Involvement of homeobox transcription factor Mohawk in palatogenesis.

Palatogenesis is affected by many factors, including gene polymorphisms and exposure to toxic chemicals during sensitive developmental periods. Cleft palate is one of the most common congenital anomalies, and ongoing efforts to elucidate the molecular mechanisms underlying palatogenesis are providing useful insights to reduce the risk of this disorder. To identify novel potential regulators of palatogenesis, we analyzed public transcriptome datasets from a mouse model of cleft palate caused by selective deletion of transforming growth factor-ß (TGFß) receptor type 2 in cranial neural crest cells. We identified the homeobox transcription factor Mohawk (Mkx) as a gene downregulated in the maxilla of TGFß knockout mice compared with wild-type mice. To examine the role of mkx in palatogenesis, we used CRISPR/Cas9 editing to generate zebrafish with impaired expression of mkxa and mkxb, the zebrafish homologs of Mkx. We found that mkx crispants expressed reduced levels of gli1, a critical transcription factor in the Sonic hedgehog (SHH) signaling pathway that plays an important role in the regulation of palatogenesis. Furthermore, we found that mkxa-/- zebrafish were more susceptible than mkxa+/+ zebrafish to the deleterious effects of cyclopamine, an inhibitor of SHH signaling, on upper jaw development. These results suggest that Mkx may be involved in palatogenesis regulated by TGFß and SHH signaling, and that impairment in Mkx function may be related to the etiology of cleft palate.

Generation of a Transgenic Zebrafish Line for In Vivo Assessment of Hepatic Apoptosis.

Hepatic apoptosis is involved in a variety of pathophysiologic conditions in the liver, including hepatitis, steatosis, and drug-induced liver injury. The development of easy-to-perform and reliable in vivo assays would thus greatly enhance the efforts to understand liver diseases and identify associated genes and potential drugs. In this study, we developed a transgenic zebrafish line that was suitable for the assessment of caspase 3 activity in the liver by using in vivo fluorescence imaging. The larvae of transgenic zebrafish dominantly expressed Casper3GR in the liver under control of the promoter of the phosphoenolpyruvate carboxykinase 1 gene. Casper3GR is composed of two fluorescent proteins, tagGFP and tagRFP, which are connected via a peptide linker that can be cleaved by activated caspase 3. Under tagGFP excitation conditions in zebrafish that were exposed to the well-characterized hepatotoxicant isoniazid, we detected increased and decreased fluorescence associated with tagGFP and tagRFP, respectively. This result suggests that isoniazid activates caspase 3 in the zebrafish liver, which digests the linker between tagGFP and tagRFP, resulting in a reduction in the Förster resonance energy transfer to tagRFP upon tagGFP excitation. We also detected isoniazid-induced inhibition of caspase 3 activity in zebrafish that were treated with the hepatoprotectants ursodeoxycholic acid and obeticholic acid. The transgenic zebrafish that were developed in this study could be a powerful tool for identifying both hepatotoxic and hepatoprotective drugs, as well as for analyzing the effects of the genes of interest to hepatic apoptosis.