RESUMEN
The cabbage stem flea beetle (CSFB, Psylliodes chrysocephala) is a key pest of oilseed rape. The ban on neonicotinoids in the European Union due to environmental concerns and the emergence of pyrethroid-resistant populations have made the control of CSFB extremely challenging. In search of a solution, we have recently shown that RNA interference (RNAi) has potential in the management of CSFB. However, the previously tested target genes for RNAi-mediated pest control (subsequently called target genes) exhibited moderate and slow-acting lethal effects. In this study, 27 double-stranded RNAs (dsRNAs) were orally delivered to identify highly effective target genes in CSFB adults by leveraging the findings of a genome-wide RNAi screen in Tribolium castaneum. Our screen using 500 ng of dsRNA identified 10 moderately effective (> 50% mortality) and 4 highly effective target genes (100% mortality in 8-13 days). The latter mainly included proteasome subunits. Gene expression measurements confirmed target gene silencing and dose-response studies revealed LD50 values as low as ~20 ng in 14 days following a single exposure to dsRNA. Four highly effective dsRNAs also inhibited leaf damage (up to ~75%) and one affected locomotion. The sequences of promising target genes were subjected to in silico target prediction in non-target organisms, for example, beneficials such as honeybees, to design environmentally friendly dsRNAs. Overall, the study provides valuable insights for the development of dsRNA-based insecticides against CSFB.
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Animals from all major clades have evolved a segmented trunk, reflected in the human spine or the insect segments. These units emerge during embryogenesis from a posterior segment addition zone (SAZ), where repetitive gene activity is regulated by a mechanism described by the clock and wavefront/speed gradient model. In the red flour beetle Tribolium castaneum, RNA interference (RNAi) has been used to continuously knock down the function of primary pair-rule genes (pPRGs), caudal or Wnt pathway components, which has led to the complete breakdown of segmentation. However, it has remained untested, if this breakdown was reversible by bringing the missing gene function back to the system. To fill this gap, we established a transgenic system in T. castaneum, which allows blocking an ongoing RNAi effect with temporal control by expressing a viral inhibitor of RNAi via heat shock. We show that the T. castaneum segmentation machinery was able to reestablish after RNAi targeting the pPRGs Tc-eve, Tc-odd, and Tc-runt was blocked. However, we observed no rescue after blocking RNAi targeting Wnt pathway components. We conclude that the insect segmentation system contains both robust feedback loops that can reestablish and labile feedback loops that break down irreversibly. This combination may reconcile conflicting needs of the system: Labile systems controlling initiation and maintenance of the SAZ ensure that only one SAZ is formed. Robust feedback loops confer developmental robustness toward external disturbances.
Asunto(s)
Tipificación del Cuerpo , Interferencia de ARN , Tribolium , Animales , Tribolium/genética , Tipificación del Cuerpo/genética , Regulación del Desarrollo de la Expresión Génica , Retroalimentación Fisiológica , Animales Modificados Genéticamente , Relojes Biológicos/genéticaRESUMEN
Gene expression has been employed for homologizing body regions across bilateria. The molecular comparison of vertebrate and fly brains has led to a number of disputed homology hypotheses. Data from the fly Drosophila melanogaster have recently been complemented by extensive data from the red flour beetle Tribolium castaneum with its more insect-typical development. In this review, we revisit the molecular mapping of the neuroectoderm of insects and vertebrates to reconsider homology hypotheses. We claim that the protocerebrum is non-segmental and homologous to the vertebrate fore- and midbrain. The boundary between antennal and ocular regions correspond to the vertebrate mid-hindbrain boundary while the deutocerebrum represents the anterior-most ganglion with serial homology to the trunk. The insect head placode is shares common embryonic origin with the vertebrate adenohypophyseal placode. Intriguingly, vertebrate eyes develop from a different region compared to the insect compound eyes calling organ homology into question. Finally, we suggest a molecular re-definition of the classic concepts of archi- and prosocerebrum.
