Single cell RNA-seq analysis reveals temporally-regulated and quiescence-regulated gene expression in Drosophila larval neuroblasts

Abstract

The mechanisms that generate neural diversity during development remains largely unknown. Here, we use scRNA-seq methodology to discover new features of the Drosophila larval CNS across several key developmental timepoints. We identify multiple progenitor subtypes - both stem cell-like neuroblasts and intermediate progenitors - that change gene expression across larval development, and report on new candidate markers for each class of progenitors. We identify a pool of quiescent neuroblasts in newly hatched larvae and show that they are transcriptionally primed to respond to the insulin signaling pathway to exit from quiescence, including relevant pathway components in the adjacent glial signaling cell type. We identify candidate "temporal transcription factors" (TTFs) that are expressed at different times in progenitor lineages. Our work identifies many cell type specific genes that are candidates for functional roles, and generates new insight into the differentiation trajectory of larval neurons.

Keywords: Insulin signaling; Intermediate neural progenitor; Neuroblast; Quiescence; Single cell RNA-sequencing; Temporal transcription factor.

Fig. 1

Larval atlas shows distinct cell identities and differentiating neural progenitor axis. A An atlas of 97,845 cells collected from 1h, 24h and 48h ALH larvae was built. These cells were analyzed with Seurat and clustered to identify major cell types such as neural progenitors, glial, mature neurons and other features to validate the atlas in clustering by cell type. B Feature plots of Dpn and Hey show a differentiating neural progenitor axis. The mature neuronal marker nSyb shows limited expression in progenitors that extends into mature neuronal clusters. The glial marker repo shows glial cells separated from progenitors and mature neurons. C Validated cell identity markers label distinct progenitor cell types within a developmental axis. D Progenitor atlas was made with a subset from the whole atlas of 33,458 neural progenitor and immature neuron cells. Black line indicates expected developmental trajectory. E UMAPs of progenitor clusters from 1h-48h ALH

Fig. 2

Quiescent neuroblasts and glial cells show enriched markers for regulating the TOR and insulin pathway. A UMAP of CNS cell types with quiescent neuroblasts in cluster 8 (circled). B UMAP of cluster from 1h-48h ALH. C Dot plot of top cluster defining genes alongside validated cell identity markers for quiescent neuroblasts. D Dot plot of genes involved with cell cycle regulation including the insulin signaling, AKT and TOR pathways. E UMAP re-clustered of 11,004 glia cells from a subset of the whole atlas. Diagram adapted from Kremer et al, 2017 [73]. F Validated glial cell type markers. G Temporal expression of signaling molecules involved in neuroblast quiescence within glial subtypes. H Validation of InR and Foxo expression in Dpn+ quiescent neuroblasts. Scale bar, 5 uM. I Model depicting cell growth and cell cycle genes identified as significantly enriched or depleted in the quiescent neuroblast cluster at 1h ALH, placed in the context of known signaling pathways

Fig. 3

Type I neuroblasts shows candidate novel markers and temporal transcription factors. A UMAP of type I neuroblasts highlighted. B UMAP of cluster from 1h-48h ALH. C Dot plot of top cluster defining genes and validated markers for type I neuroblasts. D Dot plot of differentially expressed transcription factors between 24h ALH and 48h ALH type I neuroblasts

Fig. 4

Type II progenitor cluster contains type II neuroblasts that show candidate temporal transcription factors. A UMAP of type II progenitors highlighted. B UMAP of cluster from 1h-48h. C  UMAP of sub clustered type II progenitors. D UMAP of type II neuroblasts from 1h-48h. E Dot plot of validated markers between type II neuroblasts and nonannotated progenitors. F Dot plot of top cluster defining genes and validated markers for type II neuroblasts. G Dot plot of differentially expressed TTFs between 24h alh and 48h type II neuroblasts

Fig. 5

INPs show candidate novel markers. A UMAP of INPs highlighted. B UMAP of cluster from 1h-48h alh. C Dot plot of top cluster defining genes and validated markers for INPs. D Dot plot of differentially expressed genes between type II neuroblasts, INPs and GMCs. E Dot plot of differentially expressed genes between type I neuroblasts and INPs.

Fig. 6

GMCs, new-born neurons and immature neurons show candidate novel markers. A UMAP of GMCs highlighted. B UMAP of GMCs from 1h-48h alh. C UMAP of new-born neurons highlighted. D UMAP of new-born neurons from 1h-48h alh. E UMAP of immature neurons highlighted. F UMAP of immature neurons from 1h-48h alh. G-I Dot plot of top cluster defining genes and validated markers for: G GMCs H New-born neurons I Immature neurons. J Differentially expressed genes between type I neuroblasts, GMCs, new-born neurons and immature neurons.

Fig. 7

Mature neuron conclusion. A An atlas of mature neurons (Brp and nSyb positive) was made with a subset of 51,596 cells from the whole atlas. B UMAP of atlas from 1h-48h alh with clusters 4 and 5 outlined. (C) Validated cell identity markers label distinct neuronal cell types. D Dot plot of differentially expressed factors between cluster 4 and 5. E Dot plot of top differentially* expressed genes between 1h alh, 24h alh and 48h alh in labeled mature neurons. F Dot plot of differentially* expressed cell surface molecules between 1h alh and 24h alh in labeled mature neurons. 48h alh contained no differentially expressed cell surface molecule genes. G Dot plot of differentially* expressed transcription factors between 1h alh, 24h alh and 48h alh in labeled mature neurons. *Genes found differentially expressed in at least 2 out of 9 annotated clusters of differentiated neuron cell types.