Assessment of changes in place of death of older adults who died from dementia in the United States, 2000-2014: a time-series cross-sectional analysis.

BACKGROUND: As the mortality attributable to dementia-related diseases in the United States escalates, providing quality and equitable end-of-life care for dementia patients across care settings has become a major public health challenge. Previous research suggests that place of death may be an indicator of quality of end-of-life care. This study aims to examine the geographical variations and temporal trends in place of death of dementia decedents in the US and the relationships between place of death of dementia decedents and broad structural determinants. METHODS: Using nationwide death certificates between 2000 and 2014, we described the changes in place of death of dementia decedents across states and over time. Chi-square test for trend in proportions was used to test significant linear trend in the proportion of dementia decedents at difference places. State fixed effects models were estimated to assess the relationships between the proportion of dementia decedents at difference places and state-level factors, particularly availability of care facility resources and public health insurance expenditures. RESULTS: Dementia decedents were more likely to die at home and other places and less likely to die at institutional settings over the study period. There was wide inter-state and temporal variability in the proportions of deaths at different places. Among state-level factors, availability of nursing home beds was positively associated with rates of nursing home/long term care deaths and negatively associated with rates of home deaths. Medicaid expenditure on institutional long term supports and services was positively associated with rates of nursing home/long term care deaths and negatively associated with rates of home deaths. Medicaid expenditure on home and community based services, however, had a positive association with rates of home deaths. CONCLUSIONS: There was a persistent shift in the place of death of dementia decedents from institutions to homes and communities. Increased investments in home and community based health services may help dementia patients to die at their homes. As home becomes an increasingly common place of death of dementia patients, it is critical to monitor the quality of end-of-life care at this setting.

Inversion of Lake Bathymetry through Integrating Multi-Temporal Landsat and ICESat Imagery.

Lake bathymetry provides valuable information for lake basin planning and treatment, lake watershed erosion and siltation management, water resource planning, and environmental protection. Lake bathymetry has been surveyed with sounding techniques, including single-beam and multi-beam sonar sounding, and unmanned ship sounding. Although these techniques have high accuracy, most of them require long survey cycles and entail a high degree of difficulty. On the contrary, optical remote sensing inversion methods are easy to implement, but tend to provide less accurate bathymetry measures, especially when applied to turbid waters. The present study, therefore, aims to improve the accuracy of bathymetry measurements through integrating Landsat Thematic Mapper imagery, the Ice, Cloud, and Land Elevation Satellite's Geoscience Laser Altimeter System (ICESat/GLAS) data, and water level data measured at hydrological stations. First, the boundaries of a lake at multiple dates were derived using water extraction, initial boundary extraction, and Landsat Thematic Mapper/Enhanced Thematic Mapper (TM/ETM+) strip removal processing techniques. Second, ICESat/GLAS data were introduced to obtain additional topographic information of a lake. The striped topography of a lake's area was then obtained through eliminating and correcting erroneous points and interpolating the values of unknown points. Third, the entire bathymetry of the lake was obtained through interpolating water level values of lake boundary points in various dates. Experiments show that accurate bathymetry (±1 m) can be successfully derived.

Detailed Urban Land Use Land Cover Classification at the Metropolitan Scale Using a Three-Layer Classification Scheme.

Urban Land Use/Land Cover (LULC) information is essential for urban and environmental management. It is, however, very difficult to automatically extract detailed urban LULC information from remote sensing imagery, especially for a large urban area. Medium resolution imagery, such as Landsat Thematic Mapper (TM) data, cannot uncover detailed LULC information. Further, very high resolution (VHR) satellite imagery, such as IKONOS and QuickBird data, can only be applied to a small area, largely due to the data unavailability and high computation cost. As a result, little research has been conducted to extract detailed urban LULC information for a large urban area. This study, therefore, developed a three-layer classification scheme for deriving detailedurban LULC information by integrating newly launched Chinese GF-1 (medium resolution) and GF-2 (very high resolution) satellite imagery and synthetically incorporating geometry, texture, and spectral information through multi-resolution image segmentation and object-based image classification (OBIA). Homogeneous urban LULC types such as water bodies or large areas of vegetation could be derived from GF-1 imagery with 16 m and 8 m spatial resolutions, while heterogeneous urban LULC types such as industrial buildings, residential buildings, and roads could be extracted from GF-2 imagery with 3.2 m and 0.8 m spatial resolutions. The multi-resolution segmentation method and a random forest algorithm were employed to perform image segmentation and object-based image classification, respectively. An analysis of the results suggests an overall accuracy of 0.89 and 0.87 were achieved for the second and third level urban LULC classification maps, respectively. Therefore, the three-layer classification scheme has the potential to derive high accuracy urban LULC information through integrating medium and high-resolution remote sensing imagery.

