Gap Gene Research Articles

Repression activity of Tailless on h 1 and eve 1 pair-rule stripes

We investigated the hypothesis that several transcriptional repressors are necessary to set the boundaries of anterior pair-rule stripes in Drosophila. Specifically, we tested whether Tailless (Tll) is part of a repression mechanism that correctly sets the anterior boundaries of hairy 1 (h 1) and even-skipped 1 (eve 1) stripes. Single mutant tll embryos displayed subtle deviations from the normal positions of h 1 and eve 1 stripes. Moreover, we observed stronger stripe deviations in embryos lacking both Tll and Sloppy-paired 1 (Slp 1), a common repressor for anterior pair-rule stripes. Using h 1 and eve 1 reporter constructs in the genetic assays, we provided further evidence that interference with normal mechanisms of stripe expression is mediated by Tll repression. Indeed, Tll represses both h 1 and eve 1 reporter stripes when misexpressed. Investigating the expression of other anterior gap genes in different genetic backgrounds and in the misexpression assays strengthened Tll direct repression in the regulation of h 1 and eve 1. Our results are consistent with tll being a newly-identified component of a combinatorial network of repressor genes that control pair-rule stripe formation in the anterior blastoderm of Drosophila.

Staphylococci isolated from ready-to-eat meat – Identification, antibiotic resistance and toxin gene profile

The aim of this study was to analyse the staphylococci isolated from ready-to-eat meat products, including pork ham, chicken cold cuts, pork sausage, salami and pork luncheon meat, sliced in the store to the consumer's specifications, along with species identification and determination of antibiotic resistance. Genes encoding staphylococcal enterotoxins, staphylococcal enterotoxin-like proteins, exfoliative toxins, and toxic shock syndrome toxin 1 were also investigated. From the 41 samples, 75 different staphylococcal isolates were obtained. Based on PCR-RFLP analysis of the gap gene using AluI and HpyCH4V restriction enzymes, the isolates were identified as Staphylococcus equorum (28%), S. vitulinus (16%), S. carnosus (14%), S. succinus (11%), S. xylosus (11%), S. saprophyticus (9%), S. warneri (9%), S. haemolyticus (1%) and S. pasteuri (1%). The incidence and number of resistances to antimicrobials was found to be species but not source of isolation dependent. All S. xylosus, S. saprophyticus, S. haemolyticus and S. pasteuri isolates showed antibiotic resistance. A lower percentage of resistance was recorded for S. warneri (71%) and S. vitulinus (58%), followed by S. equorum (57%), S. carnosus (50%) and S. succinus (50%). The most frequent resistance was observed to fusidic acid (43%). The mecA gene was amplified in 4% of the staphylococci. However, phenotypic resistance to methicillin was not confirmed in any of these isolates. On the other hand, the mecA gene was not detected in any of 9% of the isolates resistant to cefoxitin. It was also found that among 75 isolates, 60 (80%) harbored from 1 to 10 out of 21 analyzed superantigenic toxin genes. The most prevalent genes were: sei (36% isolates) among enterotoxins, seln (32% isolates) among enterotoxin-like proteins and eta encoding exfoliative toxin A (37% isolates). The findings of this study further extend previous observations that, when present in food, not only S. aureus but also other species of staphylococci could be of public health significance.

Abstract 1529: RASAL2 promotes tumor progression and metastasis in colorectal cancer

