Roles of Wnt pathway genes wls, wnt9a, wnt5b, frzb and gpc4 in regulating convergent-extension during zebrafish palate morphogenesis.

The Wnt signaling pathway is crucial for tissue morphogenesis, participating in cellular behavior changes, notably during the process of convergent-extension. Interactions between Wnt-secreting and receiving cells during convergent-extension remain elusive. We investigated the role and genetic interactions of Wnt ligands and their trafficking factors Wls, Gpc4 and Frzb in the context of palate morphogenesis in zebrafish. We describe that the chaperon Wls and its ligands Wnt9a and Wnt5b are expressed in the ectoderm, whereas juxtaposed chondrocytes express Frzb and Gpc4. Using wls, gpc4, frzb, wnt9a and wnt5b mutants, we genetically dissected the Wnt signals operating between secreting ectoderm and receiving chondrocytes. Our analysis delineates that non-canonical Wnt signaling is required for cell intercalation, and that wnt5b and wnt9a are required for palate extension in the anteroposterior and transverse axes, respectively.

Spalt-like 4 promotes posterior neural fates via repression of pou5f3 family members in Xenopus.

Amphibian neural development occurs as a two-step process: (1) induction specifies a neural fate in undifferentiated ectoderm; and (2) transformation induces posterior spinal cord and hindbrain. Signaling through the Fgf, retinoic acid (RA) and Wnt/ß-catenin pathways is necessary and sufficient to induce posterior fates in the neural plate, yet a mechanistic understanding of the process is lacking. Here, we screened for factors enriched in posterior neural tissue and identify spalt-like 4 (sall4), which is induced by Fgf. Knockdown of Sall4 results in loss of spinal cord marker expression and increased expression of pou5f3.2 (oct25), pou5f3.3 (oct60) and pou5f3.1 (oct91) (collectively, pou5f3 genes), the closest Xenopus homologs of mammalian stem cell factor Pou5f1 (Oct4). Overexpression of the pou5f3 genes results in the loss of spinal cord identity and knockdown of pou5f3 function restores spinal cord marker expression in Sall4 morphants. Finally, knockdown of Sall4 blocks the posteriorizing effects of Fgf and RA signaling in the neurectoderm. These results suggest that Sall4, activated by posteriorizing signals, represses the pou5f3 genes to provide a permissive environment allowing for additional Wnt/Fgf/RA signals to posteriorize the neural plate.

Vascular Robo4 restricts proangiogenic VEGF signaling in breast.

Formation of the vascular system within organs requires the balanced action of numerous positive and negative factors secreted by stromal and epithelial cells. Here, we used a genetic approach to determine the role of SLITs in regulating the growth and organization of blood vessels in the mammary gland. We demonstrate that vascularization of the gland is not affected by loss of Slit expression in the epithelial compartment. Instead, we identify a stromal source of SLIT, mural cells encircling blood vessels, and show that loss of Slit in the stroma leads to elevated blood vessel density and complexity. We examine candidate SLIT receptors, Robo1 and Robo4, and find that increased vessel angiogenesis is phenocopied by loss of endothelial-specific Robo4, as long as it is combined with the presence of an angiogenic stimulus such as preneoplasia or pregnancy. In contrast, loss of Robo1 does not affect blood vessel growth. The enhanced growth of blood vessels in Robo4(-/-) endothelium is due to activation of vascular endothelial growth factor (VEGF)-R2 signaling through the Src and FAK kinases. Thus, our studies present a genetic dissection of SLIT/ROBO signaling during organ development. We identify a stromal, rather than epithelial, source of SLITs that inhibits blood vessel growth by signaling through endothelial ROBO4 to down-regulate VEGF/VEGFR2 signaling.

Histologic analysis of fetal bovine derived acellular dermal matrix in tissue expander breast reconstruction.

BACKGROUND: This study seeks to determine human host response to fetal bovine acellular dermal matrix (ADM) in staged implant-based breast reconstruction. METHODS: A prospective study was performed for patients undergoing immediate breast reconstruction with tissue expander placement and SurgiMend acellular fetal bovine dermis. At the time of exchange for permanent implant, we obtained tissue specimens of SurgiMend and native capsule. Histological and immunohistochemical assays were performed to characterize the extent of ADM incorporation/degradation, host cell infiltration, neovascularization, inflammation, and host replacement of acellular fetal bovine collagen. RESULTS: Seventeen capsules from 12 patients were included in our study. The average "implantation" time of SurgiMend was 7.8 months (range, 2-23 months). Histological analysis of the biopsy of tissue revealed rare infiltration of host inflammatory cells, even at 23 months. One patient had an infection requiring removal of the tissue expander at 2 months. Contracture, inflammatory changes, edema, and polymorphonuclear leukocyte infiltration were rare in the ADM. An acellular capsule was seen in many cases, at the interface of SurgiMend with the tissue expander. CONCLUSIONS: SurgiMend demonstrated a very infrequent inflammatory response. An antibody specific to bovine collagen allowed for direct identification of bovine collagen separate from human collagen. Cellular infiltration and neovascularization of SurgiMend correlated with the quality of the mastectomy skin flap rather than the duration of implantation. Future studies are needed to further characterize the molecular mechanisms underlying tissue incorporation of this product.

