Matthew C. Salanga

Mechanical Forces Alter Zyxin Unbinding Kinetics Within Focal Adhesions of Living Cells

The formation of focal adhesions that mediate alterations of cell shape and movement is controlled by a mechanochemical mechanism in which cytoskeletal tensional forces drive changes in molecular assembly; however, little is known about the molecular biophysical basis of this response. Here, we describe a method to measure the unbinding rate constant kOFF of individual GFP‐labeled focal adhesion molecules in living cells by modifying the fluorescence recovery after photobleaching (FRAP) technique and combining it with mathematical modeling. Using this method, we show that decreasing cellular traction forces on focal adhesions by three different techniques—chemical inhibition of cytoskeletal tension generation, laser incision of an associated actin stress fiber, or use of compliant extracellular matrices—increases the kOFF of the focal adhesion protein zyxin. In contrast, the kOFF of another adhesion protein, vinculin, remains unchanged after tension dissipation. Mathematical models also demonstrate that these force‐dependent increases in zyxins kOFF that occur over seconds are sufficient to quantitatively predict large‐scale focal adhesion disassembly that occurs physiologically over many minutes. These findings demonstrate that the molecular binding kinetics of some, but not all, focal adhesion proteins are sensitive to mechanical force, and suggest that force‐dependent changes in this biophysical parameter may govern the supramolecular events that underlie focal adhesion remodeling in living cells. J. Cell. Physiol. 207: 187–194, 2006.

Krüppel-like factor 2 cooperates with the ETS family protein ERG to activate Flk1 expression during vascular development

The VEGF receptor, FLK1, is essential for differentiation of the endothelial lineage and for embryonic vascular development. Using comparative genomics, we have identified conserved ETS and Krüppel-like factor (KLF) binding sites within the Flk1 enhancer. In transgenic studies, mutation of either site results in dramatic reduction of Flk1 reporter expression. Overexpression of KLF2 or the ETS transcription factor ERG is sufficient to induce ectopic Flk1 expression in the Xenopus embryo. Inhibition of KLF2 function in the Xenopus embryo results in a dramatic reduction in Flk1 transcript levels. Furthermore, we show that KLF2 and ERG associate in a physical complex and that the two proteins synergistically activate transcription of Flk1. Since the ETS and KLF protein families have independently been recognized as important regulators of endothelial gene expression, cooperation between the two families has broad implications for gene regulation during development, normal physiology and vascular disease.

ETS family protein ETV2 is required for initiation of the endothelial lineage but not the hematopoietic lineage in the Xenopus embryo

Transcription factors of the ETS family are important regulators of endothelial and hematopoietic development. We have characterized the Xenopus orthologue of the ETS transcription factor, ETV2. Expression analysis shows that etv2 is highly expressed in hematopoietic and endothelial precursor cells in the Xenopus embryo. In gain‐of‐function experiments, ETV2 is sufficient to activate ectopic expression of vascular endothelial markers. In addition, ETV2 activated expression of hematopoietic genes representing the myeloid but not the erythroid lineage. Loss‐of‐function studies indicate that ETV2 is required for expression of all endothelial markers examined. However, knockdown of ETV2 has no detectable effects on expression of either myeloid or erythroid markers. This contrasts with studies in mouse and zebrafish where ETV2 is required for development of the myeloid lineage. Our studies confirm an essential role for ETV2 in endothelial development, but also reveal important differences in hematopoietic development between organisms. Developmental Dynamics 239:1178–1187, 2010.

The myocardin-related transcription factor, MASTR, cooperates with MyoD to activate skeletal muscle gene expression

The myocardin family proteins (myocardin, MRTF-A, and MRTF-B) are serum response factor (SRF) cofactors and potent transcription activators. Gene-ablation studies have indicated important developmental functions for myocardin family proteins primarily in regulation of cardiac and smooth muscle development. Using Xenopus genome and cDNA databases, we identified a myocardin-related transcription factor expressed specifically in the skeletal muscle lineage. Synteny and sequence alignments indicate that this gene is the frog orthologue of mouse MASTR [Creemers EE, Sutherland LB, Oh J, Barbosa AC, Olson EN (2006) Coactivation of MEF2 by the SAP domain proteins myocardin and MASTR. Mol Cell 23:83–96]. Inhibition of MASTR function in the Xenopus embryo by using dominant-negative constructions or morpholino knockdown results in a dramatic reduction in expression of skeletal muscle marker genes. Overexpression of MASTR in whole embryos or embryonic tissue explants induces ectopic expression of muscle marker genes. Furthermore, MASTR cooperates with the myogenic regulatory factors MyoD and Myf5 to activate transcription of skeletal muscle genes. An essential function for MASTR in regulation of myogenic development in the vertebrate embryo has not been previously indicated.

