Centrosome Regulation in Development and Dysregulation in Disease

National Heart, Lung, And Blood Institute

Investigators

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Abstract

An intimate link between centrosome function and neurogenesis was revealed by the identifying many genes with centrosome-associated functions mutated in microcephaly disorders. Consistent with the major role of the centrosome in mitosis, mutations in these centrosome-related microcephaly (CRM) genes are thought to affect neurogenesis by depleting the pool of neural progenitor cells, primarily through apoptosis as a consequence of mitotic failure, or premature differentiation as a consequence of cell cycle delay and randomization of spindle orientation. However, as suggested by the wide range of microcephaly phenotypes and the multifunctional nature of many CRM proteins, this picture of CRM gene function is incomplete. Understanding how events at the molecular and cellular scales contribute to tissue form and function is key to uncovering mechanisms driving animal development, physiology and disease. Elucidating these mechanisms has been enhanced through the study of model organisms and the use of sophisticated genetic, biochemical and imaging tools. Here we highlight how non-destructive imaging of Drosophila melanogaster at high resolution using micro computed tomography (-CT) allows for visualization of development and tissue morphogenesis at an unprecedented level of detail. To demonstrate the power of -CT, we characterized the developing brain from larval to adult stages, including the stages of pupation that have been understudied by current light microscopy methods due to the delicate nature of metamorphosing tissue. We uncover a series of novel morphogenetic changes in brain volume as pupation proceeds during normal development. We then probed a series of microcephaly genes to determine when brain development might go awry. For example, we show that mutations inspc105r (the fly ortholog of the kinetochore protein KNL1) leads to massively reduced head structures due to complete failure in pupal brain development. To demonstrate how -CT can be incorporated into existing experimental workflows and combined with traditional light microscopy and molecular genetic approaches, we characterized two models of human microcephaly by evaluating mutations in the Abnormal Spindle (asp) and WD Repeat Domain 62 (wdr62). Loss of asp leads to significant reduction in brain size, coupled with severe morphology defects of the visual processing center of the fly (retina, lamina, and optic lobes), which can be enhanced through the loss of wdr62. Structure function analysis of Asp transgenes revealed that the N-terminal of Asp (AspMF) is sufficient to rescue both the volume and morphology defects of the asp mutant. Interestingly, live imaging of mitotic divisions within developing larval brains from asp mutant animals revealed that Asp is critical for maintaining centrosome-spindle pole cohesion and spindle pole focusing of microtubule minus ends in neuroblasts, whose loss severely disrupts the ability of these stem cells to divide correctly to generate neurons and glia. Surprisingly, identical cell division defects were observed in AspMF rescue brains, suggesting that Asp contributes to proper brain development through novel mechanisms independent of its role as a mitotic regulator. Our work demonstrates that -CT is a versatile and accessible tool that complements standard imaging techniques, capable of uncovering novel biological mechanisms that have remained undocumented due to limitations of current methods. Beyond microcephaly research, my lab is also focused on mechanisms underlying proper sperm formation. The centriole, or basal body, is the center of attachment between the sperm head and tail. While the distal end of the centriole templates the cilia, the proximal end associates with the nucleus. Using Drosophila, we identify a centriole-centric mechanism that ensures proper proximal end docking to the nucleus. This mechanism relies on the restriction of pericentrin-like protein (PLP) and the pericentriolar material (PCM) to the proximal end of the centriole. PLP is restricted proximally by limiting its mRNA and protein to the earliest stages of centriole elongation. Ectopic positioning of PLP to more distal portions of the centriole is sufficient to redistribute PCM and microtubules along the entire centriole length. This results in erroneous, lateral centriole docking to the nucleus, leading to spermatid decapitation as a result of a failure to form a stable head-tail linkage. This study has now guided our ongoing screen to uncover additional proteins required for centriole attachment to the nucleus, and thus required for proper fertility.