Papers by Isabel Guerrero
During Drosophila epithelial development, signalling specialized filopodial or cytonemes establis... more 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 regul...
Specific neuropeptides regulate in arthropods the shedding of the old cuticle (ecdysis) followed ... more Specific neuropeptides regulate in arthropods the shedding of the old cuticle (ecdysis) followed by maturation of the new cuticle. In <i>Drosophila melanogaster</i>, the last ecdysis occurs at eclosion from the pupal case, with a post-eclosion behavioural sequence that leads to wing extension, cuticle stretching and tanning. These events are highly stereotyped and are controlled by a subset of crustacean cardioactive peptide (CCAP) neurons through the expression of the neuropeptide Bursicon (Burs). We have studied the role of the transcription factor Odd-paired (Opa) during the post-eclosion period. We report that <i>opa</i> is expressed in the CCAP neurons of the central nervous system during various steps of the ecdysis process and in peripheral CCAP neurons innerving the larval muscles involved in adult ecdysis. We show that its downregulation alters Burs expression in the CCAP neurons. Ectopic expression of Opa, or the vertebrate homologve Zic2<i>,&...
Specific neuropeptides regulate in arthropods the shedding of the old cuticle (ecdysis) followed ... more Specific neuropeptides regulate in arthropods the shedding of the old cuticle (ecdysis) followed by maturation of the new cuticle. In <i>Drosophila melanogaster</i>, the last ecdysis occurs at eclosion from the pupal case, with a post-eclosion behavioural sequence that leads to wing extension, cuticle stretching and tanning. These events are highly stereotyped and are controlled by a subset of crustacean cardioactive peptide (CCAP) neurons through the expression of the neuropeptide Bursicon (Burs). We have studied the role of the transcription factor Odd-paired (Opa) during the post-eclosion period. We report that <i>opa</i> is expressed in the CCAP neurons of the central nervous system during various steps of the ecdysis process and in peripheral CCAP neurons innerving the larval muscles involved in adult ecdysis. We show that its downregulation alters Burs expression in the CCAP neurons. Ectopic expression of Opa, or the vertebrate homologve Zic2<i>,&...
Nature Communications
During embryonic development, cell-cell communication is crucial to coordinate cell behavior, esp... more During embryonic development, cell-cell communication is crucial to coordinate cell behavior, especially in the generation of differentiation patterns via morphogen gradients. Morphogens are signaling molecules secreted by a source of cells that elicit concentration-dependent responses in target cells. For several morphogens, cell-cell contact via filopodia-like-structures (cytonemes) has been proposed as a mechanism for their gradient formation. Despite of the advances on cytoneme signaling, little is known about how cytonemes navigate through the extracellular matrix and how they orient to find their target. For the Hedgehog (Hh) signaling pathway in Drosophila, Hh co-receptor and adhesion protein Interference hedgehog (Ihog) and the glypicans Dally and Dally-like-protein (Dlp) interact affecting the cytoneme behavior. Here, we describe that differences in the cytoneme stabilization and orientation depend on the relative levels of Ihog and glypicans, suggesting a mechanism for cyt...
Precise genome engineering is essential for both basic and applied research, permitting the manip... more 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 Hedge...
PLOS Computational Biology, 2021
Morphogen gradients are crucial for the development of organisms. The biochemical properties of m... more Morphogen gradients are crucial for the development of organisms. The biochemical properties of many morphogens prevent their extracellular free diffusion, indicating the need of an active mechanism for transport. The involvement of filopodial structures (cytonemes) has been proposed for morphogen signaling. Here, we describe an in silico model based on the main general features of cytoneme-meditated gradient formation and its implementation into Cytomorph, an open software tool. We have tested the spatial and temporal adaptability of our model quantifying Hedgehog (Hh) gradient formation in two Drosophila tissues. Cytomorph is able to reproduce the gradient and explain the different scaling between the two epithelia. After experimental validation, we studied the predicted impact of a range of features such as length, size, density, dynamics and contact behavior of cytonemes on Hh morphogen distribution. Our results illustrate Cytomorph as an adaptive tool to test different morphoge...
