Gastrulation

Research Interests

Study of the genes and processes involved in the formation of the head field and the associated process of anterior neural induction of the vertebrate embryo. For this purpose, we use the mouse, Xenopus and chick embryos as model systems using the most up to date technologies in molecular embryology. Those include generation and analysis of KO and transgenic mouse lines, microinjection into Xenopus embryos and chick electroporation.

Group Members

Margaret Bento	External Ph.D. Student
	Tel: 21 440 7918
Marta Vitorino	External Diploma Student
	Tel: 21 440 7918
Sara Marques	Research Technician
	Tel: 21 440 7918
Ana Salgueiro	Research Technician
	Tel: 21 440 7918

Research Project

Endogenous Cerberus activity is required for anterior head specification in Xenopus.

We analyzed the endogenous requirement for Cerberus in Xenopus head development. “Knock down” of Cerberus function by antisense morpholino oligonucleotides did not impair head formation in the embryo. In contrast, targeted increase of BMP, Nodal and Wnt signaling in the Anterior-Dorsal-Endoderm (ADE) resulted in synergistic loss of anterior head structures, without affecting more posterior axial ones. Remarkably, those head phenotypes were aggravated by simultaneous depletion of Cerberus. These experiments demonstrated for the first time that endogenous Cerberus protein can inhibit BMP, Nodal and Wnt factors in vivo. Conjugates of Dorsal Ectoderm (DE) and ADE explants in which Cerberus function was “knocked down“ revealed the requirement of Cerberus in the ADE for the proper induction of anterior neural markers and repression of more posterior ones. This data supports the view that Cerberus function is required in the leading edge of the ADE for correct induction and patterning of the neuroectoderm.

Collaborators

Max-Planck-Institut fur Entwicklungsbiologie, Tuebingen, Germany
Klaus-Michael Kuerner and Herbert Steinbeisser

Research Project

Mkp3 is the strongest direct negative feedback modulator of Fgf8 signaling in the mammalian isthmic organizer.

The pivotal mechanisms that govern the correct patterning and regionalization of the distinct areas of the mammalian brain are driven by key molecules that emanate from secondary organizers at neural plate and tube stages. FGF8 is the candidate morphogenetic molecule to pattern the mesencephalon and rhombencephalon in the Isthmic organizer (IsO). Recognizable relevance has been given to the intracellular pathways by which Fgf8 is regulated and modulated. Recently, it has been demonstrated that the dual mitogen activated protein kinase phosphatase-3 (Mkp3), plays a role as a negative feedback modulator of the MAPK/ERK FGF8 signaling in chick limb bud development. We have investigated the role of the mouse Mkp3 and its functional relationship with the Fgf8 signaling pathway in the mouse IsO using gene transfer micro-electroporation assays and protein-soaked-bead experiments. Here we demonstrate that Mkp3, beyond any other known modulators, has a fast, direct and strong negative action on the MAPK/ERK-mediated FGF8 signaling in the mouse neuroepithelium.

Collaborators

Instituto de Neurociencias. University of Miguel Hernández (UMH-CSIC), Alicante. Spain
Diego Echevarria and Salvador Martínez

Research Project

Emergence of the anterior-posterior axis after implantation relates to the re-orienting symmetry of the mouse embryo rather than the uterine axis.

When the anterior-posterior axis of the mouse embryo becomes explicit at the time of gastrulation it is almost perpendicular to the long axis of the uterus. This has led to the belief that the uterus could play a key role in positioning the major future body axis. We demonstrate that when the anterior-posterior axis first emerges it does not respect the axes of the uterus, but rather the morphology of the embryo itself. Unexpectedly, the emerging anterior-posterior axis is initially aligned not with the long, but rather with the short morphological axis of the embryo. Then whether the embryo is allowed to develop either in vitro or in the uterus, the anterior-posterior axis becomes aligned with the long morphological axis of embryo just prior to gastrulation. Of three mechanisms that could account for this apparent shift in orientation of the anterior-posterior axis - cell migration, spatial change of gene expression or a change in embryo shape - lineage tracing studies on E6.0 embryos favor a change in shape accompanied by a restriction of the expression domain of anterior markers. This property of the embryo must be modulated by some interactions with the uterus as ultimately the anterior-posterior axis and long axis of the embryo become aligned with the left-right uterine axis. In conclusion, the emerging anterior-posterior axis relates to the morphology of the embryo rather than that of the uterus. The apparent shift in its orientation to become aligned with the long embryonic axis and at the same time with the uterus is associated with a change in embryo shape and a refinement of anterior gene expression pattern. This suggests a fine inter-dependence between the expression of anterior-posterior markers, the shape of the embryo and the axes of the uterus.

