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International audience; Sperm cells provide essential, if usually diminutive, ingredients to successful sexual reproduction. Despite this conserved function, sperm competition and coevolution with female traits can drive spectacular morphological change in these cells. Here, we characterize four repeated instances of convergent evolution of sperm gigantism in Caenorhabditis nematodes using phylogenetic comparative methods on 26 species. Species at the extreme end of the 50‐fold range of sperm‐cell volumes across the genus have sperm capable of comprising up to 5% of egg‐cell volume, representing severe attenuation of the magnitude of anisogamy. Furthermore, we uncover significant differences in mean and variance of sperm size among genotypes, between sexes, and within and between individuals of identical genotypes. We demonstrate that the developmental basis of sperm size variation, both within and between species, becomes established during an early stage of sperm development at the formation of primary spermatocytes, while subsequent meiotic divisions contribute little further sperm size variability. These findings provide first insights into the developmental determinants of inter‐ and intraspecific sperm size differences in Caenorhabditis. We hypothesize that life history and ecological differences among species favored the evolution of alternative sperm competition strategies toward either many smaller sperm or fewer larger sperm.

International audience; Thermal developmental plasticity represents a key organismal adaptation to maintain reproductive capacity in contrasting and fluctuating temperature niches. Although extensively studied, research on thermal plasticity has mainly focused on phenotypic outcomes, such as adult life history, rather than directly measuring plasticity of underlying developmental processes. How thermal plasticity of developmental phenotypes maps into plasticity of resulting final phenotypes, and how such mapping relationships evolve, thus remain poorly understood. Here we address these questions by quantifying thermal plasticity of Caenorhabditis hermaphrodite germline development. We integrate measurements of germline development and fertility at the upper thermal range in isolates of C. briggsae, C. elegans, and C. tropicalis. First, we compare intra- and interspecific variation in thermal germline plasticity with plasticity in reproductive output. Second, we ask whether the developmental errors leading to fertility break-down at upper thermal limits are evolutionarily conserved. We find that temperature variation modulates spermatogenesis, oogenesis and germ cell progenitor pools, yet the thermal sensitivity of these processes varies among isolates and species, consistent with evolutionary variation in upper thermal limits of hermaphrodite fertility. Although defective sperm function is a major contributor to heat-induced fertility break-down, high temperature also significantly perturbs oogenesis, germline integrity, and mitosis-meiosis progression. Remarkably, the occurrence and frequency of specific errors are strongly species- and genotype-dependent, indicative of evolutionary divergence in thermal sensitivity of distinct processes in germline development. Therefore, the Caenorhabditis reproductive system displays complex genotype-by-temperature interactions at the developmental level, which may remain masked when studying thermal plasticity exclusively at the life history level.

International audience; Sperm competition is a key force driving evolutionary diversification of sperm morphology. In Caenorhabditis nematodes, sperm size is a major factor determining sperm competitiveness: male sperm is larger and consistently outcompetes hermaphrodite sperm, and larger male sperm outcompetes smaller male sperm. To generate an in-depth overview of sperm size variation in Caenorhabditis nematodes, we quantified male sperm size in 100 C. elegans wild isolates from a world-wide collection and in the 26 currently described Caenorhabditis species, including the three androdioecious species. Analysis of C. elegans wild isolates indicates significant levels of heritable variation in mean male sperm size, but Genome-Wide Association Mapping did not allow detection of any significant QTL underlying this variation. The observed sperm size distributions across strains indicate a complex genetic basis involving many loci of small effect. This survey of intraspecific variation also uncovered a strongly reduced mean male sperm size in LSJ1 and LSJ2 strains, recently derived from the reference strain N2. We show through quantitative complementation tests that a nurf-1 deletion in LSJ strains is responsible for reduced male sperm size, suggesting a role of the Nucleosome Remodeling Factor (NURF)-like complex in C. elegans spermatogenesis. Our quantification of sperm size evolution in different Caenorhabditis species confirmed earlier reports that mean sperm size of gonochoristic species is globally larger than in males of androdioecious species, although exceptions occur. While species of the elegans group all display relatively small, and similar, male sperm size, several gonochoristic species outside this group show convergent evolution of distinct, extremely large male sperm. In addition, within-species genetic variation in male sperm size is considerable in all examined gonochoristic and androdioecious species. We present experimental results aimed at understanding how germline and sperm development shape such dramatic variation in sperm size across species, isolate, sex and individual.

