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Abstract Introduction Establishment of distinct follicle cell fates at the early stages of Drosophila oogenesis is crucial for achieving proper morphology of individual egg chambers. In Drosophila oogenesis, Notch-signaling controls proliferation and differentiation of follicular cells, which eventually results in the polarization of the anterior-posterior axis of the oocyte. Here we analyzed the functions of Tribolium Notch-signaling factors during telotrophic oogenesis, which differs fundamentally from the polytrophic ovary of Drosophila. Results We found Notch-signaling to be required for maintaining the mitotic cycle of somatic follicle cells. Upon Delta RNAi, follicle cells enter endocycle prematurely, which affects egg-chamber formation and patterning. Interestingly, our results indicate that Delta RNAi phenotypes are not solely due to the premature termination of cell proliferation. Therefore, we monitored the terminal/stalk cell precursor lineage by molecular markers. We observed that upon Delta RNAi terminal and stalk cell populations were absent, suggesting that Notch-signaling is also required for the specification of follicle cell populations, including terminal and stalk precursor cells. Conclusions We demonstrate that with respect to mitotic cycle/endocycle switch Notch-signaling in Tribolium and Drosophila has opposing effects. While in Drosophila a Delta-signal brings about the follicle cells to leave mitosis, Notch-signaling in Triboliumis necessary to retain telotrophic egg-chambers in an “immature” state. In most instances, Notch-signaling is involved in maintaining undifferentiated (or preventing specialized) cell fates. Hence, the role of Notch in Tribolium may reflectthe ancestral function of Notch-signaling in insect oogenesis. The functions of Notch-signaling in patterning the follicle cell epithelium suggest that Tribolium oogenesis may - analogous to Drosophila - involve the stepwise determination of different follicle cell populations. Moreover, our results imply that Notch-signaling may contribute at least to some aspects of oocyte polarization and AP axis also in telotrophic oogenesis.

Abstract Background Hox genes are expressed in specific domains along the anterior posterior body axis and define the regional identity. In most animals these genes are organized in a single cluster in the genome and the order of the genes in the cluster is correlated with the anterior to posterior expression of the genes in the embryo. The conserved order of the various Hox gene orthologs in the cluster among most bilaterians implies that such a Hox cluster was present in their last common ancestor. Vertebrates are the only metazoans so far that have been shown to contain duplicated Hox clusters, while all other bilaterians seem to possess only a single cluster. Results We here show that at least three Hox genes of the spider Cupiennius salei are present as two copies in this spider. In addition to the previously described duplicated Ultrabithorax gene, we here present sequence and expression data of a second Deformed gene, and of two Sex comb reduced genes. In addition, we describe the sequence and expression of the Cupiennius proboscipedia gene. The spider Cupiennius salei is the first chelicerate for which orthologs of all ten classes of arthropod Hox genes have been described. The posterior expression boundary of all anterior Hox genes is at the tagma border of the prosoma and opisthosoma, while the posterior boundary of the posterior Hox genes is at the posterior end of the embryo. Conclusion The presence of at least three duplicated Hox genes points to a major duplication event in the lineage to this spider, perhaps even of the complete Hox cluster as has taken place in the lineage to the vertebrates. The combined data of all Cupiennius Hox genes reveal the existence of two distinct posterior expression boundaries that correspond to morphological tagmata boundaries.

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., (© 2022. The Author(s).)

Background: Stem cells are undifferentiated cells with a potential for self-renewal, which are essential to support normal development and homeostasis. To gain insight into the molecular mechanisms underlying adult stem cell biology and organ evolution, we use the telotrophic ovary of the beetle Tribolium. To this end, we participated in a large-scale RNAi screen in the red flour beetle Tribolium , which identified functions in embryonic and postembryonic development for more than half of the Tribolium genes., Results: We identified TC003132 as candidate gene for the follicle stem cell linage in telotrophic Tribolium oogenesis. TC003132 belongs to the Casein Kinase 2 substrate family (CK2S), which in humans is associated with the proliferative activity of different cancers. Upon TC003132 RNAi, central pre-follicular cells are lost, which results in termination of oogenesis. Given that also Notch-signalling is required to promote the mitotic activity of central pre-follicular cells, we performed epistasis experiments with Notch and cut . In addition, we identified a putative follicle stem cell population by monitoring the mitotic pattern of wild type and TC003132 depleted follicle cells by EdU incorporations. In TC003132 RNAi these putative FSCs cease the expression of differentiation makers and are eventually lost., Conclusions: TC003132 depleted pre-follicular cells neither react to mitosis or endocycle stimulating signals, suggesting that TC003132 provides competence for differentiation cues. This may resemble the situation in C. elegans were CK2 is required to maintain the balance between proliferation and differentiation in the germ line. Since the earliest effect of TC003132 RNAi is characterized by the loss of putative FSCs, we posit that TC003132 crucially contributes to the proliferation or maintenance of follicle stem cells in the telotrophic Tribolium ovary.

