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     Molecular Biology of Development


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


  abstract

 

Development is controlled by the behaviour and properties of cells (shape / polarity / motility / proliferation / death, etc.). The spatio-temporal arrangement of cellular processes forms the basis of the formation of transient embryonic territories, from which intercellular signals are established that modify cell state. Changes in these spatio-temporal patterns lead to changes in body plans. We are using clonal analysis and imaging to study these aspects of development and their links with genetics in several chordate models, with particular emphasis on an apparently simple structure – the hair follicle – and more complex structures – the central nervous system, the surface ectoderm and the paraxial mesoderm.



  report

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The system of clonal analysis

This year, we have complemented our LaacZ clonal analysis system in two ways:

1. We have evaluated the possible use of transgenic mice with a LaacZ reporter gene under control of the promoter of a ubiquitous gene called Rosa 26 (Collaboration with V. Wilson, Centre for Stem Cell Research, Edinburgh). A library of embryonic day 10.5 clones has been produced. Several clones were found to contribute to more than one tissue (Fig. 1), thus validating the proposed system. We have also produced a novel LaacZ transgenic line with a LaacZ reporter with a 1000 bp aa duplication. This line complements the line with a 289 bp aa duplication. This strategy will make it possible to address questions about cell potentialities (for embryonic and adult stem cells in particular), lineage restrictions and cell behaviours (particularly those involved in formation of the transient structures of the embryo).

2. We have tried to establish a system of temporally induced LacZ labelling (clones) based on the use of a Cre recombinase fused to a modified oestrogen receptor. The limitations of this system have been determined by comparing several inducible CreRT transgenic mice.

The biological problems

Our past studies have clearly highlighted the problems posed by the extensive use of cell behaviour in vertebrate chordate development, by comparison with invertebrate chordates, in which cell behaviour plays only a minor role. This led us to study this problem in mice, in a simple multitissue structure: the hair follicle (Fig. 2). The morphogenesis of this structure extends over several weeks, making it ideal for experimental studies. The successive steps of the process can be detailed, notably by using a system of temporally induced clones. We have successfully defined the fate and major behaviours of all cells at the origin of this structure. Our findings seem to suggest that the extensive use of cell behaviours in the development of vertebrate chordates may involve uncoupling of the major developmental operations, based on the three-dimensional nature of embryonic structures.

Along the same lines, but in more complex structures, we are studying formation of the spinal cord and of the forebrain in chick embryo. We are using DNA electroporation and time-lapse imaging to define the sequences of movements and the formation of boundaries, in parallel with clonal analysis in the mouse.

This has led us to try to determine the developmental characteristics of the invertebrate ancestor of vertebrates. We have performed preliminary in vivo labelling experiments on embryos of Branchiostoma lanceolatum (Fig. 3), an extant cephalochordate, the cephalochordates being a sister group of vertebrates extant at the time of the Cambrian Explosion. Our goal is to use 4D confocal time-lapse microscopy to follow the behaviour of individual cells.

We are also developing genetic tools for clonal analysis in zebra fish (Danio rerio).

Figure 1: A clone contributing to multiple tissues in a Rosa LaacZ embryo.

Figure 2: Hair follicle structure. Several cell layers surround the hair shaft. They are formed by the matrix (not shown) during a morphogenetic process that lasts four weeks.

Figure 3: Gastrula of Branchiostoma (optical section). The nuclei and mitoses are visualised with a vital dye, SYTO 16.

Keywords: LaacZ, cell lineage, mouse embryo, clonal analysis, central nervous system, hair follicle, Amphioxus, zebrafish, developmental biology



  publications

puce Publications 2004 of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
  Françoise Kamel (fkamel@pasteur.fr) Jean-François Nicolas, INSERM (DR1, jfnicola@pasteur.fr)

Estelle Hirsinger, CNRS (CR2, ehirsing@pasteur.fr)

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

Elena Tzouanacou, stagiaire post-doctorale (elena@pasteur.fr)

Emilie Legué, stagiaire de doctorat (elegue@pasteur.fr)

Isabelle Roszko, stagiaire de doctorat (iroszko@pasteur.fr)

Anne-Cécile Petit, stagiaire de DEA (acpetit@pasteur.fr)

Christine Mariette, technicienne supérieure IP (mariette@pasteur.fr)

Suzanne Capgras, technicienne supérieure, IP (scapgras@pasteur.fr)

Pascal Dardenne, technicien animalier, IP (pdardenne@pasteur.fr)

Claude Mourier, aide de laboratoire, IP (cmourier@pasteur.fr)

Françoise Kamel, secrétaire, IP (fkamel@pasteur.fr)


Activity Reports 2004 - Institut Pasteur
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