Retinoic acid is required for endodermal pouch morphogenesis and not for pharyngeal endoderm specification

Abstract

Because tissues from all three germ layers contribute to the pharyngeal arches, it is not surprising that all major signaling pathways are involved in their development. We focus on the role of retinoic acid (RA) signaling because it has been recognized for quite some time that alterations in this pathway lead to craniofacial malformations. Several studies exist that describe phenotypes observed upon RA perturbations in pharyngeal arch development; however, these studies did not address whether RA plays multiple roles at distinct time points during development. Here, we report the resulting phenotypes in the hindbrain, the neural crest-derived tissues, and the pharyngeal endoderm when RA synthesis is disrupted during zebrafish gastrulation and pharyngeal arch morphogenesis. Our results demonstrate that RA is required for the postgastrulation morphogenesis and segmentation of endodermal pouches, and that loss of RA does not affect the length of the pharyngeal ectoderm or medial endoderm along the anterior-posterior axis. We also provide evidence that RA is not required for the specification of pharyngeal pouch endoderm and that the pharyngeal endoderm consists of at least two different cell populations, of which the pouch endoderm is sensitive to RA and the more medial pharyngeal endoderm is not. These results demonstrate that the developmental processes underlying pharyngeal arch defects differ depending on when RA signaling is disturbed during development.

Origin and Early Development of the Posterior Lateral Line System of Zebrafish

“…DEAB is an inhibitor of retinaldehyde, the enzyme that synthetizes retinoic acid, and can be used to deplete retinoic acid during embryogenesis (Kopinke et al, 2006). We observed that treating embryos with DEAB between 4 and 10 hpf leads to dramatic defects in the formation of the PLL system and of the inner ear.…”

“…4-(Diethylamino) benzaldehyde (DEAB; Sigma-Aldrich) was used at 50 M (Kopinke et al, 2006) by dilution of a stock solution (50 mM in DMSO). Twelve fertilized eggs were placed in 6 ml of this solution at time t, transferred to 40 ml of system water at time t ϩ 2 h, and transferred a second time 15 min later.…”

Loss of adenomatous polyposis coli (apc) results in an expanded ciliary marginal zone in the zebrafish eye

“…In situ hybridization and immunohistochemistry was performed as previously described (Kopinke et al, 2006). The following in situ probes were used: axin2 (kind gift of R. Dorsky), dkk1 (gift from C. Houart), follistatin (Bauer et al, 1998), otx1 (Li et al, 1994), atoh7 (atoh7, (Kay et al, 2001), sox2 (Okuda et al, 2006), myca and cyclinD1 , pax6a (Krauss et al, 1991), stat3 (Krauss et al, 1991), her6 (Pasini et al, 2004), meis1 (Waskiewicz et al, 2001), vsx2 (Barabino et al, 1997 …”

Regulation and function of Dbx genes in the zebrafish spinal cord

“…Previous data have suggested that RA is required for Dbx expression in other vertebrates (Pierani et al, 1999;Novitch et al, 2003). To block RA signaling in embryos, we used 4-(diethylamino)-benzaldehyde (DEAB), which has been shown to inhibit retinaldehyde dehydrogenase and subsequently the synthesis of RA (Perz-Edwards et al, 2001;Hamade et al, 2006;Kopinke et al, 2006). Embryos were treated with DEAB at 50% epiboly to test whether RA is required for the induction of Dbx expression.…”