Projects
1. Bone Morphogenetic Protein: A differential regulator of cell fate
ES cells can be differentiated to form sequential populations of early primitive ectoderm-like (EPL) cells and then definitive ectoderm (Figures 2,3). Bone Morphogenetic Protein 4 (BMP4) directs the differentiation of these populations towards different cell fates: EPL cells form mesoderm while definitive ectoderm is directed to surface ectoderm (the progenitor population for tissues such as skin, hair, cornea and the pituitary gland). We have shown that the same signalling pathway, mediated by Smad proteins, is activated in both populations. This demonstrates that a single growth factor can produce different cell fates depending on the target cell. This is a critical aspect of development, where a limited number of signalling molecules produce a wide variety of cellular responses in different tissues.
Our in vitro model system provides an opportunity to explore how this control is implemented in early embryonic cell populations, which would be difficult to access in vivo. An understanding of the molecular basis of this altered competence will not only provide insight into developmental processes but is also likely to lead to improved protocols for the directed differentiation of ES cells to pure cell types, a prerequisite for future cell-based therapies.
In this project you will identify the molecular basis for the altered competence to BMP4 between EPL and definitive ectoderm cells. This will involve identification of any differences in the BMP signalling components between the two cell types. Candidates include BMP receptors and recently discovered co-receptors, their phosphorylation status, the degree of Smad activation and the proteins that interact with Smads. You will apply a variety of approaches, including the quantitation of signalling components, their phosphorylation status and the analysis of Smad complexes utilising immunoprecipitation and proteomics.
2. Control of pluripotent cell proliferation by cellular fibronectin
Cellular fibronectin (cFN) is a protein present in the extracellular matrix that separates the extraembryonic visceral endoderm from the cells of the ICM in the early mouse embryo. The role of cFN in early embryogenesis remains unclear but results from our in vitro model suggest it is involved in (i) the burst of proliferation that results in a few dozen pluripotent ICM cells developing into several thousand primitive ectoderm cells in a period of about 48 h, and (ii) the morphological change associated with the formation of EPL cells.
This project involves determining the signalling pathway(s) cFN that effect this burst of proliferation and morphological change. Several candidate pathways exist, each of which can be manipulated either at the level of cell-surface receptors (such as integrins) or signalling intermediates (such as FAK, paxillin and integrin-linked kinase). Quantification of the activation and repression of these signalling components and their effects on the rate of proliferation and morphology will be an important part of this research.
Knowledge gained from the mouse system could be trialled on the equivalent system using human ES cells. Human ES cells proliferate much more slowly that mouse ES cells and any increase in the rate of proliferation while maintaining genetic stability would be particularly advantageous from a biotechnological perspective.
3. Formation of the embryonic endodermal lineages by directed differentiation of EPL cells.
Transplantation of human islet tissue, a route to restoration of insulin dependence in type I diabetics, is limited by a shortage of cadaveric material. Differentiation of ES cells to glucose-responsive, insulin-secreting beta cells would provide an alternative source of cells for transplantation and disease control. During mammalian development beta cells can trace their origins to the embryonic endodermal lineage formed at gastrulation. At this time, the embryonic endoderm is proposed to arise from differentiation of a bi-potential mesoderm/endoderm progenitor (mesendoderm) in response to TGFbeta signaling. To date, little has been reported on the formation of embryonic endoderm from ES cells in culture.
We have recently demonstrated the formation of mesendoderm from EPL cells during differentiation, and the ability to direct the differentiation of these cells to the mesodermal lineage and away from the endodermal lineage, demonstrating the plasticity of this progenitor. The opportunity exists to apply information within the literature to our in vitro differentiation systems to develop robust and rationale technologies to “switching” the fate of this progenitor away from mesoderm and into the embryonic endodermal lineage. This would represent a significant step towards the development of therapies.
