Person:

Ewen-Campen, Benjamin

Most genomic resources available for insects represent the Holometabola, which are insects that undergo complete metamorphosis like beetles and flies. In contrast, the Hemimetabola (direct developing insects), representing the basal branches of the insect tree, have very few genomic resources. We have therefore created a large and publicly available transcriptome for the hemimetabolous insect Gryllus bimaculatus (cricket), a well-developed laboratory model organism whose potential for functional genetic experiments is currently limited by the absence of genomic resources. cDNA was prepared using mRNA obtained from adult ovaries containing all stages of oogenesis, and from embryos samples on each day of embryogenesis. Using 454 Titanium pyrosequencing, we sequenced over four million raw reads, and assembled them into 21,512 isotigs (predicted transcripts) and 120,805 singletons with an average coverage per base pair of 51.3. We annotated the transcriptome manually for over 400 conserved genes involved in embryonic patterning, gametogenesis, and signaling pathways. BLAST comparison of the transcriptome against the NCBI non-redundant protein database (nr) identified significant similarity to nr sequences for 55.5% of transcriptome sequences, and suggested that the transcriptome may contain 19,874 unique transcripts. For predicted transcripts without significant similarity to known sequences, we assessed their similarity to other orthopteran sequences, and determined that these transcripts contain recognizable protein domains, largely of unknown function. We created a searchable, web-based database to allow public access to all raw, assembled and annotated data. This database is to our knowledge the largest de novo assembled and annotated transcriptome resource available for any hemimetabolous insect. We therefore anticipate that these data will contribute significantly to more effective and higher-throughput deployment of molecular analysis tools in Gryllus.

Background: Arthropods are the most diverse animal phylum, but their genomic resources are relatively few. While the genome of the branchiopod Daphnia pulex is now available, no other large-scale crustacean genomic resources are available for comparison. In particular, genomic resources are lacking for the most tractable laboratory model of crustacean development, the amphipod Parhyale hawaiensis. Insight into shared and divergent characters of crustacean genomes will facilitate interpretation of future developmental, biomedical, and ecological research using crustacean models. Results: To generate a transcriptome enriched for maternally provided and zygotically transcribed developmental genes, we created cDNA from ovaries and embryos of P. hawaiensis. Using 454 pyrosequencing, we sequenced over 1.1 billion bases of this cDNA, and assembled them de novo to create, to our knowledge, the second largest crustacean genomic resource to date. We found an unusually high proportion of C2H2 zinc finger-containing transcripts, as has also been reported for the genome of the pea aphid Acyrthosiphon pisum. Consistent with previous reports, we detected trans-spliced transcripts, but found that they did not noticeably impact transcriptome assembly. Our assembly products yielded 19,067 unique BLAST hits against nr (E-value cutoff e-10). These included over 400 predicted transcripts with significant similarity to D. pulex sequences but not to sequences of any other animal. Annotation of several hundred genes revealed P. hawaiensis homologues of genes involved in development, gametogenesis, and a majority of the members of six major conserved metazoan signaling pathways. Conclusions: The amphipod P. hawaiensis has higher transcript complexity than known insect transcriptomes, and trans-splicing does not appear to be a major contributor to this complexity. We discuss the importance of a reliable comparative genomic framework within which to consider findings from new crustacean models such as D. pulex and P. hawaiensis, as well as the need for development of further substantial crustacean genomic resources.

Primordial germ cell (PGC) formation in holometabolous insects like Drosophila melanogaster relies on maternally synthesised germ cell determinants that are asymmetrically localised to the oocyte posterior cortex. Embryonic nuclei that inherit this "germ plasm" acquire PGC fate. In contrast, historical studies of basally branching insects (Hemimetabola) suggest that a maternal requirement for germ line genes in PGC specification may be a derived character confined principally to Holometabola. However, there have been remarkably few investigations of germ line gene expression and function in hemimetabolous insects. Here we characterise PGC formation in the milkweed bug Oncopeltus fasciatus, a member of the sister group to Holometabola, thus providing an important evolutionary comparison to members of this clade. We examine the transcript distribution of orthologues of 19 Drosophila germ cell and/or germ plasm marker genes, and show that none of them localise asymmetrically within Oncopeltus oocytes or early embryos. Using multiple molecular and cytological criteria we provide evidence that PGCs form after cellularisation at the site of gastrulation. Functional studies of vasa and tudor reveal that these genes are not required for germ cell formation, but that vasa is required in adult males for spermatogenesis. Taken together, our results provide evidence that Oncopeltus germ cells may form in the absence of germ plasm, consistent with the hypothesis that germ plasm is a derived strategy of germ cell specification in insects.

Germ cells occupy a unique position in animal reproduction, development, and evolution. In sexually reproducing animals, only they can produce gametes and contribute genetically to subsequent generations. Nonetheless, germ line specification during embryogenesis is conceptually the same as the specification of any somatic cell type: germ cells must activate a specific gene regulatory network in order to differentiate and go through gametogenesis. While many genes with critical roles in the germ line have been characterized with respect to expression pattern and genetic interactions, it is the molecular interactions of the relevant gene products that are ultimately responsible for germ cell differentiation. This review summarizes the current state of knowledge on the molecular functions and biochemical connections between germ line gene products. We find that homologous genes often interact physically with the same conserved molecular partners across the metazoans. We also point out cases of nonhomologous genes from different species whose gene products play analogous biological roles in the germ line. We suggest a preliminary molecular definition of an ancestral “pluripotency module” that could have been modified during metazoan evolution to become specific to the germ line.

