Analyses of iHC transcriptome profiles

Mechanosensory hair cells (HCs) are the primary receptors of our senses of hearing and balance. However, very little is known about the transcriptional regulators involved in HC fate determination and differentiation. In this paper, we show that expression of three HC lineage-specific transcription factors: Gfi1, Pou4f3 and Atoh1, can induce a direct commitment towards HC fate during in vitro embryonic stem cell (ESC) differentiation. Induced HCs (iHCs) express numerous HC-specific genes and exhibit polarized membrane protusions reminiscent of stereociliary bundles. The ability to obtain purified populations of iHCs by virtue of the Myo7:mVenus reporter line (iGPA-Myo7a:mVenus) allowed us to generate highly reproducible gene expression profiles for these cells, at various phases of their development (day 8 and day 12 of cell culture). Embryoid bodies (EBs) derived from the iGPA-Myo7a:mVenus reporter ESC line at day 8 and day 12, treated with Dox alone or with Dox and RA, were used to obtain FACS-purified populations of iHCs. Unsorted EBs from the same reporter line, grown in the same conditions but without Dox or RA treatment, were used as controls. Independent RNA preparations from each of the selected time points and treatments (three biological replicates for Dox or Dox+RA treatments, two for “no treatment”) were processed and hybridized on Affymetrix whole-transcript microarrays (Mouse Genome 2.1 ST Arrays Strip).