Forward & Reverse Genetics in Zebrafish Lila Solnica-Krezel Department of Developmental Biology 1 Mutant screens help identify the essential components of embryonic development • Saturation mutagenesis – production of as many mutations as possible with related phenotypes – Complementation analysis distinguishes alleles and genes – Mutagens used to increase mutation rates – Redundant genes – genes missed in a screen because function performed by two genes Genetic Screens in Zebrafish - A Practical Approach • How mutations are induced – Germ line versus mosaic – Mutagens (chemical, retroviral, transposon) • Testing mutagenesis efficiency • How to screen? – Haploid Screens – Diploid Zygotic Screens – Maternal Screens – Enhancer and Suppressor Screens – Phenotype - You find what you are looking for • Saturation issue • Limitations of forward genetic screen approaches • Reverse Genetics in zebrafish - TILLING Alkylating Agents are Effective Mutagens EMS ENU Somatic vs Germinal Mutations Specific Locus Test Mosaic versus non-mosaic mutations Genetics 136, 1401-1420; 1994 Results of specific-locus tests 141,311 genomes screened, over thousands of crosses Improving Efficiency of ENU Mutagenesis Wilm, Kim, LSK, unpublished Figure 11.2(1) Screening Protocol for • Strategy for Identifying Mutations of Zebrafish identification of Development recessive zygotic mutations in zebrafish. • Upon ENU mutagenesis F2 generation genetic screen is performed • Every F1 fish is heterozygous for mutations in different genes Figure 11.2(2) Screening Protocol for Identifying Mutations of Zebrafish Development Homeodomain wild type bozozok Nodal Signaling Fast1/ Cripto one-eyed pinhead FoxH1 schmalspur Smad5 Alk8 BMP Signaling somitabun lost a fin Sizzled kluska ogon Noncanonical Wnt Signaling Glypican knypek Strabismus trilobite Solnica-Krezel et al., Development, 1996 Haploid Screens • Advantages • Limitations Review: Patton and Zon, Nature Rev. Genetics 2: 956-966 (2001) Skipping generations… Gynogenetic Screens Using Insertional Mutagens Why saturation mutagenesis may miss some genes Fig. 19.11 Techniques to reveal additional genes missed by saturation mutagenesis • Overexpression or misexpression screens – Usually dominant phenotypes – Redundant protein expression does not prevent appearance of phenotype • Screens for suppressor mutations – Mutation in one gene that compensates for mutation in another gene active in same process – Phenotype more similar to wild-type than mutant Suppressor mutations Fig. 19.12 Screen Design vu66 mutation enhances knypek phenotype 24hpf (Chyunue Yin) vu66 is closely linked to ugly duckling (udu) o-dianisidine staining (Liu et al., 2007) udu mutant was originally isolated in the Tubingen screen (Hammerschmidt et al., 1996) Liu et al reported that udu encodes a novel nuclear factor essential for primitive erythroid cell development. (2007) Atsushi Sawada vu66 is a new allele of udu mutant locus Atsushi Sawada udu gene is expressed maternally (Liu et al., 2007) - maternal udu expression could account for mild gastrulation defects of Zudu mutants ugu+/-;kny+/- wild type intercross Ciruna et al. Nature 2006 24 Z udu-/- 25 Atsushi Sawada pax2a expression in the pronephric tubule is lost in MZudu sibling (udu+/-) MZudu-/- Majumdar et al., 2000 10-somite cmlc2 expression in the heart primordium is lost in MZudu Sibling (udu+/-) MZudu-/- 25-somite ! Many of the SANT-domain containing proteins are involved in chromatin remodeling. (Boyer et al., 2004) Screens for Maternal Effect Mutations Mullins Pellegri Nusslein-Volhard Dev. Cell 6:771-80 (2004) TILLING (Targeting Induced Local Lesions in Genomes) PDZ-RhoGEF ~ 850bp ~670 TTTATGTTCCACT TGCAGACGCACCT T A ~540 500 450 400 350 300 250 200 150 100 Example of Tilling Gel (EP4) 64/69 confirmed (93%) 10 potential mutations: all confirmed, 1 nonsenseSeok-Hyung Kim Mutation Types Identified by TILLING Genome research, 2003 What fraction of mutations identified by forward genetic screens are nonsense mutations? Seok-Hyung Kim Efficiency of Mutagenesis Assayed by TILLING Vanderbilt Mutagenesis Regimen Mutation Rates Determined by TILLING 3.00 mM ENU x 4 1/516 kb 3.25 mM ENU x 4 1/441 kb 3.50 mM ENU x 4 1/209 kb 3.50 mM ENU x 6 1/210 kb Wienholds et al., Utrecht Laboratories 6 x 3 mM ENU 1 / 235 kb Draper and Moens, Fred Hatchinson Cancer Center, Seattle 4 x 3mM ENU 1 / 500 kb Seok-Hyung Kim http://www.fhcrc.org/tilling http://www.fhcrc.org/tilling ! 