Effects of LiCl on Zebrafish Embryonic Development & Whole Mount in situ Hybridization of Goosecoid (gsc) Gene in Zebrafish Introduction/background: [Instructors: this protocol was adapted from Stachel et al., 1993. Lithium perturbation and goosecoid expression identify a dorsal specification pathway in the pregastrula zebrafish. Development 117: 1261-1274.] In this laboratory exercise, you will assay gene expression by in situ hybridization using the zebrafish, Danio rerio. In situ hybridization is a molecular technique commonly used by developmental biologists to determine WHERE a particular gene is expressed in the embryo; in other words, in situ hybridization is used to find it - to find the location/cells where a particular RNA of a gene is present/expressed. Specifically, you will be assaying for the expression of the Goosecoid gene (abbreviated gsc). Goosecoid is a homeobox gene that is expressed in the Spemann s Organizer (aka Organizer) just prior to and during gastrulation in zebrafish (Stachel et al., 1993). It is hypothesized that LiCl is a chemical which can alter Goosecoid expression during gastrulation (Stachel et al., 1993). During this laboratory exercise you will be assaying for Goosecoid gene expression in WT and LiCl treated zebrafish embryos in order to determine if LiCl does, indeed, effect Goosecoid gene expression. General Information on Zebrafish: Zebrafish embryos are excellent for developmental studies because of the large number of transparent eggs that can be obtained from a single mating. In addition, zebrafish are a good genetic system, are easy to care for, and have a relatively short life cycle. Zebrafish are induced to mate by photoperiodic conditions; if kept in conditions of 14 hours of light and 10 hours of dark, fish will mate each dawn. Eggs are laid over a relatively short period of time, resulting in a fairly synchronous population of embryos. For additional information on zebrafish, please consult either Westerfield, M. 2007. The Zebrafish Book: a guide for the laboratory use of zebrafish (Danio rerio), Eugene, University of Oregon Press AND/OR Kimmel et al. 1995. Stages of embryonic development of the zebrafish. Developmental Dynamics 203: 253:310. Zebrafish embryo observation: You will be observing both WT zebrafish embryos and zebrafish embryos treated with LiCl. To aid in your observation of the different developmental stages of zebrafish, there are a number of resources available to help. There is an extensive collection of photographs of developing embryos and a movie is available at http://zfin.org under the heading Atlases and Resources or you can follow the following link: http://zfin.org/zf_info/zfbook/stages/index.html. You should use these photographs to become familiar with the normal stages of development. There is also a staging series for zebrafish included at the end of this handout.
Zebrafish in situ hybridization: We will be carrying out whole mount in situ hybridization using wild type embryos as well as embryos that were treated with 3M LiCl at 4 hpf (hours post fertilization). Embryos have been collected and fixed for you at 7 hpf, and gsc RNA probe has been synthesized as well. An outline of the steps we will carry out is below, followed by the detailed protocol that we will be using. We will be performing this experiment over 2 weeks of lab. Done by your instructor prior to lab: Fixation of embryos, pre-hybridization, and hybridization in RNA probe Week 1: Wash embryos Between Week 1 & Week 2: Pre-incubation of embryos and incubation in anti-dig-antibody overnight Week 2: Wash embryos in PBTw and staining solution (remove non-specific antibody) Staining/Coloration reaction Mounting & photography (may be completed outside of lab or during different lab time) Wholemount In Situ Hybridization Protocol Embryo Fixation and Dehydration (this has been done by your instructor) (1) Fix embryos in fresh 4% Fix (1:1 solution of 8% paraformaldehyde {make fresh weekly} and 2x PBS). Fix for 5 hours at RT or overnight at 4!C. Embryos should be dechorionated before fixation. (To avoid variable staining, do not pack the embryos into