Genetic and Genetic and Developmental Basis of Developmental Basis of

Evolutionary Pelvic Evolutionary Pelvic Reduction in Threespine Reduction in Threespine

Sticklebacks Sticklebacks Michael D. Shapiro, Melissa E. Marks, Catherine Michael D. Shapiro, Melissa E. Marks, Catherine

L. Peichel, Benjamin K Blackman, et al.L. Peichel, Benjamin K Blackman, et al.

Presentation By Phillip Calender and Adam GrayPresentation By Phillip Calender and Adam Gray

IntroductionIntroduction Threespine Sticklebacks are small fish native to Threespine Sticklebacks are small fish native to

northern rivers, seas, oceans and lakes worldwidenorthern rivers, seas, oceans and lakes worldwide Marine threespine sticklebacks have pelvic Marine threespine sticklebacks have pelvic

structures to protect themselves against gape-structures to protect themselves against gape-limited, soft-mouthed predators by presenting a limited, soft-mouthed predators by presenting a lacerating structure.lacerating structure.

Freshwater Sticklebacks have lost this pelvic Freshwater Sticklebacks have lost this pelvic structure as a probable response to local absence structure as a probable response to local absence of predatory fish, low calcium ion availability, to of predatory fish, low calcium ion availability, to to avoid predation by macroinvertebrates that to avoid predation by macroinvertebrates that grasp the fish by their dorsal and pelvic spines.grasp the fish by their dorsal and pelvic spines.

Introduction (cont.)Introduction (cont.) The goals of the study were to determine the The goals of the study were to determine the

number and type of genetic changes underlying number and type of genetic changes underlying this pelvic reduction.this pelvic reduction.

Aspects studied were:Aspects studied were:– Genetic architecture of pelvic reduction through Genetic architecture of pelvic reduction through

examination of the number and location of chromosome examination of the number and location of chromosome regions controlling pelvic reductionregions controlling pelvic reduction

– Isolation and mapping of candidate genesIsolation and mapping of candidate genes– Comparison of left-right asymmetry in pelvic reductionComparison of left-right asymmetry in pelvic reduction– Sequence comparison of candidate geneSequence comparison of candidate gene– Testing for possible regulatory change in the candidate Testing for possible regulatory change in the candidate

genegene– Comparison to other Threespine Stickleback populationsComparison to other Threespine Stickleback populations

Genetic architecture of pelvic Genetic architecture of pelvic reductionreduction

First, the researchers crossed a marine First, the researchers crossed a marine female with a Paxton Lake male.female with a Paxton Lake male.

375 F2 progeny were then studied and 375 F2 progeny were then studied and measured for extent of development of measured for extent of development of pelvic featurespelvic features

Scoring pelvic reduction as a qualitative Scoring pelvic reduction as a qualitative trait involved normal pelvic structures trait involved normal pelvic structures versus any form of size reduction, loss, or versus any form of size reduction, loss, or asymmetryasymmetry

Genetic architecture of pelvic Genetic architecture of pelvic reductionreduction

The scoring revealed a near 3:1 Mendelian The scoring revealed a near 3:1 Mendelian ratio of unaffected to affected fish, ratio of unaffected to affected fish, meaning that pelvic reduction is dominant.meaning that pelvic reduction is dominant.

Mapping of the qualitative trait connected Mapping of the qualitative trait connected linkage group 7, which showed strong linkage group 7, which showed strong control over all pelvic traitscontrol over all pelvic traits

Several chromosome regions with smaller Several chromosome regions with smaller effects (QTL’s) were also detectedeffects (QTL’s) were also detected

Remaining variance was deemed to be Remaining variance was deemed to be from additional minor loci with phenotypic from additional minor loci with phenotypic effects too small to detect, or to effects too small to detect, or to environmental or epistatic factorsenvironmental or epistatic factors

Genetic architecture of pelvic Genetic architecture of pelvic reductionreduction

Even fish with one Paxton and one marine allele near a Even fish with one Paxton and one marine allele near a given QTL showed significant change in the size of pelvic given QTL showed significant change in the size of pelvic structures, indicating incomplete dominance of pelvic structures, indicating incomplete dominance of pelvic reduction, and the effects of the Paxton QTL’s was additivereduction, and the effects of the Paxton QTL’s was additive

““Rare, favourable, new alleles with semi-additive effects are Rare, favourable, new alleles with semi-additive effects are expected to spread more repidly in an evolving population expected to spread more repidly in an evolving population than purely recessive alleles.”than purely recessive alleles.”

