Johannes B. Schinko

Large-scale insertional mutagenesis of a coleopteran stored grain pest, the red flour beetle Tribolium castaneum, identifies embryonic lethal mutations and enhancer traps

BackgroundGiven its sequenced genome and efficient systemic RNA interference response, the red flour beetle Tribolium castaneum is a model organism well suited for reverse genetics. Even so, there is a pressing need for forward genetic analysis to escape the bias inherent in candidate gene approaches.ResultsTo produce easy-to-maintain insertional mutations and to obtain fluorescent marker lines to aid phenotypic analysis, we undertook a large-scale transposon mutagenesis screen. In this screen, we produced more than 6,500 new piggyBac insertions. Of these, 421 proved to be recessive lethal, 75 were semi-lethal, and eight indicated recessive sterility, while 505 showed new enhancer-trap patterns. Insertion junctions were determined for 403 lines and often appeared to be located within transcription units. Insertion sites appeared to be randomly distributed throughout the genome, with the exception of a preference for reinsertion near the donor site.ConclusionA large collection of enhancer-trap and embryonic lethal beetle lines has been made available to the research community and will foster investigations into diverse fields of insect biology, pest control, and evolution. Because the genetic elements used in this screen are species-nonspecific, and because the crossing scheme does not depend on balancer chromosomes, the methods presented herein should be broadly applicable for many insect species.

Divergent functions of orthodenticle, empty spiracles and buttonhead in early head patterning of the beetle Tribolium castaneum (Coleoptera).

The head gap genes orthodenticle (otd), empty spiracles (ems) and buttonhead (btd) are required for metamerization and segment specification in Drosophila. We asked whether the function of their orthologs is conserved in the red flour beetle Tribolium castaneum which in contrast to Drosophila develops its larval head in a way typical for insects. We find that depending on dsRNA injection time, two functions of Tc-orthodenticle1 (Tc-otd1) can be identified. The early regionalization function affects all segments formed during the blastoderm stage while the later head patterning function is similar to Drosophila. In contrast, both expression and function of Tc-empty spiracles (Tc-ems) are restricted to the posterior part of the ocular and the anterior part of the antennal segment and Tc-buttonhead (Tc-btd) is not required for head cuticle formation at all. We conclude that the gap gene like roles of ems and btd are not conserved while at least the head patterning function of otd appears to be similar in fly and beetle. Hence, the ancestral mode of insect head segmentation remains to be discovered. With this work, we establish Tribolium as a model system for arthropod head development that does not suffer from the Drosophila specific problems like head involution and strongly reduced head structures.

RNAi in the Red Flour Beetle (Tribolium)

INTRODUCTION Tribolium castaneum is exceptionally amenable to gene knockdown by RNA interference (RNAi) which, in this insect, is systemic (spreading throughout the organism and to the next generation), highly penetrant, and able to phenocopy genetic null phenotypes. Hence, any gene function can be knocked down at any stage in (apparently) all tissues upon injection of double-stranded RNA (dsRNA). The RNAi effect is elicited both in the injected animal and, if female pupae or adults have been injected, transferred to the offspring. Embryonic RNAi (eRNAi) usually generates the strongest phenotypes in the injected individual, but suffers from elevated lethality caused by injection injury. Pupal RNAi (pRNAi), in which female pupae are injected and phenotypes scored in the offspring, is the easiest to perform. However, in some cases, the knockdown of a gene leads to sterility of the injected female. This problem can be circumvented in many cases by injecting adult females (aRNAi) or using eRNAi. In order to interfere with processes during metamorphosis, injection into last-stage larvae is used (lRNAi). Up to two genes in a single experiment have been successfully knocked down via RNAi. The inclusion of more than two genes usually leads to a dilution effect, which lowers phenotypic strength. This protocol describes the production of dsRNA from a polymerase chain reaction (PCR) template, injection procedures for each Tribolium life stage, and important controls for effective analysis.

