Identification of new genes that regulate cellular migration and invasion

Collective cellular migration is an evolutionarily conserved mechanism which plays a crucial role in several biological processes such as embryo development or immune system maintenance. However, this mechanism is also prominent in pathological conditions, such as cancer and metastasis. The amount of genetic strategies available in Drosophila makes it an ideal model system to study the process of cell migration.  In this study, we have used Drosophila and in particular two tissues with proliferative capacities, the wing disc and the adult fly intestine, to identify new genes regulating the acquistion of metastatic properties by tumor cells. To do this, we have used the UAS:Gal4 system to knock down a collection of random genes via RNAi in tumor cells that express an activated form of Ras, RasV12. By analysing cell morphology, proliferation and survival in fixed tissue, using confocal microscopy, we have seen that RNAis expression against 3 of these genes CG31393, CG16857 and CG33993, results in loss of tissue architecture and over-proliferation in both the wing disc and the larval intestine. Interestingly in this last case, over-proliferation is more prominent in the base of the Malpighian tubules, which is populated by cells with similar characteristics to stem cells. In addition, restrictive expression of the RNAis in groups of cells in the wing disc, either by clonal analysis or specific Gal4 expression, have shown that these cells are able to migrate outside of the tumor area, suggesting the acquisition of a metastatic behaviour. Ultimately, CRISPR was used for the generation of mutants in these genes to support data obtained by RNAi expression.Our results strongly suggest that these three genes regulate the tumurogenetic properties, proliferation and migration, of RasV12 tumor cells. In the future, we propose to analyse whether mutations in these genes phenocopy expression of the RNAis and try to identify the molecular and cellular mechanisms by which these genes regulate tumurogenesis

Influence of the 3D environment in collective cell migration

Motivation: Cell migration plays a key role in several biological processes, such as embryo development or immune system maintenance, and its alteration is involved in pathological conditions like cancer and metastasis. Cells can migrate individually or as a group, but in both cases, they migrate in vivo through a complex 3D environment, although the contribution of that environment to cell migration is still poorly understood. The migration of the border cells in the Drosophila melanogaster ovaries constitutes an ideal system to study collective cell migration in vivo. Border cells are a group of cells that detach from the follicular epithelium that surrounds the egg chamber and migrate through 15 germ cells, called nurse cells, until they reach the oocyte. In this project we use the border cell migration to study the influence of the 3D-environment over the collective migration of a group of cells.Methods: we analysed the border cell migration in both fixed and live samples in wild type and mutant conditions, which were generated either using mutants or expressing RNAis using the UAS-GAL4 system. Fixed samples were stained with some dyes and antibodies to visualize actin, DNA and the border cells, and in vivo analysis was performed using fly strains carrying fluorescently labelled proteins. In all cases, samples were analysed by confocal microscopy and images and movies were processed with ImageJ and Imaris software.Results: the aim of this project was to analyse the influence of the 3D environment (organization of the nurse cells) in border cell migration. For this purpose, the first step was the identification of two mutant alleles of a gene that, when combined, cause an abnormal organization of the nurse cells. Then, we found that in this mutant background border cell migration was affected. Furthermore, there was a correlation between border cell migration defects and aberrant distribution of nurse cells (especially in the most anterior part of the egg chamber). Conclusions: according to our findings, we can say that the organization of the 3D environment is a key factor in collective cell migration, since it is necessary to maintain the forces balance required by cells to migrate

Identification of new genes involved in tumorigenesis

Motivation: Cell proliferation and survival plays a fundamental role both during embryonic development and in the adult individual. The formation of an organ or tissue requires the proliferation of its primordial cells. This action is carried out in a controlled and coordinated way in normal cells but becomes harmful and damaging when it is acquired by tumor cells. Activation of Ras signalling occurs in ~30% of human cancers. However, activated Ras alone is insufficient to produce malignancy. Thus, the discovery of genes cooperating with Ras in cancer is imperative to understand tumoral growth driven by Ras activating mutations. In recent years, the fruit fly Drosophila Melanogaster has become an important model system for cancer studies. In particular, the wing imaginal disc, the primordial of the wing, has been successfully used to identify genes cooperating with an activated form of the Ras oncogene, RasV12, to induce tissue overgrowth and metastasis. In a previous screen in the lab the gene PVRAP was isolated as a putative enhancer of oncogenic Ras. Methods: To analyse the role of PVRAP as regulator of oncogenic Ras, the function of the gene PVRAP was knocked down using two approaches: 1) generation of a collection of Drosophila PVRAP mutants generated by the CRISPR technique and 2) used of the interference RNA (RNAi) technique. One of the tasks of this master´s thesis was to sequence the putative mutants and identified those carrying mutations in the gene PVRAP. To express the PVRAP siRNA in a RasV12 context, the UAS / Gal4 system is used, which allows expressing genes of interest in a tissue-specific manner. Results: The expression of RasV12 has been resulted in an overgrowth of the wing imaginal disc, which is of epithelial origin, observed by means of a marker that delineates the cells. This RasV12 phenotype has been found to be increased in a PVRAP mutant background. This increase is also observed when a PVRAP RNAi is expressed Additionally, several mutants have been identified that create null mutations in PVRAP. One of them, called mutant 9, expresses a greater phenotype and is currently under study. Conclusions: So far, it can be accepted that the RNAi-mediated knockdown of PVRAP resulted in an exacerbated tumorigenic capacity of RasV12 oncoprotein.

