During mammalian development, left-right (L-R) asymmetry is established by a cilia-driven leftward fluid flow within a midline embryonic cavity called the node. This ‘nodal flow’ is detected by peripherally-located crown cells that each assemble a primary cilium which contain the putative Ca2+ channel PKD2. The interaction of flow and crown cell cilia promotes left side-specific expression of Nodal in the lateral plate mesoderm (LPM). Whilst the PKD2-interacting protein PKD1L1 has also been implicated in L-R patterning, the underlying mechanism by which flow is detected and the genetic relationship between Polycystin function and asymmetric gene expression remains unknown. Here, we characterize a Pkd1l1 mutant line in which Nodal is activated bilaterally, suggesting that PKD1L1 is not required for LPM Nodal pathway activation per se, but rather to restrict Nodal to the left side downstream of nodal flow. Epistasis analysis shows that Pkd1l1 acts as an upstream genetic repressor of Pkd2. This study therefore provides a genetic pathway for the early stages of L-R determination. Moreover, using a system in which cultured cells are supplied artificial flow, we demonstrate that PKD1L1 is sufficient to mediate a Ca2+ signaling response after flow stimulation. Finally, we show that an extracellular PKD domain within PKD1L1 is crucial for PKD1L1 function; as such, destabilizing the domain causes L-R defects in the mouse. Our demonstration that PKD1L1 protein can mediate a response to flow coheres with a mechanosensation model of flow sensation in which the force of fluid flow drives asymmetric gene expression in the embryo.
Vertebrates exhibit left-right (L-R) asymmetry in positioning and patterning their internal organs and associated vasculature; abnormal L-R asymmetry can result in birth defects such as congenital heart disease. The earliest known event in mammalian L-R patterning is a leftward fluid flow across a transient embryonic structure termed the node. This ‘nodal flow’ is driven by the action of motile cilia, hair-like organelles protruding from the cell surface within the node. Nodal flow is sensed by crown cells that surround the node; this requires immotile primary cilia and the putative cation channel Polycystin-2 (PKD2). A second Polycystin protein, PKD1L1, is implicated in this pathway. We describe two principle findings: a genetic hierarchy in cilia-motility genes act upstream of Polycystin-encoding genes and in which Pkd1l1 acts upstream of, and likely represses Pkd2. We further demonstrate that PKD1L1 is sufficient to mediate a flow-induced Ca2+ response in cultured cells, and that an extracellular PKD domain is critical for both flow detection and proper L-R patterning. Together, these findings reveal a genetic pathway operating at the level of flow sensation and demonstrate that PKD1L1 is able to act to elicit flow-induced Ca2+ signals, thereby supporting the mechanosensation model of nodal flow sensation.
Grimes DT, Keynton JL, Buenavista MT, Jin X, Patel SH, Kyosuke S, et al. (2016) Genetic Analysis Reveals a Hierarchy of Interactions between Polycystin-Encoding Genes and Genes Controlling Cilia Function during Left-Right Determination. PLoS Genet 12(6): e1006070. doi:10.1371/journal.pgen.1006070
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