A single origin for nymphalid butterfly eyespots followed by widespread loss of associated gene expression

Understanding how novel complex traits originate involves investigating the time of origin of the trait, as well as the origin of its underlying gene regulatory network in a broad comparative phylogenetic framework. The eyespot of nymphalid butterflies has served as an example of a novel complex trait, as multiple genes are expressed during eyespot development. Yet the origins of eyespots remain unknown. Using a dataset of more than 400 images of butterflies with a known phylogeny and gene expression data for five eyespot-associated genes from over twenty species, we tested origin hypotheses for both eyespots and eyespot-associated genes. We show that eyespots evolved once within the family Nymphalidae, approximately 90 million years ago, concurrent with expression of at least three genes associated with early eyespot development. We also show multiple losses of expression of most genes from this early three-gene cluster, without corresponding losses of eyespots. We propose that complex traits, such as eyespots, may have originated via co-option of a large pre-existing complex gene regulatory network that was subsequently streamlined of genes not required to fulfill its novel developmental function.     

Hox genes are essential for the development of eyespots in Bicyclus anynana butterflies

The eyespot patterns found on the wings of nymphalid butterflies are novel traits that originated first in hindwings and subsequently in forewings, suggesting that eyespot development might be dependent on Hox genes. Hindwings differ from forewings in the expression of Ultrabithorax (Ubx), but the function of this Hox gene in eyespot development as well as that of another Hox gene Antennapedia (Antp), expressed specifically in eyespots centers on both wings, are still unclear. We used CRISPR-Cas9 to target both genes in Bicyclus anynana butterflies. We show that Antp is essential for eyespot development on the forewings and for the differentiation of white centers and larger eyespots on hindwings, whereas Ubx is essential not only for the development of at least some hindwing eyespots but also for repressing the size of other eyespots. Additionally, Antp is essential for the development of silver scales in male wings. In summary, Antp and Ubx, in addition to their conserved roles in modifying serially homologous segments along the anterior–posterior axis of insects, have acquired a novel role in promoting the development of a new set of serial homologs, the eyespot patterns, in both forewings (Antp) and hindwings (Antp and Ubx) of B. anynana butterflies. We propose that the peculiar pattern of eyespot origins on hindwings first, followed by forewings, could be due to an initial co-option of Ubx into eyespot development followed by a later, partially redundant, co-option of Antp into the same network.     

以发育模块重复征用和整合为基础的进化创新机制

进化新征的起源和分化是进化发育生物学研究的核心问题。通过对多细胞生物早期发育调控机制的比较分析,发现亲缘关系较远的生物所共有的一些形态特征受保守的发育调控程序调节（深同源性）。许多创新性状的发生是基于对预先存在的基因或发育调控模块的重复利用和整合。发育基因调控网络在结构和功能上高度模块化,因此不仅可以通过模块拆分和重复征用改变发育程式,而且也增强了调控网络自身的进化力。研究基因调控网络和发育系统的进化动态将有助于更深入地认识生物演化过程中创新性状发生和表型进化的分子机制。     

Distinguishing serial homologs from novel traits: Experimental limitations and ideas for improvements

One of the central but yet unresolved problems in evolutionary biology concerns the origin of novel complex traits. One hypothesis is that complex traits derive from pre‐existing gene regulatory networks (GRNs) reused and modified to specify a novel trait somewhere else in the body. This simple explanation encounters problems when the novel trait that emerges in a body is in a region that is known to harbor a latent or repressed trait that has been silent for millions of years. Is the novel trait merely a re‐emerged de‐repressed trait or a truly novel trait that emerged via a novel deployment of an old GRN? A couple of new studies sided on opposite sides of this question when investigating the origin of horns in dung beetles and helmets in treehoppers that develop in the first thoracic segment (T1) of their bodies, a segment known to harbor a pair of repressed/modified wings in close relatives. Here, I point to some key limitations of the experimental approaches used and highlight additional experiments that could be done in future to resolve the developmental origin of these and other traits.     

