Stem cells: a new route to rejuvenation.
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Aging↗
Biomedical subjects
M Azim Surani
Publications and source records attributed to M Azim Surani.
Dppa2 and Dppa4 are closely linked SAP motif genes restricted to pluripotent cells and the germ line.
Despite the enormous medical potential of ESCs, the molecular mechanisms conferring the ability to differentiate into all cell types of the embryo remain elusive. We used an in silico approach to identify genes expressed exclusively in mouse preimplantation embryos and pluripotent cell lines. Two of these genes were developmental pluripotency-associated gene 2 (Dppa2) and Dppa4, which we show are closely linked genes encoding putative nuclear SAP domain proteins expressed in human and mouse pluripotent stem cells and germ cell tumor-derived embryonal carcinoma cells. In the mouse, these genes are transcribed in germinal vesicle-stage oocytes and throughout the cleavage stages of embryogenesis. They then become restricted to the pluripotent inner cell mass of blastocysts and are subsequently downregulated. After gastrulation, Dppa2 and Dppa4 are expressed only in the developing germ line, showing that these genes mark cells of the pluripotent cycle. In the germ line, both genes are downregulated as the germ cells commit to the oogenic pathway or soon after commitment to the spermatogenic pathway. We have observed similar germ line expression profiles for other pluripotent markers, and these results are consistent with the hypothesis that pluripotent markers must be downregulated during fetal germ line development, a process that may be required to facilitate appropriate germ line differentiation. The study of expression and function of pluripotent markers such as Dppa2 and Dppa4 is likely to unveil new aspects of the regulation of pluripotency and germ line development in mammals.
Animals↗
Influence of sex chromosome constitution on the genomic imprinting of germ cells.
Germ cells in XY male mice establish site-specific methylation on imprinted genes during spermatogenesis, whereas germ cells in XX females establish their imprints in growing oocytes. We showed previously that in vitro, sex-specific methylation patterns of pluripotent stem cell lines derived from germ cells were influenced more by the sex chromosome constitution of the cells themselves than by the gender of the embryo from which they had been derived. To see whether the same situation would prevail in vivo, we have now determined the methylation status of H19 expressed from the maternal allele, and the expression and methylation status of a paternally expressed gene Peg3, in germ cells from sex-reversed and control embryos. For these imprinted genes, we conclude that the female imprint is a response of the germ cells to undergoing oogenesis, rather than to their XX chromosome constitution. Similarly, both our XY and our sex-reversed XX male germ cells clearly showed a male rather than a female pattern of DNA methylation; here, however, the sex chromosome constitution had a significant effect, with XX male germ cells less methylated than the XY controls.
Animals↗
Blimp1 associates with Prmt5 and directs histone arginine methylation in mouse germ cells.
Blimp1, a transcriptional repressor, has a crucial role in the specification of primordial germ cells (PGCs) in mice at embryonic day 7.5 (E7.5). This SET-PR domain protein can form complexes with various chromatin modifiers in a context-dependent manner. Here, we show that Blimp1 has a novel interaction with Prmt5, an arginine-specific histone methyltransferase, which mediates symmetrical dimethylation of arginine 3 on histone H2A and/or H4 tails (H2A/H4R3me2s). Prmt5 has been shown to associate with Tudor, a component of germ plasm in Drosophila melanogaster. Blimp1-Prmt5 colocalization results in high levels of H2A/H4 R3 methylation in PGCs at E8.5. However, at E11.5, Blimp1-Prmt5 translocates from the nucleus to the cytoplasm, resulting in the loss of H2A/H4 R3 methylation at the time of extensive epigenetic reprogramming of germ cells. Subsequently, Dhx38, a putative target of the Blimp1-Prmt5 complex, is upregulated. Interestingly, expression of Dhx38 is also seen in pluripotent embryonic germ cells that are derived from PGCs when Blimp1 expression is lost. Our study demonstrates that Blimp1 is involved in a novel transcriptional regulatory complex in the mouse germ-cell lineage.