Traits controlled by a relatively small number of genes Traits controlled by a relatively small number of genes should evolve more quickly than traits that require should evolve more quickly than traits that require mutation and fixation across many loci.mutation and fixation across many loci.

Results suggest that major morphological mutations in Results suggest that major morphological mutations in pelvic reduction here occurred through relatively few pelvic reduction here occurred through relatively few chromosome regions (in a natural population)chromosome regions (in a natural population)

Isolation and mapping of candidate Isolation and mapping of candidate genesgenes

Several genes are known to be expressed Several genes are known to be expressed specifically in hindlimbs.specifically in hindlimbs.

Tbx4, Pitx1, Pitx2Tbx4, Pitx1, Pitx2 BAC clones were prepared for stickleback BAC clones were prepared for stickleback

orthologues of each geneorthologues of each gene Sequenced portions of the clones were used to Sequenced portions of the clones were used to

identify polymorphic genetic markersidentify polymorphic genetic markers These markers were then used to determine the These markers were then used to determine the

segregation pattern of these genes in the F2 segregation pattern of these genes in the F2 populationpopulation

Pitx1Pitx1 mapped to linkage group 7 while mapped to linkage group 7 while Tbx4Tbx4 and and Pitx2Pitx2 mapped to linkage groups 1 and four mapped to linkage groups 1 and four respectively .respectively .

Left-right asymmetry in stickleback Left-right asymmetry in stickleback pelvic reductionpelvic reduction

Pitx1Pitx1 pelvic limb reduction in mice is pelvic limb reduction in mice is directionally asymmetric, with directionally asymmetric, with greater reduction on the right than greater reduction on the right than left side.left side.

Does this also occur in the Does this also occur in the Threespine stickleback F2 Threespine stickleback F2 population?population?

Left-right asymmetry in stickleback Left-right asymmetry in stickleback pelvic reductionpelvic reduction

YESYES

Directional asymmetry of pelvic Directional asymmetry of pelvic reduction in sticklebacks also occurs. reduction in sticklebacks also occurs. Longer spines were seen on the left Longer spines were seen on the left than the right in 78% of the animals than the right in 78% of the animals with asymmetric development (as with asymmetric development (as opposed to complete loss.opposed to complete loss.

Granted, this is a weak test for Granted, this is a weak test for Pitx1Pitx1

Pitx1Pitx1 Sequence comparison in Sequence comparison in marine and benthic fishmarine and benthic fish

Determined the exon/intron structure of the geneDetermined the exon/intron structure of the gene Sequenced the entire coding region and Sequenced the entire coding region and

exon/intron junctions of the gene in both parentsexon/intron junctions of the gene in both parents Pitx1Pitx1 in threespine sticklebacks is very identical in threespine sticklebacks is very identical

to that of other known to that of other known Pitx1Pitx1 sequences sequences But, no coding region mutations were found that But, no coding region mutations were found that

would alter this gene between the two parents.would alter this gene between the two parents. Both parents form the exact same protein Both parents form the exact same protein

product according to reverse transcriptase product according to reverse transcriptase mediated polymerase chain reaction and mediated polymerase chain reaction and sequencing studies.sequencing studies.

So why the difference?So why the difference?

Altered Altered Pitx1Pitx1 gene expression in gene expression in pelvic reduced fishpelvic reduced fish

To test for possible regulatory changes in To test for possible regulatory changes in the gene, the researchers examined the the gene, the researchers examined the spatial pattern of gene expression during spatial pattern of gene expression during normal development of the F2 progenynormal development of the F2 progeny

Pitx1Pitx1 is expressed in many different is expressed in many different regions, including the developing thymus, regions, including the developing thymus, olfactory pits, sensory neuromasts on the olfactory pits, sensory neuromasts on the head, trunk, and tail, and the ventral head, trunk, and tail, and the ventral portion of the developing caudal fin in portion of the developing caudal fin in addition to the site of the pelvic fin bud.addition to the site of the pelvic fin bud.