Functionality of the GAL4/UAS system in Tribolium requires the use of endogenous core promoters

BackgroundThe red flour beetle Tribolium castaneum has developed into an insect model system second only to Drosophila. Moreover, as a coleopteran it represents the most species-rich metazoan taxon which also includes many pest species. The genetic toolbox for Tribolium research has expanded in the past years but spatio-temporally controlled misexpression of genes has not been possible so far.ResultsHere we report the establishment of the GAL4/UAS binary expression system in Tribolium castaneum. Both GAL4Δ and GAL4VP16 driven by the endogenous heat shock inducible promoter of the Tribolium hsp68 gene are efficient in activating reporter gene expression under the control of the Upstream Activating Sequence (UAS). UAS driven ubiquitous tGFP fluorescence was observed in embryos within four hours after activation while in-situ hybridization against tGFP revealed expression already after two hours. The response is quick in relation to the duration of embryonic development in Tribolium - 72 hours with segmentation being completed after 24 hours - which makes the study of early embryonic processes possible using this system. By comparing the efficiency of constructs based on Tribolium, Drosophila, and artificial core promoters, respectively, we find that the use of endogenous core promoters is essential for high-level expression of transgenic constructs.ConclusionsWith the established GAL4/UAS binary expression system, ectopic misexpression approaches are now feasible in Tribolium. Our results support the contention that high-level transgene expression usually requires endogenous regulatory sequences, including endogenous core promoters in Tribolium and probably also other model systems.

Evolutionary conservation of the eumetazoan gene regulatory landscape

Despite considerable differences in morphology and complexity of body plans among animals, a great part of the gene set is shared among Bilateria and their basally branching sister group, the Cnidaria. This suggests that the common ancestor of eumetazoans already had a highly complex gene repertoire. At present it is therefore unclear how morphological diversification is encoded in the genome. Here we address the possibility that differences in gene regulation could contribute to the large morphological divergence between cnidarians and bilaterians. To this end, we generated the first genome-wide map of gene regulatory elements in a nonbilaterian animal, the sea anemone Nematostella vectensis. Using chromatin immunoprecipitation followed by deep sequencing of five chromatin modifications and a transcriptional cofactor, we identified over 5000 enhancers in the Nematostella genome and could validate 75% of the tested enhancers in vivo. We found that in Nematostella, but not in yeast, enhancers are characterized by the same combination of histone modifications as in bilaterians, and these enhancers preferentially target developmental regulatory genes. Surprisingly, the distribution and abundance of gene regulatory elements relative to these genes are shared between Nematostella and bilaterian model organisms. Our results suggest that complex gene regulation originated at least 600 million yr ago, predating the common ancestor of eumetazoans.

Efficient CRISPR-mediated gene targeting and transgene replacement in the beetle Tribolium castaneum

Gene-editing techniques are revolutionizing the way we conduct genetics in many organisms. The CRISPR/Cas nuclease has emerged as a highly versatile, efficient and affordable tool for targeting chosen sites in the genome. Beyond its applications in established model organisms, CRISPR technology provides a platform for genetic intervention in a wide range of species, limited only by our ability to deliver it to cells and to select mutations efficiently. Here, we test the CRISPR technology in an emerging insect model and pest, the beetle Tribolium castaneum. We use simple assays to test CRISPR/Cas activity, we demonstrate efficient expression of guide RNAs and Cas9 from Tribolium U6 and hsp68 promoters and we test the efficiency of knockout and knock-in approaches in Tribolium. We find that 55-80% of injected individuals carry mutations (indels) generated by non-homologous end joining, including mosaic bi-allelic knockouts; 71-100% carry such mutations in their germ line and transmit them to the next generation. We show that CRISPR-mediated gene knockout of the Tribolium E-cadherin gene causes defects in dorsal closure, which is consistent with RNAi-induced phenotypes. Homology-directed knock-in of marker transgenes was observed in 14% of injected individuals and transmitted to the next generation by 6% of injected individuals. Previous work in Tribolium mapped a large number of transgene insertions associated with developmental phenotypes and enhancer traps. We present an efficient method for re-purposing these insertions, via CRISPR-mediated replacement of these transgenes by new constructs. Summary: CRISPR-based technology can mediate efficient gene targeting and transgene replacement in the beetle Tribolium, which is emerging as a powerful insect model organism.

