Periodic Reporting for period 1 - TSILENCICH (Epigenetic silencing of transposable elements in Lake Malawi cichlids)
Reporting period: 2021-07-01 to 2023-06-30
Transposable elements (TEs), also known as “jumping genes”, are mobile genetic elements able to mobilise from one piece of DNA into another. The mobilisation of TEs can generate genome instability and DNA damage. To counteract the detrimental effects of their mobilisation, TEs must be silenced. Several molecular pathways have evolved in animal germ cells, cells that differentiate into gametes, to silence TEs, thus protecting future generations from genomic instability derived from TE mobilisation. A specific class of small RNAs that interact with Piwi Argonaute proteins, termed Piwi-interacting RNAs (piRNAs), is a major driver of TE silencing in animal germ cells and is generally required for animal fertility. The antagonistic relationship between TEs and TE silencing factors of the host leads to an evolutionary arms race and fast evolution of both opposing components. These interactions and fast evolution can create reproductive incompatibilities that may act as barriers to hybridisation.
Cichlid fishes of the East African Great Lakes and surrounding bodies of water are the most species-rich adaptive radiation in vertebrates. This means this group of animals has diverged explosively, into approximately two thousand species, in East Africa in the last ten million years. These fishes have adapted to all the available niches in the lakes and rivers of East Africa, acquiring along the way tremendous variation in morphology, colour, diet, and behaviour. The genetic and molecular basis of cichlid phenotypic diversity remains a topic of great interest to understand how organisms adapt fast and speciate. Furthermore, tilapias are a non-radiating assemblage of African cichlids related to the radiating cichlid lineages in East Africa. As the second most produced group of fish in aquaculture worldwide, tilapias comprise a fish group with great economic importance.
Genomic studies of East African cichlids have uncovered recurrent hybridisation during the evolutionary history of the radiations. In fact, hybridisation has been proposed to fuel the radiations. This project sought to discover how cichlids tolerate hybridisation, particularly regarding TE regulation by the piRNA pathway. First, we have conducted important groundwork to understand the transcriptional activity of TEs in cichlids as well as their epigenetic silencing by the piRNA pathway. We have performed reciprocal crosses between two cichlid species of Lake Malawi and found a degree of embryonic lethality in the F2 hybrid generation, consistent with post-zygotic incompatibilities. We are dissecting the molecular basis of the embryonic lethality, as well as its accompanying developmental defects. In conclusion, the scientific objectives of TSILENCICH successfully illuminate how animals tolerate genetic conflict and genetic incompatibilities. The training objectives of TSILENCICH were also achieved, providing a substantial positive impact in the researcher’s career. Given the current pressure exerted on biodiversity by fast climate change, it is important to understand how animals adapt to their environment and the molecular drivers or barriers to adaptation.
Cichlid fishes of the East African Great Lakes and surrounding bodies of water are the most species-rich adaptive radiation in vertebrates. This means this group of animals has diverged explosively, into approximately two thousand species, in East Africa in the last ten million years. These fishes have adapted to all the available niches in the lakes and rivers of East Africa, acquiring along the way tremendous variation in morphology, colour, diet, and behaviour. The genetic and molecular basis of cichlid phenotypic diversity remains a topic of great interest to understand how organisms adapt fast and speciate. Furthermore, tilapias are a non-radiating assemblage of African cichlids related to the radiating cichlid lineages in East Africa. As the second most produced group of fish in aquaculture worldwide, tilapias comprise a fish group with great economic importance.
Genomic studies of East African cichlids have uncovered recurrent hybridisation during the evolutionary history of the radiations. In fact, hybridisation has been proposed to fuel the radiations. This project sought to discover how cichlids tolerate hybridisation, particularly regarding TE regulation by the piRNA pathway. First, we have conducted important groundwork to understand the transcriptional activity of TEs in cichlids as well as their epigenetic silencing by the piRNA pathway. We have performed reciprocal crosses between two cichlid species of Lake Malawi and found a degree of embryonic lethality in the F2 hybrid generation, consistent with post-zygotic incompatibilities. We are dissecting the molecular basis of the embryonic lethality, as well as its accompanying developmental defects. In conclusion, the scientific objectives of TSILENCICH successfully illuminate how animals tolerate genetic conflict and genetic incompatibilities. The training objectives of TSILENCICH were also achieved, providing a substantial positive impact in the researcher’s career. Given the current pressure exerted on biodiversity by fast climate change, it is important to understand how animals adapt to their environment and the molecular drivers or barriers to adaptation.
First, we explored the transcriptional activity of TEs in East African cichlids of every Great Lake and Nile Tilapia as an outgroup to the radiation. By profiling TE expression in cichlid gonads and throughout embryogenesis, we have found hundreds of expressed TE families and dynamic expression patterns. Of note, we have identified differential expression of TE families according to sex, and a developmental period in early gastrulation where TEs are most highly expressed, consistent with a relaxed period of epigenetic silencing before zygotic production of TE silencing factors.
