MECANISMES DE REGULATION<br />DE L'HEMATOPOÏESE EMBRYONNAIRE<br />CHEZ LA DROSOPHILE

Haematopoietic development provides an excellent paradigm to address how multipotentcells generate a spectrum of cell types through the combinatorial action of transcription factors.During this process, the blood cells progenitors proliferate and differentiate to give rise todifferent specialised blood cell types. Despite the evolutionary distance between Drosophila andvertebrates, many of the molecular pathways governing haematopoiesis have been conserved. Inparticular, transcription factors of the GATA, FOG (Friend Of GATA) and RUNX families,which regulate several steps of haematopoiesis in vertebrates, also control Drosophilahaematopoiesis. Thus, the fruit fly may provide a valuable model system to gain insight into themechanisms of blood cell lineage formation in vivo. In Drosophila embryo, the blood cellprogenitors (prohemocytes) give rise to two terminally differentiated cell types : plasmatocytesand crystal cells. We have investigated the mechanisms of regulation of this process duringDrosophila embryonic development.First, we have analysed the function and the mechanism of action of the GATAtranscription factor Serpent (Srp) that is required for blood cell precursor specification and forthe formation of the two populations of blood cells (plasmatocytes and crystal cells). We haveshown that serpent encodes different isoforms that have different activities during this process.Furthermore we have shown that the activity of Srp is modulated by different cofactors duringhaematopoiesis. Notably, Srp is able to recruit the FOG cofactor U-Shaped (Ush), and to form afunctional complex with the RUNX transcription factor, Lozenge (Lz). The different isoforms ofSrp and the cooperation between Srp and different cofactors endow srp with pleiotropicfunctions during haematopoiesis.Secondly, we have undertaken an in vivo analysis of the mechanism of segregation of thetwo embryonic blood cell lineages. The differentiation of the blood cell progenitors intoplasmatocytes or crystal cells is control by lineage-specific transcription factors. The relatedproteins Glial cells missing (Gcm) and Gcm2 control plasmatocyte development, while theRUNX factor Lozenge (Lz) is specifically required for crystal cell differentiation. We found thatprohemocytes are bipotent progenitors, whose fate is determined by a dynamic interplaybetween the lineage-specific transcription factors, Gcm/Gcm2 and Lz. The resolution of bloodcell fate choice corresponds to an original two-steps process in which Gcm/Gcm2 control theinitiation and the maintenance of the crystal cell fate. Interestingly, the transition from a bipotenthaematopoietic precursor to lineage-restricted precursors in Drosophila embryos does not relyon reciprocal antagonism between two lineage-specific transcription factors, unlike some cel fate decisions in mammalian haematopoeisisL'hématopoïèse regroupe les phénomènes menant à la formation des composantescellulaires du sang. Au cours de ce processus, des cellules précurseurs vont proliférer et sedifférencier dans les multiples types cellulaires spécialisés. Le développement du systèmehématopoïétique de la Drosophile et des vertébrés présente de nombreuses similitudes aussi bienau niveau fonctionnel et ontogénique qu'au niveau des gènes qui régulent la formation descellules sanguines. Chez la Drosophile, au stade embryonnaire, les précurseurshématopoïétiques, les prohémocytes, vont générer deux types de cellules sanguines, lesplasmatocytes et les cellules à cristaux. Nous avons entrepris de caractériser les mécanismes derégulation de l'hématopoïèse embryonnaire chez la Drosophile.Dans un premier temps, nous avons analysé la fonction et le mode d'action du facteur detranscriptions de type GATA Serpent (Srp) au cours de ce processus. Nous avons mis enévidence que le gène serpent code pour deux isoformes qui ont des activités différentielles aucours de ce processus. D'autre part, nous avons montré que l'activité de Srp au cours del'hématopoïèse est modulée par recrutement de cofacteurs. Ainsi, nous avons montré que Srp estcapable de recruter U-Shaped, un cofacteur de type FOG (Friend Of GATA), mais aussi, deformer un complexe fonctionnel avec le facteur de transcription de type RUNX, Lozenge. Lacaractérisation des isoformes de Srp et la mise en évidence de l'interaction de ce facteur GATAavec différents partenaires a permis de mettre en évidence la versatilité des fonctions de srp aucours de l'hématopoïèse.Dans un second temps, nous avons entrepris de caractériser in vivo l'étape de ségrégationdes deux populations, plasmatocytes et cellules à cristaux. Nous avons mis en évidence que laségrégation de ces deux lignages à partir d'une population de prohémocytes bipotents est unprocessus très dynamique, contrôlé par un mécanisme original en deux étapes. Cette régulationqui fait intervenir les facteurs de transcription lignage-spécifiques Lozenge et Glial-Cell-Missing (Gcm) et Gcm2, contrôle précocement la détermination des précurseurs et tardivementle maintient de l'identité de ces cellules dans les phases de différenciation en cellules à cristauxversus plasmatocytes. De manière intéressante, nous avons montré que la régulation de laségrégation, ne repose pas sur un antagonisme réciproque entre les facteurs de transcriptionlignage-spécifiques. Ce mécanisme qui contrôle l'acquisition d'un destin cellulaire diffère doncdes processus de régulation de l'hématopoïèse mis en évidence chez les mammifères

