- Email: [email protected]
Investigations at the ‘Four-Front’ of Mammalian Development
TIGS 1286 No. of Pages 2
Spotlight
Investigations at the ‘Four-Front’ of Mammalian Development Amy Ralston1,* Understanding how and when cells become different during embryogenesis is a goal that is at the forefront of investigations in mammalian development. Two recent studies from the laboratories of Nicholas Plachta and Magdalena ZernickaGoetz present evidence that cellular heterogeneities detected in four-cell mouse embryos bias the process of cell fate acquisition thereafter. Deciphering the genetic mechanisms that drive cell specialization during mammalian development is a goal that is at the forefront of research in reproductive and stem cell biology. To this end, gene knockout studies in mice have been invaluable for revealing when, where, and how genes regulate cell fates during embryogenesis. Knockout studies have shown that potent cell fate-specifying genes are required after the eight-cell stage, consistent with classical studies showing that cell fates are first specified after this stage [1]. While knockout studies can reveal the absolute requirement of genes, they do not reveal when a cell first begins to exhibit differences that could influence its path to maturation. The availability of single-cell RNA sequencing has enabled researchers to describe cell-tocell gene expression differences originating earlier than the 16-cell stage [2–4]. This kind of study is joined by two more recent studies, from the laboratories of Nicholas Plachta and Magdalena Zernicka-Goetz, which describe molecular differences among cells of the embryo as early as the four-cell stage [5,6]. Together, these reports raise exciting
questions for future investigations into a correlation between cells in which SOX2 dwells longer in the nucleus at the early mammalian development. four-cell stage and acquisition of pluripoOne of the first goals of the mammalian tent cell fate at later stages. Although the embryo is to specify pluripotent epiblast nuclear dwell time of endogenous SOX2 cells, which are the progenitors of embry- was not measured at the four-cell stage, onic stem cells and the fetus. Therefore, the report points to the existence of previan understanding of the mechanisms that ously uncharacterized heterogeneity at drive cell fate specification in the early the four-cell stage. embryo could provide key insight into the origins of pluripotency. Progenitors White and colleagues then focused on of the pluripotent epiblast are first mor- identifying genes acting upstream of phologically evident at the 16-cell stage, SOX2 to regulate its nuclear dwell time when one or two cells come to occupy the in the four-cell embryo. The group examinside of the ball of cleaving cells (Figure 1). ined the role of CARM1, a histone arginine At the 16-cell stage, pluripotency proteins, methyltransferase, because it was previsuch as OCT4 and NANOG, are ously reported to regulate cell fates as expressed in all cells, although SOX2 is early as the four-cell stage [9]. Using specifically restricted to inside cells at this siRNA to knock down CARM1 expresstage [7,8], indicating that it is one of the sion, White and colleagues showed that earliest markers of pluripotent progeni- CARM1 levels correlate with SOX2-GFP tors. Curiously, however, Sox2 is not nuclear dwell time and, ultimately, the required for the formation of the inside acquisition of pluripotent cell fate. cells at the 16-cell stage [7]. The two new reports present a model positing that The study by Goolam and colleagues SOX2 activity is regulated before the 16- investigated candidates acting downcell stage, and describe genes acting stream of SOX2 to predispose cells to upstream and downstream of SOX2 to adopt a pluripotent cell fate. Goolam regulate pluripotent cell fate. Using com- and colleagues focused on the gene plementary approaches to investigate Sox21, which they proposed to be a tarwhether molecular differences are appar- get of SOX2 at the four-cell stage. ent among the cells of the four-cell stage Although this hypothesis has not been mouse embryo, both groups arrive at the tested in mouse embryos, Goolam and conclusion that the origins of pluripotent colleagues note that Sox21 is a target of cells can be traced back to the four-cell SOX2 in other contexts. The authors stage, when heterogeneity in SOX2 activ- reported that the levels of Sox21 differ dramatically among the cells of the early ity is first apparent. embryo, and correlate with Carm1 Using live imaging to track the behavior of expression levels. Accordingly, mosaic fluorescently tagged transcription factors siRNA knockdown of Sox21 at the fourinjected into early mouse embryos, White cell stage lowers the odds that knockand colleagues report that SOX2-GFP down cells will become pluripotent, conexpression exhibits cell-to-cell variation sistent with a role for SOX21 in promoting in nuclear localization dynamics at the pluripotent cell fate downstream of four-cell stage. The authors interpret this CARM1 and SOX2. This result is surprisobservation to mean that the cells of the ing, given that Sox21-knockout mice are four-cell embryo exhibit differences in reportedly born normally, exhibiting only SOX2-DNA binding and, therefore, that an alopecia phenotype [10]. Therefore, SOX2-GFP acts as a sensor of chromatin Sox21 is not required for pluripotency in differences among the cells of the four-cell vivo, but SOX21 [4_TD$IF]expression [5_TD$IF]differences embryo. By lineage tracing SOX2-GFP- [6_TD$IF]among cells [7_TD$IF]could [8_TD$IF]influence [9_TD$IF]which [10_TD$IF]cells overexpressing cells, the authors report [1_TD$IF]eventually [12_TD$IF]adopt [13_TD$IF]pluripotent [14_TD$IF]fate.
