Major phenotypic changes evolve in parallel in nature by molecular mechanisms that are largely unknown. Here, we use positional cloning methods to identify the major chromosome locus controlling armor plate patterning in wild threespine sticklebacks. Mapping, sequencing, and transgenic studies show that the Ectodysplasin (EDA) signaling pathway plays a key role in evolutionary change in natural populations and that parallel evolution of stickleback low-plated phenotypes at most freshwater locations around the world has occurred by repeated selection of Eda alleles derived from an ancestral low-plated haplotype that first appeared more than two million years ago. Members of this clade of low-plated alleles are present at low frequencies in marine fish, which suggests that standing genetic variation can provide a molecular basis for rapid, parallel evolution of dramatic phenotypic change in nature.

The genetic and molecular basis of morphological evolution is poorly understood, particularly in vertebrates. Genetic studies of the differences between naturally occurring vertebrate species have been limited by the expense and difficulty of raising large numbers of animals and the absence of molecular linkage maps for all but a handful of laboratory and domesticated animals. We have developed a genome-wide linkage map for the threespine stickleback (Gasterosteus aculeatus), an extensively studied teleost fish that has undergone rapid divergence and speciation since the melting of glaciers 15,000 years ago 1 The benthic species feeds on invertebrates near shore and has a great reduction in the amount of body armor, increased body depth, and a decreased number of gill rakers for filtering ingested food. The limnetic species more closely resembles an ancestral marine fish, with more extensive body armor, a longer and more streamlined body, and an increased number of gill rakers. Despite reproductive isolation between the two species in the wild 3-6 , it is possible to establish productive matings between the two species under laboratory conditions 2 . The resulting F1 hybrids are viable and fertile, making it possible to carry out a formal genetic analysis of the number and location of loci responsible for the adaptive morphological differences between these naturally occurring vertebrate species.To develop resources for genome-wide linkage mapping in Gasterosteus aculeatus, we used large-scale library screening and sequencing to identify a collection of genomic and cDNA clones containing microsatellite repeat sequences. Initially, we sequenced of 192 kb of random genomic clones and showed that CA dinucleotides were the most common form of microsatellite in sticklebacks, occurring approximately once every 14 kb. We subsequently screened genomic and cDNA libraries with a (GT) 15 probe, sequenced 3560 clones, and identified 1176 new microsatellite loci. Primers flanking 410 new and 18 previously identified microsatellites 7-9 were designed and used to type a genetic cross between the benthic and limnetic species from Priest Lake, British Columbia (Figure 1). For this cross, an individual Priest benthic female was mated with a single Priest limnetic male, and a single F1 male (B 1 L 1 ) was crossed to a second Priest benthic female (B 2 B 3 ) to generate 103 progeny. Of the 281 markers that amplified robust bands from the F1 and benthic parent, 227 (81%) were polymorphic, and therefore informative, in one or both parents. Higher rates of polymorphism were seen in the F1 male than the benthic female parent (71% vs. 57% of 281 markers), consistent with a greater level of genetic diversity between the distinct populations of benthic and limnetic fish than within the benthic population.The segregation patterns of the 227 informative markers were scored on 92 progeny from the cross, and the 20,884 resulting genotypes were analyzed for linkage using JoinMap software 10 . The markers were ordered into 26 linkage groups co...

How many genetic changes control the evolution of new traits in natural populations? Are the same genetic changes seen in cases of parallel evolution? Despite long-standing interest in these questions, they have been difficult to address, particularly in vertebrates. We have analyzed the genetic basis of natural variation in three different aspects of the skeletal armor of threespine sticklebacks (Gasterosteus aculeatus): the pattern, number, and size of the bony lateral plates. A few chromosomal regions can account for variation in all three aspects of the lateral plates, with one major locus contributing to most of the variation in lateral plate pattern and number. Genetic mapping and allelic complementation experiments show that the same major locus is responsible for the parallel evolution of armor plate reduction in two widely separated populations. These results suggest that a small number of genetic changes can produce major skeletal alterations in natural populations and that the same major locus is used repeatedly when similar traits evolve in different locations.

