Unit: Development Biology
Director: BABINET Charles
Our laboratory is studying mouse embryonic development. Three lines of research are conducted: 1) We are studying the role of nucleocytoplasmic interactions in mouse preimplantation development. 2) We study the function of different genes in development via gene targeting approaches. 3) We develop strategies to improve the efficiency of gene targeting.
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 contained in the zygote and the alien paternal genome. A positional cloning approach has allowed us to identify two genes 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. In addition, we have shown that the disruption of these genes (by targeted mutagenesis in ES cells) results in early embryonic lethality, thus demonstrating their essential role in embryonic development. We are now pursuing a detailed phenotypic analysis of the homozygous -/- embryo both at the molecular and cellular levels.
2. Functional analysis of HNF1β and Trapα
The function 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 then undertook an analysis of the role of HNF1β in later embryonic development, using a strategy of conditional mutagenesis. We have focused our observations to the effects of HNF1β specific inactivation in endoderm which results in defects in the morphogenesis of liver, pancreas and gut. Our first results suggest an implication of HNF1β in the regionalisation of the latter. Thus inactivation of HNF1β in the gut promotes important defects in its development. We are now developing a molecular analysis of these defects by probing in the first place various molecular markers already known to be implicated in this regionalisation.
Functional analysis of an insertion mutation resulting in defective cardiac morphogenesis (J. Barra)
Using a gene trap approach, we obtained an insertion mutation resulting in the homozygous state in perinatal embryonic lethality. We showed that the gene trap vector was inserted in the TRAPα gene which codes for a subunit of a protein complex associated with the translocon. Analysis of the phenotype of mutant embryos demonstrated a defective cardiac morphogenesis, including a cardiac hypertrophy and a septation defect of the cardiac outflow tract inducing a gap between ventricles. We are currently examining the possible implication of various cell types as well as growth factors (e.g. TGFβ2) in the observed phenotype. Preliminary results suggest a defect in the differentiation of the endocardial cushions.
3. An approach to improve the efficiency of gene targeting (M. Cohen-Tannoudji)
We have recently developped a strategy for mouse germline modifications based on the stimulation of endogenous DNA repair processes. This approach allows to enhance considerably the targeting efficiency at a given locus. It consists in the introduction of a recognition site for the meganuclease I-SceI into the locus to be targeted. I-SceI induces a double strand break in the locus which stimulates homologous recombination with an incoming repair matrix. Our aim is now to: i) to improve the targeting methods by introducing targeted modifications directly into the zygote, thus avoiding the use of ES cells. ii) Create genetic modifications in somatic cells. iii) Identify permissive sites for the targeted expression of genes involved in human pathologies or of therapeutic agents.
Creation of the Centre d'Ingénierie Génétique Murine (CIGM)
Charles Babinet was in charge of a transgenic facility which generated "gene addition" transgenic mice for the groups of the Pasteur Institute. He has obtained the extension of this facility to the generation of targetted mutations via homologous recombination in ES cells. Thus, the CIGM (Centre d'Ingénierie Génétique Murine) has been created and will comprise four personnels. The head of this new service is Dr. Francina Langa and Charles Babinet is scientific advisor.
Keywords: Mouse embryonic development, ES cells, cardiac morphogenesis, HNF1beta, Trapalpha, nucleocytoplasmic interactions, gene targeting