Cortical bone loss is an early feature of nonradiographic axial spondyloarthritis.

BACKGROUND: In the present study, we investigated bone geometry, microstructure, and volumetric bone mineral density (vBMD) in a cohort of patients with nonradiographic axial spondyloarthritis (nr-axSpA) in order to define the early bone changes occurring in axial spondyloarthritis (axSpA) and to define potential factors for deterioration of bone microstructure. METHODS: Patients with axSpA (n = 107) and healthy control subjects (n = 50) of similar age and sex were assessed for geometric, volumetric, and microstructural parameters of bone using high-resolution peripheral quantitative computed tomography (HR-pQCT) at the radius. Additionally, demographic and disease-specific characteristics of patients with axSpA were recorded. RESULTS: Patients with nr-axSpA and control subjects were comparable in age, sex, and body mass index. Geometric and microstructural analysis by HR-pQCT revealed a significantly reduced cortical area (p = 0.022) and cortical thickness (p = 0.006) in patients with nr-axSpA compared with control subjects. Total and cortical vBMD were significantly reduced in patients with nr-axSpA (p = 0.042 and p = 0.007, respectively), whereas there was no difference in trabecular vBMD. Patients with a short disease duration (< 2 years; n = 46) also showed significant reduction of cortical thickness and cortical area compared with control subjects. Patients with disease duration > 2 years (n = 55) additionally developed a decrease of cortical and total vBMD. Multiple regression models identified male sex to be associated with lower cortical vBMD and female sex to be associated with lower trabecular vBMD. CONCLUSIONS: Bone microstructure in patients with nr-axSpA is characterized primarily by deterioration of cortical bone. Cortical bone loss starts early and is evident within the first 2 years of the disease.

Effects of age and weight on the metabolic activities of the cervical, thoracic and lumbar spines as measured by fluorine-18 fluorodeoxyglucose-positron emission tomography in healthy males.

OBJECTIVE: This study aimed to explore the age and weight-related metabolic trends in the spines of healthy male subjects using fluorine-18 fluorodeoxyglucose positron emission tomography (18F-FDG PET) imaging. SUBJECTS AND METHODS: Forty three healthy male subjects (age 23-75 years, weight 50-145kg) were selected from the CAMONA study. A global assessment methodology was applied to the subjects' 18F-FDG 180 minute scans, where each region of the spine (cervical, thoracic and lumbar) was individually encapsulated in a single region of interest, and standardized uptake value (SUVmean) was calculated per respective region. RESULTS: SUVmean increased significantly with weight in both the thoracic spine (Slope=0.0066, P=0.001) and lumbar spine (Slope=0.0087, P<0.0001), but not the cervical spine. There were no significant correlations between age and SUVmean in all three regions. The cervical spine (average SUVmean=1.84±0.31) illustrated elevated activity when compared to the thoracic (average SUVmean=1.46±0.27, P<0.0001) and lumbar (average SUVmean=1.41±0.28, P<0.0001) spines. CONCLUSION: This study illustrated the ability of 18F-FDG PET to assess metabolic processes in the spine. The data provided evidence of weight dependent metabolic activity, likely related to inflammation. This study offers a methodological precedent that can be applied to studies in populations with back pain.

In vivo pre-activation of monocytes in patients with axial spondyloarthritis.

