Zebrafish as a

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Zebrafish as a Model System for Development And
Disease
Bruce Appel Dept. of Biological Sciences IGP
Lecture 2 10/26/2005
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Required reading The art and design of genetic
screens zebrafish, Patton and Zon (2001)
Nature Reviews Genetics 2, 956-966 Recommended
reading Many ribosomal protein genes are cancer
genes in zebrafish, Amsterdam et al., (2004)
PLoS 2, 0690 Web sites ZFIN
http//zfin.org/cgi-bin/webdriver?MIvalaa-ZDB_hom
e.apg Vanderbilt zebrafish labs Solnica-Krezel
lab http//www.molbio.vanderbilt.edu/solnica/lsk
page.html Appel lab http//sitemason.vanderbilt.e
du/site/kcXha8 Zhong lab http//medschool.mc.vand
erbilt.edu/facultydata/php_files/part_dept/show_fa
culty/show_partcellbiology.php?id32411 Knapik
labhttp//medschool.mc.vanderbilt.edu/facultydata
/php_files/part_dept/show_part.php?id311563 Gamse
lab https//medschool.mc.vanderbilt.edu/biosci/b
io_fac.php?id311830 Jesson lab opening soon!
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What do you want to know? (about development and
disease)
How will you figure it out?
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• Overview
• Theme Using vertebrate genetics to investigate
  vertebrate
• problems.
• Lecture 2
• The awesome power of zebrafish genetics
• Zebrafish mutant screens
• From phenotype to gene and back again
• Lecture 3
• Intro to noncanonical Wnt signaling and
  gastrulation
• Lecture 4
• Canonical Wnt signaling and anteroposterior
• patterning of the central nervous system
• Flex Time
• Investigation of microRNA function using
  zebrafish

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Zebrafish genetics provides a powerful
alternative to mouse genetics for investigating
gene function, development and disease

• FORWARD GENETICS
• (flies, worms, zebrafish)
• Phenotype driven
• Genes are mutated randomly
• Requires many animals

• REVERSE GENETICS
• (mice)
• Gene driven
• Genes mutated specifically
• Facilitated by embryonic stem cells

Wild-type organism
Gene identification
Organism with mutant germ line
ES cell with targeted mutation
Mutant phenotype
Mutant germ line
Gene identification
Mutant phenotype
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• Zebrafish are good for forward genetics because
• They are cheap (0.01/day per adult fish)
• They are small and dont need much space (20,000
  in 1,000 sf facility)
• They grow fast (2.5-3 months to sexual maturity)
• They make lots of embryos (50-500 per female
  weekly)
• Embryos develop ex-utero (outside mamma, so you
  can see them)
• Embryos are transparent

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• Mutagenesis
• ENU
• Alkylating agent similar to EMS
• High frequency
• Single gene mutations (usually point mutations)
• Different kinds of alleles
• Nulls
• Hypomorphs allelic series
• Reveals functional domains of proteins
• Hard to clone mutated gene
• Ionizing radiation (gamma)
• High frequency
• Multigene deletions and chromosomal
  rearrangements
• Null mutations
• Hard to identify/clone mutant gene responsible
  for phenotype
• Retroviral insertions
• Low frequency
• Single gene mutations
• Easy to clone mutated gene

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A pseudotyped retrovirus acts as an insertional
mutagen. Upside rapid identification of mutated
gene by plasmid rescue. Downside inefficient
mutagenesis
Patton and Zon, 01
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For geneticists, diploidy can be
inconvenient. Why? To uncover loss of function
mutations, both gene copies must be disrupted.
Strategy 1 Screen for recessive mutations in
progeny of F2 generation
Patton and Zon, 02
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Example of a real screen (headed by Prof. Lila
Solnica-Krezel as a postdoc)
Driever et al 96
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Strategy 2 Screen of gynogenetic haploid progeny
of F1 fish
Normal, diploid embryo
Haploid embryo
Patton and Zon, 01
Haploids reduce the time, effort and cost of a
screen at the expense of a normal developmental
background.
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Strategies 3a and 3b Screen of gynogenetic
diploid progeny of F1 fish
Early Pressure
Heat shock
F2

• Gynogenetic diploids reduce the
• time, cost and effort of a screen but
• have their own drawbacks
• EP screens miss genes at the ends
• of chromosomes
• 2. HS screens are inefficient

Homozygous from centromere to crossover, heterozy
gous distal to crossover
Homozygous at all loci
Patton and Zon, 01
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Alrighty. Weve got the genetics. What are they
screen assays? 1st Generation Morphology-based
screens (Tübingen and Boston screens,
Development 123, pp 1-2001, 1996 and mutants from
Eugene)
spinal cord
floor plate
notochord
dorsal aorta
Wild-type
spinal cord
notochord
dorsal aorta
deltaAdx2
Appel et al., 1998
Haffter et al. 1996
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Phenotypic classes from large-scale F2 screens
include body shape mutants and brain mutants (and
many other classes).
Solnica-Krezel et al, 96
Schier et al, 96
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• What are some of the most significant things we
  learned
• from these screens?
• Mechanisms of gastrulation (lecture 3)
• Mechanisms of anteroposterior patterning (lecture
  4)
• Mechanisms of primordial germ cell migration

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How do we get more out of our screens? Expanding
the screens idea 1 adult phenotype
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Expanding the screens idea 2 molecular markers

• In situ RNA hybridization probes reveal specific
  domains and cell types. This example shows a
• developmental brain patterning defect.

