Zebrafish, an alternative to the mouse for genetic studies of vertebrate development.

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Zebrafish, an alternative to the mouse for
genetic studies of vertebrate development.
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Use quotation marks!!!!
Avoid the use of demonstrate or prove when
describing research goals.
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Zebrafish advantages Cheaper to keep in the lab
relative to mouse ( half the cost) Large broods
(usually hundreds, but up to 1000) Can strip both
sperm and eggs from adults and fertilize in
vitro Can get new eggs every few days Rapid
development ( fertilization hatch 2 days adult
morphological features can be scored in seven
days) Reproductively capable at 3-4
months Accessible embryogenesis (external fert.
Transparent eggs) Disadvantages New organism -
no experimental history N25 1.6 x109 Possibly
tetraploid Will not address questions specific to
mammals
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Development is rapid Embryos are
transparent Embryos develop outside the
mother Cells can be marked and lineage tracing
can be done Tissue transplants are possible
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Fish
Frog
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Heterozygosity in diploid eukaryotes often makes
genetic studies cumbersome. G. Streisinger
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Making haploids
UV
sperm
eggs
Haploids develop normally up to a point
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Making haploids
UV
/m
eggs


m

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Making gynogenetic diploids
UV
/m
eggs
Meiosis II
Early pressure (EP)
Heat shock(HS) Late pressure (LPP)
Mitosis (1st cleavage)
/
m/m
/
m/m
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Kimmel (1989)TIG 5283
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• Problems with screening via haploids
• mutations that affect processes that are
  defective in haploids will not be detected.
• mutations are difficult to recover (?? Loss of
  founder fish??)

• Problems screening with diploids
• high background of abnormal embryos and larvae
  (50-70 that dont make normal-looking embryos)
• recombination masks mutations near the ends of
  the chromosomes.

m
m

m
m
m
EP



m


(not a problem for HS or LP)
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Nusslein-Volhard decides to throw her hat in the
ring. Decides that these problems with haploids
and gynogenetic diploids are serious barriers to
systematic screens for developmental
mutants. Needs to use another approach Using
standard genetics requires Devising an
efficient breeding scheme for producing
homozygous fish Methods for cleanly culturing
many lines of fish An efficient mutagen Lots of
people Lots of work (Need ways for cloning the
genes)
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Why do they use mutagenized spermatogonia rather
than mutagenized mature sperm?
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spermatogonium
Mature sperm
Mature sperm
First mitosis
Mosaic embryo
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mature sperm
Pre-meiotic sperm(spermatogonia)
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This fish also carries one mutagenized genome
Cross scheme for Haffter et al. screen
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Background work (Mullins et al. (1994) Current
Biology 4 189) Efficient system for maintaining
large numbers of fish. Efficient system for
mutagenesis Need high frequency mutagen and easy
means to deliver it X-rays and gamma rays were
used in the past. How to test a new
mutagen? Specific locus test (4 loci) ENU was
best with 1-3 mutations/locus/1000 haploid
genomes
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Results of the Haffter et al. screen
(15)
(1.3)
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Allele frequencies
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Scheme for Driever screen (identical to that of
Haffter et al.)
Driever W, Solnica-Krezel L, Schier AF, Neuhauss
SC, Malicki J, Stemple DL, Stainier DY,
Zwartkruis F, Abdelilah S, Rangini Z, Belak J,
Boggs C.(1996)A genetic screen for mutations
affecting embryogenesis in zebrafish.
Development12337-46.
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Results of Driever et al screen 2383 mutations
identified (4264) 695 mutations kept (1557) 577
finished (894) 220 loci (372) 164 single alleles
(222)
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Did they achieve saturation? No. Estimates are
between 50 and 90 (favoring the lower number).
However, I dont know the final results based on
complementation tests between the two screens
(still not done). Using the estimates from the
screen, they predict about 2400 genes that can
mutate to embryonic lethality. But they did
find a boatload of mutants - enough to fill an
entire issue of Development with phenotypic
descriptions.
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Limitations of the screens 1) redundancy 2)
maternal effect mutants only by accident 3)
missed subtle phenotypes 4) observer bias
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(24) evidence for bias in screening
(30)
Driever et al. (39/220 loci 18) Haffter et
al. (24/327 loci 7)
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How do you get the DNA corresponding to the
mutant gene?
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Cloning Map based approach a) map gene b)
obtain overlapping DNA clones of region c) high
resolution mapping (many progeny) d) identify
gene among small set of candidates 1. Rescue
of mutant phenotype 2. Identify mutant allele
by sequencing Candidate gene approach a) use
phenotype to predict gene known from other
organisms b) find that gene in zebrafish (use
ESTs and genetic map) c) test as in d
above Total of 145 genes cloned as of 2006
(Amsterdam Hopkins (2006) Trends in Gen 22 473
)
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Insertional mutagenesis provides a more rapid way
to clone genes.
Identification of 315 genes essential for
early zebrafish development Adam Amsterdam,
Robert M. Nissen, Zhaoxia Sun, Eric C. Swindell,
Sarah Farrington, and Nancy Hopkins Center for
Cancer Research and Department of Biology,
Massachusetts Institute of Technology, 77
Massachusetts Avenue, Cambridge, MA
02139 Contributed by Nancy Hopkins, June 8,
2004 This contribution is part of the special
series of Inaugural Articles by members of the
National Academy of Sciences elected on April 20,
2004.