Terminal regions of the Drosophila embryo are patterned by the localized activation of the Torso-RTK pathway, which promotes the downregulation of Capicua. In the short-germ beetle Tribolium , the function of the terminal system appears to be rather different, as the pathway promotes axis elongation and, in addition, is required for patterning the extra-embryonic serosa at the anterior. Here, we show that Torso signalling also induces gene expression by relieving Capicua-mediated repression in Tribolium Given that the majority of Torso target genes remain to be identified, we established a differential gene-expression screen. A subset of 50 putative terminal target genes was screened for functions in early embryonic patterning. Of those, 13 genes show early terminal expression domains and also phenotypes were related to terminal patterning. Among others, we found the PIWI-interacting RNA factor Maelstrom to be crucial for early embryonic polarization. Tc-mael is required for proper serosal size regulation and head morphogenesis. Moreover, Tc-mael promotes growth-zone formation and axis elongation. Our results suggest that posterior patterning by Torso may be realized through Maelstrom-dependent activation of posterior Wnt domains., (© 2017. Published by The Company of Biologists Ltd.)

Genetic screens are powerful tools to identify the genes required for a given biological process. However, for technical reasons, comprehensive screens have been restricted to very few model organisms. Therefore, although deep sequencing is revealing the genes of ever more insect species, the functional studies predominantly focus on candidate genes previously identified in Drosophila, which is biasing research towards conserved gene functions. RNAi screens in other organisms promise to reduce this bias. Here we present the results of the iBeetle screen, a large-scale, unbiased RNAi screen in the red flour beetle, Tribolium castaneum, which identifies gene functions in embryonic and postembryonic development, physiology and cell biology. The utility of Tribolium as a screening platform is demonstrated by the identification of genes involved in insect epithelial adhesion. This work transcends the restrictions of the candidate gene approach and opens fields of research not accessible in Drosophila.

The iBeetle-Base (http://ibeetle-base.uni-goettingen.de) makes available annotations of RNAi phenotypes, which were gathered in a large scale RNAi screen in the red flour beetle Tribolium castaneum (iBeetle screen). In addition, it provides access to sequence information and links for all Tribolium castaneum genes. The iBeetle-Base contains the annotations of phenotypes of several thousands of genes knocked down during embryonic and metamorphic epidermis and muscle development in addition to phenotypes linked to oogenesis and stink gland biology. The phenotypes are described according to the EQM (entity, quality, modifier) system using controlled vocabularies and the Tribolium morphological ontology (TrOn). Furthermore, images linked to the respective annotations are provided. The data are searchable either for specific phenotypes using a complex 'search for morphological defects' or a 'quick search' for gene names and IDs. The red flour beetle Tribolium castaneum has become an important model system for insect functional genetics and is a representative of the most species rich taxon, the Coleoptera, which comprise several devastating pests. It is used for studying insect typical development, the evolution of development and for research on metabolism and pest control. Besides Drosophila, Tribolium is the first insect model organism where large scale unbiased screens have been performed., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Understanding how Bacillus thuringiensis (Bt) toxins interact with proteins in the midgut of susceptible coleopteran insects is crucial to fully explain the molecular bases of Bt specificity and insecticidal action. In this work, aminopeptidase N (TcAPN-I), E-cadherin (TcCad1), and sodium solute symporter (TcSSS) have been identified by ligand blot as putative Cry3Ba toxin-binding proteins in Tribolium castaneum (Tc) larvae. RNA interference knockdown of TcCad1 or TcSSS proteins resulted in decreased susceptibility to Cry3Ba toxin, demonstrating the Cry toxin receptor functionality for these proteins. In contrast, TcAPN-I silencing had no effect on Cry3Ba larval toxicity, suggesting that this protein is not relevant in the Cry3Ba toxin mode of action in Tc. Remarkable features of TcSSS protein were the presence of cadherin repeats in its amino acid sequence and that a TcSSS peptide fragment containing a sequence homologous to a binding epitope found in Manduca sexta and Tenebrio molitor Bt cadherin functional receptors enhanced Cry3Ba toxicity. This is the first time that the involvement of a sodium solute symporter protein as a Bt functional receptor has been demonstrated. The role of this novel receptor in Bt toxicity against coleopteran insects together with the lack of receptor functionality of aminopeptidase N proteins might account for some of the differences in toxin specificity between Lepidoptera and Coleoptera insect orders.

