The zebrafish (Danio rerio) is increasingly being used to study basic vertebrate biology and human disease using a rich array of in vivo genetic and molecular tools. However, the inability to readily modify the genome in a targeted fashion has been a bottleneck in the field. Here we show that improvements in artificial transcription activator-like effector nucleases (TALENs) provide a powerful new approach for targeted zebrafish genome editing and functional genomic applications1–5. Using the GoldyTALEN modified scaffold and zebrafish delivery system, we show this enhanced TALEN toolkit demonstrates a high efficiency in inducing locus-specific DNA breaks in somatic and germline tissues. At some loci, this efficacy approaches 100%, including biallelic conversion in somatic tissues that mimics phenotypes seen using morpholino (MO)-based targeted gene knockdowns6. With this updated TALEN system, we successfully used single-stranded DNA (ssDNA) oligonucleotides (oligos) to precisely modify sequences at predefined locations in the zebrafish genome through homology-directed repair (HDR), including the introduction of a custom-designed EcoRV site and a modified loxP (mloxP) sequence into somatic tissue in vivo. We further show successful germline transmission of both EcoRV and mloxP engineered chromosomes. This combined approach offers the potential to model genetic variation as well as to generate targeted conditional alleles.

Transforming growth factor-B (TGF-B) stimulates cellular proliferation and transformation to a myofibroblast phenotype in vivo and in a subset of fibroblast cell lines. As the Smad pathway is activated by TGF-B in essentially all cell types, it is unlikely to be the sole mediator of cell type-specific outcomes to TGF-B stimulation. In the current study, we determined that TGF-B receptor signaling activates phosphatidylinositol 3-kinase (PI3K) in several fibroblast but not epithelial cultures independently of Smad2 and Smad3. PI3K activation occurs in the presence of dominant-negative dynamin and is required for p21-activated kinase-2 kinase activity and the increased proliferation and morphologic change induced by TGF-B in vitro. (Cancer Res 2005; 65(22): 10431-40)

Members of the transforming growth factor ␤ (TGF-␤) family of proteins signal through cell surface transmembrane serine/threonine protein kinases known as type I and type II receptors. The TGF-␤ signal is extended through phosphorylation of receptor-associated Smad proteins by the type I receptor. Although numerous investigations have established the sequence of events in TGF-␤ receptor (TGF-␤R) activation, none have examined the role of the endocytic pathway in initiation and/or maintenance of the signaling response. In this study we investigated whether TGF-␤R internalization modulates type I receptor activation, the formation of a functional receptor/Smad/SARA complex, Smad2/3 phosphorylation or nuclear translocation, and TGF-␤-dependent reporter gene activity. Our data provide evidence that, whereas type I receptor phosphorylation and association of SARA and Smad2 with the TGF-␤R complex take place independently of clathrin lattice formation, Smad2 or Smad3 activation and downstream signaling only occur after endocytic vesicle formation. Thus, TGF-␤R endocytosis is not simply a way to dampen the signaling response but instead is required to propagate signaling via the Smad pathway.

Transforming growth factor-β receptor recycling is regulated by the clathrin adaptor Dab2 protein. In the absence of Dab2, receptors localize in a perinuclear locale because they are unable to transit from the early endosomal antigen 1-positive early endosome to the Rab11-positive endosomal recycling compartment.

SUMMARY Transforming growth factor beta (TGF-β) family ligands are pleotropic proteins with diverse cell type-specific effects on growth and differentiation. For example, PAK2 activation is critical for the proliferative/pro-fibrotic action of TGF-β on mesenchymal cells, and yet is not responsive to TGF-β in epithelial cells. We therefore investigated the regulatory constraints that prevent inappropriate PAK2 activation in epithelial cultures. The results show that the epithelial-enriched protein Erbin controls the function of the NF2 tumor suppressor Merlin by determining the output of Merlin's physical interactions with active PAK2. While mesenchymal TGF-β signaling induces PAK2-mediated inhibition of Merlin function in the absence of Erbin, Erbin/Merlin complexes bind and inactivate GTPase-bound PAK2 in epithelia. These results not only identify Erbin as a key determinant of epithelial resistance to TGF-β signaling, but also show that Erbin controls Merlin tumor suppressor function by switching the functional valence of PAK2 binding.

Transforming growth factor beta (TGF-beta) coordinates a number of biological events important in normal and pathophysiological growth. In this study, deletion and substitution mutations were used to identify receptor motifs modulating TGF-beta receptor activity. Initial experiments indicated that a COOH-terminal sequence between amino acids 482-491 in the kinase domain of the type I receptor was required for ligand-induced receptor signaling and down-regulation. These 10 amino acids are highly conserved in mammalian, Xenopus, and Drosophila type I receptors. Although mutation or deletion of the region (referred to as the NANDOR BOX, for nonactivating non-down-regulating) abolishes TGF-beta-dependent mitogenesis, transcriptional activity, type I receptor phosphorylation, and down-regulation in mesenchymal cultures, adjacent mutations also within the kinase domain are without effect. Moreover, a kinase-defective type I receptor can functionally complement a mutant BOX expressing type I receptor, documenting that when the BOX mutant is activated, it has kinase activity. These results indicate that the sequence between 482 and 491 in the type I receptor provides a critical function regulating activation of the TGF-beta receptor complex.

TGF-␤ modulates numerous diverse cellular phenotypes including growth arrest in epithelial cells and proliferation in fibroblasts. Although the Smad pathway is fundamental for the majority of these responses, recent evidence indicates that nonSmad pathways may also have a critical role. Here we report a novel mechanism whereby the nonreceptor tyrosine focal adhesion kinase (FAK) functions as an adaptor necessary for cell type-specific responses to TGF-␤. We show that in contrast to Smad actions, non-Smad pathways, including c-Abl, PAK2, and Akt, display an obligate requirement for FAK. Interestingly, this occurs in Src null SYF cells and is independent of FAK tyrosine phosphorylation, kinase activity, and/or proline-rich sequences in the C-terminal FAT domain. FAK binds the phosphatidylinositol 3-kinase (PI3K) p85 regulatory subunit following TGF-␤ treatment in a subset of fibroblasts but not epithelial cells and has an obligate role in TGF-␤-stimulated anchorage-independent growth and migration. Together, these results uncover a new scaffolding role for FAK as the most upstream component regulating the profibrogenic action of TGF-␤ and suggest that inhibiting this interaction may be useful in treating a number of fibrotic diseases.

This study assessed the feasibility and acceptability of a novel virtual world-based CR (VWCR) program, Destination Rehab, as an extension of a conventional center-based CR program. The VWCR program is a feasible, highly acceptable, and innovative platform to potentially influence health behaviors and CV risk and may increase accessibility to disadvantaged populations with higher CV disease burdens.