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This article reprinted from: Smith, P. A. 2009. Phenotypes to genotypes using C. elegans. Pages 349-356, in Tested Studies for Laboratory Teaching, Volume 30 (K.L. Clase, Editor). Proceedings of the 30th Workshop/Conference of the Association for Biology Laboratory Education (ABLE), 403 pages. Compilation copyright © 2009 by the Association for Biology Laboratory Education (ABLE) ISBN 1-890444-12-X All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the copyright owner. Use solely at one’s own institution with no intent for profit is excluded from the preceding copyright restriction, unless otherwise noted on the copyright notice of the individual chapter in this volume. Proper credit to this publication must be included in your laboratory outline for each use; a sample citation is given above. Upon obtaining permission or with the “sole use at one’s own institution” exclusion, ABLE strongly encourages individuals to use the exercises in this proceedings volume in their teaching program. Although the laboratory exercises in this proceedings volume have been tested and due consideration has been given to safety, individuals performing these exercises must assume all responsibilities for risk. The Association for Biology Laboratory Education (ABLE) disclaims any liability with regards to safety in connection with the use of the exercises in this volume. The focus of ABLE is to improve the undergraduate biology laboratory experience by promoting the development and dissemination of interesting, innovative, and reliable laboratory exercises. Visit ABLE on the Web at: http://www.ableweb.org Phenotypes to Genotypes Using C. elegans Pliny A. Smith Biology Department Lake Forest College 555 N. Sheridan Road Lake Forest, IL 60045 [email protected] Biography Pliny A. Smith obtained his B.A. in Biology from Grinnell College in 1992 and his Ph.D. from the University of Missouri in 2001. He has been an assistant professor in the Biology Department of Lake Forest College since 2006. Pliny teaches Developmental Biology, Organism Biology, the Biology of Aging. He uses C. elegans both in the classroom as well as to train undergraduates to carry out original research. Introduction Background: This lab is a variation of the classic microbiology unknown identification labs; however, it uses the metazoan C. elegans and genetics. Students will be given a series of unidentified plates containing coded worm strains; known only to the instructor. By examining the phenotypes and other properties of the worms, the students will be instructed to identify the genotypes of each unknown from a chart of possible genotypes and phenotypes. Some of the unknowns cannot be distinguished from each other without additional testing, which the students should conduct to complete their project. These include tests for conditional alleles, dominance, and complementation of alleles. Deciding appropriate tests to conduct helps to develop the student’s ability to use available information and reagents in the design of a hypothesis. Reasons to use C. elegans for this laboratory. Caenorhabditis. elegans is an excellent organism for studying developmental genetics. As a free-living soil nematode, C. elegans is safe for undergraduate students to handle, and teaches skills including use of a microscope and manipulation of microscopic animals. The worm is simple to culture, requiring only small agar plates inoculated with non-pathogenic E. coli as food (Brenner, 1974). In addition, thousands of genetically distinct worm strains are available from the C. elegans Genetics Center at the University of Minnesota. C. elegans have the advantage over flies in that they remain on Petri dishes; they don’t fly away. C. elegans can be stored at -80˚C for years; they do not require the isolation of virgins for mating. Completion of genetic crosses takes only a few days and the crosses are easily performed by students using a stereo microscope, a wire tip (worm pick), and an alcohol burner. Many phenotypes are temperature sensitive (conditional alleles), providing another opportunity for students to test hypotheses. This protocol describes how students can identify genotypes of worms, both behavioral and anatomical, using phenotypic observations and simple experiments. 350 ABLE 2008 Proceedings Vol. 30 Student Outline Objectives: 1. Observe Caenorhabditis elegans nematode life stages, sexes, and phenotypes. 2. Identify genotypes of worms using phenotypic observations. 