Coordinated maintenance of H3K36/K27 methylation by histone demethylases preserves germ cell identity and immortality

ABSTRACT Dppa4 (developmental pluripotency-associated 4) has been identified in several high-profile screens as a gene that is expressed exclusively in pluripotent cells. It encodes a nuclear protein with an SAP-like domain and appears to be associated preferentially with transcriptionally active chromatin. Its exquisite expression pattern and results of RNA interference experiments have led to speculation that Dppa4, as well as its nearby homolog Dppa2, might play essential roles in embryonic stem (ES) cell function and/or germ cell development. To rigorously assess suggested roles, we have generated Dppa4-deficient and Dppa4/Dppa2 doubly deficient ES cells, as well as mice lacking Dppa4. Contrary to predictions, we find that Dppa4 is completely dispensable for ES cell identity and germ cell development. Instead, loss of Dppa4 in mice results in late embryonic/perinatal death and striking skeletal defects with partial penetrance. Thus, surprisingly, Dppa4-deficiency affects tissues that apparently never transcribed the gene, and at least some loss-of-function defects manifest phenotypically at an embryonic stage long after physiologic Dppa4 expression has ceased. Concomitant with targeted gene inactivation, we have introduced into the Dppa4 locus a red fluorescent marker (tandem-dimer red fluorescent protein) that is compatible with green fluorescent proteins and allows noninvasive visualization of pluripotent cells and reprogramming events.

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Testing the role of SOX15 in human primordial germ cell fate

Wellcome Open Research ◽

10.12688/wellcomeopenres.15381.2 ◽

2019 ◽

Vol 4 ◽

pp. 122

Author(s):

Merrick Pierson Smela ◽

Anastasiya Sybirna ◽

Frederick C.K. Wong ◽

M. Azim Surani

Keyword(s):

Flow Cytometry ◽

Germ Cell ◽

Primordial Germ Cell ◽

Mechanistic Study ◽

Germline Development ◽

Cell Identity ◽

Protein Levels ◽

Germ Cell Specification ◽

The Impact ◽

Early Human

Background: Potentially novel regulators of early human germline development have been identified recently, including SOX15 and SOX17, both of which show specific expression in human primordial germ cells. SOX17 is now known to be a critical specifier of human germ cell identity. There have been suggestions, as yet without evidence, that SOX15 might also play a prominent role. The early human germline is inaccessible for direct study, but an in vitro model of human primordial germ cell-like cell (hPGCLC) specification from human embryonic stem cells (hESCs) has been developed. This enables mechanistic study of human germ cell specification using genetic tools to manipulate the levels of SOX15 and SOX17 proteins to explore their roles in hPGCLC specification. Methods: SOX15 and SOX17 proteins were depleted during hPGCLC specification from hESCs using the auxin-inducible degron system, combined with a fluorescent reporter for tracking protein levels. Additionally, SOX15 protein was overexpressed using the ProteoTuner system. Protein-level expression changes were confirmed by immunofluorescence. The impact on hPGCLC specification efficiency was determined by flow cytometry at various time points. qPCR experiments were performed to determine some transcriptional effects of SOX15 perturbations. Results: We observed specific SOX15 expression in hPGCLCs by using immunofluorescence and flow cytometry analysis. Depletion of SOX15 had no significant effect on hPGCLC specification efficiency on day 4 after induction, but there was a significant and progressive decrease in hPGCLCs on days 6 and 8. By contrast, depletion of SOX17 completely abrogated hPGCLC specification. Furthermore, SOX15 overexpression resulted in a significant increase in hPGCLC fraction on day 8. qPCR analysis revealed a possible role for the germ cell and pluripotency regulator PRDM14 in compensating for changes to SOX15 protein levels. Conclusions: SOX17 is essential for hPGCLC specification, yet SOX15 is dispensable. However, SOX15 may have a role in maintaining germ cell identity.

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BMP Signaling and the Maintenance of Primordial Germ Cell Identity in Drosophila Embryos

PLoS ONE ◽

10.1371/journal.pone.0088847 ◽

2014 ◽

Vol 9 (2) ◽

pp. e88847 ◽

Cited By ~ 8

Author(s):

Girish Deshpande ◽

Elinor Willis ◽

Sandip Chatterjee ◽

Robert Fernandez ◽

Kristen Dias ◽

...

