Ben Ewen-Campen

On the origin and evolutionary diversification of beetle horns

Many scarab beetles produce rigid projections from the body called horns. The exaggerated sizes of these structures and the staggering diversity of their forms have impressed biologists for centuries. Recent comparative studies using DNA sequence-based phylogenies have begun to reconstruct the historical patterns of beetle horn evolution. At the same time, developmental genetic experiments have begun to elucidate how beetle horns grow and how horn growth is modulated in response to environmental variables, such as nutrition. We bring together these two perspectives to show that they converge on very similar conclusions regarding beetle evolution. Horns do not appear to be difficult structures to gain or lose, and they can diverge both dramatically and rapidly in form. Although much of this work is still preliminary, we use available information to propose a conceptual developmental model for the major trajectories of beetle horn evolution. We illustrate putative mechanisms underlying the evolutionary origin of horns and the evolution of horn location, shape, allometry, and dimorphism.

In Vivo Transcriptional Activation Using CRISPR/Cas9 in Drosophila.

A number of approaches for Cas9-mediated transcriptional activation have recently been developed, allowing target genes to be overexpressed from their endogenous genomic loci. However, these approaches have thus far been limited to cell culture, and this technique has not been demonstrated in vivo in any animal. The technique involving the fewest separate components, and therefore the most amenable to in vivo applications, is the dCas9-VPR system, where a nuclease-dead Cas9 is fused to a highly active chimeric activator domain. In this study, we characterize the dCas9-VPR system in Drosophila cells and in vivo. We show that this system can be used in cell culture to upregulate a range of target genes, singly and in multiplex, and that a single guide RNA upstream of the transcription start site can activate high levels of target transcription. We observe marked heterogeneity in guide RNA efficacy for any given gene, and we confirm that transcription is inhibited by guide RNAs binding downstream of the transcription start site. To demonstrate one application of this technique in cells, we used dCas9-VPR to identify target genes for Twist and Snail, two highly conserved transcription factors that cooperate during Drosophila mesoderm development. In addition, we simultaneously activated both Twist and Snail to identify synergistic responses to this physiologically relevant combination. Finally, we show that dCas9-VPR can activate target genes and cause dominant phenotypes in vivo, providing the first demonstration of dCas9 activation in a multicellular animal. Transcriptional activation using dCas9-VPR thus offers a simple and broadly applicable technique for a variety of overexpression studies.

Notch/Delta signalling is not required for segment generation in the basally branching insect Gryllus bimaculatus

Arthropods and vertebrates display a segmental body organisation along all or part of the anterior-posterior axis. Whether this reflects a shared, ancestral developmental genetic mechanism for segmentation is uncertain. In vertebrates, segments are formed sequentially by a segmentation ‘clock’ of oscillating gene expression involving Notch pathway components. Recent studies in spiders and basal insects have suggested that segmentation in these arthropods also involves Notch-based signalling. These observations have been interpreted as evidence for a shared, ancestral gene network for insect, arthropod and bilaterian segmentation. However, because this pathway can play multiple roles in development, elucidating the specific requirements for Notch signalling is important for understanding the ancestry of segmentation. Here we show that Delta, a ligand of the Notch pathway, is not required for segment formation in the cricket Gryllus bimaculatus, which retains ancestral characteristics of arthropod embryogenesis. Segment patterning genes are expressed before Delta in abdominal segments, and Delta expression does not oscillate in the pre-segmental region or in formed segments. Instead, Delta is required for neuroectoderm and mesectoderm formation; embryos missing these tissues are developmentally delayed and show defects in segment morphology but normal segment number. Thus, what initially appear to be ‘segmentation phenotypes’ can in fact be due to developmental delays and cell specification errors. Our data do not support an essential or ancestral role of Notch signalling in segment generation across the arthropods, and show that the pleiotropy of the Notch pathway can confound speculation on possible segmentation mechanisms in the last common bilaterian ancestor.