3. Perform a genetic cross of C. elegans and predict the outcomes. Introduction: This laboratory investigation connects an organism’s genotype to its phenotype. Using the information provided in Tables 1 and 2, most populations of worms can be accurately identified using a microscope and an eyelash to manipulate individuals. However, phenotypes are not always distinct to one allele of a particular gene; often alleles of different genes may affect the same trait of an organism. In such cases, a geneticist must devise experiments that determine if the predicted genotype is present. The C. elegans worms used in this laboratory are identified by a strain name, i.e., N2, which refers to the laboratory that created the strain. Genes are identified by a 3-letter code such as unc, dpy, or bli followed by a number, i.e., unc-32. The genotypes of published C. elegans strains and the meaning of every C. elegans gene name can be found at http://www.wormbase.org. Materials • Worm pick • Alcohol lamps • Known and Unknown worm strains on NGM agar plates (see Tables 1 and 2) • Fresh NGM agar plates (60 x 15 mm) inoculated with E. coli • Zoom stereo microscopes with at least 30X magnification • Parafilm and plastic boxes for plate storage Experimental Procedure. 1. Obtain three plates of mixed-stage known worm strains, N2 (Bristol wildtype), CB4856 (Hawaiian wildtype), and CB164 (dpy-17). 2. Observe each plate using a stereo microscope and record observations concerning size, movement, and morphology for each type of worm. 3. An eyelash pick can be used to lightly touch the worm to see if it responds to tactile stimuli. 4. Check for the presence of male worms. 5. Obtain a set of unknown worm strains; a coded number marks each. 6. Observe the plates using a stereo microscope as before, making careful observations. 7. Compare the worms on each plate for: a. Vulva morphology – Vulva not present, Multi-vulva, Protruding vulva. b. Coordination – Slow moving, Rolling, and Insensitive to touch. c. Body Morphology – Small, Dumpy, Round head, Blistered. 8. Compare the phenotypes of each worm to the list of genotypes and phenotypes provided in the table and enter the unknown strains code in the appropriate box. Mini Workshop 351 9. In some cases, a clear choice cannot be made initially; an experiment must be performed to identify the worm strains. 10. Design the experiment, along with appropriate controls. 11. For each experiment, write your predictions of possible outcomes, you may need to create a Punnett Square. 12. Return at the specified time to follow-up the observations for the experiment and complete Table 2 using the new data collected. Table 1. Known Worm Strains Strain Genotype CB4856 Hawaii WT clone N2 Bristol WT clone Phenotype WT, burrows more than N2; males leave a copulatory plug after mating. Wildtype – standard strain for C. elegans research 352 ABLE 2008 Proceedings Vol. 30 Table 2. Unknown Worm Strains Number Strain Genotype BA17 fem-1(hc17) IV CB1309 CB30 CB3970 CB3991 MT1201 MT3283 MT8189 SM1310 SM139 SU93 M154 CB164 Phenotype Feminization of animals at 25˚C (TS); no sperm made at higher temperatures (Nelson et al., 1978) lin-2(e1309) X Vulvaless sma-1(e30) V Adults are small with blunt head, larvae have white blunt heads, recessive (McKeown et al., 1998) unc-4(e120) bli-1(e769) Slow moving, with blistered skin II sma-8(e2111) V. Short, blunt head. (Dominant) egl-9(n571) V Egg laying defective him-5(e1490)V, egl- High incidence of males, hermaphrodites are 15(n484) X egg laying defective (Egl) (Harfe et al., 1998) lin-15(n765ts) X Muv phenotype (TS) grow at 25˚C for good multivulval phenotype (Herman et al., 1990) tbx-2(ok529)/ dpy- Dpy/Unc worms, WT, and Tbx-2 worms, all 17(e164), unc-32(e184) have ceh-22::GFP. ceh-22:GFP (tinman, III; ceh-22::GFP nkx2.5 homolog) is expressed in the pharynx, (integrated) but it is much lighter than myo-2::GFP (Smith et al., 2007; Johnstone et al., 1992; Okkema et al., 1994; Mango, 2007) dpy-7(e1324ts) X Dumpy, (TS); more dpy at higher temperatures (Gilleard et al., 1997) ajm-1::GFP pRF4 IV ajm-1::GFP – adherens junction GFP, worms MH27::GFPm roll [jds1] roll if born at > 20˚C, but not at 15˚C (Koppen, et al., 2001) sma-1(?);mIs11IV Short, blunt head (recessive), GFP expression with myo-2:GFP, pes-10::GFP, and gut::GFP. Strong GFP expression in pharynx muscle in adults; embryonic expression in body cells Dpy-17(e164) III Short, Dpy worms (Johnstone et al., 1992) Notes for Instructor Genetics of C. elegans. C. elegans have five pairs of autosomes and a pair of sex chromosomes; XX animals are hermaphroditic and self-fertile. XO animals are rarely seen unless they result from a mating between a male (XO) and a hermaphrodite (XX). Without pre-existing males in a culture, XO males may infrequently arise from spontaneous chromosomal non-disjunction of the X chromosome during meiosis. Male sperm are larger than hermaphrodite sperm and always outcompete