INTRODUCTION: Innate immune responses, including monocyte functions, seem to play an important role in the pathogenesis of axial spondyloarthritis (axSpA). Therefore, we characterized the phenotype and functional state of monocytes of patients with axSpA. METHODS: Fifty-seven patients with axSpA, 11 patients with rheumatoid arthritis (RA), and 29 healthy controls were included in the study. We determined the percentage of classic, intermediate, and non-classic monocytes according to CD14 and CD16 expression and the expression of Toll-like receptor (TLR) 1, 2, and 4 in whole blood by flow cytometry. The percentage of monocytes producing interleukin (IL)-1beta, IL-6, tumor necrosis factor alpha (TNFα), IL-12/23p40, and IL-1 receptor antagonist (IL-1ra) was detected by flow cytometry after stimulation of whole blood without and with different TLR and nucleotide-binding oligomerization domain ligands-i.e., lipopolysaccharide (LPS), fibroblast-stimulating lipopeptid-1, PAM3CSK4, and muramyl dipeptide (MDP)-for 5 h. IL-10 production was measured after 18 h of stimulation in supernatants by enzyme-linked immunosorbent assay. RESULTS: In patients with axSpA but not patients with RA, we found higher frequencies of classic monocytes than in controls (median of 90.4% versus 80.4%, P < 0.05), higher frequencies of monocytes spontaneously producing IL-1beta and IL-1ra (P < 0.05), and a higher percentage of monocytes producing IL-1beta after MDP stimulation (P < 0.05). Elevated cytokine production was confined to axSpA patients under conventional therapy (non-steroidal anti-inflammatory drugs) and not found in patients under TNFα inhibitor treatment. The LPS-induced production of IL-6 and IL-10 was lower in axSpA patients compared with controls (P < 0.05). Monocytic TLR expression was unaffected in patients with axSpA. CONCLUSION: Enhanced spontaneous and MDP-induced cytokine secretion by monocytes suggests in vivo pre-activation of monocytes in axSpA patients under conventional therapy which is reverted under TNF inhibitor treatment.

Notochord vacuoles are lysosome-related organelles that function in axis and spine morphogenesis.

The notochord plays critical structural and signaling roles during vertebrate development. At the center of the vertebrate notochord is a large fluid-filled organelle, the notochord vacuole. Although these highly conserved intracellular structures have been described for decades, little is known about the molecular mechanisms involved in their biogenesis and maintenance. Here we show that zebrafish notochord vacuoles are specialized lysosome-related organelles whose formation and maintenance requires late endosomal trafficking regulated by the vacuole-specific Rab32a and H(+)-ATPase-dependent acidification. We establish that notochord vacuoles are required for body axis elongation during embryonic development and identify a novel role in spine morphogenesis. Thus, the vertebrate notochord plays important structural roles beyond early development.

Aberrant axial mineralization precedes spinal ankylosis: a molecular imaging study in ank/ank mice.

INTRODUCTION: The diagnosis of ankylosing spondylitis is made from a combination of clinical features and the presence of radiographic evidence that may be detected only after many years of inflammatory back pain. It is not uncommon to have a diagnosis confirmed 5 to 10 years after the initial onset of symptoms. Development of a more-sensitive molecular imaging technology to detect structural changes in the joints would lead to earlier diagnosis and quantitative tracking of ankylosis progression. Progressive ankylosis (ank/ank) mice have a loss of function in the Ank gene, which codes for a regulator of PPi transport. In this study, we used these ank/ank mutant mice to assess a noninvasive, quantitative measure of joint ankylosis with near-infrared (NIR) molecular imaging in vivo. METHODS: Three age groups (8, 12, and 18 weeks) of ank/ank (15 mice) and wild-type littermates (12 +/+ mice) were assessed histologically and radiographically. Before imaging, OsteoSense 750 (bisphosphonate pamidronate) was injected i.v. Whole-body images were analyzed by using the multispectral Maestro imaging system. RESULTS: OsteoSense 750 signals in the paw joints were higher in ank/ank mice in all three age groups compared with controls. In the spine, significantly higher OsteoSense 750 signals were detected early, in 8-week-old ank/ank mice compared with controls, although minimal radiographic differences were noted at this time point. The molecular imaging changes in the ank/ank spine (8 weeks) were supported by histologic changes, including calcium apatite crystals at the edge of the vertebral bodies and new syndesmophyte formation. CONCLUSIONS: Changes in joint pathology of ank/ank mice, as evaluated by histologic and radiographic means, are qualitative, but only semiquantitative. In contrast, molecular imaging provides a quantitative assessment. Ankylosis in ank/ank mice developed simultaneously in distal and axial joints, contrary to the previous notion that it is a centripetal process. NIR imaging might be feasible for early disease diagnosis and for monitoring disease progression in ankylosing spondylitis.

