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J Rathjen

Publications and source records attributed to J Rathjen.

13 recordsLinked to original sources
PubMed2002

Transient pluripotent cell populations during primitive ectoderm formation: correlation of in vivo and in vitro pluripotent cell development.

Formation and differentiation of a pluripotent cell population is central to mammalian development, and the isolation, identification and manipulation of human pluripotent cells is predicted to be of therapeutic use. Within the early mammalian embryo, two distinct populations of pluripotent cells have been described: the inner cell mass (ICM), which differentiates to form a second pluripotent cell populations, the primitive ectoderm. Indirect evidence suggests the existence of temporally distinct intermediate pluripotent cell populations as primitive ectoderm is formed. We coupled an in vitro model of primitive ectoderm formation (the transition of embryonic stem cells to early primitive ectoderm-like (EPL) cells) with ddPCR-based techniques to identify three novel genes, Psc1, CRTR-1 and PRCE, that were expressed differently during pluripotent cell progression. Detailed mapping of these genes with Oct4, Rex1 and Fgf5 on pregastrulation embryos provided the first molecular evidence for the existence of successive, temporally distinct pluripotent cell populations in the embryo between the ICM and primitive ectoderm. No evidence was found for spatial heterogeneity within the Oct4(+) pool. The transition between populations correlated with morphological or developmental alterations in pluripotent cells in vivo. Genes that are temporally expressed during pluripotent cell progression may provide an opportunity for molecular discrimination of pluripotent cells at different stages of maturation in vivo and an understanding of the cellular origins and properties of pluripotent cell lines isolated from diverse sources. Furthermore, the strong correlation of gene expression demonstrated between EPL cell formation in vitro and primitive ectoderm formation in vivo validates EPL cells as a model for primitive ectoderm, thereby providing a model system for the investigation of pluripotent differentiation and an opportunity for directed differentiation of pluripotent cells to therapeutically useful cell populations.

Amino Acid Sequence↗
PubMed2001

Mouse ES cells: experimental exploitation of pluripotent differentiation potential.

Pluripotent ES cells can be used to generate a wide variety of cell populations in vitro in a manner resembling embryonic development. Recent advances in controlling ES cell differentiation, combined with the power of genetic and biochemical manipulation, are providing insights into cell biology and the determination of cell fate.

Animals↗
PubMed2001

Lineage specific differentiation of pluripotent cells in vitro: a role for extraembryonic cell types.

The controlled differentiation of pluripotent cells will be a prerequisite for many cell therapies. We have previously reported homogeneous conversion of embryonic stem (ES) cells in vitro to early primitive ectoderm-like (EPL) cells, equivalent to early primitive ectoderm, an obligatory differentiation intermediate between ES cells and somatic cell populations. Early primitive ectoderm-like cells differentiated within aggregates form mesodermal lineages at the expense of ectoderm. In this work we demonstrate that the failure of EPL cells to form ectodermal cell types does not reflect an inherent restriction in developmental potential. Early primitive ectoderm-like cells form ectodermal derivatives such as neurons in response to neural inducers such as retinoic acid, or when differentiated in the environment provided by ES cell embryoid bodies. This could be explained by signals from the extraembryonic cell type visceral endoderm which forms in differentiating ES cell but not EPL cell aggregates. Consistent with this possibility, culture of EPL cell aggregates in the presence of visceral endoderm-like signals did not prevent differentiation of the pluripotent cells, but resulted in suppression of mesoderm formation. These results suggest a role for visceral endoderm in regulation of germ layer specification from pluripotent cells, and can be integrated into a model for cell differentiation in vitro and in vivo.

Blotting, Northern↗
PubMed2001

Structure, sequence and function of a marsupial LIF gene: conservation of IL-6 family cytokines.