Germ Cell Specification Requires Zygotic Mechanisms Rather Than Germ Plasm in a Basally Branching Insect

BACKGROUND Primordial germ cell (PGC) specification is a universal process across animals, but the molecular mechanisms specifying PGCs are remarkably diverse. In Drosophila, PGCs are specified by maternally provided, asymmetrically localized cytoplasmic factors (germ plasm). In contrast, historical literature on most other arthropods reports that PGCs arise from mesoderm during midembryogenesis, suggesting that an arthropod last common ancestor may have specified PGCs via zygotic mechanisms. However, there has been no direct experimental evidence to date for germ plasm-independent arthropod PGC specification. RESULTS Here we show that in a basally branching insect, the cricket Gryllus bimaculatus, conserved germ plasm molecules are ubiquitously, rather than asymmetrically, localized during oogenesis and early embryogenesis. Molecular and cytological analyses suggest that Gryllus PGCs arise from abdominal mesoderm during segmentation, and twist RNAi embryos that lack mesoderm fail to form PGCs. Using RNA interference we show that vasa and piwi are not required maternally or zygotically for PGC formation but rather are required for primary spermatogonial divisions in adult males. CONCLUSIONS These observations suggest that Gryllus lacks a maternally inherited germ plasm, in contrast with many holometabolous insects, including Drosophila. The mesodermal origin of Gryllus PGCs and absence of instructive roles for vasa and piwi in PGC formation are reminiscent of mouse PGC specification and suggest that zygotic cell signaling may direct PGC specification in Gryllus and other Hemimetabola.

Optimized strategy for in vivo Cas9-activation in Drosophila

Significance Recently, a number of approaches have been developed to repurpose the CRISPR/Cas9 system as a sequence-specific transcriptional activator for gain-of-function experiments (CRISPR activators, or “CRISPRa”). While multiple CRISPRa strategies have been characterized in cell culture, little is known about their performance in vivo. We present an optimized strategy for generating a large-scale CRISPRa resource in Drosophila and show that this system has a high success rate and generates easily recognizable phenotypes in vivo. We describe a growing collection of transgenic fly lines to facilitate large-scale in vivo CRISPRa experiments. While several large-scale resources are available for in vivo loss-of-function studies in Drosophila, an analogous resource for overexpressing genes from their endogenous loci does not exist. We describe a strategy for generating such a resource using Cas9 transcriptional activators (CRISPRa). First, we compare a panel of CRISPRa approaches and demonstrate that, for in vivo studies, dCas9-VPR is the most optimal activator. Next, we demonstrate that this approach is scalable and has a high success rate, as >75% of the lines tested activate their target gene. We show that CRISPRa leads to physiologically relevant levels of target gene expression capable of generating strong gain-of-function (GOF) phenotypes in multiple tissues and thus serves as a useful platform for genetic screening. Based on the success of this CRISRPa approach, we are generating a genome-wide collection of flies expressing single-guide RNAs (sgRNAs) for CRISPRa. We also present a collection of more than 30 Gal4 > UAS:dCas9-VPR lines to aid in using these sgRNA lines for GOF studies in vivo.

Next-generation CRISPR/Cas9 transcriptional activation in Drosophila using flySAM

Significance We present flySAM, a potent system for Cas9-based transcriptional activation (CRISPRa) in Drosophila. flySAM greatly improves on existing in vivo CRISPRa techniques in terms of potency, scalability, and ease of use, and provides a simple and general method for conducting overexpression experiments and screens. flySAM will now serve as the basis for our growing collection of publicly available CRISPRa transgenic fly lines. CRISPR/Cas9-based transcriptional activation (CRISPRa) has recently emerged as a powerful and scalable technique for systematic overexpression genetic analysis in Drosophila melanogaster. We present flySAM, a potent tool for in vivo CRISPRa, which offers major improvements over existing strategies in terms of effectiveness, scalability, and ease of use. flySAM outperforms existing in vivo CRISPRa strategies and approximates phenotypes obtained using traditional Gal4-UAS overexpression. Moreover, because flySAM typically requires only a single sgRNA, it dramatically improves scalability. We use flySAM to demonstrate multiplexed CRISPRa, which has not been previously shown in vivo. In addition, we have simplified the experimental use of flySAM by creating a single vector encoding both the UAS:Cas9-activator and the sgRNA, allowing for inducible CRISPRa in a single genetic cross. flySAM will replace previous CRISPRa strategies as the basis of our growing genome-wide transgenic overexpression resource, TRiP-OE.

