Figure 5.

Regulatory effects of miR-263b on the expression of the bHLH transcription factor Atonal. A–C, Predicted regulatory effects of miR-263b on atonal expression. A, According to our regulatory model, in control conditions, miR-263b is expected to repress the expression of atonal, contributing to the maintenance of concentration values of Atonal protein within a physiological range compatible with normal sensory neuron function. B, Reduction of miR-263b function is expected to de-repress atonal expression, leading to an increase in Atonal protein concentration. C, In contrast, upregulation of miR-263b expression is predicted to reduce the levels of expression of Atonal below normal concentration. D–F, Experimental validation of the predicted regulatory effects of miR-263b on Atonal expression within the natural atonal transcriptional domain (Ato-Gal4). D, G, G', Normal expression of Atonal (red) as detected by immunohistochemistry using anti-Atonal antibodies. E, H, H', Functional reduction of miR-263b within the atonal expression domain by means of expressing a miR-263b-sponge leads to an increase in Atonal signal (red), in line with predicted de-repression effects (see B). F, I, I', Increase of miR-263b expression leads to downregulation of Atonal expression (red), as predicted by the miR-263b-dependent Atonal expression control model, see C (blue signal: DAPI). J, Quantification of the regulatory interactions between miR-263b and Atonal shows a statistically significant increase of Atonal expression when miR-263b function is reduced (ato>miR-263b-sp), and reduction of Atonal signal when miR-263b is overexpressed (ato>miR-263b). These observations validate the proposed miR-263b-dependent regulatory model for atonal expression control in sensory neurons (n ≥ 13, Mann–Whitney test).