Insights and perspectives on the enigmatic alary muscles of arthropods

International audienceThree types of muscles, cardiac, smooth and skeletal muscles are classically distinguished in eubilaterian animals. The skeletal, striated muscles are innervated multinucleated syncytia, which, together with bones and tendons, carry out voluntary and reflex body movements. Alary muscles (AMs) are another type of striated syncytial muscles, which connect the exoskeleton to the heart in adult arthropods and were proposed to control hemolymph flux. Developmental studies in Drosophila showed that larval AMs are specified in embryos under control of conserved myogenic transcription factors and interact with excretory, respiratory and hematopoietic tissues in addition to the heart. They also revealed the existence of thoracic AMs (TARMs) connecting to specific gut regions. Their asymmetric attachment sites, deformation properties in crawling larvae and ablation-induced phenotypes, suggest that AMs and TARMs could play both architectural and signalling functions. During metamorphosis, and heart remodelling, some AMs trans-differentiate into another type of muscles. Remaining critical questions include the enigmatic modes and roles of AM innervation, mechanical properties of AMs and TARMS and their evolutionary origin. The purpose of this review is to consolidate facts and hypotheses surrounding AMs/TARMs and underscore the need for further detailed investigation into these atypical muscles

Diversification of muscle types in Drosophila: upstream and downstream of identity genes.

International audienceUnderstanding gene regulatory pathways underlying diversification of cell types during development is one of the major challenges in developmental biology. Progressive specification of mesodermal lineages that are at the origin of body wall muscles in Drosophila embryos has been extensively studied during past years, providing an attractive framework for dissecting cell type diversification processes. In particular, it has been found that muscle founder cells that are at the origin of individual muscles display specific expression of transcription factors that control diversification of muscle types. These factors, encoded by genes collectively called muscle identity genes, are activated in discrete subsets of muscle founders. As a result, each founder cell is thought to carry a unique combinatorial code of identity gene expression. Considering this, to define temporally and spatially restricted expression of identity genes, a set of coordinated upstream regulatory inputs is required. But also, to realize the identity program and to form specific muscle types with distinct properties, an efficient battery of downstream identity gene targets needs to be activated. Here we review how the specificity of expression and action of muscle identity genes is acquired

Two isoforms of Serpent containing either one or two GATA zinc fingers have different roles in Drosophila haematopoiesis

serpent (srp) encodes a GATA transcription factor essential for haematopoiesis in Drosophila. Previously, Srp was shown to contain a single GATA zinc finger of C-terminal type. Here we show that srp encodes different isoforms, generated by alternative splicing, that contain either only a C-finger (SrpC) or both a C- and an N-finger (SrpNC). The presence of the N-finger stabilizes the interaction of Srp with palindromic GATA sites and allows interaction with the Friend of GATA factor U-shaped (Ush). We have examined the respective functions of SrpC and SrpNC during embryonic haematopoiesis. Both isoforms individually rescue blood cell formation that is lacking in an srp null mutation. Interestingly, while SrpC and SrpNC activate some genes in a similar manner, they regulate others differently. Interaction between SrpNC and Ush is responsible for some but not all aspects of the distinct activities of SrpC and SrpNC. Our results suggest that the inclusion or exclusion of the N-finger in the naturally occurring isoforms of Srp can provide an effective means of extending the versatility of srp function during development

Dynamics of transcriptional (re)-programming of syncytial nuclei in developing muscles