Abstract Background: Metastatic colorectal cancer (mCRC) is a heterogeneous disease with a poor prognosis. By using high resolution array comparative genomic hybridization (aCGH) and mRNA gene expression microarray of mCRC samples, we found up-regulation of RASAL2, a RAS GAP gene that is located on chromosome 1q, in colorectal tumors. Oncogenic RAS represents one of the most common molecular changes in human colorectal adenocarcinoma. However, the role of RASAL2 in colorectal adenocarcinoma metastasis and the related mechanisms are still unclear. We analyzed its aberrant expression, clinical significance and biological effects in colorectal cancer. Methods: Expression of RASAL2 was examined by QRT-PCR, western blot and immunohistochemistry in CRC cell lines, primary and metastatic CRC and the corresponding normal colonic mucosa. The biological effects of RASAL2 on proliferation, apoptosis, cell cycle, and cell motility and invasiveness were evaluated by siRNA knock down and RASAL2 reconstitution in CRC cell lines. Results: Up-regulation of RASAL2 mRNA was observed in CRC cell lines (n = 9) than normal colon mucosal tissues. Interestingly, significantly higher RASAL2 mRNA was found in cell lines derived from advanced stage tumors (n = 4, Dukes’ C and D) than those from early stage tumors (n = 5, Dukes’ A and B) (P = 0.0317). Up-regulation of RASAL2 mRNA was also found in primary CRCs (n = 77) compared with normal colon mucosa (n = 18, P<0.0001). In 15 cases that paired primary and metastatic tumors were available, 12 (80%) demonstrated higher RASAL2 in metastatic tumors than their primary counterparts. RASAL2 protein was detected in 37% (81 of 219) of CRC by immunohistochemistry. Whereas in the paired normal colonic mucosa, the positive rate is 14% (20 of 142, P<0.0001). We knocked down RASAL2 by siRNA in 2 CRC cell lines (DLD1 and HCT116) with high endogenous RASALs level. Successful knockdown was demonstrated by western blot analysis. SiRASAL2 significantly inhibited cell proliferation (P<0.05), reduced colony formation (P<0.05) and repressed cell invasion and migration ability (P<0.05) in both cell lines. Flow cytometry analysis revealed G1 arrest in cells treated with siRASAL2. Ectopic expression of RASAL2 was performed in 2 CRC cell lines with low endogenous RASAL2 (SW480 and LoVo). Over expression of RASAL2 did not change the cell proliferation and anchorage-dependent growth of these cell lines but effectively enhanced cell invasiveness by transwell invasion assay. Conclusions: Up-regulation of RASAL2 was frequently found in CRC cell lines and primary and metastatic tumors. Our experimental findings suggested that RASAL2 might play an oncogenetic role in CRC and promotes tumor invasion and metastasis. A better understanding of the molecular mechanism of RASAL2 in promoting CRC cell metastasis may lead to a more effective management of mCRC patients. Citation Format: Yi PAN, Joanna Hung Man TONG, Raymond Wai Ming LUNG, Samantha Wei Man LUN, Kwok Wai LO, Anthony Wing Hung CHAN, Ka Fai TO. RASAL2 promotes tumor progression and metastasis in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1529.

Exploring developmental gene toolkit and associated pathways in a potential new model crustacean using transcriptomic analysis.

The crustaceans are one of the largest, most diverse, and most successful groups of invertebrates. The diversity among the crustaceans is also reflected in embryonic development models. However, the molecular genetics that regulates embryonic development is not known in those crustaceans that have a short germ-band development with superficial cleavage, such as Macrobrachium olfersi. This species is a freshwater decapod and has great potential to become a model for developmental biology, as well as for evolutionary and environmental studies. To obtain sequence data of M. olfersi from an embryonic developmental perspective, we performed de novo assembly and annotation of the embryonic transcriptome. Using a pooling strategy of total RNA, paired-end Illumina sequencing, and assembly with multiple k-mers, a total of 25,636,097 pair reads were generated. In total, 99,751 unigenes were identified, and 20,893 of these returned a Blastx hit. KEGG pathway analysis mapped a total of 6866 unigenes related to 129 metabolic pathways. In general, 21,845 unigenes were assigned to gene ontology (GO) categories: molecular function (19,604), cellular components (10,254), and biological processes (13,841). Of these, 2142 unigenes were assigned to the developmental process category. More specifically, a total of 35 homologs of embryonic development toolkit genes were identified, which included maternal effect (one gene), gap (six), pair-rule (six), segment polarity (seven), Hox (four), Wnt (eight), and dorsoventral patterning genes (three). In addition, genes of developmental pathways were found, including TGF-β, Wnt, Notch, MAPK, Hedgehog, Jak-STAT, VEGF, and ecdysteroid-inducible nuclear receptors. RT-PCR analysis of eight genes related to embryonic development from gastrulation to late morphogenesis/organogenesis confirmed the applicability of the transcriptome analysis.

