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Human Gonad Developmental Cell Atlas

Periodic Reporting for period 1 - HUGODECA (Human Gonad Developmental Cell Atlas)

Reporting period: 2020-01-01 to 2020-12-31

Animals rely on genetic factors to determine sex. In humans and other mammals, chromosomes X and Y condition sex. Individuals with two X chromosomes are genetically female, while individuals carrying one X and one Y chromosome are male. The reproduction of all vertebrate species depends on the development of reproductive organs, the gonads. In human, male gonads are the testis which produce sperm and female gonads are the ovaries which produce oocytes. In human, testis and ovaries develop from the same embryonic structure, which is initially bipotential and morphologically indistinguishable in XX and XY embryos. Around 6 weeks of gestation, a distinct developmental program is activated in male and female which leads to the rapid differentiation of testis or ovaries and of male or female external genital organs.
Differences of sex development (DSD) is a unifying term which describes individuals in whom the development of chromosomal, gonadal, or anatomical sex is atypical. DSD account for 7.5% of all birth defects. These disorders have either genetic origin or can be induced by environmental factors.
The goal of the HUGODECA project is to describe the organization of the developing human gonads and to understand the molecular and cellular mechanisms underlying sex determination. HUGODECA gathers 9 teams of researchers, clinicians and companies working all around Europe.
The HUGODECA project is organized in 5 work packages dedicated to:
• The identification of the different cell types that are present in the gonads before and during human sex determination, using cutting-edge gene profiling methods.
• The construction of the first cellular atlas of the developing gonads and genital tracts at a micrometer scale resolution, using the most advanced 2D and 3D microscopy technologies.
• The implementation of novel data processing pipelines for the integration of multidimensional and complex biological data (genetic, bioinformatics, images etc)
• The development of new assays allowing to analyze the influence of environmental factors such as endocrine disruptors on gonad development
WP1: Individual gonadal cells were captured from 42 embryos spanning post-conception weeks 5 to 21. Their molecular identity was established by means of high-throughput technologies combined with bioinformatic analyses, enabling the characterization of all known major cell types as well as the discovery of new subpopulations and marker genes in the human gonads. The data are currently being integrated and analyzed.
WP2: The work in WP2 was significantly delayed due to the pandemic. Processing of fetal tissues has not yet been conducted, because of shortage in sample procurement, processing and shipments. Preparatory work has been done according to plan, so fetal tissues will be processed as soon as they arrive to the labs in Stockholm. Tests have been done on samples that were previously cleared and imaged in 3D. P02-KTH has developed a ST protocol that is compatible with formalin fixed tissue specimens, which is important for its compatibility with 3D imaged specimens. P08-CARTANA has been acquired by 10x Genomics and will leave the consortium. P03-Stockholm University will take over the tasks and budget, and we expect no substantial deviations from the work plan.
WP3: Although it was also impacted by the lack of samples during the pandemic, we could performed immunolabelling on 6 fetuses (n=3 each for male and female samples) and 40 isolated gonads stained with a variety of markers and antibody combination (up to 4 markers). Further 15 are being processed.
A 3D map of the main gonadal cell type is being constructed. AI-based strategies have been implemented to quantify the 3D image data. A first selection of eleven antibodies suited for 3D imaging and Cyclic immunofluorescence analysis has been made by P10-MB. A first batch of Human embryonic samples (testes and ovary) is being processed.
WP4: The WP team developed the deliverable D4.1 aiming at getting an enhanced 2D view of H&E, cyclic immunofluorescent and OMICS data. We successfully achieved better performances, both in terms of speed and number of support OMICS annotations that may decorate whole slide tissue sections, reaching 10+ millions of annotations (i.e. genes’ localisation).
WP5: Freshly isolated tissue from >138 human fetal gonads have been cultured ex vivo with specific compounds selected to manipulate intrinsic or extrinsic signaling. Analysis of molecular and cellular alterations as well as effects on secreted hormone levels are ongoing.
WP6: The logo, graphic chart and presentation leaflet have been finalize. The Hugodeca website and intranet are also active. A twitter account has been opened. Many activities (exchange of students/postdocs, on site-training courses and in person meetings) had to be postponed because of COVID-19. This is also the case for conferences and seminars. Online/Virtual solutions are being implemented.
WP7: A first version of the Data Management Plan, that covers all types of data to be generated during the HUGODECA project, has been established and a study management platform was set-up (https://studies.hgdc.genouest.org/).
Differences of sex determination (DSD) conditions present considerable challenges and difficulties in clinical management including surgical correction and gender assignment, as well as the associated complications of infertility and a predisposition to germ cell malignancies. Due to an incomplete understanding of the genetic programs and morphogenetic events involved in sex determination and gonad development, the majority of DSD patients lack a precise diagnosis.
The HUGODECA project will build a comprehensive cell atlas of the gonads during development that will enable to understand the molecular mechanisms of sex determination by providing a healthy reference map to understand the developmental origin of human infertility and to assess stem-cell derived gametes. The unique combination of techniques and expertise present in HUGODECA should improve our knowledge of sterility and congenital conditions affecting sex development, and identify potential new target genes and biomarkers for DSD.
HUGODECA will also advance technologies and workflow required to build cell atlases of developing human organs in both sexes.
Our long-term vision for HUGODECA extends beyond gonad development, and includes innovation and validation of advanced spatial transcriptomics and proteomics methods, together with cutting-edge data processing and viewing applications. We will ensure public dissemination of HUGODECA’s research activities to the scientific community, Human Cell Atlas (HCA) members, clinicians, obstetricians-gynecologists, medical students, patients and the general public to diffuse human embryogenesis knowledge. For example, HUGODECA will develop and implement an interactive and multi-dimensional online human embryology portal.
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