Abdominal patterning in Drosophila requires the function of Nanos (nos) and Pumilio (pum) to repress posterior translation of hunchback mRNA. Here we provide the first functional analysis of nanos and pumilio genes during blastodermal patterning of a short-germ insect. We found that nos and pum in the red flour beetle Tribolium castaneum crucially contribute to posterior segmentation by preventing hunchback translation. While this function seems to be conserved among insects, we provide evidence that Nos and Pum may also act on giant expression, another gap gene. After depletion of nos and pum by parental RNAi, Hunchback and giant remain ectopically at the posterior blastoderm and the posterior Krüppel (Kr) domain is not being activated. giant may be a direct target of Nanos and Pumilio in Tribolium and presumably prevents early Kr expression. In the absence of Kr, the majority of secondary gap gene domains fail to be activated, and abdominal segmentation is terminated prematurely. Surprisingly, we found Nos and Pum also to be involved in early head patterning, as the loss of Nos and Pum results in deletions and transformations of gnathal and pre-gnathal anlagen. Since the targets of Nos and Pum in head development remain to be identified, we propose that anterior patterning in Tribolium may involve additional maternal factors., (Copyright © 2012 Elsevier Inc. All rights reserved.)

In Drosophila, the JAK-STAT signalling pathway regulates a broad array of developmental functions including segmentation and oogenesis. Here we analysed the functions of Tribolium JAK-STAT signalling factors and of Suppressor Of Cytokine Signalling (SOCS) orthologues, which are known to function as negative regulators of JAK-STAT signalling, during telotrophic oogenesis and short-germ embryogenesis. The beetle Tribolium features telotrophic ovaries, which differ fundamentally from the polytrophic ovary of Drosophila. While we found the requirement for JAK-STAT signalling in specifying the interfollicular stalk to be principally conserved, we demonstrate that these genes also have early and presumably telotrophic specific functions. Moreover, we show that the SOCS genes crucially contribute to telotrophic Tribolium oogenesis, as their inactivation by RNAi results in compound follicles. During short-germ embryogenesis, JAK-STAT signalling is required in the maintenance of segment primordia, indicating that this signalling cascade acts in the framework of the segment-polarity network. In addition, we demonstrate that JAK-STAT signalling crucially contributes to early anterior patterning. We posit that this signalling cascade is involved in achieving accurate levels of expression of individual pair-rule and gap gene domains in early embryonic patterning., (Copyright © 2010 Elsevier Inc. All rights reserved.)

In the short-germ beetle Tribolium castaneum, the head gap gene orthodenticle (Tc-otd) has been proposed to functionally substitute for bicoid, the anterior morphogen unique to higher dipterans. In this study we reanalyzed the function of Tc-otd. We obtained a similar range of cuticle phenotypes as in previously described RNAi experiments; however, we noticed unexpected effects on blastodermal cell fates. First, we found that Tc-otd is essential for dorsoventral patterning. RNAi depletion results in lateralized embryos, a fate map change that by itself can explain the observed loss of the anterior head, which is a ventral anlage in Tribolium. We find that this effect is due to diminished expression of short gastrulation (sog), a gene essential for establishment of the Decapentaplegic (Dpp) gradient in this species. Second, we found that gnathal segment primordia in Tc-otd RNAi embryos are shifted anteriorly but otherwise appear patterned normally. This anteroposterior (AP) fate map shift might largely be due to diminished zen-1 expression and is not responsible for the severe segmentation defects observed in some Tc-otd RNAi embryos. As neither Tc-sog nor Tc-zen-1 probably requires Otd gradient-mediated positional information, we posit that the blastoderm function of Tc-Otd depends on its initial homogeneous maternal expression and that this maternal factor does not provide significant positional information for Tribolium blastoderm embryos.

