The neurodevelopmental transcriptome of the Drosophila melanogaster microcephaly gene abnormal spindle reveals a role for temporal transcription factors and the immune system in regulating brain size

Abstract

The coordination of cellular behaviors during neurodevelopment is critical for determining the form, function, and size of the central nervous system. Mutations in the vertebrate Abnormal Spindle-Like, Microcephaly Associated (ASPM) gene and its Drosophila melanogaster ortholog abnormal spindle (asp) lead to microcephaly, a reduction in overall brain size whose etiology remains poorly defined. Here we provide the neurodevelopmental transcriptional landscape for a Drosophila model for autosomal recessive primary microcephaly (MCPH) and extend our findings into the functional realm in an attempt to identify the key cellular mechanisms responsible for Asp-dependent brain growth and development. We identify multiple transcriptomic signatures, including new patterns of co-expressed genes in the developing CNS. Defects in optic lobe neurogenesis were detected in larval brains through downregulation of temporal transcription factors (tTFs) and Notch signaling targets, which correlated with a significant reduction in brain size and total cell numbers during the neurogenic window of development. We also found inflammation as a hallmark of asp MCPH brains, detectable throughout every stage of CNS development, which also contributes to the brain size phenotype. Finally, we show that apoptosis is not a primary driver of the asp MCPH phenotype, further highlighting an intrinsic Asp-dependent neurogenesis promotion mechanism that is independent of cell death. Collectively, our results suggest that the etiology of asp MCPH is complex and that a comprehensive view of the cellular basis of the disorder requires an understanding of how multiple pathway inputs collectively determine the microcephaly phenotype. AUTHOR SUMMARYAutosomal recessive primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by a reduction in brain size, intellect, and life span. Over 30 genes have been found mutated in human MCPH patients, with Abnormal Spindle-Like, Microcephaly Associated (ASPM) being the most common. Although the clinical aspects of the disorder are well-characterized, the underlying cellular and molecular mechanisms are not. The fruit fly, Drosophila melanogaster, has an ortholog of the ASPM gene named abnormal spindle (asp), and mutations also give rise to fruit flies with small brains. It had previously been suggested that mitotic spindle defects were responsible for the asp MCPH phenotype, preventing neural stem cells from dividing properly to generate the necessary number of neurons and glia in the brain. However, genetic studies in flies showed that this wasnt the case, suggesting that our knowledge of MCPH remains incomplete and must be revised. In this manuscript, we identified new pathways important for asp-dependent brain growth through transcriptional profiling. We found a number of key pathways disrupted in asp mutants, which have not been described previously. Using genetic tools in the fly, we tested a subset of these pathways to identify their contributions to the brain growth defects seen in asp mutants. Our results add to the growing number of pathways necessary for brain growth control, and provide a suitable foundation for follow-up genetic studies to assess MCPH.