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Escarabajos , Placa Neural , Animales , Drosophila melanogaster , Insectos , Encéfalo , Vertebrados/genética , Expresión GénicaRESUMEN
BACKGROUND: Functional genomics uses unbiased systematic genome-wide gene disruption or analyzes natural variations such as gene expression profiles of different tissues from multicellular organisms to link gene functions to particular phenotypes. Functional genomics approaches are of particular importance to identify large sets of genes that are specifically important for a particular biological process beyond known candidate genes, or when the process has not been studied with genetic methods before. RESULTS: Here, we present a large set of genes whose disruption interferes with the function of the odoriferous defensive stink glands of the red flour beetle Tribolium castaneum. This gene set is the result of a large-scale systematic phenotypic screen using RNA interference applied in a genome-wide forward genetics manner. In this first-pass screen, 130 genes were identified, of which 69 genes could be confirmed to cause phenotypic changes in the glands upon knock-down, which vary from necrotic tissue and irregular reservoir size to irregular color or separation of the secreted gland compounds. Gene ontology analysis revealed that many of those genes are encoding enzymes (peptidases and cytochromes P450) as well as proteins involved in membrane trafficking with an enrichment in lysosome and mineral absorption pathways. The knock-down of 13 genes caused specifically a strong reduction of para-benzoquinones in the gland reservoirs, suggesting a specific function in the synthesis of these toxic compounds. Only 14 of the 69 confirmed gland genes are differentially overexpressed in stink gland tissue and thus could have been detected in a transcriptome-based analysis. However, only one out of eight genes identified by a transcriptomics approach known to cause phenotypic changes of the glands upon knock-down was recognized by this phenotypic screen, indicating the limitation of such a non-redundant first-pass screen. CONCLUSION: Our results indicate the importance of combining diverse and independent methodologies to identify genes necessary for the function of a certain biological tissue, as the different approaches do not deliver redundant results but rather complement each other. The presented phenotypic screen together with a transcriptomics approach are now providing a set of close to hundred genes important for odoriferous defensive stink gland physiology in beetles.
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Escarabajos , Tribolium , Animales , Escarabajos/genética , Genómica , Fenotipo , Transcriptoma , Tribolium/genéticaRESUMEN
The red flour beetle Tribolium castaneum has emerged as an important insect model system for a variety of topics. With respect to studying gene function, it is second only to the vinegar fly D. melanogaster. The RNAi response in T. castaneum is exceptionally strong and systemic, and it appears to target all cell types and processes. Uniquely for emerging model organisms, T. castaneum offers the opportunity of performing time- and cost-efficient large-scale RNAi screening, based on commercially available dsRNAs targeting all genes, which are simply injected into the body cavity. Well established transgenic and genome editing approaches are met by ease of husbandry and a relatively short generation time. Consequently, a number of transgenic tools like UAS/Gal4, Cre/Lox, imaging lines and enhancer trap lines are already available. T. castaneum has been a genetic experimental system for decades and now has become a workhorse for molecular and reverse genetics as well as in vivo imaging. Many aspects of development and general biology are more insect-typical in this beetle compared to D. melanogaster. Thus, studying beetle orthologs of well-described fly genes has allowed macro-evolutionary comparisons in developmental processes such as axis formation, body segmentation, and appendage, head and brain development. Transgenic approaches have opened new ways for in vivo imaging. Moreover, this emerging model system is the first choice for research on processes that are not represented in the fly, or are difficult to study there, e.g. extraembryonic tissues, cryptonephridial organs, stink gland function, or dsRNA-based pesticides.
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Insect brains are formed by conserved sets of neural lineages whose fibers form cohesive bundles with characteristic projection patterns. Within the brain neuropil, these bundles establish a system of fascicles constituting the macrocircuitry of the brain. The overall architecture of the neuropils and the macrocircuitry appear to be conserved. However, variation is observed, for example, in size, shape, and timing of development. Unfortunately, the developmental and genetic basis of this variation is poorly understood, although the rise of new genetically tractable model organisms such as the red flour beetle Tribolium castaneum allows the possibility to gain mechanistic insights. To facilitate such work, we present an atlas of the developing brain of T. castaneum, covering the first larval instar, the prepupal stage, and the adult, by combining wholemount immunohistochemical labeling of fiber bundles (acetylated tubulin) and neuropils (synapsin) with digital 3D reconstruction using the TrakEM2 software package. Upon comparing this anatomical dataset with the published work in Drosophila melanogaster, we confirm an overall high degree of conservation. Fiber tracts and neuropil fascicles, which can be visualized by global neuronal antibodies like antiacetylated tubulin in all invertebrate brains, create a rich anatomical framework to which individual neurons or other regions of interest can be referred to. The framework of a largely conserved pattern allowed us to describe differences between the two species with respect to parameters such as timing of neuron proliferation and maturation. These features likely reflect adaptive changes in developmental timing that govern the change from larval to adult brain.