Mapping Soil Alkalinity and Salinity in Northern Songnen Plain, China with the HJ-1 Hyperspectral Imager Data and Partial Least Squares Regression.

In arid and semi-arid regions, identifying and monitoring of soil alkalinity and salinity are in urgently need for preventing land degradation and maintaining ecological balances. In this study, physicochemical, statistical, and spectral analysis revealed that potential of hydrogen (pH) and electrical conductivity (EC) characterized the saline-alkali soils and were sensitive to the visible and near infrared (VIS-NIR) wavelengths. On the basis of soil pH, EC, and spectral data, the partial least squares regression (PLSR) models for estimating soil alkalinity and salinity were constructed. The R² values for soil pH and EC models were 0.77 and 0.48, and the root mean square errors (RMSEs) were 0.95 and 17.92 dS/m, respectively. The ratios of performance to inter-quartile distance (RPIQ) for the soil pH and EC models were 3.84 and 0.14, respectively, indicating that the soil pH model performed well but the soil EC model was not considerably reliable. With the validation dataset, the RMSEs of the two models were 1.06 and 18.92 dS/m. With the PLSR models applied to hyperspectral data acquired from the hyperspectral imager (HSI) onboard the HJ-1A satellite (launched in 2008 by China), the soil alkalinity and salinity distributions were mapped in the study area, and were validated with RMSEs of 1.09 and 17.30 dS/m, respectively. These findings revealed that the hyperspectral images in the VIS-NIR wavelengths had the potential to map soil alkalinity and salinity in the Songnen Plain, China.

A Geographic Information-Assisted Temporal Mixture Analysis for Addressing the Issue of Endmember Class and Endmember Spectra Variability.

Spectral mixture analysis (SMA) is a common approach for parameterizing biophysical fractions of urban environment and widely applied in many fields. For successful SMA, the selection of endmember class and corresponding spectra has been assumed as the most important step. Thanks to the spatial heterogeneity of natural and urban landscapes, the variability of endmember class and corresponding spectra has been widely considered as the profound error source in SMA. To address the challenging problems, we proposed a geographic information-assisted temporal mixture analysis (GATMA). Specifically, a logistic regression analysis was applied to analyze the relationship between land use/land covers and surrounding socio-economic factors, and a classification tree method was used to identify the present status of endmember classes throughout the whole study area. Furthermore, an ordinary kriging analysis was employed to generate a spatially varying endmember spectra at all pixels in the remote sensing image. As a consequence, a fully constrained temporal mixture analysis was conducted for examining the fractional land use land covers. Results show that the proposed GATMA achieved a promising accuracy with an RMSE of 6.81%, SE of 1.29% and MAE of 2.6%. In addition, comparative analysis result illustrates that a significant accuracy improvement has been found in the whole study area and both developed and less developed areas, and this demonstrates that the variability of endmember class and endmember spectra is essential for unmixing analysis.

Speciation and distribution characteristics of heavy metals and pollution assessments in the sediments of Nashina Lake, Heilongjiang, China.

