Extracellular Matrix in Development and Disease

The evolution of multicellular metazoan organisms was marked by the inclusion of an extracellular matrix (ECM), a multicomponent, proteinaceous network between cells that contributes to the spatial arrangement of cells and the resulting tissue organization. The development of an ECM that provides support in larger organisms may have represented an advantage in the face of selection pressure for the evolution of the ECM

Authentication of a Novel Antibody to Zebrafish Collagen Type XI Alpha 1 Chain (Col11a1a)

Objective: Extracellular matrix proteins play important roles in embryonic development and antibodies that specifically detect these proteins are essential to understanding their function. The zebrafish embryo is a popular model for vertebrate development but suffers from a dearth of authenticated antibody reagents for research. Here, we describe a novel antibody designed to detect the minor fibrillar collagen chain Col11a1a in zebrafish (AB strain). Results: The Col11a1a antibody was raised in rabbit against a peptide comprising a unique sequence within the zebrafish Col11a1a gene product. The antibody was affinity-purified and characterized by ELISA. The antibody is effective for immunoblot and immunohistochemistry applications. Protein bands identified by immunoblot were confirmed by mass spectrometry and sensitivity to collagenase. Col11a1a knockout zebrafish were used to confirm specificity of the antibody. The Col11a1a antibody labeled cartilaginous structures within the developing jaw, consistent with previously characterized Col11a1 antibodies in other species. Col11a1a within formalin-fixed paraffin-embedded zebrafish were recognized by the antibody. The antibodies and the approaches described here will help to address the lack of well-defined antibody reagents in zebrafish research

Emerging Gene-Editing Modalities for Osteoarthritis

Osteoarthritis (OA) is a pathological degenerative condition of the joints that is widely prevalent worldwide, resulting in significant pain, disability, and impaired quality of life. The diverse etiology and pathogenesis of OA can explain the paucity of viable preventive and disease-modifying strategies to counter it. Advances in genome-editing techniques may improve disease-modifying solutions by addressing inherited predisposing risk factors and the activity of inflammatory modulators. Recent progress on technologies such as CRISPR/Cas9 and cell-based genome-editing therapies targeting the genetic and epigenetic alternations in OA offer promising avenues for early diagnosis and the development of personalized therapies. The purpose of this literature review was to concisely summarize the genome-editing options against chronic degenerative joint conditions such as OA with a focus on the more recently emerging modalities, especially CRISPR/Cas9. Future advancements in novel genome-editing therapies may improve the efficacy of such targeted treatments

Decellularized Porcine Cartilage Scaffold; Validation of Decellularization and Evaluation of Biomarkers of Chondrogenesis

Osteoarthritis is a major concern in the United States and worldwide. Current non-surgical and surgical approaches alleviate pain but show little evidence of cartilage restoration. Cell-based treatments may hold promise for the regeneration of hyaline cartilage-like tissue at the site of injury or wear. Cell–cell and cell–matrix interactions have been shown to drive cell differentiation pathways. Biomaterials for clinically relevant applications can be generated from decellularized porcine auricular cartilage. This material may represent a suitable scaffold on which to seed and grow chondrocytes to create new cartilage. In this study, we used decellularization techniques to create an extracellular matrix scaffold that supports chondrocyte cell attachment and growth in tissue culture conditions. Results presented here evaluate the decellularization process histologically and molecularly. We identified new and novel biomarker profiles that may aid future cartilage decellularization efforts. Additionally, the resulting scaffold was characterized using scanning electron microscopy, fluorescence microscopy, and proteomics. Cellular response to the decellularized scaffold was evaluated by quantitative real-time PCR for gene expression analysis

An Amino-Terminal Fragment of Apolipoprotein E4 Leads to Behavioral Deficits, Increased PHF-1 Immunoreactivity, and Mortality in Zebrafish

Although the increased risk of developing sporadic Alzheimer’s disease (AD) associated with the inheritance of the apolipoprotein E4 (APOE4) allele is well characterized, the molecular underpinnings of how ApoE4 imparts risk remains unknown. Enhanced proteolysis of the ApoE4 protein with a toxic-gain of function has been suggested and a 17 kDa amino-terminal ApoE4 fragment (nApoE41-151) has been identified in post-mortem human AD frontal cortex sections. Recently, we demonstrated in vitro, exogenous treatment of nApoE41-151 in BV2 microglial cells leads to uptake, trafficking to the nucleus and increased expression of genes associated with cell toxicity and inflammation. In the present study, we extend these findings to zebrafish (Danio rerio), an in vivo model system to assess the toxicity of nApoE41-151. Exogenous treatment of nApoE41-151 to 24-hour post-fertilization for 24 hours resulted in significant mortality. In addition, developmental abnormalities were observed following treatment with nApoE41-151 including improper folding of the hindbrain, delay in ear development, deformed yolk sac, enlarged cardiac cavity, and significantly lower heart rates. A similar nApoE31-151 fragment that differs by a single amino acid change (C\u3eR) at position 112 had no effects on these parameters under identical treatment conditions. Decreased presence of pigmentation was noted for both nApoE31-151- and nApoE41-151-treated larvae compared with controls. Behaviorally, touch-evoked responses to stimulus were negatively impacted by treatment with nApoE41-151 but did not reach statistical significance. Additionally, triple-labeling confocal microscopy not only confirmed the nuclear localization of the nApoE41-151 fragment within neuronal populations following exogenous treatment, but also identified the presence of tau pathology, one of the hallmark features of AD. Collectively, these in vivo data demonstrating toxicity as well as sublethal effects on organ and tissue development support a novel pathophysiological function of this AD associated-risk factor

Semi-quantitative scale developed to assess morphological changes following treatment of zebrafish embryos with an amino-terminal fragment of nApoE4_1-151.

