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Live imaging of collagen deposition during skin development and repair in a collagen I - GFP fusion transgenic zebrafish line.

ABSTRACT: Fibrillar collagen is a major component of many tissues but has been difficult to image in vivo using transgenic approaches because of problems associated with establishing cells and organisms that generate GFP-fusion collagens that can polymerise into functional fibrils. Here we have developed and characterised GFP and mCherry collagen-I fusion zebrafish lines with basal epidermal-specific expression. We use these lines to reveal the dynamic nature of collagen-I fibril deposition beneath the developing embryonic epidermis, as well as the repair of this collagen meshwork following wounding. Transmission electron microscope studies show that these transgenic lines faithfully reproduce the collagen ultrastructure present in wild type larval skin. During skin development we show that collagen I is deposited by basal epidermal cells initially in fine filaments that are largely randomly orientated but are subsequently aligned into a cross-hatch, orthogonal sub-epithelial network by embryonic day 4. Following skin wounding, we see that sub-epidermal collagen is re-established in the denuded domain, initially as randomly orientated wisps that subsequently become bonded to the undamaged collagen and aligned in a way that recapitulates developmental deposition of sub-epidermal collagen. Crossing our GFP-collagen line against one with tdTomato marking basal epidermal cell membranes reveals how much more rapidly wound re-epithelialisation occurs compared to the re-deposition of collagen beneath the healed epidermis. By use of other tissue specific drivers it will be possible to establish zebrafish lines to enable live imaging of collagen deposition and its remodelling in various other organs in health and disease.

SUBMITTER: Morris JL

PROVIDER: S-EPMC6080847 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Live imaging of collagen deposition during skin development and repair in a collagen I - GFP fusion transgenic zebrafish line.

Morris Josephine L JL Cross Stephen J SJ Lu Yinhui Y Kadler Karl E KE Lu Yongbo Y Dallas Sarah L SL Martin Paul P

Developmental biology 20180606 1

Fibrillar collagen is a major component of many tissues but has been difficult to image in vivo using transgenic approaches because of problems associated with establishing cells and organisms that generate GFP-fusion collagens that can polymerise into functional fibrils. Here we have developed and characterised GFP and mCherry collagen-I fusion zebrafish lines with basal epidermal-specific expression. We use these lines to reveal the dynamic nature of collagen-I fibril deposition beneath the de ...[more]

PMID: 29883658

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Project description:Type I collagen is the most abundant extracellular matrix protein in bone and other connective tissues and plays key roles in normal and pathological bone formation as well as in connective tissue disorders and fibrosis. Although much is known about the collagen biosynthetic pathway and its regulatory steps, the mechanisms by which it is assembled extracellularly are less clear. We have generated GFPtpz and mCherry-tagged collagen fusion constructs for live imaging of type I collagen assembly by replacing the α2(I)-procollagen N-terminal propeptide with GFPtpz or mCherry. These novel imaging probes were stably transfected into MLO-A5 osteoblast-like cells and fibronectin-null mouse embryonic fibroblasts (FN-null-MEFs) and used for imaging type I collagen assembly dynamics and its dependence on fibronectin. Both fusion proteins co-precipitated with α1(I)-collagen and remained intracellular without ascorbate but were assembled into α1(I) collagen-containing extracellular fibrils in the presence of ascorbate. Immunogold-EM confirmed their ultrastuctural localization in banded collagen fibrils. Live cell imaging in stably transfected MLO-A5 cells revealed the highly dynamic nature of collagen assembly and showed that during assembly the fibril networks are continually stretched and contracted due to the underlying cell motion. We also observed that cell-generated forces can physically reshape the collagen fibrils. Using co-cultures of mCherry- and GFPtpz-collagen expressing cells, we show that multiple cells contribute collagen to form collagen fiber bundles. Immuno-EM further showed that individual collagen fibrils can receive contributions of collagen from more than one cell. Live cell imaging in FN-null-MEFs expressing GFPtpz-collagen showed that collagen assembly was both dependent upon and dynamically integrated with fibronectin assembly. These GFP-collagen fusion constructs provide a powerful tool for imaging collagen in living cells and have revealed novel and fundamental insights into the dynamic mechanisms for the extracellular assembly of collagen. © 2018 American Society for Bone and Mineral Research.