Sediment core samples from Nashina Lake, Heilongjiang, China were collected using a gravity sampler. The cores were sliced horizontally at 1 cm each to determine the particle size, total concentrations and speciation of Cd, Cr, Cu, Mn, Ni, Pb, and Zn. Total concentrations of heavy metals were extracted using an acid mixture (containing hydro fluoric acid, nitric acid, and sulphuric acid) and analyzed using an inductively coupled plasma spectrometry. A sequential extraction procedure was employed to separate chemical species. Analysis of results indicate that the concentrations of heavy metals in the sediments of Nashina Lake in descending order are Mn, Cr, Zn, Pb, Ni, Cu, and Cd. The ratios of the average concentrations of four heavy metals (e.g.Cr, Cu, Ni, Zn) to their background values were >1; and those of Mn, Cd, and Pb were >1. Moreover, some toxic metals were mainly distributed in bioavailable fractions. For instance, both Cd and Mn were typically found in Acid-extractable species or Fe-Mn oxide species, and thus can be easily remobilized and enter the food chain. Finally, the analysis of geo-accumulation index showed that anthropogenic pollution levels of Cr, Cu, Mn, Ni, Zn were low, but those of Pb and Cd were at the moderate level. As both Pb and Cd are toxic metals, it is highly necessary to prohibit their transformation and accumulation in the sediments.

Impact of urbanization on natural ecosystem service values: a comparative study.

With rapid population growth and rural to-urban migration in many Chinese cities, a large amount of natural lands have been converted to urban and agricultural lands recently. During this process of land conversion, economic development and quality of life improvement are considered as major goals, and their influences on ecological systems have often been neglected. The degradation of natural ecological systems due to land use change, however, has become severe,and may require immediate attentions from urban planners and local governments. Taking HaDaQi industrial corridor, Heilongjiang Province, China,as a case study area, this paper examined the trend of land use changes during 1990­2005, and quantified their influences on natural eco system service values. In particular, this study applied two major valuation methods, and examined whether different valuation methods generate significantly different results. Analysis of results suggests that human dominated land uses (e.g., urban and agriculture)have expanded rapidly at the cost of natural lands (e.g., wetlands and forest). Due to these land use changes, the total ecosystem service value decreased 29% (2.26% annually) from 1990 to 2005 when the first method was applied, and this rate is estimated to be 15.7% (1.13% annually)with the second approach. Moreover, the annual rate of ecosystem service value decline during 2000­2005 is about four times higher than that in 1990­2000 with both methods, suggesting much more severe ecosystem degradation during 2000­2005.

Detecting spatiotemporal clusters of dementia mortality in the United States, 2000-2010.

Based on national death certificate data during 2000 and 2010, we employed the space-time scan statistic to identify spatiotemporal clusters of dementia mortality in the contiguous United States. Results revealed that, for both Alzheimer's disease and all-cause dementia mortality in the total population, the most likely clusters occurred in the Northeast region, with lower than average relative risk. The most likely excess mortality clusters were in the Pacific Northwest and Ohio River Valley and Carolinas. Temporal information of clusters suggested reduction in the relative risk of Alzheimer's disease and all-cause dementia mortality in most of the highly likely clusters. The results should propel public health agencies to evaluate the capacity of local health and social care to meet dementia patients' needs before death in the high-risk cluster areas. Further investigation of causal factors of these clusters is needed.

Cellular and molecular mechanisms of synovial joint and articular cartilage formation.

Synovial joints and articular cartilage play crucial roles in the skeletal function, but relatively little is actually known about their embryonic development. Here we first focused on the interzone, a thin mesenchymal cell layer forming at future joint sites that is widely thought to be critical for joint and articular cartilage development. To determine interzone cell origin and fate, we microinjected the vital fluorescent dye DiI at several peri-joint sites in chick limbs and monitored the behavior and fate of labeled cells over time. Peri-joint mesenchymal cells located immediately adjacent to incipient joints migrated, became part of the interzone, and were eventually found in epiphyseal articular layer and joint capsule. Interzone cells isolated and reared in vitro expressed typical phenotypic markers, including GDF-5, Wnt-14, and CD-44, and differentiated into chondrocytes over time. To determine the molecular mechanisms of articular chondrocyte formation, we carried out additional studies on the ets transcription factor family member ERG and its alternatively spliced variant C-1-1 that we previously found to be expressed in developing avian articular chondrocytes. We cloned the human counterpart of avian C-1-1 (ERGp55Delta81) and conditionally expressed it in transgenic mice under cartilage-specific Col2 gene promotor-enhancer control. The entire transgenic mouse limb chondrocyte population exhibited an immature articular-like phenotype and a virtual lack of growth plate formation and chondrocyte maturation compared to wild-type littermate. Together, our studies reveal that peri-joint mesenchymal cells take part in interzone and articular layer formation, interzone cells can differentiate into chondrocytes, and acquisition of a permanent articular chondrocyte phenotype is aided and perhaps dictated by ets transcription factor ERG.

