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Since its initial discovery in a Drosophila mutagenesis screen, the Hedgehog pathway has been revealed to be instrumental in the proper development of the vertebrate face. Vertebrates possess three hedgehog paralogs: Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh). Of the three, Shh has the broadest range of functions both in the face and elsewhere in the embryo, while Ihh and Dhh play more limited roles. The Hedgehog pathway is instrumental from the period of prechordal plate formation early in the embryo, until the fusion of the lip and secondary palate, which complete the major patterning events of the face. Disruption of Hedgehog signaling results in an array of developmental disorders in the face, ranging from minor alterations in the distance between the eyes to more serious conditions such as severe clefting of the lip and palate. Despite its critical role, Hedgehog signaling seems to be disrupted through a number of mechanisms that may either be direct, as in mutation of a downstream target of the Hedgehog ligand, or indirect, such as mutation in a ciliary protein that is otherwise seemingly unrelated to the Hedgehog pathway. A number of teratogens such as alcohol, statins and steroidal alkaloids also disrupt key aspects of Hedgehog signal transduction, leading to developmental defects that are similar, if not identical, to those of Hedgehog pathway mutations. The aim of this review is to highlight the variety of roles that Hedgehog signaling plays in developmental disorders of the vertebrate face.

Wnt/β-catenin has been described as crucial for dorsal-ventral and antero-posterior patterning, playing multiple roles at different stages of development. Cholesterol-rich membrane microdomains (CRMMs), cholesterol- and sphingolipid-enriched domains of the plasma membrane, are known as platforms for signaling pathways. Although we have demonstrated the importance of the CRMMs for head development, how they participate in prechordal plate formation and embryo axis patterning remains an open question. Moreover, the participation of the CRMMs in the Wnt/β-catenin signaling pathway activity in vivo is unclear, particularly during embryonic development. In this study, we demonstrated that CRMMs disruption by methyl-beta-cyclodextrin (MβCD) potentiates the activation of the Wnt/β-catenin signaling pathway in vitro and in vivo during embryonic development, causing head defects by expanding the Wnt expression domain. Furthermore, we also found that the action of CRMMs depends on the microenvironmental context because it also works in conjunction with dkk1, when dkk1 is overexpressed. Thus, we propose CRMMs as a further mechanism of prechordal plate protection against the Wnt signals secreted by posterolateral cells, complementing the action of secreted antagonists.

We have identified several genes that are required for various morphogenetic processes during gastrulation and tail formation. Two genes are required in the anterior region of the body axis: one eyed pinhead (oep) and dirty nose (dns). oep mutant embryos are defective in prechordal plate formation and the specification of anterior and ventral structures of the central nervous system. In dns mutants, cells of the prechordal plate, such as the prospective hatching gland cells, fail to specify. Two genes are required for convergence and extension movements. In mutant trilobite embryos, extension movements on the dorsal side of the embryo are affected, whereas in the formerly described spadetail mutants, for which two new alleles have been isolated, convergent movements of ventrolateral cells to the dorsal side are blocked. Two genes are required for the development of the posterior end of the body axis. In pipetail mutants, the tailbud fails to move ventrally on the yolk sac after germ ring closure, and the tip of the tail fails to detach from the yolk tube. Mutants in kugelig (kgg) do not form the yolk tube at the posterior side of the yolk sac.

Dickkopf1 (dkk1) encodes a secreted WNT inhibitor expressed in Spemann’s organizer, which has been implicated in head induction in Xenopus. Here we have analyzed the role of dkk1 in endomesoderm specification and neural patterning by gain-and loss-of-function approaches. We find that dkk1, unlike other WNT inhibitors, is able to induce functional prechordal plate, which explains its ability to induce secondary heads with bilateral eyes. This may be due to differential WNT inhibition since dkk1, unlike frzb, inhibits Wnt3a signalling. Injection of inhibitory antiDkk1 antibodies reveals that dkk1 is not only sufficient but also required for prechordal plate formation but not for notochord formation. In the neural plate dkk1 is required for anteroposterior and dorsoventral patterning between mes-and telencephalon, where dkk1 promotes anterior and ventral fates. Both the requirement of anterior explants for dkk1 function and their ability to respond to dkk1 terminate at late gastrula stage. Xenopus embryos posteriorized with bFGF, BMP4 and Smads are rescued by dkk1. dkk1 does not interfere with the ability of bFGF to induce its immediate early target gene Xbra, indicating that its effect is indirect. In contrast, there is cross-talk between BMP and WNT signalling, since induction of BMP target genes is sensitive to WNT inhibitors until the early gastrula stage. Embryos treated with retinoic acid (RA) are not rescued by dkk1 and RA affects the central nervous system (CNS) more posterior than dkk1, suggesting that WNTs and retinoids may act to pattern anterior and posterior CNS, respectively, during gastrulation.