Michael W. Perry

Multiple enhancers ensure precision of gap gene-expression patterns in the Drosophila embryo

Segmentation of the Drosophila embryo begins with the establishment of spatially restricted gap gene-expression patterns in response to broad gradients of maternal transcription factors, such as Bicoid. Numerous studies have documented the fidelity of these expression patterns, even when embryos are subjected to genetic or environmental stress, but the underlying mechanisms for this transcriptional precision are uncertain. Here we present evidence that every gap gene contains multiple enhancers with overlapping activities to produce authentic patterns of gene expression. For example, a recently identified hunchback (hb) enhancer (located 5-kb upstream of the classic enhancer) ensures repression at the anterior pole. The combination of intronic and 5′ knirps (kni) enhancers produces a faithful expression pattern, even though the intronic enhancer alone directs an abnormally broad expression pattern. We present different models for “enhancer synergy,” whereby two enhancers with overlapping activities produce authentic patterns of gene expression.

Neural-specific elongation of 3′ UTRs during Drosophila development

The 3′ termini of eukaryotic mRNAs influence transcript stability, translation efficiency, and subcellular localization. Here we report that a subset of developmental regulatory genes, enriched in critical RNA-processing factors, exhibits synchronous lengthening of their 3′ UTRs during embryogenesis. The resulting UTRs are up to 20-fold longer than those found on typical Drosophila mRNAs. The large mRNAs emerge shortly after the onset of zygotic transcription, with several of these genes acquiring additional, phased UTR extensions later in embryogenesis. We show that these extended 3′ UTR sequences are selectively expressed in neural tissues and contain putative recognition motifs for the translational repressor, Pumilio, which also exhibits the 3′ lengthening phenomenon documented in this study. These findings suggest a previously unknown mode of posttranscriptional regulation that may contribute to the complexity of neurogenesis or neural function.

Enhancer additivity and non-additivity are determined by enhancer strength in the Drosophila embryo.

Metazoan genes are embedded in a rich milieu of regulatory information that often includes multiple enhancers possessing overlapping activities. In this study, we employ quantitative live imaging methods to assess the function of pairs of primary and shadow enhancers in the regulation of key patterning genes-knirps, hunchback, and snail-in developing Drosophila embryos. The knirps enhancers exhibit additive, sometimes even super-additive activities, consistent with classical gene fusion studies. In contrast, the hunchback enhancers function sub-additively in anterior regions containing saturating levels of the Bicoid activator, but function additively in regions where there are diminishing levels of the Bicoid gradient. Strikingly sub-additive behavior is also observed for snail, whereby removal of the proximal enhancer causes a significant increase in gene expression. Quantitative modeling of enhancer–promoter interactions suggests that weakly active enhancers function additively while strong enhancers behave sub-additively due to competition with the target promoter. DOI: http://dx.doi.org/10.7554/eLife.07956.001

Molecular logic behind the three-way stochastic choices that expand butterfly colour vision

Butterflies rely on color vision extensively to adapt to the natural world. Most species express a broad range of color sensitive Rhodopsins in three stochastically distributed types of ommatidia (unit eyes)1–3. The retinas of Drosophila deploy just two main types, where fate is controlled by the binary stochastic decision to express the transcription factor Spineless (Ss) in R7 photoreceptors4. We investigated how butterflies instead generate three stochastically distributed ommatidial types, resulting in a more diverse retinal mosaic that provides the basis for additional color comparisons and an expanded range of color vision. We show that the Japanese Yellow Swallowtail (Papilio xuthus, Papilionidae) and the Painted Lady (Vanessa cardui, Nymphalidae) have a second R7-like photoreceptor in each ommatidium. Independent stochastic expression of Ss in each R7-like cell results in expression of a Blue (Ss-ON) or a UV (Ss-OFF) Rhodopsin. In Papilio, these choices of Blue/Blue, Blue/UV, or UV/UV in the two R7s are coordinated with expression of additional Rhodopsins in the remaining photoreceptors, and together define the three types of ommatidia. Knocking out ss using CRISPR/Cas95,6 leads to the loss of the Blue fate in R7-like cells and transforms retinas into homogeneous fields of UV/UV-type ommatidia, with all corresponding features. Hence, the three possible outcomes of Ss expression define the three ommatidial types in butterflies. This developmental strategy allowed the deployment of an additional red-sensitive Rhodopsin in Papilio, allowing for the evolution of expanded color vision with a richer variety of receptors7,8. This surprisingly simple mechanism that makes use of two binary stochastic decisions coupled with local coordination may prove to be a general means of generating an increased diversity of developmental outcomes.Butterflies rely extensively on colour vision to adapt to the natural world. Most species express a broad range of colour-sensitive Rhodopsin proteins in three types of ommatidia (unit eyes), which are distributed stochastically across the retina. The retinas of Drosophila melanogaster use just two main types, in which fate is controlled by the binary stochastic decision to express the transcription factor Spineless in R7 photoreceptors. We investigated how butterflies instead generate three stochastically distributed ommatidial types, resulting in a more diverse retinal mosaic that provides the basis for additional colour comparisons and an expanded range of colour vision. We show that the Japanese yellow swallowtail (Papilio xuthus, Papilionidae) and the painted lady (Vanessa cardui, Nymphalidae) butterflies have a second R7-like photoreceptor in each ommatidium. Independent stochastic expression of Spineless in each R7-like cell results in expression of a blue-sensitive (SpinelessON) or an ultraviolet (UV)-sensitive (SpinelessOFF) Rhodopsin. In P. xuthus these choices of blue/blue, blue/UV or UV/UV sensitivity in the two R7 cells are coordinated with expression of additional Rhodopsin proteins in the remaining photoreceptors, and together define the three types of ommatidia. Knocking out spineless using CRISPR/Cas9 (refs 5, 6) leads to the loss of the blue-sensitive fate in R7-like cells and transforms retinas into homogeneous fields of UV/UV-type ommatidia, with corresponding changes in other coordinated features of ommatidial type. Hence, the three possible outcomes of Spineless expression define the three ommatidial types in butterflies. This developmental strategy allowed the deployment of an additional red-sensitive Rhodopsin in P. xuthus, allowing for the evolution of expanded colour vision with a greater variety of receptors. This surprisingly simple mechanism that makes use of two binary stochastic decisions coupled with local coordination may prove to be a general means of generating an increased diversity of developmental outcomes.