International audience; We are interested in understanding how environmental variation modulates developmental processes and resulting phenotypic variation. We focus on the C. elegans germline, a molecularly well-tractable system that shows a high degree of environmental sensitivity. The basic germline organization is conserved among Caenorhabditis species, however reproductive schedules and fecundity are very variable between species and isolates. Yet, it remains unclear how evolution of such life history traits is explained by changes in germline properties, e.g. sperm number, oocyte quality or the sperm-oocyte switch. We aim to link environmental and evolutionary variability of the Caenorhabditis germline to better understand genotype-by-environment interactions for developmental traits underlying fitness-related characters. Our first aim was to characterize how germline proliferation and differentiation as well as fecundity are modified in animals exposed to experimental conditions mimicking ecologically relevant environments. Here we present how one specific environment -high temperature exposure (27°C)- affects germline and reproductive properties. In addition to previously reported deleterious effects on sperm function, we found that high temperature significantly impacts germline proliferation and differentiation, apparently through modifications of Distal Tip Cell morphology and signalling through the Delta/Notch pathway. Our second aim was to quantify genotype-by-environment interactions by analysing how isolates of the three hermaphroditic species (C. elegans, C. briggsae and C. sp. 11) differ in germline plasticity across different environments. We uncover significant genotype-by-environment interactions for germline and fecundity in response to high temperature. More specifically, we find that the thermal limits of reproduction are species-specific and may be associated with particular germline defects. We are now using this experimental paradigm to explore how environmental factors impact the Caenorhabditis reproductive system and how germline integrity is maintained in variable environments. Please note: Abstract shown here should NOT be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

International audience; Embryonic stem (ES) cells are pluripotent cells derived from blastocyst-stage embryos. They are characterized by their infinite self-renewal capacity and their ability to differentiate into many cell types in vitro as well as in vivo. The present protocol describes culture conditions that allow efficient differentiation of mouse ES cells toward the endothelial lineage, both in two-dimensional cultures and in three-dimensional, multicellular embryoid bodies. We also provide a protocol for establishing recombinant ES cell clones, giving the example of cells expressing green fluorescent protein and an antibiotic resistance gene under the control of an endothelial-specific promoter. These transgenes allow the visualization and the genetic selection of endothelial cells from a heterogeneous population of differentiating ES cells. Potential applications for these differentiation models are given, including the study of the endothelial cell lineage and its derivatives, the testing of molecules involved in angiogenesis, and the potential use of selected endothelial cells in vivo.

International audience; The partial sequence of the increasing capillary permeability protein (ICPP) purified from Vipera lebetina venom revealed a strong homology to vascular endothelial growth factor (VEGF)-A. We now report its complete amino acid sequence determined by Edman degradation and its biological effects on mouse and human vascular endothelial cells. ICPP is a homodimeric protein linked by cysteine disulfide bonds of 25115 Da revealed by mass spectrometry. Each monomer is composed of 110 amino acids including eight cysteine residues and a pyroglutamic acid at the N-terminal extremity. ICPP shares 52% sequence identity with human VEGF but lacks the heparin binding domain and Asn glycosylation site. Besides its strong capillary permeability activity, ICPP was found to be a potent in vitro angiogenic factor when added to mouse embryonic stem cells or human umbilical vein endothelial cells. ICPP was found to be as potent as human VEGF165 in activating p42/p44 MAPK, in reinitiation of DNA synthesis in human umbilical vein endothelial cells, and in promoting in vitro angiogenesis of mouse embryonic stem cells. All these biological actions, including capillary permeability in mice, were fully inhibited by 1 microm of a new specific VEGF receptor tyrosine kinase inhibitor (ZM317450) from AstraZeneca that belongs to the anilinocinnoline family of compounds. Indeed, up to a 30 times higher concentration of inhibitor did not affect platelet-derived growth factor, epidermal growth factor, FGF-2, insulin, alpha-thrombin, or fetal calf serum-induced p42/p44 MAPK and reinitiation of DNA synthesis. Therefore, we conclude that this venom-derived ICPP exerts its biological action (permeability and angiogenesis) through activation of VEGF receptor signaling (VEGF-R2 and possibly VEGF-R1).