Leukaemia Inhibitory Factor (LIF) is a multifunctional cytokine with an obligate role in the mouse in embryonic implantation. In this paper we demonstrate the existence of a functional LIF gene in the marsupial Sminthopsis crassicaudata, and the presence of LIF-related sequences in the monotreme Tachyglossus aculeatus (Australian echidna). Isolation of genomic and cDNA clones from S. crassicaudata, indicated that the LIF gene is highly conserved between marsupials and monotremes in terms of sequence and genomic organisation. Critical functional residues within the LIF sequence were also conserved including residues implicated in intracellular LIF activity, and in interaction with the receptor subunits LIFR and gp130. These findings suggest that the structure and biochemical function of the protein is likely to be conserved. Consistent with this, purified recombinant S. crassicaudata LIF interacted functionally with mouse receptor components and was sufficient for maintenance of mouse embryonic stem (ES) cells in the undifferentiated state. Conservation of LIF outside eutherians is intriguing given the markedly divergent reproductive strategies which include, for some marsupial species, embryonic diapause, and in monotremes, the absence of implantation. The availability of marsupial LIF probes provides an opportunity to investigate conservation of expression and function in these mammals.

Amino Acid Sequence↗
PubMed2000

Reversible programming of pluripotent cell differentiation.

We have undertaken an in vitro differentiation analysis of two related, interconvertible, pluripotent cell populations, ES and early primitive ectoderm-like (EPL) cells, which are most similar in morphology, gene expression, cytokine responsiveness and differentiation potential in vivo to ICM and early primitive ectoderm, respectively. Pluripotent cells were differentiated in vitro as aggregates (embryoid bodies) and the appearance and abundance of cell lineages were assessed by morphology and gene expression. Differentiation in EPL cell embryoid bodies recapitulated normal developmental progression in vivo, but was advanced in comparison to ES cell embryoid bodies, with the rapid establishment of late primitive ectoderm specific gene expression, and subsequent loss of pluripotent cell markers. Nascent mesoderm was formed earlier and more extensively in EPL cell embryoid bodies, and resulted in the appearance of terminally differentiated mesodermal cell types prior to and at higher levels than in ES cell embryoid bodies. Nascent mesoderm in EPL cell embryoid bodies was not specified but could be programmed to alternative fates by the addition of exogenous factors. EPL cells remained competent to form primitive endoderm even though this is not the normal fate of primitive ectoderm in vivo. The establishment of primitive ectoderm-like gene expression and inability to participate in embryogenesis following blastocyst injection is therefore not directly associated with restriction in the ability to form extra-embryonic lineages. However, the EPL cell embryoid body environment did not support differentiation of primitive endoderm to visceral endoderm, indicating the lack of an inductive signal for visceral endoderm formation deduced to originate from the pluripotent cells. Similarly, the inability of EPL cells to form neurons when differentiated as embryoid bodies was attributable to perturbation of the differentiation environment and loss of inductive signals rather than a restricted differentiation potential. Reversion of EPL cells to ES cells was accompanied by restoration of ES cell-like differentiation potential. These results demonstrate the ability of pluripotent cells to adopt developmentally distinct, stable cell states with altered differentiation potentials.

Animals↗
PubMed1999

Formation of a primitive ectoderm like cell population, EPL cells, from ES cells in response to biologically derived factors.

The primitive ectoderm of the mouse embryo arises from the inner cell mass between 4.75 and 5.25 days post coitum, around the time of implantation. Positioned at a pivotal time in development, just prior to formation of the three germ layers of the embryo proper, the primitive ectoderm responds directly to the signals generated during gastrulation. We have identified a conditioned medium, MEDII, which caused the homogeneous conversion of ES cells to a morphologically distinct cell population, termed early primitive ectoderm-like (EPL) cells. EPL cells expressed the pluripotent cell markers Oct4, SSEA1 and alkaline phosphatase. However, the formation of EPL cells was accompanied by alterations in Fgf5, Gbx2 and Rex1 expression, a loss in chimaera forming ability, changes in factor responsiveness and modified differentiation capabilities, all consistent with the identification of EPL cells as equivalent to the primitive ectoderm population of the 5.5 to 6.0 days post coitum embryo. EPL cell formation could be reversed in the presence of LIF and withdrawal of MEDII, which suggested that EPL cell formation was not a terminal differentiation event but reflected the ability of pluripotent cells to adopt distinct cell states in response to specific factors. Partial purification of MEDII revealed the presence of two separable biological activities, both of which were required for the induction and maintenance of EPL cells. We show here the first demonstration of uniform differentiation of ES cells in response to biological factors. The formation of primitive ectoderm, both in vivo and in vitro, appears to be an obligatory step in the differentiation of the inner cell mass or ES cells into cell lineages of the embryonic germ layers. EPL cells potentially represent a model for the development of lineage specific differentiation protocols and analysis of gastrulation at a molecular level. An understanding of the active components of MEDII may provide a route for the identification of factors which induce primitive ectoderm formation in vivo.