Yes-associated protein 65 (YAP) expands neural progenitors and regulates Pax3 expression in the neural plate border zone.

Yes-associated protein 65 (YAP) contains multiple protein-protein interaction domains and functions as both a transcriptional co-activator and as a scaffolding protein. Mouse embryos lacking YAP did not survive past embryonic day 8.5 and showed signs of defective yolk sac vasculogenesis, chorioallantoic fusion, and anterior-posterior (A-P) axis elongation. Given that the YAP knockout mouse defects might be due in part to nutritional deficiencies, we sought to better characterize a role for YAP during early development using embryos that develop externally. YAP morpholino (MO)-mediated loss-of-function in both frog and fish resulted in incomplete epiboly at gastrulation and impaired axis formation, similar to the mouse phenotype. In frog, germ layer specific genes were expressed, but they were temporally delayed. YAP MO-mediated partial knockdown in frog allowed a shortened axis to form. YAP gain-of-function in Xenopus expanded the progenitor populations in the neural plate (sox2(+)) and neural plate border zone (pax3(+)), while inhibiting the expression of later markers of tissues derived from the neural plate border zone (neural crest, pre-placodal ectoderm, hatching gland), as well as epidermis and somitic muscle. YAP directly regulates pax3 expression via association with TEAD1 (N-TEF) at a highly conserved, previously undescribed, TEAD-binding site within the 5' regulatory region of pax3. Structure/function analyses revealed that the PDZ-binding motif of YAP contributes to the inhibition of epidermal and somitic muscle differentiation, but a complete, intact YAP protein is required for expansion of the neural plate and neural plate border zone progenitor pools. These results provide a thorough analysis of YAP mediated gene expression changes in loss- and gain-of-function experiments. Furthermore, this is the first report to use YAP structure-function analyzes to determine which portion of YAP is involved in specific gene expression changes and the first to show direct in vivo evidence of YAP's role in regulating pax3 neural crest expression.

Jun NH2-terminal kinase (JNK) prevents nuclear beta-catenin accumulation and regulates axis formation in Xenopus embryos.

Jun NH(2)-terminal kinases (JNKs) regulate convergent extension movements in Xenopus embryos through the noncanonical Wnt/planar cell polarity pathway. In addition, there is a high level of maternal JNK activity spanning from oocyte maturation until the onset of gastrulation that has no defined functions. Here, we show that maternal JNK activation requires Dishevelled and JNK is enriched in the nucleus of Xenopus embryos. Although JNK activity is not required for the glycogen synthase kinase-3-mediated degradation of beta-catenin, inhibition of the maternal JNK signaling by morpholino-antisense oligos causes hyperdorsalization of Xenopus embryos and ectopic expression of the Wnt/beta-catenin target genes. These effects are associated with an increased level of nuclear and nonmembrane-bound beta-catenin. Moreover, ventral injection of the constitutive-active Jnk mRNA blocks beta-catenin-induced axis duplication, and dorsal injection of active Jnk mRNA into Xenopus embryos decreases the dorsal marker gene expression. In mammalian cells, activation of JNK signaling reduces Wnt3A-induced and beta-catenin-mediated gene expression. Furthermore, activation of JNK signaling rapidly induces the nuclear export of beta-catenin. Taken together, these results suggest that JNK antagonizes the canonical Wnt pathway by regulating the nucleocytoplasmic transport of beta-catenin rather than its cytoplasmic stability. Thus, the high level of sustained maternal JNK activity in early Xenopus embryos may provide a timing mechanism for controlling the dorsal axis formation.

Hox genes, homology and axis formation--the application of morphological concepts to evolutionary developmental biology.

