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  Director : Jean-François NICOLAS (jfnicola@pasteur.fr)


  abstract

 

The control of cell behaviour (modes of cell growth, division and dispersion) is a fundamental aspect of the vertebrate development. Indeed, the complex arrangement of cellular operations forms the basis of the formation of transient embryonic territories from which inter-cellular signals are organised and can be diversified. We are studying these aspects of development. To understand their roots, we decided to compare several vertebrate model systems. These studies necessitate clonal analysis (using the LaacZ system) and imagery. We are also interested in the epigenetic modifications (specific to vertebrates) that occur at specific developmental stages.



  report

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Epigenetic modifications of the genome that occur during development

We carried out a molecular study of EF1a LacZ, EF1a LagZ and EF1a LagoZ transgenic mice (previously established in the laboratory) in which LacZ, LagZ and LagoZ contain different proportions of CpG. The methylation patterns of the reporter gene and of the EF1a promoter (which is included in a CpG-rich island) have been carefully established for both somatic and germinal tissues. The comparison of these patterns lead us to propose a model that explains the extinction of gene expression. According to this model, the CpG-rich regions of LacZ and LagZ interfere with the mechanism that protects the CpG-rich island of EF1a during the de novo methylation of genomic DNA that occurs upon embryo implantation. It induces a change in promoter chromatin structure incompatible with expression.

Clonal analysis

We carried out a clonal analysis of the muscular and central nervous systems. The method used, which was designed in our laboratory, is based on the labelling of a single embryonic cell and on the analysis of its clonal descendants at a precise time during development. This necessitates the use of transgenic mice harbouring a special reporter gene named LaacZ. The subsequent generation of libraries containing several hundred clones allows the detailed description of cell behaviour.

Concerning the muscular system, the clonal analysis of the 40 segments that form the myotome at E11.5 revealed that (1) the pool of precursor cells only consists of two clonal compartments, the medial and lateral compartments, just before the production of these segments and that (2) during this production, a direct topographic relationship is conserved between the pool of producer cells and the myotomal segments. These findings are at the basis of a novel interpretation of the patterns of expression of developmental genes in these structures.

Earlier in the history of the precursors of the muscular system, during trunk formation (axiogenesis), we have shown that cells follow a coherent behaviour (i.e. few movements and few divisions) although the embryo elongates considerably. This coherent cell behaviour concerns both the antero-posterior and dorso-ventral axes. It is established before the bilateralisation of the embryonic trunk structures. As it is during this period that cells are exposed to molecular signals that mediate segmentation and determine antero-posterior positional identity (the molecular clock and the Hox genes), these analyses help us to understand how the control of cell behaviour during this period is organised, according to the constraints generated by these molecular systems (Fig. 2). The molecular bases of segmentation and the acquisition of AP positional identity may depend on the mode of production of the myotome precursor from a pool of cells that renews itself, forming a permanent structure, the spatial organisation of the cells in this pool and the synchronic production of the left and right contributions (of almost similar clonal composition).

Finally, to study the formation of the definitive muscular system, we have produced two novel libraries, containing 1200 clones found in the embryo at E14.5 and in the new born at P7, from the "LaacZ muscle" transgenic line.

Concerning the central nervous system, we are currently analysing clones in the telencephalon and in the cerebellum (obtained from the LaacZ-nerve transgenic line).

Novel system, novel models

We have explored the use of a novel method of cell labelling in the embryo. In this method, labelling is temporally induced. This system is based on the properties of a recombinase (the P1 bacteriophage Cre enzyme) fused to a mutated receptor to oestrogen and on the utilisation of an appropriate recipient mouse reporter line. This method has led to the production of libraries of induced labelling in the surface ectoderm and in the epidermis (Figure 3). Their analysis should reveal the properties (position, mode of production, lineage relationship and potentialities) of the stem cells of the tegument and their relationship with embryonic cells.