Specific neuropeptides regulate in arthropods the shedding of the old cuticle (ecdysis) followed ... more Specific neuropeptides regulate in arthropods the shedding of the old cuticle (ecdysis) followed by maturation of the new cuticle. In <i>Drosophila melanogaster</i>, the last ecdysis occurs at eclosion from the pupal case, with a post-eclosion behavioural sequence that leads to wing extension, cuticle stretching and tanning. These events are highly stereotyped and are controlled by a subset of crustacean cardioactive peptide (CCAP) neurons through the expression of the neuropeptide Bursicon (Burs). We have studied the role of the transcription factor Odd-paired (Opa) during the post-eclosion period. We report that <i>opa</i> is expressed in the CCAP neurons of the central nervous system during various steps of the ecdysis process and in peripheral CCAP neurons innerving the larval muscles involved in adult ecdysis. We show that its downregulation alters Burs expression in the CCAP neurons. Ectopic expression of Opa, or the vertebrate homologve Zic2<i>,&...
Development, 2019
During development, specialized cells produce signals that distribute among receiving cells to in... more During development, specialized cells produce signals that distribute among receiving cells to induce a variety of cellular behaviors and organize tissues. Recent studies have highlighted cytonemes, a type of specialized signaling filopodia that carry ligands and/or receptor complexes, as having a role in signal dispersion. In this Primer, we discuss how the dynamic regulation of cytonemes facilitates signal transfer in complex environments. We assess recent evidence for the mechanisms for cytoneme formation, function and regulation, and postulate that contact between cytoneme membranes promotes signal transfer as a new type of synapse (morphogenetic synapsis). Finally, we reflect on the fundamental unanswered questions related to understanding cytoneme biology.
Mechanisms of Development, 1996
The genes decapentaplegic (dpp) and wingless (wg), which encode secreted factors of the TGF-beta ... more The genes decapentaplegic (dpp) and wingless (wg), which encode secreted factors of the TGF-beta and Wnt families, respectively, are required for the proper development of the imaginal discs. The expression of these genes must be finely regulated since their ectopic expression induces overgrowth and pattern alterations in wings and legs. Genes like patched (ptc) and costal-2 (cos-2), and the gene encoding the catalytic subunit of the protein kinase A gene (pkA) are required to restrict dpp and wg expression in their proper positions. We show here that some mutations in the cubitus interruptus (ci) gene also show ectopic dpp expression in the wing disc. We have also analyzed the functional hierarchy between these genes and the gene fused (fu), in the activation of dpp by the hedgehog (hh) signal. fu is required to transmit the hh signal in imaginal discs, since fu mutations rescue the phenotype due to the ectopic hh expression or to the lack of ptc activity. fu is also required for the activation of engrailed (en) caused when hh is ectopically activated in the wing disc. By contrast, fu mutations do not rescue the phenotypic consequences of the abnormal ci, cos-2 or pkA activity. Although fu, cos-2 and ci probably form part of the same pathway that controls dpp expression, pkA probably controls dpp transcription by a different pathway.
Frontiers in Cell and Developmental Biology, 2021
Extracellular vesicles (EVs) mediate cell-to-cell crosstalk whose content can induce changes in a... more Extracellular vesicles (EVs) mediate cell-to-cell crosstalk whose content can induce changes in acceptor cells and their microenvironment. MLP29 cells are mouse liver progenitor cells that release EVs loaded with signaling cues that could affect cell fate. In the current work, we incubated 3T3-L1 mouse fibroblasts with MLP29-derived EVs, and then analyzed changes by proteomics and transcriptomics. Results showed a general downregulation of protein and transcript expression related to proliferative and metabolic routes dependent on TGF-beta. We also observed an increase in the ERBB2 interacting protein (ERBIN) and Cxcl2, together with an induction of ribosome biogenesis and interferon-related response molecules, suggesting the activation of immune system signaling.
Journal of Developmental Biology, 2016
Intercellular communication is a fundamental process for correct tissue development. The mechanis... more Intercellular communication is a fundamental process for correct tissue development. The mechanism of this process involves, among other things, the production and secretion of signaling molecules by specialized cell types and the capability of these signals to reach the target cells in order to trigger specific responses. Hedgehog (Hh) is one of the best-studied signaling pathways because of its importance during morphogenesis in many organisms. The Hh protein acts as a morphogen, activating its targets at a distance in a concentration-dependent manner. Post-translational modifications of Hh lead to a molecule covalently bond to two lipid moieties. These lipid modifications confer Hh high affinity to lipidic membranes, and intense studies have been carried out to explain its release into the extracellular matrix. This work reviews Hh molecule maturation, the intracellular recycling needed for its secretion and the proposed carriers to explain Hh transportation to the receiving cells. Special focus is placed on the role of specialized filopodia, also named cytonemes, in morphogen transport and gradient formation.