Canadian regulatory aspects of gene editing technologies

The development of gene editing techniques, capable of producing plants and animals with new and improved traits, is revolutionizing the world of plant and animal breeding and rapidly advancing to commercial reality. However, from a regulatory standpoint the Government of Canada views gene editing as another tool that will join current methods used to develop desirable traits in plants and animals. This is because Canada focusses on the potential risk resulting from the novelty of the trait, or plant or animal product entering the Canadian environment or market place, rather than the process or method by which it was created. The Canadian Food Inspection Agency is responsible for the regulation of the environmental release of plants with novel traits, and novel livestock feeds, while Health Canada is responsible for the regulation of novel foods. Environment and Climate Change Canada, in partnership with Health Canada, regulates modified animals for entry into the environment. In all cases, these novel products may be the result of conventional breeding, mutagenesis, recombinant DNA techniques or other methods of plant or animal breeding such as gene editing. This novelty approach allows the Canadian regulatory system to efficiently adjust to any new developments in the science of plant and animal breeding and allows for risk-appropriate regulatory decisions. This approach encourages innovation while maintaining science-based regulatory expertise. Canadian regulators work cooperatively with proponents to determine if their gene editing-derived product meets the definition of a novel product, and whether it would be subject to a pre-market assessment. Therefore, Canada’s existing regulatory system is well positioned to accommodate any new innovations or technologies in plant or animal breeding, including gene editing.

     

Single locus affects embryonic segment polarity and multiple aspects of an adult evolutionary novelty

Background  

The characterization of the molecular changes that underlie the origin and diversification of morphological novelties is a key challenge in evolutionary developmental biology. The evolution of such traits is thought to rely largely on co-option of a toolkit of conserved developmental genes that typically perform multiple functions. Mutations that affect both a universal developmental process and the formation of a novelty might shed light onto the genetics of traits not represented in model systems. Here we describe three pleiotropic mutations with large effects on a novel trait, butterfly eyespots, and on a conserved stage of embryogenesis, segment polarity.     

An integer optimization algorithm for robust identification of non-linear gene regulatory networks

ABSTRACT: BACKGROUND: Reverse engineering gene networks and identifying regulatory interactions are integral to understanding cellular decision making processes. Advancement in high throughput experimental techniques has initiated innovative data driven analysis of gene regulatory networks. However, inherent noise associated with biological systems requires numerous experimental replicates for reliable conclusions. Furthermore, evidence of robust algorithms directly exploiting basic biological traits are few. Such algorithms are expected to be efficient in their performance and robust in their prediction. RESULTS: We have developed a network identification algorithm to accurately infer both the topology and strength of regulatory interactions from time series gene expression data in the presence of significant experimental noise and non-linear behavior. In this novel formulism, we have addressed data variability in biological systems by integrating network identification with the bootstrap resampling technique, hence predicting robust interactions from limited experimental replicates subjected to noise. Furthermore, we have incorporated non-linearity in gene dynamics using the S-system formulation. The basic network identification formulation exploits the trait of sparsity of biological interactions. Towards that, the identification algorithm is formulated as an integer-programming problem by introducing binary variables for each network component. The objective function is targeted to minimize the network connections subjected to the constraint of maximal agreement between the experimental and predicted gene dynamics. The developed algorithm is validated using both in-silico and experimental data-sets. These studies show that the algorithm can accurately predict the topology and connection strength of the in silico networks, as quantified by high precision and recall, and small discrepancy between the actual and predicted kinetic parameters. Furthermore, in both the in silico and experimental case studies, the predicted gene expression profiles are in very close agreement with the dynamics of the input data. CONCLUSIONS: Our integer programming algorithm effectively utilizes bootstrapping to identify robust gene regulatory networks from noisy, non-linear time-series gene expression data. With significant noise and non-linearities being inherent to biological systems, the present formulism, with the incorporation of network sparsity, is extremely relevant to gene regulatory networks, and while the formulation has been validated against in silico and E. Coli data, it can be applied to any biological system.     

Effects of ornamentation and phylogeny on the evolution of wing shape in stalk‐eyed flies (Diopsidae)

Exaggerated male ornaments are predicted to be costly to their bearers, but these negative effects may be offset by the correlated evolution of compensatory traits. However, when locomotor systems, such as wings in flying species, evolve to decrease such costs, it remains unclear whether functional changes across related species are achieved via the same morphological route or via alternate changes that have similar function. We conducted a comparative analysis of wing shape in relation to eye‐stalk elongation across 24 species of stalk‐eyed flies, using geometric morphometrics to determine how species with increased eye span, a sexually selected trait, have modified wing morphology as a compensatory mechanism. Using traditional and phylogenetically informed multivariate analyses of shape in combination with phenotypic trajectory analysis, we found a strong phylogenetic signal in wing shape. However, dimorphic species possessed shifted wing veins with the result of lengthening and narrowing wings compared to monomorphic species. Dimorphic species also had changes that seem unrelated to wing size, but instead may govern wing flexion. Nevertheless, the lack of a uniform, compensatory pattern suggests that stalk‐eyed flies used alternative modifications in wing structure to increase wing area and aspect ratio, thus taking divergent morphological routes to compensate for exaggerated eye stalks.     

