PhD Position at the National Muséum of Natural History, Paris, France

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June 8, 2008

PhD Position at the National Muséum of Natural History, Paris, France
“Comparative analysis of mecanisms controlling neural stem cell status in Xenopus”
Laboratory:
Evolution des Régulations Endocriniennes
UMR CNRS 5166 – USM 501, Dpt RDDM, MNHN
7, rue Cuvier, 75231 Paris, cedex 5, France
Supervisers:
Pr. B. Demeneix, demeneix@mnhn.fr (+33 1 40 79 36 16)
Dr. L. Coen, coen@mnhn.fr, (+33 1 40 79 57 47)

Project description :
Scientific context
Embryonic stem cells (or ES cells) are characterized by their self-renewing properties
and their capacity to differentiate and give rise to all cell types in an organism. Oct3/4, Sox2
and Nanog transcription factors have been identified as the key elements in the maintenance
of ES cell pluripotency and self-renewal properties in mammals (Boyer et al., 2005, Cell 122,
947-956). Moreover, it has been shown very recently that overexpression of the four factors
Sox2, Oct3/4, Klf4 and c-Myc in a somatic cell is sufficient to generate a cell exhibiting the
characteristics of pluripotency (Takahashi and Yamanaka, 2006, Cell 126, 663-676), and that
selection for the re-expression of the Nanog factor in these cells is the sign of a return to the
ES-type cell state (Okita et al., 2007, Nature 6, 1-6).
Although ES cells have the ability to generate all the tissues of an organism
(pluripotency), they are distinguished from adult stem cells that have already committed to a
specific tissular programme (multipotency). Adult stem cells, identified in many tissues, are
capable under certain conditions of differentiating and aiding the formation and/or
regeneration of the tissues in which they occur. Currently, most studies on these cells are
carried out in vitro with the aim of ending up with cellular therapy applications. However, it
seems that the behaviour of these cells in vivo is considerably influenced by their biological
context. It is thus essential to acquire a better understanding of the physiology of stem cells in
their natural context.

Objective
The status of somatic and embryonic stem cells and understanding their regulation or
reprogramming mechanism is a central problem of fundamental and evolutionary biology.
Nevertheless, although these stem cells and the factors regulating their destiny are increasingly
studied and thus better understood in mammals, very little research has so far been devoted to
their potential roles in other vertebrate species such as amphibians.
This thesis aims to evaluate the conservation of the mechanisms that control stem cells
in vertebrates, in particular neural stem cells, by comparing two models: mouse (mammal) and
xenopus (amphibian). The role of the three key genes implicated in these mechanisms (Sox2,
Oct3/4 and Nanog) will be analyzed in vivo, by drawing on the use of somatic and germinal
transgenesis techniques in xenopus. More precisely, we wish to answer the following questions:
1- Are Oct3/4 and Sox2 genes expressed in neural stem cells in xenopus?
2- Is there conservation of control mechanisms in the determination of the stem cell status,
analogous with the situation in mammals?
3- Do these genes play a preponderant role in the maintenance of stem cells in xenopus, in
particular in the case of neural stem cells?
These comparisons will enable us to advance hypotheses as to the evolution of these
regulations in vertebrates.

Preliminary findings
We recently showed that thyroid hormones (TH) and their alpha receptor (TR?) are
necessary for the proliferation of neural stem cells in the adult mouse (Lemkine et al., 2005,
FASEB J. 19, 863-865) and that TH can regulate the expression of the genes that are
fundamental for the physiology of stem cells, namely CyclinD1, c-Myc and Sox2 (Hassani et
al., 2007, Nucleic Acids Res. 35, e65; Hassani et al., in preparation). We also showed that the
desiodases needed for the activation and inactivation of TH, as well as the TH receptors, are
present in the neurogenic zone of the xenopus tadpole (Morvan-Dubois et al., 2006,
Endocrinology 147, 4941-4949; Havis et al., 2006, EMBO J. 25, 4943-4951), the same zone
in which Sox2 is expressed. Thus our working hypothesis is that the Sox2 gene is one of the
first relays in the regulation cascades activated by TH in neurogenesis in xenopus.

Choice of model for the study
This amphibian model is increasingly appropriate for studies involving genomics,
transcriptional regulations and cellular imaging, thanks to the emergence of new tools
(Amaya, 2005, Genome Res. 15, 1683-1691). Somatic and germinal transgenic approaches
coupled with the use of fluorescent proteins opens the way to studying these transcriptional
regulations through imagining in vivo. This makes it an invaluable model for themes requiring
an integrated biological approach.

Methodology
1- The regulatory factors governing expression in stem cells and the response of the genes
Oct3/4 and Sox2 to TH will be identified and cloned in xenopus, for comparison with known
promoter sequences in the mouse.
2- These xenopus promoters placed in front of fluorescent reporter genes will be expressed by
germinal transgenesis in xenopus (Kroll and Amaya, 1996, Development 122, 3173-3183), a
technique fully mastered in the laboratory (Coen et al., 2001, 2007). The expression profile
will be followed by observing fluorescence during development (precocious embryogenesis,
tadpoles, adults).
3- In xenopus, orthologues of the Oct3/4 et Sox2 genes are present (Katoh and Katoh, 2005,
Oncol Rep 14, 797-800; Morrison and Brickman, 2006, Development 133, 2011-2022). An
orthologue of the Nanog gene has recently been described in the chicken (Lavial et al., 2007,
Development 134, 3549-3563). We envisage conducting a search in silico for the Nanog gene
as well as its promoter in existing sequencing data banks for Xenopus laevis and tropicalis,
with the aim finding out if this gene is present in these species.
4- We shall overexpress the Sox2 and Oct3/4 factors (and potentially Nanog) by means of
somatic and germinal transgenesis in xenopus, using non-viral replication plasmids intended
to allow non-integrated, long-term expression of these factors. We shall study the effect on
their overexpression – either alone or in combination –, on the state of cellular differentiation
during precocious embryogenesis in xenopus, and on the status of neural stem cells.

Candidates with a background in animal biology, genomics and molecular biology are
welcome to apply. The position is available from September 2008 for 3 years.
Applications (CV + cover letter) should be sent before 30 June 2008 to Dr Laurent Coen
(coen@mnhn.fr) who will supervise the student and from whom further details can be
obtained.

Scholarship Tags: France, History, Museum, National, Natural, Paris, PhD, Position