Final Report Summary - GSCS MAINTENANCE (Post-transcriptional regulation of Germline Stem Cells (GSCs) maintenance)
I have completed the tasks and objectives designed for the "GSCs maintenance" project of the FP7-PEOPLE-2011 IIF program. In order to implement the project I have collaborated with my host researcher Prof. Adi Salzberg at the Rappaport Faculty of medicine of the Technion Institute of Technology (Haifa, Israel). In the first year, we have launched a research group to study post-transcriptional regulation of germline stem cells (GSCs) maintenance. The project group included three MSc students in addition to the scientist in charge and me. During the first period we have generated the relevant transgenic lines and characterize their phenotypes. In the second year, we have developed a unique method to analyze the transcriptome and microRNAome of small groups of cells (stem and niche cells) that were isolated from the entire organism. We also used sensor assay to in vivo detect the expression of microRNAs (miRNAs) in the stem cell niche (An example in Fig. 1). During the project I have disseminated the knowledge and experience I previously obtained in the US and the current project, to both my immediate and neighboring scientific community. During the lifetime of the project I have published three research papers, two invited reviews and one book chapter. I presented my previous and ongoing research in multiple seminars and meetings in Israel and in Europe. I have also established a new teaching course: "Molecular mechanism of Stem cells". I teach this course to graduate students both in the host institute: The Faculty of Medicine in the Technion-Israel Institute of Technology, and in a neighboring institute: the Human Biology Department at the University of Haifa, Haifa. I will continue to teach the course also this year after the completion of the IIF program. Last but not least, I am establishing a new independent laboratory for adult stem cells research in a tenure track position at the University of Haifa, Israel.
A summary of Research objectives:
The cell turn-over of highly regenerative tissues is maintained by a small population of tissue specific stem cells. While the intrinsic properties of tissue stem cells are indeed implicated in tissue maintenance, these cells are often unable to fulfill their actions in the absence of signals that originate from the microenvironment, namely, the stem cell niche. During my post-doc in the US, we have made the ground-breaking discovery that a specific miRNA molecule let-7, which belongs to a prominent class of post-transcriptional RNA inhibitors, is a major regulator of niche aging (Toledano et al., Nature 2012). In the current project we mainly used the Drosophila testis as a model to study the function of stem cells and the niche in tissue maintenance and aging. We have employed advanced biochemical and genetic approaches to identify GSCs enriched- and niche enriched-miRNAs; to further explore the mechanisms that govern stem cells aging process and link the levels of heterochronic (controlling timing events) miRNA molecules to the declined stem cells capability in aged males.
The two specific aims of this research are:
1. To identify the molecular events by which miRNAs inhibit mRNA targets to maintain the SC niche in Drosophila testis.
We made a clearly significant progress in this specific aim and found that the hub, a major component of the stem cells niche employ a unique cell survival mechanism. We postulate that this survival mechanism is mediated by microRNAs (miRNAs), that function as antiapoptotic regulators of the hub cells.
• We found that the anti-apoptotic miRNA bantam is expressed in the stem cell niche and prevent the expression of the pro-apoptotic protein Hid (Fig. 1). To determine whether bantam is expressed in the hub we immunostained testis of transgenic flies carrying bantam sensor with niche markers (generous gift from Prof. SM Cohen, IMCB, Singapore). bantam sensor contains two perfectly complementary sites for bantam miRNA in the 3'UTR. Therefore, cells that endogenously expressed bantam are expected to inhibit GFP translation and therefore appear dark (Fig. 1).
• Preventing miRNAs production in the hub, by knocking down key regulators of miRNAs biogenesis, results with niche disappearance followed by stem cell loss and sterility. Specifically, we knocked-down drosha and dicer-1 in the hub of adult males and in both cases the niche disseminated
• Hub cells disappearance due to miRNAs loss involves cell death, suggesting that miRNAs function in the hub to prevent death of the stem cell niche.
• We developed a biochemical approach to dissociate the hub cells of the Drosophila testis and to extract the RNA for micrRNAome and transcriptome analysis.
The significance of our findings is twofold: At the cellular level, we show for the first time a mechanism whereby miRNAs regulate the survival of fully-committed post-mitotic niche cells and protect them from apoptosis. At the tissue level, we propose that protection of a specific group of niche cells from apoptosis facilitates ongoing stem cell turnover. Given that support of tissue homeostasis by stem cells is a primordial mechanism and that miRNAs are particularly conserved evolutionarily, exploring these mechanisms is expected to significantly enhance our understanding of fundamental aspects of tissue homeostasis regulation across species.
2. To characterize the self-renewal function of Nos in GSCs maintenance and to identify its mRNA targets.
nos mutants did not result in a significant GSCs loss, suggesting that in the testis Nos does not act as a self-renewal factor.