Retinoid signaling regulates CTGF expression in hypertrophic chondrocytes with differential involvement of MAP kinases.

UNLABELLED: Retinoids are important for growth plate chondrocyte maturation, but their downstream effectors remain unclear. Recently, CTGF (CCN2) was found to regulate chondrocyte function, particularly in the hypertrophic zone. The goal of the study was to determine whether CTGF is a retinoid signaling effector molecule, how it is regulated, and how it acts. INTRODUCTION: Using a combination of in vivo and in vitro approaches, we carried out a series of studies at the cellular, biochemical, and molecular level to determine whether and how retinoid signaling is related to expression and function of connective tissue growth factor (CTGF) in chondrocyte maturation and endochondral ossification. MATERIALS AND METHODS: Limbs of chick embryos in ovo were implanted with retinoic pan-antagonist RO 41-5253-filled beads, and phenotypic changes were assessed by in situ hybridization. CTGF gene expression and roles were tested in primary cultures of immature and hypertrophic chondrocytes. Cross-talk between retinoid signaling and other pathways was tested by determining endogenous levels of active ERK1/2 and p38 MAP kinases and phenotypic modulations exerted by specific antagonists of mitogen-activated protein (MAP) kinases and BMP signaling (Noggin). RESULTS: Interference with retinoid signaling blocked expression of CTGF and other posthypertrophic markers in long bone anlagen in vivo and hypertrophic chondrocyte cultures, whereas all-trans-retinoic acid (RA) boosted CTGF expression and even induced it in immature proliferating cultures. Exogenous recombinant CTGF stimulated chondrocyte maturation, but failed to do so in presence of retinoid antagonists. Immunoblots showed that hypertrophic chondrocytes contained sizable levels of phosphorylated ERK1/2 and p38 MAP kinases that were dose- and time-dependently increased by RA treatment. Experimental ERK1/2 inhibition led to a severe drop in baseline and RA-stimulated CTGF expression, whereas p38 inhibition increased it markedly. These responses were gene-specific, because the opposite was seen with other hypertrophic chondrocyte genes such as collagen X and RA receptor gamma (RARgamma). Tests with Noggin showed that RA induction of CTGF expression was negatively influenced by BMP signaling, whereas induction of collagen X expression was BMP-dependent. CONCLUSIONS: Retinoids appear to have a preeminent role in controlling expression and function of CTGF in hypertrophic and posthypertrophic chondrocytes and do so with differential cooperation and intervention of MAP kinases and BMP signaling.

Expression of Indian Hedgehog, BMP-4 and Noggin in craniosynostosis induced by fetal constraint.

Indian Hedgehog (Ihh), bone morphogenetic protein (BMP), and its antagonist Noggin play an important regulatory role in bone formation. We used an animal model to study the role of these molecules in craniosynostosis induced by fetal constraint. C57Bl/6 mice underwent cervical cerclage on the 18th day of gestation, and their pups were harvested 48 and 72 hours beyond the normal gestational period. Constrained and control calvariae were examined for expression of BMP-4, Noggin, Histone H4C, Ihh, Sonic Hedgehog (Shh), and Patched 1 (Ptch1), one of the Hh transcriptional target molecules/Hh receptors. Constraint-induced suture fusion was associated with decreased expression of Ihh and Noggin, whereas BMP-4 was expressed in both control and constrained sutures. Ptch1 colocalized with Ihh-positive osteogenic cells at the osteogenic fronts, but not with Shh transcripts, suggesting that Ihh, but not Shh, regulates Ptch1 expression in cranial suture development. Histone H4C was preferentially expressed in Ihh-positive cells, indicating that Ihh may regulate osteogenic cell proliferation at the osteogenic fronts. These results suggest a role for Ihh and Noggin signaling in constraint-induced craniosynostosis.

Temporomandibular joint formation and condyle growth require Indian hedgehog signaling.

