Visualization of skeletons and intervertebral disks in live fish larvae by fluorescent calcein staining and disk specific GFP expression
2010; Wiley; Volume: 26; Issue: 2 Linguagem: Inglês
10.1111/j.1439-0426.2010.01418.x
ISSN1439-0426
AutoresYutaka Haga, Shaojun Du, Sayaka Masui, Yuki Fujinami, Masato Aritaki, Shuichi Satoh,
Tópico(s)Reproductive biology and impacts on aquatic species
ResumoJournal of Applied IchthyologyVolume 26, Issue 2 p. 268-273 Full Access Visualization of skeletons and intervertebral disks in live fish larvae by fluorescent calcein staining and disk specific GFP expression Y. Haga, Y. Haga Department of Marine Biosciences, Faculty of Marine Science, Tokyo University of Marine Science and Technology, Minato, Tokyo, JapanSearch for more papers by this authorS. J. Du, S. J. Du Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, MD, USASearch for more papers by this authorS. Masui, S. Masui Department of Marine Biosciences, Faculty of Marine Science, Tokyo University of Marine Science and Technology, Minato, Tokyo, JapanSearch for more papers by this authorY. Fujinami, Y. Fujinami Miyako Station, National Center for Stock Enhancement, Fisheries Research Agency, Miyako, Iwate, JapanSearch for more papers by this authorM. Aritaki, M. Aritaki Miyako Station, National Center for Stock Enhancement, Fisheries Research Agency, Miyako, Iwate, JapanSearch for more papers by this authorS. Satoh, S. Satoh Department of Marine Biosciences, Faculty of Marine Science, Tokyo University of Marine Science and Technology, Minato, Tokyo, JapanSearch for more papers by this author Y. Haga, Y. Haga Department of Marine Biosciences, Faculty of Marine Science, Tokyo University of Marine Science and Technology, Minato, Tokyo, JapanSearch for more papers by this authorS. J. Du, S. J. Du Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, MD, USASearch for more papers by this authorS. Masui, S. Masui Department of Marine Biosciences, Faculty of Marine Science, Tokyo University of Marine Science and Technology, Minato, Tokyo, JapanSearch for more papers by this authorY. Fujinami, Y. Fujinami Miyako Station, National Center for Stock Enhancement, Fisheries Research Agency, Miyako, Iwate, JapanSearch for more papers by this authorM. Aritaki, M. Aritaki Miyako Station, National Center for Stock Enhancement, Fisheries Research Agency, Miyako, Iwate, JapanSearch for more papers by this authorS. Satoh, S. Satoh Department of Marine Biosciences, Faculty of Marine Science, Tokyo University of Marine Science and Technology, Minato, Tokyo, JapanSearch for more papers by this author First published: 13 April 2010 https://doi.org/10.1111/j.1439-0426.2010.01418.xCitations: 5 Author's address: Y. Haga, Department of Marine Biosciences, Faculty of Marine Science, Tokyo University of Marine Science and Technology, Konan 4-5-7, Minato, Tokyo 108-8477, Japan.E-mail: haga@kaiyodai.ac.jp AboutFiguresReferencesRelatedInformationPDFSectionsSummary Introduction Materials and methods Results Discussion AcknowledgementsReferencesCiting LiteraturePDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessClose modalShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Summary 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. Introduction Aquaculture, is presently the fastest growing production sector in aquatic foods, now accounts for approximately 50% of the global seafood supply (Nayler et al., 2009). To support increasing production, large numbers of juveniles have to be produced in hatcheries. Hatchery-raised juveniles however often show skeletal deformities (Cahu et al., 2003; Lall and Lewis-McCrea, 2007). Skeletal deformities are now a serious concern in modern aquaculture industry and often observed in commercially important species (Haga et al., 2001; Hattori et al., 2003; Lall and Lewis-McCrea, 2007; Moretti et al., 1999). Skeletal deformity potentially occurs in all species and can be induced by various factors such as nutritional, environmental, behavioural and genetic factors. The morphology of deformed skeleton resembles each other even if it is induced by different factors (Cahu et al., 2003; Lall and Lewis-McCrea, 2007; Moretti et al., 1999). To study skeletal development and to observe the morphology