Organogénese

Interesse da Investigação

Organogenesis, limb development, left-right asymmetry.

Membros do Grupo

Ana Catarina Afonso	Postdoc
	Tel: 21 446 4664
Joana Monteiro	External Ph.D. Student
	Tel: 21 446 4651
Rita Félix	External Masters Student
	Tel: 21 446 4651
Diana Pires	External Masters Student
	Tel: 21 446 4651
Rui Castanhinha	2009 PIBS
	Tel: 21 446 4651

Projecto de Investigação

Analysis of the molecular cascade for mesodermal limb chondrogenesis: Sox genes and BMP signaling.

The formation of cartilage is an essential process during skeletogenesis in vertebrates. During embryonic development, the formation of the endochondral bones is preceded by the formation of a transitory cartilaginous template. The sequence of molecular and morphological events leading to chondrogenesis has not been fully defined, but the formation of the chondrogenic aggregates involves changes in cell shape and growth kinetics, the production of a specific extracellular matrix, and the expression of adhesion molecules. We are studying how Sox genes and BMP signaling are functionally coupled during limb chondrogenesis. Using the experimental model of TGF 1-induced interdigital digits, we dissect the sequence of morphological and molecular events during in vivo chondrogenesis. Our results show that Sox8 and Sox9 are the most precocious markers of limb cartilage, and their induction is independent and precedes the activation of BMP signaling. Sox10 appears also to cooperate with Sox9 and Sox8 in the establishment of the digit cartilages. In addition, we have observed that experimental induction of Sox gene expression in the interdigital mesoderm is accompanied by loss of the apoptotic response to exogenous BMPs. L-Sox5 and Sox6 are respectively induced coincident and after the expression of Bmpr1b in the prechondrogenic aggregate, and their activation correlates with the induction of Type II Collagen and Aggrecan genes in the differentiating cartilages. The expression of Bmpr1b precedes the appearance of morphological changes in the prechondrogenic aggregate and establishes a landmark from which the maintenance of the expression of all Sox genes and the progress of cartilage differentiation becomes dependent on BMPs. In addition, we have observed that Ventroptin a novel BMP antagonist, precedes Noggin in the modulation of BMP activity in the developing cartilages.

Colaboradores

J. Universidad Autónoma de México, México.
Montero, J.A., Gañan, Y., Macias, D., Merino, R. and Hurle, J.

Projecto de Investigação

MKP3 mediates the cellular response to FGF8 signalling in the vertebrate limb.

During the development of any given structure or organ, several signalling mechanisms cooperate in providing positional information to cells in the corresponding developmental fields. In many cases, this cross-talk of signalling pathways involves interactions between different tissues of the developing embryo, with sequential transfer of positional information from one tissue to another. One of the best examples of signalling molecules involved in the complex cross-talk mechanisms that pattern developing embryos is the FGF superfamily of secreted factors. FGFs are essential during embryonic development, including the regulation of cellular proliferation and differentiation. The mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and phosphatidylinositol-3-OH kinase (PI(3)K)/Akt pathways are involved in the regulatory mechanisms of several cellular processes including proliferation, differentiation and apoptosis. We have shown that during chick, mouse and zebrafish limb/fin development, a known MAPK/ERK regulator, Mkp3, is induced in the mesenchyme by fibroblast growth factor 8 (FGF8) signalling, through the PI(3)K/Akt pathway. This correlates with a high level of phosphorylated ERK in the apical ectodermal ridge (AER), where Mkp3 expression is excluded. Conversely, phosphorylated Akt is detected only in the mesenchyme. Constitutively active Mek1, as well as the downregulation of Mkp3 by small interfering RNA (siRNA), induced apoptosis in the mesenchyme. This suggests that MKP3 has a key role in mediating the proliferative, anti-apoptotic signalling of AER-derived FGF8.

Colaboradores

The Salk Institute for Biomedical Sciences, U.S.A.
Juan Carlos Izpisua-Belmonte, Kawakami, Y., Koth, C., Büscher, D., Itoh, T., Raya, A., Ng, J., Rodriguez Esteban, C., Takahashi, S., Swarz, M. and Asahara, H.

Projecto de Investigação

Notch activity acts as a sensor for extracelluar calcium during vertebrate left-right determination.

Notch activity plays an important role in the early phases of LR determination by directly regulating Nodal expression around the node in mouse and zebrafish embryos. The activity of the Notch signalling pathway depends on the complex interplay of several receptors, ligands and modulators. In an effort to identify factors that regulate Notch activity around the node, we have characterized the dynamics of gene expression and interactions among relevant genes and proteins of this signalling pathway during early embryo gastrulation, and have generated a mathematical model to help us pinpoint those factors that are essential in LR determination. During vertebrate embryo development, the breaking of the initial bilateral symmetry is translated into asymmetric gene expression around the node and/or in the lateral plate mesoderm. The earliest conserved feature of this asymmetric gene expression cascade is the left-sided expression of Nodal, which depends on the activity of the Notch signalling pathway. We have identified the source of the asymmetric activation of Notch as a transient accumulation of extracellular calcium, which in turn depends on left–right differences in H1/K1-ATPase activity. Our results uncover a mechanism by which the Notch signalling pathway translates asymmetry in epigenetic factors into asymmetric gene expression around the node.

