Functional genomics identifies neural stem cell sub-type expression profiles and genes regulating neuroblast homeostasis

Abstract

The Drosophila larval central brain contains about 10,000 differentiated neurons and 200 scattered neural progenitors (neuroblasts), which can be further subdivided into ~95 type I neuroblasts and eight type II neuroblasts per brain lobe. Only type II neuroblasts generate self-renewing intermediate neural progenitors (INPs), and consequently each contributes more neurons to the brain, including much of the central complex. We characterized six different mutant genotypes that lead to expansion of neuroblast numbers; some preferentially expand type II or type I neuroblasts. Transcriptional profiling of larval brains from these mutant genotypes versus wild-type allowed us to identify small clusters of transcripts enriched in type II or type I neuroblasts, and we validated these clusters by gene expression analysis. Unexpectedly, only a few genes were found to be differentially expressed between type I/II neuroblasts, suggesting that these genes play a large role in establishing the different cell types. We also identified a large group of genes predicted to be expressed in all neuroblasts but not in neurons. We performed a neuroblast-specific, RNAi-based functional screen and identified 84 genes that are required to maintain proper neuroblast numbers; all have conserved mammalian orthologs. These genes are excellent candidates for regulating neural progenitor self-renewal in Drosophila and mammals.

Figure 1. Using ectopic self-renewal mutants for expression profiling of neuroblasts

Six genotypes were used which are known to cause expansions in the number of neuroblasts in Drosophila larval central brains. (A) Single-slice confocal images of wild-type (120 hours ALH) and mutant (144 hours ALH) brain lobes stained for Dpn (neuroblast marker) and Elav (neuronal marker). (B) The variable level of ectopic neuroblast number per brain lobe of each mutant genotype (n=2 for each genotype). (C) Schematic of wild-type type I and type II neuroblast divisions. Type I neuroblasts have nuclear Ase as well as diffuse cytoplasmic Pros, which is asymmetrically segregated into the GMC upon neuroblast division. Type II neuroblasts lack both Pros and Ase, both of which are expressed in INPs; Pros is then segregated asymmetrically into the GMC upon INP division. GMCs divide to generate Elav⁺ neurons. (D) High magnification image of a Dpn⁺ Ase⁺ type I neuroblast (white arrowhead) and a Dpn⁺ Ase⁻ type II neuroblast (yellow arrowhead) in the dorso-medial region of a wild-type brain. The type II neuroblast can be unambiguously identified based on the presence of GFP driven by R19H09-Gal4 (Bayraktar et al., 2010). (E) Mutant brains (120 hours ALH) stained with anti-Dpn (to mark all neuroblasts) and anti-Ase (which only marks type I neuroblasts). Inset in the Ase panel of the lgl pins brain shows that the Pros staining pattern in the same brain matches very closely to the Ase pattern. (F) Schematic of the methodology used here. Scale bars: 10 µm in (D); 100 µm in (A and E).

Figure 2. Results of cluster analysis

(A) Cluster analysis-categorized genes with expression changes in mutant compared to wild-type brains, divided into three groups (A, B, and C).The dendrogram at the top is labeled according to the mutant genotype; Roman numerals indicate the neuroblast subtype(s) enriched in each mutant (I = type I; II = type II). (B) Log₂ expression changes (mutant/wild-type) averaged over all genes in each group.