Master regulatory genes; telling them what to do

In 1995, the eyeless (ey) gene was dubbed the "master-regulator" of eye development in Drosophila. Not only is ey required for eye development, but its misexpression can convert many other tissues into eye, including legs, wings and antennae.(1) ey is remarkable for its ability to drive coordinate differentiation of the multiple cell types that have to differentiate in a very precise pattern to construct the fly eye, and for its power to override the previous differentiation programs of many other diverse tissues. Even more remarkable, the ey homolog Pax6 and homologs of other eye determination genes from Drosophila are also required for eye development in vertebrates,(2,3) prompting reassessment of the evolution of vision throughout the animal kingdom.(4,5) Now Kumar and Moses have published a study that throws a new light on ey function in Drosophila.(6) According to their work, ey becomes a master regulator of eye development much later than previously thought, and is regulated by signalling through the Notch and EGFR signaling pathways.     

Morphological variability of intercastes in the ant Temnothorax nylanderi: pattern of trait expression and modularity

Ants have distinct morphological castes (queens and workers), but aberrant queen-worker “intercastes” occasionally occur, both in wild and laboratory conditions. Intercastes are rare, however, such novel phenotypes may have evolutionary significance. Their morphology is highly variable in any given species, providing valuable information about the integration of queen traits (e.g. ocelli, wings, complex segmentation of thorax, large gaster and ovaries, spermatheca). Generally, these traits are all diminished or absent in workers. We used multivariate morphometry to analyze an exceptionally large sample of 101 intercastes of Temnothorax nylanderi. We determined distributions and correlations of traits, and confirmed the mosaic nature of intercastes. Queen-specific traits are not expressed coherently in intercastes, but the possible patterns of trait combination are limited. A large number of small-sized intercastes had disproportionately larger head, ocelli and gaster but smaller thorax. In contrast, queen-like growth of thorax and rudimentary wings only occurred in large-sized intercastes. This is the most comprehensive analysis of intercaste variability, and suggests the existence of constraints on recombination of caste-specific modular traits.     

Co-option of male courtship signals from aggressive display in bowerbirds

The pre-existing trait hypothesis suggests that females evolve a mating preference for an already existing male trait. This hypothesis poses a simple resolution to Darwin's long-standing question of how elaborate, male display traits evolve. The frequently observed convergence of aggressive and courtship displays across a wide array of species provides the only current support for this hypothesis. Here we provide much more detailed supporting evidence from bowerbird skrraa calls used in aggression and courtship. Consistent with the pre-existing trait hypothesis we show that (i) putatively co-opted skrraa calls used in courtship and aggression are homologous, (ii) skrraa calls were used in aggression in bowerbirds before being used in courtship, (iii) historically, intense, aggressive-like courtship calls were present near the time of co-option, and (iv) bower types contemporaneous with co-option emphasize design features that provide females protection from the adverse effects of intense courtship displays. These results, plus evidence for a female preference for males with intense aggressive-like courtship skrraa calls, suggest that aggressive skrraa calls have been co-opted for use in male courtship display     

遗传基因组学(Genetical genomics)的研究进展

遗传基因组学(geneticalgenomics)是将微阵列技术和数量性状座位(QTL)分析结合起来,全基因组水平上定位基因表达的QTL(eQTL).它为研究复杂性状的分子机理和调控网络提供全新的手段.遗传基因组这个概念和研究策略在2001年由Janson和Nap首先提出,到目前为止,遗传基因组学已应用于酵母、老鼠、人以及玉米等植物.研究结果表明:基因表达水平的差异是可遗传的复杂性状;eQTL可以分为顺式作用eQTL和反式作用eQTL,顺式作用eQTL就是某个基因的eQTL定位到该基因所在的基因组区域,表明可能是该基因本身的差别引起mRNA水平的差别,反式作用就是eQTL定位到其他基因组区域,表明其他基因的差别控制该基因mRNA水平的差异.将eQTL结果、基因功能注解以及多种统计分析方法相结合,不仅能更准确地鉴别控制复杂性状及其相关基因表达的候选基因,而且能构建相应的基因调控网络.     

