DYNAMIC ANALYSIS OF THE PATTERNING ACTIVITY OF HEDGEHOG IN DROSOPHILA – HHMOVE

The various potential transportation mechanisms that Hh might employ deserve a detailed analysis on a genome-wide scale to get a better understanding of the distinct mechanisms Hh might use to exit the cell. To fill this gap we carried out a genome-wide screen in Drosophila. For this, we used a collection of inducible RNA interfering transgenic lines allowing targeted gene silencing specifically in the Hh producing cells. In parallel, using immuno-electron microscopy on Drosophila wing imaginal discs, we analyzed the distribution of Hh molecule at the sub cellular level.

We could complete a genome-wide screen for Hh secretome in Drosophila which allowed us to identify a new regulator of apico-basal trafficking of Hh, the small GTPase Rab8. We could show that Hh activity is functionally distributed between apically and laterally secreted pools, each activating long- and short-range targets, respectively.
Using immuno-electron microscopy on Drosophila wing imaginal discs, we identified a cellular structure, the Hherisomes that contain the majority of intracellular Hh in Hh secreting cells. Hherisomes are recycling tubular endosomes. Our data indicate that Hherisomes are required to sustain physiological Hh activity necessary for patterning and tissue growth in the wing disc.
From a computational point of view we have developed a software to detect filopodia, on which Hh spreads, from confocal images. A statistical analysis is associated to this detection which provides the number of extensions, their length and their maximal distance from the cell membrane. Besides, we have tuned a marked point process based on software to detect Hh particles within the wing disks.

Studies in vertebrates have shown that Hh is involved in various biological processes encompassing embryonic development, stem cell biology and tissue homeostasis (Briscoe and Thérond, 2013). Deregulation of the Hh pathway was found to be responsible for congenital syndromes and persistent Hh pathway activity has pathological consequences in various cancers (Beachy, et al., 2004). Efforts are being made to understand and manipulate the aberrant secretion of Hh on the surrounding responsive tissue and the signaling cascade downstream of ligand receptor activation. Therefore, our work provide a greater understanding of regulation of Hh secretion, and could uncover novel avenues for the discovery of therapeutic tools relevant for the treatment of a wide variety of Hh-dependent pathologies.

Symposium Organisation:

- Labex Signalife Workshop “Distant Intercellular Communications” (June 24th; 2019).

3 publications are related to this work :

Tamas Matusek et al.,. Functional analysis of ESCRT-positive extracellular vesicles in the Drosophila wing imaginal disc. Methods in Molecular Biology 2019;1998:31-47, edited by Springer.

Tanvi Gore et al.,. Role of the small GTP-ase Rab8 in the establishment of Hedgehog long- and short-range activity. (Development, 2021 Mar 9;148(5).

Sandrine Pizette, et al., under press in Journal of Cell Science. Identification of a new Hedgehog-containing endocytic compartment required for high level Hedgehog signaling and tissue growth.

Among the signaling molecules involved in animal morphogenesis are the Hedgehog (Hh) family proteins which act at a short and long range to direct cell fate decisions in invertebrate and vertebrate tissues. We propose a fundamental research project aiming at a better understanding of tissue morphogenesis controlled by Hh during animal development by focusing on the mecanisms that control Hh transport to target cells in Drosophila. One of the challenge in the morphogen field is to decipher the spatio-temporal dynamics of secreted signals involved in cell fate decisions. In particular, we showed that Hh secreted proteins are not randomly distributed in the extracellular space cells but are rather differentially secreted on apical and basolateral spaces (partner 1: Pascal Thérond, IBV, Nice). We previously showed that Hh gradient is a composite of pools secreted by different routes: an apically secreted pool with long range activity and a more basolateral secreted pool with short-range activity. Moreover, several extra-cellular Hh carriers have been identified (lipoprotein particles; membrane extension called cytonemes; exovesicles) but their individual contribution to the spreading of the different Hh pools is still unresolved. Therefore, the ability to quantitatively analyze and control the spatio-temporal and dynamical properties of the secreted Hh gradient taken as a whole is essential for understanding Hh morphogenetic gradient. The overall goal of this proposal is to understand how Hh is secreted and evaluate the contribution of the multiple carriers to Hh transportation in Drosophila melanogaster.
To study these mecanisms, we will develop new methods to modify the spatio-temporal and dynamical properties of the extra-cellular Hh gradient and separate the contribution of the apical versus basal Hh pools in two different epitheliums of the drosophila larvae. This will allow us to analyze the contribution of the different carriers in the establishment of the Hh gradient (P. Thérond and partner 2: Xavier Descombes, INRIA Sophia-Antipolis). This will be completed with a genome-wide screen to identify additional genes and related cellular processes responsible for Hh release (P. Thérond). With these tools in hands, we propose to classify the multiple pools of Hh and develop accurate tracking algorithm to compare trajectories of different Hh pools transportation in live animals (X. Descombes). The particular interest of this collaboration lies in the combination of development of algorithm to analyze Hh distribution and trajectories with extremely powerfull genetics, ease of in vivo manipulation and lack of genetic redundancy of Drosophila. Our project could uncover novel avenues for the discovery of therapeutic tools relevant for human health as deregulated Hh activity impairs stem cell and tissue homeostasis.