京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24946号 / 生博第508号 / 新制||生||68(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 松田 道行, 教授 松本 智裕, 教授 原田 浩 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DGAM

During development, somatic cell cues direct sex-specific differentiation of germ cells that is characterised by two distinct cell cycle states. At 12.5 days post coitum (dpc) in a testis, XY germ cells stop proliferating and enter G1/G0 arrest. In the ovary, XX germ cells bypass G1/G0 arrest and instead enter the first phase of meiosis I from 13.5 dpc. Whilst it is hypothesised that errors in cell cycle control during development precede the formation of testicular germ cell tumours, the mechanism of cell cycle control at this time has not been thoroughly investigated. This project therefore sought to explore the mechanism of XY germ cell G1/G0 arrest using several approaches. Although cell cycle regulation for somatic cells is well established, we know very little regarding germ cell control of this process. Therefore my first aim was to profile this machinery at the transcript level using a cell cycle cDNA array. Purified populations of germ cells were isolated both before and after sex differentiation and expression of 112 cell cycle related genes was assessed. From this study a comprehensive network governing apoptosis and calcium signalling that was common to both XX and XY germ cells was observed. Importantly, the retinoblastoma family and cyclin dependent kinase inhibitor p21 was implicated in the regulation of G1/G0 arrest in XY germ cells. Lastly, XX germ cells displayed a down-regulation of genes involved in both G1 and G2 phases of the cell cycle consistent with their progression past G1 phase. This study has provided a detailed analysis of cell cycle gene expression during fetal germ cell development and identified candidate factors for future investigation in order to understand cases of aberrant cell cycle control in these specialised cells. In order to investigate several candidate genes identified within the cell cycle array, I next sought to generate a germ cell-specific Cre recombinase mouse model for use in conditional knockout studies. As current Cre lines lack specificity or appropriate temporal expression, we used the germ cell-specific regions of the fragilis promoter to drive Cre expression during germ cell specification. Eleven founder lines were generated using this construct and four were analysed using a reporter line. Although we have not achieved germ cell expression from these lines to date, analysis continues in order to identify an invaluable new tool for germ cell research. Following the implication of the retinoblastoma family in XY germ cell G1/G0 arrest, I next investigated the role of RB in these cells using the Rb null mutant. RB is a known cell cycle suppressor that controls this process in many cell types and, subsequently, mice homozygous for the Rb deletion die in utero at 14.5 dpc. Using this model we analysed developing gonads from 14.5 – 16.5 dpc using ex vivo culture techniques. At 14.5 dpc when wild type germ cells have arrested, proliferating germ cells were detected in the absence of Rb using proliferation marker Ki67. This proliferation was accompanied by a slight increase in germ cell number at 14.5 dpc, however, two days later at 16.5 dpc germ cell numbers were slightly decreased in the Rb-/- testes. During this time we could also detect increased expression of other RB family members p107 and p130, suggesting that these factors may compensate for the loss of Rb in the germ line. This investigation has implicated RB in the regulation of XY germ cell G1/G0 arrest and will form the basis for future work aimed at understanding the initiation of this cell cycle state. In addition to RB, a lesser-known transcription factor was also investigated in the initiation and maintenance of XY germ cell G1/G0 arrest. The high mobility group box transcription factor 1 (HBP1) suppresses proliferation and promotes differentiation in various cell types and was recently identified within the XY germ cells at the appropriate time of sex differentiation. In my analysis two Hbp1 transcripts were identified within the XY germ cells that display different sub-cellular localisations in vitro. Next, Hbp1-LacZ reporter lines were generated to aid in understanding the germ cell-specific regulation of these transcripts and lastly, I analysed the genetrap mutation for Hbp1. Surprisingly, this model revealed no aberrations to germ cell-cell cycle control during development. In summary, I have performed the first comprehensive study of the cell cycle machinery utilised by germ cells as they undergo the first stages of sex differentiation. Using loss-of-function models I was able to implicate the cell cycle regulator RB specifically in XY germ cell G1/G0 arrest and, conversely, demonstrate that the transcription factor HBP1 is not required for this process.

