Fibroblast growth factor 8 (Fgf8) is a secreted signaling protein expressed in numerous temporospatial domains that are potentially relevant to cardiovascular development. However, the pathogenesis of complex cardiac and outflow tract defects observed in Fgf8-deficient mice, and the specific source(s) of Fgf8 required for outflow tract formation and subsequent remodeling are unknown. A detailed examination of the timing and location of Fgf8 production revealed previously unappreciated expression in a subset of primary heart field cells; Fgf8 is also expressed throughout the anterior heart field (AHF) mesoderm and in pharyngeal endoderm at the crescent and early somite stages. We used conditional mutagenesis to examine the requirements for Fgf8 function in these different expression domains during heart and outflow tract morphogenesis. Formation of the primary heart tube and the addition of right ventricular and outflow tract myocardium depend on autocrine Fgf8 signaling in cardiac crescent mesoderm. Loss of Fgf8 in this domain resulted in decreased expression of the Fgf8 target gene Erm,and aberrant production of Isl1 and its target Mef2c in the anterior heart field, thus linking Fgf8 signaling with transcription factor networks that regulate survival and proliferation of the anterior heart field. We further found that mesodermal- and endodermal-derived Fgf8 perform specific functions during outflow tract remodeling: mesodermal Fgf8 is required for correct alignment of the outflow tract and ventricles, whereas activity of Fgf8 emanating from pharyngeal endoderm regulates outflow tract septation. These findings provide a novel insight into how the formation and remodeling of primary and anterior heart field-derived structures rely on Fgf8 signals from discrete temporospatial domains.

In order to understand how secreted signals regulate complex morphogenetic events, it is crucial to identify their cellular targets. By conditional inactivation of Fgfr1 and Fgfr2 and overexpression of the FGF antagonist sprouty 2 in different cell types, we have dissected the role of FGF signaling during heart outflow tract development in mouse. Contrary to expectation, cardiac neural crest and endothelial cells are not primary paracrine targets. FGF signaling within second heart field mesoderm is required for remodeling of the outflow tract: when disrupted, outflow myocardium fails to produce extracellular matrix and TGFβ and BMP signals essential for endothelial cell transformation and invasion of cardiac neural crest. We conclude that an autocrine regulatory loop, initiated by the reception of FGF signals by the mesoderm, regulates correct morphogenesis at the arterial pole of the heart. These findings provide new insight into how FGF signaling regulates context-dependent cellular responses during development.

To study the roles of key transcription factor networks, growth factors, and signaling molecules in the endoderm, notochord, and floorplate, we developed an inducible Cre-expressing system for altering gene function in this tissue. We generated an allele of Foxa2 that directs a tamoxifen-regulated Cre in the Foxa2 expression domain (Foxa2 mcm ). Activity of Foxa2 mcm recapitulates endogenous Foxa2 expression in endoderm, notochord, and floorplate. Efficiency of the system in a given tissue type was dose-and timingdependent. By comparing efficiency and location of Cre activity after administration of tamoxifen by oral gavage vs. intraperitoneal injection, we found that oral gavage achieves more rapid, robust recombination with less embryonic toxicity. This system will be useful for controlling the activity of floxed alleles at multiple stages of mouse embryogenesis and fetal development. Developmental Dynamics 237:447-453, 2008.

Newts have the remarkable ability to regenerate lost appendages including their forelimbs, hindlimbs, and tails. Following amputation of an appendage, the wound is rapidly closed by the migration of epithelial cells from the proximal epidermis. Internal cells just proximal to the amputation plane begin to dedifferentiate to form a pool of proliferating progenitor cells known as the regeneration blastema. We show that dedifferentiation of internal appendage cells can be initiated in the absence of amputation by applying an electric field sufficient to induce cellular electroporation, but not necrosis or apoptosis. The time course for dedifferentiation following electroporation is similar to that observed following amputation with evidence of dedifferentiation beginning at about 5 days postelectroporation and continuing for 2 to 3 weeks. Microarray analyses, real-time RT-PCR, and in situ hybridization show that changes in early gene expression are similar following amputation or electroporation. We conclude that the application of an electric field sufficient to induce transient electroporation of cell membranes induces a dedifferentiation response that is virtually indistinguishable from the response that occurs following amputation of newt appendages. This discovery allows dedifferentiation to be studied in the absence of wound healing and may aid in identifying genes required for cellular plasticity.

We targeted the reverse tetracycline controlled transactivator (rtTA) to the Foxa2 locus (Foxa2 ITA ) to generate a system for regulating Cre-recombinase activity within Foxa2 expression domains, including the endoderm, notochord, and floor plate of early mouse embryos. The use of an internal ribosomal entry site to obtain rtTA expression preserves Foxa2 function of the targeted allele. Cre activity with this system reflects the level of endogenous Foxa2 activity and is also tightly controlled by doxycycline. The location of Cre activity within the broader Foxa2 expression domain can be restricted by altering the timing of doxycycline administration. Isolated floor plate expression can be obtained in this manner. This system will provide a useful tool for manipulating gene expression in endoderm, notochord, and floor plate, all of which are tissues with important structural and patterning functions during embryogenesis. Developmental Dynamics 236:1085-1092, 2007.