Identification of Staphylococcus spp. and detection of mecA by an oligonucleotide array

Phenotypic identification of coagulase-negative staphylococci (CoNS) is difficult and many staphylococcal species carry mecA. This study developed an array that was able to detect mecA and identify 30 staphylococcal species by targeting the internal transcribed spacer regions. A total of 129 target reference strains (30 species) and 434 clinical isolates of staphylococci were analyzed. Gene sequencing of 16S rRNA, gap or tuf genes was the reference method for species identification. All reference strains (100%) were correctly identified, while the identification rates of clinical isolates of S. aureus and CoNS were 98.9% and 98%, respectively. The sensitivity and specificity for mecA detection were 99% and 100%, respectively, in S. aureus isolates, and both values were 100% in isolates of CoNS. The assay takes 6 h from a purified culture isolate, and so far it has not been performed directly on patient samples.

Predicting Ancestral Segmentation Phenotypes from Drosophila to Anopheles Using In Silico Evolution.

Molecular evolution is an established technique for inferring gene homology but regulatory DNA turns over so rapidly that inference of ancestral networks is often impossible. In silico evolution is used to compute the most parsimonious path in regulatory space for anterior-posterior patterning linking two Dipterian species. The expression pattern of gap genes has evolved between Drosophila (fly) and Anopheles (mosquito), yet one of their targets, eve, has remained invariant. Our model predicts that stripe 5 in fly disappears and a new posterior stripe is created in mosquito, thus eve stripe modules 3+7 and 4+6 in fly are homologous to 3+6 and 4+5 in mosquito. We can place Clogmia on this evolutionary pathway and it shares the mosquito homologies. To account for the evolution of the other pair-rule genes in the posterior we have to assume that the ancestral Dipterian utilized a dynamic method to phase those genes in relation to eve.

Shadow Enhancers Mediate Dynamic Shifts of Gap Gene Expression in the Drosophila Embryo

Drosophila patterning genes often contain pairs of primary and shadow enhancers that possess overlapping activities [1-5]. It has been suggested that this regulatory "redundancy" helps ensure reliable activation of gene expression under stressful conditions such as increases in temperature [4, 5]. There isalso evidence that shadow enhancers help produce sharp on/off boundaries of gene expression in response to small changes in the levels of regulatory factors, such as the maternal Bicoid gradient [6, 7]. Here, we use live-imaging methods to visualize the temporal dynamics of the gap genes Kruppel and knirps, which are essential for the patterning of the thorax and abdomen, respectively [8, 9]. Previous analyses of fixed embryos suggested anterior shifts of the Kruppel and knirps expression patterns [10]. Here, we use computational visualization methods to reveal the precise temporal dynamics of these shifts and further suggest that shadow enhancers are crucial for this process. We discuss potential mechanisms for enhancer dominance, whereby one enhancer represses the other to foster temporal dynamics.