Abstract Background A stereotyped array of body wall muscles enables precision and stereotypy of animal movements. In Drosophila, each syncytial muscle forms via fusion of one founder cell (FC) with multiple fusion competent myoblasts (FCMs). The specific morphology of each muscle, i.e. distinctive shape, orientation, size and skeletal attachment sites, reflects the specific combination of identity transcription factors (iTFs) expressed by its FC. Here, we addressed three questions: Are FCM nuclei naive? What is the selectivity and temporal sequence of transcriptional reprogramming of FCMs recruited into growing syncytium? Is transcription of generic myogenic and identity realisation genes coordinated during muscle differentiation? Results The tracking of nuclei in developing muscles shows that FCM nuclei are competent to be transcriptionally reprogrammed to a given muscle identity, post fusion. In situ hybridisation to nascent transcripts for FCM, FC-generic and iTF genes shows that this reprogramming is progressive, beginning by repression of FCM-specific genes in fused nuclei, with some evidence that FC nuclei retain specific characteristics. Transcription of identity realisation genes is linked to iTF activation and regulated at levels of both transcription initiation rate and period of transcription. The generic muscle differentiation programme is activated independently. Conclusions Transcription reprogramming of fused myoblast nuclei is progressive, such that nuclei within a syncytial fibre at a given time point during muscle development are heterogeneous with regards to specific gene transcription. This comprehensive view of the dynamics of transcriptional (re)programming of post-mitotic nuclei within syncytial cells provides a new framework for understanding the transcriptional control of the lineage diversity of multinucleated cells

Additional file 9: Figure S3. of Dynamics of transcriptional (re)-programming of syncytial nuclei in developing muscles

Separate transcription of col and duf in DA3 syncytial nuclei. (A–C) FISH against nascent duf transcripts (green) coupled to Mef2 (red) and Col (blue), immunostaining of all muscle and DA3 nuclei, respectively, in stage 13, 14 and 15 wt embryos. (D) Double FISH of nascent col (red) and duf (green) transcripts in stage 14 wt embryos immunostained for Col (blue); (D’) Col staining alone; (D”) same as (D), showing DAPI staining (blue) of all nuclei. Single Z sections are shown. The yellow arrow points to a nucleus with low Col protein level transcribing duf and not col. (E) Box plots showing the number of nuclei transcribing either col, duf or both, or either col or sns, relative to the total number of DA3 nuclei. (PDF 2537 kb