Trends in Genetics, Month Year, Vol. xx, No. yy
1
TIGS 1286 No. of Pages 2
4-cell
8-cell
to future studies to address these exciting questions.
CARM1 Acknowledgments
H3R26Me2
I would like to acknowledge Tristan Frum and Yojiro Yamanaka for discussion, and the National Institute of
SOX2+DNA
General Medical Sciences for funding by R01 GM104009. 1
Department of Biochemistry and Molecular Biology,
Michigan State University, East Lansing, MI 48823, USA
16-cell
64-cell
SOX21 *Correspondence: [email protected] (A. Ralston). http://dx.doi.org/10.1016/j.tig.2016.05.005
Inside Outside Pluripotent cells
Biased contribuon to pluripotent lineage
Figure 1. Heterogeneities at the Four-Cell Stage Bias Cell Fate Allocation in the Early Mouse Embryo. Evidence presented in two recent studies leads to a model in which the CARM1 histone arginine methyltransferase increases access of SOX2 to its[3_TD$IF] transcriptional targets, such as Sox21, which then biases cells to adopt a pluripotent cell fate.
Ultimately, these two reports propose that CARM1 is a key regulator of cell fate, and raise several interesting questions. First, why do Carm1-null embryos survive early embryonic development? Does oocyteexpressed Carm1 function during the first few cleavages? Second, what causes the heterogeneous expression of Carm1, and is this mechanism and the Carm1 expression pattern stereotyped among embryos? Finally, a question that applies to a broad
2
Trends in Genetics, Month Year, Vol. xx, No. yy
range of biomedical investigations: does heterogeneity matter? Since pluripotent progenitors can form in the complete absence of many of the genes described above, this argues that heterogeneous gene expression is not important. While the heterogeneity may not be necessary for development, it may exist to create biases that influence the developmental trajectories during development. Either way, the field will be looking ‘four-ward’
References 1. Chazaud, C. and Yamanaka, Y. (2016) Lineage specification in the mouse preimplantation embryo. Development 143, 1063–1074 2. Shi, J. et al. (2015) Dynamic transcriptional symmetrybreaking in pre-implantation mammalian embryo development revealed by single-cell RNA-seq. Development 142, 3468–3477 3. Biase, F.H. et al. (2014) Cell fate inclination within 2-cell and 4-cell mouse embryos revealed by single-cell RNA sequencing. Genome Res. 24, 1787–1796 4. Piras, V. et al. (2014) Transcriptome-wide variability in single embryonic development cells. Sci. Rep. 4, 7137 5. White, M.D. et al. (2016) Long-lived binding of Sox2 to DNA predicts cell fate in the four-cell mouse embryo. Cell 165, 75–87 6. Goolam, M. et al. (2016) Heterogeneity in Oct4 and Sox2 targets biases cell fate in 4-cell mouse embryos. Cell 165, 61–74 7. Wicklow, E. et al. (2014) HIPPO pathway members restrict SOX2 to the inner cell mass where it promotes ICM fates in the mouse blastocyst. PLoS Genet. 10, e1004618 8. Guo, G. et al. (2010) Resolution of cell fate decisions revealed by single-cell gene expression analysis from zygote to blastocyst. Dev. Cell 18, 675–685 9. Torres-Padilla, M. et al. (2007) Histone arginine methylation regulates pluripotency in the early mouse embryo. Nature 445, 214–218 10. Kiso, M. et al. (2009) The disruption of Sox21-mediated hair shaft cuticle differentiation causes cyclic alopecia in mice. Proc. Natl. Acad. Sci. U.S.A 106, 9292–9297
Spotlight
Investigations at the ‘Four-Front’ of Mammalian Development Amy Ralston1,* Understanding how and when cells become different during embryogenesis is a goal that is at the forefront of investigations in mammalian development. Two recent studies from the laboratories of Nicholas Plachta and Magdalena ZernickaGoetz present evidence that cellular heterogeneities detected in four-cell mouse embryos bias the process of cell fate acquisition thereafter. Deciphering the genetic mechanisms that drive cell specialization during mammalian development is a goal that is at the forefront of research in reproductive and stem cell biology. To this end, gene knockout studies in mice have been invaluable for revealing when, where, and how genes regulate cell fates during embryogenesis. Knockout studies have shown that potent cell fate-specifying genes are required after the eight-cell stage, consistent with classical studies showing that cell fates are first specified after this stage [1]. While knockout studies can reveal the absolute requirement of genes, they do not reveal when a cell first begins to exhibit differences that could influence its path to maturation. The availability of single-cell RNA sequencing has enabled researchers to describe cell-tocell gene expression differences originating earlier than the 16-cell stage [2–4]. This kind of study is joined by two more recent studies, from the laboratories of Nicholas Plachta and Magdalena Zernicka-Goetz, which describe molecular differences among cells of the embryo as early as the four-cell stage [5,6]. Together, these reports raise exciting