Follistatin interacts with Noggin in the development of the axial skeleton.

When compared to single mutants for Follistatin or Noggin, we find that double mutants display a dramatic further reduction in trunk cartilage formation, particularly in the vertebral bodies and proximal ribs. Consistent with these observations, expression of the early sclerotome markers Pax1 and Uncx is diminished in Noggin;Follistatin compound mutants. In contrast, Sim1 expression expands medially in double mutants. As the onset of Follistatin expression coincides with sclerotome specification, we argue that the effect of Follistatin occurs after sclerotome induction. We hypothesize that Follistatin aids in maintaining proper somite size, and consequently sclerotome progenitor numbers, by preventing paraxial mesoderm from adopting an intermediate/lateral plate mesodermal fate in the Noggin-deficient state.

Wnt3a reestablishes osteogenic capacity to bone grafts from aged animals.

BACKGROUND: Age-related fatty degeneration of the bone marrow contributes to delayed fracture-healing and osteoporosis-related fractures in the elderly. The mechanisms underlying this fatty change are unknown, but they may relate to the level of Wnt signaling within the aged marrow cavity. METHODS: Transgenic mice were used in conjunction with a syngeneic bone-graft model to follow the fates of cells involved in the engraftment. Immunohistochemistry along with quantitative assays were used to evaluate Wnt signaling and adipogenic and osteogenic gene expression in bone grafts from young and aged mice. Liposomal Wnt3a protein (L-Wnt3a) was tested for its ability to restore osteogenic potential to aged bone grafts in critical-size defect models created in mice and in rabbits. Radiography, microquantitative computed tomography (micro-CT) reconstruction, histology, and histomorphometric measurements were used to quantify bone-healing resulting from L-Wnt3a or a control substance (liposomal phosphate-buffered saline solution [L-PBS]). RESULTS: Expression profiling of cells in a bone graft demonstrated a shift away from an osteogenic gene profile and toward an adipogenic one with age. This age-related adipogenic shift was accompanied by a significant reduction (p < 0.05) in Wnt signaling and a loss in osteogenic potential. In both large and small animal models, osteogenic competence was restored to aged bone grafts by a brief incubation with the stem-cell factor Wnt3a. In addition, liposomal Wnt3a significantly reduced cell death in the bone graft, resulting in significantly more osseous regenerate in comparison with controls. CONCLUSIONS: Liposomal Wnt3a enhances cell survival and reestablishes the osteogenic capacity of bone grafts from aged animals. CLINICAL RELEVANCE: We developed an effective, clinically applicable, regenerative medicine-based strategy for revitalizing bone grafts from aged patients.

Mechanosensing by the primary cilium: deletion of Kif3A reduces bone formation due to loading.

Primary cilia, solitary microtubule-based structures that grow from the centriole and extend into the extracellular space, have increasingly been implicated as sensors of a variety of biochemical and biophysical signals. Mutations in primary cilium-related genes have been linked to a number of rare developmental disorders as well as dysregulation of cell proliferation. We propose that primary cilia are also important in mechanically regulated bone formation in adults and that their malfunction could play a role in complex multi-factorial bone diseases, such as osteoporosis. In this study, we generated mice with an osteoblast- and osteocyte-specific knockout of Kif3a, a subunit of the kinesin II intraflagellar transport (IFT) protein; IFT is required for primary cilia formation, maintenance, and function. These Colα1(I) 2.3-Cre;Kif3a(fl/fl) mice exhibited no obvious morphological skeletal abnormalities. Skeletally mature Colα1(I) 2.3-Cre;Kif3a(fl/fl) and control mice were exposed to 3 consecutive days of cyclic axial ulna loading, which resulted in a significant increase in bone formation in both the conditional knockouts and controls. However, Colα1(I) 2.3-Cre;Kif3a(fl/fl) mice did exhibit decreased formation of new bone in response to mechanical ulnar loading compared to control mice. These results suggest that primary cilia act as cellular mechanosensors in bone and that their function may be critical for the regulation of bone physiology due to mechanical loading in adults.