During development, somatic cell cues direct sex-specific differentiation of germ cells that is characterised by two distinct cell cycle states. At 12.5 days post coitum (dpc) in a testis, XY germ cells stop proliferating and enter G1/G0 arrest. In the ovary, XX germ cells bypass G1/G0 arrest and instead enter the first phase of meiosis I from 13.5 dpc. Whilst it is hypothesised that errors in cell cycle control during development precede the formation of testicular germ cell tumours, the mechanism of cell cycle control at this time has not been thoroughly investigated. This project therefore sought to explore the mechanism of XY germ cell G1/G0 arrest using several approaches. Although cell cycle regulation for somatic cells is well established, we know very little regarding germ cell control of this process. Therefore my first aim was to profile this machinery at the transcript level using a cell cycle cDNA array. Purified populations of germ cells were isolated both before and after sex differentiation and expression of 112 cell cycle related genes was assessed. From this study a comprehensive network governing apoptosis and calcium signalling that was common to both XX and XY germ cells was observed. Importantly, the retinoblastoma family and cyclin dependent kinase inhibitor p21 was implicated in the regulation of G1/G0 arrest in XY germ cells. Lastly, XX germ cells displayed a down-regulation of genes involved in both G1 and G2 phases of the cell cycle consistent with their progression past G1 phase. This study has provided a detailed analysis of cell cycle gene expression during fetal germ cell development and identified candidate factors for future investigation in order to understand cases of aberrant cell cycle control in these specialised cells. In order to investigate several candidate genes identified within the cell cycle array, I next sought to generate a germ cell-specific Cre recombinase mouse model for use in conditional knockout studies. As current Cre lines lack specificity or appropriate temporal expression, we used the germ cell-specific regions of the fragilis promoter to drive Cre expression during germ cell specification. Eleven founder lines were generated using this construct and four were analysed using a reporter line. Although we have not achieved germ cell expression from these lines to date, analysis continues in order to identify an invaluable new tool for germ cell research. Following the implication of the retinoblastoma family in XY germ cell G1/G0 arrest, I next investigated the role of RB in these cells using the Rb null mutant. RB is a known cell cycle suppressor that controls this process in many cell types and, subsequently, mice homozygous for the Rb deletion die in utero at 14.5 dpc. Using this model we analysed developing gonads from 14.5 – 16.5 dpc using ex vivo culture techniques. At 14.5 dpc when wild type germ cells have arrested, proliferating germ cells were detected in the absence of Rb using proliferation marker Ki67. This proliferation was accompanied by a slight increase in germ cell number at 14.5 dpc, however, two days later at 16.5 dpc germ cell numbers were slightly decreased in the Rb-/- testes. During this time we could also detect increased expression of other RB family members p107 and p130, suggesting that these factors may compensate for the loss of Rb in the germ line. This investigation has implicated RB in the regulation of XY germ cell G1/G0 arrest and will form the basis for future work aimed at understanding the initiation of this cell cycle state. In addition to RB, a lesser-known transcription factor was also investigated in the initiation and maintenance of XY germ cell G1/G0 arrest. The high mobility group box transcription factor 1 (HBP1) suppresses proliferation and promotes differentiation in various cell types and was recently identified within the XY germ cells at the appropriate time of sex differentiation. In my analysis two Hbp1 transcripts were identified within the XY germ cells that display different sub-cellular localisations in vitro. Next, Hbp1-LacZ reporter lines were generated to aid in understanding the germ cell-specific regulation of these transcripts and lastly, I analysed the genetrap mutation for Hbp1. Surprisingly, this model revealed no aberrations to germ cell-cell cycle control during development. In summary, I have performed the first comprehensive study of the cell cycle machinery utilised by germ cells as they undergo the first stages of sex differentiation. Using loss-of-function models I was able to implicate the cell cycle regulator RB specifically in XY germ cell G1/G0 arrest and, conversely, demonstrate that the transcription factor HBP1 is not required for this process.