An Engineered orco Mutation Produces Aberrant Social Behavior and Defective Neural Development in Ants

Ants exhibit cooperative behaviors and advanced forms of sociality that depend on pheromone-mediated communication. Odorant receptor neurons (ORNs) express specific odorant receptors (ORs) encoded by a dramatically expanded gene family in ants. In most eusocial insects, only the queen can transmit genetic information, restricting genetic studies. In contrast, workers in Harpegnathos saltator ants can be converted into gamergates (pseudoqueens) that can found entire colonies. This feature facilitated CRISPR-Cas9 generation of germline mutations in orco, the gene that encodes the obligate co-receptor of all ORs. orco mutations should significantly impact olfaction. We demonstrate striking functions of Orco in odorant perception, reproductive physiology, and social behavior plasticity. Surprisingly, unlike in other insects, loss of OR functionality also dramatically impairs development of the antennal lobe to which ORNs project. Therefore, the development of genetics in Harpegnathos establishes this ant species as a model organism to study the complexity of eusociality.

The evolutionary diversity of insect retinal mosaics: common design principles and emerging molecular logic.

Independent evolution has resulted in a vast diversity of eyes. Despite the lack of a common Bauplan or ancestral structure, similar developmental strategies are used. For instance, different classes of photoreceptor cells (PRs) are distributed stochastically and/or localized in different regions of the retina. Here, we focus on recent progress made towards understanding the molecular principles behind patterning retinal mosaics of insects, one of the most diverse groups of animals adapted to life on land, in the air, under water, or on the water surface. Morphological, physiological, and behavioral studies from many species provide detailed descriptions of the vast variation in retinal design and function. By integrating this knowledge with recent progress in the characterization of insect Rhodopsins as well as insight from the model organism Drosophila melanogaster, we seek to identify the molecular logic behind the adaptation of retinal mosaics to the habitat and way of life of an animal.

Genetic Basis of Melanin Pigmentation in Butterfly Wings

Butterfly wing patterns are a model system for studying the evolution and development of adaptive traits. Zhang et al. combine RNA-seq and CRISPR/Cas9... Despite the variety, prominence, and adaptive significance of butterfly wing patterns, surprisingly little is known about the genetic basis of wing color diversity. Even though there is intense interest in wing pattern evolution and development, the technical challenge of genetically manipulating butterflies has slowed efforts to functionally characterize color pattern development genes. To identify candidate wing pigmentation genes, we used RNA sequencing to characterize transcription across multiple stages of butterfly wing development, and between different color pattern elements, in the painted lady butterfly Vanessa cardui. This allowed us to pinpoint genes specifically associated with red and black pigment patterns. To test the functions of a subset of genes associated with presumptive melanin pigmentation, we used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing in four different butterfly genera. pale, Ddc, and yellow knockouts displayed reduction of melanin pigmentation, consistent with previous findings in other insects. Interestingly, however, yellow-d, ebony, and black knockouts revealed that these genes have localized effects on tuning the color of red, brown, and ochre pattern elements. These results point to previously undescribed mechanisms for modulating the color of specific wing pattern elements in butterflies, and provide an expanded portrait of the insect melanin pathway.