Axis Patterning by BMPs: Cnidarian Network Reveals Evolutionary Constraints

Summary BMP signaling plays a crucial role in the establishment of the dorso-ventral body axis in bilaterally symmetric animals. However, the topologies of the bone morphogenetic protein (BMP) signaling networks vary drastically in different animal groups, raising questions about the evolutionary constraints and evolvability of BMP signaling systems. Using loss-of-function analysis and mathematical modeling, we show that two signaling centers expressing different BMPs and BMP antagonists maintain the secondary axis of the sea anemone Nematostella. We demonstrate that BMP signaling is required for asymmetric Hox gene expression and mesentery formation. Computational analysis reveals that network parameters related to BMP4 and Chordin are constrained both in Nematostella and Xenopus, while those describing the BMP signaling modulators can vary significantly. Notably, only chordin, but not bmp4 expression needs to be spatially restricted for robust signaling gradient formation. Our data provide an explanation of the evolvability of BMP signaling systems in axis formation throughout Eumetazoa.

Genetics, development and composition of the insect head – A beetle’s view

Many questions regarding evolution and ontogeny of the insect head remain open. Likewise, the genetic basis of insect head development is poorly understood. Recently, the investigation of gene expression data and the analysis of patterning gene function have revived interest in insect head development. Here, we argue that the red flour beetle Tribolium castaneum is a well suited model organism to spearhead research with respect to the genetic control of insect head development. We review recent molecular data and discuss its bearing on early development and morphogenesis of the head. We present a novel hypothesis on the ontogenetic origin of insect head sutures and review recent insights into the question on the origin of the labrum. Further, we argue that the study of developmental genes may identify the elusive anterior non-segmental region and present some evidence in favor of its existence. With respect to the question of evolution of patterning we show that the head Anlagen of the fruit fly Drosophila melanogaster and Tribolium differ considerably and we review profound differences of their genetic regulation. Finally, we discuss which insect model species might help us to answer the open questions concerning the genetic regulation of head development and its evolution.

Single and Double Whole-Mount In Situ Hybridization in Red Flour Beetle (Tribolium) Embryos

INTRODUCTION The red flour beetle, Tribolium castaneum, has emerged as an important model system for studying the evolution of development. Studies with Tribolium complement the vast amount of research done with Drosophila. Developmental features that are conserved between Drosophila and Tribolium, such as body segmentation, are achieved by quite different means, and thus comparison of developmental mechanisms between these two insects can address many interesting questions concerning the evolution of morphology and other characters. Most in situ protocols used for Tribolium have been adapted from Drosophila studies. Whole-mount in situ hybridization is a standard technique to visualize the activity of genes in embryos. The single and double staining protocol presented here uses two nonfluorescent stains to reveal gene activity. The development of both stains can be monitored visually, allowing the strength of the signal to be adjusted as needed. Cells that express both of the genes under investigation are readily detected using a microscope. The use of EGTA during fixation increases the proportion of embryos that devitellinize upon methanol treatment.

Heat shock-mediated misexpression of genes in the beetle Tribolium castaneum

Insect gene function has mainly been studied in the fruit fly Drosophila melanogaster because in this species many techniques and resources are available for gene knock down and the ectopic activation of gene function. However, in order to study biological aspects that are not represented by the Drosophila model, and in order to test to what degree gene functions are conserved within insects and what changes in gene function accompanied the evolution of novel traits, the establishment of respective tools in other insect species is required. While gene knock down can be induced by RNA interference in many insects, methods to misexpress genes are much less developed. In order to allow misexpression of genes in a timely controlled manner in the red flour beetle Tribolium castaneum, we have established a heat shock-mediated misexpression system. We show that endogenous heat shock elements perform better than artificial heat shock elements derived from vertebrates. We carefully determine the optimal conditions for heat shock and define a core promoter for use in future constructs. Finally, using this system, we study the effects of misexpressing the head patterning gene Tc-orthodenticle1 (Tc-otd1), We show that Tc-otd1 suppresses Tc-wingless (Tc-wg) in the trunk and to some degree in the head.