tubes -- use <20/tube for stages lacking tails, <30/tube for stages with tails). (2) Fixed embryos were washed several times (x 15 min) in 100% Methanol (MeOH) at RT. The MeOH removes all the water from the embryos, resulting in dehydration. Dehydrated embryos were then stored at -20!C for several days. [Instructors: If you contact the author of this submission, you can obtain gsc plasmid, detailed instructions for making the probe, and other details associated with performing the in situ.] Embryo Rehydration and Hybridization (this has been done by your instructor) (1) Embryos stored in MeOH must be rehydrated before continuing to step 3: wash 1 x 5 min in 66% MeOH: 33% PBTw followed by 1 x 5 min in 33% MeOH: 66% PBTw (2) Wash 5 x 3 min with PBTw. (3) Incubate embryos in hybridization solution for 1-4 hours at 65!C. Use 500 ul per tube. WARM the hyb solution to RT before adding. 2
(4) Incubate embryos in hybridization solution (100 500 ul per tube) with diluted RNA probe overnight at 65!C. The dilution for the probe we will be using is 1:100 in prehyb solution. WARM the HYB mix to 65!C before adding. [Instructors: diluted probe can be saved and reused again] (5) Wash the embryos quickly in hybridization solution at 65!C. -----------------------------THIS IS WHERE YOU START WEEK ONE---------------------- Embryo Stringency Washes: (NOTE: Please WEAR gloves during these washing steps; Add 0.5 ml of each wash to each tube of embryos. You will have2 tubes: WT and LiCl treated.) (1) 1 x 15 min in 75% hybridization mix- 25% 2XSSC at 65!C (prewarmed solution) (2) 1 x 15 min in 50% hybridization mix- 50% 2XSSC at 65!C (prewarmed solution) (3) 1 x 15 min in 25% hybridization mix- 75% 2XSSC at 65!C (prewarmed solution) (4) 1 x 15 min in 2XSSC at 65!C (prewarmed solution) (5) 2 x 30 min in 0.2x SSC at 65!C (prewarmed solution) (6) 1 x 5 min in 75% 0.2x SSC-25% PBTw at ROOM TEMP (RT) on rotator (7) 1 x 5 min in 50% 0.2x SSC-50% PBTw at RT on rotator (8) 1 x 5 min in 25% 0.2x SSC-75% PBTw at RT on rotator (9) 2 x 5 min in 100% PBTw at RT on rotator (10) Store embryos in 4 o C fridge in PBTw solution until Week 2. Antibody binding (incubation) and washes: (This will be performed by your instructor) (1) Incubate in blocking/preincubation solution (PBTw plus 2% normal goat serum, 2 mg/ml Bovine Serum Albumin, BSA) for 1-2 hours at RT on rotator. (2) Remove blocking/preincubation solution (3) Incubate embryos in antibody solution overnight at 4 o C with gentle shaking on rotator. Antibody solution is a 1:4000 dilution of anti-digoxigenin-ap antibody, diluted in blocking/preincubation solution. ------------------------------THIS IS WHERE YOU START WEEK TWO---------------------- (4) Wash 2 x rapidly with PBTw. (5) Wash 5 x 15 min in PBTw. Staining/Coloration Reaction: (1) Wash 3 x 5 min in staining buffer. (2) In final wash of staining buffer, transfer embryo to 9-well glass dishes. Remember which wells contain which embryos! There is the word Pyrex written in the middle of the plate for orientation. (3) Remove any excess staining buffer. This is easiest if you press the glass pipette completely against bottom of glass dish and slowly pipette-off any remaining solution. 3
(4) Add 750 ul of staining solution with NBT and BCIP per well. Your instructor will prepare this solution for you to use. Store dishes in the dark at RT and monitor every 15-30 min, depending upon the signal intensity. You are looking for a blue/purple reaction product. NOTE: Use gloves when handling the NBT and BCIP because it will stain your skin. (5) Stop reactions by washing 3 x PBTw. Then wash 2 x PBS. Mounting for photography: Photograph your embryos as soon as possible. If you cannot photograph your embryos within a couple of days, then remove excess liquid and add 1 ml of 2% paraformaldehyde fix and store at 4 o C. WEAR GLOVES when handing the 2% fix. Remember to print copies of the images to document your data for your lab notebooks. --------------------YOU WILL MAKE OBSERVTIONS DURING WEEK 1-------------------- Observation of Untreated and LiCl Treated Zebrafish Embryos (Fixed) Your instructor has WT (untreated) zebrafish embryos at approximately 28 hpf (hours post fertilization) and LiCl treated zebrafish embryos also at 28 hpf. The WT untreated embryos have been dechorionated while the LiCl treated embryos have not. The chorion is the protective covering around the embryo; the embryo will naturally break-free the of the chorion by 2-3 dpf (days post fertilization). Make sure that you make drawings of what you observe. How are the WT and LiCl treated embryos different phenotypically? What do you hypothesize went wrong in the treated embryos? 4
