Hedgehog (Hh) signal molecules play a fundamental role in development, adult stem cell maintenance and cancer. Hh can signal at a distance, and we have proposed that its graded distribution across Drosophila epithelia is mediated by filopodia-like structures called cytonemes. Hh reception by Patched (Ptc) happens at discrete sites along presenting and receiving cytonemes, reminiscent of synaptic processes. Here, we show that a vesicle fusion mechanism mediated by SNARE proteins is required for Ptc placement at contact sites. Transport of Ptc to these sites requires multivesicular bodies (MVBs) formation via ESCRT machinery, in a manner different to that regulating Ptc/Hh lysosomal degradation after reception. These MVBs include extracellular vesicle (EV) markers and, accordingly, Ptc is detected in the purified exosomal fraction from cultured cells. Blockage of Ptc trafficking and fusion to basolateral membranes result in low levels of Ptc presentation for reception, causing an extended and flattened Hh gradient.

The conserved family of Hedgehog (Hh) signaling proteins plays a key role in cell-cell communication during development, tissue repair and cancer progression, inducing distinct concentration-dependent responses in target cells located at short and long distances. One simple mechanism for long distance dispersal of the lipid modified Hh is the direct contact between cell membranes through filopodia-like structures known as cytonemes. Here we have analyzed in Drosophila the interaction between the glypicans Dally and Dally-like protein, necessary for Hh signaling, and the adhesion molecules and Hh coreceptors Ihog and Boi. We describe that glypicans are required to maintain the levels of Ihog, but not of Boi. We also show that the overexpression of Ihog, but not of Boi, regulates cytoneme dynamics through their interaction with glypicans, the Ihog fibronectin III domains being essential for this interaction. Our data suggest that the regulation of glypicans over Hh signaling is specifically given by their interaction with Ihog in cytonemes. Contrary to previous data, we also show that there is no redundancy of Ihog and Boi functions in Hh gradient formation, being Ihog, but not of Boi, essential for the long-range gradient.

Precise genome engineering is essential for both basic and applied research, permitting the manipulation of genes and gene products in predictable ways. The irruption of the CRISPR/Cas technology accelerated the speed and ease by which defined exogenous sequences are integrated into specific loci. To this day, a number of strategies permit gene manipulation. Nevertheless, knock-in generation in multicellular animals remains challenging, partially due to the complexity of insertion screening. Even when achieved, the analysis of protein localization can still be unfeasible in highly packed tissues, where spatial and temporal control of gene labeling would be ideal. Here, we propose an efficient method based on homology-directed repair (HDR) and single-strand annealing (SSA) repair pathways. In this method, HDR mediates the integration of a switchable cassette. Upon a subsequent CRISPR-triggered repair event, resolved by SSA, the cassette is seamlessly removed. By engineering the Hedgehog (Hh) pathway components, we demonstrated fast and robust knock-in generation with both fluorescent proteins and short protein tags in tandem. The use of homology arms as short as 30 base pairs further simplified and cheapened the process. In addition, SSA can be triggered in somatic cells, permitting conditional gene labeling in different tissues. Finally, to achieve conditional labeling and manipulation of proteins tagged with short protein tags, we have further developed a toolbox based on rational engineering and functionalization of the ALFA nanobody.

The conserved family of Hedgehog (Hh) signaling proteins plays a key role in cell-cell communication in development, tissue repair and cancer progression. These proteins can act as morphogens, inducing responses dependent on the ligand concentration in target cells located at a distance. Hh proteins are lipid modified and thereby have high affinity for membranes, which hinders the understanding of their spreading across tissues. Direct contact between cell membranes by filopodia-like structures (also known as cytonemes) could be the simplest explanation for Hh dispersal. To better understand this signaling mechanism, we have analyzed in Drosophila the interaction between the glypicans that, besides for other pathways, are necessary for Hh signaling, plus the adhesion molecules and Hh coreceptors Ihog and Boi. We describe that glypicans (Dally and Dally-like protein) are required to maintain Ihog, but not Boi, protein levels. We also show that ectopic Ihog stabilizes cytonemes through its interaction with glypicans, and we determine that two Ihog fibronectin III domains are essential for this interaction. Our data suggest that this interaction with Ihog in cytonemes confers the specificity of glypicans for Hh signaling.

During Drosophila epithelial development, signalling specialized filopodial or cytonemes establish direct contacts between distant cells to facilitate the formation of the Hedgehog (Hh) signalling gradient. However, the regulatory mechanisms for Hh cytoneme initiation and their dynamic behaviour are still unknown. Here we show that Hh cytoneme dynamics in Drosophila epithelia depends on the Epidermal Growth Factor (EGF) signalling pathway through its ligand Spitz. We describe that the EGF pathway is required to maintain basal plasma membrane levels of the adhesion protein and Hh co-receptor Ihog, able to modify cyoneme behaviour. Furthermore, we describe that EGF signalling promotes Ihog interaction with actin cytoskeleton regulators, such as the actin cross-linker Filamin A, Cheerio (Che) in Drosophila. Cheerio is recruited by Ihog towards focalized small regions of the plasma membrane to contribute to the cytoneme stabilization function of Ihog. Finally, we describe that EGF regulation and PI3K signalling act synergically on Hh cytoneme behaviour, revealing a new form of cooperation between major tissue organizers.

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El lenguaje de las campanas representa un patrimonio cultural inmaterial y litúrgico genuino de cada pueblo. Las campanas han servido desde hace siglos como medio de comunicación y cohesión social, marcando los ritmos diarios de la vida en el pueblo. Por desgracia, en las últimas décadas, el abandono rural, los cambios litúrgicos y la mecanización de las campanas, han repercutido negativamente en la conservación de este patrimonio. En Torre de Juan Abad (Ciudad Real) se han conservado unas grabaciones de todos los toques originales de la parroquia de los años 60, lo que supone la posibilidad de estudiar y documentar todo el repertorio de toques que se ejecutaba en dicho pueblo, permitiendo, en un futuro, implementarlos nuevamente en la parroquia.