Visualization of skeletons and intervertebral disks in live fish larvae by fluorescent calcein staining and disk specific GFP expression |
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| Authors: | Y. Haga S. J. Du S. Masui Y. Fujinami M. Aritaki S. Satoh |
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| Affiliation: | 1. Department of Marine Biosciences, Faculty of Marine Science, Tokyo University of Marine Science and Technology, Minato, Tokyo, Japan;2. Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, MD, USA;3. Miyako Station, National Center for Stock Enhancement, Fisheries Research Agency, Miyako, Iwate, Japan |
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| Abstract: | Zebrafish and medaka have become popular models for studying skeletal development because of high fecundity, shorter generation period, and transparency of fish embryo. The first step to study skeletal development is visualizing bone and cartilage. Live animal staining with fluorescent calcein have several advantages over the standard skeletal staining protocol by using alizarin red and alcian blue for bone and cartilage. However, there is no detailed study examining skeletal development of live marine fish larvae by calcein staining. Here we applied calcein staining to examine skeletal development in red sea bream larvae. In addition, green fluorescent protein (GFP) reporter zebrafish was employed to trace lineage analysis of intervertebral disk cells in live fish larvae. Calcein staining of red sea bream larvae successfully visualized development of craniofacial skeletons as well as urinary calculus. Histochemical detection of alkaline phosphatase (ALP) activity revealed that abnormal segmentation of notochord induced by RA during vertebral development in zebrafish. Immunohistochemistry clearly revealed that GFP‐positive cells in intervertebral space was nucleus polposus like cell in twhh‐GFP transgenic zebrafish. It was demonstrated usefulness of calcein and ALP staining and twhh‐GFP transgenic zebrafish for studying skeletal development in live fish larvae. |
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