Analyses of iHC transcriptome profiles. Mus musculus

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). Overall design: 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).

机械感觉毛细胞（mechanosensory hair cells, HCs）是我们听觉与平衡觉的主要感受器。然而，目前对于参与HC命运决定与分化的转录调控因子仍知之甚少。
本研究证实，过表达三种HC谱系特异性转录因子——Gfi1、Pou4f3与Atoh1——可在体外胚胎干细胞（embryonic stem cell, ESC）分化过程中，直接诱导细胞向HC命运特化。诱导产生的HCs（induced HCs, iHCs）可表达多种HC特异性基因，并呈现出类似静纤毛束的极化膜突起。
借助Myo7:mVenus报告细胞系（iGPA-Myo7a:mVenus），我们可分离得到纯化的iHC群体，进而在细胞发育的不同阶段（细胞培养第8天与第12天）获取这些细胞重复性极佳的基因表达谱。

实验整体设计：从iGPA-Myo7a:mVenus报告ESC系诱导得到的胚胎体（embryoid bodies, EBs），在培养至第8天与第12天时，分别经多西环素（doxycycline, Dox）单独处理，或经Dox与视黄酸（retinoic acid, RA）联合处理，随后通过荧光激活细胞分选（fluorescence-activated cell sorting, FACS）获取纯化的iHC群体。同一报告细胞系在相同培养条件下未经Dox或RA处理的未分选EBs，则作为对照样本。
针对每个选定的时间点与处理组，我们独立制备RNA样本（Dox处理组与Dox+RA处理组各设3次生物学重复，"未处理组"设2次生物学重复），随后将样本处理后与Affymetrix全转录组微阵列（Mouse Genome 2.1 ST Arrays Strip）进行杂交。