of skeletons in fish, two major methodologies are generally employed; double staining with alcian blue and alizarin red and soft X ray photography. In general, alcian blue and alizarin red staining of fixed specimens are used to observe the skeleton in fish (Gavaia et al., 2000; Kawamura and Hosoya, 1991; Walker and Kimmel, 2007). However, it is reported that the acidic solution used in the staining procedure often leads to demineralization of the skeleton, eventually resulting in unsuccessful staining of the specimens (Gavaia et al., 2000; Walker and Kimmel, 2007). Soft X-ray exposure requires taking several series of photographs since it allows the visualization of hard tissues in planar sections in specimens. In addition, the X-ray approach is not very useful for fish larvae and specimens <10 cm total length (TL). Recent studies by Du et al. (2001) and Inohaya et al. (2007) have shown that calcein staining is a powerful method to visualize bone and cartilage during skeletal development of zebrafish Danio rerio and medaka Oryzias latipes. Calcein staining has several advantages such as simple methodology applicable to use in commercial hatcheries and high sensitivity allowing observation of subtle changes in bone shape that precedes severe bone deformity. However, use of calcein staining for marine fish larvae has not been reported in detail. Therefore, we examined whether calcein staining can be used to observe skeletal development of red sea bream Pagrus major. In addition to calcein staining, alkaline phosphatase (ALP) activity is often used as a marker of osteoblasts in teleost bone development (Grotmol et al., 2005; Inohaya et al., 2007; Suzuki et al., 2003). ALP activity on tissue section was examined in these studies to determine the localization of osteoblasts. However, it is needed to obtain several series of sections and to reconstruct the three dimensional distributions to overview ALP activity. Therefore, we also tried to visualize ALP activity in whole fish larvae specimens during vertebral development. The vertebral skeleton is composed by a metameric pattern of the vertebral column and intervertebral disks. Abnormal vertebral skeletons are well described in cultured fish and numerous papers focused on abnormal development in affected fish (Haga et al., 2001; Lall and Lewis-McCrea, 2007; Nagano et al., 2007). However, much less attention has been paid to study the intervertebral disk development. In embryos, the unsegmented notochord supports the entire body as a backbone. During early embryogenesis, signals from the notochord induce the migration and proliferation of the sclerotome to form the unsegmented perichordal tube around the notochord. Mesenchyme in the perichordal tube acquires metameric pattern of densely packed as well as loosely packed areas. Densely packed cells fuse with the loosely packed cells to form the vertebral body. Notochord cells located in the vertebral body start to relocate into intervertebral regions and undergo hypertrophy to form the inner gelatinous core of the intervertebral disk (nucleus pulposus) (Rufai et al., 1995; Semba et al., 2006). However, very little is known about the molecular mechanisms that control intervertebral disk development. From the late 1990s, medaka and zebrafish have become popular model system to study skeletal development of vertebrates because of several advantages such as direct accessibility to internal organs, high fecundity, and availability of number of genetic markers (Du and Haga, 2004; Renn et al., 2006). In addition, green fluorescent protein (GFP) reporter systems in transparent fish embryo are employed in many areas of research including analyses of gene expression patterns and tissue/organ development, dissection of promoters/enhancers, etc. Various types of GFP transgenic fish also have been developed for effective screening of mutants showing skeletal deformities (Du and Haga, 2004; Inohaya et al., 2007). However, only one transgenic fish was reported to have GFP expression specifically in intervertebral space (Inohaya et al., 2007). Here we described the development of another transgenic fish, tiggy winkle hedgehog (twhh)-GFP transgenic zebrafish which carries GFP specifically in intervertebral