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Evolutionary and developmental analysis of germline formation in simple chordate embryos

ZHENG TAO 東北大学

2020.03.25

概要

Germ cell formation and its protection from taking on somatic fates are critical for the continuity of species. In many animals, primordial germ cells (PGCs) form during embryogenesis by one of the two mechanisms: an ancestral epigenesis mechanism or a derived preformation mode. The epigenesis mechanism requires cell-cell interaction, while the preformation mode depends on maternal determinants in the germ plasm.

Posterior end mark (Pem) mRNA is a component of the ascidian germ plasm, known as Centrosome-Attracting Body (CAB). Pem protein is translated in CAB and nuclear Pem protein functions to repress the activity of RNA polymerase II (RNAPII), thereby globally suppressing germline transcription. Although this Pem-mediated germline transcriptional repression is required to prevent germline cells from somatic differentiation, the decrease of Pem protein level is observed from around the 64-cell stage. This downregulation is mediated by another maternally localized factor ZF-1 and is a pre-requisition for the initiation of zygotic expression of germline genes at the 110-cell to gastrula stages. But then a question arises as to how the transcriptionally competent germline cells remain silent in somatic gene expression. To study this issue, I specially asked the following two questions using an ascidian species Halocynthia roretzi: 1) does Pem protein still repress somatic gene expression in later stage germline cells? 2) are there any other factors that repress the transcription of somatic genes following the decrease of Pem protein?

I report here that Pem knockdown resulted in an elevated RNAPII activity at the 110-cell stage, suggesting that Pem still takes part in transcriptional repression in later stage germline cells. However, the percentage of embryos showing ectopic somatic gene expression in the germline cells upon Pem knockdown was small, suggesting that there might be another factor(s) repressing germline transcription. I found that H3K27me3, a repressive transcription-related chromatin mark, became enriched in germline cells from the 64-cell stage onwards, hinting that it may function to repress somatic gene expression in the germline. In fact, Pem knockdown and treatment with an inhibitor for H3K27me3 resulted in significantly more embryos showing ectopic expression of muscle-related genes (MA4 and Snail) in germline cells at the 110-cell stage, but not of genes essential for fate determination of other tissue types such as notochord and nerve cord. Interestingly, the muscle lineage is the last somatic lineage that separates from the germline during ascidian embryogenesis. I found that the ectopic expression in the Pem- and H3K27me3-deficient embryos is dependent on a maternally localized muscle determinant, Macho-1, whose mRNA is inherited only by germline cells but protein is thought to be present in a broad region of the posterior part of the embryo including muscle lineage cells. In addition, Clone 22, which is expressed in all the cells including germline’s sister cells, but not in germline cells at the 110-cell stage, is also ectopically expressed upon the double treatments. Therefore, I propose that Pem- and H3K27me3-dependent mechanisms repress gene expression normally activated in somatic lineages that have been shared with germline until late stages because there is a high chance that germline inherits transcription factors activating the gene expression. Taken together, my results show that a chromatin-based mechanism comes in to repress somatic gene expression when the Pem amount is downregulated. Using a chromatin-based mechanism following a mechanism mediated by a maternally localized factor is conserved among ascidian, Caenorhabditis elegans, and Drosophila.

For the second topic of the current study, I studied evolutionary aspects of germline development using several simple chordate/tunicate species such as an appendicularian species Oikopleura dioica and an ascidian species Ciona savignyi. It is known that there is no Pem in the genome of O. dioica. I specifically asked the following three questions: 1) does O. dioica use preformation or epigenesis mechanism to segregate germline? 2) do O. dioica germline cells experience transcriptional repression even without Pem? 3) does Pem from different ascidian species function differently?

Here, by using another ascidian species C. savignyi, I found that C. savignyi Pem has transcriptional repression activity just like H. roretzi Pem, but that domains required for the transcriptional repression within the Pem proteins are different between H. roretzi and C. savignyi. I also report that a conserved germline marker Vasa protein was detected in O. dioica germline cells at the 4- and 16-cell stages, supporting the notion that O. dioica uses the preformation mechanism. Considering the proposed most basal phylogenetic position of appendicularians among the tunicate species, the evolution of preformation mechanism likely preceded the acquirement of the Pem gene in the ascidian lineage. I also noticed that the RNAPII activity was absent in O. dioica germline cells at the 32-cell stage, indicating that O. dioica uses a mechanism that does not involve Pem and is different from ascidian species to target RNAPII for germline transcriptional regulation.

These results suggest that mechanisms by which germline gene expression is repressed can be diversified even between closely related species. Taken together, studying simple chordate species has provided us an interesting opportunity to better understand the evolutionary aspects of animal germline development.

In summary, the present study focusing on germline transcriptional regulation in simple chordate animals reveals both conserved and diverse mechanisms of germline development among animal species.

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