Expression profiling of prospero in the Drosophila larval chemosensory organ: Between growth and outgrowth

Abstract

Background: The antenno-maxilary complex (AMC) forms the chemosensory system of the Drosophila larva and is involved in gustatory and olfactory perception. We have previously shown that a mutant allele of the homeodomain transcription factor Prospero (prosVoila1, V1), presents several developmental defects including abnormal growth and altered taste responses. In addition, many neural tracts connecting the AMC to the central nervous system (CNS) were affected. Our earlier reports on larval AMC did not argue in favour of a role of pros in cell fate decision, but strongly suggested that pros could be involved in the control of other aspect of neuronal development. In order to identify these functions, we used microarray analysis of larval AMC and CNS tissue isolated from the wild type, and three other previously characterised prospero alleles, including the V1 mutant, considered as a null allele for the AMC.

Results: A total of 17 samples were first analysed with hierarchical clustering. To determine those genes affected by loss of pros function, we calculated a discriminating score reflecting the differential expression between V1 mutant and other pros alleles. We identified a total of 64 genes in the AMC. Additional manual annotation using all the computed information on the attributed role of these genes in the Drosophila larvae nervous system, enabled us to identify one functional category of potential Prospero target genes known to be involved in neurite outgrowth, synaptic transmission and more specifically in neuronal connectivity remodelling. The second category of genes found to be differentially expressed between the null mutant AMC and the other alleles concerned the development of the sensory organs and more particularly the larval olfactory system. Surprisingly, a third category emerged from our analyses and suggests an association of pros with the genes that regulate autophagy, growth and insulin pathways. Interestingly, EGFR and Notch pathways were represented in all of these three functional categories. We now propose that Pros could perform all of these different functions through the modulation of these two antagonistic and synergic pathways.

Conclusions: The current data contribute to the clarification of the prospero function in the larval AMC and show that pros regulates different function in larvae as compared to those controlled by this gene in embryos. In the future, the possible mechanism by which Pros could achieve its function in the AMC will be explored in detail.

Figure 1

AMC region from third instar larvae observed by optical microscopy. (A) Bright-field view of the larval AMC region (dorsal view, anterior down), the hooks appear in dark. Cells that constitute the AMC are located on either side of the hooks. (B) 3D reconstruction of AMC (TO +DO), labelled with Pros (red) and Elav (green) that labels neuronal cells. (B'1-3) Zoomed view of a confocal section of the framed region in B showing respectively the Pros (B'1), Pros/Elav (B'2) and Elav (B'3) staining; Anti-Prospero labels two types of Pros expressing cells (Pros+): large (arrowheads in B'1) and small cells (arrows in B'1). Some of the small Pros+ cells express Elav (B'2). Scale bars represent 10 μm.

Figure 2

Gene expression analyses. (A) Hierarchical clustering of 5950 genes for a total of 17 samples relative to the larval AMC and CNS of the different prosV mutants. Each row represents a gene and each column a sample. For each organ, the samples at the top of the image are classified according to the severity of their phenotypes (from wild type to the most severe phenotype: V14, V13, V24 and V1). Each cell in the matrix corresponds to the expression level of one gene in a sample (see colour scale at the bottom of the image). The yellow frames represent the AMC tissue specific signature and contain the genes that are differentially expressed between AMC and the CNS independently of Pros expression. (B) Discriminating scores (DS) smoothed in a window of 100 genes, calculated between V1 and other prosV in the AMC (in red), and between V1 and V14 in the CNS (in blue), among the gene clusters. In the AMC, three peaks, annotated 1, 2 and 3 (black bars), appear to be enriched in differentially expressed genes, they have been associated respectively to "cell fate commitment", "proteasome complex" and "signal transduction" ontologies. (C) Hierarchical clustering of the 306 genes present in peak 1 in the AMC. Pink frame zooms on a set of highly correlated (r>0.9) genes that are differentially expressed between V1 and all other alleles in the AMC. These genes are referenced on the right according to the Drosophila nomenclature (see also Table 3). The dendrogram on the left represents correlation distances between the profiles of the studied genes. Differentially expressed genes indicated in red were common with CNS. (D) Same as (C) in CNS samples. The Pink framed region contains 86 genes which are referenced on the right according to the Drosophila nomenclature. Differentially expressed genes indicated in red were common with AMC. (E) Motif found in the promoting region of the 28 genes common to AMC and CNS (genes indicated in red).

Figure 3

Schematic representation of the overlapping function attributed to the AMC putative Pros target genes. The functional categories were established using a manual annotation (the criteria used for this annotation are indicated in the text, see also for further phenotypic description and corresponding references [Additional file 5: Supplemental Tables S2-S4]). The three functional groups identified are represented by three distinct colored sets. The genes located at the intersection between two sets can assume both functions. It should be noted that the genes indicated in black (EGFR, Notch, Ash2 and prosβ2) belong to the three functional groups: neurite outgrowth, sensory organ development, and growth/autophagy.