Cabecera 2019 CBMSO CSIC UAM

Monday, 19th August 2019

Biophysics and Systems Biology


Grupo-400

 


 

 

David Míguez

 CSciStaff

CPublications

 

 

 

 

Research summary:

Our lab focuses on the study of the dynamics of biological systems combing experimental, theoretical and computational tools. Our main interests are:

  • Regulation of stem cell differentiation during developmental processes
    The cellular machinery is governed by interacting proteins, genes and metabolites that form complex and highly interconnected networks of interactions. This way, extracellular stimuli triggers pathways of biological events that regulate gene expression, protein activity, and ultimately, cell response. We combine in vivo and in silico approaches to understand how the wiring of the pathways determines the role of the proteins that regulate the differentiation of neurons. We also study how the interplay between mode and rate of division of stem cells is orchestrated. To do that, we use in toto microscopy combined with theoretical tools and algorithms developed in the lab to quantify the effect of key regulators in the balance between proliferation and differentiation during vertebrate neurogenesis.
  • Nonlinear regulation in pathways and its impact on disease treatment
    Small molecule inhibitors display significant potential as treatment for diseases that involve the deregulation of signal transduction pathways. These inhibitors are developed based on their target specificity and binding affinity. We focus on the fact that the numerous signaling proteins and feedback loops in signaling pathways strongly influence the efficiency of pharmacological treatment. The existence of several regulatory positive and negative feedback loops either creates complex dose-responses, desensitization to periodic treatments, or modulation of the drug effect in combinatorial treatments. Our experiments show that the effect of inhibitors strongly depends on the architecture of the targeted pathway, and a detailed characterisation of these nonlinear effects can be useful when designing optimal treatment strategies.
  • Stochasticity and effect of perturbations in biological networks
    Biological networks control cellular behaviour both at the intracellular and at intercellular level. These highly interconnected networks need to perform in the continuously fluctuating and changing cellular microenvironment. Disruption of these networks often leads to aberrant cell behaviour and disease. This leads to some broad questions that we try to address in the lab at different levels: How can these highly nonlinear biological networks integrate and process information? How can they operate robustly in the presence of noise and fluctuations? What are the mechanisms at the network level underlying the adaptation to the different sources of extrinsic and intrinsic noise ? To answer these questions, we use a synthetic biology approach to analyse experimental and computationally the dynamics of fluctuations in minimal networks motifs.

 

figure1

Figure 1: Confocal sections of neural tube of G. Gallus at different developmental times.
Blue: Dapi. Red: Differentiated cells. Green: progenitor cells

Epithelial cell polarity and cancer

 group400px

 


 

 

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.

 fig01.300px

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).

 fig02.300px  

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.

--

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. 

 fig03.300px

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

 

Development and Differentiation

      Structure and evolution of centromeres and telomeres

 

  

web 

 

 

  

 Alfredo Villasante

CSciStaff

CPublications


 

 

Research Summary:

The behaviour of the eukaryotic chromosome relies on the function of two chromosomal elements: the centromere and the telomere. Malfunction of these elements lead to inviable cells and/or genetic diseases. Many types of cancer and most abortions happen due to an abnormal chromosome segregation, which is caused by a defective centromere. Telomeres are also involved in cancer and aging. These crucial chromosomal loci are included in heterochromatic regions. Unfortunately, the heterochromatin remains poorly characterized due to its enrichment in repetitive sequences.

The recurrent homogenization/amplification undergone by any tandemly repeated DNA sequence explains the lack of homology between centromeric sequences in distant species. However, in accordance with the conserved centromeric function, most centromeric proteins are conserved. Interestingly, the centromeric histone H3 variant CENP-A shows rapid evolution and seems to coevolve with centromeric satellites. The "centromere paradox" may well be explained by the formation of conserved noncanonical structures such as quadruplexes or other secondary structures. Although the prevalent model assumes that centromeres are epigenetically defined by CENP-A, we have proposed that both sequence-independent structural motifs and epigenetic modifications contribute to centromere identity.

Understanding of the evolutionary origin of centromeres and telomeres may elucidate aspects of their function. With such intention, we have also hypothesized that centromeres were derived from telomeres during the evolution of the eukaryotic chromosome. In support of this hypothesis we have recently shown that the centromeric region of the D. melanogaster Y chromosome evolved from a telomere.

To gain insights into the involvement of structural determinants in the function of centromeres and telomeres, it would be necessary to investigate the structural behavior of DNA and RNA oligonucleotides from telomeric and centromeric repeats. Currently, in collaboration with Carlos Gonzalez's group (IQFR, Madrid) and Ricardo Arias-Gonzalez's group (IMDEA Nanociencia, Madrid), we are using biophysical techniques and single molecule analysis to determine the three-dimensional structure and dynamics of centromeric and telomeric nucleic acids.

