Embryonic Patterning Research

Distribution of two painted endoderm populations in a mouse embryo, 7.0 days after conception, showing extensive anterior-posterior movement.  The picture is a digital compilation of red, green and bright-field images and was shown on the cover of Development volume 134 issue 2 (Tam et al., 2007).









A significant milestone in early development is gastrulation, when three germ layers - known as ectoderm, mesoderm and endoderm - are formed.  Cells of these three layers will subsequently give rise to all the tissues of the foetus.  Our work addresses a fundamental issue of embryonic development at the start of life:  the molecular activity controlling the formation of major body parts of the embryo.

Cell grafting and tracking techniques are performed on mouse embryos that are grown for 1-3 days in culture to map the developmental fates of cells in the embryo.  Information has been collated of the location of the precursors of different types of tissue in these cell layers, and the pattern of movement of these cells as they are allocated to specific regions of the body.

To track the fates of the endoderm cells we have pioneered the use of whole embryo electroporation in mouse embryos.  This allows us to introduce marker genes into cells.  We also use fluorescent stains that are painted on the cells.  This way we are delineating the origin of the different parts of the embryonic gut in the endodermal layer.

We have now fully mapped the cell fates in all three germ layers: the ectoderm (that gives rise to the nervous system and skin), the mesoderm (forming the muscle, bone and connective tissues and the endoderm, which is the precursor of the digestive system and the associated organs such as the thyroid, pancreas and liver).

Current projects:

Control of cell differentiation during mouse embryogenesis
Cell movement in germ layer formation
Gene function in gut endoderm development

Control of cell differentiation during embryogenesis

The goal of our research is to gain insight into how progenitor cells for fetal tissues may be maintained and determine the conditions that facilitate differentiation into germ layer derivatives.

The knowledge of how to maintain, expand and differentiate stem cells is essential for the realization of clinical cell-based therapies for the replacement and repair of diseased tissues.  Cells of the early embryo are capable of generating many cell types, hence are regarded as pluripotent cells.  As the embryo develops, there is a progressive restriction on the ability of the cells to do so.  Cells in more advanced embryos will give rise to an increasingly limited set of cell types.  Recently, stem cells have been derived from mouse embryos at the post-implantation stage.  These epiblast stem cells differ from the conventional embryonic stem cells, regarding the culture conditions for maintenance and differentiation, suggesting that they are a different type of stem cell that are already predisposed for more specialized differentiation.  This project will examine if these self-renewable cells may be induced to differentiate more efficiently into typical embryonic tissues by the signal activity normally present in the mouse embryo in vivo.

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Cell movement in germ layer formation

Mice that have lost the activity of genes such as Lhx1, Sox17, Mixl1 and Dkk1 are lacking in endodermal cells.  Experiments performed on embryos with mutations of Lhx1, Mixl1 or Dkk1 gene showed that the mutant cells are fully capable of differentiation into endoderm, but are not able to move normally to populate the correct region of the gut.  Our studies highlight failure of the cells to move rather than a loss of potential to differentiate as a potential cause of the developmental defects.

We are pursuing the molecular mechanism that controls the movement of the mesoderm and endoderm, with specific focus on the signalling and morphogenetic determinants that are activated during gastrulation of the mouse embryo.

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Gene function in gut endoderm development

The epithelium of the primitive gut is formed from definitive endoderm and the muscle and connective tissues are mesoderm-derived.  The foregut forms the liver, pancreas, the epithelium of the digestive tract and lungs, the thymus, thyroid and parathyroid glands.  Our goal is to understand fully the molecular basis for the formation, organization and differentiation of these organs.  As a first step towards this, we compared the genes expressed in the foregut endoderm of mouse embryos with tissues that do not contain endoderm using expression profiling microarrays.

	Cell junctions in the endoderm cell layer of the embryonic gut revealed by an antibody that detects the E-cadherin protein.

We have searched for novel or uncharacterized genes with gut endoderm-specific expression by comparing the gene expression profiles of microdissected gut endoderm tissue with non-endodermal tissues by microarray analysis.  Endodermal expression of selected novel genes is being confirmed by in situ hybridization to whole mouse embryos.  Genes whose expression is confirmed as endoderm-specific will be further analysed to understand their functional role in development of the endoderm.  From this analysis we identified a set of genes that are predominantly expressed in the endoderm and which do not, as yet, have any known function in early development.

We are now using a variety of approaches to study the functions of some of these genes during development of the endoderm and its derivative organs.  In this project, the effects of reduced or absent gene function (by knockdown, gene-targeting or gene-trap) and overexpression (by electroporation, transfection and transgenic methods) will be tested in mouse embryos, embryonic stem cells and other appropriate cell models.



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