Altered Altered Pitx1Pitx1 gene expression in gene expression in pelvic reduced fishpelvic reduced fish

Altered Altered Pitx1Pitx1 gene expression in gene expression in pelvic reduced fishpelvic reduced fish

For the freshwater fish, no For the freshwater fish, no Pitx1Pitx1 expression was seen expression was seen in the prospective pelvic in the prospective pelvic region, but the gene was region, but the gene was expressed in all other expressed in all other normal regions.normal regions.

But the caudal fins are But the caudal fins are normal, so the altered normal, so the altered expression cannot be due expression cannot be due solely to an absence of solely to an absence of some structuressome structures

The expression was also The expression was also not due to a not due to a developmental delay or a developmental delay or a difference in timing. No difference in timing. No expression was ever expression was ever detected in the freshwater detected in the freshwater larvaelarvae

Altered Altered Pitx1Pitx1 gene expression in gene expression in pelvic reduced fishpelvic reduced fish

Parallel Evolution of Pelvic Parallel Evolution of Pelvic ReductionReduction

Complementation cross was created Complementation cross was created between Paxton fish and Lake between Paxton fish and Lake Vifilsstadavatn (Iceland) fish to see if Vifilsstadavatn (Iceland) fish to see if the genetic basis of pelvic loss is the genetic basis of pelvic loss is similar in both locations.similar in both locations.

The pelvic reduction alleles in the The pelvic reduction alleles in the two populations failed o complement two populations failed o complement and restore normal pelvic and restore normal pelvic morphology.morphology.

Parallel evolution of pelvic Parallel evolution of pelvic reductionreduction

Cross between Icelandic fish (same lake, Cross between Icelandic fish (same lake, this is just easier to pronounce) produced this is just easier to pronounce) produced F1 fish that had strong pelvic features, F1 fish that had strong pelvic features, implying that the failure of pelvic implying that the failure of pelvic development in the complementary cross development in the complementary cross (Paxton X Iceland) was not due to (Paxton X Iceland) was not due to dominant genetic changes in the Hard-to-dominant genetic changes in the Hard-to-pronounce lake fish. pronounce lake fish.

Pelvic Reduction in these two distinct Pelvic Reduction in these two distinct Pacific and Atlantic basin populations likely Pacific and Atlantic basin populations likely results from defects in similar genesresults from defects in similar genes

DiscussionDiscussion CisCis-acting regulatory mutations in -acting regulatory mutations in

Pitx1Pitx1 are a major cause of pelvic are a major cause of pelvic reduction in this rapidly evolving reduction in this rapidly evolving system (10,000 yrs old, many system (10,000 yrs old, many conserved non-gene regions around conserved non-gene regions around the the Pitx1Pitx1 locus) locus)

DiscussionDiscussion Paxton Benthic fish show no alterations in Paxton Benthic fish show no alterations in

Pitx1 Pitx1 coding sequences and changes in coding sequences and changes in gene regulation disrupt expression only at gene regulation disrupt expression only at specific sites in developing larvae.specific sites in developing larvae.

Regulatory mutations in key Regulatory mutations in key developmental control genes may provide developmental control genes may provide a general mechanism to selectively alter a general mechanism to selectively alter expression in specific structures yet expression in specific structures yet preserve expression at all other sitespreserve expression at all other sites

DiscussionDiscussion This variation has been seen in many This variation has been seen in many

other species, but DNA sequences other species, but DNA sequences pertaining to tissue specific expression are pertaining to tissue specific expression are still unknownstill unknown

Pitx1Pitx1 gene is flanked by a 300+kb region gene is flanked by a 300+kb region that is highly enriched in conserved non-that is highly enriched in conserved non-coding sequencescoding sequences

Future research will be to identify these Future research will be to identify these regions and compare them between regions and compare them between stickleback populationsstickleback populations

DiscussionDiscussion Complementation cross suggests Complementation cross suggests

that this mode of pelvic reduction is that this mode of pelvic reduction is the mechanism used in populations the mechanism used in populations 5,700 km apart. 5,700 km apart.

May be similar in other species, and May be similar in other species, and the methods of this paper can be the methods of this paper can be used in other species.used in other species.

THANK YOU FOR THANK YOU FOR COMING!COMING!

Questions?Questions?