A small zebra fish facility and a unit for the electroporation of chick embryos have been established to allow comparative embryology experiments to be carried out on vertebrates.

Figure 1 : The LaacZ labeling system of clones. A) The nls LaacZ reporter gene. A nls LacZ reporter gene, in which a duplication has rendered the enzyme inactive, is linked to a muscle specific promoter, MSP. A spontaneous homologous recombination between the duplicated " aa " sequences re-establishes the b-galactosidase coding frame (in blue). B) Production of clones in the progeny of a cross between a LaacZ transgenic male and a wild-type female. During development, a spontaneous homologous recombination occurs between the duplicated sequences of LaacZ, thus creating a functional LacZ gene in the cell. This event initiates the clonal labeling (coloured in blue) in the embryo. In the a-2 transgenic line, the frequency of myogenic b-gal+ clones at E11.5 is 5x10-2 per embryo.

Figure 2 : Model for the organisation of the pool of myotomal precursors in the primitive streak. Posterior is above and anterior is below. The primitive streak (PS) : presomitic mesoderm (PSM) and somites (S), and then dermomyotome (D) and myotome (M) are represented independently of the adjacent structures. The pool of self-renewing cells involved in axiogenesis is located in the primitive streak. The colour gradient symbolizes AP regionalisation. Most anterior cells (black circles) contribute to medial part of the paraxial mesoderm, from the presomitic mesoderm to the dermomyotome and myotome (black arrows). More posterior cells (open circles) contribute more laterally to the paraxial mesoderm (white arrows). This result in a reorientation of the axis from AP to ML, symbolized by the change of the gradient orientation. Maintenance of the regionalisation established in the primitive streak until formation of dermomyotome and myotome is represented by the conservation of the gradient in these structures. Note the symmetry of the gradient between the left and right parts of the paraxial mesoderm. Medial cells in the pool of self-renewing cells (black and open circles) contribute bilaterally to the paraxial mesoderm (black and white arrows), and the most lateral cells (grey circle) contribute only to the side where they are located (grey arrow). Clonal separation between medial and lateral parts of the somite which persists in the myotome, is superimposed on the mediolateral regionalisation of the paraxial mesoderm established in the self-renewing cell pool in the primitive streak. A, anterior; P, posterior; l, lateral; m, medial; PS, primitive streak; PSM, presomitic mesoderm; S, somite; D, dermomyotome; M, myotome.

Figure 3 : Temporally induced LacZ labelling. A. Labelling was carried out at E8.5. Dorsal view of the embryo six days after labelling: surface ectoderm growth was oriented along the dorso-lateral axis. B. Labelling was carried out at P60. View of the hair follicles, 14 days after labelling. This experiment indicates that the renewal of the hair follicles involves the recruitment of several stem cells of different clonal origins.

Keywords: LaacZ , cell lineage , mouse embryo , clonal analysis , muscular system , central nervous system , methylation of ADN , CpG , épigénétics , LagoZ , developmental biology



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
  Françoise kamel, IP (fkamel@pasteur.fr), Secretary Jean-François NICOLAS, DR1 INSERM (jfnicola@pasteur.fr)

Luc MATHIS, CNRS (lmathis@pasteur.fr)

Sylvie FORLANI, postdoc (sforlani@pasteur.fr)

Sophie ELOY-TRINQUET, postdoc (eloytrin@moka.ccr.jussieu.fr)

Johan SIEUR, PhD student

Emilie LEGUE, PhD student (elegue@pasteur.fr)

Isabelle ROSZKO, PhD student (iroszko@pasteur.fr)

Aurore JEANDON, student

Christine MARIETTE, technician IP (mariette@pasteur.fr)

Suzanne CAPGRAS, technician, IP (scapgras@pasteur.fr)

Pascal DARDENNE, animal technician, IP (pdardenne@pasteur.fr)

Claude MOURIER

Françoise KAMEL, secrétary, IP (fkamel@pasteur.fr)


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