Charles Babinet

A cell surface glycoprotein involved in the compaction of embryonal carcinoma cells and cleavage stage embryos

Fab fragments of rabbit anti-embryonal carcinoma cells IgG dramatically perturb cell-cell interactions between embryonal carcinoma cells and between early mouse embryo blastomeres. These antibodies prevent compaction of preimplantation embryos (or trigger their decompaction) and have similar effects on embryonal carcinoma cells. They probably act through the masking of specific molecules (Fab targets) involved in the mechanisms of recognition between cells during compaction. Fab target molecules have been extracted from embryonal carcinoma cell membranes and purified using their property to inhibit the effects mediated by anti-embryonal carcinoma Fab. The solubilization of the Fab targets could be achieved using both detergent extraction and trypsin treatment of membranes. In the latter case, a glycoprotein of 84,000 daltons could be purified which has all the properties expected from the Fab target and accounts for most of the Fab-inhibiting activity of embryonal carcinoma cell membranes.

Cell-cell interactions in early embryogenesis: A molecular approach to the role of calcium

Compaction, a process of cell-cell adhesion between mouse blastomeres or between embryonal carcinoma (EC) cells requires calcium ions. A decompaction effect similar to that observed in the absence of Ca2+ is triggered by Fab fragments of rabbit anti-EC IgG. This effect occurs through the recognition of a specific cell-surface glycoprotein named uvomorulin. An 84,000 dalton fragment of uvomorulin (UMt) has been previously extracted by trypsin from EC cell membranes and purified. WE present evidence that effects of Ca2+ on compaction are transmitted through conformational changes in uvomorulin. First, Ca2+ protects UMt from further proteolysis by trypsin. Mn2+ and Sr2+ have similar effects, whereas this protection is reversed by La3+. Second, UMt can bind the monoclonal antibody De1 only in the presence of Ca2+ (half-binding at 10(-5) M Ca2+). This antigenic exposure also takes place in the presence of Mn2+ or Sr2+ and is reversed by La3+. Third, metal ions (Ca2+, Mn2+, Sr2+) that promote trypsin resistance and recognition by DE1 are found to trigger the compaction of morulae and EC cells. Metal ions (La3+) that reduce trypsin resistance and affinity for DE1 result in decompaction.

Inactivation of CUG-BP1/CELF1 Causes Growth, Viability, and Spermatogenesis Defects in Mice

ABSTRACT CUG-BP1/CELF1 is a multifunctional RNA-binding protein involved in the regulation of alternative splicing and translation. To elucidate its role in mammalian development, we produced mice in which the Cugbp1 gene was inactivated by homologous recombination. These Cugbp1−/− mice were viable, although a significant portion of them did not survive after the first few days of life. They displayed growth retardation, and most Cugbp1−/− males and females exhibited impaired fertility. Male infertility was more thoroughly investigated. Histological examination of testes from Cugbp1−/− males showed an arrest of spermatogenesis that occurred at step 7 of spermiogenesis, before spermatid elongation begins, and an increased apoptosis. A quantitative reverse transcriptase PCR analysis showed a decrease of all the germ cell markers tested but not of Sertoli and Leydig markers, suggesting a general decrease in germ cell number. In wild-type testes, CUG-BP1 is expressed in germ cells from spermatogonia to round spermatids and also in Sertoli and Leydig cells. These findings demonstrate that CUG-BP1 is required for completion of spermatogenesis.

The Cell Cycle of Early Mammalian Embryos: Lessons from Genetic Mouse Models

Genes coding for cell cycle components predicted to be essential for its regulation have been shown to be dispensable in mice, at the whole organism level. Such studies have highlighted the extraordinary plasticity of the embryonic cell cycle and suggest that many aspects of in vivo cell cycle regulation remain to be discovered. Here, we discuss the particularities of the mouse early embryonic cell cycle and review the mutations that result in cell cycle defects during mouse early embryogenesis, including deficiencies for genes of the cyclin family (cyclin A2 and B1), genes involved in cell cycle checkpoints (Mad2, Bub3, Chk1, Atr), genes involved in ubiquitin and ubiquitin-like pathways (Uba3, Ubc9, Cul1, Cul3, Apc2, Apc10, Csn2) as well as genes the function of which had not been previously ascribed to cell cycle regulation (Cdc2l, E4F and Omcg1).