Collaborators

Wellcome Trust/Cancer Research Institute, Cambridge, UK
Daniel Mesnard and Magdalena Zernicka-Goetz

Research Project

Study of the genetic interaction between cerberus-like and cripto

Nodal encodes a TGF-ß superfamily ligand essential for mesoderm specification and anterior-posterior (A-P) axis formation in the mouse embryo (for review see Shier and Shen, 2000). Nodal signals by binding to type I receptor ALK4 and the type II receptors Activin receptor (ActR) IIa and ActR IIB. Cripto is an EGF-CFC protein located extracellularly and bound to the membrane through a GPI anchor (Minchiotti et al, 2000). Cripto directly binds nodal and interacts with ALK4 and ALK7, both type I serine/threonine kinase receptors, permitting in the first case and enhancing in the second the ability to respond to nodal signal. cripto null mutants display severe gastrulation defects, resembling a “head without a trunk” (Ding et al, 1998). In these mutants early markers of the AVE (hex and cer-l) are mislocalized in the distal visceral endoderm and early markers of the primitive streak (fgf-8 and brachyury) are mislocalized proximally. So, cripto seems essential for the correct localization of the anterior and posterior organizing centers and, consequently, rotation of anterior-posterior axis fails to occur. In this project we will identify and characterize genetic interactions between members of nodal pathway by producing double mutant mice. Both cer-l and cripto are actively involved in Nodal transduction pathway: cripto modulates the signaling and cer-l inhibits it, since the binding of cer-l and Nodal prevents the activation of the Nodal receptors. Our hypothesis is that the inactivation of the Nodal inhibitor cer-l in a cripto null background could result in a less severe phenotype than the cripto-/- one and our proposal is to generate a double null mutant cer-l;cripto to test it.

Collaborators

International Institute of Genetics and Biophysics, Naples, Italy
Giovanna Liguori and Graziella M. Persico

Research Project

The novel secreted factor cerberus-like2 is involved in the genetic pathway determinating the left-right asymmetry in the mouse.

By sequence homology analysis, we have identified a novel mouse gene of the Cerberus-like family, that we designated cerberus-like 2 (cerl-2). The genomic region and full-length cDNA were cloned and sequenced .The mouse cerl-2 gene encodes a 185 amino acid protein that contains a typical hydrophobic signal sequence at its amino terminus and at the carboxy terminus, a cysteine-rich domain closely related to the Xcerberus and mouse cerberus-like ones. This secreted molecule is only expressed at the perinodal region between E7.0-8.0. In Xenopus assays, cerl-2 mRNA is able to fully mimic the activity of Xcer including the induction of ectopic head-like structures as Xcer does. Inactivation by homologous recombination in ES cells demonstrated that cerl-2 is involved in the correct establishment of the left/right body asymmetry during embryonic development. Expression of nodal and Lefty2 in the left LPM is absent in cerl-2 -/- mutants, whereas Pitx2 is randomized. These defects in gene expression lead to sinus inversus of abdominal organs and to morphological and laterality defects of the heart. Biochemical, functional and genetic analyses uncovered the cerl-2 mechanism of activity, which is crucial for the genetic pathway determinating the left-right asymmetry.

