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

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 post-gastrulation 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.