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



A key operation of embryogenesis is to organize the repartition of cells in territories. We studied this aspect of development using clonal analysis in LaacZ/LacZ mosaïc mice. The analyses of clones in the central nervous system and in the muscular system led to a description of clonal patterns and of their links with genetic and clonal patterning during the elaboration of the body plan of the organism.

During development, gene expression is controlled by transacting elements and by stable cis-epigenetic modifications of the DNA. Exploitation of LacZ reporter genes modified as to their of CpG dinucleotides content, the target of DNA methyltransferases, facilitated a study of the consequences of some of the cis modifications of DNA at crucial steps of development.



Clonal patterning and genetic patterning of the mouse embryo

During development, the state of cells varies continuously. At a given time, the state of a cell is dependent on the integration of many extrinsic and intrinsic signals received by its ancestors. As these signals are spatiotemporally restricted, the state of a given cell is a function of the different positions that its ancestors have occupied during development. As a consequence, an important operation of embryogenesis is the continuous control of cell repartition and dispersion, often by forming territories. It is likely that it is by forming territories that the embryo coordinates morphogenesis and epigenesis.

We are engaged in studying the formation of the embryonic territories and with a view to this, we use a genetic method of clonal analysis which enables us to obtain clones randomly produced during development (Fig. 1). This method provides an access to clones representative of any ancestral cell of a given structure. The analyses can be focused on specific structures by using tissue-specific promoters without losing the possibility of observing even very early events.

This year, the targeting of clones in the central nervous system (CNS), analysed at embryonic day 12.5 (E12.5) and in the muscular system, analysed at E11.5, has facilitated an approach to the problem of the establishment of embryonic territories during elongation of the embryon (axiogenesis). A comparison of early clones in both systems has revealed three distinct periods of clonal organization : a wide cell dispersion of precursor cells along the antero-posterior (AP) axis, the arrest of this dispersion and next an organization along the medio-lateral (ML) axis. Therefore, clonal organization of the embryo concerns successively and in part independently the AP and ML axes.

Comparative analyses of all clones which recapitulate the whole history of the organization of the two systems has revealed the modalities of clonal organization.

As concerns the CNS, the pattern of clones reveals that after an initial organization in a unique pool whose cells contribute to the whole structure from anterior to posterior, this pool is regionalized ; one part produces the structures of the anterior CNS and the other part the structures of the posterior CNS. To distinct modes of clonal patterning are associated with this early regionalization. The anterior CNS is organised according to a regional mode. This leads to a structure in which spatial relationships are clonal and clonal diversity between units is important. The posterior CNS is organized according to a temporal mode from two pools of self-renewing cells which produce the AP axis from anterior to posterior (Fig. 2). With such a mode of production, spatial relationships within the structure are temporal, not clonal, and the clonal diversity between units (territories) is minimal.

Concerning the muscular system, an early regionalisation is also observed between the anterior (the head) and the posterior (the trunk). The posterior is produced by a unique pool of self-renewing cells which constructs synchronously the left and the right side of the embryo. A detailed analysis of cell organization indicates that this unique system of production of territories generates a structure (the presomitic mesoderm) which remains relatively static until the individualization of the somites (Fig. 3).

For both the CNS and the muscular system, the clonal organization in the territories of the embryo prefigures the future clonal organization.

Several important conclusions can be derived from this analysis.

  1. Clonal and genetic patterning go together.

  2. Different systems of genetic patterning (for the anterior under the control of the head organizer and for the posterior under the control of the trunk organizer) correspond to different systems of clonal patterning (regional versus temporal).

  3. Embryonic territories immediately posterior to the organizer (the node) contain pools of self-renewing cells for the posterior CNS and paraxial mesoderm. This region of the gastrula therefore has specific properties distinct from more anterior regions.

  4. Cell properties of paraxial mesoderm territories and of the system from which it is derived fit remarkably well with those expected for the very elaborate systems of genetic specification (by Hox genes) and of segmentation (by a molecular clock and a determination front). This is an amazing example of coordination between cell behaviour (morphogenesis) and activation of specification genes. The phylogenetic history of this coordination will show whether if it constitutes a constraint for the elements of the phylotypic stage of chordates.

  5. The maintenance of clonal organization of the body plan of the organism, at the phylotypic stage in the adult, is a strong indication of the privileged character of this stage.

Epigenetic modifications during development and their impact on expression

Our experimental approach to this problem is based on a comparison of the expression of transgenes with different levels of CpG dinucleotides. Thus from the LacZ reporter gene containing 291 CpGs, a LagZ gene containing only 52 CpGs and a LagoZ containing only 2 CpG, were engineered. Combined with the promoter of E1Fa, a gene with no tissue-specificity, these three transgenes were introduced by transgenesis into the genome of mice. Analysis of their expression pattern has shown that from a certain level the presence of CpG is highly repressive in somatic cells. In contrast, this level is almost nonrepressive in germ cells. Curiously, a differential expression in relation with the parental origin is observed in the first stages of development of the mice but only for some of the combinations tested. As, in these contructions, only the coding region of the gene was modified, we are now mainly interested in understanding the molecular basis of the repression. Subsequent to the comparison of the different states of expressions and of the different states of methylation of the promoter and reporter regions of the transgenes we shall be able to distinguish between several equally probable hypotheses.

Photos :

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. A model for cellular events during development of the neural tube. The model concerns the organization of territories and global cell movements, as well as the development of individual cell lineages. Before the onset of gastrulation (E6), the pool of CNS founders (in green) is not regionalized, and due to the important cell intermingling occuring before gastrulation and during neural plate elongation (E5-E7), individual precursors produce descendants distributed along the entire neuraxis. From the onset of gastrulation (E7), individual clones have a lower probability of producing descendants in both anterior and posterior CNS, due to the posterior oriented cell movements of the self-renewing pool of cells and an anterior coherent growth. Consequently, the pools for spinal cord (in blue) and brain (red-yellow) become separated (earliest regionalisation). Subsequently (E7-E8), important cell intermingling continues in the SC primordium, due to an anterior to posterior regression of the self-renewing pool of stem cells. In contrast, due to coherent growth in the anterior neural plate, the A-P order of precursors is rapidly established, leading to an early regionalisation of brain territories (red-to-yellow gradient). After E8, A-P cell mixing in the brain and anterior SC becomes more limited and is followed by a phase of D-V cell dispersion.

Figure 3 : 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.


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  Office staff Researchers Scientific trainees Other personnel

KAMEL Françoise ; IP, fkamel@pasteur.fr

NICOLAS, Jean-François(DR1 INSERM, jfnicola@pasteur.fr)

HENRY Isabelle (CR1 INSERM, ihenry@pasteur.fr)

MATHIS Luc (CR2 CNRS, lmathis@pasteur.fr)

FORLANI Sylvie, stagiaire post-doctorale

ELOY-TRINQUET Sophie, stagiaire de Doctorat

SIEUR Johan, stagiaire de Doctorat

TEICHERT Arnaud, stagiaire de DEA

LEGUE Emilie, stagiaire de DEA

ROSZKO Isabelle, stagiaire de DEA

FONTAINE David, stagiaire de Maîtrise

MARIETTE Christine (technicienne supérieure de laboratoire), IP, mariette@pasteur.fr

CAPGRAS Suzanne (technicienne de laboratoire), IP, scapgras@pasteur.fr

DARDENNE Pascal (technicien animalier), IP, dardenne@pasteur.fr

FAUCHER Sylvie (aide de laboratoire)


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