Posterior expression of Caudal is required for early embryonic development in nematodes, arthropods, and vertebrates. In Drosophila, ectopic Caudal in anterior cells can induce head defects, and in Caenorhabditis the absence of Caudal in anterior embryonic cells is required for proper development. Anterior Caudal repression in these species is achieved through unrelated translational repressors, the homeodomain protein Bicoid and the KH domain factor Mex-3, respectively. Here we report that the Mex-3 ortholog in the flour beetle Tribolium plays a crucial role in head formation and that Caudal in this species is repressed by the combined activities of Mex-3 and Zen-2, a protein sharing common ancestry with the dipteran morphogen Bicoid. We propose that Mex-3 represents an ancient "anterior" promoting factor common to all Ecdysozoa (and maybe all Bilateria), whose role has been usurped in higher dipterans by Bicoid.

INTRODUCTIONThe spider Cupiennius salei, commonly known as the American wandering spider, is a particularly useful laboratory model for embryological studies because of the availability of tools to study and manipulate its embryonic development. Cupiennius is used to study axis formation, segmentation, appendage development, neurogenesis, and silk production. These studies contribute to our understanding of the evolution of these processes, but they also help us to understand the origin and diversification of evolutionary novelties. Comparisons between spiders and insects can show the degree of conservation and divergence of developmental mechanisms during arthropod evolution. Any embryological feature conserved between spiders and insects is likely to represent an ancestral feature for arthropods. Comparative molecular embryological work in insects and spiders should eventually allow us to define a molecular archetype for the phylum Arthropoda. This in itself will be a necessary cornerstone for comparing the different metazoan phyla, including chordates. The discovery of RNA interference (RNAi), in which double-stranded RNA (dsRNA) suppresses the translation of homologous mRNA, has had a huge impact on evolutionary developmental biology by enabling the analysis of loss-of-function phenotypes in organisms in which classical genetic analysis is laborious or not possible. This protocol describes the application of RNAi to embryos of the spider C. salei.

INTRODUCTIONThe spider Cupiennius salei, commonly known as the American wandering spider, is particularly useful for embryological studies because of the availability of tools to study and manipulate its embryonic development. Cupiennius is used to study axis formation, segmentation, appendage development, neurogenesis, and silk production. These studies contribute to our understanding of the evolution of these processes, but they also help us to understand the origin and diversification of evolutionary novelties. Comparisons between spiders and insects can show the degree of conservation and divergence of developmental mechanisms during arthropod evolution. Any embryological feature conserved between spiders and insects is likely to represent an ancestral feature for arthropods. Comparative molecular embryological work in insects and spiders should eventually allow us to define a molecular archetype for the phylum Arthropoda. This will be a necessary cornerstone for comparing the different metazoan phyla, including chordates. This protocol describes the detection of proliferating cells in whole-mount Cupiennius embryos. When labeled nucleotides are introduced into mitotically dividing cells, these cells incorporate the labels into the newly synthesized DNA. Thus, only cells that have synthesized DNA after the addition of the label will be detected. This protocol uses 5-bromo-2'-deoxy-uridine (BrdU) as a label that is subsequently detected by immunocytochemistry. BrdU labeling is a relatively easy way to detect cells that have recently synthesized DNA. The main advantage of this technique is that the label accumulates over time and, by varying the incubation time before fixation, an increasingly cumulative picture of cell proliferation activity can be obtained.

INTRODUCTIONThe spider Cupiennius salei, commonly known as the American wandering spider, is a particularly useful laboratory model for embryological studies because of the availability of tools to study and manipulate its embryonic development. Cupiennius is used to study axis formation, segmentation, appendage development, neurogenesis, and silk production. These studies contribute to our understanding of the evolution of these processes, but they also help us to understand the origin and diversification of evolutionary novelties. Comparisons between spiders and insects can show the degree of conservation and divergence of developmental mechanisms during arthropod evolution. Any embryological feature conserved between spiders and insects is likely to represent an ancestral feature for arthropods. Comparative molecular embryological work in insects and spiders should eventually allow us to define a molecular archetype for the phylum Arthropoda. This in itself will be a necessary cornerstone for comparing the different metazoan phyla, including chordates. This protocol describes the collection and fixation of embryos from C. salei. The fixed embryos can be stored at -20°C for prolonged periods and used for in situ hybridization, in studies of apoptosis using terminal deoxynucleotidyl-transferase-mediated dUTP-digoxigenin nick-end labeling (TUNEL), and for immunohistochemistry.