 Fig01-500
Schematic representation of a possible evolutionary scenario for the origin of eukaryotic chromosomes.
----

Relevant publications:

  • Garavís, M., Bocanegra, R., Herrero-Galán, E., González, C., Villasante, A., Arias-Gonzalez, J.R. (2013) Mechanical unfolding of long human telomeric RNA (TERRA). Chem Commun. 49:6397-9.
  • Méndez-Lago, M., Wild, J., Whitehead, S.L., Tracey, A., de Pablos, B., Rogers, J., Szybalski, W., Villasante, A. (2009). Novel sequencing strategy for repetitive DNA in a Drosophila BAC clone reveals that the centromeric region of the Y chromosome evolved from a telomere. Nucleic Acids Res. 7:2264-73.
  • Villasante, A., Abad, J.P., Planelló, R., Méndez-Lago, M., Celniker, S.E., de Pablos, B. (2007). Drosophila telomeric retrotransposons derived from an ancestral element that was recruited to replace telomerase. Genome Res. 17:1909-18.
  • Villasante, A., Abad, J.P., Méndez-Lago, M. (2007). Centromeres were derived from telomeres during the evolution of the eukaryotic chromosome. Proc Natl Acad Sci U S A. 104:10542-7.
  • Hoskins, R.A., Carlson, J.W., Kennedy, C., Acevedo, D., Evans-Holm, M., Frise, E., Wan, K.H., Park, S., Mendez-Lago, M., Rossi, F., Villasante, A., Dimitri, P., Karpen, G.H., Celniker, S.E. (2007). Sequence finishing and mapping of Drosophila melanogaster heterochromatin. Science. 316:1625-8.
figura 2 web final
Mechanical unfolding and refolding of long human telomeric RNA (TERRA) by optical tweezers.

Genetic control of morphogenesis


Grupo-400

 


Ginés Morata Pérez

CSciStaff

CPublications

 

Research summary:

In recent years the research of our group has focussed on two major research lines: 1) the study of cell competition, especially in relation with apoptosis and tumorigenesis, and 2) experimental analysis of regeneration in the imaginal discs.

Regarding cell competition, we have shown that it functions as a tumour suppressor mechanism, inducing apoptosis in oncogenic cells. We are presently analysing the mechanisms by which tumour cells may evade the control by cell competition and proceed to develop a tumour. Recent results (Ballesteros-Arias et al 2014) indicate that they can evade cell competition by forming a protective microenvironment of about 500-600 cells. In this situation, cell competition induces apoptosis in the cells at the periphery of the tumour, but it being a short-range phenomenon, tumour cells in the centre of the group are beyond its range and continue proliferating. Under these circumstances cell competition reverses its normal anti-tumour role and functions as a tumour stimulating factor, due to the proliferative signals that emanate from the tumour cells in apoptosis at the border of the tumour. We are presently trying to identify the factors involved in the tumorigenesis by apoptotic cells. The connection between apoptosis and tumour growth may have relevant clinical implications.

The second research line has been the analysis of regeneration in the imaginal discs; the overall aim is the study of the genetic reprogramming mechanisms during the reconstruction of structures that have been damaged or eliminated. Our recent publications (Herrera et al 2013; Herrera and Morata, 2014, see also review in Morata and Herrera 2016) have demonstrated that during regeneration the epigenetic control of cell identities breaks down transiently, allowing for changes in the identity of the affected cells, which reconstruct the missing organ. These cells become reprogrammed by a novel mechanism that requires interactions with their neighbours.

In forthcoming years we plan to carry out a comprehensible study of regeneration of the different body parts of Drosophila using new tracking methods to follow the regeneration processes. We will be paying special attention to the regenerative response of different regions within the imaginal discs, in particular the distinction between body trunk and appendages, an issue that has been hitherto neglected.

fig1

Drosophila wing disc containing a marked clone (green) deficient for the pro-apoptotic genes and over-expressing the JNK pathway. The cells of the clone express the metalloprotease 1, which degrades extracellular matrix. Note the presence of F-actin protrusions (blue) at the basal region of the cells

fig2

Wing imaginal disc in which posterior compartment cells (right side) have suffered a treatment of 2 days of apoptosis induction followed by 3 days of recovery. The cells belonging to the posterior lineage are labeled in yellow, while the anterior compartment is labeled in magenta. Note the presence of groups of cells (asterisks) that were originated in the posterior compartment and now belong to the anterior compartment. This result suggests that the ablation treatment provokes a transient collapse of the compartments boundary and a change in cell identity

 

 

Latest publications:

  • Herrera, S. Martin, R. and Morata. G. (2013) "Tissue homeostasis in the wing disc of Drosophila: immediate response to massive damage during development" PLoS Genet 9 (4), e10034446
  • Morata, G and Herrera, S. (2013) "Eiger triggers death from afar" eLife 2:e01388. doi: 10.7554/eLife.01388
  • Herrera, S and Morata, G. (2014) "Transgressions of compartment boundaries and cell reprogramming during regeneration of the imaginal discs of Drosophila" eLife. 3: e01831. doi: 10.7554/eLife.01831
  • Ballesteros-Arias, L., Saavedra V, and Morata, G. (2014) "Cell competition may function either as tumour-suppressing or as tumour‐stimulating factor in Drosophila". Oncogene 33, 4377-4384
  • Morata, G. and Struhl, G. (2014) "Tethered wings" Nature 505, 162-163
  • Morata, G. and Ballesteros-Arias L. (2014) "Death to the losers. Cell competition is linked to innate immunity mechanisms to eliminate unwanted cells and maintain healthy tissue". Science 346, 1181-1182
  • Morata, G. and Ballesteros-Arias, L. (2015) "Cell competition, apoptosis and tumour development" Int. J. Dev. Biol. 59, 79-86
  • Morata G. and Herrera, S.C. (2016) "Cell reprogramming during regeneration in Drosophila: transgression of compartment boundaries" Current Opinion in Genetics & Development 40, 11-16
  • Calleja, M. Morata, G and Casanova, J. (2016) "The tumorigenic properties of Drosophila epithelial cells mutant for lethal giant larvae". Dev. Dynamics in press

 


 

Doctoral theses:

  • Evgeny Shlevklov (2011) Nuevos mecanismos de regulación de la apoptotis en Drosophila melanogaster. Universidad Autónoma de Madrid. Director: Ginés Morata Pérez.
  • Javier Ménendez González (2011) Competición celular y desarrollo de tumores en discos imaginales de Drosophila. Universidad Autónoma de Madrid. Director: Ginés Morata Pérez.