The temporomandibular joint (TMJ) is essential for jaw function, but the mechanisms regulating its development remain poorly understood. Because Indian hedgehog (Ihh) regulates trunk and limb skeletogenesis, we studied its possible roles in TMJ development. In wild-type mouse embryos, Ihh expression was already strong in condylar cartilage by embryonic day (E) 15.5, and expression of Ihh receptors and effector genes (Gli1, Gli2, Gli3, and PTHrP) indicated that Ihh range of action normally reached apical condylar tissue layers, including polymorphic chondroprogenitor layer and articular disc primordia. In Ihh(-/-) embryos, TMJ development was severely compromised. Condylar cartilage growth, polymorphic cell proliferation, and PTHrP expression were all inhibited, and growth plate organization and chondrocyte gene expression patterns were abnormal. These severe defects were partially corrected in double Ihh(-/-)/Gli3(-/-) mutants, signifying that Ihh action is normally modulated and delimited by Gli3 and Gli3(R) in particular. Both single and double mutants, however, failed to form an articular disc primordium, normally appreciable as an independent condensation between condylar apex and neighboring developing temporal bone in wild-type. This failure persisted at later stages, leading to complete absence of a normal functional disc and lubricin-expressing joint cavities. In summary, Ihh is very important for TMJ development, where it appears to regulate growth and elongation events, condylar cartilage phenotype, and chondroprogenitor cell function. Absence of articular disc and joint cavities in single and double mutants points to irreplaceable Ihh roles in formation of those critical TMJ components.

Public health data collection and sharing using HIPAA messages.

Public health information has significant value for doctors, public health officials, epidemiological researchers, the general public, and government agencies. Unfortunately, these data are difficult to obtain and are typically collected on as-needed basis and maintained locally. This localized process unavoidably limits the access to important public health data by its users. Moreover, the diversity of data transmission standards and collection techniques make the collected data less usable. This paper proposes a new standardized public health information system based on the HIPAA (Health Insurance Portability and Accountability Act) messages, which are the standard transactions between hospitals and insurance companies. In particular, this paper explores the applicability of HIPAA messages as a data source and transmission standard, and proposes a prototype design of a new system to collect and share public health data using HIPAA messages.

Chick limbs with mouse teeth: an effective in vivo culture system for tooth germ development and analysis.

Mouse tooth germ development is currently studied by three main approaches: in wild-type and mutant mouse lines, after transplantation of tooth germs to ectopic sites, and in organ culture. The in vivo approaches are the most physiological but do not provide accessibility to tooth germs for further experimental manipulation. Organ cultures, although readily accessible, do not sustain full tooth germ development and are appropriate for short-term analysis. Thus, we sought to establish a new approach that would combine experimental accessibility with sustained development. We implanted fragments of embryonic day 12 mouse embryo first branchial arch containing early bud stage tooth germs into the lateral mesenchyme of day 4-5 chick embryo wing buds in ovo. Eggs were reincubated, and implanted tissues were examined by histochemistry and in situ hybridization over time. The tooth germs underwent seemingly normal growth, differentiation, and morphogenesis. They reached the cap, bell, and crown stages in approximately 3, 6, and 10 days, respectively, mimicking in a striking manner native temporal patterns. To examine mechanisms regulating tooth germ development, we first implanted tooth germ fragments, microinjected them with neutralizing antibodies to the key signaling molecule Sonic hedgehog (Shh), and examined them over time. Tooth germ development was markedly delayed, as revealed by poor morphogenesis and lack of mature ameloblasts and odontoblasts displaying characteristic traits such as an elongated cell shape, nuclear relocalization, and amelogenin gene expression. These phenotypic changes began to be reversed upon further incubation. The data show that the limb bud represents an effective, experimentally accessible as well as economical system for growth and analysis of developing tooth germs. The inhibitory effects of Shh neutralizing antibody treatment are discussed in relation to roles of this signaling pathway proposed by this and other groups previously.

Indian hedgehog and syndecans-3 coregulate chondrocyte proliferation and function during chick limb skeletogenesis.

