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Periodic Report Summary 1 - Notch and polarity (Modulation of the Notch receptor activity through the regulation of the epithelial apical-basal polarity in Drosophila melanogaster)

Quality validation date:2012-04-26

Abstract
Several preliminary observations indicate that the apical-basal organization of epithelial cells influence the activity of the Notch (N) signaling pathway. In Drosophila, N regulates fate decisions within epithelia which display a clear Apical-Basal Polarity (ABP). N and its ligands Delta (Dl) and Serrate (Ser) colocalize at the adherens junctions (AJ) level in epithelial cells, suggesting that signaling may take place at the AJ level in these epithelial cells.
Studies in Drosophila have shown multiple instances of modulation of N activity by ABP and AJ components like Crumbs, Par-1, DE-Cadherin, Echinoid or ZO-1.

MAIN OBJECTIVE
The aim of this project is to systematically analyze the mechanisms of N regulation by ABP components using a genetic clonal approach in Drosophila.
We make use of sensory organ development as a model system to study N activity. During development of sensory organs in the dorsal thorax of Drosophila, the Sensory Organ Precursor (SOP) is singled out from a cluster of equivalent epithelial cells by a process called lateral inhibition which is very sensitive to N-activity levels.

EXPERIMENTAL DESIGN

We generate clones of cells expressing dsRNA against a specific gene product within the notum and observe the propensity of SOPs located along the clone border to be selected from cells within this clone, indicative of the relative levels of N activity of the cells in the clone vs. the cells outside the clone. We label both the clones and the SOP with fluorescent markers that can be followed in vivo and are suitable for computer-based analysis of the results.

1) Generation of clones. We have obtained a collection of RNAi lines that target potential regulators of cell polarization. This collection consists of 279 lines targeting 184 genes.

The clones are monitored by GFP labeling whereas the SOPs are labeled by RFP (See Fig. 1). We have set up the necessary Drosophila stocks to generate clones. 57 dsRNA have been analyzed so far. 4 lines corresponding to 4 different genes, two of them completely uncharacterized, have resulted in phenotypes affecting SOP specification, indicative of the feasibility of the screen (See Fig. 2).

2) Digital analysis of the resulting images. In collaboration with the Dynamic Imaging Platform at the Pasteur Institute, we are establishing an automated image analysis system. The goal is to identify automatically clone borders and SOP positions in order to describe and compare the distribution of SOPs. We are still working on the complex software necessary to properly annotate the images.

3) Study of the mechanisms. The subset of genes identified as N regulators in the previous experiments will be further studied genetically and molecularly in order to address their role in N signaling or SOP specification. As we are still acquiring images and generating primary data for the dsRNA lines, the study of the mechanisms by which the potential positive genes act will be done on the second part of the project period, as already planned.

EXPECTED RESULT AND IMPACT

The immediate expected result arising from the screen is the identification of new genes involved in N regulation. Further analysis of these genes might unveil new mechanisms controlling N activity, which in turn is one of the main actors controlling development and tissue homeostasis in animals.

Moreover, these mechanisms underlying Notch regulation may be potentially involved directly in other signaling pathways (for example, the Alzheimer disease-causing APP) or indirectly as membrane sorting mechanisms are used by multiple signaling routes. On a more practical view, N signaling has been related to several congenital diseases as well as malignancies, mainly leukemia. Understanding the mechanisms of proper N regulation is fundamental to design therapeutical strategies against these diseases.

Collaboration sought:N/A

Source of support:CEC

Programme	Project reference	Project title
FP7-PEOPLE	231009	Modulation of the Notch receptor activity through the regulation of the epithelial apical-basal polarity in Drosophila melanogaster

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