Identification and characterization of genes abnormally expressed in wing-deficient mutant (flügellos) of the silkworm,Bombyx mori

The wing-deficient mutant, flügellos (fl), of the silkworm lacks four wings in the pupa and the adult, due to aberrant wing morphogenesis during metamorphosis. To elucidate the mechanisms of wing-specific deficiencies in the fl mutant, we used mRNA differential display and identified five genes abnormally expressed in the fl wing discs. Northern blot and RT-PCR analyses revealed that four genes were overexpressed, but the fifth one was not transcribed in the fl wing discs. The expression level of ribosome-associated protein p40 in the fl wing discs was elevated approximately 10 times compared to the wild-type (WT) discs. Another overexpressed gene CB10 encodes a novel wing-specific protein with a putative zinc-finger motif. Overexpression of two components of extracellular matrix, cuticle protein 18 (BMCP18) and a fibrillin-like protein AD10, may result in the abnormal wing morphogenesis in the fl mutant. In contrast, a novel member of multifunctional Ca2+-binding protein annexins, designated as annexin b13 (Anx b13), was expressed dominantly in the wing discs of WT but completely repressed in the fl tissues. Strong expression of Anx b13 in wing discs during the fourth and fifth instar indicates that ANX B13 plays an important role in wing morphogenesis.     

Drosophila DREF acting via the JNK pathway is required for thorax development

The Drosophila Jun N-terminal kinase (JNK) gene basket (bsk) promoter contains a DNA replication-related element (DRE)-like sequence, raising the possibility of regulation by the DNA replication-related element-binding factor (DREF). Chromatin immunoprecipitation assays with anti-DREF IgG showed the bsk gene promoter region to be effectively amplified. Luciferase transient expression assays revealed the DRE-like sequence to be important for bsk gene promoter activity, and knockdown of DREF decreased the bsk mRNA level and the bsk gene promoter activity. Furthermore, knockdown of DREF in the notum compartment of wing discs by pannier-GAL4 and UAS-DREFIR resulted in a split thorax phenotype. Monitoring of JNK activity in the wing disc by LacZ expression in a puckered (puc)-LacZ enhancer trap line revealed the reduction in DREF knockdown clones. These findings indicate that DREF is involved in regulation of Drosophila thorax development via actions on the JNK pathway.     

Molecular Marker Identification for Relapse Prediction in 5-FU-Based Adjuvant Chemotherapy in Gastric and Colorectal Cancers

To confirm the clinical significance of NF-κB and JNK protein expression from experimentally identified candidates for predicting prognosis for patients with 5-FU treatment, we evaluated the protein expression of surgically removed specimens. A total of 79 specimens were obtained from 30 gastric and 49 colorectal cancer patients who underwent R0 resection followed by postoperative 5-FU based adjuvant chemotherapy. Immunohistochemical examinations of NF-κB and JNK on tissue microarrays (TMAs) revealed that significantly shorter time-to-relapse (TTR) in both NF-κB(+) and JNK(−) subgroups in both gastric (NF-κB(+), p = 0.0002, HR11.7. 95%CI3 3.2–43.4; JNK(−), p = 0.0302, HR4.4, 95%CI 1.2–16.6) and colon (NF-κB(+), p = 0.0038, HR36.9, 95%CI 3.2–426.0; JNK(−), p = 0.0098, HR3.2, 95%CI 1.3–7.7) cancers. These protein expression patterns also show strong discriminately power in gastric cancer patients for overall survival rate, suggesting a potential utility as prognostic or chemosensitivity markers. Baseline expression of these proteins using gastric cancer cell lines demonstrated the reciprocal patterns between NF-κB and JNK, while 5-FU exposure of these cell lines only induced NF-κB, suggesting that NF-κB plays a dominant role in the response to 5-FU. Subsequent siRNA experiments confirmed that gene knockdown of NF-κB increased 5-FU-specific sensitivity, whereas that of JNK did not affect the chemosensitivity. These results suggest that the expression of these proteins may aid in the decisions involved with adjuvant chemotherapy for gastrointestinal tract cancers.     

Gene Expression Profiling Identifies HOXB4 as a Direct Downstream Target of GATA-2 in Human CD34+ Hematopoietic Cells

Aplastic anemia is characterized by a reduced hematopoietic stem cell number. Although GATA-2 expression was reported to be decreased in CD34-positive cells in aplastic anemia, many questions remain regarding the intrinsic characteristics of hematopoietic stem cells in this disease. In this study, we identified HOXB4 as a downstream target of GATA-2 based on expression profiling with human cord blood-derived CD34-positive cells infected with control or GATA-2 lentiviral shRNA. To confirm the functional link between GATA-2 and HOXB4, we conducted GATA-2 gain-of-function and loss-of-function experiments, and HOXB4 promoter analysis, including luciferase assay, in vitro DNA binding analysis and quantitative ChIP analysis, using K562 and CD34-positive cells. The analyses suggested that GATA-2 directly regulates HOXB4 expression through the GATA sequence in the promoter region. Furthermore, we assessed GATA-2 and HOXB4 expression in CD34-positive cells from patients with aplastic anemia (n = 10) and idiopathic thrombocytopenic purpura (n = 13), and demonstrated that the expression levels of HOXB4 and GATA-2 were correlated in these populations (r = 0.6573, p<0.01). Our results suggested that GATA-2 directly regulates HOXB4 expression in hematopoietic stem cells, which may play an important role in the development and/or progression of aplastic anemia.     

