The transmembrane protein XFLRT3 forms a complex with FGF receptors and promotes FGF signalling.

Fibroblast growth factors (FGFs) signal through high-affinity tyrosine kinase receptors to regulate a diverse range of cellular processes, including cell growth, differentiation and migration, as well as cell death. Here we identify XFLRT3, a member of a leucine-rich-repeat transmembrane protein family, as a novel modulator of FGF signalling. XFLRT3 is co-expressed with FGFs, and its expression is both induced after activation and downregulated after inhibition of FGF signalling. In gain- and loss-of function experiments, FLRT3 and FLRT2 phenocopy FGF signalling in Xenopus laevis. XFLRT3 signalling results in phosphorylation of ERK and is blocked by MAPK phosphatase 1, but not by expression of a dominant-negative phosphatidyl inositol 3-OH kinase (PI(3)K) mutant. XFLRT3 interacts with FGF receptors (FGFRs) in co-immunoprecipitation experiments in vitro and in bioluminescence resonance energy transfer assays in vivo. The results indicate that XFLRT3 is a transmembrane modulator of FGF-MAP kinase signalling in vertebrates.

An atlas of differential gene expression during early Xenopus embryogenesis.

We have carried out a large-scale, semi-automated whole-mount in situ hybridization screen of 8369 cDNA clones in Xenopus laevis embryos. We confirm that differential gene expression is prevalent during embryogenesis since 24% of the clones are expressed non-ubiquitously and 8% are organ or cell type specific marker genes. Sequence analysis and clustering yielded 723 unique genes displaying a differential expression pattern. Of these, 18% were already described in Xenopus, 47% have homologs and 35% are lacking significant sequence similarity in databases. Many of them encode known developmental regulators. We classified 363 of the 723 genes for which a Gene Ontology annotation for molecular function could be attributed and found 'DNA binding' and 'enzyme' the most represented terms. The most common protein domains encoded in these embryonic, differentially expressed genes are the homeobox and RNA Recognition Motif (RRM). Fifty-nine putative orthologs of human disease genes, and 254 organ or cell specific marker genes were identified. Markers were found for nasal placode and archenteron roof, organs for which a specific marker was previously unavailable. Markers were also found for novel subdomains of various other organs. The tissues for which most markers were found are muscle and epidermis. Expression of cell cycle regulators fell in two classes, containing proliferation-promoting and anti-proliferative genes, respectively. We identified 66 new members of the BMP4, chromatin, endoplasmic reticulum, and karyopherin synexpression groups, thus providing a first glimpse of their probable cellular roles. Cluster analysis of tissues to measure tissue relatedness yielded some unorthodox affinities besides expectable lineage relationships. In conclusion, this study represents an atlas of gene expression patterns, which reveals embryonic regionalization, provides novel marker genes, and makes predictions about the functional role of unknown genes.

Heterogeneity of TT virus related sequences isolated from human tumour biopsy specimens.

TT viruses have recently been reported in serum samples from varying percentages of human blood donors and in patients with chronic liver disease. However, no association with human pathology has yet been reported. In a pilot study we analysed 162 biopsy specimens from various human cancers and from colon polyps for the presence of TT virus related sequences by polymerase chain reaction using three sets of nested primers for TT virus detection. All gels were subjected to Southern blot hybridisation, and DNA from hybridising bands was cloned and sequenced. A total of 54.3% of tumour specimens contained identifiable TT virus related sequences. Specimens from hypopharynx, larynx, endometrial, ovarian and bladder cancers were 14-35% positive and gastrointestinal cancers (oesophagus, stomach, colon, rectum) and colon polyps 57-100% positive. Lung cancers (68.4%), mammary cancers (50%), multiple myelomas (85.7%) and human leukaemias (53.3%) also revealed a high prevalence of TT virus related sequences. Since normal control tissues were not available for the tumour biopsy specimens tested, these data do not permit conclusions concerning a possible causal relationship between the virus infections and carcinogenesis. Another aspect, however, deserves attention: the heterogeneity of TT virus clones obtained from the specimens tested here was striking: 66 novel sequences, probably belonging to new types were identified. Only 16 clones corresponded by more than 97% of their sequences to established prototypes. Even individual tumours commonly contained sequences substantially diverging in nucleic acid composition. Up to five different types were identified within an individual tumour. The high variability of these virus types suggests that additional primer combinations within the highly conserved region of the genome would detect a still higher rate of positive tumours.

In situ analysis of gene expression in Xenopus embryos.

The molecular anatomy of the vertebrate embryo was systematically analysed through gene expression during early development of the Xenopus frog using whole-mount in situ hybridization. Expression patterns are documented and assembled into the database Axeldb (http://www.dkfz-heidelberg.de/abt0135/axeldb.htm). Synexpression groups representing genes with shared, complex expression pattern that predict molecular pathways involved in patterning and differentiation have been identified. These sets of co-regulated genes show a striking similarity with operons, and may be a key determinant facilitating evolutionary change leading to animal diversity.