 

Development and Regeneration

        Cellular Dynamics during morphogenesis

 

 

 Grupo-400

 


 

 

Nicole Gorfinkiel

CSciStaff

CPublications

 

 

 

 

Research summary:

 Fig01-300Tono
Schematic representation of the different stages of Dorsal Closure (dorsal view). Arrows indicate the different forces contributing to the process: the apical contraction of the AS generates a force that positively contributes to closure (red arrows) while the epidermis resist the dorsalward movement (blue arrows). A supra-cellular actin cable that forms at the interface between the AS and the epidermis also contributes to closure.
 
Fig02-300

Quantitative analysis in 2D of AS morphogenesis. Panel (1): Contraction of the AS during Dorsal Closure. Cellular membranes are labelled with E-Cadherin-GFP. Panel (2): Automatic tracking of AS cell interfaces during Dorsal Closure. Panel (3): Automatic tracking of AS cell areas during Dorsal Closure.

 

 

 

During embryonic development, groups of up to thousands of cells exhibit coordinated movements and deformations that eventually give rise to the complex three dimensional structures of organs and organisms..

The extraordinary development of microscopy techniques and the posiibility to follow morphogenetic processes in vivo using a variety of fluorescent reporters make now posible to visualize andquantify molecular, celular and tissue processes in real time. These novel approaches are starting to reveal the DYNAMICS ofdevelopmental processes and are revolutionizing the field of Developmental Biology. Moreover, mechanics –forces, motions andelasticity- has emerged as an important player during collective cell movements. One of the challenges in the área of morphogenesis isto understand how the interaction between biochemical and mechanical processes at different temporal and spatial scales gives rise tothe activity of tissues.

Research in my lab tackles these questions using the process of Dorsal Closure in Drosophila, a morphogenetic process that has emerged as a reference for the study of more complex processes occuring in vertebrates such as neural tuve closure and wound healing. DorsalClosure is the porcess whereby interactions between two epitelial tissues, the epidermis and the amnioserosa contribute to generate thefinal shape of the Drosophila larvae. We use a combination of live imaging, quantitative image analysis, theoretical modeling, ingenetically and mechanically perturbed embryos to approach these problems

Work in the lab is organized along the following areas:

1) To understand the molecular and celular mechanisms underlying apical contraction in AS cells.

2) To understand how AS cells coordinate this activity accross the whole tissue.

3) To understand how tissue mechanical properties emerge from the activity of its constituent cells.

 


 

Latest publications:

  • Gorfinkiel, N. 2013. Mechano-chemical coupling drives cell area oscillations during morphogenesis. Biophys Journal 104: 1-3. Invited review
  • Lada, K., Gorfinkiel, N. and Martinez Arias, A. 2012. "Interactions between the amnioserosa and theepidermis revealed by the function of the u-shaped gene". Biology Open 1: 353-361.
  • Gorfinkiel, N.* and Blanchard, G*. 2011. Dynamics of actomyosin contractile activity during epithelial morphogenesis. Current Opinion in Cell Biology 23: 531-9. * corresponding authors
  • Blanchard, G. B., Murugesu, S., Adams, R. J., Martinez Arias, A. and Gorfinkiel, N.* 2010. Cytoskeletal dynamics and supracellular organisation of cell shape fluctuations during dorsal closure. Development 137,2743-2752. corresponding author
  • Gorfinkiel, N.*, Blanchard, G. B., Adams, R. J. A. & Martinez-Arias, A. 2009. Mechanical control of global cell behaviour during Dorsal Closure in Drosophila. Development 136, 1889-1898. * corresponding author.

 

 

video-icon
 .mp4 Video
.mov Video

 

 

Cell fate specification in the development of the Drosophila central nervous system


Grupo-400

 


Fernando Jiménez Díaz-Benjumea

CSciStaff

CPublications

 

Research summary:

During the development of the Drosophila central nervous system neural stem cells, neuroblasts, divide asymmetrically to generate neurons and self-regenerate. In this process neuroblasts go through a series of temporal windows that define the fate of distinct neurons. These temporal windows are defined by the temporal expression of a set of transcription factors. Simultaneously, the expression of the HOX genes, along the anterior posterior axis of the embryo, generates diversity in the different segments. Our goal is to understand the mechanisms by which the HOX genes and the temporal factors interplay to define the combinatorial code of transcription factors that specify the fate of the different neurons. Our model systems are two sets of neurons characterized by the expression of neuropeptides Leucokinin and CCAP (Figure 1).

 Fig01-300
Expression pattern of Leukoquinin and CCAP neuropeptides in the Drosophila central nervous system.

