THE DEVELOPMENTAL ARCHITECTURE OF WING PATTERN VARIATION IN HELICONIUS ERATO

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0210117
• Project Start Date: Jan 1, 2007
• Project End Date: Sep 30, 2012
• Program Code: [(N/A)]- (N/A)

Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695

Performing Department
GENETICS

Non Technical Summary
There is a need to move beyond model systems and more directly explore the mechanisms by which developmental pathways diverge and generate morphological variation. We know little about how changes in genetic regulatory interactions cause morphological diversification within and among very closely related species and nothing about how these interactions are modified during an evolutionary radiation. Such information is critical to fully link developmental and evolutionary processes and fully understand the connection between micro- and macroevolutionary change. This research aims to develop butterflies of the genus Heliconius into a genetic model system to study the interface between development and adaptive change.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
304	3110	1080	100%

Knowledge Area
304 - Animal Genome;

Subject Of Investigation
3110 - Insects;

Field Of Science
1080 - Genetics;

Keywords

heliconius erato

butterfly

butterflies

adaptive change

wing pattern

color pattern

genetic architecture

adaptive radiation

pattern formation

microarray

lepidoptera

linkage map

Goals / Objectives
The long-term goal of this study is to understand the developmental basis of phenotypic evolution in Heliconius and how the striking convergent evolution of the muellerian co-mimics, H. erato and H. melpomene, is achieved. Both species have undergone a remarkable parallel radiation into some 23 different geographic races, with at least 10 distinctive color patterns. In the present study, we will develop a gene chip for H. erato composed of genes expressed during wing development and use it to study the temporal and spatial variation in gene expression during pattern development. These data will allow us to correlate differences in gene expression with differences in wing pigmentation both within and between different genetic populations. We will use this powerful new resource to address the following specific questions. (1) What are the patterns of gene expression that are associated with differences in pigmentation? The mimicry genes of Heliconius switch between alternative pigments in discrete regions of the wing. These regions are large, differ between genotypes, and involve known pigment biosynthetic pathways. We will analyze the temporal patterns of gene expression characteristic of each pattern element, and use this information to identify genes associated with pattern formation and pattern diversity in Heliconius. (2) What can patterns of variation and co-variation in gene expression tell us about the genetic control of pattern formation? The regulatory pathways that control pigmentation include upstream patterning genes and downstream genes that control the synthesis, uptake and stabilization of pigment precursors and pigments. By analyzing how expression patterns of candidate genes are clustered through time and space we will identify gene associations that are characteristic for each pattern element and location on the wing, and study how these associations change during pattern ontogeny in different elements of the color pattern. (3) What are the fine-scale patterns of gene expression of candidate loci identified through the in-silica analysis of gene expression patterns and what is their linkage relationship to "mimicry" genes? Our analysis of gene expression will identify a suite of new candidate loci and provide an important first step for elucidating gene interactions through pattern development. Based on these results, we will conduct further detailed spatial/temporal expression experiments using quantitative RT-PCR and in-situ hybridization screens. These experiments will validate conclusions and test hypotheses about pattern development drawn from our initial microarray experiments. Furthermore, we will place a subset of these candidate loci onto our growing genetic map and determine their linkage relationship relative to the known "mimicry" genes that underlie the H. erato adaptive radiation.

Project Methods
We have produced a Heliconius microarray to analyze gene expression patterns throughout wing development. The observed expression profiles will be used to i) identify genes associated with pattern formation and change, ii) reconstruct interactions between patterning genes and genes in pigment synthesis, uptake and stabilization pathways, and iii) understand how these interactions vary among genetic populations, across different areas of the wing, and throughout the ontogeny of pattern development. We have three major research objectives: Objective 1: Establish a database of genes expressed during wing disc development and create a wing disc microarray for gene expression profiling in H. erato. Objective 2: Determine correlated gene expression patterns and temporal gene expression changes in forewings of wing color pattern variants. We will conduct a series of temporal and spatial microarray experiments to elucidate the genetic networks controlling synthesis of alternative pigments on homologous areas of the wing. We are interested in the genetic mechanisms that specify regional wing color. The targeted phenotypes differ in allele variation at two major unlinked color pattern loci, the Dry and Cr loci. Both loci act on the forewing and hindwing pattern elements and control the deposition of red, yellow, and melanic pigments (Dry) or white, yellow, and melanic pigments (Cr). We will examine gene expression across the wing surface, and how these expression profiles vary across different wing pattern phenotypes and through development. We will dissect pattern elements from the three areas of forewing and collect tissue samples at seven time periods, beginning at the mid fifth-instar stage and extending through the final stages of pattern development. Forewing discs will be dissected from developing larvae and pupae at seven time intervals through wing development. RNA will be extracted from wing sections and fluorescently labeled. We will follow our routine strategy for capture, normalization, and analysis of microarray data. We will test for significant gene expression changes among regions, among races, and among time points. Our data will consist of differential gene expression patterns between successive developmental time points, in different areas of the wing. We will use clustering algorithms to discover sets of genes with expression associated with ontogeny of each portion of the pattern. Clustering diagrams will be used to determine how clusters that contain genes of interest change, and whether candidate genes later in ontogeny are added to clusters that contained early patterning genes. Objective 3: Validate expression patterns using rtPCR, in situs and candidate gene mapping. In Heliconius, large size and easy accessibility of the wings facilitate validation, allowing individuals to be resampled, thus increasing the accuracy of temporal sampling. We will examine the spatial expression patterns of a subset of potential candidate genes identified in the clustering studies, using in-situ RNA hybridization in Heliconius wing discs. Additionally, we will place 60-100 patterning candidate loci onto our linkage map of the H. erato radiation.

