In the post-natal growth plate, chondrocytes are arranged in columns parallel to the long axis of the bone. Chondrocytes divide perpendicular to this axis and then move into position one on top of another in a process called "rotation" that maintains columnar organization. Primary cilia are non-motile microtubule base appendages extending from the surface of almost all vertebrate cells. Primary cilia were described on chondrocytes almost 40 years ago but the function of these structures in cartilage biology is not known. Intraflagellar transport (IFT) is the process by which primary cilia are generated and maintained. This study tested the hypothesis that IFT plays an important role in post-natal skeletal development. Kif3a, a subunit of the Kinesin II motor complex, that is required for intraflagellar transport and the formation of cilia, was deleted in mouse chondrocytes via Col2a-Cre-mediated recombination. Disruption of IFT resulted in subsequent depletion of cilia and post-natal dwarfism due to premature loss of the growth plate likely a result of reduced proliferation and accelerated hypertrophic differentiation of chondrocytes. Cell shape and columnar orientation in the growth plate were also disrupted suggesting a defect in the process of rotation. Alterations in chondrocyte rotation were accompanied by disruption of the actin cytoskeleton and alterations in the localization of activated FAK to focal adhesion-like structures on chondrocytes. This is the first report indicating a role for IFT and primary cilia in the development of the post-natal growth plate. The results suggest a model in which IFT/cilia act to maintain the columnar organization of the growth plate via the process of chondrocyte rotation.

In this study, we address the function of Transforming Growth Factor beta (TGF-beta) and its type II receptor (Tgfbr2) in limb development in vivo. Mouse embryos were generated in which the Tgfbr2 gene was deleted in early limb mesenchyme using Prx1Cre-mediated LoxP recombination. A high level of Tgfbr2 gene deletion was verified in limb mesenchyme by PCR between E9.5 and E10.5 days in Cre expressing mice. RT-PCR assays indicated a significant depletion of Tgfbr2 mRNA by E10.5 days as a result of Cre mediated gene deletion. Furthermore, limb mesenchyme from Cre(+);Tgfbr2(f/f) mice placed in micromass culture did not respond to exogenously added TGF-beta1 confirming the functional deletion of the receptor. However, there was an unexpected increase in the number and intensity of Alcian blue stained chondrogenic nodules in micromass cultures derived from Tgfbr2-deleted limbs relative to cultures from control limbs suggesting that Tgfbr2 normally limits chondrogenesis in vitro. In vivo, early limb development and chondrocyte differentiation occurred normally in Tgfbr2-depleted mice. Later in development, depletion of Tgfbr2 in limb mesenchyme resulted in short limbs and fusion of the joints in the phalanges. Alteration in the length of the long bones was primarily due to a decrease in chondrocyte proliferation after E13.5 days. In addition, the transition from prehypertrophic to hypertrophic cells was accelerated while there was a delay in late hypertrophic differentiation leading to a reduction in the length of the marrow cavity. In the joint, cartilage cells replaced interzone cells during development. Analysis of markers for joint development indicated that the joint was specified properly and that the interzone cells were initially formed but not maintained. The results suggest that Tgfbr2 is required for normal development of the skeleton and that Tgfbr2 can act to limit chondrogenesis in mesenchymal cells like the interzone.

Wnt5a, a non-canonical Wnt ligand critical for outflow tract (OFT) morphogenesis, is expressed specifically in second heart field (SHF) progenitors in the caudal splanchnic mesoderm (SpM) near the inflow tract (IFT). Using a conditional Wnt5a gain of function (GOF) allele and Islet1-Cre, we broadly over-expressed Wnt5a throughout the SHF lineage, including the entire SpM between the IFT and OFT. Wnt5a over-expression in Wnt5a null mutants can rescue the cell polarity and actin polymerization defects as well as severe SpM shortening, but fails to rescue OFT shortening. Moreover, Wnt5a over-expression in wild-type background is able to cause OFT shortening. We find that Wnt5a over-expression does not perturb SHF cell proliferation, apoptosis or differentiation, but affects the deployment of SHF cells by causing them to accumulate into a large bulge at the rostral SpM and fail to enter the OFT. Our immunostaining suggests an inverse correlation between cell cohesion and Wnt5a level in the wild-type SpM. Ectopic Wnt5a expression in in the rostral SpM of Wn5a-GOF mutants diminishes the upregulation of adherens junction; whereas loss of Wnt5a in Wnt5a null mutants causes premature increase in adherens junction level in the caudal SpM. Over-expression of mouse Wnt5a in Xenopus animal cap cells also reduces C-cadherin distribution on the plasma membrane without affecting its overall protein level, suggesting that Wnt5a may play an evolutionarily conserved role in controlling the cell surface level of cadherin to modulate cell cohesion during tissue morphogenesis. Collectively, our data indicate that restricted expression of Wnt5a in the caudal SpM is essential for normal OFT morphogenesis, and uncover a novel function of spatially regulated cell cohesion by Wnt5a in driving the deployment of SHF cells from the SpM into OFT.

