Final Report Summary - PGC PROLIFERATION (Somatic control of primordial germ cell proliferation)
Final Report – IRG project 230877
Introduction. Tissue-specific stem cells are at the core of homeostasis and regeneration of organs such as skin, intestine and the hematopoietic system. Understanding how stem cells maintain organ function is the first step in rational design of procedures to manipulate these cells in vivo. One leading model system used to understand the basic principles of adult stem cell biology has been germ line stem cells (GSCs) in the fruit fly Drosophila melanogaster. Intensive study of this system has shown an evolutionary conservation of molecular players and biological principles that underlie the control of stem cell maintenance and differentiation.
In female flies, two or three GSCs are attached to somatic cap cells, which maintain them in an undifferentiated state (niche cells, fig. 1A). When GSCs divide, one daughter cell (called cystoblast) is removed from the protective environment of the niche and starts to differentiate. Differentiation entails four rounds of incomplete cell division to form a 16-interconnected cell cyst. Germ line cyst differentiation is executed in close contact with another group of somatic cells – the escort cells (ECs). While many signaling pathways are known to control GSC maintenance, relatively few somatic pathways were shown to control differentiation. One signaling pathway that has been demonstrated to control GSC maintenance is Stat (Signal Transducer and Activator of Transcription) signaling. Stat is required within niche cells to maintain GSCs in both male and female flies.
Main Results. In a screen that was designed to uncover novel somatic regulators that affect GSC maintenance and differentiation, we have isolated a gene named woc (without children). When ECs are mutant for woc, cystoblasts fail to form germ line cysts, and woc-mutant ovaries fill with cystoblasts (cystoblast tumors, Fig. 1B). Analysis of woc-mutant ECs showed that while wild-type ECs send thin cell extensions that wrap dividing germ line cysts, mutant ECs do not send these extensions and do not maintain tight connections with the germ cells. Similar problems of reduced contact between somatic cells and germ cells were found earlier during development. In larval ovaries, Primordial Germ Cells (PGCs – the precursors of GSCs) develop in tight contact with somatic Intermingled Cells (ICs). ICs that are woc-mutant fail to intermingle with germ cells, and remain outside of the gonadal region where PGCs are located.
Using mutant phenotypic analysis, epistasis studies and gene expression analysis we have discovered that Woc, which is a transcription factor and chromatin binding protein, is required down-stream of Stat signaling to allow cystoblast differentiation. Stat is required within ICs and ECs for an elevated expression of the transcription factor Zfh-1. In stat- and woc- mutant ECs, Zfh-1 protein levels are reduced by 25%-40%, and mRNA levels are reduced by 50%. Indeed, reduction of Zfh-1 levels using RNAi results in similar phenotypes of dissociation of soma and germ line both in larval and adult ovaries. Work in tissue culture demonstrated that Woc is required for a Stat-mediated elevation in zfh-1 mRNA expression. In addition, we have shown that Zfh-1 is haplo-insufficient for its function in ECs, since flies carrying a deficiency uncovering the region show increased amounts of undifferentiated germ cells. Finally, expression of Zfh-1 from a heterologus promoter can rescue the woc phenotype.
Conclusions. Our study highlights several novel insights into the biology of GSCs. First, we demonstrate a novel function for Stat signaling in GSC differentiation. Thus far, Stat has been regarded as a stem cell maintenance factor. Our work demonstrates it is also important for stem cell differentiation. How can one signaling pathway perform opposite functions (regulate both GSC maintenance and differentiation)? We propose that the primary function of Stat is neither maintenance nor differentiation, but adhesion between somatic cells and germ cells. Germ cells require somatic contact and somatic input in every stage of their development. In the larvae, Stat signaling within ICs is required to maintain contact with PGCs. In the adult niche, Cap cells require Stat signaling to maintain contact with GSCs. This contact in turn is required for proper GSC maintenance. The GSC daughter cells (Cystoblsats) require contact with ECs in order to differentiate properly. Thus, Stat is fulfilling a permissive role at each stage of the germ cell life cycle by allowing contact with the appropriate somatic cell type, which elicits a different response within the germ cells.
This work also highlights a novel type of germ cell tumors. Most tumors that were described thus far are GSC tumors, and lack an expression of the major differentiation factor Bag of marbles (Bam). Indeed, Bam has been considered a necessary and sufficient factor for GSC differentiation, as mis-expression of this protein in GSCs causes their differentiation. GSCs in woc-mutant ovaries express Bam, yet they fail to differentiate. This suggests that Bam expression is not sufficient for GSC differentiation and that an additional somatic signal from ECs is required for proper GSC differentiation. What that signal might be is still unknown.
Finally, our work demonstrates a novel role for the Chromatin binding protein and transcription factor Woc. Woc has several mammalian homologues with unknown function. This work may provide clues into its function in mammals.
Potential Impact. The work may impact the field of stem cell biology by highlighting the role of adhesion as an important factor not only between niche cells and stem cells, but also between differentiating stem cell daughters and their support cells. In addition, it highlights the requirement for somatic instruction for germ cell function at every stage of their life cycle.
Woc is most closely related to the mammalian MYM-type (ZMYM: Zinc finger, MYeloproliferative and Mental retardation motif) family of zinc finger transcription factors. Aberrations in ZMYM2 (Znf198) and ZMYM3 (Znf261) proteins are associated with a myeloproliferative syndrome and with mental retardation, respectively. Interestingly, Zfhx1b, a human homologue of the fly Zfh-1 is also associated with mental retardation, since its heterozygocity causes the human Mowat-Wilson syndrome. Our findings that Woc controls Zfh-1 expression down-stream of Stat activation, open new venues for research of ZMYM2 and ZMYM3 function in mammalian development and human disease.
