Inactivation of Zeb1 in GRHL2-deficient mouse embryos rescues mid-gestation viability and secondary palate closure

摘要

Cleft lip and palate are common birth defects resulting from failure of the facial processes to fuse during development. The mammalian grainyhead-like ( Grhl1-3 ) genes play key roles in a number of tissue fusion processes including neurulation, epidermal wound healing and eyelid fusion. One family member, Grhl2 , is expressed in the epithelial lining of the first pharyngeal arch in mice at embryonic day (E)10.5, prompting analysis of the role of this factor in palatogenesis. Grhl2 -null mice die at E11.5 with neural tube defects and a cleft face phenotype, precluding analysis of palatal fusion at a later stage of development. However, in the first pharyngeal arch of Grhl2 -null embryos, dysregulation of transcription factors that drive epithelial-mesenchymal transition (EMT) occurs. The aberrant expression of these genes is associated with a shift in RNA-splicing patterns that favours the generation of mesenchymal isoforms of numerous regulators. Driving the EMT perturbation is loss of expression of the EMT-suppressing transcription factors Ovol1 and Ovol2 , which are direct GRHL2 targets. The expression of the miR-200 family of microRNAs, also GRHL2 targets, is similarly reduced, resulting in a 56-fold upregulation of Zeb1 expression, a major driver of mesenchymal cellular identity. The critical role of GRHL2 in mediating cleft palate in Zeb1sup?/?/sup mice is evident, with rescue of both palatal and facial fusion seen in Grhl2sup?/?/sup;Zeb1sup?/?/sup embryos. These findings highlight the delicate balance between GRHL2/ZEB1 and epithelial/mesenchymal cellular identity that is essential for normal closure of the palate and face. Perturbation of this pathway may underlie cleft palate in some patients. RESULTS The MXPs of Grhl2 sup?/?/sup embryos are smaller than those of wild-type littermates at E10.5 Grhl2 is expressed in the epithelium lining the maxillary and nasal processes at E10.5 ( Brouns et al., 2011 ), with expression continuing in oral epithelium until E17.5 ( Auden et al., 2006 ). This expression pattern is consistent with Grhl2 playing a role in closure of the palate. As Grhl2sup?/?/sup embryos have a cleft face, we first determined whether their maxillary and nasal processes formed normally between E9.5 and E10.5. Scanning electron micrographs revealed that the MXPs were abnormally small in Grhl2sup?/?/sup embryos at E10.5 ( Fig.?1 A-D). The mean ratio of MXP/MDP area was 0.96 for wild-type embryos and 0.78 for Grhl2sup?/?/sup embryos, although this difference was not statistically significant. Next, we dissected the first pharyngeal arch (PA1) at E10.5, and noted again that the Grhl2sup?/?/sup MXPs were reduced in size ( Fig.?1 E). Images of the head of E10.5 embryos revealed that the nasal processes were malformed in Grhl2sup?/?/sup embryos, making it infeasible to determine whether the lambdoid junction had formed ( Fig.?1 F). We cut defined section planes through the nasal and maxillary processes of E10.5 embryos and determined that the nasal pits were present in Grhl2sup?/?/sup embryos, as were the MXPs, which stained darkly with Haematoxylin and Eosin (H&E) ( Fig.?1 G,H). These findings showed that Grhl2sup?/?/sup embryos have small MXPs at E10.5. In order to determine whether Grhl2sup?/?/sup embryos had a deficiency of cranial neural crest cells, we stained coronal sections through E10.5 PA1 for SOX9. Grhl2sup?/?/sup embryos exhibited normal numbers of SOX9-positive neural crest cells in their MXP and mandibular process (MDP) mesenchyme at E10.5 ( Fig.?S1A , B ). In order to determine whether GRHL2 is required in neural crest cells for palate closure, we conditionally inactivated Grhl2 using Wnt1Cre ( Danielian et al., 1998 ). Grhl2sup+/?/sup;Wnt1Cresup+/sup × Grhl2supfl/fl/sup timed matings were performed and the embryos harvested at E17.5. Of 55 embryos harvested, 16 (29%) were Grhl2supfl/?/sup;Wnt1Cresup+/sup . This was not significantly different from the Mendelian expectation of 25%, indicating that embryos lacking Grhl2 in neural crest cells were viable to E17.5. Furthermore, skeletal preparations stained with Alizarin Red and Alcian Blue revealed normal palate closure in Grhl2supfl/?/sup;Wnt1Cresup+/sup conditional knockout embryos at E17.5 ( Fig.?S1C ). These findings indicate that GRHL2 is not required for neural crest cells to populate the first pharyngeal arch or required in neural crest cells for palate closure.