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Division cellulaire asymétrique
B I would like to first thank Elly and Brigitte for inviting me there for this meeting. I have to say that it is unusal for me, as a fly person, to come to a meeting where Drosophila is a somewhat marginal model system compared to hydra, planaria, sea cucumber or axolotl are more mainstream... Anyway, you will get to hear a little bit of Drosophila in my talk which will be on asymmetric cell divisions in Drosophila So, let me first simply start by reminding you that a division is said to be asymmetric when a mother cell divides to generate two daughter cells with distinct developmental potential.
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And I will address this question using Drosophila as a model organsim
And I will address this question using Drosophila as a model organsim. In Drosophila, each of the sensory organs that cover the dorsal thorax is composed of only four cells. These four cells, together with one associated glial cell, are produced by a single sensory organ precursor cell, called pI, via a stereotyped series of asymmetric cell divisions. These asymmetric divisions occur during metamorphosis and take place within a developping single-layered epithelium that is otherwise composed of epidermal cells that divide in a symmetrical manner. In this lineage, the division of the pI cell produces two secondary precursor cells, called pIIa and pIIb. It has been established more than 10 years ago by V Hartenstein that it is the differential activation of the Notch receptor which is responsible for the difference in fate that is seen between the pIIa and pIIb cell. Notch signaling is activated in the pIIa cell and is blocked in the pIIb cell. This difference in signaling activitys is due to the unequal segregation of a Notch inhibitor called Numb.
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Sheath Socket Shaft Neuron
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pI Glial pIIb pI pIIIb Neurone métaphase Sheath Shaft pIIa pIIb Socket
télophase A101 / a-tub / Numb
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Répartition inégale des déterminants de l’identité cellulaire
1. Polarisation 2. Distribution asymétrique des déterminants 3. Position du site de clivage (répartition inégale) One of the major finding of the last ten years was that such a difference in cell fate can result from the unequal segregation of specific molecules that act as cell fate determinant. These cell fate determinants are synthesized in the mother cell, localized at one pole of the dividing cell, and specifically segregated into one of the two daughter cells . So, what I’d like to do now is telling you about the molecular mechanisms that underlie the unequal segregation of a cell fate determinant. A B
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A A A A 1. Perte de polarité 2. Défaut dans la position
du site de clivage A A A A
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Knoblich, 2002
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pI Glial pIIb pI pIIIb Neurone métaphase Sheath Shaft pIIa pIIb Socket
télophase A101 / a-tub / Numb
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Glial pIIb pI pIIIb Neurone Sheath Shaft wild-type Socket pIIa pIIa pIIb Socket Neurone/Glial pI pI Neurone/Glial Socket Socket Neurone/Glial Socket Neurone/Glial numb Notch-Act pIIa pIIb Notch
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wild-type Numb numb Notch Notch (ts) Notch (ts) + numb Jan, 1996
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a-adaptin
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Numb a-adaptin pIIa pIIb pI pI pIIa pIIb a-adaptin Hs-numb
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a-adaptin Notch pIIa pIIb pI pI pIIa pIIb a-adaptin Notch (ts) - 29°C
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• • • • • • • pIIb pIIa Numb Notch Numb a-adaptin Notch pIIb pIIa
In the last part of my talk, I would like to switch gear and to talk about how Notch signaling is regulated following asymmetric cell division. As mentionned at the beginning of my talk, Numb antagonizes Notch in the pIIb cell. Numb is an endocytic adaptor molecule that binds to Notch as well as to the AP2 complex . These molecular interactions have suggested a model in which Numb down-regulates Notch signaling in the pIIb cell by clearing the surface of the pIIb cell from Notch receptors via endocytosis. As a consequence, the pIIb cell has a reduced level of Notch at the surface, and thereby receives a weaker inhibitory signal compared to its sister cell. This model makes no specific prediction about the localization of the Notch ligands, which are not expected to contribute to establishing asymmetry. Notch • • • • • • • pIIb pIIa
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Pon-GFP
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pI epidermal pI apical anterior posterior basal
Importantly, these asymmetric divisions take place within a single layered epithelium at the surface of the developping pupa. Moreover, each mitotic cell displays a stereotyped orientation. For instance, the pI cells divide within the plane of the epithelium and along the antero-posterior axis of the fly. This can be viewed on this movie made by Y Bellaiche in which the epithelium is viewed from above and in which three pI cells divide along the antero-posterior axis. Two GFP markers are used here: One GFP marker that colocalises with Numb, named Pon-GFP And one Histone-YFP marker. Both markers are specifically expressed in pI cells using the UAS-Gal4 system. So, in the first part of my talk, I will discuss the mechanisms by which the pI cells, which are epithelial cells with distinct a apical-basal polarity, acquire a antero-posterior polarity. Pon-GFP Histone2B-YFP
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Planar polarity in Drosophila
frizzled flamingo strabismus dishevelled prickle The pI cells are not the only cells of this tissue to be oriented along the antero-posterior axis. Actually, each epidermal cell in this tissue produces a single epidermal cell at its posterior vertex. Much is known about the genetics of planar polarity establishment in epidermal cells. One interesting property of the planar polarity proteins is that they localize asymmetrically within the plane of the epithelium. For instance, in the wing epidermis, Fz has been shown by D Strutt to localize at the distal vertex at the time when each epidermal cell secretes one epidermal hair. Fz colocalizes with Dsh at this pole, while, in contrast, Pk localizes opposite to Fz and Dsh. So, we next investigated whether these proteins also localize asymmetrically along the antero-posterior axis in the pI cell. Pk Stbm Fz Dsh
