Mitfover-expression leads to microphthalmia and coloboma inMitf-cremice

Abstract

ABSTRACTThe Microphthalmia associated transcription factor (Mitf) is a critical regulator of the melanocyte lineage and also plays an important role in eye development. Mitf activity in different cell types is controlled in part by ten alternative promoters and their resulting isoforms. A useful tool for melanocyte-based research, theMitf-cretransgene was designed to express Cre recombinase from the Mitf-M promoter, which is melanocyte specific. However,Mitf-cremice are also microphthalmic, perhaps because of insertional mutagenesis or disrupted gene expression. Here, we investigated these possibilities. We determined that the eye phenotype arises early, withMitf-creembryos at E13.5 exhibiting variable ocular sizes and abnormalities, but all with coloboma. Targeted locus amplification and next generation sequencing indicated that multiple copies of the transgene integrated into an intergenic region on chromosome 2, in betweenSpred1andMeis2. The BAC transgene used to makeMitf-crewas larger than expected, carrying three upstream alternative promoters, Mitf-H, Mitf-D, and Mitf-B, which could express their isoforms intact off the transgene. RT-qPCR using eye tissue demonstrated a 5-fold increase inMitftranscripts containing exon 1B1b, which is shared by Mitf-H, Mitf-D, and Mitf-B, whileSpred1andMeis2did not differ in their expression. These findings clarify and support the usage ofMitf-crein conditional mutagenesis in melanocytes. The specific over-expression of the Mitf-H and Mitf-D isoforms, which are preferentially expressed in the RPE, presents a unique resource for those interested in eye development and coloboma.