Epithelial Planar Bipolarity Emerges from Notch-Mediated Asymmetric Inhibition of Emx2

Abstract

Most plane-polarized tissues are formed by identically oriented cells [1,2]. A notable exception occurs in the vertebrate vestibular system and lateral-line neuromasts, where mechanosensory hair cells orient along a single axis but in opposite directions to generate bipolar epithelia [3][4][5]. In zebrafish neuromasts, pairs of hair cells arise from the division of a non-sensory progenitor [6, 7] and acquire opposing planar polarity via the asymmetric expression of the polarity-determinant transcription factor Emx2 [8][9][10][11]. Here, we reveal the initial symmetry-breaking step by decrypting the developmental trajectory of hair cells using single-cell RNA sequencing (scRNA-seq), diffusion pseudotime analysis, lineage tracing, and mutagenesis. We show that Emx2 is absent in non-sensory epithelial cells, begins expression in hair-cell progenitors, and is downregulated in one of the sibling hair cells via signaling through the Notch1a receptor. Analysis of Emx2-deficient specimens, in which every hair cell adopts an identical direction, indicates that Emx2 asymmetry does not result from auto-regulatory feedback. These data reveal a two-tiered mechanism by which the symmetric monodirectional ground state of the epithelium is inverted by deterministic initiation of Emx2 expression in hair-cell progenitors and a subsequent stochastic repression of Emx2 in one of the sibling hair cells breaks directional symmetry to establish planar bipolarity.

Emx2 regulates hair cell rearrangement but not positional identity within neuromasts

“…Consistent with the DPT analysis, the nascent HC cluster showed significantly higher number of emx2 read-counts ( Figure 3E) and percentages of emx2-positive cells ( Figure 3F) than other clusters. Taken together, our scRNA-seq results showed that, similar to previous results (Kozak et al, 2020), emx2 transcripts were found within the HC lineage except we found that the transcripts initiate as HCs transitioned from precursors to nascent HCs rather than peaking early in the precursor cell population as described previously (Kozak et al, 2020). The difference between these results is not clear and may be attributed to the difference in the age of the larvae and the number of HCs analyzed.…”

“…However, immunostaining may not be sensitive enough to detect onset of emx2 transcription. Indeed, a recent study demonstrated enriched Emx2 transcripts and immunoreactivity in HC precursors using immunostaining, in situ hybridization and scRNA-seq (Kozak et al, 2020). These results imply that the downregulation of Emx2 in one of the nascent HCs generated the asymmetry within a pair of HCs ( Figure 1C).…”

“…To validate these results, we investigated emx2 transcription within the neuromast HC lineage by analyzing our existing scRNA-seq data (not published) generated from fluorescence-activated sorted (FACS) neuromast HCs using myo6:ribotag-Gfp larvae (Matern et al, 2018) 1B,C) modeled after similar studies (Lush et al, 2019, Kozak et al, 2020, 2188 cells within the HC lineage from clusters #0,1, 3 and 4 were selected for reclustering, excluding a supporting cell cluster (Cluster #5) and a cluster with low levels of the transgene (Cluster #2). Reclustering of HC lineage cells resulted in five clusters ( Figures 3A, Figure 3 --figure supplement 1D,E).…”

“…The mechanism for generating the bidirectional orientation pattern of HCs in the neuromast is not clear but it is attributed to a HC rearrangement process that involves lateral inhibition mediated by Notch signaling (Jacobo et al, 2019, Kozak et al, 2020.…”

“…However, the timing and mechanism of Emx2's requirement in this process is not clear. For example, Emx2 could be activated early in the HC precursor stage prior to HC formation (Kozak et al, 2020) and be involved in HCs' positional acquisition ( Figure 1C, scenario 1). Alternatively, it could be activated only in one of the sibling HCs (Jacobo et al, 2019) and generates the opposite hair bundle orientation after HCs acquired their positions (scenario 2).…”