Stability-driven nonnegative matrix factorization to interpret spatial gene expression and build local gene networks

Spatial gene expression patterns enable the detection of local covariability and are extremely useful for identifying local gene interactions during normal development. The abundance of spatial expression data in recent years has led to the modeling and analysis of regulatory networks. The inherent complexity of such data makes it a challenge to extract biological information. We developed staNMF, a method that combines a scalable implementation of nonnegative matrix factorization (NMF) with a new stability-driven model selection criterion. When applied to a set ofDrosophilaearly embryonic spatial gene expression images, one of the largest datasets of its kind, staNMF identified 21 principal patterns (PP). Providing a compact yet biologically interpretable representation ofDrosophilaexpression patterns, PP are comparable to a fate map generated experimentally by laser ablation and show exceptional promise as a data-driven alternative to manual annotations. Our analysis mapped genes to cell-fate programs and assigned putative biological roles to uncharacterized genes. Finally, we used the PP to generate local transcription factor regulatory networks. Spatially local correlation networks were constructed for six PP that span along the embryonic anterior-posterior axis. Using a two-tail 5% cutoff on correlation, we reproduced 10 of the 11 links in the well-studied gap gene network. The performance of PP with theDrosophiladata suggests that staNMF provides informative decompositions and constitutes a useful computational lens through which to extract biological insight from complex and often noisy gene expression data.

Hunchback knockdown induces supernumerary segment formation in Bombyx

Insect segment number within species appears to be fixed irrespective of germ types: long vs. short/intermediate. The present study showed induction of supernumerary segment formation by the knockdown of Bombyx hunchback (Bm-hb), presumably by terminal segment addition, a short/intermediate-like-segmentation mode that is not observed in normal Bombyx embryogenesis. This suggests that Bm-hb suppresses segmentation. The results obtained also suggest that the gap gene Bm-Kr (Bombyx Krüppel) provides a permissive environment for the progression of segmentation by suppressing the expression Bm-hb, which terminates segmentation. This indicates a novel mechanism by which the gap gene is involved in segmentation. It appears that Bm-Kr and Bm-hb are involved in segment counting and their interplay contributes to the correct number of segments being formed in Bombyx. Similar mechanisms may be operating in insects that employ the non-Drosophilan mode of segmentation such as in short/intermediate-germ insects.

Development of the infection strategy of the hemibiotrophic plant pathogen, Colletotrichum orbiculare, and plant immunity

The hemibiotrophic fungus Colletotrichum orbiculare forms appressoria as infection structures and primarily establishes biotrophic infection in cucumber epidermal cells. Subsequently, it develops necrotrophic infection. In the pre-invasion stage, morphogenesis of appressoria of C. orbiculare is triggered by signals from the plant surface. We found that C. orbiculare PAG1 (Perish-in-the-Absence-of-GYP1), a component of MOR [morphogenesis-related NDR (nuclear Dbf2-related) kinase network] plays an essential role as a key component of the plant-specific signaling pathway and that hydrolysis of cutin by a spore surface esterase creates a cutin monomer that constitutes a key plant-derived signal. Development of the infection structure of C. orbiculare is strictly regulated by the cell cycle and we found that proper regulation of G1/S progression via two-component GAP genes, consisting of BUB2 (Budding-Uninhibited-by-Benomyl-2) and BFA1 (Byr-Four-Alike-1) is essential for the establishment of successful infection. In the post-invasion stage, the establishment of the biotrophic phase of hemibiotrophic fungi is crucial for successful infection. We found that C. orbiculare WHI2 (WHIsky-2), an Saccharomyces cerevisiae stress regulator homolog, is involved in the phase transition from biotrophy to necrotrophy through TOR (Target of Rapamycin) signaling, and is thus essential for full pathogenesis.

Regulation of twin of eyeless during Drosophila development

The Pax-6 protein is vital for eye development in all seeing animals, from sea urchins to humans. Either of the Pax6 genes in Drosophila (twin of eyeless and eyeless) can induce a gene cascade leading to formation of entire eyes when expressed ectopically. The twin of eyeless (toy) gene in Drosophila is expressed in the anterior region of the early fly embryo. At later stages it is expressed in the brain, ventral nerve cord and (eventually) the visual primordium that gives rise to the eye-antennal imaginal discs of the larvae. These discs subsequently form the major part of the adult head, including compound eyes. We have searched for genes that are required for normal toy expression in the early embryo to elucidate initiating events of eye organogenesis. Candidate genes identified by mutation analyses were subjected to further knock-out and miss-expression tests to investigate their interactions with toy. Our results indicate that the head-specific gap gene empty spiracles can act as a repressor of Toy, while ocelliless (oc) and spalt major (salm) appear to act as positive regulators of toy gene expression.