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Polar distribution of planar polarity proteins
Stbm H2B-YFP GFP-Stbm H2B-YFP Fz-GFP pI pI Our preliminary data suggest that Fz localizes to the posterior cortex, while Pk localizes to the opposite pole. We also found that Stbm, which behaves genetically like Pk, localizes like Pk to the anterior cortex, opposite to Fz. Fz Pk, Stbm
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Planar polarity genes orient pI cells
a-tubulin Numb random The first question we asked was whether these planar polarity genes indeed regulate the orientation of the pI cell division . We found that the loss of any one of these genes leads to a complete randomization of the orientation of the pI division. The Numb crescentno longer localizes at the anterior cortex but can be found at any position around the cortex andlikewise the mitotic spindle is randomly oriented. However, we observed that, in most cases, the mitotic spindle correctly lines up with Numb, such that the division of the pI cell is still asymmetric. We conclude from these data that planar polarity genes regulate the orientation of the pI division but are not required to create asymmetry per se. frizzled flamingo strabismus dishevelled prickle wild-type
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Stbm, Pk Fz, Dsh ? anterior posterior orientation (prior to mitosis)
So, based on these data, we propose that planar polarity molecules define two cortical domains prior to the division of the pI cell. How these two domains are established along the body axis is not well understood, but studies in epidermal cells suggest a mutual antagonism between Dsh and Fz on, one hand and Stbm and Pk on the other hand. Also, as mentionned earlier, the activity of these genes is not responsible for creating an asymmetry, it is only required to orient this asymmetry relative to the body axis. So, there must exist genes acting downstream of the planar polarity genes which are required to create an asymmetry in the pI cell Numb
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Les neuroblastes de l’embryon de Drosophile
VNE épiderme Temps neuroblaste GCM neurones
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Le déterminant Prospero
sauvage prospero Neuroblaste spécifique : dpn,ase GMC spécifique : eve,ftz
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Prospero Miranda Nbs GMC
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The mitotic spindle rotates by 0-90°
- Centrosomes are randomly positioned at separation - Centrosomes migrate by the shortest path to orient the spindle No evidence for centrosome priming
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Identification du gène inscuteable
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Inscuteable se localise au pôle apical
interphase prophase métaphase anaphase
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Inscuteable est requis pour localiser Miranda
Miranda / ADN
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Baz DmPKC DmPar6 Baz DmPKC DmPar6 Inscuteable Inscuteable Pins-GaiGDP
-Localisation de Miranda -Rotation du fuseau Inscuteable Inscuteable Pins-GaiGDP Gai/Gß Cibles?
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Drosophila neuroblasts
Chia, Doe, Jan, Knoblich, Knust ... apical cue Bazooka (PAR-3) DmPAR-6 DaPKC Mitotic spindle rotation Basal accumulation of Numb and Prospero Inscuteable Pins Gai-GDP + Gbg
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Planar polarity in Drosophila
frizzled flamingo strabismus dishevelled prickle The pI cells are not the only cells of this tissue to be oriented along the antero-posterior axis. Actually, each epidermal cell in this tissue produces a single epidermal cell at its posterior vertex. Much is known about the genetics of planar polarity establishment in epidermal cells. One interesting property of the planar polarity proteins is that they localize asymmetrically within the plane of the epithelium. For instance, in the wing epidermis, Fz has been shown by D Strutt to localize at the distal vertex at the time when each epidermal cell secretes one epidermal hair. Fz colocalizes with Dsh at this pole, while, in contrast, Pk localizes opposite to Fz and Dsh. So, we next investigated whether these proteins also localize asymmetrically along the antero-posterior axis in the pI cell. Pk Stbm Fz Dsh
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Planar polarity genes orient pI cells
a-tubulin Numb random The first question we asked was whether these planar polarity genes indeed regulate the orientation of the pI cell division . We found that the loss of any one of these genes leads to a complete randomization of the orientation of the pI division. The Numb crescentno longer localizes at the anterior cortex but can be found at any position around the cortex andlikewise the mitotic spindle is randomly oriented. However, we observed that, in most cases, the mitotic spindle correctly lines up with Numb, such that the division of the pI cell is still asymmetric. We conclude from these data that planar polarity genes regulate the orientation of the pI division but are not required to create asymmetry per se. frizzled flamingo strabismus dishevelled prickle wild-type
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Baz PAR-6 aPKC Pins Gai-GDP Gbg effectors aPKC targets Numb
bazooka and pins are required to localize Numb ... wild-type baz pins pins Numb Numb Numb
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Establishment of polarity in pI cells and in neuroblasts
Planar polarity signal Apical signal Bazooka DaPKC DmPAR-6 (posterior) Bazooka DaPKC DmPAR-6 (apical) Dlg-Pins (anterior) Inscuteable Pins pI Neuroblast Numb localises opposite to Baz
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Inscuteable reverts pI cell polarity
WT WT Dlg-Pins Numb Insc Numb Pins UAS-Insc UAS-Insc Inscuteable Bazooka DaPKC DmPAR-6 (anterior) Insc Numb Pins UAS-Insc UAS-Insc Numb (posterior) Baz Dlg
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Numb (drosophile-mammifères: 603 aa)
PTB Neurorétine
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