The TGF-␤ superfamily of secreted signaling molecules represents a group of evolutionarily-conserved proteins that control multiple cellular processes in a range of organisms (for reviews, see Massagué 1998;. The cellular responses to TGF-␤ ligands are mediated by a highly conserved signal transduction pathway involving a family of transmembrane receptor serine/threonine kinases and cytoplasmic signal transducers, the Smad proteins. The activated receptors phosphorylate the receptor-regulated Smads that form complexes with the co-Smads, translocate to the nucleus, and regulate the expression of target genes by direct interaction with DNA or other transcription factors (for reviews, see Massagué and Wotton 2000; ten Dijke 2000).There are several TGF-␤ family members in Drosophila, among which Dpp is the best-studied (for review, see Podos and Ferguson 1999). The Dpp signal is transduced to the nucleus by Smad complexes containing the receptor-regulated Mad protein and the co-Smad Medea (for review, see Raftery and Sutherland 1999). Mad and Medea have been shown to bind DNA and activate several Dpp target genes. For example, Mad and Medea bind to specific sites in the Dpp response element of the tinman (tin) gene, the tin-D enhancer (Xu et al. 1998), and these sites were shown to be essential for normal tin expression in the embryonic visceral mesoderm. Direct Mad-DNA contact also plays a role in the transcriptional activation of the Ubx gene in the developing midgut (Eresh et al. 1997) and the vestigial (vg) gene in the imaginal wing disc (Kim et al. 1996(Kim et al. , 1997.Mad/Medea binding sites contain repeats of the degenerate sequence GNCN, which is consistent with the sequence of the Smad binding element (SBE) GTCT found in the response regions of TGF-␤ and activin target genes (for review, see ten Dijke 2000). However, the low complexity of the recognition sites and their low affinity for Smad binding (Shi et al. 1998) cannot explain the highly specific target gene responses to TGF-␤ signaling. It was therefore proposed that in many cases Smad proteins achieve specific interactions with cognate DNA by interacting with DNA-binding partners (for review, see ten Dijke 2000).One interesting feature of Dpp and other members of the TGF-␤ family, such as activin and the bone morphogenic proteins (BMPs), is that they can function as morphogens (for review, see Podos and Ferguson 1999). Mor-

To generate specialized structures, cells must obtain positional and directional information. In multi-cellular organisms, cells use the non-canonical Wnt or planar cell polarity (PCP) signaling pathway to establish directionality within a cell. In vertebrates, several Wnt molecules have been proposed as permissible polarity signals, but none has been shown to provide a directional cue. While PCP signaling components are conserved from human to fly, no PCP ligands have been reported in Drosophila. Here we report that in the epidermis of the Drosophila embryo two signaling molecules, Hedgehog (Hh) and Wingless (Wg or Wnt1), provide directional cues that induce the proper orientation of Actin-rich structures in the larval cuticle. We further find that proper polarity in the late embryo also involves the asymmetric distribution and phosphorylation of Armadillo (Arm or β-catenin) at the membrane and that interference with this Arm phosphorylation leads to polarity defects. Our results suggest new roles for Hh and Wg as instructive polarizing cues that help establish directionality within a cell sheet, and a new polarity-signaling role for the membrane fraction of the oncoprotein Arm.

The dynamic rearrangement of cell-cell contacts is required for the establishment of functional epithelial cell sheets. However, the signaling pathways and cellular mechanisms that initiate and maintain this polarity are not well understood. We show that loss of the Wnt signaling component GSK3␤ results in increased levels of aPKC and leads to defects in apical-basal polarity. We find that GSK3␤ directly phosphorylates aPKC, which likely promotes its ubiquitin-mediated proteosomal degradation. aPKC increases the levels of Armadillo and stabilizes adherens junctions. These results suggest that the Wnt pathway component GSK3␤ regulates the polarity determinant aPKC, which in turn affects cell-cell contacts during the development of polarized tissues. Developmental Dynamics 239:115-125, 2010.

Generally, epithelial cells must organize in three dimensions to form functional tissue sheets. Here we investigate one such sheet, the Drosophila embryonic epidermis, and the morphogenetic processes organizing cells within it. We report that epidermal morphogenesis requires the proper distribution of the apical polarity determinant aPKC. Specifically, we find roles for the kinases GSK3 and aPKC in cellular alignment, asymmetric protein distribution, and adhesion during the development of this polarized tissue. Finally, we propose a model explaining how regulation of aPKC protein levels can reorganize both adhesion and the cytoskeleton.

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