A quantitative validated model reveals two phases of transcriptional regulation for the gap gene giant in Drosophila

Understanding eukaryotic transcriptional regulation and its role in development and pattern formation is one of the big challenges in biology today. Most attempts at tackling this problem either focus on the molecular details of transcription factor binding, or aim at genome-wide prediction of expression patterns from sequence through bioinformatics and mathematical modelling. Here we bridge the gap between these two complementary approaches by providing an integrative model of cis-regulatory elements governing the expression of the gap gene giant (gt) in the blastoderm embryo of Drosophila melanogaster. We use a reverse-engineering method, where mathematical models are fit to quantitative spatio-temporal reporter gene expression data to infer the regulatory mechanisms underlying gt expression in its anterior and posterior domains. These models are validated through prediction of gene expression in mutant backgrounds. A detailed analysis of our data and models reveals that gt is regulated by domain-specific CREs at early stages, while a late element drives expression in both the anterior and the posterior domains. Initial gt expression depends exclusively on inputs from maternal factors. Later, gap gene cross-repression and gt auto-activation become increasingly important. We show that auto-regulation creates a positive feedback, which mediates the transition from early to late stages of regulation. We confirm the existence and role of gt auto-activation through targeted mutagenesis of Gt transcription factor binding sites. In summary, our analysis provides a comprehensive picture of spatio-temporal gene regulation by different interacting enhancer elements for an important developmental regulator.

Gap Gene Regulatory Dynamics Evolve along a Genotype Network.

Developmental gene networks implement the dynamic regulatory mechanisms that pattern and shape the organism. Over evolutionary time, the wiring of these networks changes, yet the patterning outcome is often preserved, a phenomenon known as “system drift.” System drift is illustrated by the gap gene network—involved in segmental patterning—in dipteran insects. In the classic model organism Drosophila melanogaster and the nonmodel scuttle fly Megaselia abdita, early activation and placement of gap gene expression domains show significant quantitative differences, yet the final patterning output of the system is essentially identical in both species. In this detailed modeling analysis of system drift, we use gene circuits which are fit to quantitative gap gene expression data in M. abdita and compare them with an equivalent set of models from D. melanogaster. The results of this comparative analysis show precisely how compensatory regulatory mechanisms achieve equivalent final patterns in both species. We discuss the larger implications of the work in terms of “genotype networks” and the ways in which the structure of regulatory networks can influence patterns of evolutionary change (evolvability).

Temporal and spatial dynamics of scaling-specific features of a gene regulatory network in Drosophila.

A widely appreciated aspect of developmental robustness is pattern formation in proportion to size. But how such scaling features emerge dynamically remains poorly understood. Here we generate a data set of the expression profiles of six gap genes in Drosophila melanogaster embryos that differ significantly in size. Expression patterns exhibit size-dependent dynamics both spatially and temporally. We uncover a dynamic emergence of under-scaling in the posterior, accompanied by reduced expression levels of gap genes near the middle of large embryos. Simulation results show that a size-dependent Bicoid gradient input can lead to reduced Krüppel expression that can have long-range and dynamic effects on gap gene expression in the posterior. Thus, for emergence of scaled patterns, the entire embryo may be viewed as a single unified dynamic system where maternally derived size-dependent information interpreted locally can be propagated in space and time as governed by the dynamics of a gene regulatory network.

Analysis of functional importance of binding sites in the Drosophila gap gene network model.

