M1 BIOLOGIE DES EUCARYOTES
Première Session 2007
DECISIONS CELLULAIRES
Durée de l’épreuve: 2h
Il s’agit d’un petit travail de synthèse
INTRODUCTION
The tight control of cell proliferation and cell death is essential to normal tissue development,
and the loss of this control is a hallmark of cancers. Coordination between cell proliferation and
cell death is essential to maintain homeostasis in multicellular organisms. In Drosophila, these
two processes are coordinately regulated during development by the Hippo signaling pathway.
The Hippo tumor-suppressor pathway has emerged as a key signaling pathway that controls
tissue size in Drosophila. Hippo signaling restricts tissue size by promoting apoptosis and cellcycle arrest, and animals carrying clones of cells mutant for hippo develop severely overgrown
adult structures.
This pathway is so far composed of two membrane-associated Band 4.1 proteins, Expanded (Ex)
and Merlin (Mer), two serine/threonine kinases, Hippo (Hpo) and Warts (Wts), the scaffold
proteins Salvador (Sav) which binds to Hpo and Mats, a regulator of Wts activity.
Ex, Mer, Hpo, Sav, Wts, and Mats are negative regulators of growth, and loss-of-function
mutations in these genes result in dramatically overgrown tissues containing an excess number of
cells.
It has been proposed that Ex and Mer act upstream of Hpo, which in turn phosphorylates
and activates Wts. Wts negatively regulates the transcription of cell-cycle and cell-death
regulators such as cycE and diap1 by a mechanism described in the Article 1.
All of the components of the Hippo pathway are conserved in humans and several have been
implicated in human cancer, in particular Merlin, which in humans is encoded by the
Neurofibromatosis type-2 tumor suppressor gene.
ARTICLE 1
Huang J et al. Cell. 2005;122(3):421-34.
Identification of Yorkie (Yki) as a Wts Binding Protein in a two-hybrid screen for Wts binding
proteins.
-Yki is most closely related to the human yes-associated protein (YAP). YAP is a potential
oncogene in mammals. YAP has been implicated as a transcriptional coactivator, a class of
transcriptional regulators that do not bind to DNA themselves but associate with DNA binding
transcription factors and supply or stimulate transcriptional activation of the cognate
transcription factors.
-Activation of Yki leads to massive tissue overgrowth that resembles the loss-of-function
phenotype of hpo, sav, or wts whereas cells mutant for Yki grow poorly.
-Activated Yki drives growth by stimulating cell proliferation and inhibiting apoptosis.
-When Hpo signaling is reduced, for example by mutations in hpo, Yki is hypophosphorylated
and active, driving the expression of target genes that promote cell proliferation and suppress
apoptosis, resulting in tissue overgrowth.
-Yorkie is known to activate Cyclin E (CycE) and the apoptosis inhibitor DIAP1.
1
However, overexpression of these two targets is not sufficient to cause tissue overgrowth.
How Yorkie regulates growth, and thus the identities of downstream target genes that mediate
these effects of Hippo signaling, are largely unknown.
ARTICLES 2 et 3
Nolo et al. Curr Biol. 2006;16(19):1895-904.
B J. Thompson and S M. Cohen, Cell 2006; 126(4): 767–774,
The bantam locus of Drosophila was identified in a gain-of-function screen for genes that affect
tissue growth (Hipfner et al., 2002). bantam encodes a microRNA (miRNA)
Experience 1
GMR-Gal4:
UAS-hid
GMR-Gal4:
UAS-hid,UAS-bantam
GMR-Gal4:
UAS-hid;bantam-/+
SEM images of eyes of adult flies. The genotypes of the animals are indicated above the panels.
GMR drives overexpression of UAS transgenes in the eye.
Experience 2
Quantification of cell numbers in pupal retinae. Wild-type retinae or retinae with bantam
mutant clones had 1.67 cells in such regions, whereas retinae overexpressing Yki had 5.9 +/- 1.0
interommatidial cells and regions that overexpressed Yki but were mutant for bantam had 3.1+/0.5 cells. Error bars represent standard deviations.
2
Experience 3
B
A
wt
GMR:Yki
wt
GMR:Yki
(A) Northern blot probed with radiolabeled anti-bantam oligonucleotide. The same blot is probed
with an anti-tRNA radiolabeled oligonucleotide as a loading control. (B) Quantitative-PCR
analysis of bantam microRNA levels in wild-type retinae or retinae overexpressing Yki.
Experience 4
Wt
GMR-Gal4:
UAS-bantam
SEM images of eyes of adult flies and third-instar eye discs stained with antibody against
activated Caspase 3 (Casp). The genotypes of the animals are indicated above the panels.
GMR-Gal4 drives overexpression of UAS transgenes in the eye.
3
Experience 5
(A–C) show SEM images of eyes of adult flies and (A’– C’) show higher-magnification images of
the panels above them. (A’’_ C’’) show third-instar eye discs stained with antibody against Drice
to mark apoptotic cells. The genotypes of the animals are indicated above the panels. (A-A’’:
GMR-Gal4, B-B’’:GMR-Gal4:UAS- hpo, C-C’’:GMR-Gal4:UAS- hpo,UAS- ban
- Vous rappellerez le principe des expériences permettant la sur-expression ou
l’expression ectopique d’un gène chez la drosophile.
- Vous donnerez une conclusion de l’ensemble des résultats présentés ci-dessus en
insistant sur le rôle de bantam.
- Vous proposerez un modèle, accompagné d’une légende, de la voie de contrôle de la
taille des tissus par Hippo en utilisant les signes conventionnels :
signifie active et
signifie réprime
Un rappel des principaux acteurs moléculaires impliqués est donné sur la page 5
4
APOPTOSIS
CELL GROWTH
PROLIFERATION
Bantam ?
5