INTRODUCTIONThe spider Cupiennius salei, commonly known as the American wandering spider, is a particularly useful laboratory model for embryological studies because of the availability of tools to study and manipulate its embryonic development. Cupiennius is used to study axis formation, segmentation, appendage development, neurogenesis, and silk production. These studies contribute to our understanding of the evolution of these processes, but they also help us to understand the origin and diversification of evolutionary novelties. Comparisons between spiders and insects can show the degree of conservation and divergence of developmental mechanisms during arthropod evolution. Any embryological feature conserved between spiders and insects is likely to represent an ancestral feature for arthropods. Comparative molecular embryological work in insects and spiders should eventually allow us to define a molecular archetype for the phylum Arthropoda. This in itself will be a necessary cornerstone for comparing the different metazoan phyla, including chordates. Spider embryos can be studied as whole mounts under the dissection microscope. Alternatively, the embryos can be dissected and observed under the compound microscope. Preparing and dissecting spider embryos for compound microscopy is difficult due to the high amount of yolk, which makes the embryos very fragile. The following protocol describes how to make the necessary tools, then use them to obtain good preparations. Not all embryonic stages can be dissected and prepared; very young stages can only be examined as whole mounts.

INTRODUCTIONThe spider Cupiennius salei is a useful laboratory model for embryological and physiological studies. Its highly developed sensory organs also make it an excellent model for behavioral studies. Furthermore, Cupiennius has contributed greatly to the study of evolutionary developmental questions. This chelicerate arthropod is particularly useful for such studies because of its phylogenetic position and the availability of tools to study and manipulate its embryonic development.

INTRODUCTIONThe spider Cupiennius salei, commonly known as the American wandering spider, is a particularly useful laboratory model for embryological studies because of the availability of tools to study and manipulate its embryonic development. Cupiennius is used to study axis formation, segmentation, appendage development, neurogenesis, and silk production. These studies contribute to our understanding of the evolution of these processes, but they also help us to understand the origin and diversification of evolutionary novelties. Comparisons between spiders and insects can show the degree of conservation and divergence of developmental mechanisms during arthropod evolution. Any embryological feature conserved between spiders and insects is likely to represent an ancestral feature for arthropods. Comparative molecular embryological work in insects and spiders should eventually allow us to define a molecular archetype for the phylum Arthropoda. This in itself will be a necessary cornerstone for comparing the different metazoan phyla, including chordates. This protocol describes the detection of transcripts in fixed C. salei embryos using whole-mount in situ hybridization.

INTRODUCTIONThe spider Cupiennius salei, commonly known as the American Wandering Spider, is a particularly useful laboratory model for embryological studies because of the availability of tools to study and manipulate its embryonic development. Cupiennius is used to study axis formation, segmentation, appendage development, neurogenesis, and silk production. These studies contribute to our understanding of the evolution of these processes, but they also help us to understand the origin and diversification of evolutionary novelties. Comparisons between spiders and insects can show the degree of conservation and divergence of developmental mechanisms during arthropod evolution. Any embryological feature conserved between spiders and insects is likely to represent an ancestral feature for arthropods. Comparative molecular embryological work in insects and spiders should eventually allow us to define a molecular archetype for the phylum Arthropoda. This in itself will be a necessary cornerstone for comparing the different metazoan phyla, including chordates. A feature of apoptosis (i.e., cell death) is the cleavage or fragmentation of DNA that occurs in dead or dying cells. This protocol describes the detection of fragmented DNA in whole-mount Cupiennius embryos. The 3'-OH ends of these DNA fragments can be labeled with the terminal deoxynucleotidyl-transferase-mediated dUTP-digoxigenin nick-end labeling (TUNEL) technique. This protocol uses a terminal deoxynucleotidyl transferase to add labeled dUTP to the fragmented DNA, and this label is then detected by immunocytochemistry. The TUNEL technique is a relatively easy way to obtain a reliable picture of the cell death pattern during normal and abnormal development.

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 for selective insect control.