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BACKGROUND: Most of the known genes required for developmental processes have been identified by genetic screens in a few well-studied model organisms, which have been considered representative of related species, and informative-to some degree-for human biology. The fruit fly Drosophila melanogaster is a prime model for insect genetics, and while conservation of many gene functions has been observed among bilaterian animals, a plethora of data show evolutionary divergence of gene function among more closely-related groups, such as within the insects. A quantification of conservation versus divergence of gene functions has been missing, without which it is unclear how representative data from model systems actually are. RESULTS: Here, we systematically compare the gene sets required for a number of homologous but divergent developmental processes between fly and beetle in order to quantify the difference of the gene sets. To that end, we expanded our RNAi screen in the red flour beetle Tribolium castaneum to cover more than half of the protein-coding genes. Then we compared the gene sets required for four different developmental processes between beetle and fly. We found that around 50% of the gene functions were identified in the screens of both species while for the rest, phenotypes were revealed only in fly (~ 10%) or beetle (~ 40%) reflecting both technical and biological differences. Accordingly, we were able to annotate novel developmental GO terms for 96 genes studied in this work. With this work, we publish the final dataset for the pupal injection screen of the iBeetle screen reaching a coverage of 87% (13,020 genes). CONCLUSIONS: We conclude that the gene sets required for a homologous process diverge more than widely believed. Hence, the insights gained in flies may be less representative for insects or protostomes than previously thought, and work in complementary model systems is required to gain a comprehensive picture. The RNAi screening resources developed in this project, the expanding transgenic toolkit, and our large-scale functional data make T. castaneum an excellent model system in that endeavor.
Asunto(s)
Escarabajos , Tribolium , Animales , Escarabajos/genética , Drosophila , Drosophila melanogaster/genética , Pupa , Interferencia de ARN , Tribolium/genéticaRESUMEN
Identifying essential genes on a genome scale is resource intensive and has been performed for only a few eukaryotes. For less studied organisms essentiality might be predicted by gene homology. However, this approach cannot be applied to non-conserved genes. Additionally, divergent essentiality information is obtained from studying single cells or whole, multi-cellular organisms, and particularly when derived from human cell line screens and human population studies. We employed machine learning across six model eukaryotes and 60 381 genes, using 41 635 features derived from the sequence, gene function information and network topology. Within a leave-one-organism-out cross-validation, the classifiers showed high generalizability with an average accuracy close to 80% in the left-out species. As a case study, we applied the method to Tribolium castaneum and Bombyx mori and validated predictions experimentally yielding similar performances. Finally, using the classifier based on the studied model organisms enabled linking the essentiality information of human cell line screens and population studies.
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RNA interference (RNAi) has emerged as a powerful tool for knocking-down gene function in diverse taxa including arthropods for both basic biological research and application in pest control. The conservation of the RNAi mechanism in eukaryotes suggested that it should-in principle-be applicable to most arthropods. However, practical hurdles have been limiting the application in many taxa. For instance, species differ considerably with respect to efficiency of dsRNA uptake from the hemolymph or the gut. Here, we review some of the most frequently encountered technical obstacles when establishing RNAi and suggest a robust procedure for establishing this technique in insect species with special reference to pests. Finally, we present an approach to identify the most effective target genes for the potential control of agricultural and public health pests by RNAi.
