An Affordable and Easy-to-Construct Zebrafish Housing System for Stable Long-Term Laboratory Research.

Zebrafish have become a go-to model organism for in vivo studies, in part because of their reputation as being inexpensive to rear and house. Multiple do-it-yourself designs are currently available that provide laboratories with cost-effective housing systems. Unfortunately, these designs suffer from a range of issues ranging from poor water cycling rates and fragile housing tanks to inconsistent water conditions and designs that are prohibitively expensive for smaller laboratories to construct and maintain. These issues cause many of these housing systems to fall far short of the quality of commercially available zebrafish housing facilities. In this article, we present a novel, affordable, and easy-to-construct zebrafish housing system that improves upon previously published systems. The system utilizes three-dimensional printing technology to construct adaptable zebrafish tanks allowing for the housing of zebrafish at any stage of development. In addition, the water recirculation system utilizes multiple layers of filtration and no chemical adhesives, which allows for stable, long-term, housing of zebrafish in conditions suitable for research and teaching laboratories. The build described herein has been used by our laboratory to house zebrafish for over 3 years, representing multiple generations of housed fish.

Long-term Live Imaging Device for Improved Experimental Manipulation of Zebrafish Larvae.

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for live high-resolution microscopic imaging of dynamic biological phenomena in space and time with cellular resolution. However, the traditional method of agarose encapsulation for live imaging can impede larval development and tissue regrowth. Therefore, this manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which was designed and fabricated as a functionally compartmentalized device to orient larvae for high-resolution microscopy while permitting caudal fin transection within the device and subsequent unrestrained tail development and re-growth. This device allows for wounding and long-term imaging while maintaining viability. Given that the zWEDGI mold is 3D printed, the customizability of its geometries make it easily modified for diverse zebrafish imaging applications. Furthermore, the zWEDGI offers numerous benefits, such as access to the larva during experimentation for wounding or for the application of reagents, paralleled orientation of multiple larvae for streamlined imaging, and reusability of the device.

zWEDGI: Wounding and Entrapment Device for Imaging Live Zebrafish Larvae.

Zebrafish, an established model organism in developmental biology, is also a valuable tool for imaging wound healing in space and time with cellular resolution. However, long-term imaging of wound healing poses technical challenges as wound healing occurs over multiple temporal scales. The traditional strategy of larval encapsulation in agarose successfully limits sample movement but impedes larval development and tissue regrowth and is therefore not amenable to long-term imaging of wound healing. To overcome this challenge, we engineered a functionally compartmentalized device, the zebrafish Wounding and Entrapment Device for Growth and Imaging (zWEDGI), to orient larvae for high-resolution microscopy, including confocal and second harmonic generation (SHG), while allowing unrestrained tail development and regrowth. In this device, larval viability was maintained and tail regrowth was improved over embedding in agarose. The quality of tail fiber SHG images collected from larvae in the device was similar to fixed samples but provided the benefit of time lapse data collection. Furthermore, we show that this device was amenable to long-term (>24 h) confocal microscopy of the caudal fin. Finally, the zWEDGI was designed and fabricated using readily available techniques so that it can be easily modified for diverse experimental imaging protocols.

Matrix metalloproteinase 9 modulates collagen matrices and wound repair.

Acute and chronic injuries are characterized by leukocyte infiltration into tissues. Although matrix metalloproteinase 9 (Mmp9) has been implicated in both conditions, its role in wound repair remains unclear. We previously reported a zebrafish chronic inflammation mutant caused by an insertion in the hepatocyte growth factor activator inhibitor gene 1 (hai1; also known as spint1) that is characterized by epithelial extrusions and neutrophil infiltration into the fin. Here, we performed a microarray analysis and found increased inflammatory gene expression in the mutant larvae, including a marked increase in mmp9 expression. Depletion of mmp9 partially rescued the chronic inflammation and epithelial phenotypes, in addition to restoring collagen fiber organization, as detected by second-harmonic generation imaging. Additionally, we found that acute wounding induces epithelial cell mmp9 expression and is associated with a thickening of collagen fibers. Interestingly, depletion of mmp9 impaired this collagen fiber reorganization. Moreover, mmp9 depletion impaired tissue regeneration after tail transection, implicating Mmp9 in acute wound repair. Thus, Mmp9 regulates both acute and chronic tissue damage and plays an essential role in collagen reorganization during wound repair.

Inflammation and wound repair.

Wound repair requires the integration of complex cellular networks to restore tissue homeostasis. Defects in wound repair are associated with human disease including pyoderma gangrenosum, a heterogeneous disorder that is characterized by unhealed wounds and chronic inflammation of unclear etiology. Despite its clinical importance, there remain significant gaps in understanding how different types of cells communicate to integrate inflammation and wound repair. Recent progress in wound and regenerative biology has been gained by studying genetically tractable model organisms, like zebrafish, that retain the ability to regenerate. The optical transparency and ease of genetic manipulation make zebrafish an ideal model system to dissect multi-cellular and tissue level interactions during wound repair. The focus of this review is on recent advances in understanding how inflammation and wound repair are orchestrated and integrated to achieve wound resolution and tissue regeneration using zebrafish.

Early redox, Src family kinase, and calcium signaling integrate wound responses and tissue regeneration in zebrafish.

Tissue injury can lead to scar formation or tissue regeneration. How regenerative animals sense initial tissue injury and transform wound signals into regenerative growth is an unresolved question. Previously, we found that the Src family kinase (SFK) Lyn functions as a redox sensor in leukocytes that detects H(2)O(2) at wounds in zebrafish larvae. In this paper, using zebrafish larval tail fins as a model, we find that wounding rapidly activated SFK and calcium signaling in epithelia. The immediate SFK and calcium signaling in epithelia was important for late epimorphic regeneration of amputated fins. Wound-induced activation of SFKs in epithelia was dependent on injury-generated H(2)O(2). A SFK member, Fynb, was responsible for fin regeneration. This work provides a new link between early wound responses and late regeneration and suggests that redox, SFK, and calcium signaling are immediate "wound signals" that integrate early wound responses and late epimorphic regeneration.