disks. It was suggested that several population of the cells exist in intervertebral disks (Choi et al., 2008; Inohaya et al., 2007). However, it is unclear which kind of the cell expresses GFP in the intervertebral disk in twhh-GFP zebrafish. Therefore, an immunohistochemical analysis was conducted to determine the localization of GFP positive cells in the intervertebral disks of twhh-GFP zebrafish. Materials and methods Spawning and raising of red sea bream fish larvae Naturally spawned fertilized eggs of red sea bream were obtained from National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency, Hatsukaichi, Hiroshima. Eggs were transported by delivery service to the wet lab in Laboratory of Fish Nutrition, Tokyo University of Marine Science and Technology. Twenty thousand eggs were introduced into a 500 L tank filled with 400 L of artificial seawater. Water temperature was adjusted to 19.7 ± 0.5°C by a heater with thermostat. S type rotifers Brachionus rotundiformis and Artemia franciscana nauplii that were enriched with 0.5 ml marinegross (Nisshin Marinetech, Yokohama, Japan) in a 10 L culture medium for more than 24 h were fed from 5–25 days-post fertilization (dpf) and 24–25 dpf, respectively. Enriched rotifers and Artemia nauplii were fed twice a day at 9:30 and 14:00 and density of rotifers and Artemia nauplii was adjusted 5 and 0.04 individuals per ml, respectively. Calcein staining Calcein staining of red sea bream larvae was done according to the method detailed in Du et al. (2001). In brief, the staining solution was made in artificial sea water with calcein powder (Dojin, Tokyo) at 1 g L−1. pH was adjusted with NaOH to 7.0 and any residual calcein powder was removed by filtering through Kimwipes (Nippon Paper Cresia Co., Tokyo). Staining solution was made immediately before staining. In addition, we made staining solution and stored 1 year before use to test effect of long term storage of staining solution. For long term storage, we preserve staining solution in a brown bottle to protect from white light and kept at room temperature. For staining, live fish larvae were immersed in staining solution for 30 min. Stained fish was placed in calcein-free seawater for more than 15 min in order to remove nonspecific staining from muscle and membranous fin. During destaining, the container holding the fish was placed in the dark to prevent quenching of fluorescence by white light. Before observation, they were anesthetized with 1.2% phenoxyethanol (Sigma Co., St. Louis, USA) or 0.4% ethyl 3-aminobenzoate methanesulfonate (Sigma Co., St. Louis, USA). Skeletons stained with calcein were observed under a fluorescent microscope (BX-50; Olympus, Tokyo, Japan) in the dark with a BX-FLA attachment and U-MWIB2 filter set (Olympus, Tokyo, Japan). To trace the skeletal development of red sea bream larvae, larvae were stained every 5 days from 5–25 dpf. Identification of skeletal elements in red sea bream larvae was followed by Moteki (2002). For staining a small number of fish larvae (∼10 fish), a 10 ml glass pipet was used to collect individual larvae. For staining a large number of fish larvae, we used a hand net to collect approximately 50 larvae in bulk from the vat holding staining solution. ALP staining ALP staining during vertebral development of zebrafish at 23 and 27 dpf was performed according to Suzuki et al. (2003). To test effect of retinoic acid on ALP activity, fish were kept in 0.25 μm retinoic acid (RA) for 4 days from 10 dpf. Stained specimens were observed under stereomicroscope (Zoom 600B; Shimadzu Co., Kyoto, Japan) and photographed by digital camera (Moticam 100; Shimadzu Co.). Twhh-GFP transgenic zebrafish Production of twhh-GFP transgenic zebrafish was described elsewhere (Du and Dienhart, 2001). Twhh-GFP zebrafish larvae at 60 dpf were fixed in 4% paraformaldehyde. Tissue embedding, horizontal sectioning along head-tail axis and immunostaining was performed as described in Du et al. (1997). Results Visualizing skeletal development of red sea bream using calcein staining The skeletal development in red sea bream larvae was visualized by calcein staining. It was demonstrated that calcein successfully stained skeletal development in red sea bream larvae (Fig. 1a–d). Skeletal