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Forward & Reverse Genetics in Zebrafish
1
Lila Solnica-KrezelDepartment of Developmental
Biology
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
EMSENU
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Wilm, Kim, LSK, unpublished
• Strategy for identification of 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(1) Screening Protocol for Identifying Mutations of Zebrafish
Development
Figure 11.2(2) Screening Protocol for Identifying Mutations of Zebrafish Development
Solnica-Krezel et al., Development, 1996
wild type bozozok
somitabun lost a fin
ogon kluska
knypek trilobite
Homeodomain
one-eyed pinhead schmalspurCripto
Nodal SignalingFast1/FoxH1
BMP SignalingSmad5 Alk8
Sizzled
Glypican Strabismus
Noncanonical Wnt Signaling
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
(Chyunue Yin)
24hpf
(Liu et al., 2007)
vu66 is closely linked to ugly duckling (udu)
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)
o-dianisidine staining
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
24
ugu+/-;kny+/-intercross wild type
Ciruna et al. Nature 2006
25
Z udu-/-
Atsushi Sawada
pax2a expression in the pronephric tubule is lost in MZudu
10-somite
MZudu-/-sibling (udu+/-)
Majumdar et al., 2000
Sibling (udu+/-) MZudu-/-
25-somite
cmlc2 expression in the heart primordium is lost in MZudu
!
Many of the SANT-domain containing proteins are involved in chromatin remodeling.
(Boyer et al., 2004)
Screens for
Maternal Effect
Mutations
MullinsPellegriNusslein-Volhard
Dev. Cell 6:771-80 (2004)
TILLING (Targeting Induced Local Lesions in Genomes)
TGCAGACGCACCT A
~ 850bp
~670
~540
500
450
400
350
300
250
150
100
TTTATGTTCCACT T
PDZ-RhoGEF
200
Example of Tilling Gel (EP4)
64/69 confirmed (93%) 10 potential mutations:all confirmed, 1 nonsense Seok-Hyung Kim
Genome research, 2003
Mutation Types Identified by TILLING
Seok-Hyung Kim
What fraction of mutations identified by forward genetic screens are nonsense mutations?
Mutagenesis Regimen Mutation Rates Determined by TILLING3.00 mM ENU x 4 1/516 kb3.25 mM ENU x 4 1/441 kb3.50 mM ENU x 4 1/209 kb3.50 mM ENU x 6 1/210 kb
Efficiency of Mutagenesis Assayed by TILLING
Vanderbilt
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)
Monuki and Walsh, 2001
• associated with hereditary and sporadic holoprosencephaly• usually dominant• incomplete penetrance • variable expressivity
Six domain Homeodomain
Six family of transcription factors
Vertebrate Six3 is expressed early on in the anterior neuroectoderm, and subsequently in the developing forebrain and eyes.
mouse chick Xenopus Medaka
Bovolenta et al., 1998Oliver et al., 1995 Loosli et al., 1998Zhou et al., 2000
Zebrafish six3-related genes
Six domain
Six domain
Six domain
Six3a
Six3b
Six7
94% identity
97% similarity
74% identity
89% similarity
Homology alignments done against Six3a
98% identity
98% similarity
95% identity
98% similarity
HD
HD
HD
Fjose, A.; Kawakami, K., David, I.
six3-related gene expression during gastrulation
Anterior
neuroectoderm
Anterior
neuroectoderm
Prechordal
plate
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 domainIn six3 +/- and six3 -/- mutants
1-2 somite stage
Lagutin, Oliver et al., G&D, 2003
wnt1
FB Six3
MB
wnt1
Lagutin, Oliver et al., G&D, 2003
Ectopic expression of the murine Six3 can represswnt1 expression in zebrafish embryo
wnt1
MZ hdl/tcf3
MZ hdl + msix3
msix3 RNA
Six3
wnt1
FB
MB
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 InbalGeng et al., Dev Cell, 2008
Six domain HD
Six3b - 293 aa
WT
Six
168167 22749
E109Stop
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
Christina Speirs Geng et al., Dev Cell, 2008
six3b-/-;six7
Targeted gene disruption in zebrafishusing designed zinc finger nucleases
Amacher LabUniversity 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”
Miller et al. (2007) Nature Biotechnology[Szczepek et al. (2007) Nature Biotechnology]
“WT” “High-Fidelity”
How are dsDNA breaks repaired?
Modified from Kandavelou and Chandrasegaran, 2007
Strand invasionDNA synthesis
Ligation
Testing ZFNs in zebrafish:Finger design (Sangamo)
Target
DSB repair is very efficient in yeast with appropriate templates
MEL1MEL EL1Repair?
Transform With ZFNs
Induce ZFNexpression
DSB repair
Assay galreporter activity
No ZFN activity
Low ZFN activity
High ZFN activity
ZFN binding site
Testing ZFNs in zebrafish:Injection Strategy
Test loci: golden no tail/Brachyury
Inject ZFN mRNA in one-cell embryos
Analyze gametes or phenotypes at appropriate
developmental stage
RNA!
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
Lamason et al. (2005)
WT
golb1
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
Lawson labWolfe lab
Keith Cheng (Penn St)Dana Carroll (U. Utah)Maria Jasin (Sloan-Kettering)Judith Campisi (LBNL)
Yannick DoyonFyodor Urnov
Jasmine McCammonJohn Young
J MillerF FarajiC NgoG KatibahR AmoraL ZhangE RebarP Gregory