BackgroundThe statistical thermodynamics based approach provides a promising framework for construction of the genotype-phenotype map in many biological systems. Among important aspects of a good model connecting the DNA sequence information with that of a molecular phenotype (gene expression) is the selection of regulatory interactions and relevant transcription factor bindings sites. As the model may predict different levels of the functional importance of specific binding sites in different genomic and regulatory contexts, it is essential to formulate and study such models under different modeling assumptions.ResultsWe elaborate a two-layer model for the Drosophila gap gene network and include in the model a combined set of transcription factor binding sites and concentration dependent regulatory interaction between gap genes hunchback and Kruppel. We show that the new variants of the model are more consistent in terms of gene expression predictions for various genetic constructs in comparison to previous work. We quantify the functional importance of binding sites by calculating their impact on gene expression in the model and calculate how these impacts correlate across all sites under different modeling assumptions.ConclusionsThe assumption about the dual interaction between hb and Kr leads to the most consistent modeling results, but, on the other hand, may obscure existence of indirect interactions between binding sites in regulatory regions of distinct genes. The analysis confirms the previously formulated regulation concept of many weak binding sites working in concert. The model predicts a more or less uniform distribution of functionally important binding sites over the sets of experimentally characterized regulatory modules and other open chromatin domains.

The in-vitro effects of cAMP and cGMP modulators on inter-cellular dye transfer and gene expression levels in rat cumulus cell – oocyte complexes

Supplementation of in-vitro maturation medium with reagents that inhibit meiotic resumption whilst supporting normal function of cumulus cell–oocyte complexes (COC) is challenging. This study compared the in-vitro effects of synthetic and physiologically-relevant reagents on meiotic resumption, gap junction activity and gene expression of rat COC. Higher doses of forskolin reduced gap junction activity. Whilst addition of phosphodiesterase inhibitors initially promoted gap junction activity, this decreased with time in-vitro. Moreover despite oocytes remaining in meiotic arrest, there was a concomitant decline in expression of genes critical for oocyte maturation, and evidence of a reduction in overall transcription rate. Similarly, supplementing media with C-type natriuretic peptide and/or oestradiol delayed meiotic resumption and only initially maintained gap junction activity. In contrast, several key genes were stimulated and overall transcription rates remained constant with time in-vitro. In summary, supplementation of media with physiologically-relevant reagents may better enable normal functions of the COC.

Distinct roles of the RasGAP family proteins in C. elegans associative learning and memory.

The Ras GTPase activating proteins (RasGAPs) are regulators of the conserved Ras/MAPK pathway. Various roles of some of the RasGAPs in learning and memory have been reported in different model systems, yet, there is no comprehensive study to characterize all gap genes in any organism. Here, using reverse genetics and neurobehavioural tests, we studied the role of all known genes of the rasgap family in C. elegans in associative learning and memory. We demonstrated that their proteins are implicated in different parts of the learning and memory processes. We show that gap-1 contribute redundantly with gap-3 to the chemosensation of volatile compounds, gap-1 plays a major role in associative learning, while gap-2 and gap-3 are predominantly required for short- and long-term associative memory. Our results also suggest that the C. elegans Ras orthologue let-60 is involved in multiple processes during learning and memory. Thus, we show that the different classes of RasGAP proteins are all involved in cognitive function and their complex interplay ensures the proper formation and storage of novel information in C. elegans.

Molecular identification of coagulase-negative staphylococci isolated from bovine mastitis and detection of β-lactam resistance.