Habitat Type is Related to Nest Mass and Fledging Success of Arctic Warblers

Abstract Relatively little is known about Arctic Warblers (Phylloscopus borealis) that breed in central Alaska. We monitored Arctic Warbler populations in two adjacent but distinct habitat types in central Alaska (high elevation, ‘open shrub’ and lower elevation, ‘dense shrub’). We collected 95 nests over three breeding seasons to learn more about nest-building behavior, nest mass, composition, fledging success, and nest parasites. Females were the sole builders of ground nests, which were primarily comprised of moss, grass, and a lining of moose (Alces alces) hair. Dry weight of nests was ∼20 g, but differed up to ∼3-fold within each habitat type each season. Nests from open shrub habitats were more massive and contained less moose hair lining than nests in dense shrub. Open shrub nests fledged more young during the most productive breeding season. We report the first record of the parasitic blowfly Protocalliphora tundrae in Arctic Warbler nests and for Alaska. Blowfly parasitism (55% of nests with hatchlings) was similar in both habitat types and did not correlate with fledging success, or nest mass. Nests with greater amounts of moss tended to have lower levels of blowfly infestation.

ovoD Co-selection: A Method for Enriching CRISPR/Cas9-Edited Alleles in Drosophila

Screening for successful CRISPR/Cas9 editing events remains a time consuming technical bottleneck in the field of Drosophila genome editing. This step can be particularly laborious for events that do not cause a visible phenotype, or those which occur at relatively low frequency. A promising strategy to enrich for desired CRISPR events is to co-select for an independent CRISPR event that produces an easily detectable phenotype. Here, we describe a simple negative co-selection strategy involving CRISPR-editing of a dominant female sterile allele, ovoD1. In this system (“ovoD co-selection”), the only functional germ cells in injected females are those that have been edited at the ovoD1 locus, and thus all offspring of these flies have undergone editing of at least one locus. We demonstrate that ovoD co-selection can be used to enrich for knock-out mutagenesis via nonhomologous end-joining (NHEJ), and for knock-in alleles via homology-directed repair (HDR). Altogether, our results demonstrate that ovoD co-selection reduces the amount of screening necessary to isolate desired CRISPR events in Drosophila.

04-P012 Germ line specification in the milkweed bug, Oncopeltus fasciatus (Hemiptera)

The body axes of many animals are initially patterned by the asymmetric distribution of maternal mRNAs and proteins in the oocyte. In Drosophila, these maternal factors are synthesized by the 15 nurse cells that connect to each oocyte within the ovary. However, insect ovaries fall into three anatomical categories, of which Drosophila represents only one. The telotrophic meroistic ovaries of the true bugs (Hemiptera), for example, possess a single group of syncytial nurse cells that connect to all oocytes simultaneously via extended nutritive tubes. The panoistic ovaries of basal insects including crickets (Orthoptera) do not possess nurse cells. Little is known about the production or localization of maternal factors within the developing oocytes of insects possessing these different ovary types. To address this issue, we have begun to characterize the ovarian and embryonic transcriptomes of a Hemipteran (the milkweed bug, Oncopeltus fasciatus) and an Orthopteran (the cricket Gryllus bimaculatus) using 454 pyrosequencing. Both of these species have rich histories as laboratory species, and both possess large ovaries that facilitate live imaging and microinjection. Our sequence data will allow us to identify orthologs of candidate genes involved in a variety of embryonic patterning processes, and will lay the groundwork for future studies on the development and evolution of insect body plans. Additionally, these sequences will augment the relatively few genomic resources available for hemimetabolous insects, thereby contributing to our understanding of genomic evolution during the 330 million years since the divergence of Hemimetabola and Holometabola from their last common ancestor.

04-P011 Oocyte patterning in non-model insects: Creating transcriptomes of the ovaries and embryos of two insect species using 454 sequencing

The body axes of many animals are initially patterned by the asymmetric distribution of maternal mRNAs and proteins in the oocyte. In Drosophila, these maternal factors are synthesized by the 15 nurse cells that connect to each oocyte within the ovary. However, insect ovaries fall into three anatomical categories, of which Drosophila represents only one. The telotrophic meroistic ovaries of the true bugs (Hemiptera), for example, possess a single group of syncytial nurse cells that connect to all oocytes simultaneously via extended nutritive tubes. The panoistic ovaries of basal insects including crickets (Orthoptera) do not possess nurse cells. Little is known about the production or localization of maternal factors within the developing oocytes of insects possessing these different ovary types. To address this issue, we have begun to characterize the ovarian and embryonic transcriptomes of a Hemipteran (the milkweed bug, Oncopeltus fasciatus) and an Orthopteran (the cricket Gryllus bimaculatus) using 454 pyrosequencing. Both of these species have rich histories as laboratory species, and both possess large ovaries that facilitate live imaging and microinjection. Our sequence data will allow us to identify orthologs of candidate genes involved in a variety of embryonic patterning processes, and will lay the groundwork for future studies on the development and evolution of insect body plans. Additionally, these sequences will augment the relatively few genomic resources available for hemimetabolous insects, thereby contributing to our understanding of genomic evolution during the 330 million years since the divergence of Hemimetabola and Holometabola from their last common ancestor.