Embryonic development represents a source of inspiration for the design of active polymer materials capable of working out-of-equilibrium, using energy to perform user-defined tasks. During development, the cortex – a thin reticulated polymer gel composed of actin filaments and myosin motors – is assembled beneath the cell surface. Fueled by ATP, this active material drives the cell shape changes that underlie morphogenesis from cell to organism. Here, we propose to combine engineering and biology approaches to address the biological mechanisms that underlie morphogenesis. Focusing on two key aspects, energy consumption and system size, we will infer key physical parameters of the system and engineer mimetic systems capable of reproducing developmental morphogenetic behaviors. At the interface of polymer material science and biology, our work will both help us to understand the mechanisms underlying morphogenesis in vivo and lead to the development of morphogenetic materials in vitro.

Haploid gametes, named oocytes and spermatozoa, are generated through two specialized cell divisions without intervening S-phase, from a diploid precursor cell. In the first division, named meiosis I, paired chromosomes are segregated and distributed into two daughter cells, whereas in meiosis II and mitosis, sister chromatids are segregated. Missegregations of the genetic material in meiosis have severe consequences, because they lead to the generation of aneuploid gametes (harboring the wrong number of chromosomes). In humans, meiosis is error prone, with an estimated 20 % of fertilizable oocytes being aneuploid. Trisomies, such as trisomy 21, are due to missegregations of a chromosome in meiosis. Furthermore, oocytes of women closer to menopause show a dramatic age-dependent increase in meiotic missegregations. Using ascidian and mouse oocytes we aim at elucidating the mechanisms of chromosome segregation during female meiosis to understand why it can go awry. We study how chromosomes are held together through a protein complex named Cohesin, which has to be removed in a two-step process during the two meiotic divisions. A pool of Cohesin is protected from removal in meiosis I, but not meiosis II. Whereas the mechanisms of Cohesin protection start to be better understood, its "deprotection" is largely unknown. We will determine how this "deprotection" in meiosis II takes place.

Transposable Elements (TEs) account for 20% of the Drosophila genome (50% in human). Their mobilization in germ cells has dramatic consequences since it induces mutations and chromosomal rearrangements transmitted to the next generation. The activity of TE is thus strongly silenced in these cells by piRNAs, a class of 23-28nt long PIWI-associated small RNAs. However, to create genomic diversity the germ cell DNA information transmitted to the next generation must also be partially reshuffled. It was suggested that a supervised activity of TEs which could be achieved through a partial release of TE repression during oogenesis, might help generating this diversity. We have found that repression of P-element and Idefix TEs is strongly weakened at the most anterior part of the ovariole, in a structure called germarium that contains the dividing cysts. In addition, we found that Piwi is down-regulated in this part of the germarium accordingly called the “Piwiless pocket” (pilp). In contrast, in later oogenesis, a clear random on/off bimodal distribution of repression is observed. This variegation suggests that no transition between on and off states occurs after germarium stages. Thus, TE repression can show both plasticity in early oogenesis and cellular memory in late oogenesis. Our 1st objective is to study how modulation of the piRNA silencing pathway may regulate TE repression along germ cell differentiation, allowing an appropriate balance between genome protection and genome plasticity. To this aim, we will perform 1- in situ analyses of piRNA pathway gene expression as well as of TE expression in the pilp, 2- Micro-dissection of pilp and RNA deep-sequencing analysis, 3- Ectopic expression of Piwi in the pilp and analysis of its impact on oogenesis, gene expression and TE activity (including telomeric TE), 4- Investigation of egg chamber silencing stability using GFP-based sensors. 5- Transplantation of single germarium GSC and live monitoring of silencing capacity in later oogenesis. The biogenesis of piRNAs is still poorly understood. In a screen for novel genes involved in germ cells differentiation, we isolated a mutation of the rpp30 gene that disrupts piRNAs biogenesis. The Rpp30 protein, is a subunit of the RNase P complexe, involved in the endonucleolytic maturation of pre-tRNAs. Our 2nd objective is to understand the role of Rpp30 in piRNA biogenesis. We will test 2 hypotheses. First, RNase P could be directly required to cleave long piRNA precursors. Secondly, tRNA fragments (tRFs) generated by Rpp30 could directly silence TEs, or participate in piRNA amplification. tRFs are a novel class of small RNAs conserved in vertebrates, but their function is unknown. Analyses will involve 1- characterization of Rpp30 sub-cellular localization and of its expression during oogenesis, 2- analysis of proteins and of cellular RNAs associated to Rpp30 by mass spectrometry and deep-sequencing, 3- profiling of tRFs in WT and piRNA mutants, 4- investigation of tRFs roles in TE repression (injection of antisense tRFs in embryos pole cells). The third objective is transverse to Objectives 1 and 2. It is aimed at 1- managing, 2- mining all the sequencing datasets generated by the project to profile piRNAs, tRFs and mRNAs, 3- providing new visualization tools, 4- developing a new algorithm to analyze the piRNA signatures at genome-wide level. Thus, this project will contribute to better understand the plasticity of piRNA-mediated TE repression during oogenesis and to decipher the functional interactions between piRNAs and the new class of tRFs. In addition it provides the opportunity to develop new bioinformatic tools for small RNA deep sequencing analyses and to as well as a pilot structuration for collaborative projects that require the mining of large biological datasets.

Non-coding RNAs are emerging actors in the adaptation of plants to environmental constraints. Indeed, alteration in gene regulation, linked to the non-coding portion of the genome, rather than changes in protein coding genes, may be major forces acting in evolution and adaptation. Phosphorus is an essential mineral for plants and very often a limiting factor for crop yield. World phosphorus resources are expected to be exhausted before the end of the XXI century. Molecular and genetic approaches have revealed several regulatory elements controlling many responses triggered in plants to cope with phosphate starvation, notably root architecture, and significant differences were found among Arabidopsis ecotypes on root growth in phosphate depleted soils. We performed QTL studies that revealed the root tip as a central organ for Pi-sensing and the control of root growth. Furthermore, a consequent genetic approach identified many mutants affected in this root growth response. In parallel, the importance of transcriptomic control and of specific non coding RNAs in plant responses to phosphate starvation was established. In the present project we propose to analyze the root apex response to an environmental constraint using global genomic analysis on different Arabidopsis ecotypes showing contrasting adaptation of their root growth to phosphate starvation. We will analyze complete transcriptomes of mRNAs, non-coding RNAs and small RNAs in root apexes submitted to low and high phosphate and establish regulatory correlations between them. The advantages of the well-studied accessions of Arabidopsis together with advanced genomics approaches (RNAseq) will serve to address the impact of non-coding RNAs in adaptation to environmental constraints and in the evolution of gene regulation. We will assess the degree of evolution of expressed non-coding RNAs among ecotypes to link root adaptive traits to phosphate starvation and specific non-coding RNAs. Using the mutant collection in the Columbia ecotype affected in the responses of roots to phosphate starvation, the proposed regulatory interactions between non-coding RNAs and root growth will be further confirmed. Functional analysis of the identified key non-coding RNAs based on their inactivation and cell-specific modification will be used to define their role in the control of root architecture adaptation to low phosphate. This innovative project uses genome-wide comparisons of differently adapted genotypes to identify regulatory cascades linking root growth and phosphate nutrition. The fundamental and applied data that we expect to obtain would hopefully shed light on global regulatory mechanisms controlling root growth in phosphate-depleted soils and may have a direct impact on crop productivity through novel original patents in this field. More generally, this project will give novel insights on the role of regulatory non-coding RNAs in the evolution of gene regulation in response to the environment.