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International audience; The establishment of neuronal connectivity relies on the microtubule (MT) cytoskeleton, which provides mechanical support, roads for axonal transport and mediates signalling events. Fine-tuned spatiotemporal regulation of MT functions by tubulin post-translational modifications and MT-associated proteins is critical for the coarse wiring and subsequent refinement of neuronal connectivity. The defective regulation of these processes causes a wide range of neurodevelopmental disorders associated with connectivity defects. This review focuses on recent studies unravelling how MT composition, post-translational modifications and associated proteins influence MT functions in axon guidance and/or pruning to build functional neuronal circuits. We here summarise experimental evidence supporting the key role of this network as a driving force for growth cone steering and branch-specific axon elimination. We further provide a global overview of the MT-interactors that tune developing axon behaviours, with a special emphasis on their emerging versatility in the regulation of MT dynamics/structure. Recent studies establishing the key and highly selective role of the tubulin code in the regulation of MT functions in axon pathfinding are also reported. Finally, our review highlights the emerging molecular links between these MT regulation processes and guidance signals that wire the nervous system.

Skeletal dysplasias comprise a large spectrum of mostly monogenic disorders affecting bone growth, patterning, and homeostasis, and ranging in severity from lethal to mild phenotypes. This study aimed to underpin the genetic cause of skeletal dysplasia in three unrelated families with variable skeletal manifestations. The six affected individuals from three families had severe short stature with extreme shortening of forelimbs, short long-bones, and metatarsals, and brachydactyly (family 1); mild short stature, platyspondyly, and metaphyseal irregularities (family 2); or a prenatally lethal skeletal dysplasia with kidney features suggestive of a ciliopathy (family 3). Genetic studies by whole genome, whole exome, and ciliome panel sequencing identified in all affected individuals biallelic missense variants in KIF24, which encodes a kinesin family member controlling ciliogenesis. In families 1 and 3, with the more severe phenotype, the affected subjects harbored homozygous variants (c.1457A>G; p.(Ile486Val) and c.1565A>G; p.(Asn522Ser), respectively) in the motor domain which plays a crucial role in KIF24 function. In family 2, compound heterozygous variants (c.1697C>T; p.(Ser566Phe)/c.1811C>T; p.(Thr604Met)) were found C-terminal to the motor domain, in agreement with a genotype-phenotype correlation. In vitro experiments performed on amnioblasts of one affected fetus from family 3 showed that primary cilia assembly was severely impaired, and that cytokinesis was also affected. In conclusion, our study describes novel forms of skeletal dysplasia associated with biallelic variants in KIF24. To our knowledge this is the first report implicating KIF24 variants as the cause of a skeletal dysplasia, thereby extending the genetic heterogeneity and the phenotypic spectrum of rare bone disorders and underscoring the wide range of monogenetic skeletal ciliopathies. (c) 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

International audience; Purpose: To correlate the degree of foveal hypoplasia in congenital aniridia with visual acuity, iris phenotype, and PAX6 mutations.Design: Retrospective case series.Methods: 95 consecutive patients with high quality Spectral-domain optical coherence tomography (SD-OCT) records and available genotype were included in a single referral center. Iris hypoplasia was classified as complete, presence of iris root or remnants and mild atypical aniridia. SD-OCT images were assessed to classify foveal hypoplasia as Grade 1 to 4 and to determine mean thicknesses for retinal layers. For statistical analysis one eye for each patient have been used and one member of the same family has been included (n=76 eyes).Results: Most eyes (93.5%) showed variable degree of foveal hypoplasia. PAX6-positive patients presented higher degree of foveal hypoplasia than patients negative for PAX6 (p

The melanocortin system plays a critical role in the central regulation of food intake and energy balance. This system consists of neurons producing pro-opiomelanocortin (POMC), melanocortin receptors (MC4Rs), and the endogenous antagonist agouti-related peptide (AgRP). Pomc and Mc4r deficiency in rodents and humans causes early onset of obesity, whereas a loss of Agrp function is associated with leanness. Accumulating evidence shows that many chronic diseases, including obesity, might originate during early life. The melanocortin system develops during a relatively long period beginning during embryonic life with the birth of POMC and AgRP neurons and continuing postnatally with the assembly of their neuronal circuitry. The development of the melanocortin system requires the tight temporal regulation of molecular factors, such as transcription factors and axon guidance molecules, and cellular mechanisms, such as autophagy. It also involves a complex interplay of endocrine and nutritional factors. The disruption of one or more of these developmental factors can lead to abnormal maturation and function of the melanocortin system and has profound metabolic consequences later in life.

Xenopus provides a simple and efficient model system to study nephrogenesis and explore the mechanisms causing renal developmental defects in human. Hnf1b (hepatocyte nuclear factor 1 homeobox b), a gene whose mutations are the most commonly identified genetic cause of developmental kidney disease, is required for the acquisition of a proximo-intermediate nephron segment in Xenopus as well as in mouse. Genetic networks involved in Hnf1b expression during kidney development remain poorly understood. We decided to explore the transcriptional regulation of Hnf1b in the developing Xenopus pronephros and mammalian renal cells. Using phylogenetic footprinting, we identified an evolutionary conserved sequence (CNS1) located several kilobases (kb) upstream the Hnf1b transcription start and harboring epigenomic marks characteristics of a distal enhancer in embryonic and adult renal cells in mammals. By means of functional expression assays in Xenopus and mammalian renal cell lines we showed that CNS1 displays enhancer activity in renal tissue. Using CRISPR/cas9 editing in Xenopus tropicalis, we demonstrated the in vivo functional relevance of CNS1 in driving hnf1b expression in the pronephros. We further showed the importance of Pax8-CNS1 interaction for CNS1 enhancer activity allowing us to conclude that Hnf1b is a direct target of Pax8. Our work identified for the first time a Hnf1b renal specific enhancer and may open important perspectives into the diagnosis for congenital kidney anomalies in human, as well as modeling HNF1B-related diseases.

SummaryNatural quantitative variation in developmental processes must be driven by allelic variation. Yet, the genotype-phenotype relationships underlying developmental system variation are understudied due to their inherent complexity. Taking advantage of the simpleCaenorhabditis elegansgermline stem cell system, we characterized natural differences in the germ stem cell niche activity of two distinct wild isolates—measured as differences in germline progenitor zone (PZ) size. Through quantitative trait locus (QTL) analysis, we detected multiple candidate causal loci, including two large-effect QTL on chromosomes II and V. Resolving the chromosome V QTL, we show that the isolate with a smaller PZ exhibits a unique 148 bp deletion in the promoter region of the Notch ligand,lag-2, a central signal promoting germ stem cell fate and proliferation. As predicted, introducing this deletion into the isolate with a large PZ resulted in a smaller PZ. Unexpectedly, re-introducing the deleted ancestral sequence in the isolate with a smaller PZ further reduced PZ size. Using allelic replacement lines, we show that these contradictory phenotypic effects are due to epistatic interactions among thelag-2promoter, the chromosome II QTL, and additional loci in the genome. Although thelag-2deletion appeared to explain natural variation in germ stem cell niche activity, its effects across multiple genetic backgrounds were unpredictable due to higher-order epistasis. Studying the genetic architecture of quantitative developmental systems without taking into account its natural variation may be misleading, emphasizing the need for a better integration of developmental and quantitative genetics.