Here, expression of shh, pax2 and krox20
reveal loss of hindbrain segment r5 in valentino
mutant embryos.
Moens et al, 96
2) Antibody labeling also reveals specific cell
types. Here, an antibody that labels mitotic
cells was used to identify a mutant that has
excess proliferative cells in early development.
This mutation disrupts the bmyb gene and mutant
embryos have genomic instability. Consequently,
this mutant provides a new cancer model.
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Expanding the screens idea 3 Fluorescent
reporters reveal physiological processes and
cell types in living embryos
Lipid processing reporter
Patton and Zon, 01 from Farber et al, 01
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Expanding the screens idea 4 Transgenic
fluorescent reporters reveal cell types and
behaviors in living embryos
2 dpf
Tgolig2DsRed2 Tgnkx2.2amegfp
Photo courtesy of Jimann Shin, IGP student
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Tg(nkx2.2amegfp) Movie courtesy of Brandon
Kirby, IGP student
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An F2 screen reveals three classes of
oligodendrocyte phenotype
Deficit
Excess
Ectopic migration
David Mawdsley, Heather Snell, Hae-Chul Park
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Expanding the screens idea 5 behavior
See movies
space cadet mutant embryos have abnormal escape
responses
Laurent et al, 01
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Alrighty. Weve got mutations, how do we find the
genes? step 1 genetic mapping
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How do we construct a genetic map and use it to
clone genes? Think waaaaay back to some things
David Greenstein told you
A/a, B/b and C/c can be phenotypic variants
(red eye, white eye) or DNA sequence variants
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DNA Markers the modern alleles in a haplotype

• Genetic variation exists between individuals
• This variation can be within genes dif. alleles
• Individuals can also harbor differences in the
  sequences between genes.
• Any such difference can be exploited as a DNA
  marker, or DNA polymorphism
• These polymorphic markers are used to follow
  differences
• between individuals (to map genes)
• between populations (to study the diversity of
  the population)

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• Microsatellite markers
• a class of variable number tandem repeats (VNTRs)
• consist of two parts 1) a tandemly repeated,
  variable sequence
• flanked by 2) unique sequences
• Detects up to two alleles per individual but many
  alleles within a
• Population
• Detected by PCR

1
2
3
msA4
p1
msA8
CA
CA
CA
CA
msA7
GT
GT
GT
GT
msA6
p2
p1
msA4
CA
CA
CA
CA
CA
CA
msA2
GT
GT
GT
GT
GT
GT
p2
msA6
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Mapping searches for linkage between DNA marker
variants and phenotypic variants
Chromosome 2
Chromosome 1
msA4
ntl-
msB3
msA6
msB9
ntl
Which alleles are linked? Assume these loci are
separated by 5 cM, that you mate 2 individuals
having this genotype and you recover 100 progeny.
You isolate genomic DNA, perform PCR and run
products on a gel. What are the genotypic classes
you expect to observe and in what frequencies
should they occur?
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So, what good does this do you anyway? Step 2
gene identification Heres the key each
microsatellite marks a known position in the
genome. Once you establish linkage of your
phenotypic variant to a microsatellite Locus, you
know where to search for your gene of interest.
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• After mapping, you probably have several gene
  candidates. How do
• you know which is the right one?
• 4 step program to genetic enlightenment
• Search candidate genes for function-altering
  mutations
• Ask if the candidate gene is expressed at a time
  and place
• consistent with its phenotype
• Ask if functional knockdown phenocopies the
  mutation
• Ask if expression of the wild-type form of the
  gene suppresses the
• mutant phenotype

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Zebrafish mutations provide models of human
disease
Jekyll encodes uridine 5-diphosphate-glucose
dehydrogenase, which is required for production
of glycosaminoglycans and cardiac valve formation
Walsh and Stainier, 2001
weissherbst encodes the iron exporter Ferroportin
1. weiss mutant embryos are anemic
Donovan et al, 2000
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Expanding the tool kit
Reverse Genetics TILLING (Targeting Induced
Local Lesions IN Genomes)
(about 6,000 fish)
MJ Basestation
Mutant selection from isolated pools
Heat and cool
Mutation confirmation by Sequencing
CEL-1 digestion
Mutant F2 generation
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For those times you just cant get a
mutation Loss-of-function tests using morpholino
antisense oligonucleotides
Morpholine rings give antisense
oligonucleotides high stability and low toxicity
phosphorodiamidate linkage
MOs with sequence complementary to translation
start site of mRNA block translation
MOs specific to no tail phenocopy the no tail
mutation
Inject MOs into zygote
AUG
wt
AAAAAAA
MO
Ntl MO
ntl-
Nasevicius and Ekker, 00
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When Timing is Everything Heat-inducible
promoters drive conditional gene expression
GFP for marking cells cDNA for gene
overexpression Mutant cDNA for dominant
interfering proteins GAL4 for modular expression
systems
hsp70 promoter
Laser activation of GFP expression in single
cells of transgenic hsp70GFP embryos
Halloran et al, 00
Heat induction of constitutively active Notch
using GAL4/UAS system blocks neurogenesis in eye
Scheer et al., 01
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More Useful Tools I Fate Mapping
365 nm light from laser
inject caged fluorescein into zygote
6 hpf
Fate mapping reveals that different cell types
arise from the zebrafish shield
notochord
floor plate
hypochord
D. Latimer
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More Useful Tools II Cell Transplantation
Remove cells from donor embryo
Inject cells into host embryo
Inject dye into zygote

• Used to
• Analyze cell behavior
• Create genetic mosaics
• Test cell autonomy
• Identify signaling sources
• Test cell fate commitment

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Where are zebrafish headed? Heres one idea a
whole animal assay system for drug discovery
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