Single cell RNA-Seq reveals distinct stem cell populations that drive sensory hair cell regeneration in response to loss of Fgf and Notch signaling

Abstract

Loss of sensory hair cells leads to deafness and balance deficiencies. In contrast to mammalian hair cells, zebrafish ear and lateral line hair cells regenerate from poorly characterized, proliferating support cells. Equally ill-defined is the gene regulatory network underlying the progression of support cells to cycling hair cell progenitors and differentiated hair cells. We used single cell RNA-Sequencing (scRNA-Seq) of lateral line sensory organs and uncovered five different support cell types, including quiescent and activated stem cells. In silico ordering of support cells along a developmental trajectory identified cells that self-renew and new groups of genes required for hair cell differentiation. scRNA-Seq analyses of fgf3 mutants, in which hair cell regeneration is increased, demonstrates that Fgf and Notch signaling inhibit proliferation of support cells in parallel by inhibiting Wnt signaling. Our scRNA-Seq analyses set the foundation for mechanistic studies of sensory organ regeneration and is crucial for identifying factors to trigger hair cell production in mammals. As a resource, we implemented a shiny application that allows the community to interrogate cell type specific expression of genes of interest.

Parallel control of mechanosensory hair cell orientation by the PCP and Wnt pathways

“…and fzd7a/b are strongly expressed in support and not hair cells in neuromasts ((Mark E. Lush, 2018), Fig. 6, Fig.…”

High-resolution single cell transcriptome analysis of zebrafish sensory hair cell regeneration

“…Figure S3A shows in which cell types different placodal genes are enriched. Prior studies showed that Notch and Fgf signaling are downregulated to allow for Wnt-dependent cell proliferation (Lush et al, 2019;Romero-Carvajal et al, 2015). Notch downregulation is also required to initiate hair cell differentiation (Ma et al, 2008;Romero-Carvajal et al, 2015;Wibowo et al, 2011).…”

“…To gain a better understanding of the lineage trajectories that central support cells (central SCs) undergo during regeneration and to identify associated genes, we aligned central support-, progenitor-and hair cells along pseudotime using Monocle 3 (Cui et al, 2020;Qiu et al, 2017;Trapnell et al, 2014). We chose to limit the analysis to these cell types, as in vivo lineage tracing showed that central support cells are the direct precursors to hair cells (Lush et al, 2019). Although D/V and A/P cells can also give rise to hair cells (Romero-Carvajal et al, 2015;Thomas and Raible, 2019;Wibowo et al, 2011), it still needs to be determined whether A/P and D/V cells first acquire a central support cell fate.…”

“…However, this study lacked spatio-temporal resolution (Jiang et al, 2014). To identify the cell populations involved in regeneration, we analyzed the different cell populations that respond to hair cell death in time lapse and BrdU analyses and have characterized all cell populations in homeostatic lateral line sensory organs (neuromasts) using scRNA-Seq (Figures 1B-E; (Lush et al, 2019;Romero-Carvajal et al, 2015)). Lateral line sensory organs consist of sensory hair cells surrounded by support cells and an outermost ring of mantle cells.…”

“…During regeneration support cells divide before differentiating into hair cells (López-Schier and Hudspeth, 2006;Ma et al, 2008;Mackenzie and Raible, 2012;Wibowo et al, 2011). Central support cells are the immediate precursors of hair cells (Lush et al, 2019;Romero-Carvajal et al, 2015). D/V and A/P support cells also give rise to hair cells but it is not known if they first acquire a central support cell fate or if they directly differentiate into hair cells during regeneration.…”