Notch-Mediated Determination of Hair-Bundle Polarity in Mechanosensory Hair Cells of the Zebrafish Lateral Line.

Adrian Jacobo,Anna Erzberger,Agnik Dasgupta,Kimberly Siletti,A.J Hudspeth

doi:10.1016/j.cub.2019.08.060

Adrian Jacobo, Anna Erzberger + Show 3 more

Open Access

https://doi.org/10.1016/j.cub.2019.08.060

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Abstract

The development of mechanosensory epithelia, such as those of the auditory and vestibular systems, results in the precise orientation of mechanosensory hair cells. After division of a precursor cell in the zebrafish's lateral line, the daughter hair cells differentiate with opposite mechanical sensitivity. Through a combination of theoretical and experimental approaches, we show that Notch1a-mediated lateral inhibition produces a bistable switch that reliably gives rise to cell pairs of opposite polarity. Using a mathematical model of the process, we predict the outcome of several genetic and chemical alterations to the system, which we then confirm experimentally. We show that Notch1a downregulates the expression of Emx2, a transcription factor known to be involved in polarity specification, and acts in parallel with the planar-cell-polarity system to determine the orientation of hair bundles. By analyzing the effect of simultaneous genetic perturbations to Notch1a and Emx2, we infer that the gene-regulatory network determining cell polarity includes an undiscovered polarity effector.

Highlights

Using the lateral line of the zebrafish as a model system, we have studied how the correct number of oppositely polarized hair cells is established during the formation of mechanosensory epithelia
We have discovered a novel role for Notch-mediated lateral inhibition in determining the polarity of sensory hair cells during development and regeneration
The polarity of a hair cell is morphologically determined by its hair bundle, which protrudes from the cell’s apical surface and confers directional sensitivity to mechanical stimuli [5]

Summary

Methods

METHOD DETAILSTreatments of larvae To ablate mature hair cells in some experiments, we treated 3 dpf larvae for 2 hr at 28C with 1 mM CuSO4 in E3 medium. The animals were thoroughly washed and maintained overnight in E3 medium, fixed for immunofluorescence imaging on the following day. One day later the larvae were thoroughly washed and fixed for immunofluorescence imaging. Immunofluorescence microscopy For the immunofluorescence labeling of wholemounted Emx, Emx2-CE, and NICD-CE larvae, 4 dpf larvae were fixed overnight at 4C in 4% formaldehyde in phosphate-buffered saline solution (PBS) with 1% Tween-20 (1% PBST). Larvae were washed four times with 1% PBST for 15 min each, placed for 2 hr in blocking solution containing PBS, 2% normal donkey serum (NDS), 0.5% Tween-20, and 1% BSA. Primary antibodies were diluted in fresh blocking solution and incubated with the specimens overnight at 4C. Larvae were washed four times with 0.1% PBST for 15 min each. Larvae were washed four times in 0.2% PBST for 15 min each and stored at 4C in VectaShield (Vector Laboratories)

Results

Discussion

Conclusion