Evolution of regulatory interactions controlling floral asymmetry

A key challenge in evolutionary biology is to understand how new morphologies can arise through changes in gene regulatory networks. For example, floral asymmetry is thought to have evolved many times independently from a radially symmetrical ancestral condition, yet the molecular changes underlying this innovation are unknown. Here, we address this problem by investigating the action of a key regulator of floral asymmetry, CYCLOIDEA (CYC), in species with asymmetric and symmetric flowers. We show that CYC encodes a DNA-binding protein that recognises sites in a downstream target gene RADIALIS (RAD) in Antirrhinum. The interaction between CYC and RAD can be reconstituted in Arabidopsis, which has radially symmetrical flowers. Overexpression of CYC in Arabidopsis modifies petal and leaf development, through changes in cell proliferation and expansion at various stages of development. This indicates that developmental target processes are influenced by CYC in Arabidopsis, similar to the situation in Antirrhinum. However, endogenous RAD-like genes are not activated by CYC in Arabidopsis, suggesting that co-option of RAD may have occurred specifically in the Antirrhinum lineage. Taken together, our results indicate that floral asymmetry may have arisen through evolutionary tinkering with the strengths and pattern of connections at several points in a gene regulatory network.     

Exaggerated Trait Allometry,Compensation and Trade-Offs in the New Zealand Giraffe Weevil (Lasiorhynchus barbicornis)

Sexual selection has driven the evolution of exaggerated traits among diverse animal taxa. The production of exaggerated traits can come at a cost to other traits through trade-offs when resources allocated to trait development are limited. Alternatively some traits can be selected for in parallel to support or compensate for the cost of bearing the exaggerated trait. Male giraffe weevils (Lasiorhynchus barbicornis) display an extremely elongated rostrum used as a weapon during contests for mates. Here we characterise the scaling relationship between rostrum and body size and show that males have a steep positive allometry, but that the slope is non-linear due to a relative reduction in rostrum length for the largest males, suggesting a limitation in resource allocation or a diminishing requirement for large males to invest increasingly into larger rostra. We also measured testes, wings, antennae, fore- and hind-tibia size and found no evidence of a trade-off between these traits and rostrum length when comparing phenotypic correlations. However, the relative length of wings, antennae, fore- and hind-tibia all increased with relative rostrum length suggesting these traits may be under correlational selection. Increased investment in wing and leg length is therefore likely to compensate for the costs of flying with, and wielding the exaggerated rostrum of larger male giraffe weevils. These results provide a first step in identifying the potential for trait compensation and trades-offs, but are phenotypic correlations only and should be interpreted with care in the absence of breeding experiments.     

Integrating genetic and network analysis to characterize genes related to mouse weight

Systems biology approaches that are based on the genetics of gene expression have been fruitful in identifying genetic regulatory loci related to complex traits. We use microarray and genetic marker data from an F2 mouse intercross to examine the large-scale organization of the gene co-expression network in liver, and annotate several gene modules in terms of 22 physiological traits. We identify chromosomal loci (referred to as module quantitative trait loci, mQTL) that perturb the modules and describe a novel approach that integrates network properties with genetic marker information to model gene/trait relationships. Specifically, using the mQTL and the intramodular connectivity of a body weight–related module, we describe which factors determine the relationship between gene expression profiles and weight. Our approach results in the identification of genetic targets that influence gene modules (pathways) that are related to the clinical phenotypes of interest.     

Sensitivity and specificity of inferring genetic regulatory interactions from microarray experiments with dynamic Bayesian networks

MOTIVATION: Bayesian networks have been applied to infer genetic regulatory interactions from microarray gene expression data. This inference problem is particularly hard in that interactions between hundreds of genes have to be learned from very small data sets, typically containing only a few dozen time points during a cell cycle. Most previous studies have assessed the inference results on real gene expression data by comparing predicted genetic regulatory interactions with those known from the biological literature. This approach is controversial due to the absence of known gold standards, which renders the estimation of the sensitivity and specificity, that is, the true and (complementary) false detection rate, unreliable and difficult. The objective of the present study is to test the viability of the Bayesian network paradigm in a realistic simulation study. First, gene expression data are simulated from a realistic biological network involving DNAs, mRNAs, inactive protein monomers and active protein dimers. Then, interaction networks are inferred from these data in a reverse engineering approach, using Bayesian networks and Bayesian learning with Markov chain Monte Carlo. RESULTS: The simulation results are presented as receiver operator characteristics curves. This allows estimating the proportion of spurious gene interactions incurred for a specified target proportion of recovered true interactions. The findings demonstrate how the network inference performance varies with the training set size, the degree of inadequacy of prior assumptions, the experimental sampling strategy and the inclusion of further, sequence-based information. AVAILABILITY: The programs and data used in the present study are available from http://www.bioss.sari.ac.uk/~dirk/Supplements     

