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TWIST1, a basic helix-loop-helix transcription factor is essential for the development of cranial mesoderm and cranial neural crest-derived craniofacial structures. We have previously shown that, in the absence of TWIST1, cells within the cranial mesoderm adopt an abnormal epithelial configuration via a process reminiscent of a mesenchymal to epithelial transition (MET). Here, we show by gene expression analysis that loss of TWIST1 in the cranial mesoderm is accompanied by a reduction in the expression of genes that are associated with cell-extracellular matrix interactions and the acquisition of mesenchymal characteristics. By comparing the transcriptional profiles of cranial mesoderm-specific Twist1 loss-of-function mutant and control mouse embryos, we identified a set of genes that are both TWIST1-dependent and predominantly expressed in the mesoderm. ChIP-seq was used to identify TWIST1-binding sites in an in vitro model of a TWIST1-dependent mesenchymal cell state, and the data were combined with the transcriptome data to identify potential target genes. Three direct transcriptional targets of TWIST1 (Ddr2, Pcolce and Tgfbi) were validated by ChIP-PCR using mouse embryonic tissues and by luciferase assays. Our findings reveal that the mesenchymal properties of the cranial mesoderm are likely to be regulated by a network of TWIST1 targets that influences the extracellular matrix and cell-matrix interactions, and collectively they are required for the morphogenesis of the craniofacial structures., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The basic helix-loop-helix transcription factor Twist1 is a key regulator of craniofacial development. Twist1-null mouse embryos exhibit failure of cephalic neural tube closure and abnormal head development and die at E11.0. To dissect the function of Twist1 in the cranial mesoderm beyond mid-gestation, we used Mesp1-Cre to delete Twist1 in the anterior mesoderm, which includes the progenitors of the cranial mesoderm. Deletion of Twist1 in mesoderm cells resulted in loss and malformations of the cranial mesoderm-derived skeleton. Loss of Twist1 in the mesoderm also resulted in a failure to fully segregate the mesoderm and the neural crest cells, and the malformation of some cranial neural crest-derived tissues. The development of extraocular muscles was compromised whereas the differentiation of branchial arch muscles was not affected, indicating a differential requirement for Twist1 in these two types of craniofacial muscle. A striking effect of the loss of Twist1 was the inability of the mesodermal cells to maintain their mesenchymal characteristics, and the acquisition of an epithelial-like morphology. Our findings point to a role of Twist1 in maintaining the mesenchyme architecture and the progenitor state of the mesoderm, as well as mediating mesoderm-neural crest interactions in craniofacial development., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Using a Cre-mediated conditional deletion approach, we have dissected the function of Twist1 in the morphogenesis of the craniofacial skeleton. Loss of Twist1 in neural crest cells and their derivatives impairs skeletogenic differentiation and leads to the loss of bones of the snout, upper face and skull vault. While no anatomically recognizable maxilla is formed, a malformed mandible is present. Since Twist1 is expressed in the tissues of the maxillary eminence and the mandibular arch, this finding suggests that the requirement for Twist1 is not the same in all neural crest derivatives. The effect of the loss of Twist1 function is not restricted to neural crest-derived bones, since the predominantly mesoderm-derived parietal and interparietal bones are also affected, presumably as a consequence of lost interactions with neural crest-derived tissues. In contrast, the formation of other mesodermal skeletal derivatives such as the occipital bones and most of the chondrocranium are not affected by the loss of Twist1 in the neural crest cells.