Molecular organization of Drosophila neuroendocrine cells by Dimmed

Abstract

Background: In Drosophila, the basic-helix-loop-helix protein DIMM coordinates the molecular and cellular properties of all major neuroendocrine cells, irrespective of the secretory peptides they produce. When expressed by nonneuroendocrine neurons, DIMM confers the major properties of the regulated secretory pathway and converts such cells away from fast neurotransmission and toward a neuroendocrine state.

Results: We first identified 134 transcripts upregulated by DIMM in embryos and then evaluated them systematically using diverse assays (including embryo in situ hybridization, in vivo chromatin immunoprecipitation, and cell-based transactivation assays). We conclude that of eleven strong candidates, six are strongly and directly controlled by DIMM in vivo. The six targets include several large dense-core vesicle (LDCV) proteins, but also proteins in non-LDCV compartments such as the RNA-associated protein Maelstrom. In addition, a functional in vivo assay, combining transgenic RNA interference with MS-based peptidomics, revealed that three DIMM targets are especially critical for its action. These include two well-established LDCV proteins, the amidation enzyme PHM and the ascorbate-regenerating electron transporter cytochrome b(561-1). The third key DIMM target, CAT-4 (CG13248), has not previously been associated with peptide neurosecretion-it encodes a putative cationic amino acid transporter, closely related to the Slimfast arginine transporter. Finally, we compared transcripts upregulated by DIMM with those normally enriched in DIMM neurons of the adult brain and found an intersection of 18 DIMM-regulated genes, which included all six direct DIMM targets.

Conclusions: The results provide a rigorous molecular framework with which to describe the fundamental regulatory organization of diverse neuroendocrine cells.

Figure 1

A schematic overview of the workplan for this study.A genome wide search for dimm targets performed by Genechip, then evaluated by three downstream analyses of DIMM regulation, and further evaluated by a functional in vivo assay.

Figure 2

RNA in situ hybridization in control embryos and embryos that over-express dimm Left - control (w¹¹¹⁸); Right - elav> dimm; Probes: A, B) dimm; C,D) Phm; E,F) CG13248; G,H) CG11254 (mael); I,J) CG17293; K,L) CG6522. See also Figure S1.

Figure 3

Trans-activation by DIMM of genomic fragments of candidate genes in Drosophila BG3-c2 neuronal cell lines.Fold ratios represent Luciferase levels with dimm co-transfection divided by those without. Histograms represents means and SEMs of at least three independent replicate assays. (A). Results of testing ten DIMM targets. (B) Results from analysis of E box sequence requirements within the CG13248 regulatory region. E1, E2 and E3 indicate three separate E boxes, which were mutated singly, doubly or in triple-format. In (A), * p<0.05; ** p< 0.01 vs empty vector, by student's T-test. In (B), * p< 0.01 vs CG13248 WT sequence, by student's T-test. See also Figure S3 which illustrates E box positions in and around these candidate targets.

Figure 4

ChIP analysis in vivo of putative DIMM binding at E-boxes within dimm-dependent candidates genes.Darker histograms show the level of enrichment of the putative DIMM binding sites defined by the value in experimental versus control genotypes (see Methods). Lighter histograms show the level of enrichment of arbitrarily chosen sites ~6 kB upstream of the putative DIMM-binding sites, in the same experimental versus control genotypes. Histograms represents average and SEMs at least two independent assays (two biological replicates).

Figure 5

MS-based label-free quantitative analysis of alterations in MII peptide accumulation in photoreceptors following RNA interference of candidate DIMM targets.(A) Mass spectra of MII in the control (GMR>UAS-ppMII; UAS-dimm, red) and experimental (GMR>UAS-ppMII; UAS-dimm; UAS-PHM-RNAi, blue) samples. The intensity ratio of MII in experimental versus control samples in these spectra is 0.54. B) Mass spectra of MT2 in the control and experimental samples. The intensity ratio of MT2 in experimental to control in these spectra is 1.02. C) Exogenous MII level in the experimental samples with RNAi compared to that in the control samples. Histograms represents means and SEMs, * p<0.05; vs control (GMR>UAS-dimm), by student's T-test. N, biological replicates. See also Figure S3 for analysis of MS-based quantitation of the endogenous peptide Drm-MT2.

Figure 6

Enrichment of CG13248 (CAT-4) in DIMM neurons and its regulation by dimm in vivo A) DIMM-like and CAT-4-like IR are extensively co-localized in the adult brain. c929>UAS-GFP (anti-GFP, green), anti-CAT-4 (red); bottom: merged image. B–D) CAT-4-like IR following RNAi knock-down of dimm. B, B') parental control (386Y-GAL4); C, C') 386Y>UAS-DCR2/UAS-CAT-4-RNAi; D, D') 386Y>DCR2/ dimm-RNAi; (B–D) anti-DIMM (green), (B'–D') anti-CAT-4 (red). (E–F) CAT-4-like IR is normally present in the two DIMM-positive neurons of the four-cell Tv-cluster; following DIMM mis-expression throughout the cluster, CAT-4 appears in all four cells. E) ap> GFP, anti-GFP (green), anti-CAT-4 (red), F) ap> dimm, anti-MYC (=DIMM) (green); anti-CAT-4 (red). See also Figure S4 for high power images of cDAT-4-like immunoreactivity in different adult brain DIMM neurons.

Figure 7

Comparison of transcripts upregulated by DIMM in embryos with transcripts enriched in DIMM-positive peptidergic neurons of the adult brain Top: A Venn diagram illustrating the identification of 18 genes (13% of 134) of those up-regulated by DIMM over-expression among the 537 normally enriched in DIMM-positive large LNv [29]. Bottom lists the 18-gene intersection, asterisks mark those genes that were shown to be direct DIMM targets by current experiments.