Healthy mice with an altered c-myc gene: role of the 3' untranslated region revisited.

c-Myc is a protooncogene involved in the control of cellular proliferation, differentiation and apoptosis. Like many other early response genes, regulation of c-myc expression is mainly controlled at the level of mRNA stability. Multiple cis-acting destabilizing elements have been described that are located both in the protein-coding region and in the 3′ untranslated region (3′ UTR). However, it is not known when they function during development and whether they act as partly redundant or independent elements to regulate c-myc mRNA level of expression. To begin to address these questions, we created a series of c-myc alleles modified in the 3′ UTR, using homologous recombination and the Cre/loxP system, and analysed the consequences of these modifications in ES cells and transgenic animals. We found that deletion of the complete 3′ UTR, including runs of Us and AU-rich elements proposed, on the basis of cell-culture assays, to be involved in the control of c-myc mRNA stability, did not alter the steady-state level of c-myc mRNA in any of the various situations analysed in vivo. Moreover, mice homozygous for the 3′ UTR-deleted gene were perfectly healthy and fertile. Our results therefore strongly suggest that the 3′ UTR of c-myc mRNA does not play a major role in the developmental control of c-myc expression.

Impaired Mitotic Progression and Preimplantation Lethality in Mice Lacking OMCG1, a New Evolutionarily Conserved Nuclear Protein

ABSTRACT While highly conserved through evolution, the cell cycle has been extensively modified to adapt to new developmental programs. Recently, analyses of mouse mutants revealed that several important cell cycle regulators are either dispensable for development or have a tissue- or cell-type-specific function, indicating that many aspects of cell cycle regulation during mammalian embryo development remain to be elucidated. Here, we report on the characterization of a new gene, Omcg1, which codes for a nuclear zinc finger protein. Embryos lacking Omcg1 die by the end of preimplantation development. In vitro cultured Omcg1-null blastocysts exhibit a dramatic reduction in the total cell number, a high mitotic index, and the presence of abnormal mitotic figures. Importantly, we found that Omcg1 disruption results in the lengthening of M phase rather than in a mitotic block. We show that the mitotic delay in Omcg1 −/− embryos is associated with neither a dysfunction of the spindle checkpoint nor abnormal global histone modifications. Taken together, these results suggest that Omcg1 is an important regulator of the cell cycle in the preimplantation embryo.

The Murine Ortholog of Notchless, a Direct Regulator of the Notch Pathway in Drosophila melanogaster, Is Essential for Survival of Inner Cell Mass Cells

ABSTRACT Notch signaling is an evolutionarily conserved pathway involved in intercellular communication and is essential for proper cell fate choices. Numerous genes participate in the modulation of the Notch signaling pathway activity. Among them, Notchless (Nle) is a direct regulator of the Notch activity identified in Drosophila melanogaster. Here, we characterized the murine ortholog of Nle and demonstrated that it has conserved the ability to modulate Notch signaling. We also generated mice deficient for mouse Nle (mNle) and showed that its disruption resulted in embryonic lethality shortly after implantation. In late mNle−/− blastocysts, inner cell mass (ICM) cells died through a caspase 3-dependent apoptotic process. Most deficient embryos exhibited a delay in the temporal down-regulation of Oct4 expression in the trophectoderm (TE). However, mNle-deficient TE was able to induce decidual swelling in vivo and properly differentiated in vitro. Hence, our results indicate that mNle is mainly required in ICM cells, being instrumental for their survival, and raise the possibility that the death of mNle-deficient embryos might result from abnormal Notch signaling during the first steps of development.

A simplified method for mouse blastocyst injection

Abstract A simplified method for blastocyst injection is described. Two instruments only are used: one micropipette for holding the blastocyst and another for injection. Both instruments are easily made by cutting a pulled capillary. No bevelling or polishing is needed. The instruments may be used for many successive injections.