ABSTRACT
Purpose: Sjögren's syndrome (SS) is an autoimmune disease associated with ocular surface inflammation. The goals of this study were to establish a novel bead-based protein microarray for simultaneous analysis of 11 cytokines from tear fluid collected with Schirmer strips from patients with SS.Methods: Three to ten microliter of tear fluid was collected with Schirmer strips from both eyes of 13 healthy controls and 12 SS patients. Tear fluid was eluted from the Schirmer strips, total protein and concentrations of 11 different cytokines were analyzed.Results: The multiplex assay demonstrated high assay sensitivity with LoDs between 1.4 and 55.3 pg/µl with mean CVs between 3.7% and 9.7%. Statistically significant upregulation (p < .005) of eight cytokines was observed in SS patients compared to controls. Additionally, four patient cytokine values showed a significant inverse correlation (r<-0.7) with Schirmer strip readings.Conclusion: The assay offers analytical reliability for quantification of biomarkers in small amounts of tear fluid with potential utility for treatment monitoring of SS and other types of Dry Eye Disease.
Subject(s)
Biomarkers/metabolism , Cytokines/metabolism , Eye Proteins/metabolism , Sjogren's Syndrome/metabolism , Tears/metabolism , Adult , Female , Humans , Immunoassay , Male , Middle Aged , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
During vertebrate neurulation, cranial neural crest cells (CNCCs) undergo epithelial to mesenchymal transition (EMT), delaminate from the neural plate border, and migrate as separate streams into different cranial regions. There, they differentiate into distinct parts of the craniofacial skeleton. Canonical Wnt signaling has been shown to be essential for this process at different levels but the involved receptors remained unclear. Here we show that the frizzled co-receptor low-density-lipoprotein (LDL) receptor-related protein 5 (Lrp5) plays a crucial role in CNCC migration and morphogenesis of the cranial skeleton. Early during induction and migration of CNCCs, lrp5 is expressed ubiquitously but later gets restricted to CNCC derivatives in the ventral head region besides different regions in the CNS. A knock-down of lrp5 does not interfere with induction of CNCCs but leads to reduced proliferation of premigratory CNCCs. In addition, cell migration is disrupted as CNCCs are found in clusters at ectopic positions in the dorsomedial neuroepithelium after lrp5 knock-down and transient CRISPR/Cas9 gene editing. These migratory defects consequently result in malformations of the craniofacial skeleton. To date, Lrp5 has mainly been associated with bone homeostasis in mammals. Here we show that in zebrafish, lrp5 also controls cell migration during early morphogenetic processes and contributes to shaping the craniofacial skeleton.
Subject(s)
Cell Movement/physiology , Low Density Lipoprotein Receptor-Related Protein-5/metabolism , Neural Crest/cytology , Neural Crest/metabolism , Skull/cytology , Zebrafish Proteins/metabolism , Animals , Cell Movement/genetics , Gene Expression Regulation, Developmental/genetics , Gene Expression Regulation, Developmental/physiology , Low Density Lipoprotein Receptor-Related Protein-5/genetics , Signal Transduction/genetics , Signal Transduction/physiology , Zebrafish , Zebrafish Proteins/geneticsABSTRACT
Somitogenesis is controlled by a genetic network consisting of an oscillator (clock) and a gradient (wavefront). The "hairy and Enhancer of Split"- related (her) genes act downstream of the Delta/Notch (D/N) signaling pathway, and are crucial components of the segmentation clock. Due to genome duplication events, the zebrafish genome, possesses two gene copies of the mouse Hes7 homologue: her1 and her7. To better understand the functional consequences of this gene duplication, and to determine possible independent roles for these two genes during segmentation, two zebrafish mutants her1(hu2124) and her7(hu2526) were analyzed. In the course of embryonic development, her1(hu2124) mutants exhibit disruption of the three anterior-most somite borders, whereas her7(hu2526) mutants display somite border defects restricted to somites 8 (+/-3) to 17 (+/-3) along the anterior-posterior axis. Analysis of the molecular defects in her1(hu2124) mutants reveals a her1 auto regulatory feedback loop during early somitogenesis that is crucial for correct patterning and independent of her7 oscillation. This feedback loop appears to be restricted to early segmentation, as cyclic her1 expression is restored in her1(hu2124) embryos at later stages of development. Moreover, only the anterior deltaC expression pattern is disrupted in the presomitic mesoderm of her1(hu2124) mutants, while the posterior expression pattern of deltaC remains unaltered. Together, this data indicates the existence of an independent and genetically separable anterior and posterior deltaC clock modules in the presomitic mesdorm (PSM).
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Biological Clocks/genetics , Mutant Proteins/metabolism , Somites/embryology , Somites/metabolism , Transcription Factors/metabolism , Zebrafish Proteins/metabolism , Alleles , Animals , Base Sequence , Basic Helix-Loop-Helix Transcription Factors/genetics , Body Patterning/genetics , Embryonic Development/genetics , Exons , Gene Expression Regulation, Developmental , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mutant Proteins/genetics , Phenotype , Transcription Factors/genetics , Zebrafish/embryology , Zebrafish/genetics , Zebrafish/metabolism , Zebrafish Proteins/geneticsABSTRACT
Different from tetrapods, teleost vertebral centra form without prior establishment of a cartilaginous scaffold, in two steps: First, mineralization of the notochord sheath establishes the vertebral centra. Second, sclerotome derived mesenchymal cells migrate around the notochord sheath. These cells differentiate into osteoblasts and deposit bone onto the mineralized notochord sheath in a process of intramembranous bone formation. In contrast, most skeletal elements of the cranial skeleton arise by chondral bone formation, with remarkably similar mechanisms in fish and tetrapods. To further investigate the role of osteoblasts during formation of the cranial and axial skeleton, we generated a transgenic osx:CFP-NTR medaka line which enables conditional ablation of osterix expressing osteoblasts. By expressing a bacterial nitroreductase (NTR) fused to Cyan Fluorescent Protein (CFP) under control of the osterix promoter these cells become sensitive towards Metronidazole (Mtz). Mtz treatment of stable osx:CFP-NTR transgenic medaka for several consecutive days led to significant loss of osteoblasts by apoptosis. Live staining of mineralized bone matrix revealed reduced ossification in head skeletal elements such as cleithrum and operculum, as well as in the vertebral arches. Interestingly in Mtz treated larvae, intervertebral spaces were missing and the notochord sheath was often continuously mineralized resulting in the fusion of centra. We therefore propose a dual role for osx-positive osteoblasts in fish. Besides a role in bone deposition, we suggest an additional border function during mineralization of the chordal centra. After termination of Mtz treatment, osteoblasts gradually reappeared, indicating regenerative properties in this cell lineage. Taken together, the osx:CFP-NTR medaka line represents a valuable tool to study osteoblast function and regeneration at different stages of development in whole vertebrate specimens in vivo.