This article focuses on the interphyletic comparison of gene expression patterns. By means of the hypothesis of the inversion of the dorsoventral axis during the evolution of the Bilateria, it is demonstrated, that evolutionary developmental biologists use similarities in spatial and temporal gene expression patterns as evidence for the formulation of hypotheses of homology concerning either developing structures or body regions. The molecular genetic and morphogenetic evidence used is discussed within the framework of a cladistic-phylogenetic analysis based on the phylogenetic tree of the Bilateria. I argue that similarity of spatial and temporal gene expression patterns is not a sufficient criterion for homology inference. Therefore, gene expression patterns should be coded as characters. Their homology should be tested in concert with other characters. Furthermore, it is demonstrated, that spatial and temporal similar gene expression patterns, indicating similar molecular genetic mechanisms, were interpreted as an analytical criterion of homology, offering the possibility to identify similar structures. In contrast to this, the evolutionary developmental biologists have not developed a causal-analytically extended concept of shape, from which a causal-analytical concept of homology could be deduced. Instead, the homology concept from evolutionary morphology is used.

Cloning of vertebrate Protogenin (Prtg) and comparative expression analysis during axis elongation.

A murine cDNA encoding Protogenin, which belongs to the DCC/Neogenin family, was cloned in a screen performed to identify novel cDNAs regionally expressed in the neural plate. Isolation of the putative zebrafish orthologues allowed a comparative analysis of the expression patterns of Protogenin genes during embryogenesis in different vertebrate species. From mid-gastrulation to early somite stages, Protogenin expression is restricted to posterior neural plate and mesoderm, with an anterior limit at the level of the rhombencephalon in mouse, chicken, and zebrafish. During somitogenesis, the expression profiles in the three species share features in the neural tube but present also species-specific characteristics. The initiation of Protogenin expression just before somitogenesis and its maintenance in the neural tube and paraxial mesoderm during this process suggest a conserved role in axis elongation.

Analysis of axis induction mutant embryos reveals morphogenetic events associated with zebrafish yolk extension formation.

We analyze patterning and morphogenetic events during somitogenesis in hecate mutant embryos, which exhibit early axis induction defects. The posterior region, in the absence of a dorsal axis, is capable of forming organized gene expression patterns. The aberrant morphogenesis of mutant embryos is associated with anteriorly directed cell movements, underlying the enveloping layer, from the posterior region. In both wild-type and mutant embryos, these changes result in an accumulation of cells, whose location correlates with a constriction in the posterior yolk cell, which in the wild-type corresponds to the yolk extension. The region encompassing the constriction corresponds to a region of expression of zangptl2 in the yolk syncytial layer, which expands anteriorly together with the anteriorly migrating tail bud-derived cell population. Our data indicate that yolk extension formation is associated with coordinated changes involving the anterior migration of cells from the posterior region, changes in surface cellular layers, and inductive gene expression events in the YSL.

Genomic analysis of Xenopus organizer function.

BACKGROUND: Studies of the Xenopus organizer have laid the foundation for our understanding of the conserved signaling pathways that pattern vertebrate embryos during gastrulation. The two primary activities of the organizer, BMP and Wnt inhibition, can regulate a spectrum of genes that pattern essentially all aspects of the embryo during gastrulation. As our knowledge of organizer signaling grows, it is imperative that we begin knitting together our gene-level knowledge into genome-level signaling models. The goal of this paper was to identify complete lists of genes regulated by different aspects of organizer signaling, thereby providing a deeper understanding of the genomic mechanisms that underlie these complex and fundamental signaling events. RESULTS: To this end, we ectopically overexpress Noggin and Dkk-1, inhibitors of the BMP and Wnt pathways, respectively, within ventral tissues. After isolating embryonic ventral halves at early and late gastrulation, we analyze the transcriptional response to these molecules within the generated ectopic organizers using oligonucleotide microarrays. An efficient statistical analysis scheme, combined with a new Gene Ontology biological process annotation of the Xenopus genome, allows reliable and faithful clustering of molecules based upon their roles during gastrulation. From this data, we identify new organizer-related expression patterns for 19 genes. Moreover, our data sub-divides organizer genes into separate head and trunk organizing groups, which each show distinct responses to Noggin and Dkk-1 activity during gastrulation. CONCLUSION: Our data provides a genomic view of the cohorts of genes that respond to Noggin and Dkk-1 activity, allowing us to separate the role of each in organizer function. These patterns demonstrate a model where BMP inhibition plays a largely inductive role during early developmental stages, thereby initiating the suites of genes needed to pattern dorsal tissues. Meanwhile, Wnt inhibition acts later during gastrulation, and is essential for maintenance of organizer gene expression throughout gastrulation, a role which may depend on its ability to block the expression of a host of ventral, posterior, and lateral fate-specifying factors.

