Nature has evolved detailed cellular mechanisms by which multipotent cells come to possess particular developmental competencies as well as by which inductive signals and response networks elicit cell type specification. Further elucidation of the marks of developmental competence and their potential mechanisms of action will ultimately allow the prediction of differentiation capacity of a given progenitor or stem cell population. Thus, we wish to better understand chromatin states in embryonic endoderm cells, their progenitors, and in different stages of their descendants to the liver and pancreatic fates. To this end, we are currently employing fluorescence activated cell sorting of different endodermal and embryonic liver and pancreas cell populations to isolate progenitors, for detailed chromatin analysis. Given that a major difficulty with current stem cell differentiation protocols is to develop cells that fully express hepatocyte and beta cell phenotypes, we anticipate that the information we obtain can be used to assess whether cells at early stages in the differentiation protocol have been properly programmed; e.g. by possession of the appropriate chromatin competence marks. This could provide a novel dimension to prospectively programming stem cells to desired fates.
We are also taking a different perspective on how the engagement of pioneer transcription factors at silent genes may mark or endow the potential for gene activity. That is, we have performed a sub-genome wide location analysis of FoxA2 bound sites in the adult mouse liver, and focused on the approximately one-third of FoxA2 targets that occur at silent genes (J. Watts and KZ, in preparation). Typically, these silent FoxA2 bound genes are active in other endoderm-derived cell types. Notably, at least one of the targets is a regulatory gene whose activation can cause cell type conversion, or metaplasia, among gut tissues. Further analysis has revealed a network of repressive transcription factors that help keep the FoxA2 target gene silent in liver cells and that may be disrupted during gut pathologies associated with metaplasia. These studies reveal how pioneer factors enable dysregulated cell differentiation events that may underlie disease.
With regard to our fate mapping of the foregut endoderm, revealing different endoderm domains that together give rise to the embryonic liver bud: we have performed laser-capture microdissection of different endodermal domains, followed by RNA isolation, amplification, and microarray analysis. With the resulting RNA expression profiles, we subtracted the expression profiles of adjacent mesodermal tissue, yolk sac, and the total embryo, providing us with lists of genes whose expression is highly enriched in different domains of the foregut endoderm. We are now using bacterial artificial chromosomes harboring these genes to drive the expression of CRE recombinase in different endodermal domains of transgenic mice. This will allow us to perform genetic lineage marking analysis and determine whether descendants of different endodermal progenitors have different growth, regenerative, and stem cell capacities in the adult liver and pancreas.
A major application of developmental biology studies is to use the tissue-inductive signals that were identified from studies of embryos to prospectively program stem cells. Furthermore, understanding the signal transduction pathways that mediate tissue induction events, and how the pathways interact to form a network, can reveal agonist, antagonist, and other small molecule targets to promote efficient stem cell differentiation. To this end, we are investigating the signal transduction pathways and interactions within endoderm cells during the period preceding and culminating in liver and pancreas induction, as well as within cells at the subsequent steps of differentiation. Understanding how such pathways converge on pioneer factors at target genes and other chromatin parameters and induce new regulatory events leading to cell type specification will provide a cohesive view of how to control cell fates at will.