The cerebellum (Cb) is implicated in contributing to cognitive and social functions, in addition to having a critical role in skilled motor performance. Accordingly, the Cb is associated with many debilitating developmental diseases including autism. One gene that regulates development of the Cb and has been implicated in autism is engrailed 2 (EN2), based on human studies and the finding that En2 null mice not only have deficits in motor control, but also in social behaviors and cognition. Before we can begin to understand higher order functions of the Cb, we must gain more insight into the basic cellular and genetic processes that regulate Cb development. Our approach is to use the two EN homeobox transcription factors as molecular entry points to study Cb development, as we discovered that En1/2 conditional mutants have defects in Cb morphology, molecular patterning and afferent circuitry. We will now direct our studies towards distinguishing the cellular processes regulated by En1/2 and identifying EN2 target genes critical for these processes that could be susceptibility loci for complex behavioral diseases. We will focus on the granule neurons (GNs) that comprise the main recipients of input to the Cb and the deep cerebellar nuclei (DCN) that generate the output. DCN neurons are consistently reduced in autistic patients, which could be a primary cause of some behaviors and also reflect defects elsewhere in the Cb circuit. We will apply a multi-facetted approach that combines novel genetic techniques in mice to study normal and mutant behaviors of GNs and DCN projection neurons, including a mosaic mutant analysis using our MASTR technique and a new method to precisely target over- expression of EN2 to GNs and the DCN to test sufficiency of EN2 to alter differentiation. We will then apply both mutant approaches to live imaging of GNs as a different approach to study Cb morphogenesis and cell proliferation/differentiation. We will also address the question of whether feed back loops ensure the correct proportion of cell types is produced by studying the interaction between En1/2 and the sonic hedgehog (SHH) pathway. Finally, in order to identify the first direct targets of EN2 in the brain, we are engineering new mouse strains expressing a tagged form of EN2.
Aim 1. Study the cellular behaviors regulated by En1/2 in developing GNs and DCN projection neurons using conditional genetics and characterizing cellular behaviors in vivo, and in vitro with live imaging.
Aim 2. Identify critical target genes o EN1/2 in GN precursors and DCN projection neurons using comparative microarray analysis and ChIP-seq.
Given the involvement of the Cb in social, emotional and cognitive functions, it is critical to understand how key developmental genes regulate cellular processes that underlie Cb development and circuit formation. Our identification of EN2 target genes should provide additional genes for diagnosing diseases involving the Cb, as well as proteins for which drugs should be more feasible to develop against than for EN2. PUBLIC HEALTH RELEVANCE: The cerebellum is associated with many debilitating developmental diseases including autism, as it is critical for motor coordination and also modulates language, reasoning, social and affective processes, and has more neurons than the rest of the human brain put together. Multiple studies have found an association between the gene encoding the EN2 transcription factor and autism, and mice lacking En2 have abnormal social behavior. An identification of genes and cellular processes regulated by EN2 should provide additional genetic loci for diagnosing diseases involving the cerebellum, as well as proteins for which drugs should be more feasible to develop against than for EN2.
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