During development, somatic cell cues direct sex-specific differentiation of germ cells that is characterised by two distinct cell cycle states. At 12.5 days post coitum (dpc) in a testis, XY germ cells stop proliferating and enter G1/G0 arrest. In the ovary, XX germ cells bypass G1/G0 arrest and instead enter the first phase of meiosis I from 13.5 dpc. Whilst it is hypothesised that errors in cell cycle control during development precede the formation of testicular germ cell tumours, the mechanism of cell cycle control at this time has not been thoroughly investigated. This project therefore sought to explore the mechanism of XY germ cell G1/G0 arrest using several approaches. Although cell cycle regulation for somatic cells is well established, we know very little regarding germ cell control of this process. Therefore my first aim was to profile this machinery at the transcript level using a cell cycle cDNA array. Purified populations of germ cells were isolated both before and after sex differentiation and expression of 112 cell cycle related genes was assessed. From this study a comprehensive network governing apoptosis and calcium signalling that was common to both XX and XY germ cells was observed. Importantly, the retinoblastoma family and cyclin dependent kinase inhibitor p21 was implicated in the regulation of G1/G0 arrest in XY germ cells. Lastly, XX germ cells displayed a down-regulation of genes involved in both G1 and G2 phases of the cell cycle consistent with their progression past G1 phase. This study has provided a detailed analysis of cell cycle gene expression during fetal germ cell development and identified candidate factors for future investigation in order to understand cases of aberrant cell cycle control in these specialised cells. In order to investigate several candidate genes identified within the cell cycle array, I next sought to generate a germ cell-specific Cre recombinase mouse model for use in conditional knockout studies. As current Cre lines lack specificity or appropriate temporal expression, we used the germ cell-specific regions of the fragilis promoter to drive Cre expression during germ cell specification. Eleven founder lines were generated using this construct and four were analysed using a reporter line. Although we have not achieved germ cell expression from these lines to date, analysis continues in order to identify an invaluable new tool for germ cell research. Following the implication of the retinoblastoma family in XY germ cell G1/G0 arrest, I next investigated the role of RB in these cells using the Rb null mutant. RB is a known cell cycle suppressor that controls this process in many cell types and, subsequently, mice homozygous for the Rb deletion die in utero at 14.5 dpc. Using this model we analysed developing gonads from 14.5 – 16.5 dpc using ex vivo culture techniques. At 14.5 dpc when wild type germ cells have arrested, proliferating germ cells were detected in the absence of Rb using proliferation marker Ki67. This proliferation was accompanied by a slight increase in germ cell number at 14.5 dpc, however, two days later at 16.5 dpc germ cell numbers were slightly decreased in the Rb-/- testes. During this time we could also detect increased expression of other RB family members p107 and p130, suggesting that these factors may compensate for the loss of Rb in the germ line. This investigation has implicated RB in the regulation of XY germ cell G1/G0 arrest and will form the basis for future work aimed at understanding the initiation of this cell cycle state. In addition to RB, a lesser-known transcription factor was also investigated in the initiation and maintenance of XY germ cell G1/G0 arrest. The high mobility group box transcription factor 1 (HBP1) suppresses proliferation and promotes differentiation in various cell types and was recently identified within the XY germ cells at the appropriate time of sex differentiation. In my analysis two Hbp1 transcripts were identified within the XY germ cells that display different sub-cellular localisations in vitro. Next, Hbp1-LacZ reporter lines were generated to aid in understanding the germ cell-specific regulation of these transcripts and lastly, I analysed the genetrap mutation for Hbp1. Surprisingly, this model revealed no aberrations to germ cell-cell cycle control during development. In summary, I have performed the first comprehensive study of the cell cycle machinery utilised by germ cells as they undergo the first stages of sex differentiation. Using loss-of-function models I was able to implicate the cell cycle regulator RB specifically in XY germ cell G1/G0 arrest and, conversely, demonstrate that the transcription factor HBP1 is not required for this process.