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SIX3 (sine oculis -related) • associated with hereditary and sporadic holoprosencephaly • usually dominant • incomplete penetrance • variable expressivity Monuki and Walsh, 2001 Six family of transcription factors Six domain Homeodomain Vertebrate Six3 is expressed early on in the anterior neuroectoderm, and subsequently in the developing forebrain and eyes. mouse chick Xenopus Medaka Oliver et al., 1995 Bovolenta et al., 1998 Zhou et al., 2000 Loosli et al., 1998 Zebrafish six3-related genes Six domain HD Six3a 94% identity 98% identity 97% similarity 98% similarity Six domain HD Six3b 74% identity 95% identity 89% similarity 98% similarity Six domain HD Six7 Homology alignments done against Six3a Fjose, A.; Kawakami, K., David, I. six3-related gene expression during gastrulation Anterior neuroectoderm Prechordal plate Anterior neuroectoderm Prechordal plate six3-related gene expression during segmentation six3 mutant mice lack forebrain Lagutin, Oliver et al., G&D, 2003 Progressive expansion of wnt1 expression domain In six3 +/- and six3 -/- mutants wnt1 1-2 somite stage FB Six3 MB wnt1 Lagutin, Oliver et al., G&D, 2003 Ectopic expression of the murine Six3 can repress wnt1 expression in zebrafish embryo msix3 RNA wnt1 FB Six3 MB wnt1 MZ hdl/tcf3 MZ hdl + msix3 Lagutin, Oliver et al., G&D, 2003 Testing activity of HPE- associated Six3 mutant proteins Christina Speirs in Geng et al., Dev Cell, 2008 Testing activity of HPE-associated Six3 mutant proteins Christina Speirs & Adi Inbal Geng et al., Dev Cell, 2008 Six3b - 293 aa 49 167 168 227 W Six domain HD T E109 Six Stop TILLING: Seok-Hyung Kim Combined six3b/six7 loss-of-function results in reduced or no eyes 2 dpf Inbal et al., Neuron, 2007 Testing activity of HPE-associated Six3 mutant proteins six3b-/-; six7 Christina Speirs Geng et al., Dev Cell, 2008 Targeted gene disruption in zebrafish using designed zinc finger nucleases Amacher Lab University of California, Berkeley Zinc Finger Nucleases Carroll et al., 2006 • Individual fingers recognize DNA triplets • Fingers are modular and can be hooked together • When ZFNs dimerize, the FokI endonuclease makes a dsDNA break in the spacer region Two FokI endonuclease “flavors” “WT” “High-Fidelity” Miller et al. (2007) Nature Biotechnology [Szczepek et al. (2007) Nature Biotechnology] How are dsDNA breaks repaired? Strand invasion DNA synthesis Ligation Modified from Kandavelou and Chandrasegaran, 2007 Testing ZFNs in zebrafish: Finger design (Sangamo) Induce ZFN Assay gal No ZFN activity expression reporter activity Low ZFN activity Transform DSB repair High ZFN activity With ZFNs ZFN binding site MEL Target EL1 MEL1 Repair? DSB repair is very efficient in yeast with appropriate templates Testing ZFNs in zebrafish: Injection Strategy Inject ZFN mRNA in one-cell embryos Analyze gametes or phenotypes at appropriate developmental stage RNA! Test loci: golden no tail/Brachyury Testing ZFNs in zebrafish: Particulars… • ZFNs designed and made by Sangamo • All ZFNs used contain 4-finger ZF motifs • In vivo cleavage activity tested in yeast • Used high-fidelity, obligate heterodimer form for almost all experiments Target locus #1: the golden gene WT golb1 Lamason et al. (2005) Induced mutations are typical of NHEJ mutagenic repair ZFN mutations are transmitted through the germline WT ntl Complementation cross of Founder A with a ntlb195 heterozygote ZFN mutations are transmitted through the germline 11/18 founders screened to date show germline transmission Germline transmission of 1 - 55%, Avg = 20% Acknowledgements J Miller F Faraji C Ngo G Katibah R Amora L Zhang E Rebar P Gregory Jasmine McCammon Yannick Doyon John Young Fyodor Urnov Keith Cheng (Penn St) Dana Carroll (U. Utah) Maria Jasin (Sloan-Kettering) Judith Campisi (LBNL) Lawson lab Wolfe lab.