A vertebrate model of viral and hereditary microcephaly

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PROJECT SUMMARY/ABSTRACT Microcephaly, in which head and brain development are severely inhibited, can result in extreme intellectual disability. Microcephaly has many different causes, including gene mutation, pathogen infection and chemical exposure. While the causes of microcephaly may be broad, there may be shared molecular mechanisms involved. Recently published results show how Zika virus non-structural protein 4A (NS4A) inhibits brain development in fruit flies by inhibiting the same ANKLE2 protein whose function is disrupted by gene mutation in a hereditary form of microcephaly. This suggests that there may be key similarities in the molecular mechanisms contributing to a viral and hereditary form of microcephaly. Studying these molecular similarities in vertebrate models is essential to understanding disease in humans since vertebrates have different brain structure and genes that regulate brain development. However, the tools to study this, namely a strong vertebrate model system, do not exist. The primary goal of the proposed project is to fill these major gaps in knowledge by developing a vertebrate model of viral and hereditary microcephaly acting through the ANKLE2 pathway. For the hereditary model, ankle2 will be mutated using CRISPR/Cas9 genome editing. For the viral model, Zika virus NS4A protein will be expressed using Tol2 transgenesis. Defects in development, including various externally measured morphology metrics, brain size, structure and cellular defects in proliferation and survival will be assayed using anatomical measurement and immunofluorescence microscopy. When completed, this new model system will lay the foundation for a molecular and cellular level understanding of ANKLE2 function during vertebrate brain development and how it is disrupted by gene mutation and viral infection. In the long-term, it will enable high- throughput screening of chemical sensitizers and inhibitors of microcephaly, allow the exploration vertebrate- specific mechanisms of brain development in vivo, and behavioral studies.