The left-right axis in the mouse: from origin to morphology.

The past decade or so has seen rapid progress in our understanding of how left-right (LR) asymmetry is generated in vertebrate embryos. However, many important questions about this process remain unanswered. Although a leftward flow of extra-embryonic fluid in the node cavity (nodal flow) is likely to be the symmetry-breaking event, at least in the mouse embryo, it is not yet known how this flow functions or how the asymmetric signal generated in the node is transferred to the lateral plate. The final step in left-right patterning - translation of the asymmetric signal into morphology - is also little understood.

Ets2 is necessary in trophoblast for normal embryonic anteroposterior axis development.

Although the trophoblast is necessary for the growth, viability and patterning of the mammalian embryo, understanding of its patterning role is still rudimentary. Expression of the transcription factor Ets2 is restricted to the trophoblast in early postimplantation stages and Ets2 mutants have been previously shown to have defects in trophoblast development. We show here that Ets2 is necessary in the trophoblast for fundamental aspects of anteroposterior (AP) epiblast axis initiation, including mesoderm initiation at the primitive streak, establishment of posterior character in the epiblast and appropriate spatial restriction of the anterior visceral endoderm (AVE). Most homozygous Ets2 mutants also show highly reduced development of the trophoblast with an absence of extraembryonic ectoderm (EXE) markers. Embryos in which the EXE has been physically removed before culture in vitro phenocopy the patterning defects of Ets2 mutants. These defects cannot be rescued by providing Ets2 mutants with wild-type epiblast in tetraploid aggregations. Thus, EXE-derived signals are necessary for normal embryonic patterning. Ets2 is likely to be required in the EXE downstream of epiblast signals, such as Fgf, and, in turn, helps to regulate signals from the EXE that signal back to the epiblast to promote proper primitive streak and AVE development. This study provides new insights about the genetic and cellular basis of the patterning role and development of the early trophoblast.

Differential expression of RARbeta isoforms in the mouse striatum during development: a gradient of RARbeta2 expression along the rostrocaudal axis.

The retinoic acid receptor RARbeta is highly expressed in the striatum of the ventral telencephalon. We studied the expression pattern of different RARbeta isoforms in the developing mouse striatum by in situ hybridization. We found a differential ontogeny of RARbeta2 and RARbeta1/3 in embryonic day (E) 13.5 lateral ganglionic eminence (striatal primordium). RARbeta2 mRNA was detected primarily in the rostral and ventromedial domains, whereas RARbeta1/3 mRNAs were enriched in the caudal and dorsolateral domains. Notably, by E16.5, a prominent decreasing gradient of RARbeta2 mRNA was present in the developing striatum along the rostrocaudal axis, i.e., RARbeta2 was expressed at higher levels in the rostral than the caudal striatum. No such gradient was found for RARbeta1/3 and RARbeta3 mRNAs. The rostrocaudal RARbeta2 gradient gradually disappeared postnatally and was absent in the adult striatum. The differential expression pattern of RARbeta isoforms in the developing striatum may provide an anatomical basis for differential gene regulation by RARbeta signaling.