1. Mutagenize by injecting retrovirus into fish
   embryos
2. Virus inserts randomly into genome creating some
   gene disruptions.
3. Screen for disruptions of essential genes (lethal
   mutations)
4. Outcross the mutations to eliminate extraneous
   inserts
5. Use inverse PCR to amplify flanking DNA
6. If no exon, then sequential inverse PCR until an
   exon is hit.

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Hopkins screen Amsterdam et al. (1999) Genes
Development 132713 Amsterdam et al. (2004) PNAS
101 1293 Amsterdam Hopkins (2006) Trends in
Gen 22 473 (review)
Inject virus into about 250,000 embyros to
generate about 36,000 founder fish. Pair-mate
founders to produce F1 with many
insertions Raise 10,000 F1 families and test
each family by PCR to identify fish with gt 3
unique inserts. Interbreed F1s with inserts to
make 10,000 F2 families Proceed to screen F3 for
phenotypes
mutagenesis
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Results

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One BIG drawback Insertional mutagenesis is
much less efficient than ENU. Requires 15x the
person hours Need to use both approaches. Other
insertional agents are being tested Transposons
(Tol2 and Sleeping Beauty)
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The SIGNIFICANT plus Cloning mutants takes
about two weeks!! ALL mutants affecting
embryogenesis are kept (even pleiotropic ones),
thus eliminating biases about which mutants will
be most informative.
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Conclusions They have identified about 25 of
the zygotically expressed genes that are
essential for embryonic development and therefore
they estimate that there are about 1,600 of
those. Evidence They have insertions in 5/20
tRNA synthase genes, 26 of the 79 ribosomal
protein genes, 23/97 genes identified by chemical
mutation and cloned. Also estimates based on the
Poisson distribution give similar numbers.
Haffter paper - 2400 essential genes Lots of
cool genes!
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Another potential problem for zebrafish
genetics Genome duplication. Evidence from
studies of gene families, like the Hox genes and
other pattern formation genes that have been
studied in mammals show that there are twice as
many of these genes as are expected from
mammalian studies. Is this bad? Maybe.
However, in the few cases that have been
examined, the duplicated genes have different
expression patterns suggesting that they will be
mutable.
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Two orthologues of the mammalian gene Nodal,
squint and cyclops. Overlapping but not
identical expression patterns Differences in
single mutant phenotypes Double mutant has new
defect Conclusion partial redundancy
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Conclusion Although forward genetic screens are
labor intensive, they are identifying important
developmental regulators. Insertional
mutagenesis makes cloning easier, and although
the mutation rate is low, persistent effort has
paid off for about 25 of the essential genes.
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What about the maternal contribution to
development?

• Two types of screens
• Gynogenetic diploids
• Four generation screen

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Why?
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The screen is biased toward genes close to the
centromere

EP
Meiosis I
m
Gynogenetic diploid (m/m)

m
m
m
Polar body


Meiosis I

EP
m
Gynogenetic diploid (m/)
m
m
Polar body
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A pretty messy screen.
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Problems Inbred lines are required for
production of gynognetic diploids inbred lines
are not as healthy as wild lines. Because EP
suppresses meiosis II, crossing over will result
in heterozygous gynogentic diploids. The
frequency of crossing over increases away from
the centromere, so genes that are far away from
the centromere will not get sampled.
Gynogenesis also results in high frequencies of
males (Why?)
Alternative approach Bite the bullet and do a
brute force traditional screen. Mary Mullins,
Penn
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Pool and subject to PCR analysis of polymorphic
markers markers close to the mutant will be
homozyogous
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Results 605 genomes screened (400
families) 68 maternal effect mutants 21 were
early defects 15 representative mutants were
propagated (not clear why the others were not) 7
were able to be mapped by bulk segregant
analysis at least 12 of the 15 are in separate
genes 47 were late defects (at or after MBT) 36
led to massive cell death 13 representative
mutants were propagated
This is not 800 because of sampling error.
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How do you propagate a maternal effect mutant in
the absence of balancer chromosomes? Flanking
DNA markers. M1a mutant M2a M1b
M2b M1 and M2 have two alleles in the two
starting strains. Take tail snips for each
animal in the family
m/
/
m/m
??
M1a
M2b
M2a
M1b
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Take home lessons Even though it is a lot of
work, it is possible to do a standard forward
genetic screen to identify maternal effect
mutants. Most common phenotype is general cell
death - probably general cell viability functions
(housekeeping). Can obtain Mel mutants with
defects before or after zygotic transcription
begins. Can obtain paternal effect mutants -
sperm has some early contribution.
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It is difficult, but not impossible to obtain
mutations affecting the development of zebrafish.