The molecular mechanisms governing the cell behaviors underlying morphogenesis remain a major focus of research in both developmental biology and cancer biology. TGF-␤ ligands control cell fate specification via Smad-mediated signaling. However, their ability to guide cellular morphogenesis in a variety of biological contexts is poorly understood. We report on the discovery of a novel TGF-␤ signaling-mediated cellular morphogenesis occurring during vertebrate gastrulation. Activin/nodal members of the TGF-␤ superfamily induce the expression of two genes regulating cell adhesion during gastrulation: Fibronectin Leucine-rich Repeat Transmembrane 3 (FLRT3), a type I transmembrane protein containing extracellular leucine-rich repeats, and the small GTPase Rnd1. FLRT3 and Rnd1 interact physically and modulate cell adhesion during embryogenesis by controlling cell surface levels of cadherin through a dynamin-dependent endocytosis pathway. Our model suggests that cell adhesion can be dynamically regulated by sequestering cadherin through internalization, and subsequent redeploying internalized cadherin to the cell surface as needed. As numerous studies have linked aberrant expression of small GTPases, adhesion molecules such as cadherins, and TGF-␤ signaling to oncogenesis and metastasis, it is tempting to speculate that this FLRT3/Rnd1/cadherin pathway might also control cell behavior and morphogenesis in adult tissue homeostasis.[Keywords: GTPase; FLRT3; Rnd1; gastrulation; activin; Xenopus] Supplemental material is available at http://www.genesdev.org. During gastrulation, the three germ layers (ectoderm, mesoderm, and endoderm) establish new contacts, permitting new inductive interactions to specify the development of organ primordia. Some of the major movements that constitute gastrulation are convergence and extension, involution, and epiboly. The dynamic nature of cell-cell contacts in tissues undergoing these movements has been visualized using time-lapse video microscopy. Cells involved in convergence and extension movements are shown to continuously break and remake local adhesive contacts, via polarized membranous processes (e.g., lamellipodia), as they "slide" past one another during mediolateral intercalation (Davidson et al. 2002;Shook et al. 2004).Distinct adhesive properties conferred by adhesion molecules constitute a key feature of cells that undergo gastrulation movements. Type I cadherins are required for proper morphogenesis in sea urchin (Miller and McClay 1997), zebrafish (Montero et al. 2005;Shimizu et al. 2005), and mouse embryos (Riethmacher et al. 1995). Inactivation of C-cadherin, the primary mediator of adhesion in the Xenopus blastula, leads to both involution and convergent extension defects during gastrulation (Heasman et al. 1994). By changing the functional activity of C-cadherin at the cell surface, the morphogenetic elongation of "animal cap" ectodermal explants (mimicking the convergence and extension movements of the embryonic mesoderm) is altered (Brieher and Gumbiner 1994).The Wn...

Osteoclasts are unique cells that resorb bone, and are involved in not only bone remodeling but also pathological bone loss such as osteoporosis and rheumatoid arthritis. The regulation of osteoclasts is based on a number of molecules but full details of these molecules have not yet been understood. MicroRNAs are produced by Dicer cleavage an emerging regulatory system for cell and tissue function. Here, we examine the effects of Dicer deficiency in osteoclasts on osteoclastic activity and bone mass in vivo. We specifically knocked out Dicer in osteoclasts by crossing Dicer flox mice with cathepsin K-Cre knock-in mice. Dicer deficiency in osteoclasts decreased the number of osteoclasts (N.Oc/BS) and osteoclast surface (Oc.S/BS) in vivo. Intrinsically, Dicer deficiency in osteoclasts suppressed the levels of TRAP positive multinucleated cell development in culture and also reduced NFATc1 and TRAP gene expression. MicroRNA analysis indicated that expression of miR-155 was suppressed by RANKL treatment in Dicer deficient cells. Dicer deficiency in osteoclasts suppressed osteoblastic activity in vivo including mineral apposition rate (MAR) and bone formation rate (BFR) and also suppressed expression of genes encoding type I collagen, osteocalcin, Runx2, and Efnb2 in vivo. Dicer deficiency in osteoclasts increased the levels of bone mass indicating that the Dicer deficiency-induced osteoclastic suppression was dominant over Dicer deficiency-induced osteoblastic suppression. On the other hand, conditional Dicer deletion in osteoblasts by using 2.3 kb type I collagen-Cre did not affect bone mass. These results indicate that Dicer in osteoclasts controls activity of bone resorption in vivo.