Ancestral role of caudal genes in axis elongation and segmentation.

caudal (cad/Cdx) genes are essential for the formation of posterior structures in Drosophila, Caenorhabditis elegans, and vertebrates. In contrast to Drosophila, the majority of arthropods generate their segments sequentially from a posteriorly located growth zone, a process known as short-germ development. caudal homologues are expressed in the growth zone of diverse short-germ arthropods, but until now their functional role in these animals had not been studied. Here, we use RNA interference to examine the function of caudal genes in two short-germ arthropods, the crustacean Artemia franciscana and the beetle Tribolium castaneum. We show that, in both species, caudal is required for the formation of most body segments. In animals with reduced levels of caudal expression, axis elongation stops, resulting in severe truncations that remove most trunk segments. We also show that caudal function is required for the early phases of segmentation and Hox gene expression. The observed phenotypes suggest that in arthropods caudal had an ancestral role in axis elongation and segmentation, and was required for the formation of most body segments. Similarities to the function of vertebrate Cdx genes in the presomitic mesoderm, from which somites are generated, indicate that this role may also predate the origin of the Bilateria.

Multiple points of interaction between retinoic acid and FGF signaling during embryonic axis formation.

Anteroposterior (AP) patterning of the developing CNS is crucial for both regional specification and the timing of neurogenesis. Several important factors are involved in AP patterning, including members of the WNT and FGF growth factor families, retinoic acid receptors, and HOX genes. We have examined the interactions between FGF and retinoic signaling pathways. Blockade of FGF signaling downregulates the expression of members of the RAR signaling pathway, RARalpha, RALDH2 and CYP26. Overexpression of a constitutively active RARalpha2 rescues the effects of FGF blockade on the expression of XCAD3 and HOXB9. This suggests that RARalpha2 is required as a downstream target of FGF signaling for the posterior expression of XCAD3 and HOXB9. Surprisingly, we found that posterior expression of FGFR1 and FGFR4 was dependent on the expression of RARalpha2. Anterior expression was also altered with FGFR1 expression being lost, whereas FGFR4 expression was expanded beyond its normal expression domain. RARalpha2 is required for the expression of XCAD3 and HOXB9, and for the ability of XCAD3 to induce HOXB9 expression. We conclude that RARalpha2 is required at multiple points in the posteriorization pathway, suggesting that correct AP neural patterning depends on a series of mutually interactive feedback loops among FGFs, RARs and HOX genes.

Early gene expression along the animal-vegetal axis in sea urchin embryoids and grafted embryos.

The HE gene is the earliest strictly zygotic gene activated during sea urchin embryogenesis. It is transiently expressed in a radially symmetrical domain covering the animal-most two-thirds of the blastula. The border of this domain, which is orthogonal to the primordial animal-vegetal axis, is shifted towards the animal pole in Li+-treated embryos. Exogenous micromeres implanted at the animal pole of whole embryos, animal or vegetal halves do not modify the extent and localization of the HE expression domain. In grafted embryos or animal halves, the Li+ effect is not affected by the presence of ectopic micromeres at the animal pole. A Li+-induced shift of the border, similar to that seen in whole embryos, occurs in embryoids developing from animal halves isolated from 8-cell stage embryos or dissected from unfertilised eggs. Therefore, the spatial restriction of the HE gene is not controlled by the inductive cascade emanating from the micromeres and the patterning along the AV-axis revealed by Li+ does not require interactions between cells from the animal and vegetal halves. This suggests that maternal primary patterning in the sea urchin embryo is not limited to a small vegetal center but extends along the entire AV axis.

Age-reletad changes in the diabetic alterations of the hypohysis-thyroid-testicular axis in male rat

The effects of streptozotocin diabetes on the male reproductive tract were studied in young rats in different ages (25 to 60 days old). The data showed that diabetes caused atrophy of pituitary, testes and sexual accessory glands as well as testicular and pituitary functions, as evaluated by the circulating levels of LH and testosterone. In addition, an impairment of thyroidal function was detected since thyroxine serum levels were decreased. It is also seen that prolactin levels were reduced as a consequence of streptozotocin treated rats. Since younger animals were more drasticaly affected, we conclude that hypothyroidism that follows after the onset of diabetes could contribute to the picture on the basis of the importance of thyroidal function in the developing animal. Also it seems that reduction of prolactin levels due to hypothalamic TRH impairment would bring about a lack of the so important sinergistic action of this hormone and androgens in maintenance of androgen targetorgans.