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Wnt related signaling centers during patterning of the mouse embryo

maandag, 19 december, 2005 - 15:00
Campus: Brussels Humanities, Sciences & Engineering campus
Faculteit: Science and Bio-engineering Sciences
Caroline Kemp

The Wnt family of secreted glycoproteins is composed of nineteen members, which signal
via their receptors, known as Frizzleds (Fzds), of which there are ten. Eleven potential
Wnt antagonists can block Wnt signaling by either directly binding to Wnts, preventing
interaction with their Fzd receptors, or by binding to the Wnt coreceptors. After binding to
Fzd, intracellular Wnt signaling occurs through at least three different pathways. The best
known pathway, the canonical pathway, leads to the stabilization of ?-Catenin, which can
then activate target gene transcription in conjunction with Tcf factors. Activation of the
two noncanonical pathways, the planar cell polarity pathway and Wnt/Ca+2 pathway, can
lead to changes in cell polarity, adhesion or motility.

Gastrulation is the process by which mesoderm is formed, at which time the anteriorposterior
axis of the embryo becomes morphologically distinct. During mouse
development, Wnt signaling is necessary for proper gastrulation. Expression pattern
analyses and gene inactivation have also revealed that Wnt genes are active during
numerous other developmental processes, and that a level of functional redundancy exists
between these nineteen genes. In the adult, Wnt signaling continues to play a role in cell
proliferation, differentiation and movement.

The first part of this thesis describes the first systematic expression level analysis of all the
Wnts, Frizzleds and potential antagonists during early mouse development. Expression
pattern studies were also pursued to better understand the molecular patterning of the
embryo. Wnt2b and Wnt7b are shown to be expressed in the future posterior and
extraembryonic regions, respectively, of the pregastrula embryo. Expression of Wnt
antagonists such as Dkk1, Sfrp1 and Sfrp5, on the other hand, is localized to the anterior
region of the pregastrula, in the anterior visceral endocerm. In addition three Frizzled
receptors are shown to be expressed in the pregastrula embryo: Fzd5 in the anterior
visceral endoderm, Fzd7 in the embryonic ectoderm and Fzd10 in the future posterior
embryonic ectoderm. A model to hypothesize anterior-posterior patterning during
gastrulation is proposed to involve a balance of Wnt signals in the posterior, opposed by
antagonistic signals from the anterior. The Wnt morphogen gradient thus established
would be intracellularly transduced by the Fzd receptors to determine the more anterior or
posterior fates of the ectoderm.

In this study, for the first time, the expression patterns of Wnt1, Wnt3a, Wnt6, Wnt7b,
Wnt9a and Wnt10b and two Wnt antagonists, Sfrp1 and Dkk1, in the blastocyst stage
embryo are described. The restricted expression of Wnt9a in the cells surrounding the
blastocoele cavity is indicative of a role in patterning this preimplantation stage embryo.
In the second part of this thesis, a conditional expression system, the GeneSwitch System,
is evaluated in cell culture and in the mouse. In this system, a driver, Switch, activates
the responding gene of interest when an inducing molecule is present. The intention was
to develop this system in transgenic mice in order to induce the ectopic expression of
Wnt1 or Frzb (a Wnt antagonist) to further reveal the functions of the Wnt pathway during
gastrulation. As a first step, four founding transgenic lines for the Switch driver were
generated. Characterization of embryos and adult mice led us to the conclusion that the
Switch driver was poorly, if at all, expressed and not active in the four different transgenic
Switch lines.

In a second step, transgenic mice were generated for the inducible responding genes of
interest, Wnt1 and Frzb. Due to misexpression of the Wnt1 transgene in line 24, a
spontaneous phenotype is observed and briefly described. With a previously characterized
driver mouse line, Wnt1-Gal4, mice transgenic for the inducible Wnt1 construct were
evaluated. Initial observations indicate an increased size of the fore- and midbrain when
Wnt1 is overexpressed. Proving that the transgenic mice can respond to the Wnt1-Gal4
driver, the doors are now open for further investigating the effects of overexpressing
Wnt1or Frzb in the Wnt1 expression domain.