Yoshiro Nakano

Mechanisms and functions of Hedgehog signalling across the metazoa.

Hedgehog proteins constitute one of a small number of families of secreted signals that have a central role in the development of metazoans. Genetic analyses in flies, fish and mice have uncovered the major components of the pathway that transduces Hedgehog signals, and recent genome sequence projects have provided clues about its evolutionary origins. In this Review we provide an updated overview of the mechanisms and functions of this signalling pathway, highlighting the conserved and divergent features of the pathway, as well as some of the common themes in its deployment that have emerged from recent studies.

Contrasting distributions of patched and hedgehog proteins in the Drosophila embryo

The segment polarity genes patched (ptc) and hedgehog (hh) are thought to encode a receptor and signal molecule respectively, components of a signal transduction pathway that regulates the transcription of the wingless gene in the Drosophila embryo. Here we describe the production of antibodies specific for the products of these two genes and the patterns of protein distribution that they reveal in the developing embryo. The results are consistent with the hh protein being secreted by cells in which it is expressed and support a role for ptc in the reception of the putative hh encoded signal.

Mutations in the Novel Membrane Protein Spinster Interfere with Programmed Cell Death and Cause Neural Degeneration in Drosophila melanogaster

ABSTRACT Mutations in the spin gene are characterized by an extraordinarily strong rejection behavior of female flies in response to male courtship. They are also accompanied by decreases in the viability, adult life span, and oviposition rate of the flies. Inspin mutants, some oocytes and adult neural cells undergo degeneration, which is preceded by reductions in programmed cell death of nurse cells in ovaries and of neurons in the pupal nervous system, respectively. The central nervous system (CNS) of spinmutant flies accumulates autofluorescent lipopigments with characteristics similar to those of lipofuscin. The spinlocus generates at least five different transcripts, with only two of these being able to rescue the spin behavioral phenotype; each encodes a protein with multiple membrane-spanning domains that are expressed in both the surface glial cells in the CNS and the follicle cells in the ovaries. Orthologs of the spin gene have also been identified in a number of species from nematodes to humans. Analysis of the spin mutant will give us new insights into neurodegenerative diseases and aging.

Functional domains and sub-cellular distribution of the Hedgehog transducing protein Smoothened in Drosophila.

The Hedgehog signalling pathway is deployed repeatedly during normal animal development and its inappropriate activity is associated with various tumours in human. The serpentine protein Smoothened (Smo) is essential for cells to respond to the Hedeghog (Hh) signal; oncogenic forms of Smo have been isolated from human basal cell carcinomas. Despite similarities with ligand binding G-protein coupled receptors, the molecular basis of Smo activity and its regulation remains unclear. In non-responding cells, Smo is suppressed by the activity of another multipass membrane spanning protein Ptc, which acts as the Hh receptor. In Drosophila, binding of Hh to Ptc has been shown to cause an accumulation of phosphorylated Smo protein and a concomitant stabilisation of the activated form of the Ci transcription factor. Here, we identify domains essential for Smo activity and investigate the sub-cellular distribution of the wild type protein in vivo. We find that deletion of the amino terminus and the juxtamembrane region of the carboxy terminus of the protein result in the loss of normal Smo activity. Using Green Fluorescent Protein (GFP) and horseradish peroxidase fusion proteins we show that Smo accumulates in the plasma membrane of cells in which Ptc activity is abrogated by Hh but is targeted to the degradative pathway in cells where Ptc is active. We further demonstrate that Smo accumulation is likely to be a cause, rather than a consequence, of Hh signal transduction.

Hedgehog signal activation in oesophageal cancer patients undergoing neoadjuvant chemoradiotherapy.

