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Transcriptome analysis reveals novel patterning and pigmentation genes underlying Heliconius butterfly wing pattern variation.

ABSTRACT: Heliconius butterfly wing pattern diversity offers a unique opportunity to investigate how natural genetic variation can drive the evolution of complex adaptive phenotypes. Positional cloning and candidate gene studies have identified a handful of regulatory and pigmentation genes implicated in Heliconius wing pattern variation, but little is known about the greater developmental networks within which these genes interact to pattern a wing. Here we took a large-scale transcriptomic approach to identify the network of genes involved in Heliconius wing pattern development and variation. This included applying over 140 transcriptome microarrays to assay gene expression in dissected wing pattern elements across a range of developmental stages and wing pattern morphs of Heliconius erato.We identified a number of putative early prepattern genes with color-pattern related expression domains. We also identified 51 genes differentially expressed in association with natural color pattern variation. Of these, the previously identified color pattern "switch gene" optix was recovered as the first transcript to show color-specific differential expression. Most differentially expressed genes were transcribed late in pupal development and have roles in cuticle formation or pigment synthesis. These include previously undescribed transporter genes associated with ommochrome pigmentation. Furthermore, we observed upregulation of melanin-repressing genes such as ebony and Dat1 in non-melanic patterns.This study identifies many new genes implicated in butterfly wing pattern development and provides a glimpse into the number and types of genes affected by variation in genes that drive color pattern evolution.

SUBMITTER: Hines HM

PROVIDER: S-EPMC3443447 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Transcriptome analysis reveals novel patterning and pigmentation genes underlying Heliconius butterfly wing pattern variation.

Hines Heather M HM Papa Riccardo R Ruiz Mayte M Papanicolaou Alexie A Wang Charles C Nijhout H Frederik HF McMillan W Owen WO Reed Robert D RD

BMC genomics 20120629

<h4>Background</h4>Heliconius butterfly wing pattern diversity offers a unique opportunity to investigate how natural genetic variation can drive the evolution of complex adaptive phenotypes. Positional cloning and candidate gene studies have identified a handful of regulatory and pigmentation genes implicated in Heliconius wing pattern variation, but little is known about the greater developmental networks within which these genes interact to pattern a wing. Here we took a large-scale transcrip ...[more]

PMID: 22747837

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Project description:Heliconius butterfly wing pattern diversity offers a unique opportunity to investigate how natural genetic variation can drive the evolution of complex adaptive phenotypes. Here we took a large-scale transcriptomic approach to identify the network of genes involved in Heliconius wing pattern development and variation. This included applying 147 microarrays representing the Heliconius transcriptome to assay shifts in gene expression across pupal development among several wing pattern morphs of Heliconius erato. We focused in particular on genes differentially expressed relative to the gene optix, which controls red pattern elements in wings. We combined expression results from three hindwing morphs from Peru and from dissected basal to apical wing elements in two forewing morphs to uncover two main classes of genes. First we looked for candidate upstream regulators of optix by determining transcripts expressed differently across basal to apical sections of the forewing prior to optix expression. Second, we assessed how optix regulates downstream gene expression by targeting transcripts with differential expression similar to optix, where expression differs among red wing pattern elements of both the forewing and hindwing. This study is an analysis of two distinct datasets generated using the same microarray platform. One dataset involved comparative analysis of forewing sections of different color morphs, while the other compared whole hindwings with different color patterns. For the forewing analysis we compared proximal, medial, and distal wing sections of two color pattern morphs: H. erato petiverana and a hybrid H. himera x H. erato etylus. The proximal section in H. erato petiverana is black and the hybrid form orange/red, the medial section is red in H. erato petiverana and pale yellow in the hybrid form, and the distal section is black in both races. For the hindwing analysis, we compared hindwing color pattern gene expression in three races that meet in a hybrid zone in Peru. H. erato emma has a rayed hindwing, H. erato favorinus has a yellow-barred hindwing, and H. erato amphritrite has a black hindwing. Wings were dissected at five time intervals: 1, 3, and 5 days after pupation, when orange/red ommochrome pigments were beginning to be expressed (~7 days after pupation), and when black melanin pigments were starting to pepper the center of the wings (~8 days after pupation). In the forewings, Days 1, 3, and 5 were at 12, 36, and 60 hours post-pupation. In the hindwings these stages were sampled at 24, 48, and 72 hours. Samples hybridized to microarrays included three replicates each of each race, stage, and wing section for forewings (3 replicates x 2 morphs x 3 wing sections x 5 stages, with one replicate wing missing for Day 1 H. e. petiverana = 87 samples) and four replicates of each stage and race for hindwings (4 replicates x 3 races x 5 stages = 60 samples). Total RNA was extracted and converted to cDNA. Cy3-labeling of samples, hybridization, and array scanning was performed according to NimbleGen protocols (2008): for the forewings this was performed at the City of Hope Functional Genomics Core, while the hindwings were run separately at NCSU.Samples were hybridized to NimbleGen HD2 12-plex arrays. These arrays include 12 identical subarrays with 135,000 60 bp probes each, each hybridizing a separate sample. Samples were distributed across arrays to prevent repeat conditions as much as possible and to space similar conditions in different regions of the slide. The array design involved two classes of probes. First there was a tiling component involving 89,310 probes tiled across three genomic intervals. Results from the tiling data were used for the initial discovery of the optix gene and are not the focus of the present study. The second component involved a representation of a set of 12,450 transcript contigs at 1-6X coverage for a total of 40,046 probes, with a mean coverage of 3-4 probes per contig. The number of probes for each contig depended on the ability to create suitable probes according to NimbleGen probe selection criteria and was limited by the small size of some transcripts and the minimum spacing criterion of 15 bp apart. Sequences of low complexity and high repeats with the rest of the genome (>5X representation), determined by comparison against 1.6 MB of genomic sequence available at the time, were avoided for designing probes. An additional 3,248 random probes were placed on the array for quality control.