Background: RNA interference (RNAi) is a highly conserved cellular mechanism. In some organisms, such as Caenorhabditis elegans, the RNAi response can be transmitted systemically. Some insects also exhibit a systemic RNAi response. However, Drosophila, the leading insect model organism, does not show a robust systemic RNAi response, necessitating another model system to study the molecular mechanism of systemic RNAi in insects., Results: We used Tribolium, which exhibits robust systemic RNAi, as an alternative model system. We have identified the core RNAi genes, as well as genes potentially involved in systemic RNAi, from the Tribolium genome. Both phylogenetic and functional analyses suggest that Tribolium has a somewhat larger inventory of core component genes than Drosophila, perhaps allowing a more sensitive response to double-stranded RNA (dsRNA). We also identified three Tribolium homologs of C. elegans sid-1, which encodes a possible dsRNA channel. However, detailed sequence analysis has revealed that these Tribolium homologs share more identity with another C. elegans gene, tag-130. We analyzed tag-130 mutants, and found that this gene does not have a function in systemic RNAi in C. elegans. Likewise, the Tribolium sid-like genes do not seem to be required for systemic RNAi. These results suggest that insect sid-1-like genes have a different function than dsRNA uptake. Moreover, Tribolium lacks homologs of several genes important for RNAi in C. elegans., Conclusion: Although both Tribolium and C. elegans show a robust systemic RNAi response, our genome-wide survey reveals significant differences between the RNAi mechanisms of these organisms. Thus, insects may use an alternative mechanism for the systemic RNAi response. Understanding this process would assist with rendering other insects amenable to systemic RNAi, and may influence pest control approaches.

Background: Hox genes are expressed in specific domains along the anterior posterior body axis and define the regional identity. In most animals these genes are organized in a single cluster in the genome and the order of the genes in the cluster is correlated with the anterior to posterior expression of the genes in the embryo. The conserved order of the various Hox gene orthologs in the cluster among most bilaterians implies that such a Hox cluster was present in their last common ancestor. Vertebrates are the only metazoans so far that have been shown to contain duplicated Hox clusters, while all other bilaterians seem to possess only a single cluster., Results: We here show that at least three Hox genes of the spider Cupiennius salei are present as two copies in this spider. In addition to the previously described duplicated Ultrabithorax gene, we here present sequence and expression data of a second Deformed gene, and of two Sex comb reduced genes. In addition, we describe the sequence and expression of the Cupiennius proboscipedia gene. The spider Cupiennius salei is the first chelicerate for which orthologs of all ten classes of arthropod Hox genes have been described. The posterior expression boundary of all anterior Hox genes is at the tagma border of the prosoma and opisthosoma, while the posterior boundary of the posterior Hox genes is at the posterior end of the embryo., Conclusion: The presence of at least three duplicated Hox genes points to a major duplication event in the lineage to this spider, perhaps even of the complete Hox cluster as has taken place in the lineage to the vertebrates. The combined data of all Cupiennius Hox genes reveal the existence of two distinct posterior expression boundaries that correspond to morphological tagmata boundaries.

In the long germ insect Drosophila, all body segments are determined almost simultaneously at the blastoderm stage under the control of the anterior, the posterior, and the terminal genetic system . Most other arthropods (and similarly also vertebrates) develop more slowly as short germ embryos, where only the anterior body segments are specified early in embryogenesis. The body axis extends later by the sequential addition of new segments from the growth zone or the tail bud . The mechanisms that initiate or maintain the elongation of the body axis (axial growth) are poorly understood . We functionally analyzed the terminal system in the short germ insect Tribolium. Unexpectedly, Torso signaling is required for setting up or maintaining a functional growth zone and at the anterior for the extraembryonic serosa. Thus, as in Drosophila, fates at both poles of the blastoderm embryo depend on terminal genes, but different tissues are patterned in Tribolium. Short germ development as seen in Tribolium likely represents the ancestral mode of how the primary body axis is set up during embryogenesis. We therefore conclude that the ancient function of the terminal system mainly was to define a growth zone and that in phylogenetically derived insects like Drosophila, Torso signaling became restricted to the determination of terminal body structures.

