Conserved mode of endoderm induction acts to promote context dependent embryonic and extra-embryonic lineage specification
Anderson, Kathryn Gayle Victoria
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In mammalian development, endoderm formation occurs in two phases and the fate of these populations is different. In the blastocyst, inner cell mass (ICM) cells generate the primitive endoderm (PrE), which will give rise to the extra-embryonic parietal (PE) and visceral endoderm (VE). Hematopoietically expressed homeobox (Hhex) protein is initially expressed throughout the PrE and subsequently becomes restricted to the anterior visceral endoderm (AVE), one of two important early embryonic signalling centres in the mouse. During gastrulation a second wave of endoderm differentiation occurs, the definitive endoderm (DE), generating the foregut. Immediately following the induction of DE, regional identity is initially established in the anterior region with the expression of Hhex. One of the earliest specification events in this lineage is the specification of anterior fate by Hhex, this time in a second signalling centre, the anterior definitive endoderm (ADE). The ADE is both important for embryonic patterning, and as the precursor population for differentiating to the foregut and its derivatives the thyroid, liver and pancreas. The literature surrounding these early embryonic patterning events is covered in depth in chapter 1. Embryonic stem cells (ESCs) are normal cell lines derived from the mammalian blastocyst at the time that it is making PrE. A number of laboratories have generated protocols to make endoderm from ESCs and in my thesis I define approaches to distinguish between PrE and DE. I generated a new ESC reporter line utilising a gene normally expressed in both the PrE and later in hepatic endoderm; this reporter contains a GFP in the first exon of the Hnf4α locus. This was combined with a second fluorescent reporter containing DSRed in the Hhex locus. This cell line is described and characterised in chapter 3. As Hnf4α is initially expressed in PrE prior to Hhex, but in the DE following Hhex, I was able to use the temporal expression of this reporter to distinguish the induction of PrE from DE. As Activin and Wnt are known to induce endoderm from ESCs, I was then able to ask what sort of endoderm the combination of these two signals induced. In chapter 4 I found that normal ESCs would readily differentiate to iPrE in the presence of Activin and Wnt3a. While this has not been described previously, my analysis suggests that ESC protocols applying these cytokines directly to ESCs have produced PrE. Given that ESCs are derived from the blastocyst, the generation of iPrE from Wnt3a/Activin treatment fits with developmental paradigms. However, Act/Wnt3a is used routinely on Human ESCs (hESCs) and so I attempted to reconcile these observations. HESCs, while derived from the blastocyst, appear to progress developmentally in vitro, to a stage closer to the epiblast, immediately prior to gastrulation. I therefore assessed the effect of Activin and Wnt3a on mouse stem cell lines derived from the epiblast (Epiblast Stem Cells, EpiSCs), that are grown under similar conditions to hESCs. When Wnt3a/Act is applied to these cells I found that they made DE rather than PrE, which I describe in chapter 4. Taken together my observations suggest that Act/Wnt3a are general endoderm inducers that induce context specific differentiation in vitro. The cell type derived in response to this treatment depends on the developmental stage of the starting stem cell culture. During the course of this work, I also observed that PrE was growing under Activin/Wnt3a treatment. As a number of cell culture systems have been established that reflect PE, but not truly bipotent PrE, I investigated the conditions under which PrE can be expanded. In chapter 5 I characterize a new PrE culture system, in which bipotent extra-embryonic endoderm can be expanded indefinitely in culture. I also explore a bit more precisely the nature of the starting cells that initially become exposed to Activin/Wnt3a treatment. Previous work has extensively characterized the existence of a primed population of PrE in ESC culture and in chapter 6 I explore the existence of a primed DE population in EpiSC culture. Taken together, my thesis is the first demonstration that Activin/Wnt3a can induce different endoderm populations in different embryonic stem cell populations. It underlies the notion that the evolutionary origin of both cell types is the same and that the pathways evolved for extra-embryonic development in mammals just exploit the ancient modes of germ layer specification that evolved with gastrulation.