Rôle des facteurs à domaine BTB/POZ du groupe Tramtrack dans le recrutement des niches à cellules souches lors de la morphogenèse de l ovaire chez D. melanogaster

Chez la drosophile, la production d ovocytes dépend de la présence et du nombre de niches à cellules souches germinales (GSC) ainsi que de la division des GSC. Chez l insecte, chaque niche est intégrée à une sous-unité fonctionnelle, l ovariole. Chez D. melanogaster, la morphogenèse d une ovariole débute, chez la larve, par la formation d une pile de 8 à 10 cellules triées par sorting out, le filament terminal (TF). Le TF est le centre organisateur de la niche durant le stade larvaire et la jeune pupe. J ai cherché à déterminer les bases génétiques de deux processus développementaux. 1) La limitation du nombre de niches, 2) la formation de la niche. J ai montré que pipsqueak, Trithorax-like, batman et le locus bric-àbrac (bab), codant des facteurs à domaine BTB/POZ du groupe Tramtrack sont impliqués dans la limitation du nombre d ovarioles. Au moins deux processus sont affectés lorsque l expression de ces gènes est diminuée. Le sorting out des cellules de TF (TFC) est régulé positivement par le locus bab, alors que la prolifération et/ou la spécification des TFC est régulée négativement par psq. Nos résultats montrent donc que le nombre de niches formées est sous le contrôle d un réseau de facteurs BTB/POZ. D autre part, nous montrons que la formation de la niche est sous le contrôle du locus bab. Dans les TFC les deux paralogues bab1 et bab2 ont un rôle redondant dans la régulation de l expression d engrailed, permettant l aplatissement et l empilement des TFC. Dans les autres cellules somatiques, bab2 réprime bab1. Nous émettons l hypothèse que cette répression bloque un effet potentiel de bab1 sur l identité des cellules somatiques ou sur la migration des swarm cells.The potential to produce new cells during adult life depends on the presence and the number of stem cell niches and the division of stem cells. In the insect ovary, each germline stem cell (GSC) niche is embedded in a functional unit called an ovariole. In D. melanogaster, morphogenesis of ovarioles starts in larvae with the formation of terminal filaments (TF), each made of 8 10 cells that pile up and sort in stacks. TF constitute organizers of GSC niches during larval and early pupal development. Here I address the genetic bases of two developmental processes 1) The limitation of GSC niche recruitment 2) the formation of the niche. First we show that pipsqueak, Trithorax-like, batman and the bric-à-brac (bab) locus, all encoding BTB/POZ factors of the Tramtrack Group, are involved in limiting the number of ovarioles. At least two processes are perturbed by reducing the function of these genes. We found that when the bab dose is reduced, sorting of TF cells (TFC) was affected. In contrast, when the psq dose is reduced, more proliferation and/or specification of TFC occurs. Our results indicate that two parallel genetic pathways under the control of a network of BTB factors are combined in order to control the number of GSC niches. Second we show that morphogenesis of ovary is under control of bab locus in two different manners. In TFC the two paralogues bab1 and bab2 have a redundant function in activating engrailed expression, which is necessary to flatten and pile up the TFC. In other somatic cells bab2, represses bab1 expression. We hypothesize that this repression prevents a potential effect of bab1 expression on somatic cell identity or on swarm cells migration.VERSAILLES-BU Sciences et IUT (786462101) / SudocSudocFranceF

Amélioration de la consommation à froid d'un véhicule par optimisation des flux énergétiques

Ce travail a pour objectif de décrire le véhicule automobile d'un point de vue énergétique dans le but de proposer une solution technologique innovante permettant d'améliorer la consommation à froid d'un véhicule en optimisant la répartition de ses flux d'énergie. L'étude de la répartition de l'énergie dans le véhicule lors d'un fonctionnement à froid met en évidence les principaux facteurs de surconsommation. Cette analyse montre la forte influence de la température d'huile moteur. Sur la base de cette constatation un banc d'essais spécifique démontre le fort potentiel d'une réduction de la consommation à froid par préchauffage de ce fluide. L'état de l'art des systèmes d'optimisation des flux énergétiques associé à une modélisation du véhicule permet de cibler une solution technologique permettant la mise en place du concept de préchauffage. Le dimensionnement de cette solution aboutit à l'implantation d'un prototype sur véhicule et à la validation des gains de consommation à froid.With the increasing fuel prices and the increasing greenhouse gases, fuel consumption is become an essential criterion in car design. The purpose of this work is to describe a vehicle from an energetic point of view and to reduce the fuel consumption during cold start. The first part of the document analyzes the vehicle life situations, describes the vehicle energetic fluxes and shows the main influence of oil temperature during cold start on fuel consumption. In the second part this main influence is shown with a serie of tests on a test bench. In the third part a model is build which can simulate the engine temperature rise and its fuel consumption during this phase. In a fourth part this model is used to evaluate the efficacity of different technologic solutions. The most promising one is further studied and dimensioned to be tested on vehicle.VALENCIENNES-BU Sciences Lettres (596062101) / SudocSudocFranceF

