Faculty Bibliography

Large T antigen is the replicative helicase of simian virus 40. Its specific binding to the origin of replication and oligomerization into a double hexamer distorts and unwinds dsDNA. In viral replication, T antigen acts as a functional homolog of the eukaryotic minichromosome maintenance factor MCM. T antigen is also an oncoprotein involved in transformation through interaction with p53 and pRb. We obtained the three-dimensional structure of the full-length T antigen double hexamer assembled at its origin of replication by cryoelectron microscopy and single-particle reconstruction techniques. The double hexamer shows different degrees of bending along the DNA axis. The two hexamers are differentiated entities rotated relative to each other. Isolated strands of density, putatively assigned to ssDNA, protrude from the hexamer-hexamer junction mainly at two opposite sites. The structure of the T antigen at the origin of replication can be understood as a snapshot of the dynamic events leading to DNA unwinding. Based on these results a model for the initiation of simian virus 40 DNA replication is proposed

Basic fibroblast growth factor (FGF-2) and matrix metalloproteinases (MMPs) play key roles in vascular remodeling. Because FGF-2 controls a number of proteolytic activities in various cell types, we tested its effect on vascular endothelial cell expression of MMP-3 (stromelysin-1), a broad-spectrum proteinase implicated in coronary atherosclerosis. Endothelial cells (EC) from FGF-2-/- mice are highly responsive to exogenous FGF-2 and were therefore used for this study. The results showed that treatment of microvascular EC with human recombinant FGF-2 results in strong induction of MMP-3 mRNA and protein expression. Upregulation of MMP-3 mRNA by FGF-2 requires de novo protein synthesis and activation of the ERK-1/2 pathway. FGF-2 concentrations (5-10 ng/ml) that induce rapid and prolonged (24 h) activation of ERK-1/2 upregulate MMP-3 expression. In contrast, lower concentrations (1-2 ng/ml) that induce robust but transient (<8 h) ERK-1/2 activation are ineffective. Inhibition of ERK-1/2 activation at different times (-0.5 h to +8 h) of EC treatment with effective FGF-2 concentrations blocks MMP-3 upregulation. Thus, FGF-2 induces EC expression of MMP-3 with a threshold dose effect that requires sustained activation of the ERK-1/2 pathway. Because FGF-2 controls other EC functions with a linear dose effect, these features indicate a unique role of MMP-3 in vascular remodeling

The apical ectodermal ridge (AER) is a critical signaling center at the tip of the limb that promotes outgrowth. In mouse, formation of the AER involves a gradual restriction of AER gene expression from a broad ventral preAER domain to the tip of the limb, as well as progressive thickening of cells to form a multilayered epithelium. The AER is visible from embryonic day 10.5 to 13.5 (E10.5-E13.5) in the mouse forelimb. Previous short-term fate mapping studies indicated that, once a cell is incorporated into the AER, its descendents remain within the AER. In addition, some preAER cells appear to become incorporated into the ventral ectoderm. In the present study, we used an inducible CreER/loxP fate mapping approach in mouse to examine the long-term contribution of preAER cells to limb ventral ectoderm, as well as the ultimate fate of the mature AER cells. We used a CreER transgene that contains Msx2 regulatory sequences specific to the developing AER, and demonstrate by marking preAER cells that, at stage 2 of mouse limb bud development, the majority of the ventral ectoderm that protrudes from the body wall later covers only the paw. Furthermore, when Msx2-CreER-expressing preAER cells are marked after the onset of preAER gene expression, a similar domain of paw ventral ectoderm is marked at E16.5, in addition to the AER. Strikingly, mapping the long-term fate of cells that form the mature AER showed that, although this structure is indeed a distinct compartment, AER-derived cells are gradually lost after E12.5 and no cells remain by birth. A distinct dorsal/ventral border nevertheless is maintained in the ectoderm of the paw, with the distal-most border being located at the edge of the nail bed. These studies have uncovered new aspects of the cellular mechanisms involved in AER formation and in partitioning the ventral ectoderm in mouse limb

Slow-twitch muscle fibers of the zebrafish myotome undergo a unique set of morphogenetic cell movements. During embryogenesis, slow-twitch muscle derives from the adaxial cells, a layer of paraxial mesoderm that differentiates medially within the myotome, immediately adjacent to the notochord. Subsequently, slow-twitch muscle cells migrate through the entire myotome, coming to lie at its most lateral surface. Here we examine the cellular and molecular basis for slow-twitch muscle cell migration. We show that slow-twitch muscle cell morphogenesis is marked by behaviors typical of cells influenced by differential cell adhesion. Dynamic and reciprocal waves of N-cadherin and M-cadherin expression within the myotome, which correlate precisely with cell migration, generate differential adhesive environments that drive slow-twitch muscle cell migration through the myotome. Removing or altering the expression of either protein within the myotome perturbs migration. These results provide a definitive example of homophilic cell adhesion shaping cellular behavior during vertebrate development.

Cell fate diversity can be achieved through the asymmetric segregation of cell fate determinants. In the Drosophila embryo, neuroblasts divide asymmetrically and in a stem cell fashion. The determinants Prospero and Numb localize in a basal crescent and are partitioned from neuroblasts to their daughters (GMCs). Here we show that nonmuscle myosin II regulates asymmetric cell division by an unexpected mechanism, excluding determinants from the apical cortex. Myosin II is activated by Rho kinase and restricted to the apical cortex by the tumor suppressor Lethal (2) giant larvae. During prophase and metaphase, myosin II prevents determinants from localizing apically. At anaphase and telophase, myosin II moves to the cleavage furrow and appears to "push" rather than carry determinants into the GMC. Therefore, the movement of myosin II to the contractile ring not only initiates cytokinesis but also completes the partitioning of cell fate determinants from the neuroblast to its daughter.

Early patterning of the vertebrate midbrain and cerebellum is regulated by a mid/hindbrain organizer that produces three fibroblast growth factors (FGF8, FGF17 and FGF18). The mechanism by which each FGF contributes to patterning the midbrain, and induces a cerebellum in rhombomere 1 (r1) is not clear. We and others have found that FGF8b can transform the midbrain into a cerebellum fate, whereas FGF8a can promote midbrain development. In this study we used a chick electroporation assay and in vitro mouse brain explant experiments to compare the activity of FGF17b and FGF18 to FGF8a and FGF8b. First, FGF8b is the only protein that can induce the r1 gene Gbx2 and strongly activate the pathway inhibitors Spry1/2, as well as repress the midbrain gene Otx2. Consistent with previous studies that indicated high level FGF signaling is required to induce these gene expression changes, electroporation of activated FGFRs produce similar gene expression changes to FGF8b. Second, FGF8b extends the organizer along the junction between the induced Gbx2 domain and the remaining Otx2 region in the midbrain, correlating with cerebellum development. By contrast, FGF17b and FGF18 mimic FGF8a by causing expansion of the midbrain and upregulating midbrain gene expression. This result is consistent with Fgf17 and Fgf18 being expressed in the midbrain and not just in r1 as Fgf8 is. Third, analysis of gene expression in mouse brain explants with beads soaked in FGF8b or FGF17b showed that the distinct activities of FGF17b and FGF8b are not due to differences in the amount of FGF17b protein produced in vivo. Finally, brain explants were used to define a positive feedback loop involving FGF8b mediated upregulation of Fgf18, and two negative feedback loops that include repression of Fgfr2/3 and direct induction of Spry1/2. As Fgf17 and Fgf18 are co-expressed with Fgf8 in many tissues, our studies have broad implications for how these FGFs differentially control development