The IIF project provided me with notable opportunities for transferring the knowledge of GSCs post‐transcriptional regulation that I acquired during my post‐doctoral training at the Salk Institute, La Jolla, CA, USA to Israel and my host institute, the Technion, specifically. During the project I have initiated collaborations with scientists in the Technion and in academic institutes outside the host that I am continuing to follow. For example, with Prof. Erel Levine, a faculty member at Department of Physics and FAS Center for Systems Biology at Harvard University, that uses bioinformatics to identify mRNA targets for microRNAs that regulate the niche.
The grant allowed me to settle back into the Israeli scientific sphere after my return from abroad, and helped me to develop as a researcher and to be recognized by the scientific community. Attesting to this are my recent tenure track position at the University of Haifa, my nomination as a faculty member for the F1000 prime in the aging section, invitations to review papers in leading journals (including Nature) and numerous invitations from leading conferences to present our research as described below.
A summary of Research objectives:
The cell turn-over of highly regenerative tissues is maintained by a small population of tissue specific stem cells. While the intrinsic properties of tissue stem cells are indeed implicated in tissue maintenance, these cells are often unable to fulfill their actions in the absence of signals that originate from the microenvironment, namely, the stem cell niche. During my post-doc in the US, we have made the ground-breaking discovery that a specific miRNA molecule let-7, which belongs to a prominent class of post-transcriptional RNA inhibitors, is a major regulator of niche aging (Toledano et al., Nature 2012). In the current project we mainly used the Drosophila testis as a model to study the function of stem cells and the niche in tissue maintenance and aging. We have employed advanced biochemical and genetic approaches to identify GSCs enriched- and niche enriched-miRNAs; to further explore the mechanisms that govern stem cells aging process and link the levels of heterochronic (controlling timing events) miRNA molecules to the declined stem cells capability in aged males.
The two specific aims of this research are:
1. To identify the molecular events by which miRNAs inhibit mRNA targets to maintain the SC niche in Drosophila testis.
We made a clearly significant progress in this specific aim and found that the hub, a major component of the stem cells niche employ a unique cell survival mechanism. We postulate that this survival mechanism is mediated by microRNAs (miRNAs), that function as antiapoptotic regulators of the hub cells.
• We found that the anti-apoptotic miRNA bantam is expressed in the stem cell niche and prevent the expression of the pro-apoptotic protein Hid (Fig. 1). To determine whether bantam is expressed in the hub we immunostained testis of transgenic flies carrying bantam sensor with niche markers (generous gift from Prof. SM Cohen, IMCB, Singapore). bantam sensor contains two perfectly complementary sites for bantam miRNA in the 3'UTR. Therefore, cells that endogenously expressed bantam are expected to inhibit GFP translation and therefore appear dark (Fig. 1).
• Preventing miRNAs production in the hub, by knocking down key regulators of miRNAs biogenesis, results with niche disappearance followed by stem cell loss and sterility. Specifically, we knocked-down drosha and dicer-1 in the hub of adult males and in both cases the niche disseminated
• Hub cells disappearance due to miRNAs loss involves cell death, suggesting that miRNAs function in the hub to prevent death of the stem cell niche.
• We developed a biochemical approach to dissociate the hub cells of the Drosophila testis and to extract the RNA for micrRNAome and transcriptome analysis.
The significance of our findings is twofold: At the cellular level, we show for the first time a mechanism whereby miRNAs regulate the survival of fully-committed post-mitotic niche cells and protect them from apoptosis. At the tissue level, we propose that protection of a specific group of niche cells from apoptosis facilitates ongoing stem cell turnover. Given that support of tissue homeostasis by stem cells is a primordial mechanism and that miRNAs are particularly conserved evolutionarily, exploring these mechanisms is expected to significantly enhance our understanding of fundamental aspects of tissue homeostasis regulation across species.
2. To characterize the self-renewal function of Nos in GSCs maintenance and to identify its mRNA targets.
nos mutants did not result in a significant GSCs loss, suggesting that in the testis Nos does not act as a self-renewal factor.
The IIF project provided me with notable opportunities for transferring the knowledge of GSCs post‐transcriptional regulation that I acquired during my post‐doctoral training at the Salk Institute, La Jolla, CA, USA to Israel and my host institute, the Technion, specifically. During the project I have initiated collaborations with scientists in the Technion and in academic institutes outside the host that I am continuing to follow. For example, with Prof. Erel Levine, a faculty member at Department of Physics and FAS Center for Systems Biology at Harvard University, that uses bioinformatics to identify mRNA targets for microRNAs that regulate the niche.
The grant allowed me to settle back into the Israeli scientific sphere after my return from abroad, and helped me to develop as a researcher and to be recognized by the scientific community. Attesting to this are my recent tenure track position at the University of Haifa, my nomination as a faculty member for the F1000 prime in the aging section, invitations to review papers in leading journals (including Nature) and numerous invitations from leading conferences to present our research as described below.