To define the TE silencing pathways of cichlids, we first identified the cichlid proteins homologous to known TE silencing factors in humans, mouse, and zebrafish, and profiled their expression in gonads and brain. TE silencing factors of the piRNA pathway were expressed in gonads, which harbour germ cells, suggesting that piRNA-driven TE silencing is in place in germ cells, much like in other animals. Conversely, we could not detect consistent expression of piRNA pathway factors in the brain, arguing against a robust piRNA-driven TE silencing pathway in cichlid brains. Interestingly, we have discovered an expansion of piwil1, a key element of the piRNA pathway, in Lake Malawi cichlids. These fishes encode three piwil1 genes, one full-length and two other truncated versions carrying only the Piwi catalytic domain. We have reconstructed the evolutionary history of this expansion and found that the truncated proteins may have retained catalytic activity. The presence of TEs directly flanking the truncated piwil1 genes, but not the full-length copy, suggests that this expansion may have been mediated by TEs.
Also, we profiled small RNAs in the gonads of representative cichlid species of each East African Great Lake, using Nile Tilapia as an outgroup to the radiation. We have identified abundant small RNA populations with length and sequence signatures consistent with piRNAs. We mapped the origins of piRNAs and found that they are produced from TEs and intergenic regions. We further refined this analysis and identified fast evolving piRNA clusters. Overall, these findings strongly indicate that an active and fast evolving piRNA pathway is in place in cichlid gonads to silence TEs.
To understand how cichlids tolerate hybridisation, we have created two reciprocal crosses between two cichlid species of Lake Malawi. We found a degree of embryonic lethality in the F2 hybrid generation of both crosses, consistent with post-zygotic incompatibilities. We are currently dissecting the molecular basis of the embryonic lethality, as well as its accompanying developmental defects.
These results were presented at several international conferences, and local meetings. Publications resulting from this work will be published in preprint repositories (namely BioRxiv) and in peer-reviewed journals (with open access). All mass spectrometry, mRNA- and small RNA-sequencing datasets generated in this study will be made publicly available on online repositories (GEO or SRA). EU funding was acknowledged in all dissemination opportunities so far, and will be further acknowledged in all future dissemination/communications related to this work.
To define the TE silencing pathways of cichlids, we first identified the cichlid proteins homologous to known TE silencing factors in humans, mouse, and zebrafish, and profiled their expression in gonads and brain. TE silencing factors of the piRNA pathway were expressed in gonads, which harbour germ cells, suggesting that piRNA-driven TE silencing is in place in germ cells, much like in other animals. Conversely, we could not detect consistent expression of piRNA pathway factors in the brain, arguing against a robust piRNA-driven TE silencing pathway in cichlid brains. Interestingly, we have discovered an expansion of piwil1, a key element of the piRNA pathway, in Lake Malawi cichlids. These fishes encode three piwil1 genes, one full-length and two other truncated versions carrying only the Piwi catalytic domain. We have reconstructed the evolutionary history of this expansion and found that the truncated proteins may have retained catalytic activity. The presence of TEs directly flanking the truncated piwil1 genes, but not the full-length copy, suggests that this expansion may have been mediated by TEs.
Also, we profiled small RNAs in the gonads of representative cichlid species of each East African Great Lake, using Nile Tilapia as an outgroup to the radiation. We have identified abundant small RNA populations with length and sequence signatures consistent with piRNAs. We mapped the origins of piRNAs and found that they are produced from TEs and intergenic regions. We further refined this analysis and identified fast evolving piRNA clusters. Overall, these findings strongly indicate that an active and fast evolving piRNA pathway is in place in cichlid gonads to silence TEs.
To understand how cichlids tolerate hybridisation, we have created two reciprocal crosses between two cichlid species of Lake Malawi. We found a degree of embryonic lethality in the F2 hybrid generation of both crosses, consistent with post-zygotic incompatibilities. We are currently dissecting the molecular basis of the embryonic lethality, as well as its accompanying developmental defects.
These results were presented at several international conferences, and local meetings. Publications resulting from this work will be published in preprint repositories (namely BioRxiv) and in peer-reviewed journals (with open access). All mass spectrometry, mRNA- and small RNA-sequencing datasets generated in this study will be made publicly available on online repositories (GEO or SRA). EU funding was acknowledged in all dissemination opportunities so far, and will be further acknowledged in all future dissemination/communications related to this work.
Our work advances substantially the understanding of TE transcriptional activity and piRNA-driven TE silencing in East African cichlids. These insights are invaluable to keep pushing the state-of-the-art and further dissect how the interaction between TEs and the piRNA pathway shaped cichlid radiations. Our ongoing work on cichlid hybrids will inform on the regulatory mismatches arising upon hybridisation that may create genetic and epigenetic diversity to fuel rapid diversification. This is, to the best of our knowledge, the first systematic approach to probe the effects of TE activity and piRNA pathway in the context of cichlid hybridisation. Our work will be impactful for the worldwide community exploring the molecular basis of the phenotypic diversity of cichlids, as well as researchers interested in speciation, epigenetic silencing, TEs and genome evolution. An enhanced understanding of cichlid evolution may have economic implications for Tilapia production, the second most farmed fish worldwide.