Progress 01/01/07 to 09/30/12

Outputs
OUTPUTS: Outputs of the research were primarily disseminated through peer-reviewed publications and presentations a scientific conferences. Undergraduate and graduate student training and postdoctoral scholar mentoring were also used to disseminate the results from the research activities. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Research outcome focused on the genetic and developmental basis of wing pattern variation in Heliconius butterflies. Heliconius is a species-rich group of tropical butterflies characterized by dramatic variation in their vividly colored wing patterns and is an ideal system to study the interface between genomes, development, and the ecology and evolution of functional diversity. This natural diversity was exploited through growing genomic resources, and emerging genomic technologies to directly explore how adaptive phenotypes evolve and influence the architecture of genomic variation within natural populations. Remarkably, we found that few genomic intervals control much of the color pattern variation across the genus. These intervals behave as genomic "hotspots" of evolution that regulate both convergent and highly divergent wing pattern variation within and between species. We also positionally cloned these "hotspots" and used a combination of approaches to identify the molecular changes that cause phenotypic variation, linking them to the gene networks responsible for the formation of wing patterns, and gained a functional understanding of how they work to modulate pattern variation.

Publications

• Diversification of complex butterfly wing patterns by repeated regulatory evolution of a Wnt ligand. Martin A, Papa R, Nadeau NJ, Hill RI, Counterman BA, Halder G, Jiggins CD, Kronforst MR, Long AD, McMillan WO, Reed RD. Proc Natl Acad Sci U S A. 2012 Jul 31;109(31):12632-7.
• Transcriptome analysis reveals novel patterning and pigmentation genes underlying Heliconius butterfly wing pattern variation. Hines HM, Papa R, Ruiz M, Papanicolaou A, Wang C, Nijhout HF, McMillan WO, Reed RD. BMC Genomics. 2012 Jun 29;13(1):288.
• Adaptive introgression across species boundaries in Heliconius butterflies. Pardo-Diaz C, Salazar C, Baxter SW, Merot C, Figueiredo-Ready W, Joron M, McMillan WO, Jiggins CD. PLoS Genet. 2012 Jun;8(6):e1002752.
• Wing patterning gene redefines the mimetic history of Heliconius butterflies. Hines HM, Counterman BA, Papa R, Albuquerque de Moura P, Cardoso MZ, Linares M, Mallet J, Reed RD, Jiggins CD, Kronforst MR, McMillan WO. Proc Natl Acad Sci U S A. 2011 Dec 6;108(49):19666-71.
• optix drives the repeated convergent evolution of butterfly wing pattern mimicry. Reed RD, Papa R, Martin A, Hines HM, Counterman BA, Pardo-Diaz C, Jiggins CD, Chamberlain NL, Kronforst MR, Chen R, Halder G, Nijhout HF, McMillan WO. Science. 2011 Aug 26;333(6046):1137-41.
• Highly conserved gene order and numerous novel repetitive elements in genomic regions linked to wing pattern variation in Heliconius butterflies. Papa R, Morrison CM, Walters JR, Counterman BA, Chen R, Halder G, Ferguson L, Chamberlain N, Ffrench-Constant R, Kapan DD, Jiggins CD, Reed RD, McMillan WO. BMC Genomics. 2008 Jul 22;9:345.
• Gene expression underlying adaptive variation in Heliconius wing patterns: non-modular regulation of overlapping cinnabar and vermilion prepatterns. Reed RD, McMillan WO, Nagy LM. Proc Biol Sci. 2008 Jan 7;275(1630):37-45.
• Mate preference across the speciation continuum in a clade of mimetic butterflies. Merrill RM, Gompert Z, Dembeck LM, Kronforst MR, McMillan WO, Jiggins CD. Evolution. 2011 May;65(5):1489-500.
• Dissecting comimetic radiations in Heliconius reveals divergent histories of convergent butterflies. Quek SP, Counterman BA, Albuquerque de Moura P, Cardoso MZ, Marshall CR, McMillan WO, Kronforst MR. Proc Natl Acad Sci U S A. 2010 Apr 20;107(16):7365-70.
• Genomic hotspots for adaptation: the population genetics of Mullerian mimicry in Heliconius erato. Counterman BA, Araujo-Perez F, Hines HM, Baxter SW, Morrison CM, Lindstrom DP, Papa R, Ferguson L, Joron M, Ffrench-Constant RH, Smith CP, Nielsen DM, Chen R, Jiggins CD, Reed RD, Halder G, Mallet J, McMillan WO. PLoS Genet. 2010 Feb 5;6(2):e1000796.
• Genomic hotspots for adaptation: the population genetics of Mullerian mimicry in the Heliconius melpomene clade. Baxter SW, Nadeau NJ, Maroja LS, Wilkinson P, Counterman BA, Dawson A, Beltran M, Perez-Espona S, Chamberlain N, Ferguson L, Clark R, Davidson C, Glithero R, Mallet J, McMillan WO, Kronforst M, Joron M, Ffrench-Constant RH, Jiggins CD. PLoS Genet. 2010 Feb 5;6(2):e1000794.
• Convergent evolution in the genetic basis of Mullerian mimicry in heliconius butterflies. Baxter SW, Papa R, Chamberlain N, Humphray SJ, Joron M, Morrison C, ffrench-Constant RH, McMillan WO, Jiggins CD. Genetics. 2008 Nov;180(3):1567-77.
• Butterfly genomics eclosing. Beldade P, McMillan WO, Papanicolaou A. Heredity (Edinb). 2008 Feb;100(2):150-7.