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Gene regulatory mechanisms that specify subtype identity of central complex (CX) neurons are the subject of intense investigation. The CX is a compartment within the brain common to all insect species and functions as a 'command center' that directs motor actions. It is made up of several thousand neurons, with more than 60 morphologically distinct identities. Accordingly, transcriptional programs must effect the specification of at least as many neuronal subtypes. We demonstrate a role for the transcription factor Shaking hands (Skh) in the specification of embryonic CX neurons in Tribolium. The developmental dynamics of skh expression are characteristic of terminal selectors of subtype identity. In the embryonic brain, skh expression is restricted to a subset of neurons, many of which survive to adulthood and contribute to the mature CX. skh expression is maintained throughout the lifetime in at least some CX neurons. skh knockdown results in axon outgrowth defects, thus preventing the formation of an embryonic CX primordium. The previously unstudied Drosophila skh shows a similar embryonic expression pattern, suggesting that subtype specification of CX neurons may be conserved.
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Axones/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas de Insectos/metabolismo , Proyección Neuronal , Factores de Transcripción/metabolismo , Tribolium/metabolismo , Animales , Axones/fisiología , Ganglios de Invertebrados/citología , Ganglios de Invertebrados/metabolismo , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Proteínas de Insectos/química , Proteínas de Insectos/genética , Dominios Proteicos , Factores de Transcripción/química , Factores de Transcripción/genética , Tribolium/embriología , Tribolium/genéticaRESUMEN
RNA interference (RNAi) is a promising, selective pest control technology based on the silencing of targeted genes mediated by the degradation of mRNA after the ingestion of double-stranded (ds) RNA. However, the identification of the best target genes remains a challenge, because large scale screening is only feasible in lab model systems and it remains unclear, to what degree such data can be transferred to pest species. Here, we report on our efforts to transfer target genes found in a lab model to the mustard leaf beetle, Phaedon cochleariae. The mustard leaf beetle can be reared easily and resource-efficient in large quantities all year round and is an established chrysomelid pest for higher throughput screening approaches in the crop protection industry. Mustard leaf beetle transcriptome sequencing and assembly revealed genes orthologous to those previously described as highly efficient RNAi targets in the model beetle Tribolium castaneum. First, we observed mortality after injection of dsRNA targeting the respective orthologous genes in 2nd instar mustard beetle larvae. Next, we adopted a robust, automated multi-well plate foliar RNAi screening procedure with 2nd instar larvae of the mustard leaf beetle to assess those genes. Indeed, foliar application and oral uptake of dsRNA targeting the same genes resulted in larval mortality as well. The most effective target genes with a strong (lethal) phenotype - at dsRNA doses as low as 300 ng/leaf disc (equal to 9.6 g/ha) - were srp54k, rop, αSNAP, rpn7 and rpt3. Rather limited effects were observed after application of dsRNA targeting cactus, shibire and PP-α, though they had previously been shown to be highly lethal in red flour beetle. Importantly, our experiments demonstrated that the overall efficacy pattern obtained after oral dsRNA application was well correlated with the results obtained after dsRNA injection. RT-qPCR confirmed significant target gene knock-down after normalization by employing three reference genes shown to be stably expressed across life stages. In summary, several RNAi targeted genes elicited a strong lethal phenotype and significant target gene knock-down after feeding, suggesting P. cochleariae as a potential coleopteran screening model for foliarly applied exogenous RNAi.
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Escarabajos , Tribolium , Animales , Escarabajos/genética , Larva , Planta de la Mostaza , Interferencia de ARN , ARN Bicatenario/genética , Tribolium/genéticaRESUMEN
Animal behavior is guided by the brain. Therefore, adaptations of brain structure and function are essential for animal survival, and each species differs in such adaptations. The brain of one individual may even differ between life stages, for instance, as adaptation to the divergent needs of larval and adult life of holometabolous insects. All such differences emerge during development, but the cellular mechanisms behind the diversification of brains between taxa and life stages remain enigmatic. In this study, we investigated holometabolous insects in which larvae differ dramatically from the adult in both behavior and morphology. As a consequence, the central complex, mainly responsible for spatial orientation, is conserved between species at the adult stage but differs between larvae and adults of one species as well as between larvae of different taxa. We used genome editing and established transgenic lines to visualize cells expressing the conserved transcription factor retinal homeobox, thereby marking homologous genetic neural lineages in both the fly Drosophila melanogaster and the beetle Tribolium castaneum. This approach allowed us for the first time to compare the development of homologous neural cells between taxa from embryo to the adult. We found complex heterochronic changes including shifts of developmental events between embryonic and pupal stages. Further, we provide, to our knowledge, the first example of sequence heterochrony in brain development, where certain developmental steps changed their position within the ontogenetic progression. We show that through this sequence heterochrony, an immature developmental stage of the central complex gains functionality in Tribolium larvae.
