Mammalian mitochondrial DNA (mtDNA) is a small, maternally inherited genome that codes for 13 essential proteins in the respiratory chain. Mature oocytes contai

Background: Understanding the dynamic cellular behaviors and underlying molecular mechanisms that drive morphogenesis is an ongoing challenge in biology. Live imaging provides the necessary methodology to unravel the synergistic and stereotypical cell and molecular events that shape the embryo. Genetically-encoded reporters represent an essential tool for live imaging. Reporter strains can be engineered by placingcis-regulatory elements of interest to direct the expression of a desired reporter gene. In the case of canonical Wnt signaling, also referred to as Wnt/β-catenin signaling, since the downstream transcriptional response is well understood, reporters can be designed that reflect sites of active Wnt signaling, as opposed to sites of gene transcription, as is the case with many fluorescent reporters. However, even though several transgenic Wnt/β-catenin reporter strains have been generated, to date, none provides the single-cell resolution favored for live imaging studies.Results: We have placed six copies of a TCF/Lef responsive element and anhsp68minimal promoter in front of a fluorescent protein fusion comprising human histone H2B to GFP and used it to generate a strain of mice that would report Wnt/β-catenin signaling activity. Characterization of developmental and adult stages of the resultingTCF/Lef:H2B-GFPstrain revealed discrete and specific expression of the transgene at previously characterized sites of Wnt/β-catenin signaling. In support of the increased sensitivity of theTCF/Lef:H2B-GFPreporter, additional sites of Wnt/β-catenin signaling not documented with other reporters but identified through genetic and embryological analysis were observed. Furthermore, the sub-cellular localization of the reporter minimized reporter perdurance, and allowed visualization and tracking of individual cells within a cohort, so facilitating the detailed analysis of cell behaviors and signaling activity during morphogenesis.Conclusion: By combining the Wnt activity read-out efficiency of multimerized TCF/Lef DNA binding sites, together with the high-resolution imaging afforded by subcellularly-localized fluorescent fusion proteins such as H2B-GFP, we have created a mouse transgenic line that faithfully recapitulates Wnt signaling activity at single-cell resolution. TheTCF/Lef:H2B-GFPreporter represents a unique tool for live imaging thein vivoprocesses triggered by Wnt/β-catenin signaling, and thus should help the formulation of a high-resolution understanding of the serial events that define the morphogenetic process regulated by this signaling pathway.

Hepatocyte nuclear factors (HNFs) are a heterogeneous class of evolutionarily conserved transcription factors that are required for cellular differentiation and metabolism. Mutations in HNF-1α and HNF-4α genes impair insulin secretion and cause type 2 diabetes. Regulation of HNF-4/HNF-1 expression by HNF-3α and HNF-3β was studied in embryoid bodies in which one or both HNF-3α or HNF-3β alleles were inactivated. HNF-3β positively regulated the expression of HNF-4α/HNF-1α and their downstream targets, implicating a role in diabetes. HNF-3β was also necessary for expression of HNF-3α . In contrast, HNF-3α acts as a negative regulator of HNF-4α/HNF-1α demonstrating that HNF-3α and HNF-3β have antagonistic transcriptional regulatory functions in vivo. HNF-3α does not appear to act as a classic biochemical repressor but rather exerts its negative effect by competing for HNF-3 binding sites with the more efficient activator HNF-3β . In addition, the HNF-3α/HNF-3β ratio is modulated by the presence of insulin, providing evidence that the HNF network may have important roles in mediating the action of insulin.

Recent studies have demonstrated that the LIM homeodomain transcription factor Islet1 (Isl1) marks pluripotent cardiovascular progenitor cells and is required for proliferation, survival, and migration of recently defined second heart field progenitors. Factors that are upstream of Isl1 in cardiovascular progenitors have not yet been defined. Here we demonstrate that β-catenin is required for Isl1 expression in cardiac progenitors, directly regulating the Isl1 promoter. Ablation of β- catenin in Isl1-expressing progenitors disrupts multiple aspects of cardiogenesis, resulting in embryonic lethality at E13. β-Catenin is also required upstream of a number of genes required for pharyngeal arch, outflow tract, and/or atrial septal morphogenesis, including Tbx2 , Tbx3 , Wnt11 , Shh , and Pitx2 . Our findings demonstrate that β-catenin signaling regulates proliferation and survival of cardiac progenitors.

Successful implantation relies on precisely orchestrated and reciprocal signaling between the implanting blastocyst and the receptive uterus. We have examined the role of the Wnt/β-catenin signaling pathway during the process of implantation and demonstrate that this pathway is activated during two distinct stages. Wnt/β-catenin signaling is first transiently activated in circular smooth muscle forming a banding pattern of activity within the uterus on early day 4. Subsequently, activation is restricted to the luminal epithelium at the prospective site of implantation. Activation at both sites requires the presence of the blastocyst. Furthermore, inhibition of Wnt/β-catenin signaling interferes with the process of implantation. Our results demonstrate that the Wnt/β-catenin signaling pathway plays a central role in coordinating uterus–embryo interactions required for implantation.