The role of macrophages in cancer progression

In recent years, it has been demonstrated that the tumor microenvironment is a fundamental piece in the progression of cancer and thus, it emerges as a potential therapeutic target. This microenvironment contains non-malignant cell lines, including tumor-associated macrophages (TAMs), which play a key role in the control or promotion of tumor growth, metastasis and angiogenesis. Therefore, understanding how TAMs interact with tumor cells is critical to understand cancer development. The fruit fly Drosophila melanogaster is an excellent genetically tractable model system to address the complex cell interactions and genetic cooperation that lead to tumor formation and progression. In this respect, Drosophila embryonic macrophages (hemocytes) have emerged as a powerful system to study the innate immune response against the early stages of tumor development. Deriving from the head mesoderm, hemocytes are highly migratory cells, achieving an even distribution throughout the embryo by the end of embryogenesis. The aim of this study is to create a tumor in an embryonic tissue and analyse the response of embryonic macrophages to the presence of tumor. To do so, we have used the Gal4/UAS system to test several of the most representative oncogenes in human cancers, such as Ras, Notch, EGFR, PI3K or Yorkie, for their ability to generate a tumor in the salivary glands. The choice of this tissue relies on the fact that salivary glands develop in the head region of the embryo, the place where embryonic macrophages emerge from. Thus, one could test whether tumors recruit macrophages by analysing if they halt at salivary glands containing a tumor. Our analysis in fixed tissue shows that indeed macrophages stop their migration at salivary glands-containing tumors and wrap them. We are now in the process of analysing this behaviour at the cellular level in vivo by confocal microscopy. This project also proposes to compare at a genetic level the behaviour of wild type and TAMs macrophages. To perform this a protocol for the isolation of macrophages by flow cytometry is being established. Once control and experimental macrophages are isolated, RNA-seq will be carried out to analyse the gene expression profile of both types of macrophages.This will allow us to identify genes and pathways involved in the regulation of tumor progression by macrophages

α-Spectrin and integrins act together to regulate actomyosin and columnarization, and to maintain a monolayered follicular epithelium.

The spectrin cytoskeleton crosslinks actin to the membrane, and although it has been greatly studied in erythrocytes, much is unknown about its function in epithelia. We have studied the role of spectrins during epithelia morphogenesis using the Drosophila follicular epithelium (FE). As previously described, we show that α-Spectrin and β-Spectrin are essential to maintain a monolayered FE, but, contrary to previous work, spectrins are not required to control proliferation. Furthermore, spectrin mutant cells show differentiation and polarity defects only in the ectopic layers of stratified epithelia, similar to integrin mutants. Our results identify α-Spectrin and integrins as novel regulators of apical constriction-independent cell elongation, as α-Spectrin and integrin mutant cells fail to columnarize. Finally, we show that increasing and reducing the activity of the Rho1-Myosin II pathway enhances and decreases multilayering of α-Spectrin cells, respectively. Similarly, higher Myosin II activity enhances the integrin multilayering phenotype. This work identifies a primary role for α-Spectrin in controlling cell shape, perhaps by modulating actomyosin. In summary, we suggest that a functional spectrin-integrin complex is essential to balance adequate forces, in order to maintain a monolayered epithelium.BFN was supported by Singapore Ministry of Education; GKS by the BBSRC; IAG by the Wellcome Trust, the BBSRC and the Isaac Newton Trust (Cambridge, UK); CSCM and MDMB by the “Ministerio de Economía y Competiti vidad”,the FEDER programme (BFU2013-48988-C2-1-P), and Junta de Andalucía (Proyecto de Excelencia P09-CVI-5058); IG by a JAE-DOC (CSIC); and IMP by the Wellcome Trust, the BBSRC, the Department of Zoology (Cambridge) and the University of Cambridge.This is the final version of the article. It first appeared from the Company of Biologists via https://doi.org/10.1242/dev.13007

Participación del gen l'sc en los mecanismos que determinan la neurogénesis temprana de Drosophila

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, 1992-03-1

LanB1 Cooperates With Kon-Tiki During Embryonic Muscle Migration in Drosophila.