questions for future investigations into a correlation between cells in which SOX2 dwells longer in the nucleus at the early mammalian development. four-cell stage and acquisition of pluripoOne of the first goals of the mammalian tent cell fate at later stages. Although the embryo is to specify pluripotent epiblast nuclear dwell time of endogenous SOX2 cells, which are the progenitors of embry- was not measured at the four-cell stage, onic stem cells and the fetus. Therefore, the report points to the existence of previan understanding of the mechanisms that ously uncharacterized heterogeneity at drive cell fate specification in the early the four-cell stage. embryo could provide key insight into the origins of pluripotency. Progenitors White and colleagues then focused on of the pluripotent epiblast are first mor- identifying genes acting upstream of phologically evident at the 16-cell stage, SOX2 to regulate its nuclear dwell time when one or two cells come to occupy the in the four-cell embryo. The group examinside of the ball of cleaving cells (Figure 1). ined the role of CARM1, a histone arginine At the 16-cell stage, pluripotency proteins, methyltransferase, because it was previsuch as OCT4 and NANOG, are ously reported to regulate cell fates as expressed in all cells, although SOX2 is early as the four-cell stage [9]. Using specifically restricted to inside cells at this siRNA to knock down CARM1 expresstage [7,8], indicating that it is one of the sion, White and colleagues showed that earliest markers of pluripotent progeni- CARM1 levels correlate with SOX2-GFP tors. Curiously, however, Sox2 is not nuclear dwell time and, ultimately, the required for the formation of the inside acquisition of pluripotent cell fate. cells at the 16-cell stage [7]. The two new reports present a model positing that The study by Goolam and colleagues SOX2 activity is regulated before the 16- investigated candidates acting downcell stage, and describe genes acting stream of SOX2 to predispose cells to upstream and downstream of SOX2 to adopt a pluripotent cell fate. Goolam regulate pluripotent cell fate. Using com- and colleagues focused on the gene plementary approaches to investigate Sox21, which they proposed to be a tarwhether molecular differences are appar- get of SOX2 at the four-cell stage. ent among the cells of the four-cell stage Although this hypothesis has not been mouse embryo, both groups arrive at the tested in mouse embryos, Goolam and conclusion that the origins of pluripotent colleagues note that Sox21 is a target of cells can be traced back to the four-cell SOX2 in other contexts. The authors stage, when heterogeneity in SOX2 activ- reported that the levels of Sox21 differ dramatically among the cells of the early ity is first apparent. embryo, and correlate with Carm1 Using live imaging to track the behavior of expression levels. Accordingly, mosaic fluorescently tagged transcription factors siRNA knockdown of Sox21 at the fourinjected into early mouse embryos, White cell stage lowers the odds that knockand colleagues report that SOX2-GFP down cells will become pluripotent, conexpression exhibits cell-to-cell variation sistent with a role for SOX21 in promoting in nuclear localization dynamics at the pluripotent cell fate downstream of four-cell stage. The authors interpret this CARM1 and SOX2. This result is surprisobservation to mean that the cells of the ing, given that Sox21-knockout mice are four-cell embryo exhibit differences in reportedly born normally, exhibiting only SOX2-DNA binding and, therefore, that an alopecia phenotype [10]. Therefore, SOX2-GFP acts as a sensor of chromatin Sox21 is not required for pluripotency in differences among the cells of the four-cell vivo, but SOX21 [4_TD$IF]expression [5_TD$IF]differences embryo. By lineage tracing SOX2-GFP- [6_TD$IF]among cells [7_TD$IF]could [8_TD$IF]influence [9_TD$IF]which [10_TD$IF]cells overexpressing cells, the authors report [1_TD$IF]eventually [12_TD$IF]adopt [13_TD$IF]pluripotent [14_TD$IF]fate.