Colaboradores

TheSalk Institute for Biomedical Sciences, U.S.A.
Juan Carlos Izpisua-Belmonte, Raya, A., Kawakami, Y., Rodríguez-Esteban, C., Ibañes, M., Rasskin-Gutman D., and Büscher, D.

Projecto de Investigação

Intracellular pathways conroling outgrowth during vertebrate limb development.

Intracellularly, Wnt activity is transferred through, at least, three different pathways. The canonical pathway involves ß-catenin activation, which eventually results in trancriptional activation of target genes in the nucleus. The planar cell polarity pathway triggers Rho GTPases activity, thus inducing JNK activation and eliciting cytoskeletal rearrangements. Finally, the Calcium pathway increases the levels of intracellular calcium and decreases the level of intracellular cyclic guanosine monophosphate (cGMP). This last action is mediated by the activation of intracellular phosphodiesterase (PDE). The role of cGMP in the last pathway is the less understood. In this pathway, Wnts seem to be a regulator of the ratio of intracellular cGMP which is synthetised after guanylyl cyclase activation by Nitric Oxide (NO). These different Wnt intracellular pathways are activated by different Wnt molecules that bind to several receptors, named frizzleds, what introduces an additional level of complexity in the pathway. There are evidences that support that Wnt-Calcium-cGMP pathway antagonises the wnt ß-catenin pathway but other show a crosstalk between both intracellular responses. Wnt signaling plays a major role during limb development. It is responsible for AER formation, p-d outgrowth, chondrogenesis and muscle development. However, it has been reported that Wnt molecules acting through a non-ß-catenin mediated pathway also play a role in limb outgrowth. In particular, inactivation of the wnt5a gene resulted in shortened limbs. We now want to better understand the role of Wnt5a in limb develpoment. We want to focus on the action of Calcium, NO and cGMP during limb bud development by using the chicken embryo as a model. We are characterising by in situ hybridization and immunohistochemistry the expression pattern of the different components of the Wnt Calicum-cGMP pathway, including Prickle proteins, Calcium calmodulin dependent protein Kinase II (CamKII) and Siah family members. In addition, inhibitors for guanylyl cyclase and PDEs are also avaliable and can be delivered in different limb areas by bead application followed by the study of the limb phenotype and different genetic markers.

Colaboradores

The Salk Institute for Biomedical Sciences, U.S.A.
Juan Carlos Izpisua-Belmonte, Kawakami, Y.

Publicações

Kawakami, Y., Rodriguez-Leon, J. *, Koth, C.M., Büscher, D., Itoh, T., Raya. A., Ng, J., Rodríguez Esteban, C., Takahashi, S., Henrique, D., SchwartZ, M., Asahara, H. and Izpisúa Belmonte, J.C. * Co-authorship. (2003). MKP3 Mediates the Cellular Response to FGF8 Signalling in the Vertebrate Limb. Nature Cell Biology 5 :513-519

Chimal-Monroy, L., Rodriguez-Leon, J. *, Montero, J.A., Gañan, Y., Macias, D., Merino, R. and Hurle, J.M. * Co-authorship. (2003). Analysis of the Molecular Cascade Responsible for Mesodermal Limb ChondroGenesis: sox genes and bmp signaling. Developmental Biology 257 :292-301

Groppe, J., Greenwald, J., Wiater, E., Rodriguez-Leon, J., Economides, A.N., Kwiatkowski, W., Baban, K., Affolter, M., Vale, W.W., Izpisua-Belmonte, J.C. and Choe, S. (2003). Structural Basis of Bmp Signaling Inhibition by Noggin, a Novel Twelve-Membered Cystine Knot Protein. The Jnl. of Bone and Joint Surgery 85-A, Suppl.3 :52-58

Kawakami, Y., Capdevila, J., Büscher, D., Itoh, T., Rodríguez-Esteban, C., Izpisua-Belmonte, J.C. (2001). WNT signals control FGF-dependent limb initiation and AER induction in the chick embryo. Cell 104 (6) :891-900

Capdevila, J., Vogan, K.J., Tabin, C.J., Izpisua-Belmonte, J.C. (2000). Mechanisms of left-right determination in vertebrates. Cell 31;101 :9-21

Merino, R., Macias, D., Gañan, Y., Rodriguez-Leon, J., Eonomides, A.N., Rodriguez-Esteban, C., Izpisua-Belmonte, J.C., Hurle, J.M. (1999). Control Of Digit Formation By Activin Signaling. Development 126(10) :2161-2170

Rodriguez-Leon, J., Merino, R., Macias, D., Gañan, Y., Santesteban, E., Hurle, J.M. (1999). Retinoic Acid Regulates Programmed Cell Death Through Bmp Signaling. Nature Cell Biology 1, (2) :125-126

Merino, R., Rodriguez-Leon, J., Macías, D., Gañán, Y., Economides, A.N., Hurlé, J.M. (1999). The Bmp Antagonist Gremlin Regulates Outgrowth, Chondrogenesis And Programmed Cell Death In The Developing Limb. Development 126 :5515-5522

Rodríguez-Esteban, C., Tsukui, T., Yonei, S., Magallon, J., Tamura, K., Izpisua-Belmonte, J.C. (1999). The T-box genes Tbx4 and Tbx5 regulate limb outgrowth and identity. Nature 398 :814-18