An epidermotropic canine papillomavirus with malignant potential contains an E5 gene and establishes a unique genus.

A novel canine papillomavirus, CfPV-2, was cloned from a footpad lesion of a golden retriever. Unlike the known canine oral papillomavirus (COPV), which has a double-stranded DNA genome size of 8607 bps, the genome of CfPV-2 is 8101 bps. Some of this size difference is due to an abbreviated early-late region (ELR), which is 1200 bps shorter than that of COPV. However, CfPV-2 has other differences from COPV, including the presence of an E5 ORF between the E2 gene and the ELR and an enlarged E4 ORF (one of the largest PV E4 open reading frames). The genome of CfPV-2 shares low homology with all the other papillomaviruses and, even in the most highly conserved ORF of L1, the nucleotide sequence shares only 57% homology with COPV. Due to this highly divergent DNA sequence, CfPV-2 establishes a new PV genus, with its closest phylogenetic relatives being amongst the Xi and Gamma genuses. CfPV-2 also has unique biological features; it induces papillomas on footpads and interdigital regions which, if infection is persistent, can progress to highly metastatic squamous cell carcinoma. CfPV-2 does not induce oral papillomas in immunocompetent animals and antibodies generated against COPV and CfPV-2 are type-specific. The availability of a new canine papillomavirus with differing genetic and biological properties now makes it possible to study type-specific host immune responses, tissue tropism and the comparative analysis of viral gene functions in the dog.

Equine papillomavirus type 1: complete nucleotide sequence and characterization of recombinant virus-like particles composed of the EcPV-1 L1 major capsid protein.

Equus caballus papillomavirus type 1 (EcPV-1) was isolated from a cutaneous papilloma, the most common neoplasm in horses. The complete EcPV-1 nucleotide sequence and genomic organization were determined. Phylogenetic analysis showed that EcPV-1 is a close-to-root papillomavirus, with only distant relationships to the fibropapillomaviruses and the benign cutaneous papillomaviruses. To produce EcPV-1 virus-like particles (VLPs), the EcPV-1 L1 major capsid protein was expressed in insect cells using a recombinant baculovirus vector. The self-assembled EcPV-1 VLPs were morphologically indistinguishable from wild type papillomavirus virions. Monoclonal antibodies were developed against intact and denatured EcPV-1 VLPs. When tested by ELISA, all monoclonal antibodies produced against intact (#18) and some against denatured EcPV-1 VLPs (#16) reacted with intact EcPV-1 VLPs only, demonstrating that the VLPs carry type-specific conformational as well as linear epitopes on their surface. Recombinant EcPV-1 VLPs offer the potential of a noninfectious vaccine to prevent and eradicate equine cutaneous papillomatosis.

Nucleotide sequence and phylogenetic classification of candidate human papilloma virus type 92.

From a basal cell carcinoma (BCC) the complete genome of candidate human papillomavirus (HPV) type 92 was characterized. Phylogenetically, the candidate HPV 92 was relatively distantly related to other cutaneous HPV types within the B1 group. By quantitative real time PCR, 94 viral copies were present per cell in the BCC and another BCC contained 1 viral copy per cell. Lower copy numbers were found in two solar keratoses (1 copy per 33 cells and 1 copy per 60 cells) and two squamous cell carcinomas (1 copy per 436 cells and 1 copy per 1143 cells). The high viral load of HPV 92 in two BCCs differs from the low amount of HPV DNA reported from nonmelanoma skin cancers.

Kremen proteins are Dickkopf receptors that regulate Wnt/beta-catenin signalling.

The Wnt family of secreted glycoproteins mediate cell cell interactions during cell growth and differentiation in both embryos and adults. Canonical Wnt signalling by way of the beta-catenin pathway is transduced by two receptor families. Frizzled proteins and lipoprotein-receptor-related proteins 5 and 6 (LRP5/6) bind Wnts and transmit their signal by stabilizing intracellular beta-catenin. Wnt/beta-catenin signalling is inhibited by the secreted protein Dickkopf1 (Dkk1), a member of a multigene family, which induces head formation in amphibian embryos. Dkk1 has been shown to inhibit Wnt signalling by binding to and antagonizing LRP5/6. Here we show that the transmembrane proteins Kremen1 and Kremen2 are high-affinity Dkk1 receptors that functionally cooperate with Dkk1 to block Wnt/beta-catenin signalling. Kremen2 forms a ternary complex with Dkk1 and LRP6, and induces rapid endocytosis and removal of the Wnt receptor LRP6 from the plasma membrane. The results indicate that Kremen1 and Kremen2 are components of a membrane complex modulating canonical Wnt signalling through LRP6 in vertebrates.