Faculty Bibliography

Reduced litter sizes in mice missing pentraxin 3 (Ptx3) have been attributed to fertilization failure. However, our global gene expression studies showed high uterine Ptx3 expression at the implantation site in mice, suggesting its role in blastocyst implantation. We initiated molecular and genetic studies in mice to explore the importance of uterine Ptx3 in this process. We found that Ptx3 is expressed in a unique and transient fashion at implantation sites. With the initiation of implantation on midnight of Day 4 of pregnancy, Ptx3 is expressed exclusively in stromal cells at the site of blastocysts. On Day 5, its expression is more intense in decidualizing stromal cells, but it disappears on Day 6. The expression again becomes evident in the deciduum on Day 7, followed by a more robust expression on Day 8, particularly at the antimesometrial pole. From Day 9, with the initiation of placentation, Ptx3 expression becomes undetectable. These results suggest a role for PTX3 in implantation and decidualization. Indeed, deletion of Ptx3 results in both compromised implantation and decidualization. Interleukin 1B (IL1B), a known inducer of Ptx3, is also transiently expressed in stromal cells at the implantation site, suggesting that IL1B is an inducer of uterine Ptx3 expression. In fact, uterine Ptx3 expression follows that of Il1b induced by lipopolysaccharide treatment on Day 7 of pregnancy. Collectively, these findings provide evidence for an important role for PTX3 in implantation and decidualization. This study has clinical implications, since PTX3 is expressed in the receptive endometrium, and trophoblast cells influence decidual Ptx3 expression in humans.

Cell motility makes essential contributions to normal embryonic development and homeostasis. It is also thought to contribute in important ways to tumor metastasis. Because of this dual importance, cell migration has been extensively studied. The fruit fly Drosophila melanogaster has served as an important model organism for genetic analysis of many aspects of developmental biology, including cell migration. Here we describe the various types of cell movements that have been studied in detail, which represent models for epithelial-to-mesenchymal transition, transepithelial migration, inflammation, wound healing and invasion. We summarize what has been learned about the molecular control of cell migration from genetic studies in the fly. In addition, we describe recent efforts to model tumor metastasis directly in Drosophila by expressing oncogenes and/or mutating tumor suppressor genes. Together these studies suggest that Drosophila has much to offer as a model for varied aspects of tumor metastasis.

As membrane transporter proteins, VGLUT1-3 mediate the uptake of glutamate into synaptic vesicles at presynaptic nerve terminals of excitatory neural cells. This function is crucial for exocytosis and the role of glutamate as the major excitatory neurotransmitter in the central nervous system. The three transporters, sharing 76% amino acid sequence identity in humans, are highly homologous but differ in regional expression in the brain. Although little is known regarding their three-dimensional structures, hydropathy analysis on these proteins predicts 12 transmembrane segments connected by loops, a topology similar to other members in the major facilitator superfamily, where VGLUT1-3 have been phylogenetically classified. In this work, we present a three-dimensional model for the human VGLUT1 protein based on its distant bacterial homolog in the same superfamily, the glycerol-3-phosphate transporter from Escherichia coli. This structural model, stable during molecular dynamics simulations in phospholipid bilayers solvated by water, reveals amino acid residues that face its pore and are likely to affect substrate translocation. Docking of VGLUT1 substrates to this pore localizes two different binding sites, to which inhibitors also bind with an overall trend in binding affinity that is in agreement with previously published experimental data

At early stages of development, the faces of vertebrate embryos look remarkably similar, yet within a very short timeframe they adopt species-specific facial characteristics. What are the mechanisms underlying this regional specification of the vertebrate face? Using transgenic Wnt reporter embryos we found a highly conserved pattern of Wnt responsiveness in the developing mouse face that later corresponded to derivatives of the frontonasal and maxillary prominences. We explored the consequences of disrupting Wnt signaling, first using a genetic approach. Mice carrying compound null mutations in the nuclear mediators Lef1 and Tcf4 exhibited radically altered facial features that culminated in a hyperteloric appearance and a foreshortened midface. We also used a biochemical approach to perturb Wnt signaling and found that in utero delivery of a Wnt antagonist, Dkk1, produced similar midfacial malformations. We tested the hypothesis that Wnt signaling is an evolutionarily conserved mechanism controlling facial morphogenesis by determining the pattern of Wnt responsiveness in avian faces, and then by evaluating the consequences of Wnt inhibition in the chick face. Collectively, these data elucidate a new role for Wnt signaling in regional specification of the vertebrate face, and suggest possible mechanisms whereby species-specific facial features are generated.

BACKGROUND: Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is characterized by ventricular arrhythmias, sudden death, and fatty or fibrofatty replacement of right ventricular myocytes. Recent studies have noted an association between human ARVD/C and molecular remodeling of intercalated disc structures. However, progress has been constrained by limitations inherent to human studies. OBJECTIVE: We studied the molecular composition of the intercalated disc structure in a naturally occurring animal model of ARVD/C (Boxer dogs). METHODS: We studied hearts from 12 Boxers with confirmed ARVD/C and 2 controls. Ventricular sections from 4 animals were examined by immunofluorescent microscopy. Frozen tissue samples were used for Western blot analysis. Proteins investigated were N-cadherin, plakophilin 2, desmoplakin, plakoglobin, desmin, and connexin 43 (Cx43). RESULTS: In control dogs, all proteins tested by immunofluorescence analysis yielded intense localized signals at sites of end-to-end cell apposition. In contrast, myocardial tissues from ARVD/C-afflicted Boxers showed preservation of N-cadherin staining but loss of detectable signal for Cx43 at the intercalated disc location. Western blots indicated that the Cx43 protein was still present in the samples. Gene sequencing analysis showed no mutations in desmoplakin, plakoglobin, Cx43, or plakophilin 2. CONCLUSION: Mutation(s) responsible for ARVD/C in Boxers lead, directly or indirectly, to severe modifications of mechanical and electrical cell-cell interactions. Furthermore, significant reduction in gap junction formation may promote a substrate for malignant ventricular arrhythmias. This model may help to advance our understanding of the molecular basis, pathophysiology, and potential therapeutic approach to patients with ARVD/C

Fibrillins are the structural components of extracellular microfibrils that impart physical properties to tissues, alone or together with elastin as elastic fibers. Genetic studies in mice have revealed that fibrillin-rich microfibrils are also involved in regulating developmental programs and homeostatic processes through the modulation of TGF-beta/BMP signaling events. A new paradigm has thus emerged whereby the spatiotemporal organization of microfibrils dictates both the cellular activities and physical properties of connective tissues. These observations have paved the way to novel therapeutic approaches aimed at counteracting the life-threatening complications in human conditions caused by dysfunctions of fibrillin-rich microfibrils.

The vulnerability or destabilization of atherosclerotic plaques has been directly linked to plaque composition. Imaging modalities, such as magnetic resonance (MR) imaging, that allow for evaluation of plaque composition at a cellular and molecular level, could further improve the detection of vulnerable plaque and may allow for monitoring the efficacy of antiatherosclerotic therapies. In this review we focus on MR imaging strategies for the detection and evaluation of atherosclerotic plaques and their composition. We highlight recent advancements in the development of MR pulse sequences, computer image analysis, and the use of commercially available MR contrast agents, such as gadopentic acid (Gd-DTPA), for plaque characterization. We also discuss molecular imaging strategies that are currently being used to design specific imaging probes targeted to biochemical and cellular markers of atherosclerotic plaque vulnerability.