Behavioral Profiling of Human Transitional Cell Carcinoma Ex vivo

Outcome studies of many types of cancer have revealed that tumors of indistinguishable histologic appearance may differ significantly in aggressiveness and in their response to therapy. A strategy that would enable early identification of patients at high risk for disease progression and allow screening of multiple therapeutic agents simultaneously for efficacy would improve clinical management. We have developed an orthotopic organ culture model of bladder cancer in which quantum dot-based fluorescent imaging approaches are used to obtain quantitative measurements of tumor cell behavior. Human transitional cell carcinoma (TCC) cells are labeled with quantum dot nanoparticles, and the cells instilled into the rat bladder in vivo, after which the bladder is excised and cultured ex vivo. Cell implantation, proliferation, and invasion into the organ wall are monitored using epifluorescence imaging and two-photon laser scanning confocal microscopy. Using this approach, we were able to assign distinct phenotypes to two metastatic bladder cancer cell lines based on different patterns of invasiveness into the bladder wall. We also showed that established tumor cell masses regressed following intravesical administration of the chemotherapeutic drug thiotepa. Collectively, these findings suggest that this assay system, which we have named EViTAS (for ex vivo tumor assay system), can recapitulate salient aspects of tumor growth in the host and is amenable to behavioral profiling of human cancer.

Generation of a Xenopus laevis F1 albino J strain by genome editing and oocyte host-transfer

Completion of the Xenopus laevis genome sequence from inbred J strain animals has facilitated the generation of germline mutant X. laevis using targeted genome editing. In the last few years, numerous reports have demonstrated that TALENs are able to induce mutations in F0 Xenopus embryos, but none has demonstrated germline transmission of such mutations in X. laevis. In this report we used the oocyte host-transfer method to generate mutations in both tyrosinase homeologs and found highly-penetrant germline mutations; in contrast, embryonic injections yielded few germline mutations. We also compared the distribution of mutations in several F0 somatic tissues and germ cells and found that the majority of mutations in each tissue were different. These results establish that X. laevis J strain animals are very useful for generating germline mutations and that the oocyte host-transfer method is an efficient technique for generating mutations in both homeologs.

Tissue-specific Gene Inactivation in Xenopus laevis: Knockout of lhx1 in the Kidney with CRISPR/Cas9

Xenopus laevis is a classic developmental model, but its allotetraploid genome has limited our ability to perform genetic manipulations. The advance of... Studying genes involved in organogenesis is often difficult because many of these genes are also essential for early development. The allotetraploid frog, Xenopus laevis, is commonly used to study developmental processes, but because of the presence of two homeologs for many genes, it has been difficult to use as a genetic model. Few studies have successfully used CRISPR in amphibians, and currently there is no tissue-targeted knockout strategy described in Xenopus. The goal of this study is to determine whether CRISPR/Cas9-mediated gene knockout can be targeted to the Xenopus kidney without perturbing essential early gene function. We demonstrate that targeting CRISPR gene editing to the kidney and the eye of F0 embryos is feasible. Our study shows that knockout of both homeologs of lhx1 results in the disruption of kidney development and function but does not lead to early developmental defects. Therefore, targeting of CRISPR to the kidney may not be necessary to bypass the early developmental defects reported upon disruption of Lhx1 protein expression or function by morpholinos, antisense RNA, or dominant negative constructs. We also establish a control for CRISPR in Xenopus by editing a gene (slc45a2) that when knocked out results in albinism without altering kidney development. This study establishes the feasibility of tissue-specific gene knockout in Xenopus, providing a cost-effective and efficient method for assessing the roles of genes implicated in developmental abnormalities that is amenable to high-throughput gene or drug screening techniques.

Xenopus as a Model for GI/Pancreas Disease

Diseases affecting endodermal organs like the pancreas, lung, and gastrointestinal tract have a substantial impact on human welfare. Since many of these are congenital defects that arise as a result of defects during development, broad efforts are focused on understanding the development of these organs so as to better identify risk factors, disease mechanisms, and therapeutic targets. Studies implementing model systems, like the amphibian Xenopus, have contributed immensely to our understanding of signaling pathways (e.g., Wnt, FGF, BMP, RA) and gene regulation (e.g., hhex, ptf1a, ngn3) that underlie normal development as well as disease progression. Recent advances in genome engineering further enhance the capabilities of the Xenopus model system for pursuing biomedical research, and will undoubtedly result in a boom of new information underlying disease mechanisms ultimately leading to advancements in diagnosis and therapy.

Hedgehog signaling regulates size of the dorsal aortae and density of the plexus during avian vascular development

Signaling by the hedgehog (Hh) family of secreted growth factors is essential for development of embryonic blood vessels. Embryos lacking Hh function have abundant endothelial cells but fail to assemble vascular cords or lumenized endothelial tubes. However, the role of Hh signaling during later aspects of vascular patterning and morphogenesis is largely unexplored. We have used small molecule inhibitors and agonists to alter activity of the Hh signaling pathway in the chick embryo. When cyclopamine is added after cord formation, aortal cells form tubes, but these are small and disorganized and the density of the adjacent vascular plexus is reduced. Activation of the Hh pathway with SAG leads to formation of enlarged aortae and increased density of the plexus. The number of endothelial cell filopodia is found to correlate with Hh signaling levels. These studies show that Hh signaling levels must be tightly regulated for normal vascular patterning to be achieved. Developmental Dynamics 240:1354–1364, 2011.