hermaphrodite sperm; therefore a mating between a hermaphrodite and male results in all cross-progeny (LaMunyon et al., 1995). Mini Workshop 353 Strain Names. A C. elegans strain name consists of one to three CAPITAL letters followed by a number. This identifies the laboratory that created the strain (a genetically distinct and defined line of worms). For example, SM1310 denotes the 1310th strain that Susan E. Mango’s lab created. Gene Names and Alleles. A C. elegans gene is either named after the mutant phenotype (unc-32, let-60, sma-1), or after the class of protein it encodes (tbx-2, ins-2, ajm-1). Because a gene may have more than one allele, there is often included an allele designation. This code consists of one to three lowercase letters followed by a number that identifies the alleles source. For example, dpy-17(e164) represents the 164 allele isolated by Jonathan Hodgkin’s laboratory (e). Genotypes. When listed the genotype of a worm strain, the alleles are listed, starting from the left arm to the right arm of each chromosome, which is denoted by a Roman numeral following the alleles as in the following example: unc-4(e120) bli-1(e769) II. Heterozygous strains would add a slash mark describing the alternate alleles on the homologous chromosome. Proteins. The protein encoded by lin-15 gene is written as LIN-15 (all CAPITALS, non-italic). Phenotype. Capitalize the first letter of a phenotype description and do not use italics. For example, Uncoordinated animals such as those with a homozygous unc-32 genotype would display an Unc-32 phenotype. Descriptions of Gene Classes. The following is a list of the classes of genes suggested in this protocol. dpy "dumpy" (short and fat) body shape. Different dpy mutants have characteristic shapes from “football” to slightly fat and may be dominant or recessive. unc Uncoordinated movement. Unc mutants do not crawl the same as wild type, but this may vary from total paralysis, constant coiling of their tails, a loss of touch response, or the inability to back up. sma Small animals. Sma animals do not appear Dpy (short and fat), but are overall smaller than wild-type worms. egl Egg-laying defective. Egl worms often end their lives with the Bag (bag of worms) phenotype because the embryos hatch inside the mother who soon dies. let Lethals. These may vary from embryonic death to failure to molt into an adult. bli Blister phenotype. Adult animals have defects in their skin resulting in a bubbled or blistered appearance. lin Lineage defective. Cells fail to follow the wild type developmental program resulting in various phenotypes related to the gain or loss of cell types. fem Feminization of XX and XO animals. The Fem phenotype results in only female germ cells being generated by worms, resulting in loss of self-fertility. him High incidence of males. Chromosomal non-disjunction of the X chromosome occurs 354 ABLE 2008 Proceedings Vol. 30 more frequently than in wild type, resulting in an increased number of spontaneous males. tbx-2 T Box gene 2 of C. elegans. An evolutionarily conserved gene necessary for foregut development in C. elegans. Homozygous lethal. Maintaining worms. Worms are best grown on 30 mm or 60 mm NGM agar plates inoculated with the E. coli strain OP50 that serves as the worm’s food (Wood, 1988). The plates should be stored in a plastic box to prevent drying. Worms are ectotherms, and their development is affected substantially by temperature. C. elegans are grown at 15ºC, 20ºC, or 25ºC. At 20ºC, it takes approximately 3 days for a freshly laid embryo to develop into a reproductive adult. At 15ºC it takes about 6 days to reach maturity. Higher temperatures (20ºC-25ºC) will increase the rate of development, but can affect reproductive success (Wood, 1988). Worm populations grow exponentially; each hermaphrodite is self-fertile and will produce up to 300 offspring in a few days. Single worms can be moved to a new plate with a worm pick or larger numbers can be transferred using sterile water and a pipette. Care should be taken that the worms being transferred to new plates display the correct phenotype. Staging the worms. C. elegans are readily staged and sexed using a dissecting microscope.. The embryos are oval in shape, with a length of ~100 µm and a width of about 60 µm. The embryos remain constant in size during development, but the size of the cells within the embryo decreases as cell divisions take place. Larval worms are distinguished by size and morphology. L1s are smaller than L2s, which are smaller than L3s, which in turn are smaller than L4s. Adults are largest and are morphologically distinct from L4s by containing either male or female external genitalia. The adult hermaphrodite contains developing