elements were not observed in red sea bream larvae until 6 dpf. From 6 dpf, premaxilla, and cleithrum were observed in red sea bream larvae (Fig. 1a). Strong calcein staining was observed in the dentary and the most posterior part of Meckel's cartilage at 12 dpf (Fig. 1b). Opercule and preopercule were also visualized in larvae from 12 dpf onward (Fig. 1b). Interopercule and occipital vehicle were stained with calcein in fish from 15 and 21 dpf, respectively (Fig. 1c). In addition, a calculus was observed in the urinary bladder in larvae at 21 dpf (Fig. 1e). Figure 1Open in figure viewerPowerPoint Development of hard tissue of red sea bream larvae visualized by calcein. Live fish larvae were stained with solution dissolved 0.5–1 g L−1 calcein powder (pH 7.0). Fish were anesthetized with 1.2% phenoxyethanol and observed under a fluorescent microscope. Red sea bream larvae at 6 dpf (a), at 12 dpf (b), at 15 dpf (c), and at 21 dpf (d, e). Note the calcein staining visualizes the otolith (d) and urinary calculus (e). Bars are 500 μm (a–d) and 100 μm (e). c, cleithrum; ca, calculus; d, dentary; io, inter-opercule; Mc, Meckel's cartilage; ot, otolith; pm, premaxilla; pop, pre-opercule Our studies also revealed several expected as well as new findings. As reported in freshwater fish, no gross mortality was observed during the staining procedure. We observed very rapid quenching of fluorescence in stained skeletal elements when fish died during observation. The reason of this quenching is unknown but this happened even when the skeleton of fish was vitally stained. It was found that MS-222 stimulated melanophore dispersion and often masked visual accessibility. Since there is no effect on skin melanophores by phenoxyethanol, we found that phenoxyethanol was more appropriate for anesthetizing fish larvae before observation. We examined whether calcein solution could be used after long term storage. We found that calcein solution can still successfully label skeletons even if it was made 1 year before. However, reduced pH was found in the calcein staining solution after long term storage. It was found that usage of the hand net could save time and prevent mortality of larvae from stress by pipetting individually. The remaining calcein solution in the gut is lost within 12–18 h after staining. It was found that evacuation of remaining calcein in the gut can be accelerated by stimulating swimming activity by keeping fish at a warmer than optimal temperature (25°C) for red sea bream and feeding Artemia nauplii during destaining. Visualizing skeletal deformity in RA treated zebrafish larvae using ALP staining Alkaline phosphatase (ALP) activity is often used as a marker of osteoblasts in teleost bone development (Grotmol et al., 2005; Inohaya et al., 2007; Suzuki et al., 2003; Haga et al., 2003). Our previous studies have demonstrated that treating fish larvae with RA disrupted the segmented pattern of vertebrae in fish (Haga et al., 2001, 2009). We also assessed ALP activity during vertebral development in RA treated and normal zebrafish larvae. ALP staining of zebrafish larvae at 23 dpf showed staining observed in segmented patterns on the notochord and later expands to cover entire developing vertebral centrum of fish at 27 dpf. However, RA treatment induced partial loss of ALP staining along the notochord. It is suggested that RA treatment disrupts distribution of ALP positive osteoblasts along the notochord in zebarfish. Transgenic zebrafish model for visualizing intervertebral disks To date, numerous transgenic fish carrying GFP have been produced for various purposes. However, there is a very few GFP transgenic fish produced to visualize notochord and intervertebral disks (Table 1). Inohaya et al. (2007) reported that twist-GFP transgenic medaka Oryzias latipes showed sclerotomal cell specific GFP expression. They observed that GFP positive sclerotomal cells localized in intervertebral spaces in larval medaka and suggest that these cells give rise to osteoblasts that ossify the centre. In twist-GFP transgenic medaka, GFP positive cells located in a cluster at the surface of intervertebral ligaments. There is no GFP expression in the notochord and nucleus pulposi-like cell in the intervertebral