Active Transport, Cell Nucleus↗
MicroRNA expression profiling of single whole embryonic stem cells.
MicroRNAs (miRNAs) are a class of 17-25 nt non-coding RNAs that have been shown to have critical functions in a wide variety of biological processes during development. Recently developed miRNA microarray techniques have helped to accelerate research on miRNAs. However, in some instances there is only a limited amount of material available for analysis, which requires more sensitive techniques that can preferably work on single cells. Here we demonstrate that it is possible to analyse miRNA in single cells by using a real-time PCR-based 220-plex miRNA expression profiling method. Development of this technique will greatly facilitate miRNA-related research on cells, such as the founder population of primordial germ cells where rapid and dynamic changes occur in a few cells, and for analysing heterogeneous population of cells. In these and similar cases, our method of single cell analysis is critical for elucidating the diverse roles of miRNAs.
Animals↗
Generation of stella-GFP transgenic mice: a novel tool to study germ cell development.
The relationship between germ cells and pluripotent embryonic stem (ES) cells is of particular interest, together with approaches to generate primordial germ cell (PGCs) from ES cells. A critical requirement in these experiments is the ability to unambiguously detect PGCs with the use of, for example, reporter genes. The currently available transgenic reporters do not show exclusive expression in PGCs at their earliest developmental stages. Here we describe the use of germline-restricted expression of stella, which is currently the best marker gene for PGCs. We generated two stella-GFP reporters and show that both transgenes surpass other reporters in terms of timing and specificity of expression in PGCs. Additionally, we demonstrate the usefulness of stella-GFP during the derivation of PGCs from ES cells.
Animals↗
Loss of TSLC1 causes male infertility due to a defect at the spermatid stage of spermatogenesis.
Tumor suppressor of lung cancer 1 (TSLC1), also known as SgIGSF, IGSF4, and SynCAM, is strongly expressed in spermatogenic cells undergoing the early and late phases of spermatogenesis (spermatogonia to zygotene spermatocytes and elongating spermatids to spermiation). Using embryonic stem cell technology to generate a null mutation of Tslc1 in mice, we found that Tslc1 null male mice were infertile. Tslc1 null adult testes showed that spermatogenesis had arrested at the spermatid stage, with degenerating and apoptotic spermatids sloughing off into the lumen. In adult mice, Tslc1 null round spermatids showed evidence of normal differentiation (an acrosomal cap and F-actin polarization indistinguishable from that of wild-type spermatids); however, the surviving spermatozoa were immature, malformed, found at very low levels in the epididymis, and rarely motile. Analysis of the first wave of spermatogenesis in Tslc1 null mice showed a delay in maturation by day 22 and degeneration of round spermatids by day 28. Expression profiling of the testes revealed that Tslc1 null mice showed increases in the expression levels of genes involved in apoptosis, adhesion, and the cytoskeleton. Taken together, these data show that Tslc1 is essential for normal spermatogenesis in mice.
Animals↗
The role of exogenous fibroblast growth factor-2 on the reprogramming of primordial germ cells into pluripotent stem cells.
The germ cell lineage is a specified cell population that passes through a series of differentiation steps before giving rise, eventually, to either eggs or sperm. We have investigated the manner in which primordial germ cells (PGCs) are reprogrammed in vitro to form pluripotent stem cells in response to exogenous fibroblast growth factor-2 (FGF-2). The response is dependent on time of exposure and concentration of FGF-2. PGCs isolated in culture show a motile phenotype and lose any expression of a characteristic germ cell marker, mouse vasa homolog. Subsequently, some but not all of the cells show further changes of phenotype, accompanied by changes in expression of endogenous FGF-2 and up-regulation of its receptor, fibroblast growth factor receptor-3, in the nucleus. We propose that it is from this reprogrammed component of the now heterogeneous PGC population that pluripotent stem cells arise.
Animals↗
Blimp1 and the emergence of the germ line during development in the mouse.
