Integration of differentiation signals during indirect flight muscle formation by a novel enhancer of Drosophila vestigial gene

AbstractThe gene vestigial (vg) plays a key role in indirect flight muscle (IFM) development. We show here that vg is controlled by the Notch anti-myogenic signaling pathway in myoblasts and is regulated by a novel 822 bp enhancer during IFM differentiation. Interestingly, this muscle enhancer is activated in developing fibers and in a small number of myoblasts before the fusion of myoblasts with the developing muscle fibers. Moreover, we show that this enhancer is activated by Drosophila Myocyte enhancing factor 2 (MEF2), Scalloped (SD) and VG but repressed by Twist, demonstrating a sensitivity to differentiation in vivo. In vitro experiments reveal that SD can directly bind this enhancer and MEF2 can physically interact with both SD and TWI. Cumulatively, our data reveal the interplay between different myogenic factors responsible for the expression of an enhancer activated during muscle differentiation

Etude des interactions entre MEF2 et la voie de signalisation Notch au cours de la myogenèse adulte chez Drosophila melanogaster

La myogenèse des muscles indirects du vol (IFM) chez Drosophila melanogaster suit un schéma développemental précis. Au cours de l'embryogenèse, un groupe de cellules, les Précurseurs adultes Musculaires (AMP) se spécifient. Ces cellules deviennent des myoblastes qui prolifèrent au cours des stades larvaires et donneront par la suite les IFM adultes. Nos travaux se sont concentrés sur les interactions requises lors de la transition de myoblastes qui prolifèrent au statut de myoblaste différencié prêt à fusionner à la fibre musculaire. Il a été montré que les myoblastes qui prolifèrent ont une voie Notch active et que cette voie est inhibée dans les fibres en cours de différenciation. De plus, il a également été montré que les facteurs de transcription Myocyte Enhancer Factor 2 (MEF2), Vestigial (VG) et Scalloped (SD) sont nécessaires pour le développement des IFM et que VG est requis pour la répression de la voie Notch dans les fibres. Cette étude porte sur les interactions entre la voie Notch et MEF2 et les mécanismes mis en jeu pour réprimer la voie au cours de la différenciation. Nous avons montré que MEF2 peut réprimer la voie Notch dans des contexgtes non-musculaires. En utilisant un crible récent pour identifier des cibles potentielles de MEF2, nous avons cherché ceux qui sont également des cibles de SD. Parmi les résultats, deux cibles ont présenté un intérêt particulier, Delta et neuralized, deux composants de la voie de signalisation Notch. Nos résultats montrent dans un contexte ex vivo que les séquences enhancers de DI et neur sont régulées par MEF2/SD et MEF2/NOTCH respectivement. In vivo ces enhancers sont actifs dans les fibres des IFM en cours de différenciation pour DI et au cours de la différentiation tardive pour neur. Au cours de ma thèse, j'ai pu étudier l'effet de MEF2 sur la régulation de ces cibles pour comprendre leur rôle au cours de la différentiation des IFM.Myogenesis of indirect flight muscles (IFM) in Drosophila melanogaster follows a well defined cellular developmental scheme. During embryogenesis, a subset of cells, the Adult Muscle Precursors (AMPs), are specified. These cells will become proliferating myoblasts during the larval stages which will then give rise to the adult IFM. Our work focused on the interactions required during the transition between proliferating myoblasts to differentiated myoblasts ready to fuse to the muscle fiber. It has been previously shown that proliferating myoblasts express the Notch pathway, and that this pathway is inhibited in developing muscle fibers. On the other hand, it has also been shown that the Myocyte Enhancing Factor 2 (MEF2), Vestigial (VG) and Scalloped (SD) transcription factors are necessary for IFM development and that VG is required for Notch pathway repression in differentiating fibers. Our study focuses on the interactions between Notch and MEF2 and mechanisms by which the Notch pathway is inhibited during differentiation. Here we show that MEF2 is capable of inhibiting the Notch pathway in non myogenic cells. A previous screen for MEF2 potential targets identified Delta and Neuralized, two components of the Notch pathway. Both are expressed in developing fibers where MEF2, SD and VG are expressed. Our preliminary results show that MEF2 is required for Delta expression in developing IFMs and that this regulation is potentially dependent on an enhancer to which MEF2 and SD bind. We have identified a similar neuralized enhancer that seems to be potentially regulated by MEF2 and NICD. During my thesis I studied the effect of MEF2 on these targets in vivo and in vitro to understand the rote they play during IFM differentiation.PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Etude du rôle du gène vestigial au cours de la myogenèse adulte chez Drosophila Melanogaster

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Mef2 Interacts with the Notch Pathway during Adult Muscle Development in Drosophila melanogaster

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Repression of Notch signaling by Mef2 at the DV boundary of the wing disc.

