Project Outline

In sexually reproducing organisms, eggs and sperm are produced from specialized cells of the germline, which are essential for reproduction and survival of the species. In most vertebrates, sex determination leads to the development of a single gonad (ovary or testis) from germline progenitors. Genetic as well as environmental factors are thought to control gonad and sexual development. However, the mechanisms underlying germline development and sex determination in many vertebrates is not understood.

In the tropical freshwater fish, Zebrafish (Danio rerio), reproduction requires two distinct sexes (males and females). Germline progenitors (i.e. stem cells) develop and form a bipotential gonad, which differentiates into either testis or ovary. By contrast, Kryptolebias marmoratus (Kmar), a small egg-laying killifish from neotropical mangrove swamps, show a different mode of reproduction and can self-fertilize. Kmar adults can be distinguished as either hermaphrodites or males (Harrington, 1961).

What governs germline and sexual development in these fish is not known, and the role of genetic versus environmental factors is not well understood. The overarching goal of this project is to identify common versus distinct modes of fish reproduction and development by taking comparative cell, developmental biology and genomic approaches.

Fish are an important source of food across the globe. Given the pressures of climate change, better understanding of germline and sexual development in these fish with different reproductive modes and habitats can provide useful insights for maintaining balanced stocks across a range of fish species.

The mini-project aims are to study early killifish germline progenitor development, juvenile and adult reproductive tissues at two or three selected stages by morphological criteria and analysis of selected marker genes, and compare to zebrafish.

For the doctoral project, the student will build upon and extend the mini-project studies to include embryonic, larval and adult stages. Bulk sequencing and single cell transcriptomic analysis at selected time points will be performed on developing gonads from mangrove killifish at various stages and compared to Zebrafish. Finally, environmental stressors and/or chemical manipulations will be applied and the effect on sexual development and gene expression will be examined.

Supervisory arrangement: The experimental work and in particular, zebrafish and killifish work will be supervised by Sampath; Computational and transcriptomic analysis will be overseen by Hebenstreit. The student will meet with the main supervisor at least 1 x per week, and both supervisors 1x per month (or more frequently, when required) to discuss the project.

Methods

• Zebrafish & Killifish methods: Mating of live fish to produce eggs, collection and examining developing embryonic germline/gonads at various stages.
• Cell & Molecular Biology: Immunofluorescence, in situ hybridization, PCR, cloning, RT-PCR.
• Quantitative Image analysis
• Transcriptomics, Data Analysis

Techniques

• Molecular biology: Standard molecular biology including DNA and RNA extraction, RNA synthesis, PCR and qRT-PCR
• Zebrafish embryonic development and genetics, cell biology techniques including microinjections, genetic crosses, WISH and FISH (in situ hybridization)
• Imaging: standard light and fluorescence microscopy, spinning disc confocal microscopy and multiphoton FLIM
• Data analysis
  • Transcriptomic data analysis: basic command-line scripting, NGS data analysis of zebrafish YSL and human syncytiotrophoblasts, statistical analysis using R, pathway analysis, GO analysis, visualization methods (clustering, heatmaps), multivariate statistics (PCA), comparative genomics
  • Image analysis: 3D segmentation and image analysis, Scripting with Fiji/ImageJ