Knockdown of arl13b impairs posture, locomotion, and cerebellar morphology in zebrafish larvae. A Wild-type sibling larvae remain vertically oriented at 5 days post-fertilization (dpf), with both eyes visible from a top view (arrows). B In contrast, larvae injected with arl13b MO (subthreshold dose, 7.3 ng) often lie on their side at the bottom of the dish, with only one eye visible (arrows). Note that the body of the subthreshold-dose arl13b morphants are relatively straight and only slightly curved. C Statistics of the posture of zebrafish larvae at 5 dpf. D Wild-type larvae perform stereotyped spontaneous swimming with small bending angles. E The arl13b morphants (subthreshold dose, 7.3 ng) swim slower and exhibit greater bending angles. F–H Immunostaining with acetylated tubulin antibody outlines the cerebellum of larvae at 3 dpf. Comparing the dorsal view of wild-type embryos (F) with arl13b mutants (G) reveals morphological defects of the cerebellum (arrows). The cerebellar defects were also present in embryos injected with arl13b MO (H) (cb, cerebellum). I Statistics of the embryos with morphological defects of the cerebellum. The number of embryos examined in each condition is indicated above each column. A–E, Scale bar 1 cm. F–H, Scale bar 100 μm.

The expression of markers of cerebellar granule cell progenitors is impaired in arl13b-deficient embryos. A–I’ Representative images of in situ hybridization illustrate that the three paralogues of atoh1 (atoh1a, 1b and 1c) are expressed in distinct populations of cerebellar granule cell progenitors. atoh1a is expressed in the URL and LRL. Similar expression patterns of atoh1a occur in wild-type embryos (A) and arl13b mutants (B) while its expression is absent from the oral dorsomedial URL (dashed box) in arl13b morphants (C) at 36 hpf and 48 hpf (A’–C’). The expression patterns of atoh1b remained unaffected in arl13b mutants and morphants (D–F’). The expression level of atoh1c was decreased in the URL in arl13b morphants (I and I’) (cb, cerebellum; URL, upper rhombic lip; LRL, lower rhombic lip). A–I’, Scale bar 100 μm.

Disruption of arl13b impairs the development of cerebellar granule cells. A Expression of the granule cell progenitor marker, zic1, in the cerebellum of wild-type embryos at 48 hpf revealed by in situ hybridization. B, C Expression of zic1 is reduced in the URL of both arl13b mutant and morphant embryos compared with wild-type embryos. Note that zic1 expression is severely reduced in the dorsomedial subregions of the URL (arrows). D The expression of the differentiated granule cell marker, reelin, in the cerebellum of wild-type embryos at 3 dpf. E, F Expression of reelin is reduced in the cerebellum of both arl13b mutant and morphant embryos. Note that in some embryos reelin expression is almost absent in the dorsal medial subregions of the cerebellum (colored ovals). G–G’’ In Tg(neurod1:eGFP) transgenic embryos, GFP+ granule cells are grouped into three clusters, the dorsomedial (dashed ovals), dorsoposterior, and ventrolateral subdivisions. H–H’’ The pattern of GFP+ granule cells is dramatically altered in the cerebellum, and particularly in the dorsomedial cerebellar subdivisions (dashed ovals) are severely affected in arl13b morphants. The parallel fibers connecting the two hemispheres are disrupted in arl13b morphants. Dorsal views of the embryos are shown. I–L Malformations of the dorsomedial cerebellar subdivisions (dashed ovals) and parallel fibers are also present in arl13b mutants both at 3 dpf and 4 dpf. cb, cerebellum; PF, parallel fiber. A–F, Scale bar 100 μm. G–L, Scale bar 50 μm.

Disruption of arl13b reduces both precursor and differentiated cerebellar Purkinje cells. A–I Representative images of in situ hybridization using anti-sense probes against ptf1a and roraa to label Purkinje precursors and differentiated cells, respectively. The dorsomedial clusters of precursor of Purkinje cells and differentiated Purkinje neurons are selectively reduced (arrows). J–L’’ Immunostaining of mature Purkinje neurons using anti-parvalbumin antibody reveals that the dorsomedial Purkinje neurons are reduced in arl13b-deficient embryos. A–I, Scale bar 100 μm. J–L’’ Scale bar 50 μm.

Wnt1 is selectively down-regulated in the cerebellum of arl13b mutants. wnt1 is transiently expressed in the cerebellum of WT embryos (A, A’) (arrows) while it is dramatically reduced in arl13b mutants (B, B’). A and B, dorsal view; A’ and B’, lateral view. Note that the expression of wnt1 is selectively decreased in the cerebellum while its expression at the midbrain–hindbrain boundary is intact. A–B’, Scale bar 100 μm.

Treating the arl13b mutants with lithium mitigates the morphological defects in the cerebellum. A–D Representative images of embryos treated with 50 mmol/L LiCl at 30–37 hpf, fixed at 3 dpf, and immunostained with anti-tubulin antibody to reveal cerebellar morphology. Treatment of wild-type embryos with Li⁺ does not affect the cerebellar morphology (A, B) (arrows). The morphological defects of the cerebellum in arl13b mutants treated with Li⁺ are partially rescued (C, D) (arrow). A–D, Scale bar 100 μm. E Statistics revealing that the proportion of arl13b mutant embryos with cerebellar defects is dramatically decreased by LiCl treatment. F–I’’ Representative images of Tg(neurod1:EGFP) transgenic embryos used to label granule cells. Treating wild-type transgenic embryos with Li⁺ causes no defect (F–G’’) (dashed ovals). Treating arl13b morphant transgenic embryos restores the dorsomedial cluster of granule cells (H–I’’) (dashed ovals). F–I’’, Scale bar 100 μm. J The proportion of arl13b morphant embryos with cerebellar defects in the dorsomedial clusters is dramatically reduced by LiCl treatment.