Genetic Charaterization of appendicular tendon precursors during the first steps of metamorphosis in Drosophila melanogaster : role of Krüppel-like factor Dar1 in the development of appendicular tendon precursors

La mise en place de l’architecture musculo-squelettique émerge d’un développement coordonné inter-tissulaire entre muscles et tissus conjonctifs, dont l’agencement requiert une communication permanente lors des stades embryonnaires. En dépit de l’absence de squelette interne, la patte de la drosophile possède à l’instar des vertébrés des longs tendons internes, auxquels sont rattachés les muscles. Ces sites d’attachements partagent avec mammifères, à la fois l’aspect fonctionnel des tendons à savoir rattacher les muscles et permettre la locomotion, mais également des aspects moléculaires, avec l’identification d’orthologues requis pour leur développement et homéostasie. Chez la drosophile, les tendons appendiculaires sont les seuls sites d’attachement à proposer une telle architecture. Sur la base de ces observations et connaissances, nous avons pris le parti d’étudier plus précisément quels pouvaient être les gènes responsables du développement de ces tendons. Mes travaux de thèse ont consisté à la mise en place d’une méthode cellule-spécifique adaptée à des populations rares que sont les précurseurs de tendons appendiculaires, dans le but d’étudier leur transcriptome. Les données de RNA-seq obtenues ont permis de mettre en exergue environ 900 gènes, dont 68 facteurs de transcription enrichis au sein des précurseurs de tendons. Suite à crible ARN interférent, j’ai identifié le gène dar1 qui est spécifiquement exprimé au sein des tendons de la patte au moment de la métamorphose. La perte de ce gène entraîne une perte des longs tendons appendiculaires et induit une désorganisation drastique de l’architecture musculaire. De manière intéressante, Dar1 est un représentant de la famille des Krüppel-like factor, dont les orthologues KLF4 et 5 sont retrouvés au sein de données transcriptomiques de certains tendons chez les vertébrés, sans que leurs rôles n’aient encore pu être élucidés. A travers cette étude, Dar1 est aujourd’hui proposé comme marqueur spécifique des tendons appendiculaires, et tend à mettre en lumière des relations potentiellement intéressantes entre les tendons de drosophile et les multiples tissus conjonctifs des vertébrés.Musculoskeletal development is a coordinated process that requires the integration of multiple cues and the interaction between muscles and connective tissues (CT). Despite the lack of internal skeleton, the drosophila leg, like the vertebrate limb, shows long internal tendons, which are connected with muscle fibres. These muscle attachment sites share similar function with their mammalian counterpart; they transmit the strength generated by the muscles to allow locomotion. They also share well-known molecular orthologs that are required for their development and homeostasis. Thus, the study of this long internal tendons within the drosophila leg is of great interest to understand the development of this sort of structure. Based on these observations and knowledge, we decided to investigate the genes that are responsible for the development of such particular tendons. We focused on leg tendon precursors, which in fly, develop into tube-like CT structures. We developed a cell-specific approach to isolate tendon precursors and perform RNAseq analysis. This experiment led us to identify approximately 900 transcripts enriched in tendon precursors, in which 68 of them encode for transcription factors (TF). Amongst them, the Krüppel-like factor Dar1 is specifically expressed in tendon leg precursors during the early stages of metamorphosis. Tissue sections of fly legs with attenuated dar1 expression revealed aberrant leg muscle organization with a loss of internal appendicular tendons. These results suggest that Dar1 plays a key role in tendon development. Interestingly, Dar1 orthologs KLF- 4 and 5 are also expressed in mouse tendon precursors and studies conducted on chicken explants suggest that it could impact CT development. This work allowed Dar1 to be identified as a specific marker of long tendon of the leg that could also be required for the development of connective tissues in the vertebrate limb

Reduced telomere length in amniocytes: an early biomarker of abnormal fetal development?

International audienceAbstract Telomeres protect chromosome ends and control cell division and senescence. During organogenesis, telomeres need to be long enough to ensure the cell proliferation necessary at this stage of development. Previous studies have shown that telomere shortening is associated with growth retardation and congenital malformations. However, these studies were performed in newborns or postnatally, and data on telomere length (TL) during the prenatal period are still very limited. We measured TL using quantitative PCR in amniotic fluid (AF) and chorionic villi (CV) samples from 69 control fetuses with normal ultrasound (52 AF and 17 CV) and 213 fetuses (165 AF and 48 CV) with intrauterine growth retardation (IUGR) or congenital malformations diagnosed by ultrasound. The samples were collected by amniocentesis at the gestational age (GA) of 25.0 ± 5.4 weeks and by CV biopsy at 18.1 ± 6.3 weeks. In neither sample type was TL influenced by GA or fetal sex. In AF, a comparison of abnormal versus normal fetuses showed a significant telomere shortening in cases of IUGR (reduction of 34%, P < 10−6), single (29%, P < 10−6) and multiple (44%, P < 10−6) malformations. Similar TL shortening was also observed in CV from abnormal fetuses but to a lesser extent (25%, P = 0.0002; 18%, P = 0.016; 20%, P = 0.004, respectively). Telomere shortening was more pronounced in cases of multiple congenital anomalies than in fetuses with a single malformation, suggesting a correlation between TL and the severity of fetal phenotype. Thus, TL measurement in fetal samples during pregnancy could provide a novel predictive marker of pathological development