus.

Perturbing Rab23 activity in zebrafish does not affect early nodal signaling or general morphology of the embryo.

(A-H) Rab23 overexpression has no effect on early Nodal signaling in zebrafish embryos. The Nodal target genes goosecoid (A-D) and no-tail (E-H) are expressed normally in Q68L-rab23 injected embryos at 40-50% epiboly (A,B, E, F) and 90% epiboly (C, D, G, H) compared to control embryos. (I) Overexpression or knockdown of Rab23 does not affect general size or morphology. Images of live embryos injected with Q68L-rab23 mRNA (C,D) or rab23-atgMO (E,F) at 22 hpf and 48 hpf show no morphological abnormalities compared to control embryos injected with phenol red (A,B).