Understanding developmental patterning's influence on morphogenesis

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Abstract Morphogenetic events utilize precisely timed changes in cell shape. One of the fundamental mechanisms cells use to change their shape is apical constriction. Apical constriction relies on the contraction of cortical actomyosin networks that causes the apical side of a cell to shrink, resulting in tissue morphogenesis. In humans, apical constriction aids the internalization of the future spinal cord and brain in a process known as neural tube closure. Failure of apical constriction can lead to neural tube defects, which accounts for birth defects in 1 out of every 3,000 live births. Therefore, uncovering the processes that govern apical constriction will advance our understanding of basic mechanisms underlying cell shape changes, causes, and potential treatments for neural tube defects. Despite current knowledge of developmental patterning of apical constriction, precise genetic mechanisms that govern which cells undergo apical constriction, how the apical surface is determined, and when to constrict, remain only partially understood. I plan to use Caenorhabditis elegans (C. elegans) gastrulation, a morphogenetic event driven by apical constriction, to address these issues. Gastrulation in C. elegans starts with the internalization of the two endodermal precursor cells (EPCs), which depend on the spatial and temporal precision of the expression of cell fate specification factors end-1 and end-3. However, mechanistic links between end-1,3 and the resulting apical constriction remain largely unknown. Using the genetically tractable and optically clear C. elegans, I plan to dissect the cellular mechanisms that translate developmental patterning into specific, localized, and precisely timed cell shape changes. Comparing the transcriptome of wild-type and end-3 null embryos, I identified thirty target genes whose expression depends on end-3. After screening these genes, I identified ten new genes that contribute to C. elegans gastrulation. In Aim 1, I will use a variety of cell biological approaches to identify the mechanisms by which some of these genes couple developmental patterning to changing cell shape. Aim 2 focuses on the myosin-activating kinase MRCK-1 localizes to the apical cell cortex of EPCs and is required for apical constriction. MRCK-1 is dependent on end-1,3 expression and becomes localized apically specifically in only EPCs despite MRCK-1 being present at similar levels in all cells. I will use MRCK-1 localization as a molecular foothold for understanding how a pivotal protein becomes recruited to the apical cortex in only certain cells. Aim 2 will further investigate which domains of MRCK-1 are required for this localization pattern and identify interactors with these domains that function to initiate apical constriction, to better connect cell fate regulators and intracellular localization of a key protein. Overall, I propose the use of genetic, biochemical, and imaging techniques to advance our understanding of how transcriptional networks and other developmental patterning inputs deploy localized factors that influence cell shape changes.