The Bric-à-Brac BTB/POZ transcription factors are necessary in niche cells for germline stem cells establishment and homeostasis through control of BMP/DPP signaling in the Drosophila melanogaster ovary

International audienceMany studies have focused on the mechanisms of stem cell maintenance via their interaction with a particular niche or microenvironment in adult tissues, but how formation of a functional niche is initiated, including how stem cells within a niche are established, is less well understood. Adult Drosophila melanogaster ovary Germline Stem Cell (GSC) niches are comprised of somatic cells forming a stack called a Terminal Filament (TF) and associated Cap and Escort Cells (CCs and ECs, respectively), which are in direct contact with GSCs. In the adult ovary, the transcription factor Engrailed is specifically expressed in niche cells where it directly controls expression of the decapentaplegic (dpp) gene encoding a member of the Bone Morphogenetic Protein (BMP) family of secreted signaling molecules, which are key factors for GSC maintenance. In larval ovaries, in response to BMP signaling from newly formed niches, adjacent primordial germ cells become GSCs. The bric-à-brac paralogs (bab1 and bab2) encode BTB/POZ domain-containing transcription factors that are expressed in developing niches of larval ovaries. We show here that their functions are necessary specifically within precursor cells for TF formation during these stages. We also identify a new function for Bab1 and Bab2 within developing niches for GSC establishment in the larval ovary and for robust GSC maintenance in the adult. Moreover, we show that the presence of Bab proteins in niche cells is necessary for activation of transgenes reporting dpp expression as of larval stages in otherwise correctly specified Cap Cells, independently of Engrailed and its paralog Invected (En/Inv). Moreover, strong reduction of engrailed/invected expression during larval stages does not impair TF formation and only partially reduces GSC numbers. In the adult ovary, Bab proteins are also required for dpp reporter expression in CCs. Finally, when bab2 was overexpressed at this stage in somatic cells outside of the niche, there were no detectable levels of ectopic En/Inv, but ectopic expression of a dpp transgene was found in these cells and BMP signaling activation was induced in adjacent germ cells, which produced GSC-like tumors. Together, these results indicate that Bab transcription factors are positive regulators of BMP signaling in niche cells for establishment and homeostasis of GSCs in the Drosophila ovary

Genetic Basis for Developmental Homeostasis of Germline Stem Cell Niche Number: A Network of Tramtrack-Group Nuclear BTB Factors

International audienceThe potential to produce new cells during adult life depends on the number of stem cell niches and the capacity of stem cells to divide, and is therefore under the control of programs ensuring developmental homeostasis. However, it remains generally unknown how the number of stem cell niches is controlled. In the insect ovary, each germline stem cell (GSC) niche is embedded in a functional unit called an ovariole. The number of ovarioles, and thus the number of GSC niches, varies widely among species. In Drosophila, morphogenesis of ovarioles starts in larvae with the formation of terminal filaments (TFs), each made of 8-10 cells that pile up and sort in stacks. TFs constitute organizers of individual germline stem cell niches during larval and early pupal development. In the Drosophila melanogaster subgroup, the number of ovarioles varies interspecifically from 8 to 20. Here we show that pipsqueak, Trithorax-like, batman and the bric-à-brac (bab) locus, all encoding nuclear BTB/POZ factors of the Tramtrack Group, are involved in limiting the number of ovarioles in D. melanogaster. At least two different processes are differentially perturbed by reducing the function of these genes. We found that when the bab dose is reduced, sorting of TF cells into TFs was affected such that each TF contains fewer cells and more TFs are formed. In contrast, psq mutants exhibited a greater number of TF cells per ovary, with a normal number of cells per TF, thereby leading to formation of more TFs per ovary than in the wild type. Our results indicate that two parallel genetic pathways under the control of a network of nuclear BTB factors are combined in order to negatively control the number of germline stem cell niches

Ovariole number in <i>bab</i> mutant heterozygous combinations.

<p>(A) Distribution of the number of ovarioles per ovary in different <i>bab</i> heterozygotes. For statistics (comparison between <i>bab</i> heterozygotes and Canton-S (+)) see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049958#pone.0049958.s005" target="_blank">Table S3A</a>. (B) Distribution of the number of ovarioles per ovary in genetic interaction assays between the <i>bab</i> locus and <i>psq</i>, <i>Trl</i> and <i>batman</i>. For statistics (comparison between different allelic combinations) see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049958#pone.0049958.s005" target="_blank">Table S3B</a>. The mean number of ovarioles per ovary is given on the right. All values are expressed as the mean +/− sample standard deviation. Color table as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049958#pone-0049958-g001" target="_blank">Figure 1</a>.</p

Quantitative analysis of cell number and size in <i>psq</i> and <i>bab</i> heterozygotes.

