RT-PCR analysis of ndel1a and ndel1b expression across development. ndel1a (A) and ndel1b (B) were amplified from an oligo dT primed cDNA library produced from 1 μg of total RNA at multiple stages ranging from 3 to 120 hpf. β-Actin serves as an internal control. (A) ndel1a is maternally expressed but levels dramatically decreased by 6 hpf. From 24 to 120 hpf, ndel1a expression gradually increases. In contrast, ndel1b (B) is highly expressed at 3 hpf but expression wanes to a low point at 36 hpf. Expression then gradually escalates to the last time-point tested at 120 hpf. The temporal distinction in expression was further validated by the results of the in situ hybridizations that follow.

ndel1a is expressed in the developing zebrafish CNS, eyes, and trigeminal ganglia. Lateral views of zebrafish embryos with anterior to the left. In agreement with the results of the RT-PCR analysis, ndel1a expression is low and restricted to small regions at early stages. (A) At 14 hpf, ndel1a is expressed in the ventral forebrain and eye fields. By 16 hpf (B) and continuing at 19.5 hpf (C) expression is obvious in the eyes and developing trigeminal ganglia (arrows in B and C). By 24 hpf, strong expression of ndel1a is apparent throughout the forebrain and eyes but appears to be decreased or absent in the ganglia (D). Expression in the peripheral regions subsides completely and strong ndel1a expression is observed only in the developing brain and eyes from 48 to 120 hpf (F–I). The sense control probe yielded no tissue specific expression. Representative 24 hpf embryo pictured in (E).

 ndel1a expression domains in the early post-embryonic zebrafish brain. In situ hybridization was done on 72 hpf zebrafish using the ndel1a probe as described. Larva were cryosectioned (10-12 µm) and mounted on slides. (A) ndel1a is expressed in the olfactory bulb (OB) at 72 hpf. Expression is also obvious in multiple telencephalic and diencephalic areas including the habenula (Ha), eminentia thalami (EmT), and the dorsal (DT) and ventral (VT) thalamus (B and C). The optic tectum (TeO) is also an area of high ndel1a expression (D). Cryosections also show eye specific ndel1a expression in the ganglion cell layer and inner nuclear layer (C). Abbreviations; DT, dorsal thalamus; GCL, ganglion cell layer; H, hypothalamus; Ha, habenula; INL, inner nuclear layer; OB, olfactory bulb; P, pallium; Po, preoptic area; PT, posterior tuberculum; T, midbrain tectum; TeO, optic tectum; VT, ventral thalamus.

Trigeminal ganglia expression of ndel1a is confirmed by HuC/D double labeling. At 19.5 hpf (A and A2), whole mount in situ hybridization for ndel1a (purple) followed by immunohistochemistry for a mature neuron marker (HuC/D; brown) illustrates that they both label the developing trigeminal ganglia (arrows). By 24 hpf (B) HuC/D is still expressed in the maturing trigeminal ganglia (arrow) but ndel1a expression is no longer apparent.

Expression of ndel1b during somite and brain development in the zebrafish. Lateral (A–C and D–I) and dorsal (A2–C2) views of zebrafish after ndel1b specific in situ hybridization with anterior to the left. Widespread ventral expression in the somitic mesoderm at 14 hpf persists and expands dorsally through 19.5 hpf (A–C and A2–C2). Expression then gradually becomes restricted in an anterior to posterior gradient during the maturation of the somites until ndel1b is only expressed in the tip of the tail at 24 hpf (D). At 48 hpf (F) ndel1b is again expressed in the ventral area of the trunk and tail but this expression is gone by 72 hpf (G). Instead, from 72 to 120 hpf expression shifts to the developing brain (G–I). Hybridization with the ndel1b sense probe shows no specific staining pattern. Representative 24 hpf embryo after sense probe hybridization in (E).