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Human Gonad Developmental Cell Atlas

Periodic Reporting for period 2 - HUGODECA (Human Gonad Developmental Cell Atlas)

Reporting period: 2021-01-01 to 2022-06-30

Animals rely on genetic factors to determine sex. However, sex differentiation can also be influenced by environmental factors (such as pesticides and endocrine disruptors) among others. 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 8 weeks of gestation, a distinct developmental program is activated in male and female which leads to the rapid differentiation of testes 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: To characterize the cellular composition of the human foetal gonads, we have analysed the DNA, RNA or protein content of over 1,4 million single cells from 230 human embryos/foetuses aged between 5 to 21 post-conception weeks. Our results allowed us to identify known and new cell types and to dissect the molecular mechanisms involved in the early specification of gonads into either an ovary or a testis. In addition, we could highlight specific genes and proteins that can be used as markers to map these cells inside the gonads. Several of these genes could represent new biomarkers for DSDs.

WP2: We have generated spatially resolved transcriptomic profiling data from human developmental gonads at different time points, and using three orthogonal technologies: Nanostring WTA, 10xGenomics Visium, and 10xGenomics Cartana. This data will combined with scRNA sequencing dat,a provide a much more detailed view over the spatiotemporal development of the cellular diversity of this organ during the studied time window.

WP3: A total of 156 human foetal gonads were used to assess the spatial location of gonadal cells in situ using state-of-the-art techniques implemented in WP3. We performed, fully automated cyclic immuno-fluorescence imaging of frozen sections of gonads. We also imaged in 3D, solvent-cleared human gonads, genital ducts and genitalia using light sheet fluorescence microscopy (LSFM) and fluorescent in situ hybridization. We have been able to describe the cellular composition and organization of the developing human gonads.

WP4: Keen Eye completed the deliverable for D4.2 which is an enhanced 3D viewer for image data. With respect to D4.3 and D4.4 Keen Eye rebuilt large parts of the annotation rendering engine (for handling up to 10M Annotations), performed an investigation into 3D registration algorithms and techniques, and refactored the platform to allow such registration algorithms to run. The final integration and testing on the platform was not completed.

WP5: A total of 273 human fetal gonads were cultured ex vivo and signaling were manipulated to mimic conditions of Differences of Sex Development or effects of endocrine disruptors. The induced alterations were examined at molecular and cellular levels by established endpoints in combination with state-of-the-art techniques implemented in WPs 1-4. The inclusion of novel techniques significantly improved the analysis and thus the relevance of the obtained results.

WP6: P01a and P09 have organized in Paris a tissue-clearing and 3D imaging course freely accessible to members of teams belonging to the cluster of 6 H2020/HCA consortia. A spatial transcriptomics course was organized (with EMBO support) in Solna (Sweden) by P02 and P03.We have also organized in Paris a one-day final public symposium with international speakers. A video and a short animation, describing in lay language the objectives and partners of HUGODECA were produced and released on social media.

WP7: The goal of WP7 was to establish some guidelines about the management of the data collected, generated or processed during the HUGODECA project, so as to make them Findable, Accessible, Interoperable and Re-usable (FAIR principles) by the scientific community. We defined those guidelines in the Data Management Plan, we set up a platform to share metadata templates and we took part in the establishment of pipelines and dataset preparation.
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 has built the first comprehensive cell atlas of the human 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 has developed advanced 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 have ensured 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 is implementing an interactive and multi-dimensional online human embryology portal.