SOLUTIONS: (note that all solutions will be provided to you by your instructor) 10X PBS (for 1L): NaCl 80 g KCl 2 g Na 2 HPO 4 14.4 g NaH 2 PO 4 2.4 g ph to 7.3; adjusting volume accordingly 8% paraformaldehyde (prepare in hood): Add 4 g paraformaldehyde to 45 ml sterile water. Add several drops of 1 N NaOH. Stir on a slightly warm hotplate until dissolved. The dissolved solution should be at approximately ph 7.0. Bring solution volume to 50 ml after appropriate ph has been acquired. Store in a 50 ml Falcon at 4!C. This solution should be made fresh biweekly. PBTw (for 400 mls): 40 ml 10x PBS 358 ml sterile water 2 ml 20% Tween-20 20X SSC (for 1L): NaCl Sodium citrate ph to 7.0 then adjust volume to 1L 175.3 g 88.2 g Prehybridization solution (for 50 ml, can store at -20!C for 1-2 months): 25 ml formamide *PURE GRADE* (50% final) 12.5 ml 20x SSC (5x final) 50 ul of 50 mg/ml heparin (50 ug/ml final) 500 ul of 50 mg/mg yeast trna (500 ug/ml final) 250 ul of 20% Tween-20 (0.1% final) sterile water to 50 ml 460 ul of 1M citric acid (to bring final ph to ~6.0) Blocking solution (for 50 ml, can store at 4!C for up to 3 weeks) 100 mg Bovine Serum Albumin (BSA) 1 ml normal goat serum PBTw to 50 ml Add BSA to 25 ml PBTw, rotate conical slowly to dissolve BSA, then add goat serum and adjust volume to 50 ml with PBST. (Anti-DIG antibody can be obtained from Roche #11093274910) 5
Staining Buffer (for 200 ml): 20 ml 1M Tris-HCl, ph 9.5 (100 mm final) 1M MgCl 2 (50 mm final) 4 ml 5M NaCl (100 mm final) 1 ml 20% Tween-20 (0.1% final) 165 ml sterile water ***To make staining solution with BCT/BCIP: add 50 ul of stock NBT/ BCIP solution (Roche # 11681451001) to 5 ml of staining buffer Embryo Medium 1000 ml (1L): Hanks stock #1 Hanks stock #2 Hanks stock #4 Hanks stock #5 FRESH Hanks stock #6 ddh 2 O ***ph to 7.2 Hanks stock #1 KCl NaCl Hanks stock #2 Na 2 HPO 4 (anhydrous) KH 2 PO 4 Hanks stock #4 CaCl 2 Hanks stock #5 MgSO 4 7H2O Hanks stock #6 NaHCO 3 1 ml 959 ml 100 ml 0.4 g 8 g 100 ml 0.358 g 0.6 g 100 ml 1.44 g 100 ml 2.46 g 0.35 g 6
Zebrafish (Danio rerio) Staging Series In order to repeat an experiment, it is important to use the same reagents and conditions. In development biology studies, you should perform the studies at the same developmental stage. Images of zebrafish embryos at different developmental ages are shown to the left. Please consult either The Zebrafish Book by Monte Westerfield or Kimmel et al., Developmental Dynamics. 203: 253-310 for more information about developmental staging of zebrafish embryos. Images were obtained from (http://www.swarthmore.edu/natsci/sgilbe r1). 7
Instructor Preparatory Notes: This protocol, obviously, cannot stand alone. It must be accompanied by information about gastrulation, mechanism of LiCl toxicity, gene expression, and other aspects of Zebrafish development. The student handout for this laboratory exercise is NOT meant to include all of the pertinent information for which students should be familiar. Instead it is the recipe for the experiment. The instructor will still need to discuss these developmental biology principles either in lecture or the pre-laboratory. Zebrafish mating and development: Zebrafish are a vertebrate model organism which can be stimulated to breed based on light stimulus. Most Zebrafish colonies have lights that are set on timers which allow for 14 of light and 10 hours of dark each day. If Zebrafish adults are setup the afternoon before embryos are needed, the lights turning ON in the morning will stimulate them to breed. Mating pairs are set-up with at least one female and one male in each breeding