Rare diseases are individually rare but collectively frequent, with more than 7% of living adults affected by one of the 6000 currently described diseases. An estimated 72% of rare diseases are genetic in origin. Since the next generation sequencing (NGS) technology revolution, the rare diseases diagnosis bottleneck is no longer the sequencing but the analysis of the massive amount of data produced. Despite genome sequencing accessibility in clinical routine, the majority of patients suffering from rare diseases are still undiagnosed. Using bioinformatics and data science, my thesis project aimed to manage current bottlenecks of genomic medicine to improve rare disease diagnoses. This manuscript is focused on two main projects I led during this Ph.D. with SeqOne Genomics and CHU Grenoble Alpes.First, I tackled the reinterpretation challenge of previous sequencing analysis that remained unsolved. This reinterpretation was reported manually, and the lack of human resources and automated methods made it difficult to apply in routine diagnosis. Taking advantage of the collaborative and dynamic database ClinVar of shared variant interpretation, we developed Genome Alert!, an open-source automated method that monitors ClinVar and monthly reassesses variant pathogenicity and symptom-gene associations. The re-interpretation of 4,929 analyses revealed 45 changes with potential clinical impact, leading to four additional diagnoses. This work represents a first large validation study of an automated sequencing data re-interpretation system that could become a standard in genomic medicine.Lastly, I explored the clinical data computation challenge, aiming to improve the medical coding or physician’s phenotyping use in genomic analysis. We report the first study focusing on phenotyping practices in clinical sequencing analysis, analyzing the records of 1,686 patients from four international groups. Despite the adoption of a common standard called Human Phenotype Ontology, we found a highly heterogeneous approach to phenotyping as regards the number and choice of symptoms, even for the same patients. This fluctuating description is a major challenge that has to be overcome to enable us to exploit the clinical data in medical records. As an illustration, less than half (43%) of declared symptom-gene associations in the cohort were covered in public databases.Aiming to model the medical inductive reasoning that could explain the heterogeneity of phenotyping across clinical observations, we developed methods based on the association of symptoms with the same genetic disorder. Using graph algorithms and collaborative filtering, we trained a symptom interaction model that projects clinical descriptions in HPO format including 16,600 symptoms into the dimension of interacting symptoms containing 390 groups and 1,131,886 pairs of associated symptoms in diseases. This model uncovered the missing pieces of the incomplete clinical descriptions puzzle, achieving 99.8% coverage of the medical observations with knowledge in the medical literature. To evaluate its clinical relevance, we applied this symptom interaction model to phenotype-driven gene prioritization in the cohort and improved the diagnostic performance by 42 % compared to the best current competitor. This method should enable discoveries in precision medicine by standardizing clinical descriptions.With the work described in this manuscript, I hope I succeeded in making my contribution to spreading genomic medicine awareness in the community and providing technical solutions to improve rare diseases’ patient care., Les maladies rares sont individuellement rares mais collectivement fréquentes. Plus de 7% des adultes sont affectés dans le monde par l'une des 6000 maladies actuellement décrites. 72 % des maladies rares sont d'origine génétique. Depuis l’apparition du séquençage de nouvelle génération, le diagnostic des maladies rares n'est plus limité par le séquençage en lui-même mais l'analyse des données générées par le séquençage. Malgré l'accessibilité en routine clinique du séquençage du génome, la majorité des patients souffrant de maladies rares restent sans diagnostic. Mon projet de thèse visait à résoudre des défis actuels dans l'analyse du séquençage pour améliorer le diagnostic des maladies rares. Ce manuscrit est axé sur deux principaux projets que j'ai menés au cours de ce doctorat avec l'équipe de SeqOne Genomics et le CHU Grenoble Alpes.Premièrement, je me suis attaqué au problème de la réinterprétation des données de séquençage de patients restés sans diagnostic. Cette étape de réinterprétation est manuelle, et le manque de ressources humaines la rend difficile à réaliser en routine. Nous avons développé Genome Alert!, une méthode automatisée et libre qui monitore les changements dans la base de données de partage d'interprétation des variants ClinVar. Ce monitoring permet de réévaluer mensuellement et automatiquement la pathogénicité des variants et les gènes impliqués en maladies humaines. La réinterprétation de 4 929 analyses avec cette méthode a révélé 45 changements ayant un impact clinique potentiel et a conduit à quatre diagnostics supplémentaires. Ce travail représente la première validation à grande échelle d'un système automatisé de réinterprétation des données de séquençage qui pourrait devenir un standard en médecine génomique.En seconde partie, j'ai exploré le défi de la numérisation des données cliniques, avec pour objectif d’améliorer l’utilisation du phénotypage (ou codage médical) des cliniciens dans l'analyse génomique. Nous rapportons la première étude axée sur les pratiques de phénotypage, en analysant 1 686 descriptions de patients provenant de quatre groupes internationaux. Malgré l'adoption d'une norme commune appelée Human Phenotype Ontology, nous avons constaté une approche très hétérogène du phénotypage en ce qui concerne le nombre et le choix des symptômes, et ce même pour les mêmes patients. Cette description fluctuante est un défi majeur qui doit être surmonté pour nous permettre d'exploiter les données cliniques des dossiers médicaux. En effet, moins de la moitié (43%) des associations symptôme-gène déclarées dans la cohorte étaient retrouvées dans les bases de données publiques. Dans le but de modéliser ce raisonnement médical inductif qui pourrait expliquer l'hétérogénéité du phénotypage entre les observations cliniques, nous avons développé des méthodes basées sur l'association conjointe de symptômes au sein des maladies génétiques.À l'aide d'algorithmes graphes, nous avons entraîné un modèle d'interaction des symptômes en maladies génétiques qui projette les descriptions cliniques en format HPO (16,600 symptômes) dans la dimension des symptômes en interaction contenant 390 groupes et 1 131 886 paires de symptômes. Pour évaluer la pertinence clinique de ce modèle, nous l’avons utilisé comme système de priorisation de gènes en fonction du phénotype et avons amélioré les performances de priorisation de 42 % par rapport au meilleur concurrent actuel. Ce modèle devrait permettre de nouvelles découvertes en médecine de précision par sa capacité à exploiter des descriptions cliniques hétérogènes.Au travers ce travail de thèse, j'espère avoir réussi à apporter ma pierre à l'édifice pour sensibiliser à la médecine génomique dans la communauté médicale et fournir des solutions techniques pour améliorer la prise en charge des patients atteints de maladies rares.

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International audience; Glucose homeostasis is a real challenge for extremely preterm infants (EPIs) who have both limited substrate availability and immature glucose metabolism regulation. In the first days of life, EPIs frequently develop transient glucose intolerance, which has a complex pathophysiology that associates unregulated gluconeogenesis, immature insulin secretion, and peripheral insulin resistance. In this population, glucocorticoid therapy is frequently administrated to prevent severe bronchopulmonary dysplasia. During this treatment, glucose intolerance classically increases and may lead to hyperglycemia. We report a case of neonatal hypoglycemia that was concomitant to a glucocorticoids administration, and that led to a congenital hyperinsulinism diagnosis in an EPI with a heterozygous ABCC8 variant. The variant was inherited from his mother, who had developed monogenic onset diabetes of the youth (MODY) at the age of 23. ABCC8 encodes a beta-cell potassium channel unit and causes congenital hyperinsulinism or MODY depending on the mutation location. Moreover, some mutations have been observed in the same patient to cause both hyperinsulinism in infancy and MODY in adulthood. In our case, the baby showed repeated and severe hypoglycemias, which were undoubtedly time-associated with the betamethasone intravenous administration. This hyperinsulinism was transient, and the infant has not yet developed diabetes at three years of age. We take the opportunity presented by this unusual clinical presentation to provide a review of the literature, suggesting new insights regarding the pathophysiology of the beta-pancreatic cells’ insulin secretion: glucocorticoids may potentiate basal insulin secretion in patients with ABCC8 mutation.

Mouse embryonic stem cells have an inherent propensity to explore gene regulatory states associated with either self-renewal or differentiation. This property depends on ERK, which downregulates pluripotency genes such as Nanog. Here, we aimed at identifying repressive histone modifications that would mark Nanog for inactivation in response to ERK activity. We found that the transcription factor ZFP57, which binds methylated DNA to nucleate heterochromatin, is recruited upstream of Nanog, within a region enriched for histone H3 lysine 9 tri-methylation (H3K9me3). Whereas before differentiation H3K9me3 at Nanog depends on ERK, in somatic cells it becomes independent of ERK. Moreover, the loss of H3K9me3 at Nanog, induced by deleting the region or by knocking out DNA methyltransferases or Zfp57, is associated with reduced heterogeneity of NANOG, delayed commitment into differentiation and impaired ability to acquire a primitive endoderm fate. Hence, a network axis centred on DNA methylation, ZFP57 and H3K9me3 links Nanog regulation to ERK activity for the timely establishment of new cell identities. We suggest that establishment of irreversible H3K9me3 at specific master regulators allows the acquisition of particular cell fates during differentiation.

International audience; The hypothalamus is a large brain region made of nuclei and areas involved in the control of behaviors and physiological regulations. Among them, the arcuate nucleus (ARH) and the lateral hypothalamic area (LHA) contain key neuronal populations expressing the pro-opiomelanocortin (POMC), the agouti-related peptide (AgRP), and the melanin-concentrating hormone (MCH), respectively, that are involved in goal-oriented behaviors (such as feeding behavior) and glucose homeostasis. These neuronal populations are generated from distinct parts of the germinative neuroepithelium during embryonic life, and acquire their cell fate under the influence of morphogen proteins, specific transcription factors, and epigenetic modulators. POMC and MCH neuronal development continues by sending long descending axonal projections before birth under the control of axon guidance molecules such as Netrin1 and Slit2. Later, during the postnatal period, POMC and AgRP neurons develop intra-hypothalamic projections notably to the paraventricular nucleus of the hypothalamus through the influence of other axon guidance cues such as the class3 Semaphorins. Other cellular processes, such as autophagy and primary cilia function, and hormonal cues also appear critical for the proper development of POMC neurons.

Mesenchymal stromal cells are essential components of hematopoietic stem and progenitor cell (HSPC) niches, regulating HSPC proliferation and fate decisions. Their developmental origins are largely unknown. In zebrafish, we previously found that the stromal cells of the caudal hematopoietic tissue (CHT), a niche functionally homologous to the fetal liver in mammals, arise from the ventral part of caudal somites. We have now discovered that this ventral domain is actually the sclerotome, and that two typical markers of mammalian mesenchymal stem/stromal cells, Alcam and Pdgfr-α, are distinctively expressed there and instrumental for the emergence and migration of stromal cell progenitors, which in turn conditions the proper assembly of the vascular component of the CHT niche. Furthermore, we find that the trunk somites are similarly dependent on Alcam and Pdgfr-α to produce mesenchymal stromal cells that foster the initial emergence of HSPCs from the dorsal aorta. Thus the sclerotome contributes essential stromal cells for each of the key steps of developmental hematopoiesis, and likely is the embryological origin of most if not all mesenchymal stem/stromal cells found in non-cephalic tissues.

Protein phosphatase 1 (PP1) is a key enzyme for Plasmodium development. However, the detailed mechanisms underlying its regulation remain to be deciphered. Here, we report the functional characterization of the Plasmodium berghei leucine-rich repeat protein 1 (PbLRR1), an orthologue of SDS22, one of the most ancient and conserved PP1 interactors. Our study shows that PbLRR1 is expressed during intra-erythrocytic development of the parasite, and up to the zygote stage in mosquitoes. PbLRR1 can be found in complex with PbPP1 in both asexual and sexual stages and inhibits its phosphatase activity. Genetic analysis demonstrates that PbLRR1 depletion adversely affects the development of oocysts. PbLRR1 interactome analysis associated with phospho-proteomics studies identifies several novel putative PbLRR1/PbPP1 partners. Some of these partners have previously been characterized as essential for the parasite sexual development. Interestingly, and for the first time, Inhibitor 3 (I3), a well-known and direct interactant of Plasmodium PP1, was found to be drastically hypophosphorylated in PbLRR1-depleted parasites. These data, along with the detection of I3 with PP1 in the LRR1 interactome, strongly suggest that the phosphorylation status of PbI3 is under the control of the PP1–LRR1 complex and could contribute (in)directly to oocyst development. This study provides new insights into previously unrecognized PbPP1 fine regulation of Plasmodium oocyst development through its interaction with PbLRR1.