Hedgehog proteins exert critical roles in embryogenesis and require heparan sulfate proteoglycans (HS-PGs) for action. Indian hedgehog (Ihh) is produced by prehypertrophic chondrocytes in developing long bones and regulates chondrocyte proliferation and other events, but it is not known whether it requires HS-PGs for function. Because the HS-PG syndecan-3 is preferentially expressed by proliferating chondrocytes, we tested whether it mediates Ihh action. Primary chick chondrocyte cultures were treated with recombinant Ihh (rIhh-N) in absence or presence of heparinase I or syndecan-3 neutralizing antibodies. While rIhh-N stimulated proliferation in control cultures, it failed to do so in heparinase- or antibody-treated cultures. In reciprocal gain-of-function studies, chondrocytes were made to overexpress syndecan-3 by an RCAS viral vector. Cells became more responsive to rIhh-N, but even this response was counteracted by heparinase or antibody treatment. To complement the in vitro data, RCAS viral particles were microinjected in day 4-5 chick wing buds and effects of syndecan-3 misexpression were monitored over time. Syndecan-3 misexpression led to widespread chondrocyte proliferation and, interestingly, broader expression and distribution of Ihh. In addition, the syndecan-3 misexpressing skeletal elements were short, remained cartilaginous, lacked osteogenesis, and exhibited a markedly reduced expression of collagen X and osteopontin, products characteristic of hypertrophic chondrocytes and bone cells. The data are the first to indicate that Ihh action in chondrocyte proliferation involves syndecan-3 and to identify a specific member of the syndecan family as mediator of hedgehog function.

Sonic hedgehog functions as a mitogen during bell stage of odontogenesis.

Epithelial-mesenchymal interactions are required for tissue growth and gene expression patterns during odontogenesis. We showed previously that Sonic hedgehog (SHH) is detectable in both dental epithelium and mesenchyme, while Shh transcripts are present in dental epithelium only, suggesting that SHH functions as an autocrine signal in epithelium and a paracrine signal in mesenchyme. This hypothesis was tested here. We found by in situ hybridization that the SHH autocrine receptor Ptch-2 is indeed expressed in dental epithelium whereas the paracrine receptor Ptc is expressed in mesenchyme. Bovine bell stage tooth germs were microsurgically separated into epithelial and mesenchymal portions and the resulting tissue fragments were organ-cultured. In epithelium fragments cultured by themselves, gene expression of Shh and Gli-1 (a putative transcriptional mediator of hedgehog signaling) was significantly decreased in both inner dental epithelium and stratum intermedium layers; this was accompanied by a sharp drop in epithelial cell proliferation. However, in companion control tissue fragments containing both epithelium and mesenchyme, Shh and Gli-1 expression as well as cell proliferation were maintained. Treatment of dental epithelial or mesenchymal cell populations in monolayer cultures with exogenous recombinant SHH stimulated cell proliferation. Together, the data provide clear evidence that Shh is synthesized by dental epithelium, reaches the underlying mesenchyme, and appears to act as an autocrine mitogen for epithelial cells and a paracrine mitogen for mesenchymal cells, thus exerting crucial functions in tooth germ growth, morphogenesis, and tissue-tissue interactions of bell stage of odontogenesis.

Expression, gene regulation, and roles of Fisp12/CTGF in developing tooth germs.

Odontogenesis involves multiple events, including tissue-tissue interactions, cell proliferation, and cell differentiation, but the underlying mechanisms of regulation are far from clear. Because Fisp12/CTGF is a signaling protein involved in similar events in other systems, we asked whether it is expressed in developing tooth germs and what roles it may have. Indeed, Fisp12/CTGF transcripts were first expressed by dental laminas, invaginating epithelium, and condensing mesenchyme at the bud stage, and then became abundant in enamel knot and preameloblasts. Fisp12/CTGF was present not only in inner dental epithelium but also in stratum intermedium and underlying dental mesenchyme. Fisp12/CTGF expression decreased markedly in secreting ameloblasts. Tissue reconstitution experiments showed that Fisp12/CTGF expression in dental epithelium required interaction with mesenchyme but was maintained by treatment of epithelium with transforming growth factor-1, a factor regulating Fisp12/CTGF expression in other systems, or with bone morphogenetic protein-2. Loss-of-function studies using CTGF neutralizing antibodies revealed that interference with endogenous factor action in tooth germ explants led to a severe inhibition of proliferation in both epithelium and mesenchyme and a marked delay in cytodifferentiation of ameloblasts and odontoblasts. Treatment of dental epithelial and mesenchymal cells in culture with recombinant CTGF stimulated cell proliferation, whereas treatment with neutralizing antibodies inhibited it. The data demonstrate for the first time that Fisp12/CTGF is expressed during odontogenesis. Expression is confined to specific sites and times, is regulated by epithelial-mesenchymal interactions and critical soluble factors, and appears to be needed for proliferation and differentiation along both ameloblast and odontoblast cell lineages.

