Unit: Molecular Biology of Development
Director: Jean-François Nicolas
Development depends on cell behaviour and cell properties (motility, proliferation, shape, polarity, etc.). The spatio-temporal arrangement of cellular processes is at the basis of the formation of transient embryonic territories from which inter-cellular signals are established that in turn modify the cell state. Changes in this spatio-temporal pattern underlie the evolution of the body plans. We are using clonal analysis and imaging to study these aspects of development and their links with genetics in several vertebrate models, with particular emphasis on the central nervous system, the muscular system, the surface ectoderm and its derivatives. We are also interested in the epigenetic modifications that perturb gene expression, in particular that of transgenes.
Epigenetic modifications of transgenes
Transgenes are often engineered using sequences derived from distantly related genomes. This may lead to inappropriate expression. For instance, the combination of promoters from widely expressed genes and bacterial LacZ sequences in transgenes often results in the absence of expression in the somatic tissues of transgenic mice. Several observations suggest that this is related to the CpG content of the LacZ gene. To test this hypothesis, we have combined several LacZ reporter genes with various CpG contents (from high to zero) with the promoter of a widely expressed gene, the α sub-unit of the elongation factor 1 of translation, and created transgenic mice. We have shown that the LacZ construct without any CpG is not subjected to the transgene repression observed in the somatic tissues of transgenic lines with higher CpG contents . Therefore, the CpG content in the transcribed region of transgenes may have a causal role in gene silencing. We subsequently compared the expression and methylation patterns of the LacZ transgenes. This showed that the mechanism by which the CpG island escapes de novo methylation is sensitive to the CpG content of adjacent sequences. This discovery has fundamental and practical implications.
We have continued our study of the formation of the muscular and nervous systems. One of the methods used, previously developed in our laboratory, is based on the genetic labelling of a single embryonic cell and on the analysis of its clonal descendants at a precise developmental stage. This necessitates the use of transgenic mice harbouring a special reporter gene named LaacZ. The generation of libraries containing several hundred clones representing all developmental stages makes the detailed description of cell behaviour possible. We have also used a novel method based on temporally induced labelling of embryonic cells. This method is based on the properties of a Cre recombinase fused to a mutated oestrogen receptor. This method has been successfully used to describe all the cellular events occurring during morphogenesis and the renewal of the hair follicles.
Concerning the muscular system, we have continued our analysis of libraries of LaacZ clones in embryos at E14.5 and in newborns at P7. We have shown that the foetal and the definitive muscular systems are formed from precursor cells, the behaviour of which is coherent along the rostro-caudal axis from before the bilateralisation of the trunk of the embryo . Nevertheless, some of the properties of clones at E14.5 and P7 differ from those at E11.5. Finally, we have started to study the clonal organisation of the appendicular muscles in relation with their position along the proximo-distal and dorso-ventral axes. We expect this study to improve our understanding of the occurrence of this evolutionary innovation in terrestrial tetrapods.
A study of LaacZ clones covering the whole period of cerebellar development has revealed several novel facts concerning the central nervous system. For instance, concerning the lineage relationships of neurons, we have shown that (1) the deep nuclei are formed from a pool of progenitors separated from the pool of the other cerebellar neurons, (2) Purkinje cells are systematically derived from multipotent progenitors and (3) that the Golgi cells constitute a heterogeneous population of cells that are all derived from the ventricular zone. These observations made it possible to generate a novel lineage tree for the cerebellar neurons. Concerning morphogenesis, we showed for the first time that the progenitors of the cells of the internal granular layer are already arranged in a clonal medio-lateral pattern in the neural tube at a time when their rostro-caudal dispersion is still extensive . This indicates that the antero-posterior order of the progenitors in the rhombic lip is established in the neural tube and maintained in the medio-lateral distribution of the cells of the internal granular layer. This suggests that the morphogenetic movements do not alter this initial order. It is possible that the preservation of positional information from the neural tube stage imposes a constraint on the modalities of cell dispersion.
Decreasing the CpG content in the LacZ gene abolishes transgene silencing in somatic tissues. In toto X-gal labelling of E9.5 embryos. The LacZ transgenes (CpG content 9.24 %) are repressed in YACs (a), and in transgenes (b). c) the LagZ transgenes (CpG content 1.6 %) exhibit a variegated expression pattern and d) the LagoZ transgenes (CpG content 0.06 %) are expressed at a high level.
Short bilateral clones that contribute to muscles at a similar rostro-caudal level on both sides of the embryo at E14.5 indicate that foetal muscles are derived from a pool of cells that share properties with the pool of cells at the origin of the myotome in E11.5 embryos.
This clone illustrates the type of contribution of the earliest precursors of the cerebellar neurons. In the internal granular layer (IGL) neurons are limited to a restricted medio-lateral domain. This indicates that the IGL precursors already adopt a medio-lateral arrangement in the neural tube. The complex morphogenetic movements that follow the neural tube stage do not perturb this initial arrangement.
Keywords: LaacZ, cell lineage, mouse embryo, clonal analysis, muscular system, central nervous system, methylation of ADN, CpG, epigenetics, LagoZ, developmental biology