Animals↗
PubMed1998

Properties and uses of embryonic stem cells: prospects for application to human biology and gene therapy.

Embryonic stem cells are pluripotent cells derived from the early mouse embryo that can be propagated stably in the undifferentiated state in vitro. They retain the ability to differentiate into all cell types found in an embryonic and adult mouse in vivo, and can be induced to differentiate into many cell types in vitro. Exploitation of ES cell technology for the creation of mice bearing predetermined genetic alterations has received widespread attention because of the sophistication that it brings to the study of gene function in mammals. Analysis of cell differentiation in vitro has also been of value, leading to the identification of novel bioactive factors and the elucidation of cell specification mechanisms. In this paper, we summarise the features of pluripotent cell lines and their applications, foreshadowing the impact that these systems may have on human biology. While the isolation of definitive human pluripotent cell lines has not yet been achieved, potential applications for these cells in the study of human biology, particularly cell specification, can be envisaged. Of particular interest is the possibility that human embryonic stem cells with properties similar to mouse embryonic stem cells might provide a generic system for gene therapy.

Animals↗
PubMed1998

Developmental complexity of early mammalian pluripotent cell populations in vivo and in vitro.

Early mammalian embryogenesis is characterised by the coordinated proliferation, differentiation, migration and apoptosis of a pluripotent cell pool that is able to give rise to extraembryonic lineages and all the cell types of the embryo proper. These cells retain pluripotent differentiation capability, defined in this paper as the ability to form all cell types of the embryo and adult, until differentiation into the three embryonic germ layers at gastrulation. Our understanding of pluripotent cell biology and molecular regulation has been hampered by the difficulties associated with experimental manipulation of these cells in vivo. However, a more detailed understanding of pluripotent cell behaviour is emerging from the application of molecular technologies to early mouse embryogenesis. The construction of mouse mutants by gene targeting, mapping of gene expression in vivo, and modelling of cell decisions in vitro are providing insight into the cellular origin, identity and action of key developmental regulators, and the nature of pluripotent cells themselves. In this review we discuss the properties of early embryonic pluripotent cells in vitro and in vivo, focusing on progression from inner cell mass (ICM) cells in the blastocyst to the onset of gastrulation.

Animals↗
PubMed1995

Sequence, genomic organization, and expression of the novel homeobox gene Hesx1.

Extensive analyses of homeobox gene expression and function during murine embryogenesis have demonstrated that homeobox gene products are key components in the establishment of pattern formation and regional identity during development. In this paper we report the molecular characterization and expression of a novel murine homeobox sequence, Hesx1, isolated from pluripotent embryonic stem cells. Hesx1 is expressed as two transcripts of 1.0 and 1.2 kilobases which encode an identical 185 amino acid open reading frame. The transcripts differ in the 3'-untranslated region due to the differential utilization of a weak splice donor site located immediately downstream of the translation termination codon. The Hesx1 homeodomain shared 80% identity with the Xenopus homeoprotein XANF-1 and was less than 50% related to other homeodomain sequences. Hesx1 and XANF-1 therefore constitute the founder members of a new homeodomain class. Hesx1 expression was down-regulated during embryonic stem cell differentiation and was detected in tissue-specific RNA samples derived from the embryonic liver, and at lower levels in viscera, amnion, and yolk sac. Expression in adult mice was not detected. These sites of expression are consistent with a role for Hesx1 in the regulation of developmental decisions in the early mouse embryo and during fetal hematopoiesis.