<p>When GFP is expressed along the AP boundary of the third instar larva wing disc using the <i>ptc-Gal4</i> driver (A), Ct, detected by a specific anti-Ct antibody, is normally expressed at the DV boundary (B; overlay in C). In contrast, mis-expression of Mef2 along the AP boundary using the same driver (D; detected using an anti-Mef2 antibody) severely reduces Ct expression at the AP boundary (E, arrowhead; overlay in F).</p

Reduced activation of the <i>Dl2.6</i> enhancer <i>in vivo</i> monitored by the expression of the <i>Dl2.6-GFP</i> transgene.

<p>In 21 h APF pupae, muscle fibers are visualized with DAPI staining by the specific arrangement of their nuclei (A, D, asterisks). In a wild-type pupae, <i>Dl2.6</i> enhancer is activated in developing fibers (B, overlay in C). In pupae overexpressing an <i>RNAi-Mef2</i> transgene, <i>Dl2.6</i> enhancer activation is significantly lower than in wild-type (E, overlay in F).</p

Activation of the <i>Dl2.6</i> enhancer <i>in vivo</i> monitored by the expression of the <i>Dl2.6-GFP</i> transgene.

<p>E′–H′ are magnifications of the dotted squares in E–H. In 16 h, 24 h, 30 h and 36 h APF pupae, the 22c10 antibody labels muscles and nerves (B, F, F′, J and N respectively), <i>Dl2.6</i> enhancer is activated in developing fibers of 16 h, 24 h and 30 h APF pupae (C, G, G′, K respectively) but not in myoblasts at 16 h APF (A–D) and 24 h APF (E–H, magnification in E′–H′, arrowheads). <i>Dl2.6</i> enhancer is not activated in developing fibers of 36 h APF pupa (M-P). DAPI-GFP overlay is shown in D, H, and H′, L and P. Scale bar: 100 µm.</p

Activation of Dl by Sd/Mef2 in 28 h APF pupa SOPs.

<p>In SOPs, visualized with YFP (green) expressed under the control of the <i>neur-Gal4</i> driver (arrowheads in A–D), Dl (red) is detectable at the same levels as in non-SOP cells (asterisks in A–D) of the notum. When YFP, <i>sd</i> and <i>Mef2</i> are overexpressed in SOPs using the same driver (E–H), Dl is up-regulated in SOPs (arrows in G) relative to non-SOP cells (asterisks in G) of the notum. DAPI labeling is shown in gray (A,E). Overlays are shown in D and H.</p

Vesicle Formation and Follicular Root Sheath Separation in Mice Homozygous for Deleterious Alleles at the Balding (bal) Locus

The balding (bal) mutation of the mouse is an auto-somal recessive mutation that causes alopecia and immunologic anomalies. A new allele was identified by allelism testing after using an interspecific back-cross to localize the mutation to the centromeric end of mouse chromosome 18. We investigated the skin and hair histologic lesions of two alleles (balJ and balPas) at this locus and analyzed the expression of several keratinocyte markers and the production of autoantibodies by immunofluorescence on frozen skin sections. The lesions observed included separation of the inner and outer root sheath in anagen follicles resulting in the hair fiber being very easily plucked from the follicle. Vesicles on the ventral tongue, mucocutaneous junction of the eyelid, foot pads, and rarely in skin were also evident. Separation occurred between the basal and suprabasilar cells forming an empty cleft, resembling that observed in human pemphigus vulgaris. Immunofluorescence studies did not reveal the presence of tissue-bound or circulating autoantibodies. Expression of keratinocyte markers in hair follicles was normal. Keratin 6-positive cells were found on either side of the follicular separation suggesting a molecular defect in adhesion molecules between the inner layer of the outer root sheath cells to layers on either sides. This hypothesis has been confirmed by another group who demonstrated that the balJ mutation is due to the insertion of a thymidine in the desmoglein 3 gene, resulting in a premature stop codon