disks. Recently, we observed intervetebral disk specific GFP expression in tiggy winkle hedgehog (twhh)-GFP transgenic zebrafish (Haga et al., 2009). In contrast to the GFP expression pattern in twist-GFP transgenic medaka, strong GFP expression was observed in nucleus pulposus-like cells in the intervertebral disks in twhh-GFP transgenic zebrafish. Antibody staining revealed that large vacuolated cells in nucleus pulposus-like structure were GFP positive (Fig. 2d,e). However, the central part of nucleus pulposus-like structure lacks GFP expression (Fig. 2e). In addition, we failed to observe intervertebral disk-specific GFP expression in sonic hedgehog (shh)-GFP transgenic zebrafish (Ertzer et al., 2007; Haga et al., 2009). GFP expression in the notochord in shh-GFP transgenic fish disappeared before 6 dpf. With the aid of GFP expression in intervertebral disk in twhh-GFP transgenic zebrafish, it was clearly demonstrated that RA treatment induced vertebral fusion by showing lack of GFP expression in live zebrafish larvae (Haga et al., 2009). This suggests that twhh-GFP expression in the intervertebral disks is a useful tool to study intervertebral disk development in vivo. Table 1. Transgenic fish expressing GFP in intervertebral space Name Species Promoter Expression References shh-GFP Danio rerio Sonic hedgehog Notochord (<2 dpf), floorplate Haga et al. (2009), Ertzer et al. (2007) twhh-GFP Danio rerio Tiggy winkle hedgehog Notochord, floorplate (<2 dpf), Nucleus pulposi Haga et al.(2009) twist-GFP Oryzias latipes Twist Sclerotome, scleroblasts on the surface of intervertebral ligament Inohaya et al. (2007) Figure 2Open in figure viewerPowerPoint ALP stained specimen of wild type zebrafish duing vertebral development and GFP positive cells in nucleus pulposus-like structure of intervertebral disks of twhh-GFP transgenic zebrafish. (a) larva at 23 dpf, (b) larvae at 27 dpf, (c) larvae at 25 dpf, fish was treated with 0.25 μm retinoic acid from 10 dpf for 4 days. Note the partial loss of staining along the notochord. (d, e) GFP positive cells are stained by antibody staining. Note that no staining was observed in central part of nucleus pulposus-like structure in intervertebral disk. Bars are (a–d) 100 μm and (e) 150 μm Discussion In this study, we clearly demonstrated that calcein staining was useful to visualize skeletal development in live marine fish larvae. In fact, tiny facial skeletal elements were observed as early as 6 dpf in red sea bream larvae such as cleithrum and premaxilla. It was reported that hatchery-raised amberjacks Seriola sp. and groupers (e.g. Epinephelus sp.) exhibited skeletal deformities particularly in craniopharyngeal skeletons (Nagano et al., 2007; Yamamoto et al., 2009). Calcein staining of cranial and jaw skeletons have been potentially useful for screening of deformed larvae of these species. The present study also demonstrated that calcein staining can be used to observe calculi in the urinary bladder of red sea bream larvae. Urinary calculi were composed of calcium phosphate (Yamashita, 1966; Ishioka et al., 1970). Since calcein binds to calcium, calcein stained substance in sea bream larva are most likely urinary calculi. It is reported that the occurrence of calculi is often beyond 40–90% in hatchery-raised fish (Sakai et al., 1996: Yamashita, 1966) and correlates with high mortality in early stages of gilthead sea bream (Moretti et al., 1999). Screening of fish larvae having urinary calculi by calcein would help solving mortality of fish larvae in a hatchery. It was reported that ethanol and formalin fixation of fish specimens diminished staining of urinary calculi by alizarin red (Ishioka et al., 1970). Live fish staining with calcein seems to be very useful to detect urinary calculus because this technique does not require fixation. It was found that calcein staining solution was still useful up to 1 year after it was made. This is beneficial to reduce cost of calcein staining. With the aid of live fish larvae staining by calcein, screening could be applied to find skeletal deformity in fish larvae in a hatchery. However, it should be noted that we should overcome several problems with screening of live fish by calcein staining for practical use; damage