Bovine mastitis is a frequent cause of economic loss in dairy herds. Coagulase-negative staphylococci (CoNS) are increasing in importance as causeof bovine intramammary infection (IMI) throughout the world in recent years. CoNShave been isolated from milk samples collected from cows with clinical andsubclinical mastitis in several countries. Identification of mastitis pathogensis important when selection appropriate antimicrobial therapy. A total of 93 strains of Staphylococcusspp isolated from bovine clinical andsubclinical mastitis in Argentina during 2010-2013 were identified by Restriction Fragment Length Polymorphism (RFLP)-PCR using the gap gene. The isolates were tested by PCR for the presence of blaZ gene and mecA gene and were tested by disk diffusion for the susceptibilityto penicillin and cefoxitin. The most common CoNS species was S.chromogenes 46.2% (43/93), followed by S. devriesei 11.8% (11/93) and S. haemolyticus 9.7% (9/93). The blaZ gene was detected in 19 (20.4%) but only 16 (17.2%) isolates were resistant to penicillin; the mecA was detected in6 (6.5%) isolates but only 4 (4.3) were resistant to cefoxitin. The 6 mecA-positive isolates showed oxaxillinMICs ≤ 0.5 μg/ml. CoNSare important minor mastitis pathogens and can be the cause of substantial economic losses. Despite the low resistance to PEN in Argentina, the presenceof MR isolates found in this study emphasize the importance of identificationof CoNS when an IMI is present because of the potentially risk of lateraltransfer of resistance genes between staphylococcal species.

Expression study of the hunchback ortholog in embryos of the onychophoran Euperipatoides rowelli.

Zinc finger transcription factors encoded by hunchback homologs play different roles in arthropods, including maternally mediated control, segmentation, and mesoderm and neural development. Knockdown experiments in spider and insect embryos have also revealed homeotic effects and gap phenotypes, the latter indicating a function of hunchback as a "gap gene". Although the expression pattern of hunchback has been analysed in representatives of all four major arthropod groups (chelicerates, myriapods, crustaceans and insects), nothing is known about its expression in one of the closest arthropod relatives, the Onychophora (velvet worms). We therefore examined the expression pattern of hunchback in embryos of the onychophoran Euperipatoides rowelli. Our transcriptomic and phylogenetic analyses revealed only one hunchback ortholog in this species. The putative Hunchback protein contains all nine zinc finger domains known from other protostomes. We found no indication of maternally contributed transcripts of hunchback in early embryos of E. rowelli. Its initial expression occurs in the ectodermal tissue of the antennal segment, followed by the jaw, slime papilla and trunk segments in an anterior-to-posterior progression. Later, hunchback expression is seen in the mesoderm of the developing limbs. A second "wave" of expression commences later in development in the antennal segment and continues posteriorly along each developing nerve cord. This expression is restricted to the neural tissues and does not show any segmental pattern. These findings are in line with the ancestral roles of hunchback in mesoderm and neural development, whereas we find no evidence for a putative function of hunchback as a "gap gene" in Onychophora.

Analyses of interactions among pair-rule genes and the gap gene Krüppel in Bombyx segmentation

In the short-germ insect Tribolium, a pair-rule gene circuit consisting of the Tribolium homologs of even-skipped, runt, and odd-skipped (Tc-eve, Tc-run and Tc-odd, respectively) has been implicated in segment formation. To examine the application of the model to other taxa, I studied the expression and function of pair-rule genes in Bombyx mori, together with a Bombyx homolog of Krüppel (Bm-Kr), a known gap gene. Knockdown embryos of Bombyx homologs of eve, run and odd (Bm-eve, Bm-run and Bm-odd) exhibited asegmental phenotypes similar to those of Tribolium knockdowns. However, pair-rule gene interactions were similar to those of both Tribolium and Drosophila, which, different from Tribolium, shows a hierarchical segmentation mode. Additionally, the Bm-odd expression pattern shares characteristics with those of Drosophila pair-rule genes that receive upstream regulatory input. On the other hand, Bm-Kr knockdowns exhibited a large posterior segment deletion as observed in short-germ insects. However, a detailed analysis of these embryos indicated that Bm-Kr modulates expression of pair-rule genes like in Drosophila, although the mechanisms appear to be different. This suggested hierarchical interactions between Bm-Kr and pair-rule genes. Based on these results, I concluded that the pair-rule gene circuit model that describes Tribolium development is not applicable to Bombyx.