Genetic Analysis Reveals a Hierarchy of Interactions between Polycystin-Encoding Genes and Genes Controlling Cilia Function during Left-Right Determination

Abstract

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.

Left–Right Patterning: Breaking Symmetry to Asymmetric Morphogenesis

“…More complex lobation patterns (e.g. 3:2 or 1:3 lobe ratios, amongst others) are present in mutants that bilaterally activate the Nodal-Pitx2 pathway but where the strength of the signal is not equal on both sides [32]. However, the basis of asymmetric branching remains largely unexplored.…”

“…Moreover, the expression of Pkd1l1 is relatively restricted to LROs [68, 69]; while Pkd2 is expressed more broadly, it is required only in sensory cells surrounding the mouse node, which harbor immotile sensory cilia, for proper L-R patterning [13]. Finally, genetic experiments in mouse embryos revealed a pathway in which Pkd1l1 acts upstream of and genetically represses Pkd2 [32]. Thus, while the requirement for PKD1L1 and PKD2 is clear from genetic studies, further work is required to address how they act on the pathway between flow and gene expression asymmetries.…”

“…One model argues that PKD2 responds to flow-based signals by eliciting an intra-ciliary Ca 2+ signal, which may then spread to the cell body to activate downstream pathways [73]. In cell culture, PKD1L1, likely in combination with PKD2, can also act as part of a flow-responsive pathway that results in a Ca 2+ signal [32]. But in the embryo there remains no obvious connection between Ca 2+ asymmetries and asymmetric gene expression, while the idea that sensory cilia act as sensors of flow by initiating ciliary Ca 2+ signals has recently come under scrutiny [74].…”

Fluid Mechanics as a Driver of Tissue-Scale Mechanical Signaling in Organogenesis

“…Additionally, research within past few decades has identified the important role that larger scale fluid flows, generated by coordinated cilia rotations, have during development including left-right (LR) symmetry breaking and tissue specification [24,25]. In the earliest stages of mouse embryogenesis, motile cilia rotate clockwise to create and monitor a leftward fluid flow across a medial fissure, called the node [26]. Peripherally located crown cells have non-motile cilia that act as mechanosensors by detecting slight differences in forces created by the fluid flow and triggering differential expression of specialized proteins and signaling pathways, eventually leading to LR symmetry breaking [26,27].…”

“…In the earliest stages of mouse embryogenesis, motile cilia rotate clockwise to create and monitor a leftward fluid flow across a medial fissure, called the node [26]. Peripherally located crown cells have non-motile cilia that act as mechanosensors by detecting slight differences in forces created by the fluid flow and triggering differential expression of specialized proteins and signaling pathways, eventually leading to LR symmetry breaking [26,27]. …”