Research at the Labosky Lab

�Figure 1. Efficient excision of Foxd3 locus in ES cells results in reduced protein levels.

Our lab is interested in studying the genes that control normal development of the mammalian embryo and that regulate the fate of embryonic progenitor cells. We have focused this work on a group of transcription factors from the “winged helix” or “forkhead” family. Many of these genes are expressed in restricted patterns in developing mouse embryo and have been shown to play a role in the regulation of cell proliferation. Several members of the family also play roles in the progression of cancerous tumors and disease.

We have been using our studies of the Foxd3 gene in multiple tissues to try and understand how progenitor cell fate is regulated. Foxd3, previously called Hfh2 and Genesis, is expressed ubiquitously in the very early embryo and then specifically in the early premigratory neural crest cells. Foxd3 was originally cloned and characterized as Genesis because it is expressed in stem cells in vitro and, as we have shown, in vivo. Embryos carrying two null alleles for Foxd3 die around implantation. In vitro work has demonstrated that Foxd3 is required in both the embryonic stem cell (ES cell) lineage and the trophoblast stem cell (TS cell) lineage.

�Figure 2 . Deletion of Foxd3 in the neural crest causes craniofacial defects and loss of the peripheral nervous system.

Our hypothesis that Foxd3 maintains stem cell characteristics will be tested in future experiments by altering levels of Foxd3 in ES cells and determining whether artificially high or low levels of the protein alter the potential of the cells. Foxd3 protein levels will be reduced in ES cells by Cre-mediated deletion of the Foxd3 coding region (Figure 1). In addition, an inducible Foxd3-ER fusion construct will be used to determine whether artificially high expression of Foxd3 will interfere with differentiation of ES cells.

Foxd3 is also expressed later in the embryo in premigratory neural crest stem cells. This later expression pattern is particularly enticing as it makes Foxd3 one of the earliest markers of neural crest in the mouse. Overexpression of Foxd3 in chick embryos (in collaboration with Dr. Martyn Goulding at the Salk Institute) has shown that Foxd3 is sufficient to specify the neural crest cell lineage. We have generated a tissue specific deletion of Foxd3 using the Cre-LoxP system to selectively mutate the gene in only the neural crest cells. This mutation results in a catastrophic loss of neural crest derived tissues including bones of the skull, the enteric and peripheral nervous systems (Figure 2).

Most adult organs have at least a limited capacity to regenerate, and it is possible that Foxd3 might also be involved in these processes in the adult animal. We recently discovered that Foxd3 is expressed in the embryonic and adult pancreas, and expression is primarily restricted to beta cells of the islet. The protein changes cellular localization with diet and in a rat model of diabetes (Figure 3). Together these data suggest that Foxd3 may be important in pancreatic disorders such as diabetes. Experiments are underway to understand the role that Foxd3 may be playing in pancreatic maintenance and/or regeneration.

�Figure 3 . Foxd3 is expressed in the embryonic pancreas (above) and in adult islets (lower left). The protein co-localizes with a Foxd3-Gfp lineage reporter in islets (bottom right).

These projects all revolve around our interests in the maintenance of progenitor cell populations in the embryo. We have been using Foxd3 as a tool to understand the similarities between these different progenitor populations. Together, our data place Foxd3 in a unique regulatory role affecting the fate of multiple stem cell lineages (ES cells, TS cells, neural crest and perhaps pancreas) and the experiments underway in the lab will use this gene as a tool to obtain a better understanding of the molecular control of multipotency in different lineages.