The Golgi apparatus (GA) is essential for intracellular sorting, trafficking and the targeting of proteins to specific cellular compartments. Anatomically, the GA spreads all over the cell but is also particularly enriched close to the base of the primary cilium. This peculiar organelle protrudes at the surface of almost all cells and fulfills many cellular functions, in particular during development, when a dysfunction of the primary cilium can lead to disorders called ciliopathies. While ciliopathies caused by loss of ciliated proteins have been extensively documented, several studies suggest that alterations of GA and GA-associated proteins can also affect ciliogenesis. Here, we aim to discuss how the loss-of-function of genes coding these proteins induces ciliary defects and results in ciliopathies.

International audience

International audience; Abstract Several studies have reported WDR73 mutations to be causative of Galloway–Mowat syndrome, a rare disorder characterised by the association of neurological defects and renal-glomerular disease. In this study, we demonstrate interaction of WDR73 with the INTS9 and INTS11 components of Integrator, a large multiprotein complex with various roles in RNA metabolism and transcriptional control. We implicate WDR73 in two Integrator-regulated cellular pathways; namely, the processing of uridylate-rich small nuclear RNAs (UsnRNA), and mediating the transcriptional response to epidermal growth factor stimulation. We also show that WDR73 suppression leads to altered expression of genes encoding cell cycle regulatory proteins. Altogether, our results suggest that a range of cellular pathways are perturbed by WDR73 loss-of-function, and support the consensus that proper regulation of UsnRNA maturation, transcription initiation and cell cycle control are all critical in maintaining the health of post-mitotic cells such as glomerular podocytes and neurons, and preventing degenerative disease.

Modulation of gene expression during differentiation by transcription factors promotes cell diversity. Despite their role in cell fate decisions, no experimental assays estimate their regulatory activity in a high-throughput manner and at the single-cell resolution. We present FateCompass for identifying lineage-specific transcription factors across differentiation. It uses single-cell transcriptomics data to infer differentiation trajectories and transcription factor activities. We combined a probabilistic framework with RNA velocities or a differentiation potential to estimate transition probabilities and perform stochastic simulations. Also, we learned transcription factor activities using a linear model of gene regulation. Considering dynamic changes and correlations, we identified lineage-specific regulators. We applied FateCompass to an islet cell formation dataset from the mouse embryo, and we found known and novel potential cell-type drivers. Also, when applied to a differentiation protocol dataset towards beta-like cells, we pinpointed undescribed regulators of an off-target population, which were experimentally validated. Thus, as a framework for identifying lineage-specific transcription factors, FateCompass could have implications on hypothesis generation to increase the understanding of the gene regulatory networks driving cell fate choices.HighlightsWe developed FateCompass, a flexible pipeline to estimate transcription factor activities during cell-fate decision using single-cell RNA seq data.FateCompass outlines gene expression stochastic trajectories by infusing the direction of differentiation using RNA velocity or a differentiation potential when RNA velocity fails.Transcription factor dynamics allow the identification of time-specific regulatory interactions.FateCompass predictions revealed known and novel cell-subtype-specific regulators of mouse pancreatic islet cell development.Differential motif analysis predicts lineage-specific regulators of stem cell-derived human β- cells and sheds light on the cellular heterogeneity of β-cell differentiation protocols.Experimental validation supports the proposed GRN controlling SC-EC differentiation predicted by FateCompass.

Kabuki syndrome (KS) (OMIM 147920 and 300867) is a rare genetic disorder characterized by specific facial features, intellectual disability, and various malformations. Immunopathological manifestations seem prevalent and increase the morbimortality. To assess the frequency and severity of the manifestations, we measured the prevalence of immunopathological manifestations as well as genotype–phenotype correlations in KS individuals from a registry. Data were for 177 KS individuals with KDM6A or KMT2D pathogenic variants. Questionnaires to clinicians were used to assess the presence of immunodeficiency and autoimmune diseases both on a clinical and biological basis. Overall, 44.1% (78/177) and 58.2% (46/79) of KS individuals exhibited infection susceptibility and hypogammaglobulinemia, respectively; 13.6% (24/177) had autoimmune disease (AID; 25.6% [11/43] in adults), 5.6% (10/177) with ≥2 AID manifestations. The most frequent AID manifestations were immune thrombocytopenic purpura (7.3% [13/177]) and autoimmune hemolytic anemia (4.0% [7/177]). Among nonhematological manifestations, vitiligo was frequent. Immune thrombocytopenic purpura was frequent with missense versus other types of variants (p = 0.027). The high prevalence of immunopathological manifestations in KS demonstrates the importance of systematic screening and efficient preventive management of these treatable and sometimes life-threatening conditions.

International audience; The extracellular matrix (ECM) is a fundamental component of the tissue of multicellular organisms that is comprised of an intricate network of multidomain proteins and associated factors, collectively known as the matrisome. The ECM creates a biophysical environment that regulates essential cellular processes such as adhesion, proliferation and migration and impacts cell fate decisions. The composition of the ECM varies across organs, developmental stages and diseases. Interestingly, most ECM genes generate transcripts that undergo extensive alternative splicing events, producing multiple protein variants from one gene thus enhancing ECM complexity and impacting matrix architecture. Extensive studies over the past several decades have linked ECM remodeling and expression of alternatively spliced ECM isoforms to cancer, and reprogramming of the alternative splicing patterns in cells has recently been proposed as a new hallmark of tumor progression. Indeed, tumor-associated alternative splicing occurs in both malignant and non-malignant cells of the tumor environment and growing evidence suggests that expression of specific ECM splicing variants could be a key step for stromal activation. In this review, we present a general overview of alternative splicing mechanisms, featuring examples of ECM components. The importance of ECM variant expression during essential physiological processes, such as tissue organization and embryonic development is discussed as well as the dysregulation of alternative splicing in cancer. The overall aim of this review is to address the complexity of the ECM by highlighting the importance of the yet-to-be-fully-characterized "alternative" matrisome in physiological and pathological states such as cancer.

International audience; Over the last century, westernization of dietary habits has led to a dramatic reduction in dietary intake of n-3 polyunsaturated fatty acids (n-3 PUFAs). In particular, low maternal intake of n-3 PUFAs throughout gestation and lactation causes defects in brain myelination. Microglia are recognized for their critical contribution to neurodevelopmental processes, such as myelination. These cells invade the white matter in the first weeks of the post-natal period, where they participate in oligodendrocyte maturation and myelin production. Therefore, we investigated whether an alteration of white matter microglia accompanies the myelination deficits observed in the brain of n-3 PUFA-deficient animals. Macroscopic imaging analysis shows that maternal n-3 PUFA deficiency decreases the density of white matter microglia around post-natal day 10. Microscopic electron microscopy analyses also revealed alterations of microglial ultrastructure, a decrease in the number of contacts between microglia and myelin sheet, and a decreased amount of myelin debris in their cell body. White matter microglia further displayed increased mitochondrial abundance and network area under perinatal n-3 PUFA deficiency. Overall, our data suggest that maternal n-3 PUFA deficiency alters the structure and function of microglial cells located in the white matter of pups early in life, and this could be the key to understand myelination deficits during neurodevelopment. Copyright © 2022 Decoeur, Picard, St-Pierre, Greenhalgh, Delpech, Sere, Layé, Tremblay and Nadjar.

The interplay between the topological organization of the genome and the regulation of gene expression remains unclear. Depletion of molecular factors underlying genome topology, such as CTCF and cohesin, leads to modest alterations in gene expression, while genomic rearrangements involving boundaries of topologically associating domains (TADs) disrupt normal gene expression and can lead to pathological phenotypes. Here we inverted an almost entire TAD (245kb out of 300kb) within the X-inactivation centre (Xic), leaving its boundaries intact. This led to a significant rearrangement of topological contacts within the TAD, mostly in accordance to the orientation of underlying CTCF binding sites but suggesting heterogeneity in the “contact” potential of different CTCF sites. The inversion also led to increased contact insulation with the neighbouring TAD. Expression of most genes within the inverted TAD remained unaffected in mouse embryonic stem cells and during differentiation. Interestingly, expression in the neighbouring TAD of the noncoding transcript Xist, which controls X-chromosome inactivation, was ectopically upregulated. The same inversion in mouse embryos led to a bias in Xist expression, but X-inactivation choice ratios did not significantly deviate from wild type. Smaller deletions and inversions of specific clusters of CTCF sites within the TAD led to similar results: rearrangement of contacts, limited changes in local gene expression but significant changes in Xist expression. Our study suggests that the wiring of regulatory interactions within a TAD can influence the expression of genes in neighbouring TADs, highlighting the existence of mechanisms for inter-TAD communication.