<p>(A) TFC number per TF at the early pupal stage. (B) TFC volume at the early pupal stage. (C) TFC number per ovary at the early pupal stage. (D) TFC number per TF in adults. (E) CC number per germarium in adults. Canton-S flies were used as a wild-type reference. Genotypes are given below each chart. In heterozygotes, +indicates wild type chromosomes from the Canton-S stock. For each genotype, values are expressed as the mean number (A, C–E), or mean volume (B) and sample size is given in parenthesis. Significant differences between genotypes are marked by a bracket and asterisk. All p values are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049958#pone.0049958.s006" target="_blank">Table S4</a>.</p

Ovariole number in ttk-BTB group heterozygous combinations.

<p>Distribution of the number of ovarioles per ovary in females carrying <i>psq</i> (A), <i>batman</i> (B) and <i>Trl</i> (C) mutant alleles, and in double and triple heterozygotes (C,D). The wild-type control (+) corresponds to Canton-S. Each phenotypic class corresponding to the indicated number of ovarioles per ovary was attributed a color as defined on the color table shown in E. The same color table was used in all representations of ovariole number distribution in this Figure and in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049958#pone-0049958-g002" target="_blank">Figures 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049958#pone-0049958-g003" target="_blank">3</a>. The mean number of ovarioles per ovary is given to the right of each distribution. All values are expressed as the mean +/− sample standard deviation. The sample size was 30 ovaries except for data presented in the A panel for which sample size was 20. All statistics are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049958#pone.0049958.s003" target="_blank">Table S1</a>.</p

Detection of TFCs in the pupal and in the adult ovary.

<p>(A–D) Wild type control (Canton-S). (E–G) <i>psq^0115/+</i>; <i>bab^P/</i>+ heterozygotes. EN positive (green) cells include two cell populations, TFCs (white stars in B, D, F), and CC (arrowheads in B, F, circled by a dotted white line in D). At the early pupal stage (A, B, E, F), confocal projections through the anterior/posterior axis of ovaries (A, E) allows counting of the number of TF per ovary (each TF is marked by a black star). Sections through individual early pupal TFs (B, F) allow discrimination between TFCs with a flattened nucleus (white stars) and CCs with a more rounded nucleus (arrowheads in B, F) located posterior to TFCs. Germ cells are marked by an arrow. In one-day-old flies (C, D, G), counting of TFs and TFCs was performed on confocal 3D stacks from whole mount ovaries stained with anti-En (green), anti DE-Cadherin (red, in D) and TO-PRO-3® (purple, in D, G). TFs protrude at the tip of the germarium. In the germarium, CCs (circled by a dotted white line) accumulate high levels of DE-cadherin (red), whereas the adjacent basal TFCs do not. Anterior to the right. Scale bar : (A, E) 10 µm, (B,F) 2.5 µm, (C, G) 40 µm, (D) 5 µm.</p

Expression of ttk group proteins in the late third instar larval ovary.

<p>(A) Expression of PSQ (green in A, A’’). TFCs are marked with anti-EN (red in A’, A’’) and cell perimeters with F-Actin (white in all images). (B, C) PSQ levels from <i>bab>psqIR</i> (<i>UAS-psqIR/+; bab-Gal4/+</i>) larval ovaries (C) compared to those of Canton-S (+, B) from the same experimental series. Signal intensity is visualized using the Fire Lookup Table (ImageJ, NIH, inset on the right). The UAS/Gal4 constructs used in this experiment allow expression of Gal4 in somatic cells but not in the germ cells. In <i>bab>psqIR,</i> when compared to + ovaries, signal intensity decreases in somatic cells, including TFs (brackets) and remains constant in germ cells (arrowheads). Scale bar: 20 µm. (D) Distribution of the number of ovarioles per ovary in <i>bab-Gal4</i> and <i>UAS-psqIR</i> combinations. Color table as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049958#pone-0049958-g001" target="_blank">Figure 1</a>. The mean number of ovarioles per ovary is given on the right. All values are expressed as the mean +/− sample standard deviation. The sample size was 30 ovaries. All statistics are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049958#pone.0049958.s004" target="_blank">Table S2A</a>. (E,F) Batman (green, E, E’’) and TRL/GAF (green, F, F’’) expression in L3 ovaries. TFCs are marked with anti-EN (red in E’, E’’, F’, F’’) and cell perimeters with F-Actin (white in all images). Scale bar : 20 µm.</p