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This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and behavioural research. As a vertebrate, zebrafish share considerable genetic sequence similarity with humans and are being used as an animal model for various human disease conditions. The advantages of zebrafish in comparison to other common vertebrate models include high fecundity, low maintenance cost, transparent embryos, and rapid development. Due to the spur of interest in zebrafish research, the need to establish and maintain a productive zebrafish housing facility is also increasing. Although literature is available for the maintenance of a zebrafish laboratory, a concise video protocol is lacking. This video illustrates the protocol for regular housing, feeding, breeding and raising of zebrafish larvae. This process will help researchers to understand the natural behaviour and optimal conditions of zebrafish husbandry and hence troubleshoot experimental issues that originate from the fish husbandry conditions. This protocol will be of immense help to researchers planning to establish a zebrafish laboratory, and also to graduate students who are intending to use zebrafish as an animal model. Video LinkThe video component of this article can be found at https://www.jove.com/video/4196/ Protocol 1. System Maintenance 1. Zebrafish are kept in a circulating system that continuously filters and aerates the system water to maintain the water quality required for a healthy aquatic environment. The circulating system also helps to filter excess food and fish excreta. Different companies provide zebrafish systems but we use systems from Aquatic Habitats, USA in our laboratory. The room temperature or the tank temperature is generally maintained between 26-28.5 °C and the lighting conditions are 14:10 hr (light: dark). A zebrafish system from Aquatic Habitats (e.g., Benchtop system) costs ~9,000 USD. This benchtop system with two shelves can hold six 10-liter, twelve 3-liter, or twenty 1.5-liter tanks on each shelf. Multiple lines of fish (e.g., transgenic, mutant, wild type) can also be housed on the same system. 2. A set of different kinds of filters are used in the system. In our system, water from all the tanks passes through a 120-micron filter pad, 50-micron canister filter, biological filter , active carbon absorption filter and UV disinfection filter before being circulated back into the tank. Dechlorinated/aged water is used in the zebrafish system. Water can be de-chlorinated by ageing for at least 48 hr. Under ideal conditions, water should be kept in a reservoir with a pump circulating the water to keep it warm, and expedite the de-chlorination. 3. The pH of the system water should be checked daily and maintained between 6.8 and 7.5. When necessary, sodium bicarbonate should be used to increase the pH. 4. Fish tanks should be cleaned regularly. To clean a fish tank, close the water flow to this tank, drain excess water by tilting the tank backwards...

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Notch 1, Notch 2, and Notch 3 are three highly conserved mammalian homologues of the Drosophila Notch gene, which encodes a transmembrane protein important for various cell fate decisions during development. Little is yet known about regulation of mammalian Notch gene expression, and this issue has been addressed in the developing rodent tooth during normal morphogenesis and after experimental manipulation. Notch 1, 2, and 3 genes show distinct cell-type specific expression patterns. Most notably, Notch expression is absent in epithelial cells in close contact with mesenchyme, which may be important for acquisition of the ameloblast fate. This reveals a previously unknown prepatterning of dental epithelium at early stages, and suggests that mesenchyme negatively regulates Notch expression in epithelium. This hypothesis has been tested in homo- and heterotypic explant experiments in vitro. The data show that Notch expression is downregulated in dental epithelial cells juxtaposed to mesenchyme, indicating that dental epithelium needs a mesenchyme-derived signal in order to maintain the downregulation of Notch. Finally, Notch expression in dental mesenchyme is upregulated in a region surrounding beads soaked in retinoic acid (50-100 micrograms/ml) but not in fibroblast growth factor-2 (100-250 micrograms/ml). The response to retinoic acid was seen in explants of 11-12-d old mouse embryos but not in older embryos. These data suggest that Notch genes may be involved in mediating some of the biological effects of retinoic acid during normal development and after teratogenic exposure.