Rubric for developmental abnormalities was accomplished in 48 hpf zebrafish following 24-hour treatment with 25 μg/ml of nApoE41-151. This scale was established to quantify the effects of nApoE41-151 as compared to untreated, control embryos. Identified hallmark defects that appeared consistently following treatment with nApoE41-151 included inflation of pericardial cavity, enlarged hearts, pigmentation alterations, and delays or lack of development in ear and brain structures. Data are representative of 10 embryos treated with 25 μg/ml nApoE41-151 per trial for a total of 30 embryos.</p

Description of antibodies used for immunofluorescence experiments.

Description of antibodies used for immunofluorescence experiments.</p

The presence of nApoE4_1-151 within tail regions of zebrafish embryos.

A-C: Representative images from confocal immunofluorescence in 5 mm paraffin-embedded sections of non-treated control 48 hpf zebrafish embryos that were stained with DAPI (A), anti-His antibody (1:500) (B), and the merged image together with PHF-1 (1:250) in Panel (C). There was no detection of any nApoE41-151 fragments in untreated control sections as indicated by the lack of labeling in Panel B. D-F: Identical to Panels A-D with the exception that embryos were exogenously treated for 24 hours with 25 μg/ml of nApoE41-151. In this case punctate staining of the fragment was observed that appeared cytoplasmic. PHF-1 labeling was identified in muscle cells that exhibited abnormal morphology (arrows, Panel F). All scale bars represent 20 μm.</p

A sublethal concentration of nApoE4_1-151 leads to morphological abnormalities in zebrafish embryos at the hatching phase.

Representative light phase contrast microscopic images following live imaging of embryos at 48 hpf following a 24-hour period with respective treatments (Control, 25 μg/ml of nApoE31-151, or 25 μg/ml of nApoE41-151. A: Arrows point to consistent morphological changes as compared to untreated controls that are healthy and categorized as having a developmental abnormality score of B: Exogenous treatment with nApoE31-151 impacted pigmentation pattern in otherwise healthy embryos. Embryos in this category were most likely to receive a score of C: 24-hour incubation of a sublethal concentration of nApoE41-151 resulted in developmental abnormality scores >3. Embryos in this category were typically observed to be delayed in development with limited hindbrain folding (orange arrow), limited or lacking pigmentation (blue arrow), as well as enlargement of the cardiac cavity (red arrow). D. Quantitative developmental abnormality scores for each treatment group following treatment of embryos for 24 hours with respective fragments at low E3 (orange bar), E4 (pink bar) concentrations (25 μg/ml) or at high concentrations (50 μg/ml) E3 (gray bar), E4 (yellow bar). The nApoE41-151 25 μg/ml-treated groups were significantly different from controls (H(4) = -2.43, p = 0.0074). At 50 μg/ml both nApoE41-151 (H(4) = -3.32, p = 0.0004) and nApoE31-151-treatment groups (H(4) = -1.77, p = 0.037) were significantly different from controls. Errors bars represent ± S.E.M. *pE. Heart rate data obtained from live microscope analyses in 25 μg/ml and 50 μg/ml treatment groups nApoE31-151 and nApoE41-151 compared to non-treated controls. nApoE41-151 (pink bar, 25 μg/ml) was significantly different from controls (H(4) = 1.77, p = 0.038). nApoE41-151 (yellow bar, 50 μg/ml) was significantly different from nApoE31-151 25 μg/ml (H(4) = 1.94, p = 0.026) and controls (H(4) = 2.665, p = 0.0036). Errors bars represent ±S.E.M. All other comparisons were insignificant. *p<0.05, **p<0.01, ***p<0.001.</p

Negative trends in motor behavior in zebrafish following treatment with nApoE4_1-151.

A. Groups for non-treated controls (green bar), nApoE31-151 25 μg/ml (orange bar), and ApoE41-151 25 μg/ml (blue bar)) were assessed via video monitoring to determine number of spontaneous tail flicks per minute that were then averaged per group for each trial. Data are representative of N = 5 trials, for a total of 25 embryos per group, ±SEM. Data depicted show limited spontaneous tail flick activation from every group with no difference detectable between groups (F(2,27) = 1.24, p = 0.305). B. Results from the touch-evoked response motor behavior experiment. Non-treated controls had a 90% response rate to the evoked, tactile stimulus, whereas for nApoE41-151-treated groups responded to fewer than 50% of stimuli. No significant difference was observed (F(12) = 1.482, p = 0.266). C and D. Results from TEMR analyses similar to Panels A and B with the exception that in this case, total distance traveled (C) or the total time swimming (D) were recorded via video monitoring and using Noldus tracking software. For Panel C, representative heat maps of individual larvae representing either wild-type controls (left Panel), or a low-performing nApoE41-151-treated zebrafish (right Panel). Each bar represents the average total distance traveled or averaged cumulative duration during 5 independent trials, for a total of 15 embryos per group, (±SEM). No significant differences were observed, with for example the Ctl group vs. nApoE41-151 having a p value = 0.08 in Panel C. P-values for Panel D were Ctl vs. E3 fragment = 0.86 and Ctl vs. E4 fragment = 0.06.</p