Laser capture microdissection of gonads from juvenile zebrafish

Reproductive Biology and Endocrinology Laser capture microdissection of gonads from juvenile zebrafish Anne Jrgensen 1 2 John E Nielsen 0 Jane E Morthorst 1 Poul Bjerregaard 1 Henrik Leffers 0 0 University Department of Growth and Reproduction, Rigshospitalet , Blegdamsvej 9, 2100 Copenhagen , Denmark 1 Institute of Biology, University of Southern Denmark , Campusvej 55, DK-5230 Odense M , Denmark 2 Department of Science , Systems and Models , Roskilde University , Universitetsvej 1, DK-4000 Roskilde , Denmark Background: Investigating gonadal gene expression is important in attempting to elucidate the molecular mechanism of sex determination and differentiation in the model species zebrafish. However, the small size of juvenile zebrafish and correspondingly their gonads complicates this type of investigation. Furthermore, the lack of a genetic sex marker in juvenile zebrafish prevents pooling gonads from several individuals. The aim of this study was to establish a method to isolate the gonads from individual juvenile zebrafish allowing future investigations of gonadal gene expression during sex determination and differentiation. Methods: The laser capture microdissection technique enables isolation of specific cells and tissues and thereby removes the noise of gene expression from other cells or tissues in the gene expression profile. A protocol developed for laser microdissection of human gonocytes was adjusted and optimised to isolate juvenile zebrafish gonads. Results: The juvenile zebrafish gonad is not morphologically distinguishable when using dehydrated cryosections on membrane slides and a specific staining method is necessary to identify the gonads. The protocol setup in this study allows staining, identification, isolation and subsequent RNA purification and amplification of gonads from individual juvenile zebrafish thereby enabling gonadal gene expression profiling. Conclusion: The study presents a protocol for isolation of individual juvenile zebrafish gonads, which will enable future investigations of gonadal gene expression during the critical period of sex differentiation. Furthermore, the presented staining method is applicable to other species as it is directed towards alkaline phosphatase that is expressed in gonocytes and embryonic stem cells, which is conserved among vertebrate species. - Background Zebrafish is used extensively as a model species for studies on vertebrate development and for assessing effects of endocrine disrupting chemicals on reproduction. Despite this, the molecular mechanisms controlling zebrafish sex determination and gonadal differentiation are poorly understood [1-3] In order to determine gene expression during the early gonadal development it is necessary to investigate the first 20 days post hatch (dph) [4]. However, due to the small size of the juvenile zebrafish it is difficult if not impossible to dissect gonads from individual fish and as gonads from different individuals cannot be pooled due to the lack of an early sex marker, an alternative strategy is needed. Therefore, the aim of the present study was to establish a method that allows identification, isolation and subsequent RNA purification of the gonads from individual juvenile zebrafish thereby allowing investigation of gene expression during the expected time of sex determination and differentiation. Microdissection is a powerful tool to isolate specific cells or tissues and thereby ensure a specific gene expression profile without noise from other cells or tissues. When cryosections are used it is possible to avoid total degradation of RNA, however, when microdissecting tissue from frozen and dehydrated juvenile zebrafish, the morphology is impaired and it is difficult to distinguish between the different tissues. The widely used haematoxylin eosin (HE) staining is not sufficient for identification of the juvenile zebrafish gonads for microdissection and therefore a specific staining protocol is necessary. Previous studies have shown that fetal germ cells (gonocytes) have embryonic stem cell like properties including expression of alkaline phosphatase [5-10] Alkaline phosphatase can be detected by staining with Nitro-Blue tetrazolium chloride and 5-Bromo-4-chloro-3-indolyl phosphate (NBT BCIP) and this has previously been applied for identification of human gonocytes followed by microdissection, RNA purification and linear amplification [11]. Methods Animals Juvenile zebrafish originated from a brood population of fish. In the evening breeding boxes were placed in an aquarium with parent fish and eggs were collected the following morning. Non-fertilised eggs were removed while the fertilised eggs were placed in 900 ml glass beakers and kept at 26 1C and a light-dark period of 14:10 h. In the interval 3-22 dph the larvae were fed two times daily with powdered dry food (Sera Micron) and one time daily with newly hatched artemia sp. nauplii (Intr Ryba GmbH, Germany). At 5, 10, 15 and 20 dph zebrafish were frozen individually in liquid nitrogen and stored at -80C until cryosectioning and NBT BCIP staining. Zebrafish used for in situ hybridisation (5, 10, 15 and 20 dph) were fixed in Stieves fixative (solution I: 90 g HgCl2 in 1.5 L H2O; Solution II: 400 g formaldehyde and 80 g glacial acetic acid in 1 L H2O; just before use mix 38 ml Solution I and 12 ml Solution II) at room temperature for 24 hr (fixatives from VWR, Copenhagen, Denmark). NBT BCIP staining for alkaline phosphatase in gonads Zebrafish were embedded in Tissue-Tech Optimal Cutting Temperature (OCT) compound (Sakura Fintek Europe, Zoeterwonde, NL), rapidly frozen in isopentan on dryice and stored at -80C until further analysis. The frozen tissue was cut in 20 m serial sections on a Cryostat and either mounted on RNase free membrane slides (Molecular Machines & Industries, Glatbrugg, Switzerland) for microdissection or on Superfrost slides for HE staining. The membrane slides were fixed immediately in 75% ethanol for 10 min. at room temperature, stored in 100% ethanol in -80C and was then stained with NBT BCIP as described previously [11]. In short, membrane slides were placed: 10 sec in incubation buffer, 90-120 sec. in NBT BCIP solution [262.5 g/mL p-Nitro-Blue tetrazolium chloride; 225 g/mL 5-Bromo-4-chloro-3-indolyl phosphate dipotassium salt (both from Sigma-Aldrich, St Louis, MO, USA); dissolved in 70% and 100% dimethyl formamide, respectively], 10 sec in DEPC water, followed by dehydration in ethanol (10 sec 62% ethanol, 2 10 sec 96% ethanol and 2 10 sec 100% ethanol). The stained germ cells and the surrounding area corresponding to the gonads were dissected using Olympus SmartCut microdissection system according to manufacturers instructions. Lysis buffer from RNAqueous-Micro Kit (Ambion, Austin, TX, USA) was added to the microdissected tissue as fast as possible and samples were stored at -80C until further analysis. RNA was purified using RNAqueous-Micro Kit according to t (...truncated)