Research Project

Transcriptional Regulation of Caronte during Embryonic Development

Xenopus Xcer, mouse Cer-l and chick Car are expressed in equivalent embryonic structures such as the anterior endomesoderm, anterior visceral endoderm, and hypoblast, respectively. In mouse and chick embryos, these genes are also expressed in the anterior definitive mesendoderm. However, at later stages, Xcer transcripts are no longer detected, mouse Cer-l RNA is found in the rostral domain of nascent somites and presomitic mesoderm, and chick Car is expressed in the left lateral plate and paraxial mesoderm. The general aim of this project is to dissect the transcriptional regulatory mechanisms that establish these similarities and differences in the expression patterns of the Cerberus-like gene family. In this second year, the specific steps taken to pursue this project were the following: (1) To identify the transcriptional cis-regulatory elements that control Car expression in the different tissues of the chick embryo; (2) To investigate the regulation of Car reporter constructs by signalling molecules known to repress or activate Car asymmetric expression (i.e., BMP4 and SHH); (3) To analyse the evolutionary conservation of Cerberus-like gene regulation in cross-species experiments. In order to determine if the regulatory mechanisms that control Car expression are conserved in mouse embryos, we have generated and analysed reporter gene expression in transgenic mice carrying chick Car regulatory regions (Car2.5-EGFP).

Publications

Kimura-Yoshida, C., Nakano, H., Okamura, D., Nakao, K., Yonemura, S., Belo, J.A., Aizawa, S., Matsui, Y. and Matsuo, I. (2005). Canonical Wnt signaling and its antagonist regulate anterior-posterior axis polarization by guiding cell migration in mouse visceral endoderm. Dev. Cell 9 :639-650

Echevarria, D., Martínez, S., Marques, S., Lucas-Teixeira, V. and Belo, J.A. (2005). Mkp3 is a negative feedback modulator of Fgf8 signaling in the mammalian isthmic organizer. Dev. Biology 277(1) :114-128

Mesnard, D.*, Filipe, M.*, Belo, J.A. and Zernicka-Goetz, M. (* equal authors) (2004). Emergence of the anterior-posterior axis after implantation relates to the re-orienting symmetry of the mouse embryo rather than the uterine axis. Current Biology 14 :184-196

Marques, S., Borges, A.C., Silva, A.C., Freitas, S., Cordenonsi, M., and Belo, J.A. (2004). The activity of the Nodal antagonist Cerl-2 in the mouse node is required for correct L/R body axis. Genes & Development 18 :2342-2347

Silva, A., Filipe, M., Kuerner, K., Steinbeisser, H. and Belo, J.A. (2003). Endogenous Cerberus activity is required for anterior head induction in Xenopus. Development 130 :4943-4953

Borges, A.C., Marques, S. and Belo, J.A (2002). Goosecoid and cerberus-like do not interact during mouse embryogenesis Int. J. Dev. Biol 46 :259-262

Borges, A.C., Marques, S. and Belo, J.A. (2001). The BMP antagonists cerberus-like and noggin do not interact during mouse forebrain development. Int. J. Dev. Biol. 45 :441-443

Bachiller, D., Klingensmith, J., Kemp, C., Belo, J.A., Anderson, R.M., May, S.R., McMahon, J.A., McMahon, A.P., Harland, R.M., Rossant, J. and De Robertis, E.M. (2000). The organizer secreted factors chordin and noggin are required for forebrain development in the mouse. Nature 403 :658-661

Belo, J.A., Bachiller ,D., Agius, E., Kemp, C., Borges, A.C., Marques, S., Piccolo, S. and De Robertis, E.M. (2000). Cerberus-like is a BMP4 and Nodal antagonist not essential for mouse development. Genesis 26 :259-264

Zhu, L., Belo, J.A., De Robertis, E.M. and Stern, C.D. (1999). Neural inducing strength of mouse organizer controlled by goosecoid dosage. Dev. Biol. 216 :276-281

Belo, J.A., Leyns, L., Yamada, G. and De Robertis, E.M. (1998). The prechordal midline of the chondrocranium is defective in Goosecoid-1 mouse mutants. Mech. Dev. (issue cover) 72 :15-25

Belo, J.A., Bouwmeester, T., Leyns, L., Kertesz, N., Gallo, M., Folletie, M., and De Robertis, E.M. (1997). Cerberus-like is a secreted factor with neuralizing activity expressed in the anterior primitive endoderm of the mouse gastrula. Mech. Dev. (issue cover) 68 :45-57

De Robertis, E.M., Kim, S., Leyns, L., Piccolo, S., Bachiller, D., Agius, E., Belo, J.A. et al. (1997). Patterning by genes expressed in the Spemann’s organizer. Cold Spring Harbor Symp. Quant. Biol. 62 :169-174