Progress 10/01/07 to 09/30/08

Outputs
OUTPUTS: The goal of this grant was to develop a gene chip for H. erato composed of genes expressed during wing development and use it to study the temporal and spatial variation in gene expression during pattern development. With this goal in mind, we have focused research efforts along several complementary fronts including: Characterizing genes expressed during H. erato wing development- Over the past year, we extended our original EST screen and are awaiting data from a 454 run on normalized cDNA products. The transcriptome pool for our 454 analysis was made from mRNA expressed though wing disc development in several different pattern races of H. erato. The 454 run is expected to produce roughly 200,000,000 bases of sequence data (in 200 pb reads) or about 20 times the size of our current EST dataset. These new data will greatly extend gene discovery and ensure that we describe as finely as possible the transcriptome of the developing butterfly wing. The sequences will be made publicly available on our dedicated and web-accessible database, ButterflyBase (via http://www.heliconius.org). Positional cloning of color pattern genes- Using a combination of bulk segregant analysis and fine-scale mapping, we have localize two color pattern loci, D and Cr, to two roughly 300kb genomic intervals. We are currently examining variation across genes in these intervals to explore patterns of LD in natural populations of Heliconius erato and to look for nucleotide positions associated with particular phenotypes. Studying gene expression- We have performed the first microarray experiment looking at gene expression patterns through development. The experiment was a medium-sized pilot array project examining gene expression profiles through hindwing development of two races of H. erato (H. e. petiverana and H. e. hydara) that differ in the presence of a yellow bar. The two races hybridize in Panama and the phenotypic differences are caused by allelic substitution at the Cr locus. Our experiment assayed expression changes in the full complement of putative gene objects identified in our EST scan. In addition, we examined expression across a genomic tile path that covered the entire Cr interval. We are waiting for the new 454 transcriptome data to perform the second and larger microarray experiment described in the grant. The new technology greatly extended gene discovery and we felt that it was prudent to delay this experiment somewhat to guarantee that we produced the highest quality final product. Examining variation in gene expression in candidate genes- We are screening expression patterns of candidate genes. These genes include i) putative open reading frames identified in our two genomic intervals that contain the major color pattern switch genes in H. erato and ii) loci identified in our EST screen, including patterning genes and pigment synthesis genes. We are continuing to refine methods for batch-wise semi-high-throughput in situ expression analysis. PARTICIPANTS: Senior Personnel: McMillan, W. Owen Nijhout, H. Frederik Halder, Georg Chen, Rui Reed, Robert Postdocs: Papa, Riccardo Counterman, Brian Graduate Student Morrison, Clay Merchan, H. Alejandro Maldonaldo, Karla Araugo Perez, Felix Laurin Dembeck Undergraduate Students: Vinnie Izzi Kenda Freeman Organizational Partners: Baylor College of Medicine University of Texas, M.D. Anderson Cancer Center University of Edinburgh Smithsonian Tropical Research Institute Cambridge University University of California Irvine Duke University Training and development: The project is both technically and intellectually challenging and over the past year we have trained several new students in modern genomic techniques and provided them with a solid background in Heliconus ecology. Several students are worth mentioning: Karla Maldonaldo is a MS student at the University of Puerto Rico. She has largely finished collecting the molecular and morphological data set that will produce the first QTL analysis of Heliconius color pattern genes. Karla spent a semester at NCSU and was part of a Student Exchange Program developed in several NSF grants. Felix Arajo Perez is a also MS student at the University of Puerto Rico. He similarly spent a semester away from UPR at NCSU. He is measuring linkage disequilibrium among loci in individuals collected on either end of a narrow hybrid zone between two Peruvian races of H. erato, H. e. flavorinus and H. e. emma. H. Alejandro Mechan is a Ph. D. student at the North Carolina State University and is looking at the dvelopmental genetics of convergent pattern change. He spent last semester at the Heliconius Facility at the Smithsonian Tropical Research Institute, in Panama. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