Expression and roles of connective tissue growth factor in Meckel's cartilage development.

Meckel's cartilage is a prominent feature of the developing mandible, but its formation and roles remain unclear. Because connective tissue growth factor (CTGF, CCN2) regulates formation of other cartilages, we asked whether it is expressed and what roles it may have in developing mouse Meckel's cartilage. Indeed, CTGF was strongly expressed in anterior, central, and posterior regions of embryonic day (E) 12 condensing Meckel's mesenchyme. Expression decreased in E15 newly differentiated chondrocytes but surged again in E18 hypertrophic chondrocytes located in anterior region and most-rostral half of central region. These cells were part of growth plate-like structures with zones of maturation resembling those in a developing long bone and expressed such characteristic genes as Indian hedgehog (Ihh), collagen X, MMP-9, and vascular endothelial growth factor. At each stage examined perichondrial tissues also expressed CTGF. To analyze CTGF roles, mesenchymal cells isolated from E10 first branchial arches were tested for interaction and responses to recombinant CTGF (rCTGF). The cells readily formed aggregates in suspension culture and interacted with substrate-bound rCTGF, but neither event occurred in the presence of CTGF neutralizing antibodies. In good agreement, rCTGF treatment of micromass cultures stimulated both expression of condensation-associated macromolecules (fibronectin and tenascin-C) and chondrocyte differentiation. Expression of these molecules and CTGF itself was markedly up-regulated by treatment with transforming growth factor-beta1, a chondrogenic factor. In conclusion, CTGF is expressed in highly dynamic manners in developing Meckel's cartilage where it may influence multiple events, including chondrogenic cell differentiation and chondrocyte maturation. CTGF may aid chondrogenesis by acting down-stream of transforming growth factor-beta and stimulating cell-cell interactions and expression of condensation-associated genes.

The Wnt antagonist Frzb-1 regulates chondrocyte maturation and long bone development during limb skeletogenesis.

The Wnt antagonist Frzb-1 is expressed during limb skeletogenesis, but its roles in this complex multistep process are not fully understood. To address this issue, we determined Frzb-1 gene expression patterns during chick long bone development and carried out gain- and loss-of-function studies by misexpression of Frzb-1, Wnt-8 (a known Frzb-1 target), or different forms of the intracellular Wnt mediator LEF-1 in developing limbs and cultured chondrocytes. Frzb-1 expression was quite strong in mesenchymal prechondrogenic condensations and then characterized epiphyseal articular chondrocytes and prehypertrophic chondrocytes in growth plates. Virally driven Frzb-1 misexpression caused shortening of skeletal elements, joint fusion, and delayed chondrocyte maturation, with consequent inhibition of matrix mineralization, metalloprotease expression, and marrow/bone formation. In good agreement, misexpression of Frzb-1 or a dominant-negative form of LEF-1 in cultured chondrocytes maintained the cells at an immature stage. Instead, misexpression of Wnt-8 or a constitutively active LEF-1 strongly promoted chondrocyte maturation, hypertrophy, and calcification. Immunostaining revealed that the distribution of endogenous Wnt mediator beta-catenin changes dramatically in vivo and in vitro, from largely cytoplasmic in immature proliferating and prehypertrophic chondrocytes to nuclear in hypertrophic mineralizing chondrocytes. Misexpression of Frzb-1 prevented beta-catenin nuclear relocalization in chondrocytes in vivo or in vitro. The data demonstrate that Frzb-1 exerts a strong influence on limb skeletogenesis and is a powerful and direct modulator of chondrocyte maturation, phenotype, and function. Phases of skeletogenesis, such as terminal chondrocyte maturation and joint formation, appear to be particularly dependent on Wnt signaling and thus very sensitive to Frzb-1 antagonistic action.