Efficient reverse-engineering of a developmental gene regulatory network

Understanding the complex regulatory networks underlying development and evolution of multi-cellular organisms is a major problem in biology. Computational models can be used as tools to extract the regulatory structure and dynamics of such networks from gene expression data. This approach is called reverse engineering. It has been successfully applied to many gene networks in various biological systems. However, to reconstitute the structure and non-linear dynamics of a developmental gene network in its spatial context remains a considerable challenge. Here, we address this challenge using a case study: the gap gene network involved in segment determination during early development of Drosophila melanogaster. A major problem for reverse-engineering pattern-forming networks is the significant amount of time and effort required to acquire and quantify spatial gene expression data. We have developed a simplified data processing pipeline that considerably increases the throughput of the method, but results in data of reduced accuracy compared to those previously used for gap gene network inference. We demonstrate that we can infer the correct network structure using our reduced data set, and investigate minimal data requirements for successful reverse engineering. Our results show that timing and position of expression domain boundaries are the crucial features for determining regulatory network structure from data, while it is less important to precisely measure expression levels. Based on this, we define minimal data requirements for gap gene network inference. Our results demonstrate the feasibility of reverse-engineering with much reduced experimental effort. This enables more widespread use of the method in different developmental contexts and organisms. Such systematic application of data-driven models to real-world networks has enormous potential. Only the quantitative investigation of a large number of developmental gene regulatory networks will allow us to discover whether there are rules or regularities governing development and evolution of complex multi-cellular organisms.     

Did sexually dimorphic dorsal coloration evolve by a pre-existing bias in males in the lizard Sceloporus minor?

Theory and empirical evidence indicate that male secondary sex traits can evolve by co-option of pre-existing biases in females. However, relatively few studies have explored whether male pre-existing biases could drive the evolution of traits important in male contests. Male spiny lizards (Sceloporus) are characterized by the expression of sexually dimorphic blue throat and abdominal patches. These features are revealed to conspecifics during social interactions, and variation in ventral color can predict the outcome of male contests in some species of spiny lizards. In Sceloporus minor, males in some populations also express bright blue color on the dorsal surfaces. Given the significance of blue color in intrasexual signaling in other species of Sceloporus, blue dorsal color may have evolved in S. minor by co-option of a male sensory bias for the color blue. We tested this hypothesis in a population that exhibits an ancestral phenotype for male dorsal color (brown/orange), and lacks males with bright blue dorsal coloration. Resident territorial males were presented with one of three types of intruder males manipulated in dorsal color by painting. Orange males mimicked the ancestral dorsal phenotype found at the study site; blue males resembled those from a population with the derived (blue) form of this trait; and green males represented a novel stimulus control. If blue dorsal color evolved in S. minor in part due to co-option of a male sensory bias, we predicted that resident males would exhibit either increased or decreased levels of aggression to blue intruders relative to controls. We found no difference in resident aggressive behavior across all treatments, thus failing to support the predictions of a pre-existing bias. We discuss these findings in the context of social behavior in Sceloporus, and propose directions for further study in this species.     

Gene-based association analysis for bivariate time-to-event data through functional regression with copula models

Several gene-based association tests for time-to-event traits have been proposed recently to detect whether a gene region (containing multiple variants), as a set, is associated with the survival outcome. However, for bivariate survival outcomes, to the best of our knowledge, there is no statistical method that can be directly applied for gene-based association analysis. Motivated by a genetic study to discover the gene regions associated with the progression of a bilateral eye disease, age-related macular degeneration (AMD), we implement a novel functional regression (FR) method under the copula framework. Specifically, the effects of variants within a gene region are modeled through a functional linear model, which then contributes to the marginal survival functions within the copula. Generalized score test statistics are derived to test for the association between bivariate survival traits and the genetic region. Extensive simulation studies are conducted to evaluate the type I error control and power performance of the proposed approach, with comparisons to several existing methods for a single survival trait, as well as the marginal Cox FR model using the robust sandwich estimator for bivariate survival traits. Finally, we apply our method to a large AMD study, the Age-related Eye Disease Study, and to identify the gene regions that are associated with AMD progression.