Rathjen Laboratory

Early Mammalian Embryogenesis

The various tissues, organs and cells of the mammalian embryo and adult are all derived from a pluripotent (or ‘founder’) cell population, the inner cell mass (ICM). The ICM is a small group of cells present at one pole the blastocyst (Figure 1). Subsequently, the cells of the ICM undergo a highly dynamic program of proliferation, differentiation, migration, apoptosis and reorganisation to give rise to a second pluripotent cell population termed primitive ectoderm. This pluripotent population then undergoes gastrulation in which the three germ layers of the embryo (ectoderm, mesoderm and endoderm) are formed. Gastrulation, and subsequent development of the embryo, is tightly regulated in both time and space. This regulation is essential for correct signaling between cells, ensuring cells go to or remain in their proper locations, and adopt appropriate cell fate. Errors in these processes can result from genetic defects or environmental factors (e.g., drugs, exposure to chemicals, radiation, etc.) and lead to developmental defects and disease.

The research in our lab is two-fold:

1. To understand the molecular mechanisms of embryogenesis by analyzing the embryo itself and by using in vitro models of embryogenesis.


2. To use this information to direct the differentiation of cells in culture to specific cell types that can be used in the treatment of human disease.

Forming Cell Lineages

Many key cell populations, such as primitive ectoderm, ectoderm, mesoderm and endoderm, are formed during embryogenesis but little is known of the molecular mechanisms that control these events. At least two complementary processes are required: Inductive signals, which regulate cell type, and signals determining positional information within the embryo (patterning). Both these processes work together to produce the correct cell types at the correct time and place and hence the proper development of the embryo.

Es Cells as a Model for Mammalian Development

We have developed a unique in vitro model of mammalian embryogenesis. Using this model we have discovered many key regulators of development. These regulators include extracellular ligand and matrix proteins, cell-surface receptors, signalling pathways, transcription factors and RNA processing proteins. These regulators control many different aspects of embryogenesis such as pluripotence, proliferation, cell migration, differentiation and patterning. Often a single regulator is involved in multiple events at different places and times during the development of the embryo and on into adulthood.

Our model involves the use of embryonic stem (ES) cells isolated from the ICM of the embryo. Like their embryonic counterparts, ES cells are pluripotent, since they can differentiate to a wide range of cell types in vitro and contribute to all cell populations of the embryo and adult in vivo.

We have identified factors which, when added to cultures of ES cells, homogeneously convert them to early primitive ectoderm-like (EPL) cells. EPL cells are analogous to the pluripotent primitive ectoderm of the embryo. By adding further factors to the culture medium in the correct combinations and the correct times, we are able to direct the differentiation of EPL cells to specific cell types. Figure 2 shows some examples, which include pure or highly enriched populations of progenitor and fully differentiated cell types found in the developing embryo and/or the adult. Thus, we have developed protocols that allow us to manipulate developmental ‘switches’ through the addition of small and large nontoxic molecules to the culture that control the activity of specific signalling pathways.

The key features of our in vitro model of embryogenesis include:

1. The ability to examine pluripotence, directed differentiation and patterning at the molecular level.


2. The production of pure or highly enriched cell types for use in animal models of disease such as Parkinson’s disease.


3. The synchronous formation of cell populations, which allows us to identify and characterise transient progenitor populations.


4. The manipulation of signalling pathways, providing an understanding of what processes contribute to normal and abnormal development.

Microarray Analysis of Stem Cells and Progenitor Cells

Because we can synchronously and homogeneously produce a series of closely related cell types, our system lends itself extremely well to microarray analysis. Recently, we have subjected four closely related cell populations to this analysis, ES cells, EPL cells, definitive ectoderm, and neurectoderm, using a chip representing 22 000 mouse genes (Figure 3). This has allowed us to identify candidate sets of genes involved in embryogenesis with particular regard to pluripotence and the switch to ectoderm formation. It has also allowed us to identify genes and their products that can be used to ‘mark’ specific cell types, which previously could not be readily recognized. This microarray analysis, and other analyses to follow, represent an important step in mapping the interrelated processes that control development and in pinpointing signaling systems whose activity can be modulated by chemical means to alter cell fate effectively.

Significance of this Research

Basic science: The availability of in vitro cell differentiation systems that mimic formation of cell lineages during embryogenesis provides enormous experimental advantages. It is possible to identify molecules and their molecular mechanisms of action that specify different cell fates. It is also possible to discriminate between pathways that instruct cell differentiation from those that pattern existing populations.

Commercial Applications

It’s predicted that the treatment of human disease and injury will be revolutionised by the development of cell-based therapeutics to correct states involving cell loss (such as stroke, Parkinson’s disease, juvenile diabetes, spinal and head injury) and cell dysfunction (such as the thalassaemias and muscular dystrophies). Additionally, the use of cells as delivery systems for therapeutic agents, like proteins, or production of virally resistant cell populations for the treatment of chronic viral infections, such as HIV, can be envisaged. ES cells are ideally suited to these purposes as they are immortal and proliferate in culture, can be differentiated into theoretically any cell type, and technologies exist for their precise genetic modification. The recent isolation of human ES cells has increased interest and confidence in the possible applications of ES cell-based therapeutics.