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The role of cytonemes in the Hh secretion in Drosophila melanogaster

Abstract : Our laboratory studies a morphogen molecule called Hedgehog (Hh) using Drosophila melanogaster as an animal model. The Hh signaling pathway is evolutionarily conserved from invertebrates to vertebrates, and plays regulatory roles in various aspects of animal development and tissue homeostasis, such as stem cell renewal, tissue repair, and organ regeneration. Hh is a dually lipidated molecule modified by cholesterol at its C-terminus and palmitic acid at its N-terminus, and therefore tightly binds the plasma membrane. Although the hydrophobic nature of the molecule, Hh exerts its function over a long-range of distance. One particular way cells adopted to communicate over long distances is through a new mechanism based on direct cell-cell contacts via long actin-based filopodia extensions, called cytonemes. This new modality for information transfer is at the core of my present project. In this work, we studied this Hh transport mechanism in a polarized epithelial tissue, called the wing imaginal disc, a larval precursor tissue from which the adult wings develop. Cytonemes have been extensively studied in this tissue with the use of the overexpression of a fluorescently tagged protein called Interference of hedgehog (Ihog). The expression of Ihog protein in the wing disc is necessary and sufficient to stabilize these long plasma membrane extensions, otherwise they would be too fragile and easy to be disrupted by conventional fixatives. Here we present Ihog, Hh and Disp as important players in cytoneme growth. In absence of different Ihog domains, we found a significant reduction of cytoneme length and numbers. In addition, Ihog/Boi loss of function was able to reduce the number and length of wild type cytonemes, marked with mCD8GFP. Further, we saw that in loss of function and gain of function genotype for Hh, cytoneme length was reduced and increased respectively, without any change at the cytoneme numbers. With this work, we suggest that Hh has a novel, non-canonical function in the cytoneme growth. To understand the role of cytonemes in Hh secretion we also analyzed discs entirely mutant for Dispatched (Disp). In disp mutants, the Hh gradient is strongly restrained, with most of Hh targets expressed only in anterior cells juxtaposing the A/P border. This phenotype could not be rescued by Ihog overexpression, despite the fact that Hh was very abundant on cytonemes in the disp mutant. Additionally, in the absence of Disp, we observed a reduction of cytoneme length, which suggests a role for Disp in the formation of these filaments, which is likely independent from its function in Hh secretion. In conclusion, although the aforementioned proteins contribute to the structure of cytonemes, we could not measure any direct correlation between the expression of Hh target genes and the simultaneous manipulation of cytoneme length in any mutant condition checked. Finally, we wanted to correlate the time of cytoneme initiation with the establishment of the Hh gradient. In order to do that, we have introduced in the laboratory an alternative system to study cytoneme formation: the abdominal histoblast model, which allowed us to directly analyze the formation and dynamics of membrane extensions in live animals. In particular, we looked at two separated dorsal histoblast nests of the pupal stage (distanced by 30 microns at the onset of pupariation) where the posterior group of cells produces Hh and the anterior cells show expression of various Hh targets. Approximately 15 hours after the onset of the metamorphosis, the abdominal histoblast cells begin to divide and migrate while simultaneously inducing apoptosis in the surrounding larval cells. Our results indicate that the establishment of the Hh gradient occurs before the juxtaposition of the two nests. Nevertheless, we could only observe cytoneme formation after the juxtaposition of the anterior and posterior histoblast cells and not when the two nests were still separated.
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Caterina Novelli. The role of cytonemes in the Hh secretion in Drosophila melanogaster. Molecular biology. Université Côte d'Azur, 2019. English. ⟨NNT : 2019AZUR4015⟩. ⟨tel-02490361v2⟩

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