Project description:Metazoans utilize a handful of highly conserved signaling pathways to create a signaling backbone that governs all stages of development, by providing spatial and temporal cues that influence gene expression. How these few signals have such a versatile developmental action is of significance to evolution, development, and disease. Their versatility likely depends upon the larger-scale network they form through integration. Such integration is exemplified by cross-talk between the Notch and the Receptor Tyrosine Kinase (RTK) pathways. We examined the transcriptional output of Notch-RTK cross-talk during Drosophila development and present in vivo data that supports a role for selected mutually-regulated genes as potentially important nodal points for signal integration. We find the complex interplay between these pathways involves their mutual regulation of numerous core components of RTK signaling in addition to targets that include components of all the major signalling pathways (TGF-Î², Hh, Jak/Stat, Nuclear Receptor and Wnt). Interestingly, Notch-RTK integration did not lead to general antagonism of either pathway, as is commonly believed. Instead, integration had a combinatorial effect on specific cross-regulated targets, which unexpectedly included the majority of Ras-responsive genes, suggesting Notch can specify the response to Ras activation. Experiment Overall Design: To identify nodal points that interlink the Notch and RTK pathways, we examined the transcriptional output of each during Drosophila embryonic development and identified common transcriptional targets. Pathway activation was achieved through ubiquitous GAL4-mediated expression of activated Notch (UAS-NotchICD) or through an activated form of the RTK signal mediator Ras1 (UAS-Ras1V12), either alone or in combination, under the control of the armadillo promoter, which drives GAL4 at moderate physiological levels. Homozygous armadillo-GAL4 virgin females were crossed either to: 1) homozygous UAS-NotchICD males; 2) males homozygous for both UAS-Ras1V12 and UAS-NotchICD; or 3) homozygous UAS-Ras1V12 males. As a control for GAL4 dependent effects, homozygous armadillo-GAL4 virgin females were crossed to w1118 males. Embryos resulting from these crosses were collected on apple juice plates for one hour, incubated at 25Â°C for 5 hours and 30 minutes for the first time point and 6 hours and 45 minutes for the second time point; dechorionated by treatment with 50% Chlorox bleach at room temperature for 1 minute with agitation, washed with 0.5X PBS, 1% Tween, rinsed with ddH2O. Embryos were then shock frozen and stored in liquid nitrogen at 5:45-6:45 hours After Egg Lay (AEL) for the first time point and 7-8 hours AEL for the second. All collections were performed in parallel sets but staggered to equalize the length of embryo submersion in liquid. Each collection contained approximately 500 embryos. Frozen embryos were ground by pestle in the Trizol reagent (Invitrogen). RNA was extracted via standard methods, with the inclusion of an additional Trizol extraction prior to RNA precipitation to increase RNA purity. Total RNA was prepared from three independent experiments for each described embryonic genotype at each of the two time points. RNA samples were subjected in triplicate to analysis by Affymetrix high-density oligonucleotide arrays using the DrosGenome1 array (Affymetrix) that contains 14,010 probe sets specific to 13,108 Drosophila genes. Probe synthesis and microarray hybridization were performed according to standard Affymetrix protocols. External standards were included to control for hybridization efficiency and sensitivity. Following washing, the chips were scanned with a Hewlett-Packard GeneArray laser scanner. Signal levels were obtained and statistical analysis performed using GC-Robust Multi-Array expression measure (GC-RMA) and LInear Modes for MicroArray (LIMMA) data packages and the affylmGUI graphical user interface in the R programming environment.

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Drosophila melanogaster

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