tank. Fertilization is external and embryos can be collected after a successful mating. Special breeding tanks are required so that the embryos which are ultimately produced can be separated from the adults. Otherwise, the adults may cannibalize the embryos. Note that those without access to a Zebrafish colony, will not be able to perform this experiment easily. Unlike some other model organisms, Zebrafish require more care and specific habitat conditions that may not be possible for someone without access to an already established colony. Alternatively, many researchers in the Zebrafish community may be willing to send instructors recently fertilized embryos and/or some fixed embryos in order to perform the experiment. In addition, if you live close enough to an established colony, you may be able to pick up fertilized embryos from these researchers. To determine if there is a local Zebrafish lab in your area go to the following website link (http://zfin.org/cgi-bin/webdriver?mival=aa-labselect.apg&select_from=lab) and type your State/Province into the Lab Address contains search field option. Contact information is listed for the pertinent results. For those who are willing to try their skills at Zebrafish husbandry and mating, several resources are available. First an online version of The Zebrafish Book (Westerfield, M. 2000. The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). 4th ed., Univ. of Oregon Press, Eugene. ) can be found by clicking on the following link: http://zfin.org/zf_info/zfbook/zfbk.html. A second resource, found in hard-copy form, with additional information on Zebrafish husbandry and development is Zebrafish: A Practical Approach edited by Nusslein-Volhard and Dahm, 2002. Embryos can be kept in Embryo Media (see recipe above) in simple petri dishes and should be incubated at 28 o C for optimal development. To change the rate of development, the embryos can be slowed down by incubating them at room temperature. By 6 days after fertilization, the embryos/larvae should be euthanized by freezing. Alternative Procedural Notes: Depending on the time that you have for laboratory exercises and the flexibility of the students in your courses, you may be able to have some of the students perform some of the instructor steps such as adding the antibody solution and/or treating and fixing the embryos for themselves. This protocol is designed to encompass 2-3 weeks of laboratory time (where students meet for only 3 hours per week in the laboratory). Obviously, depending on your unique laboratory schedule and student flexibility, the 8
timeline for performing tasks outside of the normal laboratory time is at the instructor s discretion. In addition, this protocol can be adapted to allow for students to perform an inquiry-based experiment where they might change the dose of LiCl, length of exposure, etc. Ordering Information: Various salts (NaCl, KCl, Na 2 HPO 4, NaH 2 PO 4, etc) can be ordered from any scientific supply company (ie Fisher Scientific). Other, more specific, items are listed below with ordering information. Many of these items can be found with other suppliers/companies as well. Anti-DIG antibody Roche #11093274910 DIG-RNA labeling mix Roche #11093274910 NBT/BCIP Roche # 11681451001 Tween-20 Fisher BP337-100 trna Roche 10109495001 heparin Fisher BP2524-10 BSA Calbiochem 12659 Formamide Fisher Scientific BP227-500 Normal goat serum Fisher NC9660079 or 005-000-121 Goosecoid Function and LiCl Teratology: The Organizer is group of cells necessary dorsal cell fates and also for gastrualtion movements. Goosecoid (gsc) is a transcription factor expressed in the Organizer. Activation and stabilization of the "-catenin pathway ultimately leads to gsc expression. LiCl inhibits GSK-3, a molecule important for regulating activity of "-catenin. When LiCl is present, an overabundance of active "-catenin occurs, ultimately leading to an overexpression of gsc. (For more background information on this topic see Gilbert, Developmental Biology, Sinauer & Associates, 2006) Example Results (WT-untreated on left & LiCl treated on right; Images courtesy of M. McWhorter): 9