A primary research objective is to identify the genomic regions that modulate color pattern variation in H. erato and to link changes in gene expression across these regions to changes in gene expression in the pigment (and scale maturation) pathways that are ultimately responsible for patterning the adult wing. We have focused our positional cloning efforts on two major color pattern loci, D/r and Cr. The two are unlinked and regulate different aspects of the patterning machinery. We have made great progress in identifying zero recombinant windows and obtaining BAC sequences spanning these regions for both gene regions. For both regions, this window is roughly 300kb in size and contains a number of interesting "candidate" genes. We have now generated quantitative expression profiles for most obvious candidate genes across the two regions. In our sampling, we have examined gene expression both through wing development and across different wing pattern races of H. erato. Although most genes show strong expression changes through development, only unkempt shows significant differences between racial types and thus is a good candidate for modulating adaptive differences in wing pattern (Papa et al., in prep). Most of what is known about butterfly wing pattern development concerns eyespot color patterns. At least eight different proteins involved with signaling and/or transcriptional regulation have been implicated in eyespot development. This line of research has produced a body of high-profile publications, and the eyespots have become a textbook example of evo-devo research. To date, however, very little remains known about the developmental basis of the proximal-distal stripe patterns - also known as the symmetry systems - that underlie the majority of butterfly color patterns. Indeed, it appears that most or all of the color patterns in Heliconius are derived from this stripe patterning system. We found that a Pax 3/7 homeodomain transcription factor is expressed in a stripe pattern on the forewing of Heliconius and other butterflies. Because the expression pattern is not perfectly associated with a specific color pattern we speculate that it is a pre-pattern selector gene that specifies a region in the middle of the proximal-distal axis. In this respect, it may be significant that the proximal boundary of the Pax 3/7 expression domain marks a conserved color pattern boundary within the discal cell of Heliconius. This boundary is important for three reasons: (1) it is the distal edge of the red "dennis" color pattern controlled by the D locus, (2) it marks a color pattern boundary that is highly conserved across the whole genus Heliconius, and (3) it represents the first molecular evidence for a whole-wing proximal-distal pattern formation system in butterfly wings. We are now working to produce an expression pattern time series from multiple butterfly species, and to analyze transcription of this gene through in situ hybridization.

Publications

• Papa, R., Clayton Morrison, J. R. Walters, B. A. Counterman, R. Chen, G. Halder, L. Roberts, D. D. Kapan, C. D. Jiggins, R. D. Reed, and W. O. McMillan. (2008) Highly conserved gene order and numerous novel repetitive elements in genomic regions linked to wing pattern variation in Heliconius butterflies. BMC Genomics 9: 345.
• Baxter, S., R. Papa, N. Chamberlain, S. J. Humphray, M. Joron, R. H. ffrench-Constant, W. O. McMillan and C. D. Jiggins. (2008) Convergent evolution in the genetic basis of Mullerian mimicry in Heliconius butterflies. Genetics 180: 1567.
• Reed, R. D., W. O. McMillan, and L. M. Nagy. (2008) Gene expression underlying adaptive variation in Heliconius wing patterns: non-modular regulation of overlapping cinnabar and vermilion patterns. Proceedings of the Royal Society of London, Series B 275: 37.
• Reed, R.D. P.-H. Chen H.F. Nijhout. (2007) Cryptic variation in butterfly eyespot development: the importance of sample size in gene expression studies. Evolution & Development 9:2.
• Papanicolaou, A., S. Gebauer-Jung, M. L. Blaxter, W. O. McMillan and C. D. Jiggins. (2007) ButterflyBase: a platform for Lepidoptera genomics. Nucleic Acids Research 36: D582.
• Beldade, P., A. Papanicolaou, and W. O. McMillan. (2007) Butterfly Genomics Eclosing. Heredity 10:150.