 

At the end of the embryonic neurogenesis, neuroblasts either die by apoptosis or enter quiescence. Later in larval stages, neuroblasts resume proliferation and generate, in a second neurogenesis, the adult nervous system. Little is known about the mechanisms controlling entry into quiescence and maintenance of cell fate during quiescence (Figure 2).

 Fig02-300
Upon entering quiescence neuroblasts undergo marked changes in shape, here detected by immunostaning in stages 14, 15 and 16 of embryogenesis.

 

 

 

 

 

 

 

 

 

 

 

In abdominal segments only 3 out of 30 neuroblasts per hemisegment undergo quiescence, the rest die by apoptosis. We chose abdominal neuroblasts as model system to study first, how these two fates are genetically regulated, and secondly, which are the cellular changes involving the entry into a quiescence stage.


 

Latest publications:

Signalling mechanisms during development

 

 Lab414 Isabel Guerrero400px

 


Isabel Guerrero

CSciStaff

CPublications

 

Lab web link: http://guerreroslab.cbm.uam.es

Research summary:

Secreted signaling molecules of the TGF-beta, Wnt, and Hedgehog (Hh) families have been shown to play essential roles in cell fate specification during development. In many developmental contexts, they act as morphogens that emanate from localized sources and form extracellular gradients, which differentially regulate cell fates in a concentration-dependent manner. Our group is studying the function of these morphogens during Drosophila development with higher input in the molecular and cellular mechanisms of Hh signaling. Hh is a molecule highly modified by lipids, which confer to Hh a high affinity for cell membranes. Despite these modifications, Hh protein can signal to cells distant from the source of its production. Presently, we are analyzing how Hh is released and transported through the extracellular matrix. The functional analysis of several proteins with a possible function in this process, such as Ihog, Boi, Dispatched, glypicans, and Shifted/DmWif, suggest that Hh is release through the basolateral side of polarized epithelia, with a previous recycling process from apical tobasolateral (Fig. 1). We are now working on the hipótesis that Hh is then transported in exovesicles attached to cellular extensions called "citonemas" (Fig. 2).

 

   

Fig02-300  

Ihog-RFP and Hh-GFP protein expression in the wing imaginal disc epithelium. Note that Hh-GFP is located in filopodia extensions, also called "cytonemes", and exovesicles in the wing imaginal disc.

 

Fig01-300  -------

 

Model for processing and release Hh.
A) In the producing cells Hh is translated, cleavage, lipid modified and externalized (red beads). B) Hh is then internalized and sorted to endosomes and to the apical recycling endosomes. In this process Hh probably interacts with Disp (blue discontinue line), Dlp, Dally, Boi (yellow background), and Ihog (green line). Hhis then transported to the basolateral membrane probably within multivesicular endosomes. C) These multivesicular endosomes fuse their membranes with the basolateral plasma membrane to form the cytonemes and release exovesicles. Hh is then transported in exovesicles to its target cells along cytonemes.

 

 


 

Relevant publications:

  • Callejo A, Bilioni A, Mollica E, Gorfinkiel N, Andrés G, Ibáñez C, Torroja C, Doglio L, Sierra J, Guerrero I. Dispatched mediates Hedgehog basolateral release to form the long-range morphogenetic gradient in the Drosophila wing disk epithelium. Proc Natl Acad Sci USA. (2011). Aug 2;108(31)12591-8. doi 10.1073pnas.1106881108. Epub 2011 Jun 20. Special feature article.  http://www.pnas.org/content/108/31/12591.long
  • Sánchez-Hernández D, Sierra J, Ortigão-Farias JR,GuerreroI. The WIF domain of the human and Drosophila Wif-1 secreted factors confers specificity for Wnt or Hedgehog. Development. 2012 Oct;139(20):3849-58. Epub 2012 Sep 5. Erratum in: Development. 2013 Nov;140(22):4645. *Equally contributing.   http://dev.biologists.org/content/139/20/3849.long
  • Bilioni A*, Sánchez-Hernández D*, Callejo A, Gradilla AC, Ibáñez C, Mollica E, Carmen Rodríguez-Navas M, Simon E,GuerreroI.Balancing Hedgehog, a retention and release equilibrium given by Dally, Ihog, Boi and shifted/DmWif. Dev Biol. 2013 Apr 15;376(2):198-212. doi: 10.1016/j.ydbio.2012.12.013. Epub 2012 Dec 29.  http://www.sciencedirect.com/science/article/pii/S0012160612006793
  • Bischoff, M*., Gradilla, A.C.*, Seijo, I., Andrés G., Rodríguez-Navas, C., González- Méndez, L., and Guerrero, I. Cytonemes are required for the establishment of a normal Hedgehog morphogen gradient in Drosophila epithelia. Nature Cell Biol. (2013) Nov;15(11)1269-81. doi 10.1038ncb2856. Epub 2013 Oct 13. *Equally contributing.   http://www.nature.com/ncb/journal/v15/n11/full/ncb2856.html
  • Gradilla AC, González E, Seijo I, Andrés G, Bischoff M, González-Mendez L, Sánchez V, Callejo A, Ibáñez C, Guerra M, Ortigão-Farias JR, Sutherland JD, González M, Barrio R, Falcón-Pérez JM,Guerrero I. (2014) Exosomes as Hedgehog carriers in cytoneme-mediated transport and secretion. Nat Commun. Dec 4;5:5649. doi: 10.1038/ncomms6649.   http://www.nature.com/ncomms/2014/141204/ncomms6649/full/ncomms6649.html   


 

Doctoral Theses:

1. Papel del gen patched en el desarrollo embrionario e imaginal del Drosophila melanogaster Javier Capedvila Moya. Universidad: Autónoma de Madrid, Facultad de Ciencias, UAM. Abril 1995.