The zinc finger protein glioma-associated oncogene homologue 1 (Gli-1) is a critical component of the Hedgehog (Hh) signalling pathway, which is essential for morphogenesis and stem-cell renewal, and is dysregulated in many cancer types. As data were not available on the role of Gli-1 expression in oesophageal cancer progression, we analysed whether it could be used to predict disease progression and prognosis in oesophageal cancer patients undergoing neoadjuvant chemoradiotherapy (CRT). Among 69 patients with histologically confirmed oesophageal squamous cell carcinomas (ESCCs), 25 showed a pathological complete response after preoperative CRT. Overall survival (OS) was significantly associated with lymph-node metastasis, distant metastasis, and CRT, and was further correlated with the absence of both Gli-1 nuclear expression and residual tumour. All patients with Gli-1 nuclear expression (10.1%) had distant or lymph-node metastasis, and six out of seven died within 13 months. Furthermore, patients with Gli-1 nuclear-positive cancers showed significantly poorer prognoses than those without (disease-free survival: mean DFS time 250 vs 1738 months, 2-year DFS 0 vs 54.9%, P=0.009; OS: mean OS time 386 vs 1742 months, 2-year OS 16.7 vs 54.9%, P=0.001). Our study provides the first evidence that Gli-1 nuclear expression is a strong and independent predictor of early relapse and poor prognosis in ESCC after CRT. These findings suggest that Hh signal activation might promote cancer regrowth and progression after CRT.

Changes in temperature preferences and energy homeostasis in dystroglycan mutants.

Temperature affects the physiology, behavior, and evolution of organisms. We conducted mutagenesis and screens for mutants with altered temperature preference in Drosophila melanogaster and identified a cryophilic (cold-seeking) mutant, named atsugari (atu). Reduced expression of the Drosophila ortholog of dystroglycan (DmDG) induced tolerance to cold as well as preference for the low temperature. A sustained increase in mitochondrial oxidative metabolism caused by the reduced expression of DmDG accounted for the cryophilic phenotype of the atu mutant. Although most ectothermic animals do not use metabolically produced heat to regulate body temperature, our results indicate that their thermoregulatory behavior is closely linked to rates of mitochondrial oxidative metabolism and that a mutation in a single gene can induce a sustained change in energy homeostasis and the thermal responses.

The Hippo pathway transcriptional co-activator, YAP, confers resistance to cisplatin in human oral squamous cell carcinoma

Cisplatin (CDDP) is widely used to treat oral squamous cell carcinoma (OSCC), however, many patients exhibit acquired drug resistance. Yes-associated protein (YAP) is a transcriptional co-activator of the Hippo pathway that regulates organ size and promotes cell proliferation. YAP overexpression correlates with epithelial-mesenchymal transition and nodal metastasis, resulting in anti-tubulin drug resistance. Whether YAP overexpression is the cause of CDDP resistance in cancer cells is unclear, therefore, we investigated the correlation between YAP expression and CDDP sensitivity. We established three CDDP-resistant cell lines (OSC-19-R, SCCKN-R and HSC-3-R) from the OSCC parental cell lines. We also examined the expression levels of ATP7B, GST-π and ERCC1, which are strongly associated with CDDP resistance, and Hippo pathway-related proteins by western blotting. Using immunocytochemistry, we examined the cellular localization of YAP. Additionally, following knockdown of YAP using short interfering RNAs (siRNAs), we analyzed changes in sensitivity to CDDP. Compared with parental OSC-19 cells, OSC-19-R cells were obviously larger. Expression levels of YAP were not significantly different between OSC-19 and OSC-19-R. However, expression levels of phosphorylated YAP in OSC-19-R were decreased. We observed translocation of YAP from the cytoplasm to the nucleus in OSC-19-R cells. Knockdown of YAP using siRNAs revealed that sensitivity to CDDP was significantly increased. Translocation of YAP correlated with the acquisition of CDDP resistance. YAP could be a new therapeutic target for the treatment of patients with cancer that are resistant to CDDP.

Evolution of cuticular hydrocarbons of Hawaiian Drosophilidae.

