The PEZ project investigated how elevated environmental temperatures affect the fate and molecular landscape of primordial germ cells (PGCs) in zebrafish, with a focus on understanding sex ratio biases relevant to both aquaculture and natural populations under climate change. Despite early termination of the action (10.5 months before the official end date) due to parental leave and a tenured appointment at ICM-CSIC, key technical and scientific objectives were successfully implemented and are ongoing under institutional continuity.
In Work Package 1, a robust breeding program for the transgenic zebrafish line tg(Buc:GFP) was established. Ten Buc families were raised, and offspring from the family with a balanced sex ratio were reared under control (28°C) and thermal stress (36°C) conditions in triplicate. Developmental staging was synchronised between treatments by modifying light/dark cycles, and direct visual inspection of adult sex ratios allowed the identification of optimal breeding pairs for downstream experiments.
In Work Package 2, to characterise PGC transcriptomic dynamics under heat stress, publicly available RNA-seq datasets were reanalysed to define early developmental and germline-specific markers. This analysis informed the selection of 1,000 relevant genes for spatial transcriptomics and validated the computational pipeline. Fluorescent PGCs from tg(Buc:GFP) fish were isolated via FACS, and Drosophila 1182-4H haploid cells were cultured as spike-ins for calibrated RNA-seq. While library generation is ongoing due to material limitations, the sequencing pipeline has been successfully tested using zebrafish datasets, and the transcriptomic data for control samples have been fully processed.
In Work Package 3, low-input epigenomic protocols (CUT&RUN, CUT&Tag, and ATAC-seq) were adapted and validated for zebrafish PGCs using limited cell numbers. Public data on zebrafish chromatin accessibility and PGC-specific profiles were reanalysed to inform protocol optimisation. Initial thermal exposure experiments were conducted, yielding promising CUT&RUN pilot results. Further biological replicates are needed and will be generated beyond the grant period to finalise epigenetic analyses. The adapted protocols were successfully validated in a practical course during the Chromatin Explorer Summer School in July 2025.
In Work Package 4, although the planned secondment was not completed, groundwork for spatial transcriptomics was laid. A list of 1,000 genes relevant to PGC biology and early development was sent for MERSCOPE chip printing (completed July 2024). Sample preparation protocols were tested successfully, including permeabilisation and RNA integrity verification using MERFISH-style probes. Control and thermally treated embryos are scheduled for full spatial analysis using the MERSCOPE platform from September 2025.
Scientific training activities directly relevant to the technical work included FELASA-equivalent certification in animal handling, bioinformatics training, and workshops on RNA-seq, epigenomics, and data processing pipelines. Outcomes of the action include the establishment of reliable models and protocols for studying temperature-induced developmental changes in PGCs, early identification of transcriptomic markers responsive to heat stress, and foundational implementation of spatial and epigenomic technologies in zebrafish germline research. Two manuscripts are in preparation—one on transcriptomic effects of thermal stress and another on somatic–germline spatial interactions. Research continuity is ensured through the fellow’s transition to Principal Investigator at ICM-CSIC and ongoing access to experimental platforms and collaborations.
In Work Package 5, all planned technical training was completed, including FELASA-equivalent animal handling certification, which is essential for zebrafish experiments. Bioinformatics training in HPC and Data Science with R supported transcriptomic and epigenomic data analysis. The fellow organised and taught the Chromatin Explorer Summer School (July 2025), which included hands-on training in CUT&RUN and ATAC-seq. Additionally, I followed informal learning on spatial transcriptomic techniques and analysis, reinforcing my technical expertise.
In Work Package 6, scientific dissemination included presentations at the 12th ISPRF (May 2023) and the third EPIMAR conference (May 2025), focusing on transcriptomic and epigenomic findings. Internal seminars at ICM-CSIC and IDAEA-CSIC facilitated technical discussions on developmental timing and spatial transcriptomics. Two manuscripts covering thermal stress transcriptomics and spatial germline-soma interactions will be the end point for this project.
In Work Package 7, the fellow secured a tenured PI position at ICM-CSIC (Dec 2024), ensuring continuity of the research program and access to necessary resources. Integration into EU research networks (EPIMAR, ERGA) strengthened collaboration and methodological exchange. Leadership was demonstrated through organisation of the Chromatin Explorer Summer School, which supported the project's long-term scientific impact, and by being appointed coordinator of a future research hub within her institution.