2. El papel del gen Distal-less en la identidad de los apèndices ventrales de Drosophila melanogaster. Nicole Gorfinkiel Haim, Universidad: Autónoma de Madrid, Facultad de Ciencias, Mayo 1998.

3. Estudio del papel de la vía de Señalización de Hedgehog en la determinación del patrón morfogenético del ala de Drosophila. : José Luis Mullor SanJosé. Universidad: Autónoma de Madrid. Facultad de Ciencias; UAM . Noviembre 1999.

4. Papel del gen patched en el mecanismo de señalización de Hedgehog. Carlos Torroja Fungairiño Universidad: Autónoma de Madrid, Facultad de Ciencias, UAM, Diciembre 2003.

5. Función del gen shf/Dwif en la vía de señalización de Hedgehog en Drosophila. Javier Sierra Isturiz. Universidad: Autónoma de Madrid. Facultad de Ciencias. Abril 2007.

6. Función de los genes lines y los genes de la familia Odd-Skipped en el desarrollo de los apéndices de Drosophila melanogaster.. Elvira Benítez de Gracia Universidad: Autónoma de Madrid. Facultad de Ciencias. Febrero 2008.

7. Papel de Ihog y Boi en la señalización de Hedgehog. David Sánchez Hernández Universidad: Autónoma de Madrid. Facultad de Ciencias. Enero 2014.

8. Control of two morphogenetic processes during Drosophila melanogaster metamorphosis: fusion of imaginal disc and ecdysis. Eleanor Simon Universidad: Autónoma de Madrid. Facultad de Ciencias. Noviembre 2016.

 Development and Differentiation

          Cellular Plasticity in Development and Cancer

 

 

 Grupo-400

 


 

 

César Cobaleda

 CSciStaff

CPublications

 

 

 

 

Research Summary:

Research in our group is focused on the study of cellular plasticity in the hematopoietic system and how it is controlled both in pathological conditions (cancer and developmental syndromes) and in normal development. As experimental tool we use genetically engineered mouse models in which we modify the levels and windows of expression of transcription factors and epigenetic regulators, either normal or oncogenic.

In humans, in general, the oncogene responsible for the tumoral origin is present also in all the tumoral cells. The cancer stem cell model (postulating that cancer is a hierarchically organized tissue maintained by malignant cancer stem cells), however, suggests the possibility that the genetic information responsible for tumor generation might only be needed at the level of these cancer stem cells. We have generated mouse models of hematopoietic neoplasms like multiple myeloma or lymphomas, and their characterization has allowed us to prove that it is indeed possible to induce tumor development in the mouse by restricting oncogene expression (e.g. the MafB oncogene) to the stem/progenitor cells responsible for tumor origin and maintenance.

 

Fig01-300  -------  Fig02-300
Lytic bone lesions, characteristic of human multiple myeloma, in Sca1-MafB transgenic mice (from Vicente-Dueñas et al., 2012).  

Ectopic expression of MafB reprogrammes stem cells into tumor plasma cells. (A) Normal lymphoid development. (B) Currently, MafB oncogenic effects are thought to occur in differentiated cells. The nature of the myeloma cell-of-origin is unknown. (C) Stem expression of MafB causes latent epigenetic changes that will become active at the terminal differentiation, leading to the appearance of tumor plasma cells.

 

Multiple myeloma is an incurable neoplasm characterized by the accumulation of malignant clonal plasma cells, and only a minority of patients can be cured with current therapies. Traditionally, it has been assumed that myeloma was originated from differentiated cells. Our data provide the first evidence showing the generation of myeloma by a mechanism similar to reprogramming to pluripotency, and they challenge the currently accepted mode of action of oncogenes in cancer, since they are the first practical demonstration showing that myeloma is hierarchically organized as a tissue. This explains patients' relapses and indicates that the targets to destroy the cancer-maintaining cells cannot be identified by studying differentiated tumor cells (even if these are the most abundant cells in the tumor).


 

Publications:

  • Vicente-Dueñas, C., Romero-Camarero, I., González-Herrero, I., Alonso-Escudero, E., Abollo-Jiménez, F., Jiang, X., Gutiérrez, N.C., Orfao, A., Marín, N., Villar, L.M., Criado, M.C., Pintado, B., Flores, T., Alonso-López, D., De Las Rivas, J., Jiménez, R., Criado, F.J., Cenador, M.B., Lossos, I.S., Cobaleda, C. and Sánchez-García, I. (2012) A novel molecular mechanism involved in multiple myeloma development revealed by targeting MafB to haematopoietic progenitors. EMBO J. 31, 3704-3717.
  • Vicente-Dueñas, C., Romero-Camarero, I., García-Criado, F., Cobaleda, C. and Sánchez-García, I. (2012) The cellular architecture of multiple myeloma. Cell Cycle 11, 2961-2962.
  • Vicente-Dueñas, C. , Fontán, L., González-Herrero, I., et al. (2012) Expression of MALT1 Oncogene in Hematopoietic Stem / Progenitor Cells Recapitulates the Pathogenesis of Human Lymphoma in Mice. Proc Natl Acad Sci U S A. 109, 10534-10539.
  • Vicente-Dueñas, C., Cobaleda, C., Martínez-Climent, J.A. and Sánchez-García, I. (2012) MALT lymphoma meets stem cells. Cell Cycle 15, 2961-2962.
  • Campos-Sánchez, E., Sánchez-García, I. and Cobaleda, C. (2011) Plasticity and Tumorigenicity. Atlas of Genetics & Cytogenetics in Oncology and Haematology. 2012; 16(3), DOI: 10.4267/2042/47289.