Development is controlled by a complex series of events requiring sequential gene activation. Understanding the logic of gene networks during development is necessary for a complete understanding of how genes contribute to phenotype. Pioneering work initiated in the sea urchin and Drosophila has demonstrated that reasonable transcriptional regulatory network diagrams representing early development in multicellular animals can be generated through use of appropriate genomic, genetic, and biochemical tools. Establishment of similar regulatory network diagrams for vertebrate development is a necessary step. The amphibian Xenopus has long been used as a model for vertebrate early development and has contributed greatly to the elucidation of gene regulation. Because the best and most extensively studied transcriptional regulatory network in Xenopus is that underlying the formation and function of Spemann's organizer, we describe the current status of our understanding of this gene regulatory network and its relationship to mesodermal patterning. Seventy-four transcription factors currently known to be expressed in the mesoendoderm of Xenopus gastrula were characterized according to their modes of action, DNA binding consensus sequences, and target genes. Among them, nineteen transcription factors were characterized sufficiently in detail, allowing us to generate a gene regulatory network diagram. Additionally, we discuss recent amphibian work using a combined DNA microarray and bioinformatics approach that promises to accelerate regulatory network studies.gastrulation ͉ mesoderm ͉ organizer ͉ transcription factor ͉ gene regulation

Mechanosensing is one of the crucial components of the biological events. In bone, as observed in unloading-induced osteoporosis in bed ridden patients, mechanical stress determines the levels of bone mass. Many molecules have been suggested to be involved in sensing mechanical stress in bone, while the full pathways for this event has not yet been identified. We examined the role of TRPV4 in unloading-induced bone loss. Hind limb unloading induced osteopenia in wild-type mice. In contrast, TRPV4 deficiency suppressed such unloading-induced bone loss. As underlying mechanism for such effects, TRPV4 deficiency suppressed unloading-induced reduction in the levels of mineral apposition rate and bone formation rate. In these mice, unloading-induced increase in the number of osteoclasts in the primary trabecular bone was suppressed by TRPV4 deficiency. Unloading-induced reduction in the longitudinal length of primary trabecular bone was also suppressed by TRPV4 deficiency. TRPV4 protein is expressed in both osteoblasts and osteoclasts. These results indicated that TRPV4 plays a critical role in unloading-induced bone loss.

The complex gene regulatory networks governed by growth factor signaling are still poorly understood. In order to accelerate the rate of progress in uncovering these networks, we explored the usefulness of interspecies sequence comparison (phylogenetic footprinting) to identify conserved growth factor response elements. The promoter regions of two direct target genes of Bone Morphogenetic Protein (BMP) signaling in Xenopus, Xvent2 and XId3, were compared with the corresponding human and/or mouse counterparts to identify conserved sequences. A comparison between the Xenopus and human Vent2 promoter sequences revealed a highly conserved 21 bp sequence that overlaps the previously reported Xvent2 BMP response element (BRE). Reporter gene assays using Xenopus animal pole ectodermal explants (animal caps) revealed that this conserved 21 bp BRE is both necessary and sufficient for BMP responsiveness. We combine the same phylogenetic footprinting approach with luciferase assays to identify a highly conserved 49 bp BMP responsive region in the Xenopus Id3 promoter. GFP reporters containing multimers of either the Xvent2 or XId3 BREs appear to recapitulate endogenous BMP signaling activity in transgenic Xenopus embryos. Comparison of the Xvent2 and the XId3 BRE revealed core sequence features that are both necessary and sufficient for BMP responsiveness: a Smad binding element (SBE) and a GC-rich element resembling an OAZ binding site. Based on these findings, we have implemented genome scanning to identify over 100 additional putative target genes containing 2 or more BRE-like sequences which are conserved between human and mouse. RT-PCR and in situ analyses revealed that this in silico approach can effectively be used to identify potential BMP target genes.

