title: The bHLH Transcription Factor NeuroD Governs Photoreceptor Genesis and Regeneration Through Delta-Notch Signaling.
abstract: OBJECTIVE
  Photoreceptor genesis in the retina requires precise regulation of progenitor cell competence, cell cycle exit, and differentiation, although information around the mechanisms that govern these events currently is lacking. In zebrafish, the basic helix-loop-helix (bHLH) transcription factor NeuroD governs photoreceptor genesis, but the signaling pathways through which NeuroD functions are unknown. The purpose of this study was to identify these pathways, and during photoreceptor genesis, Notch signaling was investigated as the putative mediator of NeuroD function.
  
  
  METHODS
  In embryos, genetic mosaic analysis was used to determine if NeuroD functions is cell- or non-cell-autonomous. Morpholino-induced NeuroD knockdown, CRISPR/Cas9 mutation, and pharmacologic and transgenic approaches were used, followed by in situ hybridization, immunocytochemistry, and quantitative RT-PCR (qRT-PCR), to identify mechanisms through which NeuroD functions. In adults, following photoreceptor ablation and NeuroD knockdown, similar methods as above were used to identify NeuroD function during photoreceptor regeneration.
  
  
  RESULTS
  In embryos, NeuroD function is non-cell-autonomous, NeuroD knockdown increases Notch pathway gene expression, Notch inhibition rescues the NeuroD knockdown-induced deficiency in cell cycle exit but not photoreceptor maturation, and Notch activation and CRISPR/Cas9 mutation of neurod recapitulate NeuroD knockdown. In adults, NeuroD knockdown prevents cell cycle exit and photoreceptor regeneration and increases Notch pathway gene expression, and Notch inhibition rescues this phenotype.
  
  
  CONCLUSIONS
  These data demonstrate that during embryonic development, NeuroD governs photoreceptor genesis via non-cell-autonomous mechanisms and that, during photoreceptor development and regeneration, Notch signaling is a mechanistic link between NeuroD and cell cycle exit. In contrast, during embryonic development, NeuroD governs photoreceptor maturation via mechanisms that are independent of Notch signaling.
authors.affiliation: Department of Ophthalmology and Visual Sciences University of Michigan, W. K. Kellogg Eye Center, Ann Arbor, Michigan, United States.
authors.forename: Scott M
authors.initials: SM
authors.lastname: Taylor
authors.affiliation: Department of Biological Science, Florida State University, Tallahassee, Florida, United States.
authors.forename: Karen
authors.initials: K
authors.lastname: Alvarez-Delfin
authors.affiliation: Department of Biological Science, Florida State University, Tallahassee, Florida, United States.
authors.forename: Carole J
authors.initials: CJ
authors.lastname: Saade
authors.affiliation: Departments of Anatomy/Cell Biology and Ophthalmology, Wayne State University, Detroit, Michigan, United States.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Thomas
authors.affiliation: Departments of Anatomy/Cell Biology and Ophthalmology, Wayne State University, Detroit, Michigan, United States.
authors.forename: Ryan
authors.initials: R
authors.lastname: Thummel
authors.affiliation: Department of Biological Science, Florida State University, Tallahassee, Florida, United States.
authors.forename: James M
authors.initials: JM
authors.lastname: Fadool
authors.affiliation: Department of Ophthalmology and Visual Sciences University of Michigan, W. K. Kellogg Eye Center, Ann Arbor, Michigan, United States.
authors.forename: Peter F
authors.initials: PF
authors.lastname: Hitchcock
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D051792:Basic Helix-Loop-Helix Proteins / Q000096:biosynthesis / Q000235:genetics*; D002454:Cell Differentiation; D002478:Cells, Cultured; D018507:Gene Expression Regulation, Developmental; D018257:Helix-Loop-Helix Motifs; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics*; D010786:Photoreceptor Cells / Q000166:cytology / Q000378:metabolism*; D012313:RNA / Q000235:genetics*; D051880:Receptors, Notch / Q000378:metabolism*; D012038:Regeneration; D015027:Zebrafish / Q000196:embryology*
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D009419:Nerve Tissue Proteins; D012313:RNA; D051880:Receptors, Notch; C093564:Neurogenic differentiation factor 1