 

Other activities:
"Ad hoc" member of the committees of the German Federal Office for Radiation Protection (BfS) and the French Institute for Radiation Protection and Nuclear Safety (IRSN), in charge of defining future research programs on the role of environmental factors in the pathogenesis of childhood leukemia.

More links about our work:

http://arimmora-fp7.eu/
http://atlasgeneticsoncology.org/Deep/PluripotencyID20103.html
http://www.scitopics.com/Cancer_Stem_Cells.html


 

Patents:

Cobaleda Hernández, C., Sánchez García, I., Martínez Climent, J.A., Fontán Gabás, L., Vicente Dueñas, C. "A non-human animal model of mucosa-associated lymphoid tissue (MALT) lymphoma." EP 11382319, 11/10/2011. CSIC e Instituto Científico y Tecnológico de Navarra, S.A. (ICTDP).


 

Doctoral theses:

Fernando Abollo Jiménez (2011). Identificación y caracterización funcional de nuevos factores de transcripción implicados en la progresión leucémica y la hematopoyesis. Universidad de Salamanca. Directores: César Cobaleda Hernández e Isidro Sánchez García.

Molecular and cellular basis of Drosophila organogenesis

 

 Grupo400

 


 

 

Sonsoles Campuzano

 CSciStaff

CPublications

 

 

 

 

Research summary:

The Iroquois (iro) complex genes araucan, caupolican and mirror, encode highly related transcription factors that play a plethora of functions during organogenensis. By means of the generation of novel iro mutants, we have determined that their functions are not totally redundant. Thus, araucan and caupolican, but not mirror, define the lateral transverse muscle fate in the embryo while only mirror is involved in the specification of the dorso-ventral embryonic axis..

 Fig01-300

Model for the cis-regulation of the Iroquois Complex. The genomic DNA of the Iroquois Complex harbours enhancer sequences (red and green bars) that drive expression of reporter genes in several domains of the wing imaginal disc. These enhancers should control expression of araucan and caupolican while insulator elements (grey bar) would prevent their action on the mirror promoter.

 

Conversely, the three iro genes act as tumour suppressor modulating cell cycle progression at the level of the G1 to S transition. In addition, we have shown that the linkage, evolutionarily conserved, of iro genes (Irx in vertebrates) with the functionally unrelated sowah gene is due to the presence at sowah introns of enhancers that drive expression of araucan and caupolican. Action of these and other iro enhancers over mirror should be precluded by insulator sequences that we are in the process of studying (Figure 1). On the other hand, we have initiated the characterization of the iro gene regulatory network having identified two of their target genes.

 Fig02-300

Modulation of the activity of the Notch signalling pathway by DaPKC. DaPKC loss of function in the follicular epithelium prevents adequate Notch pathway activity (A, B). In the wing disc, enhanced activity of the Notch pathway was found associated to constitutive activation of DaPKC (C, D, the domain where constitutively active DaPKC is expressed is GFP labelled). Activity of the Notch pathway was monitored by expression of its target genes hnt and E(spl)m. Ooc, oocyte; wt, wild type folliclar epithelium.

Apico-basal polarity of the epithelial cells is required for development and function of numerous organs. We have recently shown how the apical determinant Crumbs contributes to trachea formation. On the other hand, loss of polarity and hyper proliferation are two hallmarks of tumour cells. Epithelial cells mutant for crumbs or the atypical protein kinase C (DaPKC) display these two traits. Thus, to investigate the causal relationship between loss of cell polarity and uncontrolled proliferation, we are studying which signalling pathways are deregulated in those mutant conditions. We have observed malfunction of the Hippo and Notch signalling pathways (Figure 2), in the latter case in association to a defective intracellular trafficking of the Notch receptor..


 

Latest publications:

  • Letizia, A., Sotillos, S., Campuzano, S. and Llimargas, M. (2011) Crb regulated accumulation controls apical constriction and invagination in Drosophila tracheal cells. J. Cell Sci. 124, 240- 251.
  • Carrasco-Rando, M., Tutor, A.S., Prieto-Sánchez, S., González-Perez, E., Barrios, N., Letizia, A., Martín, P., S. Campuzano and Ruiz-Gomez, M. (2011) Drosophila Araucan and Caupolican integrate in muscle precursors intrinsic and signalling inputs for the acquisition of the lateral transverse fate. PLOS Genet 7(7):e1002186.
  • Andreu, M. J., Gonzalez-Perez, E., Ajuria, L., Samper, N., Gonzalez-Crespo, S., Campuzano, S. and Jimenez, G. (2012) Mirror represses pipe expression in follicle cells to initiate DV axis formation in Drosophila. Development 139, 1110 -1114.
  • Andreu, M. J., Ajuria, L., Samper, N., González-Pérez, E., Campuzano, S., González-Crespo, S. and Jiménez, G. (2012) EGFR-dependent downregulation of Capicua and the establishment of Drosophila dorsoventral polarity. Fly 6, 234-239.
  • Maeso, I., lrimia, M., Tena, J. J, González-Pérez*, E., Trans, D., Ravi, V., Venkatesh, B., Campuzano, S., Gómez-Skarmeta J. L. and Garcia-Fernandez, J. (2012) An ancient genomic regulatory block conserved across bilaterians and its dismantling in tetrapods by retrogene replacement. Genome Res. 22, 642-655.

