INVESTIGATING MAMMALIAN DEVELOPMENT USING MOUSE FORWARD GENETICS
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Forward genetics allows the identification of novel genes involved in a particular biological process. We performed an ENU forward mutagenesis screen in mice aimed at identifying genes which regulate early organ morphogenesis during development. Fifteen mouse mutants were identified in the screen which possess cardiovascular, craniofacial, extraembryonic, or general body morphology defects. After performing preliminary characterization of all 15 lines, we identified a likely causative mutation in 14 of the 15 mutants. 1D and 13B mutants both exhibit craniofacial defects and were selected for in-depth characterization to identify the molecular mechanisms whereby they regulate embryogenesis. 1D mutants are characterized by an open neural tube in the hindbrain region. This phenotype is similar to human embryos with exencephaly, a congenital birth malformation of high incidence in the human population. Positional cloning of 1D identified a mutation in SPCA1, a Golgi-localized pump that controls calcium homeostasis. Results from the molecular characterization of mouse 1D mutants, as well as from time lapse microscopy of chicken embryos, revealed that calcium is tightly regulated during neural tube closure and that calcium homeostasis is required to promote apical constriction of neuroepithelial cells. These results show that SPCA1 activity is required to regulate the actomyosin dynamics that propel apical constriction, and that the actin severing protein, Cofilin 1, is a key mediator of SPCA1 function. Together, my findings provide the first genetic evidence that calcium homeostasis is needed for neural tube closure, opening a new window into understanding the etiology of human neural tube defects. 13B mutants have neural tube defects accompanied by a suite of other malformations including randomized L-R patterning and maxillary overgrowth. Molecular characterization revealed that all 13B phenotypes result from the absence of cilia, an organelle important for neural tube patterning and for the establishment of L-R asymmetry. Exome sequencing of 13B embryos identified a nonsense mutation in Pibf1. I show that PIBF1 is required for ciliogenesis during early embryonic development and identify a novel role for PIBF1 in regulating centrosome duplication. These findings highlight the importance of PIBF1 in regulating multiple aspects of centrosome biology, and provide a model for understanding how defects in these processes contribute to human ciliopathies.
Supplemental file(s) description: High magnification view of a field of cells in the cranial neural plate of an HH6 embryo expressing GCaMP6s (top panel, green) and F-tractin (bottom panel, red)., Unicellular Ca[2+] pulses during cranial chick neurulation at HH6, Unicellular and multicellular Ca[2+] pulses during caudal chick neurulation at HH7, High magnification view of a field of cells in the cranial neural plate of an HH6 embryo expressing GCaMP6s (top panel, green) and F-tractin (bottom panel, red).
calcium; Actomyosin; Apical Constriction; Cofilin 1; Neural Tube; SPCA1; Developmental biology; Genetics; Molecular biology
Garcia-Garcia, Maria J.
Schimenti, John C.; Kurpios, Natasza
Genetics and Development
Ph. D., Genetics and Development
Doctor of Philosophy
Attribution 4.0 International
dissertation or thesis
Except where otherwise noted, this item's license is described as Attribution 4.0 International