Simple Summary Maternal gene products accumulated during oogenesis as mRNAs and proteins, which are referred to as maternal contributions, play essential roles in proper development before and after the transcription of the zygotic genome. Zebrafish represent an attractive model for the systematic characterization of genetic mutants using the CRISPR/Cas9 genome editing technology. The traditional approach for generating maternal mutants to study the functions of maternal gene products consists of creating founder fish with germ-line transmission of mutated genes, followed by successive crossing to obtain zygotic homozygous mutant females, which can produce offspring lacking the corresponding maternal gene products. This is time-consuming and normally requires three generations. Nevertheless, zygotic mutations of many genes often lead to embryonic lethality or adult sterility, making it impossible to study their maternal functions. Different approaches were used to overcome this difficulty and allowed for the characterization of several important maternal-effect genes. However, they are often technically challenging or lack versatile applications. Recently, an oocyte-specific knockout strategy has been developed, which inactivates genes of interest in the developing oocytes. It is particularly accessible, generating maternal mutants in one fish generation. By further improving its efficiency, this method can be used for the large-scale analysis of maternal gene functions. Abstract Maternal gene products accumulated during oogenesis are essential for supporting early developmental processes in both invertebrates and vertebrates. Therefore, understanding their regulatory functions should provide insights into the maternal control of embryogenesis. The CRISPR/Cas9 genome editing technology has provided a powerful tool for creating genetic mutations to study gene functions and developing disease models to identify new therapeutics. However, many maternal genes are also essential after zygotic genome activation; as a result, loss of their zygotic functions often leads to lethality or sterility, thus preventing the generation of maternal mutants by classical crossing between zygotic homozygous mutant adult animals. Although several approaches, such as the rescue of mutant phenotypes through an injection of the wild-type mRNA, germ-line replacement, and the generation of genetically mosaic females, have been developed to overcome this difficulty, they are often technically challenging and time-consuming or inappropriate for many genes that are essential for late developmental events or for germ-line formation. Recently, a method based on the oocyte transgenic expression of CRISPR/Cas9 and guide RNAs has been designed to eliminate maternal gene products in zebrafish. This approach introduces several tandem guide RNA expression cassettes and a GFP reporter into transgenic embryos expressing Cas9 to create biallelic mutations and inactivate genes of interest specifically in the developing oocytes. It is particularly accessible and allows for the elimination of maternal gene products in one fish generation. By further improving its efficiency, this method can be used for the systematic characterization of maternal-effect genes.

International audience; iPSC-derived brain and retinal organoids represent biologically relevant 3D models. A new step in the field of brain and retinal organoids was reached a few months ago with the simultaneous development of brain and eye structures from human iPS cells within the same organoid. Single-cell mRNA sequencing analyses allowed the identification of various ocular and cerebral neuronal populations and electrophysiological recordings confirm the presence of functional and electrically active neurons. The coexistence within the same organoid of different cell types from visual and brain regions and the establishment of connections between these regions raise the intriguing question of its real or potential functionality and its ability to process higher-level visual information. This unique model could also be used to further understand the development of the human visual system and associated developmental diseases.

Genomic imprinting refers to the mono-allelic and parent-specific expression of a subset of genes. While long recognized for their role in embryonic development, imprinted genes have recently emerged as important modulators of postnatal physiology, notably through hypothalamus-driven functions. Here, using mouse models of loss, gain and parental inversion of expression, we report that the paternally expressed Zdbf2 gene controls neonatal growth in mice, in a dose-sensitive but parent-of-origin-independent manner. We further found that Zdbf2-KO neonates failed to fully activate hypothalamic circuits that stimulate appetite, and suffered milk deprivation and diminished circulating Insulin Growth Factor 1 (IGF-1). Consequently, only half of Zdbf2-KO pups survived the first days after birth and those surviving were smaller. This study demonstrates that precise imprinted gene dosage is essential for vital physiological functions at the transition from intra- to extra-uterine life, here the adaptation to oral feeding and optimized body weight gain.

The canonical Wnt/β-catenin pathway governs a multitude of developmental processes in various cell lineages, including the melanocyte lineage. Indeed, β-catenin regulates transcription of Mitf-M, the master regulator of this lineage. The first wave of melanocytes to colonize the skin is directly derived from neural crest cells, whereas the second wave of melanocytes is derived from Schwann cell precursors (SCPs). We investigated the influence of β-catenin in the development of melanocytes of the first and second waves by generating mice expressing a constitutively active form of β-catenin in cells expressing tyrosinase. Constitutive activation of β-catenin did not affect the development of truncal melanoblasts but led to marked hyperpigmentation of the paws. By activating β-catenin at various stages of development (E8.5-E11.5), we showed that the activation of β-catenin in bipotent SCPs favored melanoblast specification at the expense of Schwann cells in the limbs within a specific temporal window. Furthermore, in vitro hyperactivation of the Wnt/β-catenin pathway, which is required for melanocyte development, induces activation of Mitf-M, in turn repressing FoxD3 expression. In conclusion, β-catenin overexpression promotes SCP cell fate decisions towards the melanocyte lineage.

Research on the sensory reveals a twofold reality. One is that all human beings have innate sensory aptitudes whose inter-individual variability remains limited by a complexion specific to the species. The other is that social groups implement varied sensory modalities. Consequently, if sensory perception is an attribute of our biology, it is also learned. It is not only the encounter of the biological and the cultural, it is a biocultural phenomenon. Indeed, sensory perception is defined and limited by the information to which our sense organs are naturally sensitive, but the very sensitivity of the latter is partly modulated and shaped by the social and cultural environment. Considering sensory learning obliges us to go beyond the classic scheme opposing sensation of biological order to perception of cultural order.This book, with twenty chapters that draw mainly on anthropology, but also on sociology, psychology, neuroscience, primatology and archaeology, proposes a vast panorama of the forms of sensory sharing by human beings. It shows on the one hand that the discovery and learning of the world through the senses do not imply either a radical cognitive relativism, nor the confinement of each individual in a singular Umwelt, on the other hand that sensory and multisensory learning are always the object of a cultural modulation. We learn the senses, we learn by the senses., La recherche sur le sensoriel dévoile une double évidence. L’une est que tous les êtres humains disposent d’aptitudes sensorielles innées dont la variabilité interindividuelle reste bornée par une complexion propre à l’espèce. L’autre est que les groupes sociaux mettent en œuvre des modalités sensorielles variées. Par conséquent, si la perception sensorielle est un attribut de notre biologie, elle s’apprend aussi. Elle n’est pas seulement la rencontre du biologique et du culturel, elle est un phénomène bioculturel. En effet, la perception sensorielle est définie et limitée par les informations auxquelles sont naturellement sensibles nos organes des sens, mais la sensibilité même de ces derniers est en partie modulée et modelée par l’environnement social et culturel. Considérer les apprentissages sensoriels enjoint à dépasser le schéma classique opposant sensation d’ordre biologique à perception d’ordre culturel.Cet ouvrage, de vingt chapitres qui puisent surtout en anthropologie, mais aussi en sociologie, psychologie, neurosciences, primatologie et archéologie, propose un vaste panorama des formes du partage du sensoriel par les êtres humains. Il montre d’une part que la découverte et l’apprentissage du monde par les sens n’impliquent ni un relativisme cognitif radical, ni l’enfermement de chaque individu dans un Umwelt singulier, d’autre part que les apprentissages sensoriels et multisensoriels sont toujours l’objet d’une modulation culturelle. On apprend les sens, on apprend par les sens.

Atypical teratoid rhabdoid tumors (ATRT) are rare and aggressive malignancies of the central nervous system (CNS) affecting infants and young children and characterized by a biallelic inactivation of SMARCB1 tumor suppressor gene in an otherwise very simple genome. Based on methylation and expression profiling, recent studies have pointed out the molecular diversity of these tumors, that are now divided in at least three subgroups, i.e. the so-called MYC-, TYR-, and SHH-ATRTs. This molecular diversity is suggestive of various cells of origin for each sub-type, though none of these putative cells of origin is undoubtedly identified at present time. In this respect, the analysis of expression profiling has been weakly informative, giving at most some insights on the lineage (neurogenic or melanogenic features for the SHH and TYR subtypes respectively) or on some recurrently expressed developmental genes and pathways (SHH and NOTCH pathway for the SHH subtype; HOX clusters for the MYC subtype). In this work, we have provided a detailed description of different anatomical ATRT locations to obtain precise information about the putative tissue of origin. Then, we have performed integrative analysis of anatomical location, transcriptomic and epigenetic data. This analysis has allowed us to describe and characterize 4 distinct anatomico-molecular ATRT subgroups: cranial nerves, cortical and spinal cord tumors, CNCS-ATRT; Basal ganglia and Intraventricular tumors, BG/IV-ATRT; cerebellar anterior lobe tumors, CAL-ATRT; middle cerebellar peduncle and inferior cerebellar lobe tumors, MCP/ICV-ATRT. Next, we have sought to investigate the relevance of our mouse models for human disease. We have found that the Rosa26: MYC mouse model perfectly recapitulates molecular features and clinical phenotypes of a subset of human ATRT (CNCS-ATRT) and support the hypothesis of an origin in neural crest cells. The Rosa26: SHH tumors are phenotypically similar to the BG_IV-ATRT and are characterized by the expression of gene markers from the ganglionic eminences. Finally, the CAL-ATRT are characterized by the expression of midbrain-hindbrain boundary (MHB) genes. Although we don’t have a faithful mouse model for this location, the integrative analysis has allowed us to identify the CAL-ATRT subgroup and lead us to perform single cell analysis of three human samples coming exclusively from this location. Consecutively, we have demonstrated at the single-cell level, by the first time so far, the dedifferentiation of neuronal progenitor cells and the involvement of Notch pathway in the malignant transformation of CAL-ATRT., Les tumeurs rhabdoïdes tératoïdes atypiques (ATRT) sont des tumeurs malignes rares et agressives du système nerveux central (SNC) affectant les nourrissons et les jeunes enfants et caractérisées par une inactivation bialélique du gène suppresseur de tumeur SMARCB1 dans un génome par ailleurs très simple. Sur la base du profil de méthylation et d'expression, des études récentes ont mis en évidence la diversité moléculaire de ces tumeurs, qui sont maintenant divisées en au moins trois sous-groupes, à savoir les tumeurs dites MYC-, TYR- et SHH-ATRT. Cette diversité moléculaire suggère l'existence de diverses cellules d'origine pour chaque sous-type, bien qu'aucune de ces cellules d'origine putatives ne soit sans doute identifiée à l'heure actuelle. L'analyse du profil d'expression a été faiblement informative sur le tissue d'origine mais donnée des indications sur le lignage (neurogéniques ou mélanogènique pour les sous-types SHH et TYR respectivement) ou sur certains gènes et voies de développement exprimés de manière récurrente (SHH et voie NOTCH pour le sous-type SHH ; groupes HOX pour le sous-type MYC). Dans ce travail, nous avons fourni une description détaillée des différentes localisations anatomiques des ATRT afin d'obtenir des informations précises sur le tissu d'origine. Ensuite, nous avons réalisé une analyse intégrative de la localisation anatomique, des données transcriptomiques et épigénétiques. Cette analyse nous a permis de décrire et de caractériser 4 sous-groupes anatomico-moléculaires d'ATRT : tumeurs des nerfs crâniens, du cortex et de la moelle épinière, CNCS-ATRT ; ganglions de la base et tumeurs intraventriculaires, BG/IV-ATRT ; tumeurs du lobe antérieur du cervelet, CAL-ATRT ; tumeurs du pédoncule cérébelleux moyen et du lobe cérébelleux inférieur, MCP/ICV-ATRT. Ensuite, nous avons cherché à étudier la pertinence de nos modèles murins chez l'homme. Nous avons constaté que le modèle de souris Rosa26 : MYC récapitule parfaitement les caractéristiques moléculaires et les phénotypes cliniques d'un sous-ensemble d'ATRT humains (CNCS-ATRT) et soutient l'hypothèse d'une origine dans les cellules de la crête neurale. Les tumeurs Rosa26 : SHH sont phénotypiquement similaires aux BG_IV-ATRT et sont caractérisées par l'expression de marqueurs génétiques des éminences ganglionnaires. Enfin, les tumeurs CAL-ATRT sont caractérisées par l'expression de gènes typiques de la region entre le cerveau moyen et le cerveau postérieur pendant le développement embryonnaire. Bien que nous ne disposions pas d'un modèle de souris récapitulant ce localisation, l'analyse intégrative nous a permis d'identifier le sous-groupe CAL-ATRT et nous a conduits à effectuer une analyse RNAseq cellule-unique de trois échantillons humains provenant exclusivement de cette localisation. Nous avons consécutivement démontré au niveau cellule-unique, pour la première fois à ce jour, la dédifférenciation des cellules progénitrices neuronales et l'implication de la voie Notch dans la transformation maligne des CAL-ATRT.

