Genetics of leftright asymmetry in the zebrafish brain

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Genetics of left-right asymmetry in the zebrafish
brain
Josh Gamse, Vanderbilt University
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Vertebrate brains differ in left versus right
neuroanatomy
adapted from Bear, Conners, and Paradiso, 2001
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Anatomical asymmetries in the zebrafish dorsal
diencephalon
left habenula (lh) expresses more leftover than
the right (rh)
parapineal (pp) found on the left side of the
brain
4 day larva, dorsal view
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The left-sided parapinealis revealed by gene
expression
otx5 expression
foxd3gfp expression
L R
L R
3 days old
4 days old
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The leftover gene reveals asymmetry in the
habenular nuclei
L R
leftover gene expression
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Left habenula fate does not develop if the
parapineal is destroyed
otx5/leftover
control n54 pp destroyed n62
4 d, dorsal views
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Mutagenesis to generate brain asymmetry mutants
Adapted from Haffter et al, 1996
Look at leftover
WT
mutant
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The pineal complex is symmetric in the from
beyond mutant
Wild type from beyond mutant
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Why the gene was named from beyond
pineal
fby -/-
R L
From Beyond (1986), directed by Stuart Gordon,
adapted from an H.P. Lovecraft story
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Parapineal-fated neurons are absent in fby
mutants
Parapineal specific marker gfi-1, dorsal view
L R
L R
WT fby
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The from beyond mutation affects the develoment
of the parapineal
pineal complex habenulae (2 days
old) (4 days old)
left habenula
wildtype from beyond homozygote
leftover
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Generating a polymorphic mapping strain
AB (fby heterozygote)
mapping strain

zebrafish haploid genome
X
WIK (wild type)

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Examples of zebrafish simple sequence length
polymorphism (SSLP) markers, a.k.a. Z-markers
marker 2
marker 1

AB WIK
AB WIK
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Genetic mapping of fby
Gametes
Crossovers during Meiosis I in map cross parents
AB
WIK


Intercross male and female map cross parents
Genotyping of progeny


recombinant
non-recombinant
WIK allele AB allele
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Recombination events are identified by AB/WIK
heterozygocity of an SSLP
WIK allele
Marker z11320, 4 agarose gel
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Chromosome 15
SSLP (Recombinations) Physical distance
z11320 (29/1241) 1.4Mb away
z3358 (22/1241) 1.0Mb away
z22430 (2/1241) 96kb away
from beyond mutation
z21165 (8/178) 2.7Mb away
boz
Z9189 (11/118) 5.6Mb away
In zebrafish, 1 recombination (1 centimorgan)
600 Kb
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The tbx2b transcription factor gene is the
correct distance and direction from the z22430
SSLP
100 kilobases
z22430
tbx2b
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tbx2b is expressed in a subset of pineal complex
precursors
6 somites
10 somites
L R
tbx2b
tbx2b flh
dorsal views
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The transcription factor tbx2 is mutated in from
beyond
1 93 265 678
T-box
tbx2b
244 245 246
tbx2b tbx2bfby
Y245-gtstop
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tbx2b MOs phenocopy fby
R L
R L
WT fby tbx2b MO
foxd3gfp, frontal view
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Mutation of the flh transcription factor results
in normal parapineal but a greatly reduced pineal
flh
WT
L R
L R
otx5, 48 h
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The tbx2b and flh transcription factors are
required for different cell types
tbx2b
parapineal
pineal
flh
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tbx2b represses pineal fate in the parapineal
Foxd3GFP/Zpr1
zpr1 cells
fby
WT
flh MO
fby flh MO
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tbx2b represses pineal fate in the parapineal
tbx2b
parapineal
pineal
flh
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Acknowledgments
Vanderbilt Corey Snelson Lijun Bian Erin
Booton

• Carnegie Institution
• Marnie Halpern
• Kiran Santhakumar
• Michelle Macurak
• Lea Fortuno

IGBMC, France Bernard and Christine Thisse
Picasso, 1944