Chemical Discrimination and Aggressiveness via Cuticular Hydrocarbons in a Supercolony-Forming Ant,Formica yessensis

Background

Territorial boundaries between conspecific social insect colonies are maintained through nestmate recognition systems. However, in supercolony-forming ants, which have developed an extraordinary social organization style known as unicoloniality, a single supercolony extends across large geographic distance. The underlying mechanism is considered to involve less frequent occurrence of intraspecific aggressive behaviors, while maintaining interspecific competition. Thus, we examined whether the supercolony-forming species, Formica yessensis has a nestmate recognition system similar to that of the multicolonial species, Camponotus japonicus with respect to the cuticular hydrocarbon-sensitive sensillum (CHC sensillum), which responds only to non-nestmate CHCs. We further investigated whether the sensory system reflects on the apparent reduced aggression between non-nestmates typical to unicolonial species.

Methodology/Principal Findings

F. yessensis constructs supercolonies comprising numerous nests and constitutes the largest supercolonies in Japan. We compared the within-colony or between-colonies’ (1) similarity in CHC profiles, the nestmate recognition cues, (2) levels of the CHC sensillar response, (3) levels of aggression between workers, as correlated with geographic distances between nests, and (4) their genetic relatedness. Workers from nests within the supercolony revealed a greater similarity of CHC profiles compared to workers from colonies outside it. Total response of the active CHC sensilla stimulated with conspecific alien CHCs did not increase as much as in case of C. japonicus, suggesting that discrimination of conspecific workers at the peripheral system is limited. It was particularly limited among workers within a supercolony, but was fully expressed for allospecific workers.

Conclusions/Significance

We demonstrate that chemical discrimination between nestmates and non-nestmates in F. yessensis was not clear cut, probably because this species has only subtle intraspecific differences in the CHC pattern that typify within a supercolony. Such an incomplete chemical discrimination via the CHC sensilla is thus an important factor contributing to decreased occurrence of intraspecific aggressive behavior especially within a supercolony.     

Pancreatic cancer cells enhance the ability of collagen internalization during epithelial-mesenchymal transition

Background

Extracellular matrix (ECM) remodeling is predominantly mediated by fibroblasts using intracellular and extracellular pathways. Although it is well known that extracellular degradation of the ECM by proteases derived from cancer cells facilitates cellular invasion, the intracellular degradation of ECM components by cancer cells has not been clarified. The aim of this study was to characterize collagen internalization, which is the initial step of the intracellular degradation pathway in pancreatic cancer cells, in light of epithelial–mesenchymal transition (EMT).

Methodology/Principal Findings

We analyzed the function of collagen internalization in two pancreatic cancer cell lines, SUIT-2 and KP-2, and pancreatic stellate cells (PSCs) using Oregon Green 488-gelatin. PSCs had a strong ability for collagen uptake, and the pancreatic cancer cells also internalized collagen although less efficiently. The collagen internalization abilities of SUIT-2 and KP-2 cells were promoted by EMT induced by human recombinant transforming growth factor β1 (P<0.05). Expression of Endo180, a collagen uptake receptor, was high in mesenchymal pancreatic cancer cell lines, as determined by EMT marker expression (P<0.01). Quantitative RT-PCR and western blot analyses showed that Endo180 expression was also increased by EMT induction in SUIT-2 and KP-2 cells. Endo180 knockdown by RNA interference attenuated the collagen uptake (P<0.01) and invasive abilities (P<0.05) of SUIT-2 and KP-2 cells.

Conclusions/Significance

Pancreatic cancer cells are capable of collagen internalization, which is enhanced by EMT. This ECM clearance system may be a novel mechanism for cellular invasion and a potential therapeutic target in pancreatic cancer.     

A conserved non-reproductive GnRH system in chordates

Gonadotropin-releasing hormone (GnRH) is a neuroendocrine peptide that plays a central role in the vertebrate hypothalamo-pituitary axis. The roles of GnRH in the control of vertebrate reproductive functions have been established, while its non-reproductive function has been suggested but less well understood. Here we show that the tunicate Ciona intestinalis has in its non-reproductive larval stage a prominent GnRH system spanning the entire length of the nervous system. Tunicate GnRH receptors are phylogenetically closest to vertebrate GnRH receptors, yet functional analysis of the receptors revealed that these simple chordates have evolved a unique GnRH system with multiple ligands and receptor heterodimerization enabling complex regulation. One of the gnrh genes is conspicuously expressed in the motor ganglion and nerve cord, which are homologous structures to the hindbrain and spinal cord of vertebrates. Correspondingly, GnRH receptor genes were found to be expressed in the tail muscle and notochord of embryos, both of which are phylotypic axial structures along the nerve cord. Our findings suggest a novel non-reproductive role of GnRH in tunicates. Furthermore, we present evidence that GnRH-producing cells are present in the hindbrain and spinal cord of the medaka, Oryzias latipes, thereby suggesting the deep evolutionary origin of a non-reproductive GnRH system in chordates.