Introduction. Tissue-specific stem cells are at the core of homeostasis and regeneration of organs such as skin, intestine and the hematopoietic system. Understanding how stem cells maintain organ function is the first step in rational design of procedures to manipulate these cells in vivo. One leading model system used to understand the basic principles of adult stem cell biology has been germ line stem cells (GSCs) in the fruit fly Drosophila melanogaster. Intensive study of this system has shown an evolutionary conservation of molecular players and biological principles that underlie the control of stem cell maintenance and differentiation.
In female flies, two or three GSCs are attached to somatic cap cells, which maintain them in an undifferentiated state (niche cells, fig. 1A). When GSCs divide, one daughter cell (called cystoblast) is removed from the protective environment of the niche and starts to differentiate. Differentiation entails four rounds of incomplete cell division to form a 16-interconnected cell cyst. Germ line cyst differentiation is executed in close contact with another group of somatic cells – the escort cells (ECs). While many signaling pathways are known to control GSC maintenance, relatively few somatic pathways were shown to control differentiation. One signaling pathway that has been demonstrated to control GSC maintenance is Stat (Signal Transducer and Activator of Transcription) signaling. Stat is required within niche cells to maintain GSCs in both male and female flies.
Main Results. In a screen that was designed to uncover novel somatic regulators that affect GSC maintenance and differentiation, we have isolated a gene named woc (without children). When ECs are mutant for woc, cystoblasts fail to form germ line cysts, and woc-mutant ovaries fill with cystoblasts (cystoblast tumors, Fig. 1B). Analysis of woc-mutant ECs showed that while wild-type ECs send thin cell extensions that wrap dividing germ line cysts, mutant ECs do not send these extensions and do not maintain tight connections with the germ cells. Similar problems of reduced contact between somatic cells and germ cells were found earlier during development. In larval ovaries, Primordial Germ Cells (PGCs – the precursors of GSCs) develop in tight contact with somatic Intermingled Cells (ICs). ICs that are woc-mutant fail to intermingle with germ cells, and remain outside of the gonadal region where PGCs are located.
Using mutant phenotypic analysis, epistasis studies and gene expression analysis we have discovered that Woc, which is a transcription factor and chromatin binding protein, is required down-stream of Stat signaling to allow cystoblast differentiation. Stat is required within ICs and ECs for an elevated expression of the transcription factor Zfh-1. In stat- and woc- mutant ECs, Zfh-1 protein levels are reduced by 25%-40%, and mRNA levels are reduced by 50%. Indeed, reduction of Zfh-1 levels using RNAi results in similar phenotypes of dissociation of soma and germ line both in larval and adult ovaries. Work in tissue culture demonstrated that Woc is required for a Stat-mediated elevation in zfh-1 mRNA expression. In addition, we have shown that Zfh-1 is haplo-insufficient for its function in ECs, since flies carrying a deficiency uncovering the region show increased amounts of undifferentiated germ cells. Finally, expression of Zfh-1 from a heterologus promoter can rescue the woc phenotype.
Conclusions. Our study highlights several novel insights into the biology of GSCs. First, we demonstrate a novel function for Stat signaling in GSC differentiation. Thus far, Stat has been regarded as a stem cell maintenance factor. Our work demonstrates it is also important for stem cell differentiation. How can one signaling pathway perform opposite functions (regulate both GSC maintenance and differentiation)? We propose that the primary function of Stat is neither maintenance nor differentiation, but adhesion between somatic cells and germ cells. Germ cells require somatic contact and somatic input in every stage of their development. In the larvae, Stat signaling within ICs is required to maintain contact with PGCs. In the adult niche, Cap cells require Stat signaling to maintain contact with GSCs. This contact in turn is required for proper GSC maintenance. The GSC daughter cells (Cystoblsats) require contact with ECs in order to differentiate properly. Thus, Stat is fulfilling a permissive role at each stage of the germ cell life cycle by allowing contact with the appropriate somatic cell type, which elicits a different response within the germ cells.
This work also highlights a novel type of germ cell tumors. Most tumors that were described thus far are GSC tumors, and lack an expression of the major differentiation factor Bag of marbles (Bam). Indeed, Bam has been considered a necessary and sufficient factor for GSC differentiation, as mis-expression of this protein in GSCs causes their differentiation. GSCs in woc-mutant ovaries express Bam, yet they fail to differentiate. This suggests that Bam expression is not sufficient for GSC differentiation and that an additional somatic signal from ECs is required for proper GSC differentiation. What that signal might be is still unknown.
Finally, our work demonstrates a novel role for the Chromatin binding protein and transcription factor Woc. Woc has several mammalian homologues with unknown function. This work may provide clues into its function in mammals.
Potential Impact. The work may impact the field of stem cell biology by highlighting the role of adhesion as an important factor not only between niche cells and stem cells, but also between differentiating stem cell daughters and their support cells. In addition, it highlights the requirement for somatic instruction for germ cell function at every stage of their life cycle.
Woc is most closely related to the mammalian MYM-type (ZMYM: Zinc finger, MYeloproliferative and Mental retardation motif) family of zinc finger transcription factors. Aberrations in ZMYM2 (Znf198) and ZMYM3 (Znf261) proteins are associated with a myeloproliferative syndrome and with mental retardation, respectively. Interestingly, Zfhx1b, a human homologue of the fly Zfh-1 is also associated with mental retardation, since its heterozygocity causes the human Mowat-Wilson syndrome. Our findings that Woc controls Zfh-1 expression down-stream of Stat activation, open new venues for research of ZMYM2 and ZMYM3 function in mammalian development and human disease.