Transforming growth factor β (TGFβ) is known to play important roles in multiple developmental processes. One of the main functions is in skeletal development. Our previous studies demonstrated that loss of Tgfbr2 in Prx1Cre-expressing limb mesenchyme results in defects in the long bones and joints of mice. Here we show that loss of Tgfbr2 also results in defects in the development of the skull vault indicating Tgfbr2 has a critical role in intramembranous bone formation as well as endochondral bone formation. Mutant mice did not survive after birth and demonstrated an open skull. The first signs of skull defects were observed at E14.5 day. Prx1Cre+/Tgfbr2f/f embryos showed significantly reduced cell proliferation in the developing mesenchyme of the skull by E14.5 day without any detectable alteration in apoptosis suggesting that reduced cell proliferation in Prx1Cre+/Tgfbr2f/f embryos was at least partially responsible for the defects observed. Immunofluorescent staining showed a significant reduction in the expression of Runx2/Cbfa1 and Osterix/Sp7 in Prx1Cre+/Tgfbr2f/f embryos suggesting that osteoblast differentiation was also altered in Prx1Cre+/Tgfbr2f/f embryos. To distinguish between the effects of losing Tgfbr2 on mesenchymal proliferation versus osteoblast differentiation, osteoprogenitor cells from the skulls of Tgfbr2f/f embryos were cultured under conditions of high cell density and Tgfbr2 was deleted from the cells using Adeno-Cre virus. RT-PCR analysis showed that the mRNA level of Runx2 and Osterix as well as Dlx5 and Msx2 were down-regulated in Tgfbr2-deleted cultures compared to control cultures indicating that Tgfbr2 regulates osteoblast differentiation independent of regulating proliferation. Together, these results suggest that Tgfbr2 is required for normal development of the skull.

Convergent extension (CE) is a fundamental morphogenetic mechanism that underlies numerous processes in vertebrate development, and its disruption can lead to human congenital disorders such as neural tube closure defects. The dynamic, oriented cell intercalation during CE is regulated by a group of core proteins identified originally in flies to coordinate epithelial planar cell polarity (PCP). The existing model explains how core PCP proteins, including Van Gogh (Vang) and Dishevelled (Dvl), segregate into distinct complexes on opposing cell cortex to coordinate polarity among static epithelial cells. The action of core PCP proteins in the dynamic process of CE, however, remains an enigma. In this report, we show that Vangl2 (Vang-like 2) exerts dual positive and negative regulation on Dvl during CE in both the mouse and Xenopus. We find that Vangl2 binds to Dvl to cell-autonomously promote efficient Dvl plasma membrane recruitment, a pre-requisite for PCP activation. At the same time, Vangl2 inhibits Dvl from interacting with its downstream effector Daam1 (Dishevelled associated activator of morphogenesis 1), and functionally suppresses Dvl → Daam1 cascade during CE. Our finding uncovers Vangl2-Dvl interaction as a key bi-functional switch that underlies the central logic of PCP signaling during morphogenesis, and provides new insight into PCP-related disorders in humans.

Extracellular trafficking of tumor necrosis factor receptor superfamily (TNFRSF) is tightly regulated, disruption of which triggers various autoinflammatory disorders, including TNF receptor-associated periodic syndrome (TRAPS). Here, we provide thus far unraveled molecular basis of noncysteine mutations in TNFR1 ectodomain where loss of an aromatic moiety in cysteine-rich domain (CRD) 2 results in TRAPS disease-associated phenotype. Our study characterized that a missense mutation on phenylalanine residue located in CRD2 (TNFR1 F60V) causes a delay in TNFR1 transport to cell membrane, leading to sustained receptor responsiveness and downstream NF-κB activation, characteristic of clinical manifestation of a prolonged fever. By creating and characterizing identical mutations on structurally conserved ectodomains of osteoprotegerin (OPG) and decoy receptor 3, other two secreted forms of TNFRSF, we further identified that a conserved aromatic residue at the A1 submodule of CRD2 (A1CRD2) confers structural integrity of ectodomain where aromatic sidechain deletion increases thermal instability, interfering with efficient posttranslational modification and subsequent receptor secretion. Interestingly, our functional analyses indicated that this particular noncysteine mutation is not associated with either protein misfolding or loss of function. Finally, by using a synthetic agonist, we demonstrated gain-of-function of the trafficking defect, suggesting the possibility of rescuing affected pathology in related disorders. Given the structural and topological similarities present in the ectodomains of TNFRSF members, our findings provide mechanistic insights of defects in subcellular trafficking of TNF receptors, reported in various TNFRSF-associated diseases.

TGF‐β enhances Sox9 protein expression as well as downstream targets that regulate extracellular matrix production in bovine articular chondrocytes (BACs). The extracellular matrix protein 3‐Prime‐Phoshoadenosine 5‐Prime‐Phosphosulfate Synthase 2 (Papss2) has been associated with spondyloepimetaphyseal dysplasias in humans and is required for proper sulfation of proteoglycans in cartilage. Our work and that of others has shown that TGF‐β can regulate Papss2 expression in articular cartilage. We aim to identify whether Sox9 is involved downstream of TGF‐β in the regulation of Papss2 mRNA expression. Using primary BACs in micromass cultures, we analyzed the effect of TGF‐β (5ng/ml) on chondrocyte phenotype, Sox9 protein expression, stability, cellular localization and activity. We also assessed the effect of TGF‐β and/or Sox9 overexpression on the mRNA expression of Papss2, Prg4 and GAPDH via quantitative real‐time PCR. We found that treatment with TGF‐β enhanced proteoglycan content and delayed chondrocyte hypertrophic differentiation in BACs. We also observed an increase in Sox9 protein levels and stability, without changes in mRNA. The increase in Sox9 protein correlated with enhanced Col2a‐reporter activity. Importantly, we found Papss2 to be upregulated via Sox9 when wild‐type (WT)‐Sox9 was overexpressed via adenoviral transduction (50 MOI/cell). Interestingly, treatment +/‐ TGF‐β when Sox9 was overexpressed did not further increase Papss2 mRNA, supporting the hypothesis that Sox9 works downstream of TGF‐ β. However, TGF‐β, but not Sox9, increased Prg4 mRNA expression, suggesting specificity of signaling. A GAPDH control was examined and remained unchanged in response to either treatment. The regulation of Papss2 by Sox9 represents a novel paradigm involving TGF‐β‐dependent signaling in articular chondrocytes. Grant Funding Source: Supported by NIAMS‐ NIH