Generation and Evolution of Neural Cell Types and Circuits: Insights from the Drosophila Visual System

The Drosophila visual system has become a premier model for probing how neural diversity is generated during development. Recent work has provided deeper insight into the elaborate mechanisms that control the range of types and numbers of neurons produced, which neurons survive, and how they interact. These processes drive visual function and influence behavioral preferences. Other studies are beginning to provide insight into how neuronal diversity evolved in insects by adding new cell types and modifying neural circuits. Some of the most powerful comparisons have been those made to the Drosophila visual system, where a deeper understanding of molecular mechanisms allows for the generation of hypotheses about the evolution of neural anatomy and function. The evolution of new neural types contributes additional complexity to the brain and poses intriguing questions about how new neurons interact with existing circuitry. We explore how such individual changes in a variety of species might play a role over evolutionary timescales. Lessons learned from the fly visual system apply to other neural systems, including the fly central brain, where decisions are made and memories are stored.

Opposing feedbacks on ras tune receptor tyrosine kinase signaling

The identification of Rau reveals how signaling activity that directs neuronal and glial fate is maintained in the Drosophila eye. Signaling in development is not always on or off; often, distinct intensity and duration of signaling leads to distinct outcomes. This is true for receptor tyrosine kinase (RTK) signaling in many contexts, where negative feedback often plays a role. Although such negative feedback might reduce or even turn off signaling output over time, continued signaling is often maintained for proper cell fate specification. In this issue, Sieglitz et al. identify a positive regulator of Ras-mediated RTK signaling that they name Rau. Rau is necessary to achieve specific signaling intensity for the differentiation of photoreceptors and of glia that wrap axons in the developing Drosophila eye disc. Both the negative regulator Sprouty and Rau influence signaling through the guanosine triphosphatase Ras; specifically, Rau forms a positive feedback loop important for counteracting the Sprouty negative feedback loop.

A female alternative life-history strategy arose via novel recruitment of homeobox gene, BarH-1

Colias butterflies (the “clouded sulphurs”) often occur in mixed populations where females exhibit two color morphs, yellow/orange or white. White females, known as Alba [A-], reallocate resources from colored pigment synthesis to reproductive and somatic development. Due to this tradeoff Alba females develop faster and have higher fecundity than orange females. However while colored pigments are costly to produce, males preferentially mate with orange females and transfer nutrient rich spermatophores during mating. Thus the wing color morphs represent alternative life history strategies (ALHS) that are female-limited, wherein tradeoffs, due to divergent resource investment, result in distinct phenotypes with associated fitness consequences. Here we map the genetic basis of Alba in Colias croceus to a transposable element insertion downstream of the Colias homolog of BarH-1. We use CRISPR/Cas9 to validate BarH-1’s functional role in the wing color switch and antibody staining confirms expression differences in the scale building cells of pupal wings. We use scanning electron microscopy to determine that BarH-1 expression in the wings causes a reduction in pigment granules within wing scales, and thereby gives rise to the white color. Further lipid and transcriptome analyses reveal additional physiological differences that arise due to the Alba, suggesting pleiotropic effects beyond the wing color. While male-limited ALHS are well documented, comparatively few examples of female-limited ALHS are known. This is either due to biological reality or our lack of understanding of how ALHS manifest in females, highlighting the need for mechanistic insights, as none currently exist. These findings provide, to our knowledge, the first known mechanism for a female ALHS and support an alternative view of color polymorphism as indicative of pleiotropic effects with life history consequences.Tradeoffs affect resource allocation during development and result in fitness consequences that drive the evolution of life history strategies. Yet despite their importance, we know little about the mechanisms underlying life history tradeoffs in wild populations. Many species of Colias butterflies exhibit an alternative life history strategy (ALHS) where females divert resources from wing pigment synthesis to reproductive and somatic development. Due to this reallocation, a wing color polymorphism is associated with the ALHS: individuals have either yellow/orange or white wings. Here we map the genetic basis of the ALHS switch in Colias crocea to a transposable element insertion downstream of the Colias homolog of BarH-1, a homeobox transcription factor. Using CRISPR/Cas9 gene editing, antibody staining, and electron microscopy we find morph-specific specific expression of BarH-1 suppresses the formation of pigment granules in wing scales. Lipid and transcriptome analyses reveal physiological differences associated with the ALHS. These findings characterize a novel mechanism for a female-limited ALHS and show that the switch arises via recruitment of a transcription factor previously known for its function in cell fate determination in pigment cells of the retina.