This dissertation investigates the embryonic specification of a specific group of cells: the germ cells. Germ cells, which give rise to sperm and egg, are the only cells in sexually-reproducing animals that directly contribute hereditary information to the next generation. Germ cells are therefore a universal cell type across animals, and represent a profound novelty that likely arose near the base of the animal phylogeny. Yet despite their conserved, essential function in all animals, there is surprising diversity in the mechanisms that specify these cells during embryonic development. In this dissertation, I address the diversity of germ cell specification mechanisms in insects. I focus on two species, the milkweed bug Oncopeltus fasciatus (Hemiptera) and the cricket Gryllus bimaculatus (Orthoptera), which both branch basally to the Holometabola (those insects which undergo metamorphosis, including the well-studied fruit fly Drosophila melanogaster), and thus provide important phylogenetic breadth to our understanding of germ cell specification across insects. Using functional genetic approaches, I show that germ cell specification in both Oncopeltus and Gryllus differs fundamentally from germ cell specification in Drosophila. Specifically, I provide evidence that germ cells arise via inductive cell signaling during mid-embryogenesis, rather than via maternally-supplied cytoplasmic determinants localized in the oocyte, as is the case for Drosophila. These data suggest that Drosophila employs an evolutionarily derived mode of germ cell specification. In further support of this hypothesis, I show that several of the genes required for Drosophila germ cell specification perform other functions in both Oncopeltus and Gryllus. I demonstrate that one of these genes, oskar, which is the only gene both necessary and sufficient for germ cell specification in Drosophila, instead functions in nervous system of the cricket, both during embryonic development and in the adult brain. I suggest that the evolution of the derived mode of germ cell specification seen in Drosophila may have involved co-opting oskar into the germ cell specification pathway from an ancestral role in the nervous system.

Background. Most evolutionary developmental ("evo-devo") studies of emerging model organisms focus on small numbers of candidate genes cloned individually using degenerate PCR. However, newly available sequencing technologies such as 454 pyrosequencing have recently begun to allow for massive gene discovery in animals without sequenced genomes. Within insects, although large volumes of sequence data are available for holometabolous insects, developmental studies of basally branching hemimetabolous insects typically suffer from low rates of gene discovery. Results. We used 454 pyrosequencing to sequence over 500 million bases of cDNA from the ovaries and embryos of the milkweed bug Oncopeltus fasciatus, which lacks a sequenced genome. This indirectly developing insect occupies an important phylogenetic position, branching basal to Diptera (including fruit flies) and Hymenoptera (including honeybees), and is an experimentally tractable model for short-germ development. 2,087,410 reads from both normalized and non-normalized cDNA assembled into 21,097 sequences (isotigs) and 112,531 singletons. The assembled sequences fell into 16,617 unique gene models, and included predictions of splicing isoforms, which we examined experimentally. Discovery of new genes plateaued after assembly of ~1.5 million reads, suggesting that we have sequenced nearly all transcripts present in the cDNA sampled. Many transcripts have been assembled at close to full length, and there is a net gain of sequence data for over half of the pre-existing O. fasciatus accessions for developmental genes in GenBank. We identified 10,775 unique genes, including members of all major conserved metazoan signaling pathways and genes involved in several major categories of early developmental processes. We also specifically address the effects of cDNA normalization on gene discovery in de novo transcriptome analyses. Conclusions. Our sequencing, assembly and annotation framework provide a simple and effective way to achieve high-throughput gene discovery organisms lacking a sequenced genome. These data will have applications to the study of the evolution of arthropod genes and genetic pathways, and to the wider evolution, development and genomics communities working with emerging model organisms. [The sequence data from this study have been submitted to GenBank under study accession number SRP002610.1. Custom scripts generated are available at http://www.extavourlab.com/protocols/index.html. Seven Additional files are available.]

Many model organisms specify germ cells using maternally supplied germ-line determinants. In contrast, mice rely on embryonic cell–cell signaling to induce cells to become germ cells. Molecular evidence for inductive germ-line specification had previously been provided only for the mouse. Here we provide functional evidence for inductive germ cell specification in an invertebrate, by showing that bone morphogenetic protein (BMP) signaling, which induces mouse germ cell specification, is required for establishment of embryonic germ cells in a cricket. BMP pathway knockdown causes reduction or loss of germ cells, and elevated levels of BMP signaling cause supernumerary and ectopic germ cells. BMP-based germ cell induction in mice and crickets suggests that this may be a shared ancestral mechanism in animals.

The rapid rise of CRISPR as a technology for genome engineering and related research applications has created a need for algorithms and associated online tools that facilitate design of on‐target and effective guide RNAs (gRNAs). Here, we review the state of the art in CRISPR gRNA design for research applications of the CRISPR‐Cas9 system, including knockout, activation, and inhibition. Notably, achieving good gRNA design is not solely dependent on innovations in CRISPR technology. Good design and design tools also rely on availability of high‐quality genome sequence and gene annotations, as well as on availability of accumulated data regarding off‐targets and effectiveness metrics.

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.