Amino Acid Sequence↗
PubMed1991

DNA binding of CPF1 is required for optimal centromere function but not for maintaining methionine prototrophy in yeast.

The centromere and promoter factor 1 (CPF1) binds specifically in vitro and in vivo to an octanucleotide (RTCACRTG). This sequence is found in the centromere DNA element I (CDEI) of yeast centromeres and upstream from a number of transcription units including MET25, GAL2 and TRP1. Inactivation of the CPF1 gene results in three phenotypes; slow growth, a partial loss of centromere function and methionine auxotrophy. These phenotypes correlate well with the known binding sites for CPF1 and have led to the suggestion that CPF1 functions as a kinetochore protein at centromeres and as a transcriptional activator at promoters such as MET25. By analysing transcription from the MET25, GAL2, and TRP1 genes in cpf1 strains, we demonstrate that CPF1 plays no direct role in their transcriptional regulation. Further evidence in support of this comes from the analysis of point mutations in the basic region of CPF1 that affect DNA binding. A strain expressing a non-DNA bound form of CPF1 is phenotypically Met+, shows normal growth rate but has sub-optimal centromere function. We conclude that a DNA-bound form of CPF1 is required for the kinetochore function but not for maintaining methionine prototrophy.

Amino Acid Sequence↗
PubMed1990

Characterisation of sequences required for RNA initiation from the PGK promoter of Saccharomyces cerevisiae.

In the phosphoglycerate kinase (PGK) gene of yeast, as in other highly expressed yeast genes, the sequences surrounding the site of RNA initiation have a loosely conserved structure of a CT rich stretch followed by the tetranucleotide CAAG. Using internal deletions and insertions we have identified the elements in the PGK promoter which are required for correct RNA initiation at the CAAG sequence at -39. The results indicate that two different components of the PGK promoter contribute to correct RNA initiation, the TATA homologies, located at -152 and -113, and the sequences at the site of initiation. Both TATA elements can function in RNA initiation. Deletion of the upstream TATA element, TATAI, results in slightly heterogeneous RNA initiation, but the majority of the RNA initiates correctly. Deletion of both the PGK TATA elements results in the majority of the RNA initiating at sites downstream from the wild-type I site, within the structural gene between +40 to +80. The CT rich box is not essential for correct mRNA initiation as shown by deletion analysis. The site of RNA initiation in the PGK promoter appears to be determined by sequences located immediately 5' of the CAAG sequence motif. This short sequence, ACAGATC, when located the correct distance from the TATA elements may be sufficient to determine a discrete initiation site.

Base Sequence↗
PubMed1990

CPF1, a yeast protein which functions in centromeres and promoters.

Centromeres and several promoters of Saccharomyces cerevisiae contain a highly conserved octanucleotide, RTCACRTG, called CDEI. Using biochemical, genetic and structural analyses, we show that the same protein binds in vivo to CDEI sites in centromeres and in promoters. This protein, called CPF1 for centromere promoter factor, binds DNA as a dimer. Inactivation of the gene is not lethal but leads to a partial loss of the centromere function and to a Met- phenotype. Changes of the chromatin structure due to inactivation of CPF1 are seen at centromeres and at several CDEI-carrying promoters (e.g. MET25, TRP1, GAL2). However promoter activities are affected in diverse ways making it presently difficult to describe a function for CPF1 in gene expression. The sequence of the cloned gene reveals in the carboxy-terminal part two potential amphipathic helices preceded by a positively charged stretch of amino acids very similar to the helix-loop-helix domains recently identified in factors controlling tissue specific transcription in higher eukaryotes. Carboxy-terminal truncations of CPF1 lacking this domain no longer bind to CDEI. The amino-terminal half of CPF1 carries two clusters of negatively charged amino acid residues. Surprisingly, deletions of these clusters still render cells Met+ and lead only to a marginal decrease in centromere activity.

Amino Acid Sequence↗