eLife, 2017
Morphogens regulate tissue patterning through their distribution in concentration gradients. Emer... more Morphogens regulate tissue patterning through their distribution in concentration gradients. Emerging research establishes a role for specialized signalling filopodia, or cytonemes, in morphogen dispersion and signalling. Previously we demonstrated that Hedgehog (Hh) morphogen is transported via vesicles along cytonemes emanating from signal-producing cells to form a gradient in Drosophila epithelia. However, the mechanisms for signal reception and transfer are still undefined. Here, we demonstrate that cytonemes protruding from Hh-receiving cells contribute to Hh gradient formation. The canonical Hh receptor Patched is localized in these cellular protrusions and Hh reception takes place in membrane contact sites between Hh-sending and Hh-receiving cytonemes. These two sets of cytonemes have similar dynamics and both fall in two different dynamic behaviours. Furthermore, both the Hh co-receptor Interference hedgehog (Ihog) and the glypicans are critical for this cell-cell cytoneme m...
eLife, 2017
Morphogens regulate tissue patterning through their distribution in concentration gradients. Emer... more Morphogens regulate tissue patterning through their distribution in concentration gradients. Emerging research establishes a role for specialized signalling filopodia, or cytonemes, in morphogen dispersion and signalling. Previously we demonstrated that Hedgehog (Hh) morphogen is transported via vesicles along cytonemes emanating from signal-producing cells to form a gradient in Drosophila epithelia. However, the mechanisms for signal reception and transfer are still undefined. Here, we demonstrate that cytonemes protruding from Hh-receiving cells contribute to Hh gradient formation. The canonical Hh receptor Patched is localized in these cellular protrusions and Hh reception takes place in membrane contact sites between Hh-sending and Hh-receiving cytonemes. These two sets of cytonemes have similar dynamics and both fall in two different dynamic behaviours. Furthermore, both the Hh co-receptor Interference hedgehog (Ihog) and the glypicans are critical for this cell-cell cytoneme m...
Methods in Molecular Biology, 2015
Hedgehog (Hh) molecules act as morphogens directing cell fate during development by activating va... more Hedgehog (Hh) molecules act as morphogens directing cell fate during development by activating various target genes in a concentration dependent manner. Hitherto, modeling morphogen gradient formation in multicellular systems has employed linear diffusion, which is very far from physical reality and needs to be replaced by a deeper understanding of nonlinearities. We have developed a novel mathematical approach by applying flux-limited spreading (FLS) to Hh morphogenetic actions. In the new model, the characteristic velocity of propagation of each morphogen is a new key biological parameter. Unlike in linear diffusion models, FLS modeling predicts concentration fronts and correct patterns and cellular responses over time. In addition, FLS considers not only extracellular binding partners influence, but also channels or bridges of information transfer, such as specialized filopodia or cytonemes as a mechanism of Hh transport. We detect and measure morphogen particle velocity in cytonemes in the Drosophila wing imaginal disc. Indeed, this novel approach to morphogen gradient formation can contribute to future research in the field.
Nature communications, Jan 4, 2014
The Hedgehog signalling pathway is crucial for development, adult stem cell maintenance, cell mig... more The Hedgehog signalling pathway is crucial for development, adult stem cell maintenance, cell migration and axon guidance in a wide range of organisms. During development, the Hh morphogen directs tissue patterning according to a concentration gradient. Lipid modifications on Hh are needed to achieve graded distribution, leading to debate about how Hh is transported to target cells despite being membrane-tethered. Cytonemes in the region of Hh signalling have been shown to be essential for gradient formation, but the carrier of the morphogen is yet to be defined. Here we show that Hh and its co-receptor Ihog are in exovesicles transported via cytonemes. These exovesicles present protein markers and other features of exosomes. Moreover, the cell machinery for exosome formation is necessary for normal Hh secretion and graded signalling. We propose Hh transport via exosomes along cytonemes as a significant mechanism for the restricted distribution of a lipid-modified morphogen.
The conserved family of Hedgehog (Hh) signaling proteins plays a key role in cell-cell communicat... more 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 throug...
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Papers by Isabel Guerrero