Unit: Molecular Biology of Development
Director: Jean-François Nicolas
The central themes of the unit are cell behaviour (in particular cell movements) and developmental strategies. Cell movements and complex developmental strategies linked to genetic patterning are characteristic of elaborate development (vertebrates, arthropods). By contrast, most invertebrate development primarily relies on cell-cell interactions that specify the cell types before any cell rearrangement. The transition from the invertebrate to the vertebrate type of development raises complex questions because cell movements disrupt the cell-cell interaction that are the basis of cell specification. This modification may have necessitated changes in the logic of development. Answering this requires (1) the study of cell movements and the behaviour of the extant representative invertebrate non-derived chordates, the amphioxus; (2) a better understanding of the movements and developmental strategies occurring in the mouse embryo; and (3) a more direct comparison of key aspects of mouse, zebrafish and amphioxus development.
Methods and model systems
The unit develops methods to analyse cell behaviour in several model systems: mouse, zebrafish and amphioxus. This year, we have validated the two novel clonal analysis systems that we are developing to complement the tissue-restricted LaacZ method.
In the first method (the ubiquitous LaacZ system), the expression of the LaacZ reporter gene is not restricted to any specific tissue. Therefore, we can detect all the cells of the clones, allowing us to address questions about cell behaviour and also cell potentialities.
In the second method (the system of temporal induction of clones), we can control when the clonal labelling starts. Therefore, we can generate saturated clonal representations of any stage of development (starting from gastrulation). Depending on the genetic setting used, the cells in which the clonal labelling is initiated can be either targeted or not. Depending on the reporter gene used, a global topographic analysis can be carried out at a precise time of development (LacZ reporter) or can be followed in vivo in clones (EGFP reporter). This second possibility allows us to image embryos at cell resolution either in static 3D or dynamic 4D. We will also use the 4D imaging to follow all the cells during the development of the mouse, the zebrafish and the amphioxus. 4D reconstructions obtained over a long period of time would allow in silico clonal analysis of all the cells of a single embryo. These will also provide information about the cell shape changes that occur during most developmental transitions.
The biological problems
The main biological problems studied this year concern cell behaviour a) during the elongation of the embryo, during which the organs are positioned in relation to both the AP and the DV axes; b) during the formation of two three-dimensional structures, which are the layers of neuron generated by the ventricular zone in the SNC and the epithelial sheaths produced by the stem cells of the hair follicle (HF). The identified diverse behaviour of cells play an crucial role in shaping these structures.The exploitation of the ubiquitous LaacZ system and our system of temporal induction of clones targeted to brachyury expressing cells (cells of the primitive streak and of the tail bud (Fig. 1) has allowed us to identify precisely the modes of divisions (successively stem and proliferative) and the potentialities of the cells involved in the elongation of the embryo.
A more detailed analysis of the elongation of the spinal cord in the chick and of the surface ectoderm in the mouse have demonstrated the crucial role of cell dispersion, convergence and intercalation. Oriented cell division and the modes of growth also play a role (Fig. 2).
We have shown (in the chick) using time-lapse confocal microscopy that after the individualization of the neuro-epithelium, the RhoA is involved in maintaining the orientation of the mitotic spindle parallel to the plane of the neuro-epithelium. This is important because the orientation of the cell division controls their fate by asymmetrically distributing some of their products.
The detailed study of hair follicle renewal using our system of temporal induction of clones has revealed that there is a particular layer of cells in the matrix. This layer is juxtaposed to the dermal papilla and has the properties of a germinative layer (Fig. 3). The cells divide following a stem mode. Our analysis has also revealed that the orientation of the division is related to the proximo-distal position. The cells produced by the germinative layer undergo a radial intercalation and a precise positioning in a predetermined clonal column. All these factors contribute to the shaping of the hair follicle. We have established a relationship between the determination of the fate of the cells and the control of cell behaviour. These two fundamental developmental operations are uncoupled and use the shape of the matrix. This uncoupling may be an example of a general developmental strategy that simplifies the genetic organisation of morphogenesis.
Figure 1: The system of temporal induction of clones. A chemical agent is used to induce a genetic recombination that activates the expression of a LacZ reporter gene. In this case, the induction system has been targeted to the cells of the primitive streak (induction at E7.5 and E8.5) and of the tail bud (induction at E16.5). LacZ staining in E12.5 embryos reveals their contribution to development. The arrowheads show the most rostral limit of this contribution (collaboration with V. Wilson, Edinburgh, Scotland). Photo E. Tzouanacou.
Figure 2: Cell behaviour in the surface ectoderm (SE). This clonal labelling shows that SE cells first disperse and intercalate along the whole anterior-posterior axis of the embryo and then undergo local oriented growth (for example, medio-laterally in the trunk). E14.5 embryo, LacZ staining. Photo A.C. Petit.
Figure 3: Cell behaviour in the hair follicle matrix (A) Cell outlines were drawn from an optical confocal section from a farnesylated-GFP transgenic mouse in which the cell membranes are labelled by GFP (A. Medvinsky, Edinburgh, Scotland). The cell behaviour is organised along the radial dimension of the matrix. The stem cells form a germinative layer juxtaposed to the dermal papilla (dark colours). They renew themselves and generate transient progenitors in the second layer (light colours). Cell fate is decided according to the position of the stem cells long the proximo-distal axis; (B) Clonal hierarchy in the matrix. Photo E. Legué.
Keywords: LaacZ, cell lineage, mouse embryo, clonal analysis, central nervous system, hair follicle, Amphioxus, zebrafish , developmental biology