|Director : BABINET Charles (email@example.com)|
Our laboratory is studying mouse embryonic development. On the one hand, we study a mouse strain carrying a conditional lethal mutation with parental effect and inducing early embryonic death. On the other hand, we are studying the function of different genes in embryonic development. In both cases, we privilege a functional genetic approach.
1. Cloning of the Ovum mutant mutation (Om) which induces embryonic death around the blastocyst stage and entails a parental effect (M. Cohen-Tannoudji)
The DDK inbred strain of mice carries a conditional lethal mutation (we call the death of the embryos: "DDK syndrome") which is manifested in outcrosses and depends on the direction of the cross. We showed that the DDK syndrome entails an interaction between a DDK cytoplasmic product already present in the oocyte and the alien paternal genome. A positional cloning approach has allowed us to identify two genes, Ovum mutant candidate gene 1 (Omcg1) and Notchless(Nle) which appear as privileged candidates to be involved in the DDK syndrome: indeed they are expressed in the oocyte and the testis; furthermore, they exhibit a difference in their open reading frame which is unique to DDK.
To validate the functional signification of the mutations we found, we are now using gene targeting to introduce DDK alleles of Nle and Omcg1 genes into the 129Sv mouse strain. This approach will allow us to determine whether the mutations introduced are sufficient to induce the DDK syndrome.
In addition, we are characterizing the in vivo function of Nle and Omcg1. Omcg1 codes for a nuclear zinc finger protein and its function was yet unknown. Nle was identified in drosophila as a direct modulator of Notch signalling activity. Interestingly, we have shown that the disruption of either Nle or Omcg1 results in early embryonic lethality, thus demonstrating their essential role in embryonic development. Ongoing analyses indicate that Nle is important for the maintenance or the proliferation of the Inner Cell Mass while Omcg1 is a key regulator of early embryonic cell cycle. We are now pursuing the characterization of the function of these genes using several approaches such as the generation of conditional alleles and the establishment of ES cells in which the expression of Nle and Omcg1 can be modulated in a controlled manner.
2. Functional analysis of HNF1β (J. Barra in collaboration with the group of Dr. M. Yaniv, Pasteur Institute)
We had shown that embryos homozygous for a null mutation in HNF1β gene die around day 7 of gestation, due to a defect in visceral endoderm differentiation. We have pursued the functional analysis of HNF1β using conditional mutagenesis based on the Cre/lox system. We have specifically inactivated HNF1β in the endoderm and shown it induced defects in the morphogenesis of liver, pancreas and intestine. Two Cre strains are used, one resulting from the Cre knock-in the Mox2 gene (MORE strain, P. Soriano, Seattle), the other expressing Cre under the control of a minimal promotor and the enhancer III of a Hoxa-1 gene (Lufkin, New York). We showed that HNF1β disruption in the endoderm induces strong defects in gut development particularly in the duodenum and the colon. These observations suggest a role for HNF1β in gut regionalization. A molecular analysis is under way essentially by in situ hybridization. It shows that certain markers known to be involved in gut regionalization are indeed deregulated in HNF1β mutant.
In collaboration with G. Duester's group (la Jolla) we have shown that upon HNF1β inactivation in the epiblast, Hoxb1 expression which in normally restricted to and specific of rhombomere 4 (r4) is extended to the posterior region of the rhombencephale. These results indicate that HNF1β is necessary for Hoxb1 repression posteriorly to the r4/r5 limit.
3. Functional analysis of Trapα (J. Barra)
We study a gene trap strain which carries an insertion into the Trapα gene. In the homozygous state the insertional mutation induces a perinatal lethality. Trapα codes for a subunit of a complex (TRAP) associated to the translocon. A recent study has demonstrated the direct implication of TRAP in the translocation of certain proteins. We have shown that gene trap insertion results in the production of a fusion protein between β galactosidase and Trapα, 22 amino-acids before its C-terminal end, which nevertheless localizes in the membrane of endoplasmic reticulum.
We have derived a mouse strain carrying this insertional mutation. Mutant embryos exhibit a cardiac defects including malformation of the outflow tract resulting in ventricular septal defect as well as double outlet right ventricule and partial or total common arterial trunk. We have shown that neural crest cell migration is not affected. However endocardial cushins which appear between E13.5 and the outflow track and contribute to its septation present an important hypertrophy at E12.5. To understand the origin of this phenotype we study the proliferation of endocardiac cells as well as apoptosis in the cushins. Interestingly, this phenotype is very similar to the one exhibited by TGFβ2 knock-out mice. Indeed this factor is secreted by myocardiac cells and its translocation could thus be affected in the Trapα mutant. We therefore study TGFβ2 expression in the outflow track during development.
4. Functional analysis of EDEN-BP (Ch. Kress)
EDEN-BP gene codes for an RNA binding protein initially identified in the amphibian as a regulator of the traduction around the time of fertilization. We have generated EDEN-BP knock-out mice. The study of their phenotype is in progress.
Centre d'Ingénierie Génétique Murine (CIGM)
Charles Babinet Babinet is the scientific advisor of the CIGM, a core transgenic facility recently created in the Pasteur Institute and headed by Francina Langa.
Keywords: Mouse embryonic development, ES cells, cardiac morphogenesis, HNF1?, Trap?, nucleocytoplasmic interactions, gene targeting
|Publications 2004 of the unit on Pasteur's references database|
|Office staff||Researchers||Scientific trainees||Other personnel|
|FLEURANCE Isabelle (firstname.lastname@example.org)||BABINET Charles, CNRS and IP, (Researcher, email@example.com)
BARRA Jacqueline, IP, (Researcher, firstname.lastname@example.org)
COHEN-TANNOUDJI Michel, CNRS, (Researcher, email@example.com)
|ARTUS Jérôme, PhD student
CORMIER Sarah, Postdoc
COUMAILLEAU Franck, PhD student
SOUILHOL Céline, PhD student
|KRESS Chantal (Engineer, firstname.lastname@example.org)
MESBAH Karim (Technician, email@example.com)
VANDORMAEL-POURNIN Sandrine (Technician, firstname.lastname@example.org)