Arthropods, vertebrates, and annelids all have a segmented body. Our recent discovery of involvement of Notch-signalling in spider segmentation revived the discussion on the origin of segmented body plans and suggests the sharing of a common genetic program in a common ancestor. Here, we analysed the spider homologues of the Suppressor of Hairless and Presenilin genes, which encode components of the canonical Notch-pathway, to further explore the role of Notch-signalling in spider segmentation. RNAi silencing of two spider Suppressor of Hairless homologues and the spider Presenilin homologue causes severe segmentation phenotypes. The most prominent defect is the consistent breakdown of segmentation after the formation of three (Suppressor of Hairless) or five (Presenilin) opisthosomal segments. These phenotypes indicate that Notch-signalling during spider segmentation likely involves the canonical pathway via Presenilin and Suppressor of Hairless. Furthermore, it implies that Notch-signalling influences both the formation and patterning of the spider segments: it is required for the specification of the posterior segments and for proper specification of the segment boundaries. We argue that alternative, partly redundant, pathways might act in the formation of the anterior segments that are not active in the posterior segments. This suggests that at least some differences exist in the specification of anterior and posterior segments of the spider, a finding that may be valid for most short germ arthropods. Our data provide additional evidence for the similarities of Notch-signalling in spider segmentation and vertebrate somitogenesis and strengthen our previous notion that the formation of the segments in arthropods and vertebrates might have shared a genetic program in a common ancestor.

Pair-rule patterning forms a key step for segmentation in insects. The expression patterns of pair-rule gene orthologs in representatives of other arthropod groups imply that these genes were segmentation genes in the last common ancestor of the various arthropod groups, but almost nothing is known about the underlying mechanism in noninsect arthropods. Here, we cloned and analyzed members of the Pax group III genes from the spider Cupiennius salei. Pax group III genes comprise genes like the Drosophila genes paired, gooseberry, and gooseberry-neuro, as well as the vertebrate Pax 3 and Pax 7 genes. We recovered three Pax group III genes from the spider C. salei, Cs-pairberry-1, Cs-pairberry-2, and Cs-pairberry-3, and show that the combined expression of the three spider genes mimics the patterns in insects, suggesting an ancestral role for Pax group III genes in segmentation, neurogenesis, and appendage formation in arthropods. One of the genes, pairberry-3, is expressed in a segmental periodicity before overt morphological segmentation is visible, suggesting a single segmental periodicity for opisthosomal segment pattering in the spider. Comparisons among arthropods suggest that the underlying mechanisms for pair-rule gene orthologs are more diverged than the ones for the segment-polarity genes. We argue that there may be a correlation between the lower variation in patterns of segment-polarity genes and the phylotypic stage. The segment-polarity genes are required to define the segment borders of the embryo at the germ-band stage, the arthropod phylotypic stage. Pair-rule gene orthologs act more upstream and may display more variation in their action.

It is currently debated whether segmentation in different animal phyla has a common origin and shares a common genetic mechanism. The apparent use of different genetic networks in arthropods and vertebrates has become a strong argument against a common origin of segmentation. Our knowledge of arthropod segmentation is based mainly on the insect Drosophila, in which a hierarchical cascade of transcription factors controls segmentation. The function of some of these genes seems to be conserved among arthropods, including spiders, but not vertebrates. The Notch pathway has a key role in vertebrate segmentation (somitogenesis) but is not involved in Drosophila body segmentation. Here we show that Notch and Delta genes are involved in segmentation of another arthropod, the spider Cupiennius salei. Expression patterns of Notch and Delta, coupled with RNA interference experiments, identify many similarities between spider segmentation and vertebrate somitogenesis. Our data indicate that formation of the segments in arthropods and vertebrates may have shared a genetic programme in a common ancestor and that parts of this programme have been lost in particular descendant lineages.

Chelicerates represent a basal arthropod group, which makes them an excellent system for the study of evolutionary processes in arthropods. To enable functional studies in chelicerates, we developed a double-stranded RNA-interference (RNAi) protocol for spiders while studying the function of the Distal-less gene. We isolated the Distal-less gene from the spider Cupiennius salei. Cs-Dll gene expression is first seen in cells of the prosomal segments before the outgrowth of the appendages. After the appendages have formed, Cs-Dll is expressed in the distal portion of the prosomal appendages, and in addition, in the labrum, in two pairs of opisthosmal (abdominal) limb buds, in the head region, and at the posterior-most end of the spider embryo. In embryos, in which Dll was silenced by RNAi, the distal part of the prosomal appendages was missing and the labrum was completely absent. Thus, Dll also plays a crucial role in labrum formation. However, the complete lack of labrum in RNAi embryos may point to a different nature of the labrum from the segmental appendages. Our data show that the expression of Dll in the appendages is conserved among arthropods, and furthermore that the role of Dll is evolutionarily conserved in the formation of segmental appendages in arthropods.