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eLifeDevelopmental BiologyPeer-reviewed journal article

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

Mark E. LushiD, Daniel C. DiaziD, Nina Koenecke, Sungmin BaekiD, Helena Boldt, Madeleine St. PeteriD, Tatiana Gaitán-Escudero, Andrés Romero‐CarvajaliD, Elisabeth M. Busch‐NentwichiD, Anoja G. PereraiD, Kathryn HalliD, Allison Peak, Jeffrey S. HaugiD, Tatjana PiotrowskiiD

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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.

In-depth summary

Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (14 Dec 2018) · CC BY 4.0

Single-cell profiling of zebrafish lateral line neuromasts resolves support cell subtypes and lineages, and fgf3 mutant data show Fgf and Notch restrain Wnt-driven proliferation in parallel.

Zebrafish hair cells regenerate from support cells that are poorly defined, and bulk RNA-seq of pooled support cells masked their diversity. This work separates support cell types and lineages at single-cell resolution and uses fgf3 mutants to dissect the signaling that controls their proliferation.

Key results

  1. ShownUnsupervised clustering of homeostatic neuromast cells resolved distinct hair cell, central support, mantle and amplifying populations, and markers were checked by in situ hybridization.

    1,521 purified neuromast cells (25x coverage) split into 14 clusters, merged into 7 major cell types; the abstract reports five support cell types including quiescent and activated stem cells. · Abstract; Introduction; Single cell RNA-Seq reveals support cell…

  2. ShownProliferation during homeostasis is concentrated in the dorsal-ventral poles, while anterior-posterior poles and central cells are largely quiescent.

    BrdU labeling (24 h, 18 neuromasts): 6.5% of D/V cells proliferate vs 0-1% in the A/P poles; si:ch73-261i21.5 marks the quiescent A/P region. · Cycling cells characterize the amplifying and…; Figure legends

  3. SuggestedDendrogram and PAGA pseudotime analyses support two lineages (differentiating central, amplifying peripheral), with ribosomal gene expression low in presumed quiescent mantle and central cells and higher in dividing cells.

    Clusters 7, 9, 8, 14 and 4 form the hair cell lineage; clusters 5, 6, 12, 11, 10 and 3 the amplifying lineage; PAGA links cluster 3 to cluster 4. Inference only; confirmed by prior lineage tracing. · Heatmaps and the ordering of cells along…; Dynamics of gene expression during hair cell…; Cells can transition from an amplifying to…

  4. Shownfgf3 is expressed in central support cells, and its loss raises proliferation and regeneration; scRNA-seq of mutants found sost down and wnt10a up, and dkk1 reduced the excess proliferation.

    1,459 mutant vs 1,932 sibling cells, no cluster missing; hair cells 24 h post neomycin ***p<0.0004; total cells at 5 dpf **p<0.0084 (unpaired t-test); hs:dkk1 hair cell reduction ***p=0.001 (ANOVA/Tukey). · scRNA-seq reveals that loss of fgf3 in central…; fgf3 downregulation leads to Wnt-induced…; Figure legends

  5. ShownNotch and Fgf signaling act largely in parallel to restrain Wnt-induced proliferation, with a small Notch contribution acting through Fgf.

    Notch targets are unchanged in fgf3-/-; LY411575 slightly lowers fgf3 but not etv4, fgf10a or fgfr1a; NICD strongly curbs proliferation and hair cell numbers in fgf3-/- (legends: ****p<0.0001, ANOVA/Tukey), though the authors describe it as a bit weaker than NICD alone. · fgf3 downregulation leads to Wnt-induced…; Interactions between the Fgf and Notch pathways; Figure legends

Methods, data and reproducibility

FACS-purified GFP-positive neuromast cells were profiled with 10X Chromium, clustered with Seurat, and ordered with Scanpy PAGA pseudotime. Findings were validated by in situ hybridization, BrdU/EdU spatial analysis, and transgenic Fgf, Notch and Wnt manipulations in fgf3 mutants.

  • Homeostatic wild-type neuromast cells, 5 dpf Et(Gw57a);Tg(pou4f3) larvae, 10X Chromium scRNA-seq · Zebrafish (Danio rerio) · n = 1,521 cells analyzed (1,666 barcodes called) · GSE123241
  • fgf3 sibling neuromast cells, 5 dpf scRNA-seq · Zebrafish (Danio rerio) · n = 1,932 cells · GSE123241
  • fgf3 mutant neuromast cells, 5 dpf scRNA-seq · Zebrafish (Danio rerio) · n = 1,459 cells · GSE123241
  • BrdU spatial analysis of homeostatic neuromasts (reanalyzed data) · Zebrafish (Danio rerio) · n = 18 neuromasts
Methods & tools
10X Chromium Single Cell 3' v2, Cell Ranger 1.3.1 (wild type) and 2.1.1 (fgf3 data), GRCz10 zebrafish genome, Seurat 2.3.4, Scanpy 1.2.2 (PAGA), Seaborn 0.8.1, DAVID (GO term analysis), BD Influx cell sorter (FACS), Shiny app, Illumina HiSeq 2500
Reagents & environment
Cell Ranger 1.3.1 (wild type) and 2.1.1 (fgf3 sets); GRCz10; Seurat 2.3.4; Scanpy 1.2.2; Seaborn 0.8.1; kNN with k = 15.
Reproducibility gaps
No analysis code repository is given. Upload to gEAR is described as in progress; supplementary Data files carry marker lists and primers.
Limitations (5)
  • Lineage relationships cannot be inferred from scRNA-seq alone; pseudotime and PAGA lineages rely on prior time-lapse and lineage tracing for confirmation. · stated by the authors
  • No specific marker was found for amplifying cells: polar genes (sost, fsta, wnt2, adcyap1b) are expressed beyond the cells next to mantle cells, and polar cells are spread across several clusters. · stated by the authors
  • Whether A/P quiescent cells are true stem cells needs long-term lineage tracing; the stem cell classification of mantle and amplifying cells is a postulate. · stated by the authors
  • Bulk RNA-seq of fgf3 mutants and siblings showed too little difference, so target discovery rests on a single scRNA-seq dataset per genotype with no stated biological replicates. · noted in this summary
  • Wild-type and fgf3 datasets used different Cell Ranger versions and Seurat parameters, and the input was live or methanol-fixed sorted cells, which may add technical variation. · noted in this summary

Read this if you study hair cell regeneration, support cell or stem cell heterogeneity in zebrafish, or want a worked scRNA-seq plus genetic epistasis example for Fgf, Notch and Wnt signaling.

Citations
170
FWCI
11.73
Field percentile
top 1%

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