Project description:BackgroundUnravelling the genetic basis of polymorphic characters is central to our understanding of the origins and diversification of living organisms. Recently, supergenes have been implicated in a wide range of complex polymorphisms, from adaptive colouration in butterflies and fish to reproductive strategies in birds and plants. The concept of a supergene is now a hot topic in biology, and identification of its functional elements is needed to shed light on the evolution of highly divergent adaptive traits. Here, we apply different gene expression analyses to study the supergene P that controls polymorphism of mimetic wing colour patterns in the neotropical butterfly Heliconius numata.ResultsWe performed de novo transcriptome assembly and differential expression analyses using high-throughput Illumina RNA sequencing on developing wing discs of different H. numata morphs. Within the P interval, 30 and 17 of the 191 transcripts were expressed differentially in prepupae and day-1 pupae, respectively. Among these is the gene cortex, known to play a role in wing pattern formation in Heliconius and other Lepidoptera. Our in situ hybridization experiments confirmed the relationship between cortex expression and adult wing patterns.ConclusionsThis study found the majority of genes in the P interval to be expressed in the developing wing discs during the critical stages of colour pattern formation, and detect drastic changes in expression patterns in multiple genes associated with structural variants. The patterns of expression of cortex only partially recapitulate the variation in adult phenotype, suggesting that the remaining phenotypic variation could be controlled by other genes within the P interval. Although functional studies on cortex are now needed to determine its exact developmental role, our results are in accordance with the classical supergene hypothesis, whereby several genes inherited together due to tight linkage control a major developmental switch.

Project description:BackgroundButterfly wing color patterns are an important model system for understanding the evolution and development of morphological diversity and animal pigmentation. Wing color patterns develop from a complex network composed of highly conserved patterning genes and pigmentation pathways. Patterning genes are involved in regulating pigment synthesis however the temporal expression dynamics of these interacting networks is poorly understood. Here, we employ next generation sequencing to examine expression patterns of the gene network underlying wing development in the nymphalid butterfly, Vanessa cardui.ResultsWe identified 9, 376 differentially expressed transcripts during wing color pattern development, including genes involved in patterning, pigmentation and gene regulation. Differential expression of these genes was highest at the pre-ommochrome stage compared to early pupal and late melanin stages. Overall, an increasing number of genes were down-regulated during the progression of wing development. We observed dynamic expression patterns of a large number of pigment genes from the ommochrome, melanin and also pteridine pathways, including contrasting patterns of expression for paralogs of the yellow gene family. Surprisingly, many patterning genes previously associated with butterfly pattern elements were not significantly up-regulated at any time during pupation, although many other transcription factors were differentially expressed. Several genes involved in Notch signaling were significantly up-regulated during the pre-ommochrome stage including slow border cells, bunched and pebbles; the function of these genes in the development of butterfly wings is currently unknown. Many genes involved in ecdysone signaling were also significantly up-regulated during early pupal and late melanin stages and exhibited opposing patterns of expression relative to the ecdysone receptor. Finally, a comparison across four butterfly transcriptomes revealed 28 transcripts common to all four species that have no known homologs in other metazoans.ConclusionsThis study provides a comprehensive list of differentially expressed transcripts during wing development, revealing potential candidate genes that may be involved in regulating butterfly wing patterns. Some differentially expressed genes have no known homologs possibly representing genes unique to butterflies. Results from this study also indicate that development of nymphalid wing patterns may arise not only from melanin and ommochrome pigments but also the pteridine pigment pathway.

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Transcriptome analysis reveals novel patterning and pigmentation genes underlying Heliconius butterfly wing pattern variation.

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Transcriptome analysis reveals novel patterning and pigmentation genes underlying Heliconius butterfly wing pattern variation.

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