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The cranial neural crest and the cranial mesoderm are the source of tissues from which the bone and cartilage of the skull, face and jaws are constructed. The development of the cranial mesoderm is not well studied, which is inconsistent with its importance in craniofacial morphogenesis as a source of precursor tissue of the chondrocranium, muscles, vasculature and connective tissues, mechanical support for tissue morphogenesis, and the signaling activity that mediate interactions with the cranial neural crest. Phenotypic analysis of conditional knockout mouse mutants, complemented by the transcriptome analysis of differentially enriched genes in the cranial mesoderm and cranial neural crest, have identified signaling pathways that may mediate cross-talk between the two tissues. In the cranial mesenchyme, Bmp4 is expressed in the mesoderm cells while its signaling activity could impact on both the mesoderm and the neural crest cells. In contrast, Fgf8 is predominantly expressed in the cranial neural crest cells and it influences skeletal development and myogenesis in the cranial mesoderm. WNT signaling, which emanates from the cranial neural crest cells, interacts with BMP and FGF signaling in monitoring the switch between tissue progenitor expansion and differentiation. The transcription factor Twist1, a critical molecular regulator of many aspects of craniofacial development, coordinates the activity of the above pathways in cranial mesoderm and cranial neural crest tissue compartments.

Early development of the gut endoderm and its subsequent remodeling for the formation of organ buds are accompanied by changes to epithelial cell shape and polarity. Members of the Rho-related family of small GTPases and their interacting proteins play multiple roles in regulating epithelial morphogenesis. In this study we examined the role of Cdc42 in foregut development and organ bud formation. Ablation of Cdc42 in post-gastrulation mouse embryos resulted in a loss of apical-basal cell polarity and columnar epithelial morphology in the ventral pharyngeal endoderm, in conjunction with a loss of apical localization of the known CDC42 effector protein PARD6B. Cell viability but not proliferation in the foregut endoderm was impaired. Outgrowth of the liver, lung and thyroid buds was severely curtailed in Cdc42-deficient embryos. In particular, the thyroid bud epithelium did not display the apical constriction that normally occurs concurrently with the outgrowth of the bud into the underlying mesenchyme. SHROOM3, a protein that interacts with Rho GTPases and promotes apical constriction, was strongly expressed in the thyroid bud and its sub-cellular localization was disrupted in Cdc42-deficient embryos. In Shroom3 gene trap mutant embryos, the thyroid bud epithelium showed no apical constriction, while the bud continued to grow and protruded into the foregut lumen. Our findings indicate that Cdc42 is required for epithelial polarity and organization in the endoderm and for apical constriction in the thyroid bud. It is possible that the function of CDC42 is partly mediated by SHROOM3.

Twist1 encodes a transcription factor that plays a vital role in limb development. We have used a tamoxifen-inducible Cre transgene, Ubc-CreERT2, to generate time-specific deletions of Twist1 by inducing Cre activity in mouse embryos at different ages from embryonic (E) day 9.5 onwards. A novel forelimb phenotype of supernumerary pre-axial digits and enlargement or partial duplication of the distal radius was observed when Cre activity was induced at E9.5. Gene expression analysis revealed significant upregulation of Hoxd10, Hoxd11 and Grem1 in the anterior half of the forelimb bud at E11.5. There is also localized upregulation of Ptch1, Hand2 and Hoxd13 at the site of ectopic digit formation, indicating a posterior molecular identity for the supernumerary digits. The specific skeletal phenotypes, which include duplication of digits and distal zeugopods but no overt posteriorization, differ from those of other Twist1 conditional knockout mutants. This outcome may be attributed to the deferment of Twist1 ablation to a later time frame of limb morphogenesis, which leads to the ectopic activation of posterior genes in the anterior tissues after the establishment of anterior-posterior anatomical identities in the forelimb bud.