In dieser Doktorarbeit wurden verschiedene Methoden angewandt um ein besseres Verständnis der Ins... more In dieser Doktorarbeit wurden verschiedene Methoden angewandt um ein besseres Verständnis der Insektenkopfentwicklung zu erlangen. Als Modellorganismus wurde der Rotbraune Reismehlkäfer Tribolium castaneum ausgewählt. Dieser Organismus ist für die Analyse dieses Prozesses gut geeignet, da Larven von Tribolium im Gegensatz zu Drosophilalarven einen Insektentypischen Kopf mit allen Kopfanhängen aufweisen. Erstens habe ich eine Karte des Kopfhärchenmusters erstellt, das als Marker für Kopfdefekte dient. Zweitens habe ich die so genannten Kopf-Lücken-Gene orthodenticle, empty spiracles und buttonhead in Tribolium analysiert. Es ist bekannt, dass diese Gene eine wichtige Rolle in der Kopfentwicklung bei Drosophila spielen. Ich habe das Expressionsmuster und den Phänotyp, der durch den knock down mittels RNA Interferenz verursacht wurde analysiert und mit den Drosophila Daten verglichen. Dabei wurde herausgefunden, dass abhängig von der Injektionszeit der dsRNA zwei Funktionen von Tc-orth...
Tc-knirps plays different roles in the specification of antennal and mandibular parasegment bound... more Tc-knirps plays different roles in the specification of antennal and mandibular parasegment boundaries and is regulated by a pair-rule gene in the beetle Tribolium castaneum Peel et al. Peel et al. BMC Developmental Biology 2013, 13:25
Analysis of Tribolium head patterning by forward and reverse genetics and transgenic techniques D... more Analysis of Tribolium head patterning by forward and reverse genetics and transgenic techniques Dissertation submitted in partial fulfillment of the requirements for the degree of “Doctor rerum naturalium”
Research article Large-scale insertional mutagenesis of a coleopteran stored grain pest, the red ... more Research article Large-scale insertional mutagenesis of a coleopteran stored grain pest, the red flour beetle Tribolium castaneum, identifies embryonic lethal mutations and enhancer traps
Background: The red flour beetle Tribolium castaneum has developed into an insect model system se... more Background: The 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. Results: Here 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 Triboli...
Clonal marking techniques based on the Cre/lox and Flp/FRT systems are widely used in multicellul... more Clonal marking techniques based on the Cre/lox and Flp/FRT systems are widely used in multicellular model organisms to mark individual cells and their progeny, in order to study their morphology, growth properties and developmental fates. The same tools can be adapted to introduce specific genetic changes in a subset of cells within the body, i.e. to perform mosaic genetic analysis. Marking and manipulating distinct cell clones requires control over the frequency of clone induction, which is sometimes difficult to achieve. Here we present Valcyrie, a new method that replaces the conventional Cre or Flp recombinase-mediated excision of a marker cassette by CRISPR-mediated excision. A major advantage of this approach is that CRISPR efficiency can be tuned in a predictable fashion by manipulating the degree of sequence complementarity between the CRISPR guide RNA and its targets. We establish the method in the beetle Tribolium castaneum. We demonstrate that clone marking frequency can ...
Gene editing techniques are revolutionizing the way we conduct genetics in many organisms. The CR... more 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 knock-out 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 knock-outs; 71-100% carry such mutations in their germline and transmit ...
INTRODUCTIONThe red flour beetle, Tribolium castaneum, has emerged as an important model system f... more INTRODUCTIONThe 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.
Tribolium castaneum is a member of the most species-rich eukaryotic order, a powerful model organ... more Tribolium castaneum is a member of the most species-rich eukaryotic order, a powerful model organism for the study of generalized insect development, and an important pest of stored agricultural products. We describe its genome sequence here. This omnivorous beetle has evolved the ability to interact with a diverse chemical environment, as shown by large expansions in odorant and gustatory receptors, as well as P450 and other detoxification enzymes. Development in Tribolium is more representative of other insects than is Drosophila, a fact reflected in gene content and function. For example, Tribolium has retained more ancestral genes involved in cell-cell communication than Drosophila, some being expressed in the growth zone crucial for axial elongation in short-germ development. Systemic RNA interference in T. castaneum functions differently from that in Caenorhabditis elegans, but nevertheless offers similar power for the elucidation of gene function and identification of targets...
Despite considerable differences in morphology and complexity of body plans among animals, a grea... more 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, ...
Background The Drosophila larval head is evolutionarily derived at the genetic and morphological ... more Background The Drosophila larval head is evolutionarily derived at the genetic and morphological level. In the beetle Tribolium castaneum, development of the larval head more closely resembles the ancestral arthropod condition. Unlike in Drosophila, a knirps homologue (Tc-kni) is required for development of the antennae and mandibles. However, published Tc-kni data are restricted to cuticle phenotypes and Tc-even-skipped and Tc-wingless stainings in knockdown embryos. Hence, it has remained unclear whether the entire antennal and mandibular segments depend on Tc-kni function, and whether the intervening intercalary segment is formed completely. We address these questions with a detailed examination of Tc-kni function. Results By examining the expression of marker genes in RNAi embryos, we show that Tc-kni is required only for the formation of the posterior parts of the antennal and mandibular segments (i.e. the parasegmental boundaries). Moreover, we find that the role of Tc-kni is ...
