Study of neural tube closure using forward genetic screens in mice: Novel insight into Rb and cell cycle regulation, and enteric neural crest migration by PP1 and actin interacting protein

One of the most exciting questions in developmental biology is how the nervous system is established and interconnected to form the central nervous system (CNS) and the peripheral nervous system (PNS), the latter which originates from the neural crest, which migrate away from the dorsal aspect of the neural tube. One of the key steps in neural development involves a complex morphogenesic process to close the neural tube. Defects in neural tube closure in humans are among the most prevalent of congenital malformations with an incidence of 1 out of every 1000 births. To study neural tube closure, we undertook a forward genetic ethylnitrosurea (ENU) screen in mice. We identified a variety of mouse mutants from the screen that displayed defects in neural morphogenesis and patterning. Here, I focus on the humpty dumpty (humdy) mutant line that displays defects in neural tube, eye, and enteric nervous system (ENS) development. To determine the gene whose function is required during embryogenesis to regulate the development of these tissues, I cloned the humdy mutant gene. The humdy gene encodes Phactr4, a novel Protein Phosphatase 1 (PP1) and actin regulator family member. The humdy allele carries a missense mutation which changes Arginine (Arg) to Proline (Pro) in the conserved C-terminal region. Moreover, this mutation disrupts PP1 binding but not actin binding.;In the studies described in Chapter 2, I investigated the mechanistic basis of the defect in neural tube closure (exencephaly) and in optic fissure closure (coloboma). Phactr4 is normally expressed in the neural tube and retina and, in the humdy mutant embryos, there was greatly increased proliferation and reduced differentiation in these tissues. Regulated proliferation is particularly striking at E9.5, where there is much less proliferation in the ventral half of the neural tube, where Phactr4 is most strongly expressed, relative to the dorsal half.;In the second set of studies described in Chapter 3, I have explored another phenotype in humdy mutant embryos with relevance to human birth defects. Hirschsprung disease patients have defects in the formation of the Enteric Nervous System (ENS) and this leads to an inability to properly move material through the gut. Humdy mutant embryos display a very similar intestinal defect due to a reduced number of ENS cells in the gut. (Abstract shortened by UMI.).