Muscle development is a multistep process that involves cell specification, myoblast fusion, myotube migration, and attachment to the tendons. In spite of great efforts trying to understand the basis of these events, little is known about the molecular mechanisms underlying myotube migration. Knowledge of the few molecular cues that guide this migration comes mainly from studies in Drosophila. The migratory process of Drosophila embryonic muscles involves a first phase of migration, where muscle progenitors migrate relative to each other, and a second phase, where myotubes migrate searching for their future attachment sites. During this phase, myotubes form extensive filopodia at their ends oriented preferentially toward their attachment sites. This myotube migration and the subsequent muscle attachment establishment are regulated by cell adhesion receptors, such as the conserved proteoglycan Kon-tiki/Perdido. Laminins have been shown to regulate the migratory behavior of many cell populations, but their role in myotube migration remains largely unexplored. Here, we show that laminins, previously implicated in muscle attachment, are indeed required for muscle migration to tendon cells. Furthermore, we find that laminins genetically interact with kon-tiki/perdido to control both myotube migration and attachment. All together, our results uncover a new role for the interaction between laminins and Kon-tiki/Perdido during Drosophila myogenesis. The identification of new players and molecular interactions underlying myotube migration broadens our understanding of muscle development and disease

Integrins Cooperate With the EGFR/Ras Pathway to Preserve Epithelia Survival and Architecture in Development and Oncogenesis

Adhesion to the extracellular matrix (ECM) is required for normal epithelial cell survival. Disruption of this interaction leads to a specific type of apoptosis known as anoikis. Yet, there are physiological and pathological situations in which cells not connected to the ECM are protected from anoikis, such as during cell migration or metastasis. The main receptors transmitting signals from the ECM are members of the integrin family. However, although integrin-mediated cell-ECM anchorage has been long recognized as crucial for epithelial cell survival, the in vivo significance of this interaction remains to be weighed. In this work, we have used the Drosophila wing imaginal disc epithelium to analyze the importance of integrins as survival factors during epithelia morphogenesis. We show that reducing integrin expression in the wing disc induces caspase-dependent cell death and basal extrusion of the dead cells. In this case, anoikis is mediated by the activation of the JNK pathway, which in turn triggers expression of the proapoptotic protein Hid. In addition, our results strongly suggest that, during wing disc morphogenesis, the EGFR pathway protects cells undergoing cell shape changes upon ECM detachment from anoikis. Furthermore, we show that oncogenic activation of the EGFR/Ras pathway in integrin mutant cells rescues them from apoptosis while promoting their extrusion from the epithelium. Altogether, our results support the idea that integrins promote cell survival during normal tissue morphogenesis and prevent the extrusion of transformed cells.España MICINN (Nos. BFU2016-80797-R and PID2019-109013GB-100

Bajo el signo de la luz : la fundación de una utopía andaluza

Esta publicación obtuvo el Primer Premio Joaquín Guichot a investigaciones, experiencias y materiales sobre Andalucía y su cultura en la edición XXVI del curso 2013/2014Se trata de un proyecto intercentros en el que se pretende convertir en aula a la comunidad. Nace para dar respuesta a las inquietudes de un alumnado y una población que ignoran, en buena medida, sus orígenes y los de sus antepasados. Como respuesta a esa necesidad, se vuelve sobre las huellas del tiempo para descubrir la epopeya de las nuevas poblaciones de Sierra Morena y Andalucía; una iniciativa que respondió a los ideales del Siglo de las Luces y a la necesidad de poblar los extensos terrenos que bordeaban el Camino Real. Con el formato de aprendizaje-servicio a la comunidad, el alumnado descubrirá que sus raíces culturales, sus apellidos y tradiciones, lejos de los localismos, provienen de Europa Central y suponen un ejemplo de integración cultural. "Bajo el signo de la luz" va más allá y sueña con llevar la utopía a la educación e intenta convertirse en un paradigma de integración del trabajo de Educación Infantil, Primaria y Secundaria, o de cualesquiera otras enseñanzas, y se apoya en el deseo idealista de dar a conocer la cultura andaluza al alumnado de nuestra Comunidad Autónoma.AndalucíaES