The contractile properties of adult myofibers are shaped by their Myosin heavy chain isoform content. Here, we identify by snATAC-seq a 42 kb super-enhancer at the locus regrouping the fast Myosin genes. By 4C-seq we show that active fast Myosin promoters interact with this super-enhancer by DNA looping, leading to the activation of a single promoter per nucleus. A rainbow mouse transgenic model of the locus including the super-enhancer recapitulates the endogenous spatio-temporal expression of adult fast Myosin genes. In situ deletion of the super-enhancer by CRISPR/Cas9 editing demonstrates its major role in the control of associated fast Myosin genes, and deletion of two fast Myosin genes at the locus reveals an active competition of the promoters for the shared super-enhancer. Last, by disrupting the organization of fast Myosin, we uncover positional heterogeneity within limb skeletal muscles that may underlie selective muscle susceptibility to damage in certain myopathies.

Craniofacial and jaw bones have unique physiological specificities when compared to axial and appendicular bones. However, the molecular profile of the jaw osteoblast (OB) remains incomplete. The present study aimed to decipher the bone site-specific profiles of transcription factors (TFs) expressed in OBs in vivo. Using RNA-seq analysis, we mapped the transcriptome of confirmed OBs from two different skeletal sites: mandible (Md) and tibia (Tb). The OB transcriptome contains 709 TF genes: 608 are similarly expressed in Md-OB and Tb-OB, referred to as “OB-core”; 54 TF genes are upregulated in Md-OB, referred to as “Md-set”; and 18 TF genes are upregulated in Tb-OB, referred to as “Tb-set”. Notably, the expression of 29 additional TF genes depends on their RNA transcript variants. TF genes with no previously known role in OBs and bone were identified. Bioinformatics analysis combined with review of genetic disease databases and a comprehensive literature search showed a significant contribution of anatomical origin to the OB signatures. Md-set and Tb-set are enriched with site-specific TF genes associated with development and morphogenesis (neural crest versus mesoderm), and this developmental imprint persists during growth and homeostasis. Jaw and tibia site-specific OB signatures are associated with craniofacial and appendicular skeletal disorders as well as neurocristopathies, dental disorders and digit malformations. The present study demonstrates the feasibility of a new method to isolate pure OB populations and map their gene expression signature in the context of OB physiological environment, avoiding in vitro culture and its associated biases. Our results provide insights into the site-specific developmental pathways governing OBs and identify new major OB regulators of bone physiology. We also established the importance of the OB transcriptome as a prognostic tool for human rare bone diseases to explore the hidden pathophysiology of craniofacial malformations, among the most prevalent congenital defects in humans.

The paternally expressed gene 3 (Pw1/Peg3) is a mammalian-specific parentally imprinted gene expressed in stem/progenitor cells of the brain and endocrine tissues. Here, we compared phenotypic characteristics in Pw1/Peg3 deficient male and female mice. Our findings indicate that Pw1/Peg3 is a key player for the determination of sexual dimorphism in metabolism and behavior. Mice carrying a paternally inherited Pw1/Peg3 mutant allele manifested postnatal deficits in GH/IGF dependent growth before weaning, sex steroid dependent masculinization during puberty, and insulin dependent fat accumulation in adulthood. As a result, Pw1/Peg3 deficient mice develop a sex-dependent global shift of body metabolism towards accelerated adiposity, diabetic-like insulin resistance, and fatty liver. Furthermore, Pw1/Peg3 deficient males displayed reduced social dominance and competitiveness concomitant with alterations in the vasopressinergic architecture in the brain. This study demonstrates that Pw1/Peg3 provides an epigenetic context that promotes male-specific characteristics through sex steroid pathways during postnatal development., Author summary Pw1/Peg3 is under parental specific epigenetic regulation. We propose that Pw1/Peg3 confers a selective advantage in mammals by regulating sexual dimorphism. To address this question, we examined the consequences of Pw1/Peg3 loss of function in mice in an age- and sex-dependent context and found that Pw1/Peg3 mutants display reduced sexual dimorphism in growth, metabolism and behaviors. Our findings support the intralocus sexual conflict model of genomic imprinting where it contributes in sexual differentiation. Furthermore, our observations provide a unifying role of sex steroid signaling as a common property of Pw1/Peg3 expressing stem/progenitor cells and differentiated endocrine cells, both of which remain proliferative in response to gonadal hormones in adult life.

International audience; Retinal ganglion cell (RGC) axons provide the only link between the light sensitive and photon transducing neural retina and visual centers of the brain. RGC axon degeneration occurs in a number of blinding diseases and the ability to stimulate axon regeneration from surviving ganglion cells could provide the anatomic substrate for restoration of vision. OTX2 is a homeoprotein transcription factor expressed in the retina and previous studies showed that, in response to stress, exogenous OTX2 increases the in vitro and in vivo survival of RGCs. Here we examined and quantified the effects of OTX2 on adult RGC axon regeneration in vitro and in vivo. The results show that exogenous OTX2 stimulates the regrowth of axons from RGCs in cultures of dissociated adult retinal cells and from explants of adult retinal tissue and that RGCs respond directly to OTX2 as regrowth is observed in cultures of purified adult rat RGCs. Importantly, after nerve crush in vivo, we observed a positive effect of OTX2 on the number of regenerating axons up to the optic chiasm within 14 days post crush and a very modest level of acuity absent in control mice. The effect of OTX2 on RGC survival and regeneration is of potential interest for degenerative diseases affecting this cell type. All animal procedures were approved by the local "Comié d'éιthique en expérimentation animale n°59" and authorization n° 00702.01 delivered March 28, 2014 by the French "Ministére de l'enseignement supérieur et de la recherche".

International audience; Aedes albopictus is a major vector of arboviruses. Better understanding of its sex determination is crucial for developing mosquito control tools, especially genetic sexing strains. In Aedes aegypti, Nix is the primary gene responsible for masculinization and Nix-expressing genetic females develop into fertile, albeit flightless, males. In Ae. albopictus, Nix has also been implicated in masculinization but its role remains to be further characterized. In this work, we establish Ae. albopictus transgenic lines ectopically expressing Nix. Several are composed exclusively of genetic females, with transgenic individuals being phenotypic and functional males due to the expression of the Nix transgene. Their reproductive fitness is marginally impaired, while their flight performance is similar to controls. Overall, our results show that Nix is sufficient for full masculinization in Ae. albopictus. Moreover, the transgene construct contains a fluorescence marker allowing efficient automated sex sorting. Consequently, such strains constitute valuable sexing strains for genetic control.

Stem cells (SCs) in vertebrates typically reside in “stem cell niches” (SCNs), morphologically restricted tissue microenvironments that are important for SC survival and proliferation. SCNs are broadly defined by properties including physical location, but in contrast to vertebrates and other “model” organisms, aquatic invertebrate SCs do not have clearly documented niche outlines or properties. Life strategies such as regeneration or asexual reproduction may have conditioned the niche architectural variability in aquatic or marine animal groups. By both establishing the invertebrates SCNs as independent types, yet allowing inclusiveness among them, the comparative analysis will allow the future functional characterization of SCNs.