Figure 3. Differential expression of genes excluded from type II neuroblasts

(A) Position within group B of sub-cluster containing pros and ase as well as nine other genes with unknown expression patterns. Log₂ enrichment over wild-type is shown, averaged over all genes in the sub-cluster. (B) Confocal image of a wild-type brain lobe at 120 hours ALH. Multiple type I neuroblasts, four type II lineages, and three type II neuroblasts are visible. (C) Enlargement of the region boxed in (B). All neuroblasts are Dpn⁺; a subset of type I neuroblasts are Rx⁺, while type II neuroblasts are Rx⁻. INPs are also Rx⁻. Rx is expressed in a subset of neuronal progeny in both type I and type II lineages. (D) brat mutant brain lobe (120 hours ALH) contains many Dpn⁺ neuroblasts, but these cells do not express Rx. Rx is expressed in a few of the Pros⁺ cells, all of which in this focal plane are neurons and do not express Dpn. (E) lgl pins brain lobe (120 hours ALH) in which Rx is expressed in a subset of ectopic Dpn⁺ neuroblasts. Rx expression is limited to cells expressing Pros, which in lgl pins also express Ase (Figure 1E, inset) and are likely derived from expansion of type I neuroblasts. The Pros⁻ regions (type II-derived) are entirely Rx⁻. White arrow: Rx⁻ type I neuroblast; white arrowheads: Rx⁺ type I neuroblasts; yellow arrows: Rxtype II neuroblasts; mCD8::GFP driven by R9D11-Gal4 marks a subset of type II lineages, but not the type II neuroblasts themselves (Bayraktar et al., 2010). Shown outlined here with yellow dashed lines are several dorso-medial type II lineages [DM 2, 3, 4, 5, and 6 in (B); DM 2, 3, and 4 in (C)]. Scale bars: 100 µm in (B), (D), and (E); 10 µm in (C).

Figure 4. Identification of a cluster with type II-biased expression

(A) Position in group B of a sub-cluster in which genes are expressed higher in brat and lgl lgd than in other genotypes. Enrichment shown is averaged over all genes in the sub-cluster. (B) Wild-type brain lobe (120 hours ALH). Visible are multiple type I neuroblasts as well as several type II neuroblasts and their lineages. Most type I neuroblasts are Optix⁻, while four of the six dorso-medial type II neuroblasts are Optix⁺ (DM1,2,3,6); Optix⁻ is absent from type II neuroblasts DM 4&5 and their INPs, but present in a subset of their progeny. (C) Enlargement of the box in (B) shows both Optix⁺ and Optix type I and type II neuroblasts. Shown are type II lineages DM 2, 3, and 4. Optix is nearly absent from the entire DM4 lineage. (D) brat brain (120 hours ALH) shows that Optix is expressed in a dorso-medial region in which nearly all cells are Dpn⁺ ectopic neuroblasts. (E) lgl pins brain (120 hours ALH) exhibits Optix expression primarily in Dpn⁺ Prosregions (type II-derived ectopic neuroblasts). White arrows: Optix⁻ type I neuroblasts; white arrowheads: Optix⁺ type I neuroblasts; yellow arrow: Optix⁻ type II neuroblast; yellow arrowheads: Optix⁺ type II neuroblasts. GFP driven by R9D11-Gal4 marks dorso-medial type II lineages, but not the type II neuroblasts themselves. Shown outlined here with yellow dashed lines are several dorso-medial type II lineages [DM 2, 3, 4, 5, and 6 in (B); DM 2, 3, and 4 in (C)]. Scale bars: 100 µm in (B), (D), and (E); 10 µm in (C).

Figure 5. Gene Ontology terms enriched in each group

(A) Group A, the “neuroblast cluster” with three sub-clusters marked in which the indicated GO annotations are significantly enriched compared to all Drosophila genes. Each value in red indicates the enrichment of the GO term in all group A genes excluding the adjacent sub-cluster. (B) Chart depicting the percent of all Drosophila genes characterized by select GO annotations as well as percent of genes in each group with those annotations. Asterisks indicate significant enrichment of GO term compared with all Drosophila genes (*: p<.05; **: p<.001). (C) Group C, the “neuron cluster” with sub-clusters labeled indicating significantly enriched GO terms; each value in red indicates the enrichment of the GO term in all group C genes except the adjacent sub-cluster.

Figure 6. RNAi screen identifies neuroblast homeostasis genes

(A) Flowchart describing the selection of 691 RNAi lines used in this screen. (B) Neuroblast number gain and loss phenotypes of the 179 genes for which RNAi knock-down caused lethality. (C) Neuroblast numbers per brain lobe of the genes which were assayed for neuroblast number phenotype.