生物沼气生产利用系统建模分析及可持续性评价

生物质废弃物通过厌氧发酵方式生产生物沼气是生物质高效资源化利用的重要手段之一，具有经济和环境的双重效益，是可再生能源领域的研究热点。现有研究主要针对生物质的厌氧发酵及生物沼气的利用等关键单元技术，从全系统角度的研究较为缺乏，而该体系涉及多种技术、多个单元过程以及多种影响评价指标，是一个复杂拓扑网络系统评价问题。因此，需要借助系统工程的方法对该复杂系统进行深入剖析，了解各单元及变量对系统性能影响的机制，并对影响系统效率的多种技术采用多种指标进行综合评价，筛选出具有代表性的可持续发展路线为生物沼气技术的大规模应用提供指导。基于上述背景，本文对生物沼气全系统进行了能量、环境及经济综合评价，并对系统中的沼气提纯及厌氧发酵关键单元进行了建模与能耗分析。主要研究内容与成果如下：（1）针对粗沼气提纯分离单元，开展了变压吸附法（PSA）粗生物沼气提纯制备生物甲烷工艺的动态模拟与评价研究。分别以13X沸石（Zeolite 13X）、 3K碳分子筛（CMS-3K）和508b金属有机骨架材料（MOF-508b）为吸附剂，建立了两塔-六步的改进 Skarstrom 动态变压吸附模拟流程，对变压吸附过程的关键参数如吸脱附压力、进料吹扫比等进行了灵敏度分析，确定了优化工艺参数； 考察了吸附塔内的压力、CH4 及CO2 组分的浓度随塔高及循环时间的变化；对三种吸附剂装填条件下工艺过程的能耗、设备尺寸及吸附剂装填量进行了计算与比较。结果表明，采用MOF-508b和CMS-3K作为吸附剂时的工艺能耗比Zeolite 13X作为吸附剂时分别低56%和50%；MOF-508b及CMS-3K填充的吸附塔塔径比 Zeolite 13X 填充的吸附塔塔径分别小13%和27%。（2）针对生物质厌氧发酵单元，建立了容积产气率及单元的热平衡模型。对中温 35?C 和高温 55?C 厌氧发酵状态，根据实验数据拟合了三种二元共发酵体系（牛粪与秸秆、鸡粪与秸秆、人粪与秸秆）的容积产气率模型，模型预测结果与实验结果平均偏差在 7%以内。 对热平衡模型， 考察了沼液低温余热的回收对减少过程外供热量的影响。结果表明，发酵过程所需热量的约 89%用于加热进口的冷物料，而约 11%用于维持发酵罐的恒温。 因此， 对沼液的低温余热加以回收以减少过程热量供给是十分必要的。以沼气锅炉供热发酵罐方式为例，通过沼液余热回收， 在中温发酵和高温发酵状态下，每天可以分别减少42%和49%的沼气消耗。（3）针对生物甲烷生产全系统，开展了物流与能量分析研究。在单元过程模拟基础上，建立了该系统的能耗模型和能量效率评价指标。考虑了2种发酵技术、4种粗沼气提纯技术、2 种系统热量供给及是否进行沼液低温余热的利用等因素，设计了32种情景路线；考察了发酵温度、系统热量供给方式及沼液低温余热回收模式对系统能效的影响。结果显示，采用高温发酵技术， 粗沼气及生物甲烷转化率比中温发酵分别提高120%及110%，能量的转化效率提高1倍；高温发酵比中温发酵减少了约3.1 wt%沼液及沼渣的处理量及26%的能量损失； 对中温及高温发酵情景下系统的能效分析与比较，结果表明，采用高温发酵及加压水洗技术，系统所需的热量由外部热源供给且沼液与发酵原料换热回收低温余热，系统的能效在 32 种情景中最高（46.5%）；采用中温发酵及变压吸附技术，系统所需的热量由燃烧发酵过程自产沼气供给且沼液与发酵原料不进行换热回收低温余热，系统的能量效率在 32 种情景中最低（15.8%）；在系统的热量供给方式及沼液与发酵原料换热模式相同的情况下，高温条件下系统的能效约为中温条件下的2倍。在对系统能效影响的三个因素中，发酵温度是对系统能效影响最大的因素，其次是系统热量的供给方式，最后是沼液与发酵原料的换热模式。（4）针对三种不同沼气利用方式（提纯制备生物甲烷、热电联产、固体燃料电池）构成的生物沼气生产及利用系统，对其进行了概念设计，并分别对其能效、绿色度及净现值等指标进行了综合评价与比较。结果表明， 对于系统的能量效率，为沼气提纯 &gt;沼气 SOFCs &gt; 沼气 CHP，提纯利用方式系统能效最高，SOFCs利用方式系统的能效比 CHP 利用方式系统的能效高 2.5%；对于系统绿色度变化量，沼气SOFCs &gt; 沼气 CHP &gt; 沼气提纯；对于系统的投资回收期，沼气 CHP &gt; 沼气 SOFCs &gt;沼气提纯。</p

Additional file 1: Table S1. of Dynamics of transcriptional (re)-programming of syncytial nuclei in developing muscles

Number of Mef2- and RFP-positive nuclei in dorsolateral, lateral and ventral muscles in stage 15 embryos. The numbers of RFP- and Mef2-positive nuclei were determined in the DA3 using col ECRM -H2bRFP; col LCRM -moeGFP embryos; in the DT1, the LO1 and the VA2, using col ECRM -H2bRFP; S59-mcd8GFP embryos; and in the four LT muscles, using col ECRM -H2bRFP; UAS-mcd8GFP ; Kr GMR80H11 -Gal4 embryos, stained for GFP, RFP and Mef2. For each muscle, the average number of nuclei ± standard deviation, minimum and maximum number of nuclei are given (n = 30). (PDF 287 kb

Additional file 18: Table S13. of Dynamics of transcriptional (re)-programming of syncytial nuclei in developing muscles

Percentage of dots and nuclei localised in the antero-ventral, central and postero-dorsal DA3 subdomains. Spatial coordinates of nuclei and transcriptional dots were acquired on col LCRM -moeGFP late stage 14 embryos stained for GFP and Col. FISH were with intronic probes. For each gene, the percentage of dots localised in antero-ventral (Relative Delta Y < –1), postero-dorsal (Relative Delta X > 1) or central (Relative Delta X < 1 and Relative Delta Y > –1) position are shown; 25 muscles were analysed for each condition, the corresponding total number of spots (or nuclei) is indicated. (PDF 145 kb