How multiple actin networks coexist in a common cytoplasm while competing for a shared pool of monomers is still an ongoing question. This is exemplified by meiotic maturation in the mouse oocyte, which relies on the dynamic remodeling of distinct cortical and cytoplasmic F-actin networks. Here, we show that the conserved actin-depolymerizing factor cofilin is activated in a switch-like manner upon meiosis resumption from prophase arrest. Interfering with cofilin activation during maturation resulted in widespread elongation of microvilli, while cytoplasmic F-actin was depleted, leading to defects in spindle migration and polar body extrusion. In contrast, cofilin inactivation in metaphase II-arrested oocytes resulted in a shutdown of F-actin dynamics, along with a dramatic overgrowth of the polarized actin cap. However, inhibition of the Arp2/3 complex to promote actin cap disassembly elicited ectopic microvilli outgrowth in the polarized cortex. These data establish cofilin as a key player in actin network homeostasis in oocytes and reveal that microvilli can act as a sink for monomers upon disassembly of a competing network.

Endocrine Disruptors (EDCs) are a class of anthropogenic environmental contaminants that affect organisms by disrupting their endocrine system. Notably, the lack of detailed understanding of endocrine systems and their signaling pathway prevents understanding of the effect of PEs in invertebrates. Accordingly, this thesis aims to address the issue of endocrine disruption in marine invertebrates by exploiting the larval development of the Mediterranean mussel Mytilus galloprovincialis. By applying the logic of the Adverse Outcome Pathway-AOP, the work in this thesis attempted to characterize the mechanism of action of the model EDCs BPA, TBBPA and TBT in mussel larvae and to identify plausible neuroendocrine pathways by which they induce their deleterious biological effect. The neuroendocrine elements regulating the biogenesis of the larval shell have been characterized and the morphogenetic process established as sensitive to neuroendocrine disruption. Plausible neuroendocrine AOPs that could be linked to the biological effects of the model EDCs have been identified. In addition, the possible AOP of TBT has established a plausible relationship between nuclear receptor signaling and the neuroendocrine system of mussel larvae. The results of this thesis represent essential evidence that will help and advance the current understanding of endocrine disruption mechanisms in invertebrate organisms., Les Perturbateurs Endocriniens (PE) sont une classe de contaminants environnementaux d'origine anthropique qui affectent les organismes en perturbant leur système endocrinien. Notamment, le manque de compréhension détaillée des systèmes endocriniens et leur voie de signalisation empêche la compréhension de l’effet de PEs chez les invertébrés. Conformément, cette thèse vise à aborder la question de la perturbation endocrinienne chez les invertébrés marins en exploitant le développement larvaire de la moule méditerranéenne Mytilus galloprovincialis. En appliquant la logique de l’Adverse Outcome Pathway-AOP, le travail de cette thèse vise à caractériser le mécanisme d'action de PEs modèles BPA, TBBPA et TBT chez les larves de moule et à identifier de plausibles voies neuro-endocriniennes par lesquelles ils induisent leur effet biologique délétère. Les éléments neuroendocriniens régulant la biogenèse de la coquille larvaire ont été caractérisés et le processus morphogénétique établi comme sensible à la perturbation neuroendocrinienne. Les plausibles AOPs neuro-endocriniens qui pourraient être liées aux effets biologiques de PEs modèles ont été identifiées. En plus, l'AOP possible du TBT a établi une relation plausible entre la signalisation des Récepteurs Nucléaires et le système neuroendocrinien des larves de moule. Les résultats de cette thèse représentent des preuves essentielles qui aideront et feront avancer la compréhension actuelle des mécanismes de perturbation endocrinienne chez les organismes invertébrés.

Maintaining reproduction in highly variable, often stressful, environments is an essential challenge for all organisms. Even transient exposure to mild environmental stress may directly damage germ cells or simply tax the physiology of an individual, making it difficult to produce quality gametes. In Caenorhabditis elegans, a large fraction of germ cells acts as nurse cells, supporting developing oocytes before eventually undergoing so-called physiological germ cell apoptosis. Although C. elegans apoptosis has been extensively studied, little is known about how germline apoptosis is influenced by ecologically relevant environmental stress. Moreover, it remains unclear to what extent germline apoptosis contributes to maintaining oocyte quality, and thus offspring viability, in such conditions. Here we show that exposure to diverse environmental stressors, likely occurring in the natural C. elegans habitat (starvation, ethanol, acid, and mild oxidative stress), increases germline apoptosis, consistent with previous reports on stress-induced apoptosis. Using loss-of-function mutant alleles of ced-3 and ced-4, we demonstrate that eliminating the core apoptotic machinery strongly reduces embryonic survival when mothers are exposed to such environmental stressors during early adult life. In contrast, mutations in ced-9 and egl-1 that primarily block apoptosis in the soma but not in the germline, did not exhibit such reduced embryonic survival under environmental stress. Therefore, C. elegans germ cell apoptosis plays an essential role in maintaining offspring fitness in adverse environments. Finally, we show that ced-3 and ced-4 mutants exhibit concomitant decreases in embryo size and changes in embryo shape when mothers are exposed to environmental stress. These observations may indicate inadequate oocyte provisioning due to the absence of germ cell apoptosis. Taken together, our results show that the central genes of the apoptosis pathway play a key role in maintaining gamete quality, and thus offspring fitness, under ecologically relevant environmental conditions.

Beyond their biochemical nature, living organisms can also be considered as physical objects, experiencing mechanical constraints. Plants experience growth-derived stress, resulting from the conflict between their high internal turgor pressure (which can reach 1MPa) and the stiff cell wall surrounded plant cells. In my PhD, I focused on the role of these internal mechanical signals during plant development. More specifically, I investigated the contribution of the cell cortex in mechano-transduction. Together with my collaborators, I adapted a single cell approach to investigate the relative contribution of cell geometry and cortical tension on cortical microtubule (CMT) behavior. We found that CMTs align with the cell long axis when protoplasts are confined but not pressurized and with predicted maximal tension direction (transverse axis) when they are confined and pressurized. This suggests that mechanical signals can override geometrical cues. I also found that this response is dynamic: in absence of new mechanical stimulation, MTs align with the cell long axis, indicating that CMTs respond to tension changes rather than pressurization only. In parallel, I also contributed to the development of new genetic tool specifically removing PI(4,5)P2 from the plasma membrane. This highlighted a potential role of PI(4,5)P2 during organ initiation at the shoot apical meristem. Whether PI(4,5)P2 also affects the MT response to stress remains to be explored. CMTs are involved in cellulose synthesis through the guidance of cellulose synthase complexes at the plasma membrane, and thus ensuring anisotropic cell growth. Interestingly, plant overexpressing the phospholipase PLAIIIß display reduced cellulose content and reduced mechanical strength of aerial tissues. I investigated how plasma membrane composition could affect the cellulose synthesis machinery, opening the path to the analysis of the role of plasma membrane composition in CMT response to stress, cellulose synthesis, and development., Au-delà de leur nature biochimique, les organismes vivants sont aussi des objets physiques, subissant des contraintes mécaniques. Les plantes subissent des stress mécaniques internes, résultant du conflit entre leur pression de turgescence interne élevée (pouvant atteindre 1 MPa) et leur paroi cellulaire rigide capable d’y résister. Pendant ma thèse, je me suis intéressée aux rôles de ces signaux mécaniques internes lors du développement des plantes. A travers différents projets, j'ai étudié la contribution du cortex cellulaire dans la mécano-transduction. Avec mes collaborateurs, j'ai adapté une approche cellule unique pour étudier la contribution relative de la géométrie cellulaire et de la tension corticale sur le comportement des microtubules corticaux (CMT). Nous avons observé que les CMTs s'alignent sur l'axe long de la cellule lorsque les protoplastes sont confinés mais non pressurisés et avec la direction de tension maximale prédite (axe transversal) lorsque les protoplastes sont confinés et pressurisés. Cela suggère que les signaux mécaniques peuvent contrebalancer les contraintes géométriques. Cette réponse semble dynamique puisqu’en l'absence de nouvelle stimulation mécanique, les CMTs s'alignent sur l'axe long de la cellule : les CMTs répondent aux changements de tension plutôt qu'à la pressurisation uniquement. En parallèle, j'ai également contribué au développement d'un nouvel outil génétique éliminant spécifiquement le phosphoinositide PI(4,5)P2 de la membrane plasmique, et révélant ainsi un rôle potentiel du PI(4,5)P2 lors de l’organogénèse dans le méristème apical. Par ailleurs, les CMTs sont également impliqués dans le processus de synthèse de la cellulose, un composant majeur des parois cellulaires. Les plantes sur-exprimant la phospholipase PLAIIIß présentent une teneur en cellulose diminuée et une résistance mécanique réduite des tissus aériens. J'ai étudié comment l’altération de la composition de la membrane plasmique pouvait affecter la machinerie de synthèse de la cellulose, ouvrant la voie à l'analyse du rôle de la composition de la membrane plasmique dans la réponse des CMTs aux contraintes mécaniques, à la synthèse de la cellulose et au développement.

