Tuesday, 12th December 2017

Development and Regeneration

           Epithelial cell polarity and cancer

 

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Fernando M. Belmonte

 CSciStaff

CPublications

 

 

 

 

Research summary:

Our main scientific interest is the understanding of epithelial morphogenesis and polarity, as well as their implication in human diseases, such as cancer. We are currently using an organotypic 3-dimensional in vitro model as a basic model system for my research, as well as the zebrafish pronephros and gut epithelial morphogenesis as an in vivo system. In addition, we have initiated a new research direction by using embryonic stem cells (ES) to address these issues.

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Different stagese of cysts formation with a central lumen in MDCKII cells grown in 3D (Matrigel). Stained for Slp2a (green), actin (red) and nuclei (blue).

We are extremely interested in the development of epithelial cell polarity. On the basis of data from simple models, such as cultured mammalian cells, we are beginning to understand the mechanisms that control the establishment and maintenance of epithelial cell polarity and tissue integrity. The Madin-Darby canine kidney (3D MDCK) epithelial cell system is one of the best in vitro models for investigating cell polarity during epithelial morphogenesis (Rodriguez-Fraticelli et al., 2011). However, this model cannot reconstitute the complexity of the in vivo architecture, which includes different cell types, dynamic remodeling, and tissue homeostasis. For this reason, the use of in vivo systems would serve to validate and further characterize the phenotypes observed in vitro. Zebrafish is an excellent model for characterizing (in vivo) the mechanisms for lumen formation identified in the 3D-MDCK system. In addition, it has been recently demonstrated that the core polarity proteins govern spindle orientation in stem cells and epithelial development. Furthermore, the connection between the loss of cell polarity, defective asymmetric cell division and tumor initiation is one of the most surprising and important findings in the field of cancer biology in the past 10 years.

Thus, we are focusing on the analysis of proteins that regulate lumen formation in epithelial development, and particularly on two essential aspects: membrane trafficking and spindle orientation. We have two main ongoing projects at the moment:

1.-Functional Characterization of epithelial lumen morphogenesis using 3D-in vitro models and zebrafish

We performed a two-step screening in the 3D-MDCK model to identify candidate proteins involved in lumen formation. We found a set of 14 genes that had not been known to be required for this process. These included tight and adherens junctions, Rho GTPases, lipid signaling and membrane trafficking proteins. We have already published the role of Slp2a/Slp4 in morphogenesis in the 3D-model (Galvez-Santisteban et al., 2012).

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A) transgenic zebrafish expressing a GFP epithelial marker, stainig specifically the apical membrane of epithelial tubules, intestine and pronephros (primary kidney) B) magnification. C) Cross section of a zebrafish embryo, wherein epithelial tubules are identified forming the pronephros and intestine.

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The next step is to characterize the role of the proteins obtained in the screening in zebrafish epithelial development. There are three epithelial tubes in zebrafish suitable for this analysis: the gut, the neural tube and the pronephric ducts. One of the most interesting features of the zebrafish gut tube formation is that cells polarize, and form the lumen without apoptosis. This special feature shows a crucial similarity to the 3D-MDCK system, which in the presence of laminin forms lumens by hollowing and without apoptosis (Martin-Belmonte et al., 2008). Thus, the zebrafish gut tube formation represents an excellent model for characterizing in vivo the molecular mechanism and signaling pathways for lumen formation identified in the 3D-MDCK system. However, as the screening is being done in MDCK cells, which are kidney-derived, some of the proteins might be specific to this tissue, and for these we will characterize their role in pronephric tubules. We will use the development of the pronephric ducts as an alternative epithelial model. Furthermore, a secondary objective is to validate our transcriptional screening in physiological conditions. Transcriptional changes during development are very finely controlled and they orchestrate a range of properties from the orientation of the body axes to the size and shape of the organs. We have seen a number of genes induced during lumen formation, and we want to identify the key genetic players that regulate these transcriptional changes physiologically. It is known that caudal-related homeobox proteins (Cdx) are transcription factors that are involved in the control the architecture of organs during development, thus it is likely that gut and kidney morphogenesis in zebrafish are controlled by cdx proteins (Davidson and Zon, 2006).

