Emergence of Neuronal Diversity during Vertebrate Brain Development

Abstract

Neurogenesis in the vertebrate brain comprises many steps ranging from the proliferation of progenitors to the differentiation and maturation of neurons. Although these processes are highly regulated, the landscape of transcriptional changes and progenitor identities underlying brain development are poorly characterized. Here, we describe the first developmental single-cell RNAseq catalog of more than 200,000 zebrafish brain cells encompassing 12 stages from 12 hours post-fertilization to 15 days post-fertilization. We characterize known and novel gene markers for more than 800 clusters across these timepoints. Our results capture the temporal dynamics of multiple neurogenic waves from embryo to larva that expand neuronal diversity from ~20 cell types at 12 hpf to ~100 cell types at 15 dpf. We find that most embryonic neural progenitor states are transient and transcriptionally distinct from long-lasting neural progenitors of post-embryonic stages. Furthermore, we reconstruct cell specification trajectories for the retina and hypothalamus, and identify gene expression cascades and novel markers. Our analysis reveal that late-stage retinal neural progenitors transcriptionally overlap cell states observed in the embryo, while hypothalamic neural progenitors become progressively distinct with developmental time. These data provide the first comprehensive single-cell transcriptomic time course for vertebrate brain development and suggest distinct neurogenic regulatory paradigms between different stages and tissues.

A single-cell guide to retinal development: Cell fate decisions of multipotent retinal progenitors in scRNA-seq

“…These post-embryonic RPCs possess the capacity to generate clones of both size and composition comparable to embryonic RPCs ( Wan et al, 2016 ). Correspondingly, embryonic and post-embryonic zebrafish RPCs display similar gene expression profiles when examined at single-cell resolution ( Raj et al, 2020 ; Xu et al, 2020 ). Though mice lack the innate ability to undergo retinal regeneration or continuously generate new neurons throughout the lifespan of the animal, studies have demonstrated that Msx1 + progenitor cells derived from the proximal zone of mouse CMZ give rise to both RPCs and non-neural epithelia during embryonic periods of development ( Bélanger et al, 2017 ; Marcucci et al, 2016 ).…”

“…In order to better appreciate the mechanisms governing temporal cell fate specification, researchers have recently employed large-scale scRNA-seq to profile the in vivo development of retinas in mice, humans, chicken, and zebrafish as well as the in vitro generation of human ES-cell/iPS-cell derived retinal organoids ( Clark et al, 2019 ; Collin et al, 2019 ; Cowan et al, 2020 ; Hu et al, 2019 ; Lu et al, 2020 ; Raj et al, 2020 ; Sridhar et al, 2020 ; Xu et al, 2020 ; Yamagata et al, 2021 ). These scRNA-seq studies have answered many questions regarding RPC heterogeneity at individual ages and across development, but have yet to definitively assign competence transitions within the developing vertebrate retina.…”

“…HES4 and its corresponding zebrafish ortholog, her9 , are expressed in human and zebrafish RPCs, respectively. However, Hes4 has no ortholog within the mouse genome ( Hu et al, 2019 ; Lu et al, 2020 ; Raj et al, 2020 ; Sridhar et al, 2020 ; Xu et al, 2020 ). In zebrafish, her9 is required for the differentiation, maintenance, and survival of rods and red/green cones ( Coomer et al, 2020 ).…”

“…One study of zebrafish neurodevelopment has employed scGESTALT to record lineages through barcode editing. However, genome editing had saturated before terminal divisions in the retina, revealing little lineage relationship between the different zebrafish retinal cell types but suggesting that tuning of the system to be active at the onset of retinogenesis could prove to be fruitful for resolving retinal lineages ( Raj et al, 2020 ).…”

“…More in-depth discussions and comparisons of pseudotime techniques have previously been discussed at length ( Saelens et al, 2019 ; Tritschler et al, 2019 ). Implementation of pseudotime trajectory analysis in scRNA-seq studies have determined the gene expression dynamics that govern retinal development, from optic cup formation during zebrafish and xenopus laevis embryogenesis ( Briggs et al, 2018 ; Farrell et al, 2018 ; Wagner et al, 2018 ) through terminal cell fate specification, differentiation and maturation stages of all major classes of retinal cell types in mice, humans, and zebrafish ( Clark et al, 2019 ; Lo Giudice et al, 2019 ; Lu et al, 2020 ; Raj et al, 2020 ; Sridhar et al, 2020 ). Pseudotime techniques and their implementation within retinal development scRNA-seq datasets is highlighted in Fig.…”