Characterization of Lgr5+ Progenitor Cell Transcriptomes after Neomycin Injury in the Neonatal Mouse Cochlea

Abstract

Lgr5+ supporting cells (SCs) are enriched hair cell (HC) progenitors in the cochlea. Both in vitro and in vivo studies have shown that HC injury can spontaneously activate Lgr5+ progenitors to regenerate HCs in the neonatal mouse cochlea. Promoting HC regeneration requires the understanding of the mechanism of HC regeneration, and this requires knowledge of the key genes involved in HC injury-induced self-repair responses that promote the proliferation and differentiation of Lgr5+ progenitors. Here, as expected, we found that neomycin-treated Lgr5+ progenitors (NLPs) had significantly greater HC regeneration ability, and greater but not significant proliferation ability compared to untreated Lgr5+ progenitors (ULPs) in response to neomycin exposure. Next, we used RNA-seq analysis to determine the differences in the gene-expression profiles between the transcriptomes of NLPs and ULPs from the neonatal mouse cochlea. We first analyzed the genes that were enriched and differentially expressed in NLPs and ULPs and then analyzed the cell cycle genes, the transcription factors, and the signaling pathway genes that might regulate the proliferation and differentiation of Lgr5+ progenitors. We found 9 cell cycle genes, 88 transcription factors, 8 microRNAs, and 16 cell-signaling pathway genes that were significantly upregulated or downregulated after neomycin injury in NLPs. Lastly, we constructed a protein-protein interaction network to show the interaction and connections of genes that are differentially expressed in NLPs and ULPs. This study has identified the genes that might regulate the proliferation and HC regeneration of Lgr5+ progenitors after neomycin injury, and investigations into the roles and mechanisms of these genes in the cochlea should be performed in the future to identify potential therapeutic targets for HC regeneration.

Hedgehog Signaling Promotes the Proliferation and Subsequent Hair Cell Formation of Progenitor Cells in the Neonatal Mouse Cochlea

“…Previous experiments have shown that Lgr5 is expressed in a subset of Sox2+ supporting cells, and Lgr5+ cells isolated by flow cytometry from the neonatal Lgr5 EGFP–CreERT2 mouse cochlea can proliferate and form clonal colonies in vitro (Chai et al, 2011 , 2012 ; Waqas et al, 2016a ; Cheng et al, 2017 ; Zhang et al, 2017 ). To investigate the effect of Hedgehog signaling in regulating the proliferative ability of Lgr5+ progenitor cells, we dissociated the whole cochlear epithelium from P2–P3 Lgr5 EGFP–CreERT2 mice into single cells and isolated the Lgr5-EGFP+ cells via flow cytometry and performed the sphere-forming assay with Hedgehog agonist and antagonist.…”

“…Wnt-responsive Lgr5+ supporting cells are HC progenitors in the mouse inner ear (Chai et al, 2012 ; Shi et al, 2012 ; Li et al, 2016 ; Waqas et al, 2016a , b ; Cheng et al, 2017 ; Zhang et al, 2017 ). In the cochleae of neonatal mice, Lgr5 is expressed in a subset of supporting cells, including third-row Deiters’ cells, inner pillar cells, and medial inner phalangeal cells (Chai et al, 2011 , 2012 ).…”

“…It has been reported that Wnt-responsive Lgr5+ supporting cells are HC progenitor cells in the mouse inner ear (Chai et al, 2012 ; Shi et al, 2012 ; Li et al, 2016 ; Waqas et al, 2016a , b ; Cheng et al, 2017 ; Zhang et al, 2017 ). Lgr5+ cells isolated by flow cytometry from neonatal Lgr5 EGFP–CreERT2 mice can proliferate to form clonal colonies and can mitotically regenerate new HCs (Chai et al, 2012 ; Waqas et al, 2016a ; Cheng et al, 2017 ; Zhang et al, 2017 ). Promoting the proliferation and differentiation of Lgr5+ progenitor cells thus appears to be a promising strategy to mitotically regenerate HCs.…”

Knockdown of Foxg1 in supporting cells increases the trans-differentiation of supporting cells into hair cells in the neonatal mouse cochlea

“…It is known that a limited number of HCs can be regenerated in newborn mice from SCs and inner ear progenitor cells, and several studies have shown that many important signaling pathways are involved in HC regeneration, such as Wnt, Notch, and Shh [7,8,[11][12][13][14][80][81][82][83][84]. Many other genes and related pathways have also been shown to play important roles in HC regeneration [85,86], and these pathways might have crosstalk with each other to affect the proliferation and differentiation of SCs and Lgr5+ progenitors [13,14]. Foxg1, one of the FOX protein family members, plays important roles in brain, eye, and ear development [24,35,36,44,45,55,57,60,64,66].…”

Characterization of the Transcriptome of Hair Cell Regeneration in the Neonatal Mouse Utricle

“…To better understand the signaling events of hair cell regeneration after damage, investigating the transcriptional profiles of regenerating hair cells should be the first step. By using RNAseq, two previous studies have investigated the changes in the transcriptional profiles during hair cell regeneration in the mouse cochlea [12,13]. The high degree of differentiation and specialization of hair cells in the mammalian cochlea suggests that regeneration may be limited in this area, but hair cells in the vestibular system of mammals are most likely to regenerate due to their similarity to their counterparts in non-mammals.…”