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Escarabajos/fisiología , Drosophila melanogaster/fisiología , Animales , Agregación Celular , Linaje de la Célula , Escarabajos/citología , Escarabajos/embriología , Escarabajos/genética , Drosophila melanogaster/citología , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Embrión no Mamífero/citología , Desarrollo Embrionario , Genes Homeobox , Larva/citología , Metamorfosis Biológica , Neuronas/citología , Pupa/crecimiento & desarrollo , Factores de TiempoRESUMEN
In recent years, substantial effort was spent on the exploration and implementation of RNAi technology using double-stranded RNA (dsRNA) for pest management purposes. However, only few studies investigated the geographical variation in RNAi sensitivity present in field-collected populations of the targeted insect pest. In this baseline study, 2nd instar larvae of 14 different European populations of Colorado potato beetle (CPB), Leptinotarsa decemlineata, collected from nine different countries were exposed to a foliarly applied diagnostic dose of dsactin (dsact) to test for possible variations in RNAi response. Only minor variability in RNAi sensitivity was observed between populations. However, the time necessary to trigger a dsRNA-mediated phenotypic response varied significantly among populations, indicated by significant differences in mortality figures obtained five days after treatment. An inbred German laboratory reference strain D01 and a Spanish field strain E02 showed almost 100% mortality after foliar exposure to 30 ng dsactin (equal to 0.96 g/ha), whereas another Spanish strain E01 was least responsive and showed only 30% mortality. Calculated LD50-values for foliarly applied dsact against strains D01 (most sensitive) and E01 (least sensitive) were 9.22 and 68.7 ng/leaf disc, respectively. The variability was not based on target gene sequence divergence or knock-down efficiency. Variability in expression of the core RNAi machinery genes dicer (dcr2a) and argonaute (ago2a) was observed but did not correlate with sensitivity. Interestingly, RT-qPCR data collected for all strains revealed a strong correlation between the expression level of dcr2a and ago2a (r 0.93) as well as ago2a and stauC (r 0.94), a recently described dsRNA binding protein in Coleopterans. Overall, this study demonstrates that sensitivity of CPB to sprayable RNAi slightly varies between strains but also shows that foliar RNAi as a control method works against all tested CPB populations collected across a broad geographic range in Europe. Thus, underpinning the potential of RNAi-based CPB control as a promising component in integrated pest management (IPM) and resistance management programs.
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Escarabajos , Solanum tuberosum , Animales , Larva , Interferencia de ARN , ARN BicatenarioRESUMEN
Anterior patterning in animals is based on a gene regulatory network, which comprises highly conserved transcription factors like six3, pax6 and otx. More recently, foxQ2 was found to be an ancestral component of this network but its regulatory interactions showed evolutionary differences. In most animals, foxQ2 is a downstream target of six3 and knockdown leads to mild or no epidermal phenotypes. In contrast, in the red flour beetle Tribolium castaneum, foxQ2 gained a more prominent role in patterning leading to strong epidermal and brain phenotypes and being required for six3 expression. However, it has remained unclear which of these novel aspects were insect or arthropod specific. Here, we study expression and RNAi phenotype of the single foxQ2 ortholog of the spider Parasteatoda tepidariorum. We find early anterior expression similar to the one of insects. Further, we show an epidermal phenotype in the labrum similar to the insect phenotype. However, our data indicate that foxQ2 is positioned downstream of six3 like in other animals but unlike insects. Hence, the epidermal and neural pattering function of foxQ2 is ancestral for arthropods while the upstream role of foxQ2 may have evolved in the lineage leading to the insects.