embryos inside its uterus as well. Contamination. Care should be taken to prevent foreign material from contacting the worm plates or bacterial or fungal contamination can occur. Bacteria other than the normally used OP50 strain will make it more difficult to see the worms. Fungus will decrease the fertility, survival of the worms and obstruct microscopic observation as well. Other Resources. WormBase: http://wormbase.org WormBase is a publicly funded, online database of nematode genomics, literature, reagents, and anything else worm related. WormBase can be used to research the phenotypes of alleles that students are investigating. WormBook: http://wormbook.org Contains chapters concerning C. elegans development, genetics, cell biology, evolution, as well as worm methods and protocols Wormatlas: http://wormatlas.org A free source of diagrams, descriptions, and photographs of worm anatomy. Mini Workshop 355 C. elegans Genetic Center (CGC): http://www.cbs.umn.edu/CGC/ Source of worm and bacterial strains used in C. elegans labs. Materials • • • • • • • • • • • • • Platinum/iridium wire Standard Pasteur pipets Alcohol lamps A Bunsen burner Tooth picks Nail polish Worm strains (see Table I) NGM agar plates (60x15mm) inoculated with E. coli E. coli (the OP50 strain works best) Zoom stereo microscopes with at least 30X magnification 15˚C incubator 25˚C incubator Parafilm and plastic boxes for plate storage Protocols. Creating eyelash picks 1. Place a small drop of nail polish on the end of a toothpick 2. Pluck an eyelash or eyebrow and insert the follicular (large) end into the nail polish. 3. Let dry and do not sterilize in a flame. Creating a worm pick 1. Cut a 1.0 cm piece of platinum/iridium wire. 2. Insert 1/3 of the wire into a shorten Pasteur pipet, holding the wire in place with a pair of forceps. 3. Place the end of the pipette containing the wire into a gas flame and twist the wire with the forceps to melt the glass and close the end. 4. Let the pick cool and flatten and bend the end of the wire to form a scoop. Making NGM agar plates 1. Autoclave the following: 2.5 g Bacto Peptone 3.0 g NaCl 20 g Bacto Agar 975 mL distilled H2O 2. After autoclaving, add: 25 ml 1M Sodium Phosphate, pH 6.0 1 mL 1M CaCl2 1 mL 1M MgSO4 1 mL 10 mg/ml Cholesterol in Ethanol 3. Pour ~10 mL of media into each 60 x 15 mm plastic Petri dish, an automatic plate pouring machine is very useful for making consistent plates 356 ABLE 2008 Proceedings Vol. 30 4. Inoculate NGM plates with 50–100 µL of fresh OP50 culture and allow to grow overnight. Literature Cited Brenner, S. 1974. The genetics of Caenorhabditis elegans. Genetics, 77:71-94. Gilleard, J.S., Barry, J.D., and Johnstone, I.L. 1997. cis regulatory requirements for hypodermal cellspecific expression of the Caenorhabditis elegans cuticle collagen gene dpy-7. Molecular and Cellular Biology, 17:2301-11. Harfe, B.D., Vaz Gomes, A., Kenyon, C., Liu, J., Krause, M., and Fire, A. (1998). Analysis of a Caenorhabditis elegans Twist homolog identifies conserved and divergent aspects of mesodermal patterning. Genes and Development. 12:2623-35. Herman, R.K., and Hedgecock, E.M. 1990. Limitation of the size of the vulval primordium of Caenorhabditis elegans by lin-15 expression in surrounding hypodermis. Nature 348, 169171. Johnstone, I.L., Shafi, Y., and Barry, J.D. 1992. Molecular analysis of mutations in the Caenorhabditis elegans collagen gene dpy-7. Embo Journal. 11:3857-63. Koppen, M., Simske, J.S., Sims, P.A., Firestein, B.L., Hall, D.H., Radice, A.D., Rongo, C., and Hardin, J.D. 2001. Cooperative regulation of AJM-1 controls junctional integrity in Caenorhabditis elegans epithelia. Nature Cell Biology. 3:983-991. LaMunyon, C.W. and Ward, S. 1995 Sperm precedence in a hermaphroditic nematode (Caenorhabditis elegans) is due to the competitive superiority of male sperm. Experientia. 51:817-23. Mango, S.E. 2007. The C. elegans pharynx: a model for organogenesis. WormBook 1-26. McKeown, C., Praitis, V., and Austin, J. 1998. sma-1 encodes a betaH-spectrin homolog required for Caenorhabditis elegans morphogenesis. Development. 125:2087-98. Nelson, G.A., Lew, K.K., and Ward, S. (1978). Intersex, a temperature-sensitive mutant of the nematode Caenorhabditis elegans. Developmental Biology. 66:386-409. Okkema, P.G. and Fire, A. 1994. The Caenorhabditis elegans NK-2 class homeoprotein CEH-22 is involved in combinatorial activation of gene expression in pharyngeal muscle. Development. 120:2175-86. Smith, P.A., and Mango, S.E. 2007. Role of T-box gene tbx-2 for anterior foregut muscle development in C. elegans. Developmental Bioloy. 302:25-39. Wood, W.B. 1988. The nematode Caenorhabditis elegans Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.).