Transcript Genetic and Developmental Basis of Evolutionary Pelvic

Genetic and
Developmental Basis of
Evolutionary Pelvic
Reduction in Threespine
Sticklebacks
Michael D. Shapiro, Melissa E. Marks, Catherine
L. Peichel, Benjamin K Blackman, et al.
Presentation By Phillip Calender and Adam Gray
Introduction
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Threespine Sticklebacks are small fish native to
northern rivers, seas, oceans and lakes worldwide
Marine threespine sticklebacks have pelvic
structures to protect themselves against gapelimited, soft-mouthed predators by presenting a
lacerating structure.
Freshwater Sticklebacks have lost this pelvic
structure as a probable response to local absence
of predatory fish, low calcium ion availability, to
to avoid predation by macroinvertebrates that
grasp the fish by their dorsal and pelvic spines.
Introduction (cont.)
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The goals of the study were to determine the
number and type of genetic changes underlying
this pelvic reduction.
Aspects studied were:
– Genetic architecture of pelvic reduction through
examination of the number and location of chromosome
regions controlling pelvic reduction
– Isolation and mapping of candidate genes
– Comparison of left-right asymmetry in pelvic reduction
– Sequence comparison of candidate gene
– Testing for possible regulatory change in the candidate
gene
– Comparison to other Threespine Stickleback populations
Genetic architecture of pelvic
reduction
First, the researchers crossed a marine
female with a Paxton Lake male.
 375 F2 progeny were then studied and
measured for extent of development of
pelvic features
 Scoring pelvic reduction as a qualitative
trait involved normal pelvic structures
versus any form of size reduction, loss, or
asymmetry
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Genetic architecture of pelvic
reduction
The scoring revealed a near 3:1 Mendelian
ratio of unaffected to affected fish,
meaning that pelvic reduction is dominant.
 Mapping of the qualitative trait connected
linkage group 7, which showed strong
control over all pelvic traits
 Several chromosome regions with smaller
effects (QTL’s) were also detected
 Remaining variance was deemed to be
from additional minor loci with phenotypic
effects too small to detect, or to
environmental or epistatic factors
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Genetic architecture of pelvic
reduction
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Even fish with one Paxton and one marine allele near a
given QTL showed significant change in the size of pelvic
structures, indicating incomplete dominance of pelvic
reduction, and the effects of the Paxton QTL’s was additive
“Rare, favourable, new alleles with semi-additive effects are
expected to spread more repidly in an evolving population
than purely recessive alleles.”
Traits controlled by a relatively small number of genes
should evolve more quickly than traits that require
mutation and fixation across many loci.
Results suggest that major morphological mutations in
pelvic reduction here occurred through relatively few
chromosome regions (in a natural population)
Isolation and mapping of candidate
genes
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Several genes are known to be expressed
specifically in hindlimbs.
Tbx4, Pitx1, Pitx2
BAC clones were prepared for stickleback
orthologues of each gene
Sequenced portions of the clones were used to
identify polymorphic genetic markers
These markers were then used to determine the
segregation pattern of these genes in the F2
population
Pitx1 mapped to linkage group 7 while Tbx4 and
Pitx2 mapped to linkage groups 1 and four
respectively .
Left-right asymmetry in stickleback
pelvic reduction
 Pitx1
pelvic limb reduction in mice is
directionally asymmetric, with
greater reduction on the right than
left side.
 Does this also occur in the
Threespine stickleback F2
population?
Left-right asymmetry in stickleback
pelvic reduction
YES
Directional asymmetry of pelvic
reduction in sticklebacks also occurs.
Longer spines were seen on the left
than the right in 78% of the animals
with asymmetric development (as
opposed to complete loss.
Granted, this is a weak test for Pitx1
Pitx1 Sequence comparison in
marine and benthic fish
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Determined the exon/intron structure of the gene
Sequenced the entire coding region and
exon/intron junctions of the gene in both parents
Pitx1 in threespine sticklebacks is very identical
to that of other known Pitx1 sequences
But, no coding region mutations were found that
would alter this gene between the two parents.
Both parents form the exact same protein
product according to reverse transcriptase
mediated polymerase chain reaction and
sequencing studies.
So why the difference?
Altered Pitx1 gene expression in
pelvic reduced fish
To test for possible regulatory changes in
the gene, the researchers examined the
spatial pattern of gene expression during
normal development of the F2 progeny
 Pitx1 is expressed in many different
regions, including the developing thymus,
olfactory pits, sensory neuromasts on the
head, trunk, and tail, and the ventral
portion of the developing caudal fin in
addition to the site of the pelvic fin bud.
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Altered Pitx1 gene expression in
pelvic reduced fish
Altered Pitx1 gene expression in
pelvic reduced fish
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For the freshwater fish, no
Pitx1 expression was seen
in the prospective pelvic
region, but the gene was
expressed in all other
normal regions.
But the caudal fins are
normal, so the altered
expression cannot be due
solely to an absence of
some structures
The expression was also
not due to a
developmental delay or a
difference in timing. No
expression was ever
detected in the freshwater
larvae
Altered Pitx1 gene expression in
pelvic reduced fish
Parallel Evolution of Pelvic
Reduction
 Complementation
cross was created
between Paxton fish and Lake
Vifilsstadavatn (Iceland) fish to see if
the genetic basis of pelvic loss is
similar in both locations.
 The pelvic reduction alleles in the
two populations failed o complement
and restore normal pelvic
morphology.
Parallel evolution of pelvic
reduction
Cross between Icelandic fish (same lake,
this is just easier to pronounce) produced
F1 fish that had strong pelvic features,
implying that the failure of pelvic
development in the complementary cross
(Paxton X Iceland) was not due to
dominant genetic changes in the Hard-topronounce lake fish.
 Pelvic Reduction in these two distinct
Pacific and Atlantic basin populations likely
results from defects in similar genes
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Discussion
 Cis-acting
regulatory mutations in
Pitx1 are a major cause of pelvic
reduction in this rapidly evolving
system (10,000 yrs old, many
conserved non-gene regions around
the Pitx1 locus)
Discussion
Paxton Benthic fish show no alterations in
Pitx1 coding sequences and changes in
gene regulation disrupt expression only at
specific sites in developing larvae.
 Regulatory mutations in key
developmental control genes may provide
a general mechanism to selectively alter
expression in specific structures yet
preserve expression at all other sites
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Discussion
This variation has been seen in many
other species, but DNA sequences
pertaining to tissue specific expression are
still unknown
 Pitx1 gene is flanked by a 300+kb region
that is highly enriched in conserved noncoding sequences
 Future research will be to identify these
regions and compare them between
stickleback populations
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Discussion
 Complementation
cross suggests
that this mode of pelvic reduction is
the mechanism used in populations
5,700 km apart.
 May be similar in other species, and
the methods of this paper can be
used in other species.
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