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Proteínas de Artrópodos/metabolismo , Embrión no Mamífero/metabolismo , Proteínas del Ojo/metabolismo , Factores de Transcripción Forkhead/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neurogénesis/genética , Transducción de Señal/genética , Arañas/crecimiento & desarrollo , Animales , Proteínas de Artrópodos/genética , Evolución Biológica , Tipificación del Cuerpo/genética , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Proteínas del Ojo/genética , Factores de Transcripción Forkhead/genética , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas de Homeodominio/genética , Hibridación in Situ , Proteínas del Tejido Nervioso/genética , Fenotipo , Filogenia , Interferencia de ARN , Proteína Homeobox SIX3RESUMEN
BACKGROUND: The red flour beetle Tribolium castaneum has emerged as an important model organism for the study of gene function in development and physiology, for ecological and evolutionary genomics, for pest control and a plethora of other topics. RNA interference (RNAi), transgenesis and genome editing are well established and the resources for genome-wide RNAi screening have become available in this model. All these techniques depend on a high quality genome assembly and precise gene models. However, the first version of the genome assembly was generated by Sanger sequencing, and with a small set of RNA sequence data limiting annotation quality. RESULTS: Here, we present an improved genome assembly (Tcas5.2) and an enhanced genome annotation resulting in a new official gene set (OGS3) for Tribolium castaneum, which significantly increase the quality of the genomic resources. By adding large-distance jumping library DNA sequencing to join scaffolds and fill small gaps, the gaps in the genome assembly were reduced and the N50 increased to 4753kbp. The precision of the gene models was enhanced by the use of a large body of RNA-Seq reads of different life history stages and tissue types, leading to the discovery of 1452 novel gene sequences. We also added new features such as alternative splicing, well defined UTRs and microRNA target predictions. For quality control, 399 gene models were evaluated by manual inspection. The current gene set was submitted to Genbank and accepted as a RefSeq genome by NCBI. CONCLUSIONS: The new genome assembly (Tcas5.2) and the official gene set (OGS3) provide enhanced genomic resources for genetic work in Tribolium castaneum. The much improved information on transcription start sites supports transgenic and gene editing approaches. Further, novel types of information such as splice variants and microRNA target genes open additional possibilities for analysis.
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Genes de Insecto , Genoma de los Insectos , Genómica , Tribolium/genética , Animales , Sitios de Unión , Biología Computacional/métodos , Genómica/métodos , MicroARNs/genética , Anotación de Secuencia Molecular , Filogenia , Interferencia de ARN , Reproducibilidad de los ResultadosRESUMEN
With CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated) scientists working with Tribolium castaneum can now generate transgenic lines with site-specific insertions at their region of interest. We present two methods to generate in vivo imaging lines suitable for marking subsets of neurons with fluorescent proteins. The first method relies on homologous recombination and uses a 2A peptide to create a bicistronic mRNA. In such lines, the target and the marker proteins are not fused but produced at equal amounts. This work-intensive method is compared with creating gene-specific enhancer traps that do not rely on homologous recombination. These are faster to generate but reflect the expression of the target gene less precisely. Which method to choose, strongly depends on the aims of each research project and in turn impacts of how neural cells and their development are marked. We describe the necessary steps from designing constructs and guide RNAs to embryonic injection and making homozygous stocks.
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Edición Génica/métodos , Tribolium/crecimiento & desarrollo , Animales , Animales Modificados Genéticamente , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Sistemas CRISPR-Cas , Neuronas/citología , Neuronas/metabolismo , Tribolium/genéticaRESUMEN
The red flour beetle, Tribolium castaneum, is an emerging model system well suited to the study of embryonic brain development and evolution (see Chapters 11 and 13 ). Brain genesis is driven by specific gene products whose expression underlies a tight spatiotemporal control. Therefore, the analysis of gene expression in time and space provides valuable insights into the molecular mechanisms that govern brain development. Since Tribolium-specific antibodies are scarce, fluorescent RNA in situ hybridization is the method of choice to determine the dynamics of individual gene expression. We have modified common RNA in situ protocols to facilitate the concomitant detection of two gene-specific expression patterns (double fluorescent RNA in situ). In addition, we describe a procedure which combines fluorescent single RNA in situ and immunostaining with gene-specific antibodies. Conventional in situ using RNA probes that are complementary to mature mRNAs often produce diffuse signals. We demonstrate that RNA in situ probes complementary to intronic gene sequences facilitate single cell resolution because the fluorescent signal is restricted to the nucleus. We believe our protocols can be adapted easily to suit the analysis of brain development in other insect species.