 

Doctoral Theses:

Natalia Barrios López. (2012) Las proteínas del Complejo Iroquois de Drosophila melanogaster controlan el progreso del ciclo celular y se regulan por fosforilación dependiente de MAPK. Universidad Autónoma de Madrid. Directora: Sonsoles Campuzano

Control of cell proliferation and organ regeneration through intercellular signals

 

grupo400

 


 

 

Antonio Baonza

 CSciStaff

CPublications

 

 

 

 

Research summary:

The final size of multicellular organisms largely depends on the control of cell divisionregulate by different intercellular signals during the development. Several signalling pathways have been involved in this regulation during Drosophila development. Changes in the normal function of these signals cause variation in the normal parameters of cell proliferation. Interestingly, the members of most of these signalling pathways have been well conserved throughout evolution and play an important role in control of cell proliferation in a wide range of organisms, including human. Alterations in the activity of some of the human homologues of members of these signalling pathways are implicated in many cancers.

 Fig01-300

Pattern of cell proliferation of an imaginal wing discs over-expressingunder the regulation of patch-Gal4 a serine/threonine Kinase thatinduces an excess of cell proliferation.
In red the mitotic marker Phospho-Histone3, in green the actine marker phalloidin and in blue are shown the cells that express GFP in the domain of expression of patch.

 

 


The overall goals of my group are to understand how signalling pathways control cell proliferation through the regulation of the activity and/or expression of different transcription factor during Drosophila development; to use this knowledge to gain insight into the general mechanisms by which extrinsic signals regulate cell division.
Our results suggest that the transcriptional repressors of the Helix-loop-Helix (HLH) family play a critical role mediating the control of cell proliferation by different signalling pathways. We are interesting in to study the molecular mechanisms by which this family of proteins control normal and abnormal cell cycle progression.

Other problem in which we are interested is to understand how organ regeneration is controlled. The importance of understanding how this occurs has significance at different levels. One issue is to learn more about the normal mechanism that operate during development. Other important point is that the knowledge that we can gain about the general mechanisms that regulate regeneration may help to develop new therapeutic approach in regenerative medicine. We have developed a new method to remove a part of the wing imaginal disc inside the larva. Using this method, we can study the process of regeneration in its normal developmental context. Moreover, we can analyse the adult pattern of the regenerating structure and we can take advantage of all the genetic tools available in Drosophila.


 

Latest publications:

  • San Juan, B.P., Andrade-Zapata, I., and Baonza, A. (2012) The bHLHfactorsDpn and members of the E(spl) complexmediate the function of Notch signalling regulating cell proliferation during wing disc development. Biol Open. 1(7), 667-76.
  • San-Juán, B.P., and Baonza, A. (2011) The bHLH factor deadpanis a direct target of Notchsignaling and regulates neuroblast self-renewal in Drosophila. Dev Biol. 352(1), 70-82.

Cell signalling during imaginal development in Drosophila

 

 Group400px

 


 

 

José Félix de Celis

 CSciStaff

CPublications

 

 

 

 

Research summary:

The Drosophila wing originates from an epithelial tissue (wing imaginal disc), which growth and differentiation depends on the activity of conserved signalling pathways and transcription factors. The main focus of our work is to characterise the contribution of signalling pathways to the regulation of imaginal wing disc development.

 Fig01-300px

(A) Drosophila melanogaster wild type wing (left) and domains of activation in the wing disc (red) of the Notch (Notch), Hedgehog (Hh), Epidermal growth factor receptor (EGFR), Wingless/Wnt (Wg) and Decapentaplegic/BMP (Dpp) signalling pathways. (B) Wing phenotypes resulting from manipulations in the activity of the Insulin (InR/Tor), Notch (Notch), Hedgehog (Hh) and Epidermal growth factor receptor (EGFR) signalling pathways, either by activation (column "Pathway activation") or by inhibition (column "Pathway inhibition"). (C) Wing phenotypes resulting from manipulations in the activity of the Wingless (Wg), decapentaplegic/BMP (dpp/BMP), Transforming growth factor b (TGFb) and Salvador/Warts/Hippo (SWH) signalling pathways, either by activation (column "Pathway activation") or by inhibition (column "Pathway inhibition").

Through loss- and gain-of-function genetic screens, we identified the genes gmd, MAP4K3, kurtz, kismet and spalt, and subsequently characterised their requirements during wing development.The functional analysis we carried out consisted in the generation and analysis of loss-of-function conditions, the analysis of expression patterns using in situ hybridisation, and the determination of the sub-cellular localisation of the corresponding proteins. Our studies have established the participation of these genes in the signalling pathways Notch (gmd), Insulin (Map4K3), Hedgehog (kurtz and kismet) and TGFb (spalt). We expect that the analysis in Drosophila will uncover conserved aspects of the function of these genes, which would be relevant for normal development in vertebrates and might be related with the outcome of several human genetic disorders. Our laboratory also host two Ramon y Cajal contracts, Dr. Carlos Estella y Dra. Cristina Grande. They undertake independent projects related to pattern formation in Drosophila appendages (Dr. Carlos Estella) and body plan evolution in bilateral organisms (Dra. Cristina Grande).