On page 51726, we misleadingly stated that the central acidic domain in Dsl1p is essential for viability. Evidence for this was presented in Fig. 3C. This figure shows that a mutant carrying two Trp-to-Ala replacements in this region did not support growth of a dsl1 deletion mutant. However, we recently created a GAL-regulated TAP-tagged version of DSL1 carrying five Trp-to-Ala substitutions in this region to use as a negative control in pulldown experiments. Surprisingly, this mutant complemented the dsl1 knock-out. Even a single-copy untagged version of this allele could replace the wild-type gene. However, these cells grow poorly at all temperatures tested and show phenotypes similar to those of Dsl1p-depleted cells.Sequencing showed that the plasmid used for the growth assay in our previous work contained a C-terminal truncation in addition to the Trp-to-Ala substitutions at positions 413 and 455. A mutant clone with an intact C terminus supported growth of a dsl1 deletion mutant.Our recent data are still consistent with the notion that the outer tryptophan motifs in the acidic domain of Dsl1p ( ADDITIONS AND CORRECTIONS This paper is available online at www.jbc.orgWe suggest that subscribers photocopy these corrections and insert the photocopies in the original publication at the location of the original article. Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract) services are urged to carry notice of these corrections as prominently as they carried the original abstracts.

Renin angiotensin system (RAS) regulates circulating blood volume and blood pressure systemically, whereas RAS also plays a role in the local milieu. Previous in vitro studies suggested that RAS may be involved in the regulation of bone cells. However, it was not known whether molecules involved in RAS are present in bone in vivo. In this study, we examined the presence of RAS components in adult bone and the effects of angiotensin II type 2 (AT2) receptor blocker on bone mass. Immunohistochemistry revealed that AT2 receptor protein was expressed in both osteoblasts and osteoclasts. In addition, renin and angiotensin II-converting enzyme were expressed in bone cells in vivo. Treatment with AT2 receptor blocker significantly enhanced the levels of bone mass, and this effect was based on the enhancement of osteoblastic activity as well as the suppression of osteoclastic activity in vivo. These results indicate that RAS components are present in adult bone and that blockade of AT2 receptor results in alteration in bone mass.Osteoporosis is one of the major diseases associated with aging. This disease is based on the imbalance between the two major activities, i.e. bone formation and bone resorption. Systemic signals such as parathyroid hormone (PTH) 3 and vitamin D are the major regulators of the maintenance of bone mass and blood calcium (1-3). In addition, bone mass levels are also determined by a central nervous control through the activities of sympathetic tone on both bone formation and resorption sides (4 -7). In the local milieu of bone, two major types of cells, osteoblasts and osteoclasts, are located in close proximity, exchange their signals, and coordinately resorb and form bone matrix (8). Such events are controlled by molecules present in the local microenvironment. These include cytokines, their modulators, and matrix proteins secreted by osteoblasts and osteoclasts (9 -11). However, the bone environment is quite heterogeneous, and there are also cells other than these two types. Microvasculatures are serving as a root to supply osteoclast progenitors, which are derived from hematopoietic lineage cells. Vasculatures in bone are also considered to give rise to progenitors for osteoblastic cells from their perivascular regions (12). In addition to the anatomical relationship between the vascular cells and bone cells, these cells may be functionally involved in the coordinate regulation of bone mass.Recent clinical studies indicated that beta blockers and antihypertension drugs would reduce the risk of bone fractures in the elderly populations (13,14). This suggests a possible link between vascular and skeletal systems. Renin angiotensin system (RAS) is operating not only systemically but also locally in several tissues, and bone microenvironments have been studied in this regard (15,16). Osteoblasts and osteoclasts express angiotensin II type 1 receptor in cell cultures (17-19), suggesting the existence of local RAS in bone. However, whether RAS components are expressed in bone in vivo is not known.A...

Bone regeneration for the defects in revision surgery of joint replacement is an increasingly important issue. To repair bone defects, bone cell activation by growth factors using synthetic resorbable scaffold is a useful and safe option. We examine the efficiency of nanogel-crosslinking hydrogel as a novel synthetic scaffold for BMP to stimulate osteoblasts and to induce bone formation. Cholesterol-bearing pullulan nanogel-crosslinking hydrogel (CHPA/Hydrogel) was used to deliver BMP. The CHPA hydrogel pellets were implanted in vivo. Single implantation of CHPA/hydrogel containing low amounts of BMP induced osteoblastic activation and new bone formation in vivo. Furthermore, nanogel in a disc shape established recruitment of osteoblastic cells that vigorously formed bone to heal the calvarial defects, which did not heal spontaneously without it. In conclusion, CHPA/hydrogel serves as an efficient and versatile scaffold for the stimulation of osteoblasts to form bone and to repair defects via delivery of BMP.