Background Increased embryo loss as mares become older is a major consideration for breeders as older animals are currently used for reproduction in the equine industry. Lower embryo quality has been pointed out as partly responsible for this reduced fertility. Here, the effect of mare’s age on blastocysts’ gene expression was explored. Day 8 post ovulation embryos were collected by uterine flushing from multiparous young (YM, 6-year-old, N = 5) and older (OM, > 10-year-old, N = 6) non-nursing Saddlebred mares, inseminated with the semen of one stallion. Pure (TE_part) or inner cell mass enriched (ICMandTE) trophoblast were obtained by embryo bisection and paired end, non-oriented RNA sequencing (Illumina, NextSeq500) was performed on each hemi-embryo. To discriminate gene expression in the ICM from that in the TE, deconvolution (DeMixT R package) was used on the ICMandTE dataset. Differential expression was analyzed (DESeq2) with embryo sex and diameter as cofactors using a false discovery rate < 0.05 cutoff. Functional annotation and classification of differentially expressed genes and gene set enrichment analysis were also performed. Results :Maternal aging did not affect embryo recovery rate, embryo diameter nor total RNA quantity but modified gene expression in equine D8 embryos. The expression of genes in the ICM seemed to be more altered by maternal aging than in the TE. In both compartments, the expression of genes involved in mitochondrial function, translation and transcription due to chromatin modification were disturbed by maternal age. Mitosis, signaling and adhesion pathways and embryo development were particularly decreased in the ICM hemi-embryos from old mares. Finally, in TE, ion movement related genes were affected. Conclusions :This is the first study showing an effect of maternal age on gene expression in the equine blastocyst at Day 8 post ovulation. Maternal age, even for mares as young as 10 years old, disturbs mitosis, translation and transcription, cell signaling and adhesion as well as mitochondrial function and cell commitment of horse embryos. These perturbations may affect further embryo development and contribute to decreased fertility due to aging.

My thesis is devoted to the question of how few myogenic progenitor cells (MPCs) can give rise to the complex pattern of mammalian limb musculature.Experimental embryology supports the concept that limb MPCs possess no positional information. In this view, the developing muscle connective tissue (MCT) establishes a prepattern that is invaded by naïve MPCs, which ultimately form individual muscles. Contrary to this concept, Hox proteins, homeobox transcription factors that define the body plan, were shown to accumulate in limb MPCs, suggesting that MPCs may acquire positional identity. Bone morphogenetic proteins (BMPs), diffusible growth factors expressed in the limb mesenchyme, surround developing limb muscles and are thought to guide positioning of premuscle masses; however, whether this involves positional information remains unexplored. Most of current knowledge is based on experiments on chicken and has not been substantiated using novel transgenic mammalian models.The principal hypothesis of my thesis is that BMPs signal to embryonic limb MPCs, thereby guiding patterning of limb muscles. I asked, whether MPCs acquire positional information, and whether this is dependent on BMP signalling. I therefore formulated the following aims: i) to confirm the responsiveness of MPCs to BMP signalling, ii) to validate transgenic mouse models which conditionally abrogate BMP signalling in the myogenic lineage, iii) to determine the consequences of BMP signalling abrogation on early development and final patterning of limb muscles, and iv) to determine whether MPCs possess positional identity and whether this depends on BMP signalling.In my thesis I demonstrated that migrating limb MPCs highly accumulated phosphorylated Smad1/5/9, evidence for active BMP signalling. To test for the role of BMP signalling, I generated Lbx1Cre; Rosa26-LoxP-Stop-LoxP-huSMAD6-IRES-EGFP (Lbx1Cre;RS6) transgenic mice, which overexpressed BMP inhibitory human SMAD6 in migrating limb MPCs. Lbx1Cre;RS6 embryos showed impaired proliferation, distal migration and precocious differentiation of limb MPCs. Limb muscles appeared smaller, consisted of fewer myofibers, and mispatterned. I conducted a comparative anatomical study of Lbx1Cre;RS6 and control limbs and revealed muscle mispatterning with increasing severity from proximal towards distally. Certain muscles were either fused, absent or connected to wrong tendons. Moreover, I found that MCT adapted to the muscle pattern changes. Next, I cell-autonomously abrogated BMP signalling in differentiated limb muscles by crossing HSA-Cre mice with RS6 mice. HSA-Cre;RS6 foetues developed smaller limb muscles, however, overall pattern was preserved, suggesting that MPCs receive positional information prior to differentiation. Lastly, I presented data from single cell RNA sequencing, obtained in collaboration with external laboratories, showing that limb MPCs heterogeneously expressed genes of the Hoxa and Hoxd clusters. Hox-expressing MPCs displayed high BMP signalling activity. Using immunohistochemistry, I confirmed that early migrating limb MPCs highly expressed Hoxa11 protein, which was rapidly lost during myogenic lineage progression. I also showed that MPCs sequentially upregulated Hox proteins, and this was dependent on their distal progression and corresponded to Hox-defined proximo-distal limb regions. In Lbx1Cre;RS6 embryos, limb MPCs failed to sequentially upregulate Hoxa11 and Hoxd13 proteins, and no MPCs were detected in the presumptive Hoxd13-expressing autopod region, which explains their distal amyoplasia.In conclusion, I provide evidence that BMP signalling controls proximo-distal patterning of limb muscles, probably by regulating Hox-defined positional identity in MPCs during early embryonic limb development. I propose that proximo-distal limb muscle patterning depends on Hox gene collinearity in migrating MPC, a hypothesis that remains being explored in future., Ma thèse est consacrée à la question de savoir comment peu de cellules progénitrices myogéniques (MPC) peuvent donner naissance au schéma complexe de la musculature des membres des mammifères.La compréhension actuelle est que les MPC des membres ne possèdent aucune information de position et le tissu conjonctif musculaire (MCT) établit un pré-modèle qui est envahi par des MPC naïfs, qui forment des muscles. Contrairement, il a été démontré que les protéines Hox, qui définissent le plan corporel, s'accumulent dans les MPC, suggérant que les MPC peuvent acquérir une identité de position. Les protéines morphogénétiques osseuses (BMP), qui s'expriment dans le mésenchyme des membres, entourent les muscles en développement et guident le positionnement des masses pré-musculaires; Cependant, si cela implique des informations de position reste inexploré. La compréhension actuelle est basée sur des expériences sur le poulet et n'ont pas été étayées par de nouveaux modèles de mammifères transgéniques.L'hypothèse principale de ma thèse est que les BMP signalent aux MPC, guidant la structuration musculaire. J'ai demandé si les MPC acquièrent des informations de position et si cela dépend de la signalisation BMP. J'ai formulé ces objectifs: i) confirmer la réactivité des MPC à la signalisation BMP, ii) valider des modèles de souris transgéniques abrogeant conditionnellement la signalisation BMP dans la lignée myogénique, iii) déterminer les conséquences de l'abrogation de la signalisation BMP sur le développement précoce et la structuration finale des muscles, et iv) déterminer si les MPC possèdent une identité de position et si cela dépend de la signalisation BMP.Dans ma thèse, j'ai démontré que les MPC accumulaient les Smad1/5/9 phosphorylés, preuve d'une signalisation BMP active. Pour tester le rôle de la signalisation BMP, j'ai généré les souris transgéniques Lbx1Cre;RS6, qui ont surexprimé le SMAD6 inhibiteur de BMP dans les MPC des membres en migration. Les embryons Lbx1Cre;RS6 ont montré prolifération et migration distale altérées et une différenciation précoce des MPC. Les muscles semblaient petits, se composaient de moins de myofibres et avaient une mauvaise configuration. J'ai révélé une mauvaise configuration musculaire avec une sévérité croissante du proximal vers le distal. Certains muscles étaient soit soudés, absents ou connectés à de mauvais tendons. Le MCT s'adaptait aux changements musculaires.Ensuite, j'ai abrogé la signalisation BMP dans les muscles différenciés en en générant les souris HSA-Cre;RS6. Les foetus ont développé des muscles plus petits, cependant, le schéma a été préservé, suggérant que les MPC reçoivent des informations de position avant la différenciation.Enfin, j'ai présenté des données de séquençage d'ARN monocellulaire, en collaboration avec des laboratoires externes, montrant que les MPC exprimaient les gènes Hoxa et Hoxd. Les MPC exprimant Hox ont affiché une activité de signalisation BMP élevée. En utilisant l'immunohistochimie, j'ai confirmé que les MPC précoce exprimaient la protéine Hoxa11, qui était rapidement perdue au cours de développement. J'ai également montré que les MPC augmentaient séquentiellement les protéines Hox, et cela dépendait de leur progression distale et correspondait aux régions des membres proximo-distales définies par Hox. Dans les embryons Lbx1Cre;RS6, les MPC n'ont pas réussi à réguler à la hausse de manière séquentielle les protéines Hoxa11 et Hoxd13, et aucun MPC n'a été détecté dans la région présumée des autopodes exprimant Hoxd13, ce qui explique leur amyoplasie distale.En conclusion, je fournis la preuve que la signalisation BMP contrôle la structuration proximo-distale des muscles des membres, probablement en régulant l'identité positionnelle définie par Hox dans les MPC précoces. Je propose que la structuration des muscles des membres proximo-distaux dépend de la colinéarité du gène Hox dans la migration des MPC, une hypothèse qui reste à l'étude à l'avenir.

SummaryThe epiblast (Epi) is the source of embryonic stem (ES) cells and all embryonic tissues. It differentiates alongside the primitive endoderm (PrE) in a randomly distributed “salt and pepper” pattern from the inner cell mass (ICM) during preimplantation of the mammalian embryo. NANOG and GATA6 are key regulators of this binary differentiation event, which is further modulated by heterogeneous FGF signalling. When and how Epi and PrE lineage specification is initiated within the developing embryo is still unclear. Here we generated NANOG and GATA6 double KO (DKO) mouse embryos and performed single-cell expression analyses. We found that the ICM was unable to differentiate in theDKOmice, allowing us to characterize the ICM precursor state. The normally heterogeneous expression ofFgf4between cells was significantly reduced inDKOICMs, impairing FGF signalling. In contrast, several pluripotency markers did still display cell-to-cell expression variability inDKOICMs. This revealed a primary heterogeneity independent of NANOG, GATA6 and FGF signalling that may also be conserved in humans. We found that NANOG is key in the initiation of epiblast specification already between the 16- and 32-cell stages, enabling the cell-clustered expression of many pluripotency genes. Our data uncover previously unknown biology in the early mouse embryo with potential implications for the field of pluripotent stem cells in human and other mammals.

