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ScRNA-Seq reveals distinct stem cell populations that drive hair cell regeneration after 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 support cells. Equally ill-defined is the gene regulatory network underlying the progression of support cells to differentiated hair cells. scRNA-Seq of lateral line organs uncovered five different support cell types, including quiescent and activated stem cells. Ordering of support cells along a developmental trajectory identified self-renewing cells and 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. The data is searchable and publicly accessible via a web-based interface.

SUBMITTER: Lush ME

PROVIDER: S-EPMC6363392 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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scRNA-Seq reveals distinct stem cell populations that drive hair cell regeneration after loss of Fgf and Notch signaling.

Lush Mark E ME Diaz Daniel C DC Koenecke Nina N Baek Sungmin S Boldt Helena H St Peter Madeleine K MK Gaitan-Escudero Tatiana T Romero-Carvajal Andres A Busch-Nentwich Elisabeth M EM Perera Anoja G AG Hall Kathryn E KE Peak Allison A Haug Jeffrey S JS Piotrowski Tatjana T

eLife 20190125

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 support cells. Equally ill-defined is the gene regulatory network underlying the progression of support cells to differentiated hair cells. scRNA-Seq of lateral line organs uncovered five different support cell types, including quiescent and activated stem cells. Ordering of support cells along a developmental t ...[more]

PMID: 30681411

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Project description:During embryonic development, differentiation of cochlear progenitor cells into hair cells (HCs) or supporting cells (SCs) is partially controlled through Notch signaling. Many studies have shown that inhibition of Notch signaling allows SCs to convert into HCs in both normal and drug damaged neonatal mouse cochleae. This mechanism is also implicated during HC regeneration in non-mammalian vertebrates; however, the mechanism of spontaneous HC regeneration in the neonatal mouse cochlea is less understood. While inhibition of Notch signaling can force SCs to convert into HCs and increase the number of regenerated HCs, it is currently unknown whether this pathway is involved in spontaneous HC regeneration observed in vivo. Therefore, we investigated the role of Notch signaling during the spontaneous HC regeneration process using Atoh1-CreERTM::Rosa26loxP-stop-loxP-DTA/+ mice injected with tamoxifen at postnatal day (P) 0 and P1 to ablate HCs and stimulate spontaneous HC regeneration. Expression changes of genes in the Notch pathway were measured using immunostaining and in situ hybridization, with most changes observed in the apical one-third of the cochlea where the majority of HC regeneration occurs. Expression of the Notch target genes Hes1, Hes5, Hey1, HeyL, and Jagged1 were decreased. To investigate whether reduction of Notch signaling is involved in the spontaneous HC regeneration process, we overexpressed the Notch1 intracellular fragment (N1ICD) in cochlear SCs and other non-sensory epithelial cells in the context of HC damage. Specifically, Atoh1-CreERTM::Rosa26loxP-stop-loxP-DTA/+::Sox10rtTA::TetO-LacZ::TetO-N1ICD mice were injected with tamoxifen at P0/P1 to stimulate spontaneous HC regeneration and given doxycycline from P0-P7 to induce expression of N1ICD as well as LacZ for fate-mapping. We observed a 92% reduction in the number of fate-mapped regenerated HCs in mice with N1ICD overexpression compared to controls with HC damage but no manipulation of Notch signaling. Therefore, we conclude that increased Notch signaling prevents spontaneous HC regeneration from occurring in the neonatal mouse cochlea. Understanding which components of the Notch pathway regulates regenerative plasticity in the neonatal mouse cochlea will inform investigations focused on stimulating HC regeneration in mature cochlea and eventually in humans to treat hearing loss.

Project description:BackgroundThe mammalian testicular interstitial cells are not well-defined. The present study characterized the interstitial cell types and their turnover dynamics in adult rats. Additionally, the heterogeneity of the mesenchymal population and the effects of Leydig cell elimination on interstitial homeostasis were further analyzed by scRNA-seq datasets and immunocytochemical techniques.MethodsInterstitial cells were defined at the transcriptomic level by scRNA-seq and then confirmed and quantified with protein markers. The dividing activity of the major cell types was determined by continuous EdU labeling of the animals for one week. Some of the rats were also treated with a dose of ethylenedimethylsulfonate (EDS) to examine how the loss of Leydig cells (LCs) could affect interstitial homeostasis for three weeks.ResultsSeven interstitial cell types were identified, including cell types (percentage of the whole interstitial population) as follows: Leydig (44.6%), macrophage and dendritic (19.1%), lymphoid (6.2%), vascular endothelial (7.9%), smooth muscle (10.7%), and mesenchymal (11.5%) cells. The EdU experiment indicated that most cell types were dividing at relatively low levels (<9%) except for the mesenchymal cells (MCs, 17.1%). Further analysis of the transcriptome of MCs revealed 4 subgroups with distinct functions, including 1) glutathione metabolism and xenobiotic detoxification, 2) ROS response and AP-1 signaling, 3) extracellular matrix synthesis and binding, and 4) immune response and regulation. Stem LCs (SLCs) are primarily associated with subgroup 3, expressing ARG1 and GAP43. EDS treatment not only eliminated LCs but also increased subgroup 3 and decreased subgroups 1 and 2 of the mesenchymal population. Moreover, EDS treatment increased the division of immune cells by more than tenfold in one week.ConclusionSeven interstitial cell types were identified and quantified for rat testis. Many may play more diversified roles than previously realized. The elimination of LCs led to significant changes in MCs and immune cells, indicating the importance of LCs in maintaining testicular interstitial homeostasis.

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ScRNA-Seq reveals distinct stem cell populations that drive hair cell regeneration after loss of Fgf and Notch signaling.

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scRNA-Seq reveals distinct stem cell populations that drive hair cell regeneration after loss of Fgf and Notch signaling.

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