 

Latest publications:

  • Molnar, C., Ruiz-Gómez, A., Martín, M., Rojo, S., Mayor, F. and de Celis, J. F. (2011) Role of the Drosophila non-visual b-Arrestin Kurtz in Hedgehog signalling. PLOS Genetics 7(3): e1001335.
  • Molnar, C., Casado, M., López-Varea, A., Cruz, C. and de Celis, J.F. (2012) Genetic annotation of gain-of-function screens using interference RNA and in situ hybridization of candidate genes in the Drosophila wing. Genetics 192, 741-752.
  • Organista, M. and de Celis, J. F. (2013). The Spalt transcription factors regulate cell proliferation, survival and epithelial integrity downstream of the Decapentaplegic signalling pathway. Biology Open 15;2(1):37-48.
  • Covadonga F. Hevia and Jose F. de Celis (2013).Activation and function of TGFβ signalling during Drosophila wing development and its interactions with the BMP pathway. Dev. Biol. 377: 138-153.
  • Jose F. de Celis (2013). Understanding the determinants of Notch interactions with its ligands. Sci Signal. 6, pe.19.

 

Doctoral Theses:

Martín Resnik Docampo (2011). La proteína MAP4K3 participa, a través de mecanismos independientes, en la regulación de las rutas de señalización Tor y JNK. Universidad Autónoma de Madrid. Director Jose F. de Celis.

María Fernández Organista (2012). Funciones de las proteínas Spalt e identificación de sus genes diana durante el desarrollo del disco imaginal de ala de Drosophila melanogaster. Universidad Autónoma de Madrid. Director Jose F. de Celis.

NOTE! This site uses cookies and similar technologies.

If you not change browser settings, you agree to it. Learn more

I understand

COOKIES POLICY

What are cookies?

A cookie is a file that is downloaded to your computer when you access certain web pages. Cookies allow a web page, among other things, to store and retrieve information about the browsing habits of a user or their equipment and, depending on the information they contain and the way they use their equipment, they can be used to recognize the user.

Types of cookies

Classification of cookies is made according to a series of categories. However, it is necessary to take into account that the same cookie can be included in more than one category.

  1. Cookies according to the entity that manages them

    Depending on the entity that manages the computer or domain from which the cookies are sent and treat the data obtained, we can distinguish:

    • Own cookies: those that are sent to the user's terminal equipment from a computer or domain managed by the editor itself and from which the service requested by the user is provided.
    • Third party cookies: those that are sent to the user's terminal equipment from a computer or domain that is not managed by the publisher, but by another entity that processes the data obtained through the cookies. When cookies are installed from a computer or domain managed by the publisher itself, but the information collected through them is managed by a third party, they cannot be considered as own cookies.

  2. Cookies according to the period of time they remain activated

    Depending on the length of time that they remain activated in the terminal equipment, we can distinguish:

    • Session cookies: type of cookies designed to collect and store data while the user accesses a web page. They are usually used to store information that only is kept to provide the service requested by the user on a single occasion (e.g. a list of products purchased).
    • Persistent cookies: type of cookies in which the data is still stored in the terminal and can be accessed and processed during a period defined by the person responsible for the cookie, which can range from a few minutes to several years.

  3. Cookies according to their purpose

    Depending on the purpose for which the data obtained through cookies are processed, we can distinguish between:

    • Technical cookies: those that allow the user to navigate through a web page, platform or application and the use of different options or services that exist in it, such as controlling traffic and data communication, identifying the session, access to restricted access parts, remember the elements that make up an order, perform the purchase process of an order, make a registration or participation in an event, use security elements during navigation, store content for the broadcast videos or sound or share content through social networks.
    • Personalization cookies: those that allow the user to access the service with some predefined general characteristics based on a series of criteria in the user's terminal, such as the language, the type of browser through which the user accesses the service, the regional configuration from where you access the service, etc.
    • Analytical cookies: those that allow the person responsible for them to monitor and analyse the behaviour of the users of the websites to which they are linked. The information collected through this type of cookies is used in the measurement of the activity of the websites, applications or platforms, and for the elaboration of navigation profiles of the users of said sites, applications and platforms, in order to introduce improvements in the analysis of the data of use made by the users of the service.

Cookies used on our website

The CBMSO website uses Google Analytics. Google Analytics is a simple and easy to use tool that helps website owners to measure how users interact with the content of the site. You can consult more information about the cookies used by Google Analitycs in this link.

Acceptance of the Cookies Policy

The CBMSO assumes that you accept the use of cookies if you continue browsing, considering that it is a conscious and positive action from which the user's consent is inferred. In this regard, you are previously informed that such behaviour will be interpreted that you accept the installation and use of cookies.

Knowing this information, it is possible to carry out the following actions:

How to modify the configuration of cookies

Using your browser you can restrict, block or delete cookies from any web page. In each browser the process is different, here we show you links on this particular of the most used browsers: