Epigenetic inactivation of the RASSF10 candidate tumor suppressor gene is a frequent and an early event in gliomagenesis.

We have recently described the N-terminal RAS association domain family of genes, RASSF7-10. Previously, we cloned the N-terminal RASSF10 gene and demonstrated frequent methylation of the associated 5'-CpG island in acute lymphoblastic leukemia. To characterize RASSF10 gene expression, we demonstrate that in developing Xenopus embryos, RASSF10 shows a very striking pattern in the rhombencephalon (hind brain). It is also expressed in other parts of the brain and other organs. Due to the well-defined expression pattern in the brain of Xenopus embryos, we analyzed the methylation status of the RASSF10-associated 5'-CpG island in astrocytic gliomas. RASSF10 was frequently methylated in WHO grade II-III astrocytomas and WHO grade IV primary glioblastomas (67.5%), but was unmethylated in grade I astrocytomas and in DNA from age matched control brain samples. RASSF10 gene expression both at the mRNA and protein levels could be switched back on in methylated glioma cell lines after treatment with 5-aza-2'-deoxycytidine. In secondary glioblastomas (sGBM), RASSF10 methylation was an independent prognostic factor associated with worst progression-free survival and overall survival and occurred at an early stage in their development. In cell culture experiments, overexpression of RASSF10 mediated a reduction in the colony forming ability of two RASSF10-methylated glioma cell lines. Conversely, RNAi-mediated knockdown of RASSF10-stimulated anchorage-independent growth of U87 glioma cells, increased their viability and caused an increase in the cells' proliferative ability. We generated and characterized a RASSF10-specific antibody and demonstrated for the first time that RASSF10 subcellular localization is cell-cycle dependent with RASSF10 colocalizing to centrosomes and associated microtubules during mitosis. This is the first report demonstrating that RASSF10 can act as a tumor suppressor gene and is frequently methylated in gliomas and can potentially be developed into a prognostic marker for sGBM.

The RASSF8 candidate tumor suppressor inhibits cell growth and regulates the Wnt and NF-kappaB signaling pathways.

The Ras-assocation domain family (RASSF) of tumor suppressor proteins until recently contained six proteins named RASSF1-6. Recently, four novel family members, RASSF7-10, have been identified by homology searches for RA-domain-containing proteins. These additional RASSF members are divergent and structurally distinct from RASSF1-6, containing an N-terminal RA domain and lacking the Sav/RASSF/Hpo (SARAH) domain. Here, we show that RASSF8 is ubiquitously expressed throughout the murine embryo and in normal human adult tissues. Functionally, RNAi-mediated knockdown of RASSF8 in non-small-cell lung cancer (NSCLC) cell lines, increased anchorage-independent growth in soft agar and enhanced tumor growth in severe combined immunodeficiency (SCID) mice. Furthermore, EdU staining of RASSF8-depleted cells showed growth suppression in a manner dependent on contact inhibition. We show that endogenous RASSF8 is not only found in the nucleus, but is also membrane associated at sites of cell-cell adhesion, co-localizing with the adherens junction (AJ) component beta-catenin and binding to E-cadherin. Following RASSF8 depletion in two different lung cancer cell lines using alternative small interfering RNA (siRNA) sequences, we show that AJs are destabilized and E-cadherin is lost from the cell membrane. The AJ components beta-catenin and p65 are also lost from sites of cell-cell contact and are relocalized to the nucleus with a concomitant increase in beta-catenin-dependent and nuclear factor-kappaB (NF-kappaB)-dependent signaling following RASSF8 depletion. RASSF8 may also be required to maintain actin -cytoskeletal organization since immunofluorescence analysis shows a striking disorganization of the actin- cytoskeleton following RASSF8 depletion. Accordingly, scratch wound healing studies show increased cellular migration in RASSF8-deficient cells. These results implicate RASSF8 as a tumor suppressor gene that is essential for maintaining AJs function in epithelial cells and have a role in epithelial cell migration.

Ultrasonication as a pre-treatment method for the enhancement of the psychrophilic anaerobic digestion of aquaculture effluents.

The effectiveness of ultrasonication as a pre-treatment method for the psychrophilic anaerobic treatment of aquaculture effluents was assessed using a 4 l solids digester. Ultrasonication of aquaculture wastewater was found to enhance the removal of chemical oxygen demand by anaerobic digestion by almost 10%. There was also a concurrent increase in total biogas production from 0.29 l day(-1) to 0.45 l day(-1) with a corresponding 10% increase in methane concentration. Furthermore, there was an increase of 60% in effluent total ammonia nitrogen concentration as a result of sonication in comparison with a 45% increase for untreated digester waste.

FGF-8 stimulates neuronal differentiation through FGFR-4a and interferes with mesoderm induction in Xenopus embryos.

