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The highly-conserved Grainyhead-like (Grhl) transcription factors are critical regulators of embryogenesis that regulate cellular survival, proliferation, migration and epithelial integrity, especially during the formation of the craniofacial skeleton. Family member Grhl2 is expressed throughout epithelial tissues during development, and loss of Grhl2 function leads to significant defects in neurulation, abdominal wall closure, formation of the face and fusion of the maxilla/palate. Whereas numerous downstream target genes of Grhl2 have been identified, very little is known about how this crucial developmental transcription factor itself is regulated. Here, using in silico and in utero expression analyses and functional deletion in mice, we have identified a novel 2.4 ​kb enhancer element (mm1286) that drives reporter gene expression in a pattern that strongly recapitulates endogenous Grhl2 in the craniofacial primordia, modulates Grhl2 expression in these tissues, and augments Grhl2-mediated closure of the secondary palate. Deletion of this genomic element, in the context of inactivation of one allele of Grhl2 (through generation of double heterozygous Grhl2+/-;mm1286+/- mice), results in a significant predisposition to palatal clefting at birth. Moreover, we found that a highly conserved 325 bp region of mm1286 is both necessary and sufficient for mediating the craniofacial-specific enhancer activity of this region, and that an extremely well-conserved 12-bp sequence within this element (CTGTCAAACAGGT) substantially determines full enhancer function. Together, these data provide valuable new insights into the upstream genomic regulatory landscape responsible for transcriptional control of Grhl2 during palatal closure.

The highly-conserved Grainyhead-like (Grhl) transcription factors are critical regulators of embryogenesis that regulate cellular survival, proliferation, migration and epithelial integrity, especially during the formation of the craniofacial skeleton. Family member Grhl2 is expressed throughout epithelial tissues during development, and loss of Grhl2 function leads to significant defects in neurulation, abdominal wall closure, formation of the face and fusion of the maxilla/palate. Whereas numerous downstream target genes of Grhl2 have been identified, very little is known about how this crucial developmental transcription factor itself is regulated. Here, using in silico and in utero expression analyses and functional deletion in mice, we have identified a novel 2.4 ​kb enhancer element (mm1286) that drives reporter gene expression in a pattern that strongly recapitulates endogenous Grhl2 in the craniofacial primordia, modulates Grhl2 expression in these tissues, and augments Grhl2-mediated closure of the secondary palate. Deletion of this genomic element, in the context of inactivation of one allele of Grhl2 (through generation of double heterozygous Grhl2+/-;mm1286+/- mice), results in a significant predisposition to palatal clefting at birth. Moreover, we found that a highly conserved 325 bp region of mm1286 is both necessary and sufficient for mediating the craniofacial-specific enhancer activity of this region, and that an extremely well-conserved 12-bp sequence within this element (CTGTCAAACAGGT) substantially determines full enhancer function. Together, these data provide valuable new insights into the upstream genomic regulatory landscape responsible for transcriptional control of Grhl2 during palatal closure.

Vitamin D plays an important pleiotropic role in maintaining global homeostasis of the human body. Its functions go far beyond skeletal health, playing a crucial role in a plethora of cellular functions, as well as in extraskeletal health, ensuring the proper functioning of multiple human organs, including the skin. Genes from the Grainyhead-like (GRHL) family code for transcription factors necessary for the development and maintenance of various epithelia. Even though they are involved in many processes regulated by vitamin D, a direct link between vitamin D-mediated cellular pathways and GRHL genes has never been described. We employed various bioinformatic methods, quantitative real-time PCR, chromatin immunoprecipitation, reporter gene assays, and calcitriol treatments to investigate this issue. We report that the vitamin D receptor (VDR) binds to a regulatory region of the Grainyhead-like 1 (GRHL1) gene and regulates its expression. Ectopic expression of VDR and treatment with calcitriol alters the expression of the GRHL1 gene. The evidence presented here indicates a role of VDR in the regulation of expression of GRHL1 and correspondingly a role of GRHL1 in mediating the actions of vitamin D. [ABSTRACT FROM AUTHOR]

Defective tissue fusion during mammalian embryogenesis results in congenital anomalies, such as exencephaly, spina bifida and cleft lip and/or palate. The highly conserved transcription factor grainyhead-like 2 (Grhl2) is a crucial regulator of tissue fusion, with mouse models lacking GRHL2 function presenting with a fully penetrant open cranial neural tube, facial and abdominal clefting (abdominoschisis), and an open posterior neuropore. Here, we show that GRHL2 interacts with the soluble morphogen protein and bone morphogenetic protein (BMP) inhibitor noggin (NOG) to impact tissue fusion during development. The maxillary prominence epithelium in embryos lacking Grhl2 shows substantial morphological abnormalities and significant upregulation of NOG expression, together with aberrantly distributed pSMAD5-positive cells within the neural crest cell-derived maxillary prominence mesenchyme, indicative of disrupted BMP signalling. Reducing this elevated NOG expression (by generating Grhl2−/−;Nog+/− embryos) results in delayed embryonic lethality, partial tissue fusion rescue, and restoration of tissue form within the craniofacial epithelia. These data suggest that aberrant epithelial maintenance, partially regulated by noggin-mediated regulation of BMP-SMAD pathways, may underpin tissue fusion defects in Grhl2−/− mice. [ABSTRACT FROM AUTHOR]

Background: The highly conserved Grainyhead‐like (Grhl) family of transcription factors play critical roles in the development of the neural tube and craniofacial skeleton. In particular, deletion of family member Grainyhead‐like 2 (Grhl2) leads to mid‐gestational embryonic lethality, maxillary clefting, abdominoschisis, and both cranial and caudal neural tube closure defects. These highly pleiotropic and systemic defects suggest that Grhl2 plays numerous critical developmental roles to ensure correct morphogenesis and patterning. Results: Here, using four separate Cre‐lox conditional deletion models, as well as one genetic epistasis approach (Grhl2+/−;Edn1+/− double heterozygous mice) we have investigated tissue‐specific roles of Grhl2 in embryonic development, with a particular focus on the craniofacial skeleton. We find that loss of Grhl2 in the pharyngeal epithelium (using the ShhCre driver) leads to low‐penetrance micrognathia, whereas deletion of Grhl2 within the ectoderm of the pharynx (NestinCre) leads to small, albeit significant, differences in the proximal‐distal elongation of both the maxilla and mandible. Loss of Grhl2 in endoderm (Sox17‐2aiCre) resulted in noticeable lung defects and a single instance of secondary palatal clefting, although formation of other endoderm‐derived organs such as the stomach, bladder and intestines was not affected. Lastly, deletion of Grhl2 in cells of the neural crest (Wnt1Cre) did not lead to any discernible defects in craniofacial development, and similarly, our epistasis approach did not detect any phenotypic consequences of loss of a single allele of both Grhl2 and Edn1. Conclusion: Taken together, our study identifies a pharyngeal‐epithelium intrinsic, non‐cell‐autonomous role for Grhl2 in the patterning and formation of the craniofacial skeleton, as well as an endoderm‐specific role for Grhl2 in the formation and establishment of the mammalian lung. Key Findings: Deletion of Grhl2 in the Shh‐expressing pharyngeal epithelium leads to low‐penetrance micrognathia.Deletion of Grhl2 in the Sox17‐expressing endoderm leads to subtantial lung formation and cellularity defects, and is incompatible with life.Deletion of Grhl2 in the endoderm of intestine, bladder or other tissues in which Grhl2 expression has been reported does not result in detectable developmental defects. [ABSTRACT FROM AUTHOR]