Hawaiian Drosophila offer an excellent model for adaptive evolution. More than 500 species are reported in Hawaiian islands, and there is considerable diversity in behavior and morphology. Such diversity is mainly driven by sexual selection. In this study qualitative and quantitative chemical compositions of cuticular hydrocarbons (CHCs) in 138 flies belonging to 27 Hawaiian Drosophila species, picture-winged and non picture-winged, were analyzed regarding sexual dimorphism, differences in saturation, branching position, and lengths of CHCs. We found significant variation in the CHC patterns. In several subgroups, new species show decreases in unsaturated hydrocarbons, and gradual increases in branched compounds, monomethylalkanes and dimethylalkanes, not commonly found in Drosophila. Moreover, branching positions gradually shifted towards internal carbons, and chain lengths increased in the new species. The long-term evolution of CHCs in the light of the recent evolutionary migration and adaptation history of Hawaiian Drosophila species along the developing archipelago was discussed.

A new family of homeobox genes encoding multiple homeodomain and zinc finger motifs.

Fortini et al. (1991) reported two Drosophila genes that encode proteins containing both homeodomain and zinc-finger motifs. The product of the zfh-1 gene (zinc finger homeodomain protein 1) possesses one homeodomain (H1) and nine zinc fingers (Z1-9), and the zfh-2 product contains three homeodomains (H1-3) and 16 zinc fingers (Zl-16). These genes were isolated by screening a Drosophila retina expression library with an oligonucleotide probe bearing the Drosophila opsin promoter RCS 1 consensus sequence, CTAATTGAATT. Here we report structural similarities of these genes to a human cDNA, terms ATBF1 (AT motif binding factor 1), that we isolated from a human hepatoma expression library by screening with an oligonucleotide probe bearing a human t~fetoprotein enhancer AT-rich element, TTAATAATTAC. ATBF1 encodes a protein that contains four homeodomains (H1-4) and 17 zinc fingers (Zl-17). The homeodomains of ATBF1 show 32% or lower sequence identity with the Antennapedia-class homeobox sequences indicating the divergent nature of these homeodomains. In contrast, the third homeodomain of ATBF1 (ATBF1-H3) is 51% identical with the Zfh-1 homeodomain (Fig. 1) although other ATBF1 homeodomains show less than 36% identity. Higher levels of sequence conservation were observed between ATBF1 and Zfh-2 homeodomains. The first three homeodomains of ATBF1 (ATBF1-H1, -H2, -H3) share 77, 69, and 61% identity with the corresponding homeodomains of Zfh-2 (Zfh-2-H1, -H2, -H3), respectively (Fig 1). ATBF1-H4 shows 46% identity

Zebrafish yolk-specific not really started (nrs) gene is a vertebrate homolog of the Drosophila spinster gene and is essential for embryogenesis

By using retroviral insertional mutagenesis in zebrafish, we have identified a recessive lethal mutation in the not really started (nrs) gene. The nrs mutation disrupts a gene located in linkage group 3 that is highly homologous to the spinster gene identified in Drosophila and to spinster orthologs identified in mammals. In flies, spinster encodes a membrane protein involved in lysosomal metabolism and programmed cell death in the central nervous system and in the ovary. In nrs mutant fish embryos, we detect an opaque substance in the posterior yolk cell extension at approximately 1 day after fertilization. This material progressively accumulates and by 48 hr after fertilization fills the entire yolk. By day 3 of embryogenesis, mutant embryos are severely reduced in size compared with their wild‐type siblings and they die a few hours later. By in situ hybridization, we show that the nrs mRNA is expressed in the yolk cell at the time the mutant phenotype becomes apparent. In wild‐type embryos, nrs message is present maternally and zygotically throughout embryogenesis and is also detected in adult animals. In nrs homozygous mutant embryos, nrs transcripts are undetectable at the time the phenotype becomes apparent, indicating that the retroviral insertion has most likely abolished expression of the nrs gene. Finally, the nrs phenotype can be partially rescued by microinjection of nrs encoding DNA. These results suggest that the nrs mutation affects an essential gene encoding a putative membrane‐bound protein expressed specifically in the yolk cell during zebrafish embryogenesis.