To investigate the role of mechanical constraints in morphogenesis and development, we have developed a pipeline of techniques based on incompressible elastic sensors. These techniques combine the advantages of incompressible liquid droplets, which have been used as precise in situ shear stress sensors, and of elastic compressible beads, which are easier to tune and to use. Droplets of a polydimethylsiloxane mix, made fluorescent through specific covalent binding to a rhodamin dye, are produced by a microfluidics device. The elastomer rigidity after polymerization is adjusted to the tissue rigidity. Its mechanical properties are carefully calibrated in situ, for a sensor embedded in a cell aggregate submitted to uniaxial compression. The local shear stress tensor is retrieved from the sensor shape, accurately reconstructed through an active contour method. In vitro, within cell aggregates, and in vivo, in the prechordal plate of the zebrafish embryo during gastrulation, our pipeline of techniques demonstrates its efficiency to directly measure the three dimensional shear stress repartition within a tissue.

AROMATASE, encoded by the CYP19A1 gene, is the cytochrome enzyme responsible for the synthesis of estrogens in vertebrates. In most mammals a peak of expression of the CYP19A1 gene occurs in the fetal XX gonad when sexual differentiation starts up. To elucidate the role of this peak, we produced 3 lines of TALEN genetically edited CYP19A1 KO rabbits, that were void of any production of estradiol. All KO XX rabbits developed as females, with aberrantly small sized ovaries at adulthood, an almost empty reserve of primordial follicles and very few large antrum follicles. Ovulation never occurred. Our histological, immunohistological and transcriptomic analyses showed that the surge of estradiol in the XX fetal rabbit gonad is dispensable for its determination as an ovary, or for meiosis. However, it is mandatory for the high proliferation and differentiation of both somatic and germ cells, and consequently for the establishment of the ovarian reserve.

During pre-implantation development, the mammalian embryo forms the blastocyst, which is the structure embedding the embryo into the uterus. The shaping of the blastocyst relies in large part on contractile forces generated by the actomyosin cortex. In the mouse, on timescales of seconds, we observe pulses of actomyosin contractions traveling periodically around the cell perimeter. We call this phenomenon periodic cortical waves of contraction (PeCoWaCo), a fascinating and poorly understood manifestation of contractility. In this study, we take advantage of the slow development of the mouse embryo to study thousands of contraction pulses and of the robustness of the mouse embryo to size manipulation to explore the biophysical properties of PeCoWaCo during the cleavage stages preceding early mammalian morphogenesis. We find that, during cleavage stages, periodic movements appear occasionally at the zygote and the 2-cell stage and become systematic after the 2nd round of cleavage divisions. Interestingly, the period of oscillations progressively decreases from 200 s at the zygote stage to 80 s at the 8-cell stage. Since cells becomes successively smaller with successive cleavage divisions, cell size could be an important determinant in the initiation and regulation of PeCoWaCo. We manipulate cell size on a broad range of cell radii (10-40 µm) using fragmentation and fusion of cells and find that the initiation, persistence or properties of PeCoWaCo do not depend on cell size. Following the period of PeCoWaCo, we discover that blastomeres gradually decrease their surface tensions until the 8-cell stage and that artificially softening cells enhances PeCoWaCo prematurely. Therefore, during cleavage stages, cortical softening awakens zygotic contractility before preimplantation morphogenesis. In addition, by manipulating actomyosin contractility using mutants and drugs, we showed that the period of PeCoWaCo can be tuned by F-actin polymerization rate and myosin motor activity. Altogether our results on biophysical and molecular aspects of PeCoWaCo help us understand how actomyosin contractility awakens before preimplantation morphogenesis and how it is regulated both mechanically and molecularly., Au cours du développement préimplantatoire, l'embryon de mammifère forme le blastocyste, qui est la structure fixant l'embryon dans l'utérus. La formation du blastocyste repose en grande partie sur les forces contractiles générées par le cortex d'actomyosine. Chez la souris, sur des échelles de temps de quelques secondes, nous observons des impulsions de contractions d'actomyosine voyageant périodiquement autour du périmètre cellulaire. Nous appelons ce phénomène vagues de contraction corticale périodique (PeCoWaCo), une manifestation fascinante et mal comprise de la contractilité. Dans cette étude, nous profitons du développement lent de l'embryon de souris pour étudier des milliers d'impulsions de contraction. Nous mettons également à profit la robustesse de ce développement pour explorer les propriétés biophysiques des PeCoWaCo pendant les étapes de clivage précédant la morphogenèse précoce des mammifères.Nous constatons que, lors des étapes de clivage, des mouvements périodiques apparaissent occasionnellement au stade zygote et 2-cellules puis deviennent systématiques après le 2ème cycle de divisions de clivage. Fait intéressant, la période des oscillations diminue progressivement de 200s au stade zygote à 80s au stade 8-cellules. Étant donné que les cellules deviennent de plus en plus petites avec des divisions de clivage successives, la taille des cellules pourrait être un déterminant important dans l'initiation et la régulation des PeCoWaCo. Nous manipulons la taille des cellules sur une large gamme de rayons cellulaires (10-40 µm) en utilisant la fragmentation et la fusion des cellules et constatons que l'initiation, la persistance ou les propriétés des PeCoWaCo ne dépendent pas de la taille des cellules. Après la période des PeCoWaCo, on découvre que les tensions de surface des blastomères diminuent progressivement jusqu'au stade de 8-cellules et que l’assouplissement artificiel des cellules augmente prématurément les PeCoWaCo. Par conséquent, lors des étapes de clivage, l’assouplissement cortical réveille la contractilité zygotique avant la morphogenèse préimplantatoire. En plus, en manipulant la contractilité de l'actomyosine à l'aide de mutants et des drogues, nous avons montré que la période des PeCoWaCo peut être contrôlée par la régulation du taux de polymérisation de l'actine filamenteuse et de l'activité motrice de la myosine.Dans l'ensemble, nos résultats sur les aspects biophysiques et moléculaires du PeCoWaCo nous aident à comprendre comment la contractilité de l'actomyosine s'éveille avant la morphogenèse préimplantatoire et comment elle est régulée au niveau mécanique et moléculaire., İmplantasyon öncesi gelişim sırasında, memeli embriyosu, embriyoyu rahim içineyerleştiren yapı olan blastosisti oluşturur. Blastosistin şekillendirilmesi büyük ölçüdeaktomiyozin korteks tarafından oluşturulan kasılma kuvvetlerine dayanır. Farede, saniyelikzaman ölçeklerinde, hücre çevresinde periyodik olarak dolaşan aktomiyozin kasılmalarınındarbeleri gözlemlenebilir. Bu fenomene, kasılmanın büyüleyici ve yeterince anlaşılmamış birtezahürü olan periyodik kortikal kasılma dalgaları (periodic cortical waves of contraction:PeCoWaCo) diyoruz. Bu çalışmada, erken memeli morfogenezinden önceki bölünmeaşamaları sırasında PeCoWaCo'nun biyofiziksel özelliklerini keşfetmek ve binlerce kasılmadarbesini inceleyebilmek için fare embriyosunun yavaş gelişiminden ve fare embriyosununboyut manipülasyonuna dayanıklılığından faydalandık.Bölünme aşamaları sırasında, zigotta ve 2 hücreli aşamada periyodik hareketlerinzaman zaman ortaya çıktığını ve ikinci tur bölünmeden sonra bu hareketlerin sistematik halegeldiğini bulduk. İlginç bir şekilde, salınım periyodunun zigot aşamasında 200 saniyeden, 8hücreli aşamada 80 saniyeye sistematik olarak azaldığını gözlemledik. Hücreler ardışıkbölünmeleriyle sürekli küçüldüğünden, hücre boyutu PeCoWaCo'nun başlatılmasında vedüzenlenmesinde önemli bir belirleyici olabilir. Hücreleri geniş bir hücre yarıçapı aralığında(10-40 μm) küçük parçalara bölerek veya birbirleriyle birleştirerek PeCoWaCo'nunbaşlatılmasının, kalıcılığının veya genel özelliklerinin hücre boyutuna bağlı olmadığını bulduk.PeCoWaCo periyodunu takiben, embriyo hücrelerinin zigottan 8 hücreli aşamaya kadar yüzeygerilimini kademeli olarak azalttığını ve yapay olarak korteksleri yumuşatılan hücrelerinPeCoWaCo'yu zamanından önce geliştirdiğini keşfettik. Bu sonuçlarla bölünme aşamalarısırasında, kortikal yumuşama, ilke implantasyon öncesi morfogenezinden önce zigotikkasılmaları uyandırdığını gösterdik. Ayrıca, genetik mutantlar ve kimyasallar kullanarakaktomiyozin kasılmasını manipüle ederek, PeCoWaCo periyodunun F-aktin polimerizasyonhızı ve miyozin motor aktivitesinin düzenlenmesi ile ayarlanabileceğini gösterdik.Sonuç olarak, PeCoWaCo'nun biyofiziksel ve moleküler yönleriyle ilgili bulgularımız,aktomiyosin kontraktilitesinin implantasyon öncesi morfogenezinden önce nasıl uyandığını,ayrıca hem mekanik hem de moleküler olarak nasıl düzenlendiğini anlamamıza yardımcı olur.