Project description:BACKGROUND: Zebrafish skin is composed of enveloping and basal layers which form a first-line defense system against pathogens. Zebrafish epidermis contains ionocytes and mucous cells that aid secretion of acid/ions or mucous through skin. Previous studies demonstrated that fish skin is extremely sensitive to external stimuli. However, little is known about the molecular mechanisms that modulate skin cell apoptosis in zebrafish. METHODOLOGY/PRINCIPAL FINDINGS: This study aimed to create a platform to conduct conditional skin ablation and determine if it is possible to attenuate apoptotic stimuli by overexpressing potential apoptosis modulating genes in the skin of live animals. A transgenic zebrafish line of Tg(krt4:NTR-hKikGR)(cy17) (killer line), which can conditionally trigger apoptosis in superficial skin cells, was first established. When the killer line was incubated with the prodrug metrodinazole, the superficial skin displayed extensive apoptosis as judged by detection of massive TUNEL- and active caspase 3-positive signals. Great reductions in NTR-hKikGR(+) fluorescent signals accompanied epidermal cell apoptosis. This indicated that NTR-hKikGR(+) signal fluorescence can be utilized to evaluate apoptotic events in vivo. After removal of metrodinazole, the skin integrity progressively recovered and NTR-hKikGR(+) fluorescent signals gradually restored. In contrast, either crossing the killer line with testing lines or transiently injecting the killer line with testing vectors that expressed human constitutive active Akt1, mouse constitutive active Stat3, or HPV16 E6 element displayed apoptosis-resistant phenotypes to cytotoxic metrodinazole as judged by the loss of reduction in NTR-hKikGR(+) fluorescent signaling. CONCLUSION/SIGNIFICANCE: The killer/testing line binary system established in the current study demonstrates a nitroreductase/metrodinazole system that can be utilized to conditionally perform skin ablation in a real-time manner, and provides a valuable tool to visualize and quantify the anti-apoptotic potential of interesting target genes in vivo. The current work identifies a potential use for transgenic zebrafish as a high-throughput platform to validate potential apoptosis modulators in vivo.

Project description:The neutrophil enzyme myeloperoxidase (MPO) is a major enzyme made by neutrophils to generate antimicrobial and immunomodulatory compounds, notably hypochlorous acid (HOCl), amplifying their capacity for destroying pathogens and regulating inflammation. Despite its roles in innate immunity, the importance of MPO in preventing infection is unclear, as individuals with MPO deficiency are asymptomatic with the exception of an increased risk of candidiasis. Dysregulation of MPO activity is also linked with inflammatory conditions such as atherosclerosis, emphasising a need to understand the roles of the enzyme in greater detail. Consequently, new tools for investigating granular dynamics in vivo can provide useful insights into how MPO localises within neutrophils, aiding understanding of its role in preventing and exacerbating disease. The zebrafish is a powerful model for investigating the immune system in vivo, as it is genetically tractable, and optically transparent. To visualise MPO activity within zebrafish neutrophils, we created a genetic construct that expresses human MPO as a fusion protein with a C-terminal fluorescent tag, driven by the neutrophil-specific promoter lyz. After introducing the construct into the zebrafish genome by Tol2 transgenesis, we established the Tg(lyz:Hsa.MPO-mEmerald,cmlc2:EGFP)sh496 line, and confirmed transgene expression in zebrafish neutrophils. We observed localisation of MPO-mEmerald within a subcellular location resembling neutrophil granules, mirroring MPO in human neutrophils. In Spotless (mpxNL144) larvae-which express a non-functional zebrafish myeloperoxidase-the MPO-mEmerald transgene does not disrupt neutrophil migration to sites of infection or inflammation, suggesting that it is a suitable line for the study of neutrophil granule function. We present a new transgenic line that can be used to investigate neutrophil granule dynamics in vivo without disrupting neutrophil behaviour, with potential applications in studying processing and maturation of MPO during development.

Dataset Information

Live imaging of collagen deposition during skin development and repair in a collagen I - GFP fusion transgenic zebrafish line.

Publications

Live imaging of collagen deposition during skin development and repair in a collagen I - GFP fusion transgenic zebrafish line.

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