In this project you will be devising strategies to direct the differentiation of EPL cell-derived mesendoderm into embryonic endoderm. This will involve the expression if the TGFbeta molecule nodal in COS1 cells and using conditioned medium from these cells as a culture supplement in which to differentiate EPL cells. Morphology, using novel morphological assays we have developed, gene expression and differentiation potential will be used to assay the resultant cell populations to demonstrate enrichment in the embryonic endoderm.
The identification of embryonic endoderm is difficult and hampered by a lack of markers. The scope exists within this project to develop novel markers for the identification of the embryonic endoderm in vitro and in vivo.
4. The role of CRTR1 in pluripotent cells: Identification of CRTR1 target genes
CRTR1 is a transcriptional repressor expressed in the ICM of the mammalian blastocyst (Figure 1) and in ES cells. CRTR1 expression is downregulated as these cells convert to primitive ectoderm and EPL cells, respectively. This expression pattern suggests that CRTR1 plays a role in suppressing differentiation, thereby maintaining pluripotent ICM and ES cells. Understanding the basis of pluripotency is a major goal of our laboratory and has important applications in the development of cellular therapies. To determine whether CRTR1 plays a critical role in pluripotent cells, we are taking a number of approaches including the identification of genes that CRTR1 regulates in ES cells.
In this project you will identify genes regulated by CRTR1 using a number of techniques including ChIP, and identify aspects of the molecular mechanism of repression using reporter systems in which the activity of specific domains of CRTR1 are measured. The identification of CRTR1-regulated genes should provide insight into role of transcriptional repression in the maintenance of pluripotence.
5. Purification of cytospeckles containing a novel RNA binding protein, Psc1
Psc1 is an RNA-binding protein of unknown function. We are interested in studying Psc1 because its expression is developmentally regulated, both in the early mammalian embryo and in the ES/EPL-cell in vitro system. Expression of Psc1 is high in the ICM of the blastocyst and in ES cells, and is subsequently downregulated in primitive ectoderm and EPL cells (Figure 1). Psc1 is found in nuclear speckles, many of which contain mRNA splicing factors. Psc1 also localises to punctate regions in the cytoplasm, which we have termed cytospeckles (Figure 5). We have shown that cytospeckles are motile and move throughout the cytoplasm and into the nucleus. Localisation to speckles appears to be mediated, in part, by the RNA recognition motif of the protein, suggesting that RNA binding is central to Psc1 function. We hypothesise that Psc1 plays a central role in post-transcriptional regulation of developmentally important mRNAs. This may be at the level of mRNA splicing, transport, localisation and/or stability.
The different characteristics of nuclear speckles and cytospeckles suggest Psc1 may have different functions in these two compartments. We have shown that SART1, a factor involved in formation of the mRNA splicing complex, interacts with Psc1. Both proteins co-localise in nuclear speckles, suggesting a role for Psc1 in splicing in the nucleus. The role of Psc1 in the cytoplasm is less clear and the composition of these novel cytospeckles is unknown.
In this project you will investigate the function of Psc1 by identifying interacting proteins that are present in cytospeckles. This will be performed by immunoprecipitation of Psc1 complexes from the cytoplasm and identification of the components by mass spectroscopy. In addition, you will examine the role of Psc1 in mRNA splicing. This will involve the purification of Psc1 (and possibly Psc1 mutants) for testing using in vitro splicing assays. Splicing assays will be performed in collaboration with a specialist splicing laboratory.
Funding Sources
Our laboratory is a member of the ARC Special Research Centre for the Molecular Genetics of Development and the ARC Research Network in Genes and Environment in Development, with links to labs in Molecular Biosciences, the Child Health Research Institute, and the ANU. The lab is also a key member of the recently awarded National Centre of Excellence, the Australian Stem Cell Centre, with links to a number of other labs, including Monash University and the University of Queensland. We also have active collaborations with laboratories at the Women’s and Children’s Hospital, Flinders University, and SUNY Upstate Medical School, New York.
