A comprehensive temporal patterning gene network in Drosophila medulla neuroblasts revealed by single-cell RNA sequencing

Abstract

During development, neural stem cells are temporally patterned to sequentially generate a variety of neural types before exiting the cell cycle. Temporal patterning is well-studied in Drosophila, where neural stem cells called neuroblasts sequentially express cascades of Temporal Transcription Factors (TTFs) to control the birth-order dependent neural specification. However, currently known TTFs were mostly identified through candidate approaches and may not be complete. In addition, many fundamental questions remain concerning the TTF cascade initiation, progression, and termination. It is also not known why temporal progression only happens in neuroblasts but not in their differentiated progeny. In this work, we performed single-cell RNA sequencing of Drosophila medulla neuroblasts of all ages to study the temporal patterning process with single-cell resolution. Our scRNA-seq data revealed that sets of genes involved in different biological processes show high to low or low to high gradients as neuroblasts age. We also identified a list of novel TTFs, and experimentally characterized their roles in the temporal progression and neural fate specification. Our study revealed a comprehensive temporal gene network that patterns medulla neuroblasts from start to end. Furthermore, we found that the progression and termination of this temporal cascade also require transcription factors differentially expressed along the differentiation axis (neuroblasts -> -> neurons). Lola proteins function as a speed modulator of temporal progression in neuroblasts; while Nerfin-1, a factor required to suppress de-differentiation in post-mitotic neurons, acts at the final temporal stage together with the last TTF of the cascade, to promote the switch to gliogenesis and the cell cycle exit. Our comprehensive study of the medulla neuroblast temporal cascade illustrated mechanisms that might be conserved in the temporal patterning of neural stem cells.

A Notch-dependent transcriptional mechanism controls expression of temporal patterning factors in Drosophila medulla

“…The previous TTF Ey has been shown to be required but not sufficient for activating Slp genes' expression in medulla neuroblasts, but it was not known whether this regulation is direct. Vnd is not expressed in medulla neuroblasts according to our single-cell RNA-sequencing data 17 , but a related homologous NK-2 homeobox transcription factor Scarecrow 55 (Scro) is expressed around the same time as Slp, and loss of Scro leads to significantly reduced Slp expression level 17 . The CSL transcription factor Su(H) is the Drosophila homolog of RBP-J 56,57 , and the key DNA-binding component of the active Notch transcriptional complex.…”

“…Observed distribution of temporal patterning transcription factors may be regulated at either the transcriptional or post-transcriptional level, and the protein expression pattern is not necessarily the same as that of the mRNA transcripts. A case in point is the expression pattern of the SoxN protein recently reported to function alongside Dmrt99B and Hth to specify identities of early-born neurons in the medulla; while mRNA transcripts of soxN gene are widely distributed in neuroblasts of ages, SoxN protein is only expressed in the youngest neuroblasts, suggesting that post-transcriptional mechanisms are at work to confine the actual domain of SoxN protein expression 17 . To examine whether the expression of Slp1/2 in medulla neuroblasts is regulated at transcription we observed the distributions of slp1 and slp2 mRNA transcripts by fluorescence in situ-hybridization (Fig.…”

“…Neural progenitors called neuroblasts (NBs) have been shown to sequentially express series of temporal transcription factors (TTFs), which are each required to specify subsets of neuron fates [reviewed in 8,9 ]. For example, neuroblasts in the embryonic ventral nerve cord (vnc) are temporally patterned by a TTF cascade Hunchback (Hb), Kruppel (Kr), Nubbin/Pdm2 (Pdm), Castor (Cas) and Grainy head (Grh) [10][11][12][13] , while Drosophila optic lobe medulla neuroblasts utilize a different TTF cascade composed of Homothorax (Hth), SoxNeuro (SoxN), Doublesex-Mab related 99B (Dmrt99B), Odd paired (Opa), Eyeless (Ey), Earmuff (Erm), Homeobrain (Hbn), Sloppy-paired (Slp1,Slp2), Scarecrow (Scro), Dichaete (D), BarH1/2, Tailless (Tll), Glial cell missing (Gcm), and Nerfin-1 [14][15][16][17][18][19] . In these TTF cascades, cross-regulations were identified among TTF genes based on loss and gain of function phenotypes, and they were proposed to form a transcriptional cascade [11][12][13]19,20 that can in theory self-propagate 21 .…”

Netrins and receptors control Drosophila optic lobe organization and transmedullary neuron axon targeting

“…To examine whether there is a specific subset of Tm neurons crucial for IOC formation, we first used Gal4 drivers initiated in different temporal windows to knock down Fra. Medulla neuroblasts sequentially generate different neural types controlled by a temporal transcription factor (TTF) cascade: Hth -> Opa -> Ey+Erm -> Ey+Opa -> Slp -> D -> B-H1&2->Tll->Gcm 60 . We found knocking down fra using ey-gal4, slp-gal4, or D-gal4 does not affect the inner chiasm formation (Supplementary Fig.…”

“…Temporal patterning is mediated by a cascade of temporal transcription factors (TTF), including Homothorax (Hth), Eyeless (Ey), Sloppy paired 1 and 2 (Slp), Dichaete (D) and Tailless (Tll) in neuroblasts 15,16 . More TTFs have been found by scRNA-seq of medulla neuroblasts recently [17][18][19] . Notch-dependent binary fate choice happens in two daughters of GMCs.…”