of fish larvae after observation under UV light, stress for fish larvae during staining and observation, and effectiveness of screening each larva, etc. These technical issues should be solved for screening for large numbers of fish larvae. It was observed disruption of the metameric pattern of ALP staining on notochordal sheath. Because expressing ALP activity in the notochordal sheath is coincides well with initiation of vertebral body formation in fish (Grotmol et al., 2005), ALP staining is suitable for observing early symptoms of vertebral deformity in fish. Currently, very little is known about mechanism of intervertebral disk formation. Embryonic origin of intervertebral disks is investigated mainly by histological studies and in vitro systems. In humans, two populations of cells are reported to exist in the nucleus pulposus; small, chondrocyte-like cells and large notochordal cells. The origin of nothocordal cell is still controversial (Choi et al., 2008). Lineage analysis of precursor cell should be of great benefit to study disk development. It was reported that two transgenic fish models have been developed to have intervertebral space specific GFP expression. The first one is twist-GFP transgenic medaka which has been developed by Prof. Kudo and Inohaya's laboratory (Inohaya et al., 2007). They took advantage of developing twist-GFP medaka which express GFP in sclerotormal cells and observed intervertebral space specific GFP expression in this transgenic medaka. They suggest that GFP positive cells give rise to osteoblasts that ossify vertebral bodies (Inohaya et al., 2007). However, they failed to observe GFP expression in intervertebral disk itself. We observed GFP expression in nucleus pulposus-like structures of twhh-GFP transgenic zebrafish. Therefore, these two model fish are complimentary system to better understand developmental process of intervertebral disks. Interestingly, antibody staining revealed that the central part of nucleus pulposus-like structure of twhh-GFP transgenic fish is GFP negative. We observed that notochordal cell reside in the central part of the vertebral column in zebrafish (Haga et al., 2009). However, whether notochordal cells continuously would persist at the centre of the intervertebral disks is unclear. Recently, a mouse model was reported that allows lineage analysis of intervertebral disks (Choi et al., 2008). Lineage analyses of model animals with different phylogenies could provide insights on developmental biology of intervertebral disk during vertebrate evolution. The present study suggested that calcein and ALP staining is very useful to detect skeletal development of fish larvae. Because calcein and ALP staining is suggested to visualize ossified bone and premature osteoblasts respectively, it would be suitable to dissect ontogenic development of skeletons in fish. In addition to these methods of skeletal staining, live imaging of intervertebral disks by GFP expression in several GFP transgenic fish helps us to better understand intervertebral disk development in fish. It is expected to conduct further research by employing these imaging techniques in order to obtain key findings to elucidate mechanism of skeletal deformity in cultured fish species. Acknowledgements Authors would like to express thanks Mr Kengo Ohta, staff scientist in National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency, for his assistance in collection and sending sea bream eggs. 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Citing Literature Volume26, Issue2April 2010Pages 268-273 FiguresReferencesRelatedInformation RecommendedFaithful expression of green fluorescent protein (GFP) in transgenic zebrafish embryos under control of zebrafish gene promotersBensheng Ju, Yanfei Xu, Jiangyan He, Ji Liao, Tie Yan, Choy L. Hew, Toong Jin Lam, Zhiyuan Gong, Developmental GeneticsSpatial distribution of type II collagen gene expression in the mouse intervertebral discYulong Wei, Robert J. Tower, Zuozhen Tian, Bhavana Mohanraj, Robert L. Mauck, Ling Qin, Yejia Zhang, JOR SPINEEomes::GFP—a tool for live imaging cells of the trophoblast, primitive streak, and telencephalon in the mouse embryoGloria S. 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