2.-Analysis of Asymmetric Stem-Cell Division in Renal Tubulogenesis and Disease

Asymmetric cell division in stem cells is essential for generating cell diversity during development, as well as for maintaining epithelial tissue homeostasis. Defects in asymmetric division may contribute to the developmet of cancer or tissue degeneration/aging. In fact, asymmetric division is controlled by the machinery involved in mitotic spindle orientation, and in invertebrate models, core polarity proteins govern spindle orientation and that their deregulation may drive tumor initiation (Martin-Belmonte and Perez-Moreno, 2012). However, the lack of an appropriate vertebrate model of study has limited research in this field in recent years. 

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Formación de túbulos epiteliales in vitro cultivados en micropatterns. Los micropatterns nos perminten cultivar células que se adhieren al sustrato en un patrón determinado. Los micropatterns presentan a las células diferentes moléculas a las que las se pueden adherir, permitiendo que las células crezcan formando un patrón que hemos definido. Gracias a este sistema estamos desarrollando diferentes tipos de cultivos que nos permiten construir túbulos epiteliales in vitro. Estos túbulos nos permiten probar el efecto de drogas en el epitelio renal de manera rápida y segura, así como estudiar procesos morfogenéticos de manera simplificada.

The aim of our work is to investigate the division pattern of renal stem cells during development by the generation of an innovative 3D tubulogenesis system that should model essential aspects of the nephrogenesis process in vertebrates. Such a system, with a level of complexity close to that of the adult organ, should circumvent the limitations of the use of the current in vitro and in vivo models, facilitating the analysis of stem cells, and the study of their behavior during asymmetric cell division. By using this novel 3D system, we aim to determine whether renal stem cells undergo asymmetric cell division during renal tubulogenesis and to analyze the molecular mechanisms involved. An essential goal of the project is to uncover the cell polarity mechanisms that govern stem cell division.

The characterization of the molecular mechanisms regulating stem cell division in mammalian kidneys will be relevant not only to our understanding of developmental processes: they will also have clinical importance for therapeutic applications. It is in fact important to note the relevance of these processes in disease, such as cancer and generic kidney diseases. Congenital anomalies of the kidney and urinary tract occur in 1 in 500 humans, ultimately constituting approximately 20 to 30% of all prenatal anomalies and representing a major cause of renal failure in infants and children. Indeed, another essential goal of this project is to investigate whether the division pattern of nephron progenitor cells is affected in renal genetic diseases, such as polycystic kidney disease, and how these defects affect the 3D tubulogenesis process. We are particularly interested in those renal genetic diseases that display defects in kidney development leading to embryo lethality and therefore cannot be modeled in animals.


 

Relevant publications:

  • DBañon, I; Gálvez, M.A., Vergarajauregui, S; Bosch, M; Borreguero-Pascual, A and Martín-Belmonte F. (2013) The control of IQGAP membrane localization by EGFR regulates mitotic spindle orientation during epithelial morphogenesis. EMBO J (in press).
  • Gálvez, M.A., Rodríguez-Fraticelli, AE; Vergarajauregui; Bañon, I, Bernascone, I; Fukuda, M; Mostov, KE and Martín-Belmonte F. (2012) Synaptotagmin-Like Proteins Control Formation of a Single Apical Membrane Domain in Epithelial Cells. Nature Cell Biol 14(8):838-49.
  • Rodríguez-Fraticelli AE; Auzan, M; Alonso, MA; Bornens, M and Martín-Belmonte, F. (2012) Cell confinement controls centrosome positioning and lumen initiation during epithelial morphogenesis. J Cell Biol 17;198(6):1011-23
  • Rodríguez-Fraticelli AE, Gálvez, M.A., Mostov K. Martín-Belmonte F. (2010) ITSN2 is a RhoGEF specific for Cdc42 that controls lumen formation in epithelial morphogenesis. J Cell Biol 189 (4):725-38.
  • Martín-Belmonte, F., Gassama, A., Datta, A., Yu, W., Rescher, U. Gerke, V. and Mostov, K. (2007). PTEN-Mediated Apical Segregation of Phosphoinositides Controls Epithelial Morphogenesis through Cdc42. Cell 128(2):383-97

 

http://www.martinbelmontelab.net