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Inmunohistoquímica/métodos , Hibridación Fluorescente in Situ/métodos , Tribolium/embriología , Animales , Encéfalo/embriología , Encéfalo/metabolismo , Desarrollo Embrionario/genética , Desarrollo Embrionario/fisiología , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Intrones/genética , Tribolium/metabolismoRESUMEN
Arthropod brains are fascinating structures that exhibit great complexity but also contain conserved elements that can be recognized between species. There is a long tradition of research in insect neuroanatomy, cell biology, and in studying the genetics of insect brain development. Recently, the beetle Tribolium castaneum has gained attention as a model for insect head and brain development, and many anterior patterning genes have so far been characterized in beetle embryos. The outcome of embryonic anterior development is the larval and, subsequently, the adult brain. A basic requirement to understand genetic cell type diversity within these structures is the ability to localize mRNA and protein of neural genes. Here we detail our protocols for RNA in situ hybridization in combination with immunohistochemistry, optimized for dissected brains of larval and adult beetles.
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Hibridación Fluorescente in Situ/métodos , ARN Mensajero/análisis , Tribolium/genética , Animales , Encéfalo/embriología , Encéfalo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Inmunohistoquímica , Larva/genética , Larva/metabolismo , ARN Mensajero/metabolismo , Tribolium/embriología , Tribolium/metabolismoRESUMEN
The genetic control of anterior brain development is highly conserved throughout animals. For instance, a conserved anterior gene regulatory network specifies the ancestral neuroendocrine center of animals and the apical organ of marine organisms. However, its contribution to the brain in non-marine animals has remained elusive. Here, we study the function of the Tc-foxQ2 forkhead transcription factor, a key regulator of the anterior gene regulatory network of insects. We characterized four distinct types of Tc-foxQ2 positive neural progenitor cells based on differential co-expression with Tc-six3/optix, Tc-six4, Tc-chx/vsx, Tc-nkx2.1/scro, Tc-ey, Tc-rx and Tc-fez1. An enhancer trap line built by genome editing marked Tc-foxQ2 positive neurons, which projected through the primary brain commissure and later through a subset of commissural fascicles. Eventually, they contributed to the central complex. Strikingly, in Tc-foxQ2 RNAi knock-down embryos the primary brain commissure did not split and subsequent development of midline brain structures stalled. Our work establishes foxQ2 as a key regulator of brain midline structures, which distinguish the protocerebrum from segmental ganglia. Unexpectedly, our data suggest that the central complex evolved by integrating neural cells from an ancestral anterior neuroendocrine center.
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Encéfalo/embriología , Factores de Transcripción Forkhead/metabolismo , Regulación del Desarrollo de la Expresión Génica , Tribolium/embriología , Animales , Diferenciación Celular , Células-Madre Neurales/fisiologíaRESUMEN
Although muscle development has been widely studied in Drosophila melanogaster there are still many gaps in our knowledge, and it is not known to which extent this knowledge can be transferred to other insects. To help in closing these gaps we participated in a large-scale RNAi screen that used the red flour beetle, Tribolium castaneum, as a screening platform. The effects of systemic RNAi were screened upon double-stranded RNA injections into appropriate muscle-EGFP tester strains. Injections into pupae were followed by the analysis of the late embryonic/early larval muscle patterns, and injections into larvae by the analysis of the adult thoracic muscle patterns. Herein we describe the results of the first-pass screens with pupal and larval injections, which covered â¼8,500 and â¼5,000 genes, respectively, of a total of â¼16,500 genes of the Tribolium genome. Apart from many genes known from Drosophila as regulators of muscle development, a collection of genes previously unconnected to muscle development yielded phenotypes in larval body wall and leg muscles as well as in indirect flight muscles. We then present the main candidates from the pupal injection screen that remained after being processed through a series of verification and selection steps. Further, we discuss why distinct though overlapping sets of genes are revealed by the Drosophila and Tribolium screening approaches.