To elucidate the mechanism for the specification of primordial germ cells (PGCs) in mice, we have developed and exploited the methods of single cell analysis. Based on these studies, we proposed a molecular program associated with this process, a key event of which is the repression of homeobox genes that are, without exception, upregulated in somatic neighbors. We have now identified Blimp1, a potent transcriptional repressor of a histone methyltransferase subfamily, as a key regulator of PGC specification. Indeed, the unexpected early onset of Blimp1 expression in a few cells at the most proximal-posterior epiblast cells marks the origin of the germ cell lineage. Disruption of Blimp1 function resulted in aberrant PGC-like cells with a deregulated intrinsic gene expression program at a very early stage, which demonstrates that Blimp1 is a critical determinant of the germ line in mice.
Animals↗
Analysis of Esg1 expression in pluripotent cells and the germline reveals similarities with Oct4 and Sox2 and differences between human pluripotent cell lines.
Establishment of pluripotent epiblast cells is a critical event during early mammalian development because all somatic lineages and the primordial germ cells (PGCs) are derived from them. The epiblast and PGCs are in turn the precursors of pluripotent embryonic stem cells and embryonic germ cells, respectively. Although PGCs are specialized cells, they express several key pluripotency-related genes, such as Oct4 and Sox2. We have analyzed Esg1 expression in mouse and human cells and shown that in the mouse the gene is specifically expressed in preimplantation embryos, stem cells, and the germline. Moreover, Esg1 coexpresses with Oct4 and Sox2, confirming its identity as a marker of the pluripotent cycle. Esg1 is also expressed with Oct4 and Sox2 by human embryonic stem cells and in germ cell carcinoma tissue but not by all human embryonal carcinoma cell lines. These data suggest that together with Oct4 and Sox2, Esg1 plays a conserved role in the pluripotent pathway of mouse and human stem and germ cells.
Animals↗
Nuclear reprogramming by human embryonic stem cells.
Embryonic stem cells have two unique properties. They are capable of indefinite self-renewal and, being pluripotent, they can differentiate into all possible cell types, including germ cells. A new study by Cowan et al. (2005) published in Science shows that human embryonic stem cells are able to reprogram the nuclei of fully differentiated human somatic cells, apparently conferring on them a pluripotent state.
Cell Nucleus↗
Genomic characterisation of a Fgf-regulated gradient-based neocortical protomap.
Recent findings support a model for neocortical area formation in which neocortical progenitor cells become patterned by extracellular signals to generate a protomap of progenitor cell areas that in turn generate area-specific neurons. The protomap is thought to be underpinned by spatial differences in progenitor cell identity that are reflected at the transcriptional level. We systematically investigated the nature and composition of the protomap by genomic analyses of spatial and temporal neocortical progenitor cell gene expression. We did not find gene expression evidence for progenitor cell organisation into domains or compartments, instead finding rostrocaudal gradients of gene expression across the entire neocortex. Given the role of Fgf signalling in rostrocaudal neocortical patterning, we carried out an in vivo global analysis of cortical gene expression in Fgfr1 mutant mice, identifying consistent alterations in the expression of candidate protomap elements. One such gene, Mest, was predicted by those studies to be a direct target of Fgf8 signalling and to be involved in setting up, rather than implementing, the progenitor cell protomap. In support of this, we confirmed Mest as a direct transcriptional target of Fgf8-regulated signalling in vitro. Functional studies demonstrated that this gene has a role in establishing patterned gene expression in the developing neocortex, potentially by acting as a negative regulator of the Fgf8-controlled patterning system.
Animals↗
Altered primordial germ cell migration in the absence of transforming growth factor beta signaling via ALK5.
