Single cell RNA sequencing analysis of mouse cochlear supporting cell transcriptomes with activated ERBB2 receptor, a candidate mediator of hearing restoration mechanisms. Single cell RNA sequencing analysis of mouse cochlear supporting cell transcriptomes with activated ERBB2 receptor, a candidate mediator of hearing restoration mechanisms

One potential approach to restore hearing is to enforce regeneration of hair cells (HCs) through induction of cellular signaling pathways in supporting cells (SCs) that promote dedifferentiation and proliferation. We previously showed that the constitutive activation of ERBB2 signaling in cochlear SCs indirectly promoted SC differentiation to HCs, both in vivo and in vitro. In the current study, we aimed at identifying mechanisms and molecular pathways activated in SCs with constitutive ERBB2 signaling. To this end, we used single cell RNA sequencing (scRNA-seq) to characterize the transcriptomes of individual neonatal mouse cochlear SCs that were induced to express ERBB2. We found that induction of ERBB2 in vivo resulted in generation of a new distinct cluster of cells with unique transcriptome. This population has de novo expression of two members of the small integrin-binding ligand n-linked glycoproteins (SIBLING) family and their regulators. We confirm expression of the SIBLING Secreted phosphoprotein 1 (SPP1) as one of the most up-regulated genes in response to ERBB2 activation. SPP1 mediates signaling through the CD44 receptor, promoting survival, proliferation, and differentiation of osteoblast lineage cells. Histological analyses of cochlear samples collected from young adult mice confirmed that induction of ERBB2 after noise exposure resulted in up-regulation of both SPP1 and its receptor CD44, and the formation of mitotic sensory stem-like cell aggregates in the organ of Corti. Our results suggest that ectopic activation of ERBB2-signaling in young adult mice secondarily promotes SPP1/CD44 signaling, possibly altering the microenvironment of the organ of Corti. Overall design: The scRNA-seq analysis of supporting cells with activated ERBB2 signaling was performed using mouse line harboring an inducible “Tet-On” mutant ErbB2 transgene encoding a constitutively active ERBB2 protein (CA-ERBB2), which does not require ligand binding or heterodimerization with other ERBB partners for active signaling. To limit induction of CA-ERBB2 activation to cochlear supporting cells, CA-ERBB2 mouse line was crossed with mouse line harboring an inducible DNA Cre recombinase under control of the supporting cell gene promoter Fgfr3. Double heterozygotic mice harboring both an inducible “Tet-On” CA-ErbB2 and an Fgfr3-iCre transgene were bred to homozygous line harboring a “floxed” TA transcription factor, with an IRES-GFP to use as a lineage marker (“ROSA-rtTA-GFP”). The pups with CA-ErbB2 and Fgfr3-iCre genes (denoted here as “CA-ERBB2”) were used for analysis of transcriptome of supporting cells with activated ERBB2. Pups harboring Fgfr3-iCre were used as “Control”. Activation of iCRE was performed by two injections of tamoxifen at P0 and P1, which resulted in activation of GFP expression to label supporting cells. Induction of CA-ERBB2 expression was performed at P2 by doxycycline injection. The organs of Corti were dissected from P3 pups, and GFP+ supporting cells were purified by fluorescence-activated cell sorting (FACS). RNA cDNA libraries were prepared and sequenced from about 300 GFP+ single cells per genotype collected in three independent FACS experiments.

恢复听力的一种潜在途径是通过激活支持细胞（supporting cells, SCs）中的细胞信号通路，促进其去分化与增殖，从而强制毛细胞（hair cells, HCs）再生。我们此前已证实，在体内和体外环境中，耳蜗支持细胞组成型激活ERBB2信号通路，可间接促进支持细胞向毛细胞分化。本研究旨在明确组成型激活ERBB2信号的支持细胞中所激活的分子通路与调控机制。为此，我们采用单细胞RNA测序（single cell RNA sequencing, scRNA-seq）技术，对诱导表达ERBB2的新生小鼠耳蜗支持细胞的转录组进行表征。我们发现，体内诱导ERBB2表达可产生一个具有独特转录组特征的全新细胞簇。该细胞群首次表达小整合素结合配体N连接糖蛋白（small integrin-binding ligand n-linked glycoproteins, SIBLING）家族的两个成员及其调控因子。我们证实，分泌型磷蛋白1（Secreted phosphoprotein 1, SPP1）是响应ERBB2激活的上调最显著的基因之一。SPP1可通过CD44受体介导信号通路，促进成骨细胞谱系细胞的存活、增殖与分化。对年轻成年小鼠的耳蜗样本进行组织学分析证实，噪声暴露后诱导ERBB2表达可上调SPP1及其受体CD44的表达，并在柯蒂氏器（organ of Corti）中形成有丝分裂活性的感觉干细胞样细胞聚集体。我们的研究结果表明，年轻成年小鼠中耳内异位激活ERBB2信号通路可间接促进SPP1/CD44信号通路，可能改变柯蒂氏器的微环境。

整体实验设计：我们采用携带可诱导"Tet-On"组成型活性ERBB2（CA-ERBB2）突变转基因的小鼠品系，进行ERBB2信号激活的支持细胞单细胞RNA测序分析。该突变体无需配体结合或与其他ERBB家族成员形成异二聚体即可激活信号通路。为将CA-ERBB2的激活限定于耳蜗支持细胞，我们将该小鼠品系与携带受支持细胞特异性基因启动子Fgfr3调控的可诱导DNA Cre重组酶的小鼠品系杂交。将同时携带可诱导"Tet-On"CA-ErbB2转基因与Fgfr3-iCre转基因的双杂合小鼠，与携带"floxed"TA转录因子且带有IRES-GFP作为谱系标记的纯合ROSA-rtTA-GFP小鼠品系回交。我们将同时携带CA-ErbB2与Fgfr3-iCre基因的幼崽（本文中记为"CA-ERBB2组"）用于ERBB2信号激活的支持细胞转录组分析，仅携带Fgfr3-iCre的幼崽作为"对照组"。通过在出生后第0天（P0）和第1天（P1）两次注射他莫昔芬激活iCre重组酶，进而激活GFP表达以标记支持细胞。于出生后第2天（P2）通过注射多西环素诱导CA-ERBB2表达。于出生后第3天（P3）剥离柯蒂氏器，通过荧光激活细胞分选（fluorescence-activated cell sorting, FACS）纯化GFP阳性的支持细胞。构建RNA cDNA文库并进行测序，每个基因型的样本均来自三次独立的FACS实验，每次实验收集约300个GFP阳性单细胞。