Keyword(s):

Germ Cell ◽

Primordial Germ Cell ◽

Bmp Signaling ◽

Cell Identity ◽

Drosophila Embryos

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Testing the role of SOX15 in human primordial germ cell fate

Wellcome Open Research ◽

10.12688/wellcomeopenres.15381.1 ◽

2019 ◽

Vol 4 ◽

pp. 122 ◽

Cited By ~ 2

Author(s):

Merrick Pierson Smela ◽

Anastasiya Sybirna ◽

Frederick C.K. Wong ◽

M. Azim Surani

Keyword(s):

Flow Cytometry ◽

Germ Cell ◽

Primordial Germ Cell ◽

Mechanistic Study ◽

Germline Development ◽

Cell Identity ◽

Protein Levels ◽

Germ Cell Specification ◽

The Impact ◽

Early Human

Background: Potentially novel regulators of early human germline development have been identified recently, including SOX15 and SOX17, both of which show specific expression in human primordial germ cells. SOX17 is now known to be a critical specifier of human germ cell identity. There have been suggestions, as yet without evidence, that SOX15 might also play a prominent role. The early human germline is inaccessible for direct study, but an in vitro model of human primordial germ cell-like cell (hPGCLC) specification from human embryonic stem cells (hESCs) has been developed. This enables mechanistic study of human germ cell specification using genetic tools to manipulate the levels of SOX15 and SOX17 proteins to explore their roles in hPGCLC specification. Methods: SOX15 and SOX17 proteins were depleted during hPGCLC specification from hESCs using the auxin-inducible degron system, combined with a fluorescent reporter for tracking protein levels. Additionally, SOX15 protein was overexpressed using the ProteoTuner system. Protein-level expression changes were confirmed by immunofluorescence. The impact on hPGCLC specification efficiency was determined by flow cytometry at various time points. qPCR experiments were performed to determine some transcriptional effects of SOX15 perturbations. Results: We observed specific SOX15 expression in hPGCLCs by using immunofluorescence and flow cytometry analysis. Depletion of SOX15 had no significant effect on hPGCLC specification efficiency on day 4 after induction, but there was a significant and progressive decrease in hPGCLCs on days 6 and 8. By contrast, depletion of SOX17 completely abrogated hPGCLC specification. Furthermore, SOX15 overexpression resulted in a significant increase in hPGCLC fraction on day 8. qPCR analysis revealed a possible role for the germ cell and pluripotency regulator PRDM14 in compensating for changes to SOX15 protein levels. Conclusions: SOX17 is essential for hPGCLC specification, yet SOX15 is dispensable. However, SOX15 may have a role in maintaining germ cell identity.

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Abstract LB-355: Whole-exome sequencing of intracranial germ cell tumors reveals frequent mutations in KIT and RAS pathways and histone demethylases.

10.1158/1538-7445.am2013-lb-355 ◽

2013 ◽

Cited By ~ 1

Author(s):

Linghua Wang ◽

Shigeru Yamaguchi ◽

Keita Terashima ◽

Matthew D. Burstein ◽

Jiayi M. Sun ◽

...

Keyword(s):

Germ Cell ◽

Exome Sequencing ◽

Whole Exome Sequencing ◽

Germ Cell Tumors ◽

Histone Demethylases ◽

Whole Exome ◽

Intracranial Germ Cell Tumors ◽

Frequent Mutations

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Electron microscope study of the effects of radiation on insect fertility

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100157905 ◽

1990 ◽

Vol 48 (3) ◽

pp. 72-73

Author(s):

Judy Ju-Hu Chiang ◽

Robert Kuo-Cheng Chen

Keyword(s):

Electron Microscopy ◽

Germ Cell ◽

Germ Cells ◽

Electron Microscope Study ◽

Radiation Treatment ◽

Light And Electron Microscopy ◽

Stem Borer ◽

Metabolic Energy ◽

Rice Stem Borer ◽

Effects Of Radiation

Germ cells from the rice stem borer Chilo suppresalis, were examined by light and electron microscopy. Damages to organelles within the germ cells were observed. The mitochondria, which provide the cell with metabolic energy, were seen to disintegrate within the germ cell. Lysosomes within the germ cell were also seen to disintegrate. The subsequent release of hydrolytic enzymesmay be responsible for the destruction of organelles within the germ cell. Insect spermatozoa were seen to lose the ability to move because of radiation treatment. Damage to the centrioles, one of which is in contact with the tail, may be involved in causing sperm immobility.

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