The role of the polycystin genes, Pkd1l1 and Pkd2, in the determination of left-right asymmetry

Breaking bilateral symmetry is critical for vertebrate morphogenesis. In the mouse, overt left-right (L-R) asymmetries in the development of the organs is prefigured by asymmetric cascades of genes expression. The Nodal/Pitx2 pathway is active in the left lateral plate mesoderm (LPM) and remains absent from the right side of the embryo. Expression of Nodal in the left side is initiated downstream of a symmetry-breaking event that occurs in a midline structure called the node. Within the node, 200-300 posteriorly-tilted motile cilia rotate in the clockwise direction when viewed from the ventral aspect, and in so doing generate a unidirectional leftward flow of extra-cellular fluid across the surface of the node. This flow drives both an elevation in intracellular Ca2+ and changes in gene expression at the left side of the node, the first L-R asymmetrical chemical differences in the embryo. However, a major unanswered question concerns how leftward flow is sensed by the embryo, and what genes/pathways play a role in this process. Two mutant mouse lines were discovered as part of forward genetic screens for embryos with developmental defects. These two lines, called rks and lrm4, both exhibit severe L-R defects. Mapping of the mutants revealed lrm4 to be a new allele of Pkd2, a gene known to be required for the left-sided Ca2+ elevation which occurs downstream of flow in the node. In contrast, rks was mapped to a novel gene not previously associated with L-R determination. The protein encoded by this gene, Pkd1l1, was a candidate for being the elusive sensor of leftward flow. This thesis describes experiments aimed at testing the hypothesis that Pkd1l1 and Pkd2 act together to sense leftward flow in the node during the determination of L-R asymmetry in the mouse. The overt morphological and molecular L-R defects of Pkd1l1rks/rks and Pkd2lrm4/lrm4 mutants are compared and found to be highly similar when assessed on the same genetic background. Both mutants exhibit a loss of the normally left-sided Nodal/Pitx2 pathway and, as a consequence, right isomerism of the lungs. Pkd1l1 gene expression is localised to the ventral layer of the node where flow is generated and sensed, but neither Pkd1l1 nor Pkd2 are required for normal node morphogenesis or cilia structure/motility. Moreover, genetic experiments reveal that Pkd1l1 and Pkd2 both act downstream of leftward flow but upstream of early asymmetries in gene expression around the node. In contrast to Pkd1l1rks/rks mutants, Pkd1l1-null embryos exhibit left lung isomerism and bilateral activation of Pitx2. This surprising observation suggests that Pkd1l1 restricts Nodal to the left side during L-R determination, favouring a model in which leftward flow derepresses left-sided Nodal by inhibiting Pkd1l1. Bioinformatics and structural studies support a role for an extracellular polycystic kidney disease (PKD) domain within Pkd1l1 in the sensation of flow, whilst Pkd1l1 and Pkd2 are also shown to form physical associations mediated by intracellular coiled coil domains. Finally, Pkd2 is shown to be required within the cilial compartment for L-R determination. In conclusion, the results reported in this thesis support a model in which Pkd1l1 and Pkd2 act together for the sensation of leftward flow and specifically implicate Pkd1l1's extracellular PKD domain in the sensation mechanism.