Trends in Genetics, Month Year, Vol. xx, No. yy
1
TIGS 1286 No. of Pages 2
4-cell
8-cell
to future studies to address these exciting questions.
CARM1 Acknowledgments
H3R26Me2
I would like to acknowledge Tristan Frum and Yojiro Yamanaka for discussion, and the National Institute of
SOX2+DNA
General Medical Sciences for funding by R01 GM104009. 1
Department of Biochemistry and Molecular Biology,
Michigan State University, East Lansing, MI 48823, USA
16-cell
64-cell
SOX21 *Correspondence: [email protected] (A. Ralston). http://dx.doi.org/10.1016/j.tig.2016.05.005
Inside Outside Pluripotent cells
Biased contribuon to pluripotent lineage
Figure 1. Heterogeneities at the Four-Cell Stage Bias Cell Fate Allocation in the Early Mouse Embryo. Evidence presented in two recent studies leads to a model in which the CARM1 histone arginine methyltransferase increases access of SOX2 to its[3_TD$IF] transcriptional targets, such as Sox21, which then biases cells to adopt a pluripotent cell fate.
Ultimately, these two reports propose that CARM1 is a key regulator of cell fate, and raise several interesting questions. First, why do Carm1-null embryos survive early embryonic development? Does oocyteexpressed Carm1 function during the first few cleavages? Second, what causes the heterogeneous expression of Carm1, and is this mechanism and the Carm1 expression pattern stereotyped among embryos? Finally, a question that applies to a broad
2
Trends in Genetics, Month Year, Vol. xx, No. yy
range of biomedical investigations: does heterogeneity matter? Since pluripotent progenitors can form in the complete absence of many of the genes described above, this argues that heterogeneous gene expression is not important. While the heterogeneity may not be necessary for development, it may exist to create biases that influence the developmental trajectories during development. Either way, the field will be looking ‘four-ward’
References 1. Chazaud, C. and Yamanaka, Y. (2016) Lineage specification in the mouse preimplantation embryo. Development 143, 1063–1074 2. Shi, J. et al. (2015) Dynamic transcriptional symmetrybreaking in pre-implantation mammalian embryo development revealed by single-cell RNA-seq. Development 142, 3468–3477 3. Biase, F.H. et al. (2014) Cell fate inclination within 2-cell and 4-cell mouse embryos revealed by single-cell RNA sequencing. Genome Res. 24, 1787–1796 4. Piras, V. et al. (2014) Transcriptome-wide variability in single embryonic development cells. Sci. Rep. 4, 7137 5. White, M.D. et al. (2016) Long-lived binding of Sox2 to DNA predicts cell fate in the four-cell mouse embryo. Cell 165, 75–87 6. Goolam, M. et al. (2016) Heterogeneity in Oct4 and Sox2 targets biases cell fate in 4-cell mouse embryos. Cell 165, 61–74 7. Wicklow, E. et al. (2014) HIPPO pathway members restrict SOX2 to the inner cell mass where it promotes ICM fates in the mouse blastocyst. PLoS Genet. 10, e1004618 8. Guo, G. et al. (2010) Resolution of cell fate decisions revealed by single-cell gene expression analysis from zygote to blastocyst. Dev. Cell 18, 675–685 9. Torres-Padilla, M. et al. (2007) Histone arginine methylation regulates pluripotency in the early mouse embryo. Nature 445, 214–218 10. Kiso, M. et al. (2009) The disruption of Sox21-mediated hair shaft cuticle differentiation causes cyclic alopecia in mice. Proc. Natl. Acad. Sci. U.S.A 106, 9292–9297