The role of fibroblast growth factors (FGFs) in neural induction is controversial [1,2]. Although FGF signalling has been implicated in early neural induction [3-5], a late role for FGFs in neural development is not well established. Indeed, it is thought that FGFs induce a precursor cell fate but are not able to induce neuronal differentiation or late neural markers [6-8]. It is also not known whether the same or distinct FGFs and FGF receptors (FGFRs) mediate the effects on mesoderm and neural development. We report that Xenopus embryos expressing ectopic FGF-8 develop an abundance of ectopic neurons that extend to the ventral, non-neural, ectoderm, but show no ectopic or enhanced notochord or somitic markers. FGF-8 inhibited the expression of an early mesoderm marker, Xbra, in contrast to eFGF, which induced ectopic Xbra robustly and neuronal differentiation weakly. The effect of FGF-8 on neurogenesis was blocked by dominant-negative FGFR-4a (DeltaXFGFR-4a). Endogenous neurogenesis was also blocked by DeltaXFGFR-4a and less efficiently by dominant-negative FGFR-1 (XFD), suggesting that it depends preferentially on signalling through FGFR-4a. The results suggest that FGF-8 and FGFR-4a signalling promotes neurogenesis and, unlike other FGFs, FGF-8 interferes with mesoderm induction. Thus, different FGFs show specificity for mesoderm induction versus neurogenesis and this may be mediated, at least in part, by the use of distinct receptors.

Regional gene expression in the epithelia of the Xenopus tadpole gut.

In recent years much progress has been made in the understanding of the genes and mechanisms involved in specification of the cells of the endoderm, which give rise to the epithelium of the gut and respiratory system. However, little is known about the way in which the gut becomes patterned along its anterior-posterior axis, that is, how boundaries are established between the different epithelia of the gut tube. Here we show that the expression patterns of five genes divide the Xenopus tadpole gut epithelium into at least four regions along this axis in the undifferentiated, 3-day-old gut (stage 41), and that these divisions are maintained until at least 7 days, when cell differentiation is well under way. In addition, the restricted expression patterns of these genes clearly mark the anterior and posterior boundaries of the intestine. Xsox2 is expressed in the anterior gut, spanning the oesophagus and stomach but terminating at the stomach/intestine boundary. Xcad1 and Xcad2, two caudal-type homeobox genes, are expressed in a region with an anterior limit at this boundary and a posterior limit between the colon and proctodeum, therefore covering the whole of the small and large intestines. Intestinal fatty acid binding protein (IFABP) is expressed only in the anterior small intestine, and the even-skipped homeobox gene Xhox3 is expressed in the most posterior part of the gut, the proctodeum.

The Xenopus tadpole gut: fate maps and morphogenetic movements.

We have produced a comprehensive fate map showing where the organs of the gut and respiratory system are derived from in the early Xenopus laevis endoderm. We also show the origin of the associated smooth muscle layer on a separate fate map. Comparison of the two maps shows that for most organs of the gut the prospective epithelium and smooth muscle do not overlie each other in the early embryo but come together at a later stage. These fate maps should be useful for future studies into endoderm specification. It was not previously known how the elongation of the endoderm occurs, how the single-layered dorsal and many-layered ventral endoderm gives rise to the single layered epithelium, and whether or not the archenteron cavity actually gives rise to the gut lumen. Using a variety of labelling procedures we show firstly, that radial intercalation occurs in the gut transforming a short thick tube into a long thin tube; secondly, that the archenteron lining does not become the definitive gut lumen. Instead the archenteron cavity almost closes at tailbud stages before providing a nucleus for the definitive gut cavity, which opens up during elongation. Based on this work we present a model explaining the morphogenesis of the gut.

Development of the gut in Xenopus laevis.

The lining of the gut, together with the pancreas, liver, gall bladder, and respiratory system, is formed from the endoderm. The gut also contains smooth muscle and connective tissue of mesodermal origin. The amphibian Xenopus laevis is potentially an excellent model organism for studying how the cells of the endoderm and mesoderm become programmed to produce these internal organs. However, the anatomical complexity of the coiled gut presents a problem in studying its development. In order to overcome this problem we here present a comprehensive guide to the anatomy and histology of the developing Xenopus gut. We use a simple dissection to display its anatomy and the expression of four endodermal markers (alkaline phosphatase, IFABP, XlHbox8, and endodermin). We present schematic diagrams that show how the gut is arranged in three dimensions and how this organisation changes during development. We also present drawings of histological sections of the gut which allow any region to be identified and so represent an atlas for working with sections. Finally, we describe the histology of the cells of the various organs of the gut. This histological identification may be necessary for the identification of parts following experiments in which the normal pattern is disturbed.