In dieser Doktorarbeit wurden verschiedene Methoden angewandt um ein besseres Verständnis der Ins... more In dieser Doktorarbeit wurden verschiedene Methoden angewandt um ein besseres Verständnis der Insektenkopfentwicklung zu erlangen. Als Modellorganismus wurde der Rotbraune Reismehlkäfer Tribolium castaneum ausgewählt. Dieser Organismus ist für die Analyse dieses Prozesses gut geeignet, da Larven von Tribolium im Gegensatz zu Drosophilalarven einen Insektentypischen Kopf mit allen Kopfanhängen aufweisen. Erstens habe ich eine Karte des Kopfhärchenmusters erstellt, das als Marker für Kopfdefekte dient. Zweitens habe ich die so genannten Kopf-Lücken-Gene orthodenticle, empty spiracles und buttonhead in Tribolium analysiert. Es ist bekannt, dass diese Gene eine wichtige Rolle in der Kopfentwicklung bei Drosophila spielen. Ich habe das Expressionsmuster und den Phänotyp, der durch den knock down mittels RNA Interferenz verursacht wurde analysiert und mit den Drosophila Daten verglichen. Dabei wurde herausgefunden, dass abhängig von der Injektionszeit der dsRNA zwei Funktionen von Tc-orth...
Tc-knirps plays different roles in the specification of antennal and mandibular parasegment bound... more Tc-knirps plays different roles in the specification of antennal and mandibular parasegment boundaries and is regulated by a pair-rule gene in the beetle Tribolium castaneum Peel et al. Peel et al. BMC Developmental Biology 2013, 13:25
Analysis of Tribolium head patterning by forward and reverse genetics and transgenic techniques D... more Analysis of Tribolium head patterning by forward and reverse genetics and transgenic techniques Dissertation submitted in partial fulfillment of the requirements for the degree of “Doctor rerum naturalium”
Research article Large-scale insertional mutagenesis of a coleopteran stored grain pest, the red ... more Research article Large-scale insertional mutagenesis of a coleopteran stored grain pest, the red flour beetle Tribolium castaneum, identifies embryonic lethal mutations and enhancer traps
Background: The red flour beetle Tribolium castaneum has developed into an insect model system se... more Background: The 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. Results: Here 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 Triboli...
Clonal marking techniques based on the Cre/lox and Flp/FRT systems are widely used in multicellul... more Clonal marking techniques based on the Cre/lox and Flp/FRT systems are widely used in multicellular model organisms to mark individual cells and their progeny, in order to study their morphology, growth properties and developmental fates. The same tools can be adapted to introduce specific genetic changes in a subset of cells within the body, i.e. to perform mosaic genetic analysis. Marking and manipulating distinct cell clones requires control over the frequency of clone induction, which is sometimes difficult to achieve. Here we present Valcyrie, a new method that replaces the conventional Cre or Flp recombinase-mediated excision of a marker cassette by CRISPR-mediated excision. A major advantage of this approach is that CRISPR efficiency can be tuned in a predictable fashion by manipulating the degree of sequence complementarity between the CRISPR guide RNA and its targets. We establish the method in the beetle Tribolium castaneum. We demonstrate that clone marking frequency can ...
Gene editing techniques are revolutionizing the way we conduct genetics in many organisms. The CR... more 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 knock-out 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 knock-outs; 71-100% carry such mutations in their germline and transmit ...
INTRODUCTIONThe red flour beetle, Tribolium castaneum, has emerged as an important model system f... more INTRODUCTIONThe 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.
Tribolium castaneum is a member of the most species-rich eukaryotic order, a powerful model organ... more Tribolium castaneum is a member of the most species-rich eukaryotic order, a powerful model organism for the study of generalized insect development, and an important pest of stored agricultural products. We describe its genome sequence here. This omnivorous beetle has evolved the ability to interact with a diverse chemical environment, as shown by large expansions in odorant and gustatory receptors, as well as P450 and other detoxification enzymes. Development in Tribolium is more representative of other insects than is Drosophila, a fact reflected in gene content and function. For example, Tribolium has retained more ancestral genes involved in cell-cell communication than Drosophila, some being expressed in the growth zone crucial for axial elongation in short-germ development. Systemic RNA interference in T. castaneum functions differently from that in Caenorhabditis elegans, but nevertheless offers similar power for the elucidation of gene function and identification of targets...
Despite considerable differences in morphology and complexity of body plans among animals, a grea... more 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, ...
Background The Drosophila larval head is evolutionarily derived at the genetic and morphological ... more Background The Drosophila larval head is evolutionarily derived at the genetic and morphological level. In the beetle Tribolium castaneum, development of the larval head more closely resembles the ancestral arthropod condition. Unlike in Drosophila, a knirps homologue (Tc-kni) is required for development of the antennae and mandibles. However, published Tc-kni data are restricted to cuticle phenotypes and Tc-even-skipped and Tc-wingless stainings in knockdown embryos. Hence, it has remained unclear whether the entire antennal and mandibular segments depend on Tc-kni function, and whether the intervening intercalary segment is formed completely. We address these questions with a detailed examination of Tc-kni function. Results By examining the expression of marker genes in RNAi embryos, we show that Tc-kni is required only for the formation of the posterior parts of the antennal and mandibular segments (i.e. the parasegmental boundaries). Moreover, we find that the role of Tc-kni is ...
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