Transforming growth factor beta (TGFbeta) inhibits proliferation and promotes the migration of primordial germ cells (PGCs) towards explants of gonadal ridges in vitro. However, its effects in vivo are still unclear. Here, we analyzed the behavior of PGCs in embryos lacking TGFbeta signaling via the type I receptor ALK5. TGFbeta in vivo was neither a chemoattractant for PGCs, nor did it affect their proliferation during migration towards the gonadal ridges up to embryonic day (E)10. Unexpectedly, the absence of TGFbeta signaling in fact resulted in significant facilitation of PGC migration out of the hindgut, due to the reduced deposition of collagen type I surrounding the gut of Alk5-deficient mutant embryos. Migratory PGCs adhere strongly to collagen; therefore, reduced collagen type I along the gut may result in reduced adhesion, facilitating migration into the dorsal mesenterium and gonadal ridges. Our results provide new evidence for the role of TGFbeta signaling in migration of PGCs in vivo distinct from that described previously.
Activin Receptors, Type I↗
Blimp1 is a critical determinant of the germ cell lineage in mice.
Germ cell fate in mice is induced in pluripotent epiblast cells in response to signals from extraembryonic tissues. The specification of approximately 40 founder primordial germ cells and their segregation from somatic neighbours are important events in early development. We have proposed that a critical event during this specification includes repression of a somatic programme that is adopted by neighbouring cells. Here we show that Blimp1 (also known as Prdm1), a known transcriptional repressor, has a critical role in the foundation of the mouse germ cell lineage, as its disruption causes a block early in the process of primordial germ cell formation. Blimp1-deficient mutant embryos form a tight cluster of about 20 primordial germ cell-like cells, which fail to show the characteristic migration, proliferation and consistent repression of homeobox genes that normally accompany specification of primordial germ cells. Furthermore, our genetic lineage-tracing experiments indicate that the Blimp1-positive cells originating from the proximal posterior epiblast cells are indeed the lineage-restricted primordial germ cell precursors.
Animals↗
Initiation of epigenetic reprogramming of the X chromosome in somatic nuclei transplanted to a mouse oocyte.
The active and inactive X chromosomes have distinct epigenetic marks in somatic nuclei, which undergo reprogramming after transplantation into oocytes. We show that, despite the disappearance of Xist RNA coating in 30 min, the epigenetic memory of the inactive X persists with the precocious appearance of histone H3 trimethylation of lysine 27 (H3-3meK27), without the expected colocalization with Eed/Ezh2. Subsequently, Xist re-appears on the original inactive X, and the silent Xist on the active X undergoes re-activation, resulting in unusual biallelic Xist RNA domains. Despite this abnormal Xist expression pattern, colocalization of H3-3meK27 and Eed is thereafter confined to a single Xist domain, which is presumably on the original inactive X. These epigenetic events differ markedly from the kinetics of preferential paternal X inactivation in normal embryos. All the epigenetic marks on the X are apparently erased in the epiblast, suggesting that the oocyte and epiblast may have distinct properties for stepwise programming of the genome.
Alleles↗
Development. Programming the X chromosome.
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Animals↗
Stem cells: how to make eggs and sperm.
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Animals↗
Stella is a maternal effect gene required for normal early development in mice.
stella is a novel gene specifically expressed in primordial germ cells, oocytes, preimplantation embryos, and pluripotent cells. It encodes a protein with a SAP-like domain and a splicing factor motif-like structure, suggesting possible roles in chromosomal organization or RNA processing. Here, we have investigated the effects of a targeted mutation of stella in mice. We show that while matings between heterozygous animals resulted in the birth of apparently normal stella null offspring, stella-deficient females displayed severely reduced fertility due to a lack of maternally inherited Stella-protein in their oocytes. Indeed, we demonstrate that embryos without Stella are compromised in preimplantation development and rarely reach the blastocyst stage. stella is thus one of few known mammalian maternal effect genes, as the phenotypic effect on embryonic development is mainly a consequence of the maternal stella mutant genotype. Furthermore, we show that STELLA that is expressed in human oocytes is also expressed in human pluripotent cells and in germ cell tumors. Interestingly, human chromosome 12p, which harbours STELLA, is consistently overrepresented in these tumors. These findings suggest a similar role for STELLA during early human development as in mice and a potential involvement in germ cell tumors.
Amino Acid Sequence↗