SoxNeuro orchestrates central nervous system specification and differentiation in Drosophila and is only partially redundant with Dichaete

Abstract

Background: Sox proteins encompass an evolutionarily conserved family of transcription factors with critical roles in animal development and stem cell biology. In common with vertebrates, the Drosophila group B proteins SoxNeuro and Dichaete are involved in central nervous system development, where they play both similar and unique roles in gene regulation. Sox genes show extensive functional redundancy across metazoans, but the molecular basis underpinning functional compensation mechanisms at the genomic level are currently unknown.

Results: Using a combination of genome-wide binding analysis and gene expression profiling, we show that SoxNeuro directs embryonic neural development from the early specification of neuroblasts through to the terminal differentiation of neurons and glia. To address the issue of functional redundancy and compensation at a genomic level, we compare SoxNeuro and Dichaete binding, identifying common and independent binding events in wild-type conditions, as well as instances of compensation and loss of binding in mutant backgrounds.

Conclusions: We find that early aspects of group B Sox functions in the central nervous system, such as stem cell maintenance and dorsoventral patterning, are highly conserved. However, in contrast to vertebrates, we find that Drosophila group B1 proteins also play prominent roles during later aspects of neural morphogenesis. Our analysis of the functional relationship between SoxNeuro and Dichaete uncovers evidence for redundant and independent functions for each protein, along with unexpected examples of compensation and interdependency, thus providing new insights into the general issue of transcription factor functional redundancy.

Figure 1

SoxN functional studies. (A) Overview of the SoxN datasets generated. Each coloured box below the time line represents a single gene expression, DNA adenine methyltransferase identification (DamID) or chromatin immunoprecipitation (ChIP) experiment performed at the indicated time ranges. Above the time line, major events in neural development are indicated and illustrated with FlyBase images adapted from Volker Hartenstein, Atlas of Drosophila Development, Cold Spring Harbor Laboratory Press, 1993. (B) Partitioning of genes differentially expressed in SoxN mutants. Probes corresponding to differentially expressed genes were divided into three groups (downregulated, upregulated and variable) according to their expression trend over time. (C) Genomic profiles of SoxN binding. Window scores and binding intervals at a false discovery rate (FDR) of 1% and FDR of 25% are displayed for the SoxNDam dataset (purple), and binding intervals at FDR 25% are shown for the SoxNPA179 Early (red), SoxPA179 Late (orange), SoxND1 (blue) and SoxND2 (green) ChIP datasets. SoxN core binding intervals are displayed in black, matches to the SoxN binding motif as thin bars and the locations of know cis-regulatory modules (CRMs) as grey bars. SoxN binding in the gsb-n and gsb (top panel), nerfin-1 (middle panel) and pdm2 (bottom panel) regions are displayed.

Figure 2

Features of SoxN binding and SoxN direct targets. (A) Barplot representing genomic features hit by SoxN binding intervals. 'Mixed' indicates intervals hitting more than a single feature in different genes. (B)De novo motifs discovered in the SoxN core dataset. The top three motifs found and their associated P-values are reported. (C) Proportional Venn diagram showing the overlap between genes differentially expressed in SoxN mutants (green, left) and genes bound by SoxN (blue, right). (D) Dorsal, lateral and ventral views of SoxN expression in a stage 9-10 embryo (left, images from the Berkeley Drosophila Genome Project) and false-colour heatmaps representing the average expression pattern of SoxN direct targets at the same developmental stages. (E) Network showing known interactions between SoxN direct targets obtained by superimposing the list of SoxN direct targets with a network created from the DroID database. Genes are colour-coded according to their expression trend during the time-course (green, downregulated; red, upregulated; light blue, variable). SoxN and Dichaete are highlighted with yellow boxes.

Figure 3

Validation of SoxN direct targets. (A) Selected genes from a hierarchical clustering of expression trends highlighting three functional groups of SoxN direct targets. logFC = log₂ fold change. (B) Immunohistochemical stainings of a selection of SoxN direct targets identified in this study in control embryos (left), SoxN homozygous mutants (middle) and Kr-Gal4 UAS-SoxN embryos (right). All embryos are shown as ventral views with anterior to the left. Ind, showing normal expression in mutant and transgenic embryos, is provided as a negative control.

Figure 4

SoxN and Dichaete differential binding. (A-C) Differential binding in pairwise comparisons of the SoxNDam, DDam, D-SoxNDam and SoxN-DDam datasets as normalised probe intensities (log₂ fold change). Light grey areas are probes bound in both datasets, black regions are not bound in either. (A) SoxNDam (dark blue) and DDam (dark green); (B) SoxNDam (dark blue) and D-SoxNDam (light blue); (C) DDam (dark green) and SoxN-DDam (light green). (D-F) Representative SoxN and Dichaete binding profiles in wild-type embryos (dark blue and dark green, respectively). Matches to the SoxN binding motif are displayed as thin bars, FlyLight and REDfly enhancers are displayed in light grey. (D) SoxN and Dichaete common binding across the achaete-scute complex. (E) SoxN unique binding in proximity of robo3. (F) Dichaete unique binding in the gus and Atf6 region.

Figure 5

Profiles of SoxN and Dichaete binding in Dichaete and SoxN mutant embryos. Representative SoxN binding profile in Dichaete mutant embryos (light blue) and Dichaete binding profile in SoxN mutant embryos (light green). Matches to the SoxN binding motif are displayed as thin bars, FlyLight and REDfly enhancers are displayed in light grey. Events of (A) compensation, (B) increased binding, (C)de novo binding and (D) loss of binding are highlighted as red shaded boxes.