ROLE OF THE CHROMATIN REGULATOR SETD5 DURING ZEBRAFISH BRAIN DEVELOPMENT

Loss-of-function (LoF) mutations in one SETD5 allele in humans have been recently identified as relatively frequent mendelian causes of both intellectual disability (ID) and autistic spectrum disorders, the most common neurodevelopmental disorders (3-5% of the population). Interestingly, SETD5 falls within the critical interval deleted in the 3p25.3 microdeletion syndrome which is characterized by ID, microcephaly and congenital heart defects. These findings suggest that SETD5 haploinsufficiency is sufficient to cause the disease. SETD5 encodes for a putative histone H3 methyltransferase highly expressed in the brain, whose activity has not been clearly demonstrated yet. The aim of this study is to generate and characterize zebrafish models in which setd5 has been knocked down or knocked out. As a first approach, we have targeted zebrafish setd5 with a specific antisense morpholino oligonucleotide. External inspection and in situ hybridization analysis with district-specific markers indicate that setd5 knockdown embryos display microcephaly, cardiac edema and reduced locomotor activity response. Compared to embryos injected with a control morpholino, setd5 LoF brains, despite their remarkable reduced size, show a statistically significant increase of mitotic cells as assessed by immunostaining with anti-phospho-H3 antibodies, while immunoreactivity to the neuronal differentiation marker HuC is not changed. We are currently evaluating whether setd5 knockdown results in a possible mitotic arrest of developing brain cells followed by apoptosis. Furthermore, exploiting the Crispr/Cas9 genome editing strategy, we are planning to establish stable setd5 knockout zebrafish lines. These animal models will be extremely useful to identify the molecular mechanisms underlying SETD5 LoF phenotype and to screen for drugs and chemical compounds able to rescue or alleviate the developmental defects.