Beyond the neckless phenotype: influence of reduced retinoic acid signaling on motor neuron development in the zebrafish hindbrain

Abstract

Retinoic acid (RA) has been identified as a key signal involved in the posteriorization of vertebrate neural ectoderm. The main biosynthetic enzyme responsible for RA signaling in the hindbrain and spinal cord is Raldh2. However, neckless/raldh2-mutant (nls) zebrafish exhibit only mild degrees of anteriorization in the neural ectoderm, compared to full vitamin A deficiency in amniotes and the Raldh2-/- mouse. Here we investigated the role of RA during neuronal development in the zebrafish hindbrain and anterior spinal cord using DEAB, an inhibitor of retinaldehyde dehydrogenases. We show that the nls hindbrain and spinal cord are not fully devoid of RA, since blocking Raldh-mediated RA signaling leads to a more severe hindbrain phenotype than in nls. The anteroposterior distribution of branchiomotor neurons in the facial and more posterior nuclei depends on full RA signaling throughout early and late gastrula stages. In contrast, inhibition of RA synthesis after gastrulation reduces the number of branchiomotor neurons in the vagal nucleus, but has no effect on anteroposterior cell fates. In addition, blockage of RA-mediated signaling not only interferes with the differentiation of branchiomotor neurons and their axons in the hindbrain, but also affects the development of the posterior lateral line nerve.

Regulation and function of Dbx genes in the zebrafish spinal cord

“…5D). Both of these results are consistent with previous studies and indicate that we effectively inhibited RA signaling (Begemann et al, 2004;Maves and Kimmel, 2005).…”

“…At 18 hpf and 24 hpf, the dorsal/ ventral position of dbx1a, dbx1b, and dbx2 expression was unaffected, while at 24 hpf, dbx2 was prematurely expressed throughout the entire rostral/ caudal axis of the embryo (not shown). We did not observe a loss of dbx1a expression at 24 hpf or premature expression of dbx2 at 18 hpf after BMS493 treatment, which we believe is due to a less complete block of RA signaling compared with DEAB (Begemann et al, 2004). Nevertheless, the similar results obtained with antagonists of both ligand and receptors support the conclusion that RA is not required for Dbx induction.…”

“…Although DEAB has been suggested to provide the most complete block of RA signaling possible in zebrafish (Begemann et al, 2004), our data contrasted with a previous study in chick using pharmacological agents to block RA receptors (Pierani et al, 1999). Therefore, we treated embryos with the pan-RA receptor inhibitor BMS493, which has also been used previously in zebrafish to block RA signaling (Grandel et al, 2002;Begemann et al, 2004).…”

“…Therefore, we treated embryos with the pan-RA receptor inhibitor BMS493, which has also been used previously in zebrafish to block RA signaling (Grandel et al, 2002;Begemann et al, 2004). Embryos were soaked in 10 m BMS493, beginning at 50% epiboly and fixed at either 18 or 24 hpf, and we analyzed mRNA expression of all three Dbx family members.…”

Midkine‐b regulates cell specification at the neural plate border in zebrafish

“…Low levels of ectopic RA (Ͻ10 Ϫ8 M) enhance mdkb expression, whereas high levels of ectopic RA (Ͼ10 Ϫ7 M) inhibit its transcription (Winkler and Moon, 2001). To investigate whether mdkb expression also requires endogenous RA, we incubated embryos in DEAB, an efficient inhibitor of retinaldehyde dehydrogenases (Begemann et al, 2004). Embryos were incubated in 10 M DEAB from shield stage, shortly before endogenous mdkb expression is initiated, until 80% epiboly.…”