Cell competition in mouse NIH3T3 embryonic fibroblasts is controlled by the activity of Tead family proteins and Myc.

Cell competition is a short-range communication originally observed in Drosophila. Relatively little is known about cell competition in mammals or in non-epithelial cells. Hippo signaling and its downstream transcription factors of the Tead family, control cell proliferation and apoptosis. Here, we established an in vitro model system that shows cell competition in mouse NIH3T3 embryo fibroblast cells. Co-culture of Tead-activity-manipulated cells with normal (wild-type) cells caused cell competition. Cells with reduced Tead activity became losers, whereas cells with increased Tead activity became super-competitors. Tead directly regulated Myc RNA expression, and cells with increased Myc expression also became super-competitors. At low cell density, cell proliferation required both Tead activity and Myc. At high cell density, however, reduction of either Tead activity or Myc was compensated for by an increase in the other, and this increase was sufficient to confer 'winner' activity. Collectively, NIH3T3 cells have cell competition mechanisms similar to those regulated by Yki and Myc in Drosophila. Establishment of this in vitro model system should be useful for analyses of the mechanisms of cell competition in mammals and in fibroblasts.

Associations between aldehyde dehydrogenase 2 (ALDH2) genetic polymorphisms, drinking status, and hypertension risk in Japanese adult male workers: a case-control study.

OBJECTIVES: We sought to identify associations between aldehyde dehydrogenase 2 (ALDH2), alcohol consumption, and hypertension in Japanese men. METHODS: The study participants were 1,225 male Japanese workers. We collected lifestyle information, body measurements, blood biochemical parameters, blood pressure measurements, and ALDH2 genotyping data during medical examinations conducted between March 2004 and January 2005 at a work facility and an affiliated company. Lifestyle data on alcohol intake and smoking were collected using self-administered questionnaires at the same time as when the aforementioned measurements were obtained. RESULTS: The genotype frequencies of ALDH2 genetic polymorphisms were 62.6, 32.7, and 4.7% for *1/*1, *1/*2, and *2/*2, respectively. Systolic blood pressure and diastolic blood pressure in the *1/*2 or *2/*2 group were significantly lower than those in the *1/*1 group (P < 0.001). Multiple regression analysis (stepwise method) for blood pressure according to ALDH2 genetic polymorphism revealed that the amount of daily alcohol intake affected systolic blood pressure in participants who harbored the ALDH2 genetic polymorphism *1/*2 or *2/*2. CONCLUSIONS: The interaction between alcohol intake and ALDH2 genetic polymorphisms might affect systolic blood pressure in adult male workers.

A Japanese child with asymptomatic elevation of serum creatine kinase shows PTRF-CAVIN mutation matching with congenital generalized lipodystrophy type 4.

Congenital generalized lipodystrophy (CGL), characterized by generalized absence of adipose tissue, has heterogeneous causes. Recently, a novel type of CGL complicated by muscular dystrophy was categorized as CGL4 caused by PTRF-CAVIN deficiency. However, it is unknown whether CGL4 exhibits clinical abnormalities during the infantile period. Here, we describe the youngest Japanese case of CGL4-a Japanese girl with asymptomatic high serum creatine kinase (CK) levels at 3months old. She was referred to our hospital at 5months of age because of her elevated serum CK (2528IU/L). Generalized absence of adipose tissue was first recognized at 2years of age. Mutation analysis of genes known to be responsible for CGL1-3 failed to disclose any abnormalities. Instead, analysis of the PTRF-CAVIN gene encoding PTRF-CAVIN revealed compound heterozygous mutations, one allele contained an insertion (c.696_697insC) and the other allele harbored a novel nonsense mutation (c.512C>A). Our patient had low serum leptin and adiponectin levels and insulin resistance. Pathological studies on biopsied muscle disclosed mild dystrophic change and highly reduced expression of PTRF-CAVIN. It was concluded that our PTRF-CAVIN deficient patient showed not only CGL but also asymptomatic elevation of serum CK because of her mild muscle dystrophic change.

Molecular characterization of the 5'-UTR of retinal dystrophin reveals a cryptic intron that regulates translational activity.

PURPOSE: Mutations in the dystrophin (DMD) gene cause Duchenne or Becker muscular dystrophy (DMD/BMD). DMD contains a retina-specific promoter in intron 29. The short R-dystrophin transcript from this promoter has a retina-specific exon 1 (R1) joined to exon 30 of the DMD gene. It has been claimed that this is responsible for the ophthalmological problems observed in DMD/BMD. This research characterizes the structure of the 5'-untranslated region (5'-UTR) of human R-dystrophin. METHODS: The 5'-UTR of the human R-dystrophin transcript was amplified from human retina and 20 other human tissue RNAs by reverse transcription polymerase chain reaction (RT-PCR). Amplified products were identified by sequencing. The translational activities of transcripts bearing differing 5'-UTRs were measured using a dual luciferase assay system. RESULTS: RT-PCR amplification of the R-dystrophin transcript from the retina using a conventional primer set revealed one product comprising exon R1 and exons 30 to 32 (R-dys α). In contrast, three amplified products were obtained when a forward primer at the far 5'-end of exon R1 was employed for RT-PCR. R-dys α, and a shorter form in which 98 bp was deleted from exon R1 (R-dys ß), were the two major products. A minor, short form was also identified, in which 143 bp was deleted from exon R1 (R-dys γ). The two primary retinal products (R-dys α and ß) encoded an identical open reading frame. The 98 bp deleted in R-dys ß was identified as a cryptic intron that was evolutionarily acquired in higher mammals. The shorter R-dys ß was expressed in several tissues with a wide range in expression level, while R-dys α was retina specific. The 5'-UTRs of R-dys α and ß were examined for translational activity using a dual luciferase assay system. Unexpectedly, the 5'-UTR of R-dys ß showed lower translational activity than that of R-dys α. This lower activity was presumed to be due to the removal of internal ribosome entry sites by activation of cryptic intron splicing. CONCLUSIONS: An evolutionarily-acquired cryptic intron was identified in the 5'-UTR of the human R-dystrophin transcript. The two abundant R-dystrophin transcripts in the retina showed different translational activities in vitro owing to their differential splicing of the cryptic intron. This evolutionarily-acquired alternative splicing may act as a molecular switch that regulates translation of the R-dystrophin transcript.

Renal organs of cephalopods: a habitat for dicyemids and chromidinids.

The renal organs of 32 species of cephalopods (renal appendage of all cephalopods, and renal and pancreatic appendages in decapods) were examined for parasite fauna and for histological comparison. Two phylogenetically distant organisms, dicyemid mesozoans and chromidinid ciliates, were found in 20 cephalopod species. Most benthic cephalopods (octopus and cuttlefish) were infected with dicyemids. Two pelagic cephalopod species, Sepioteuthis lessoniana and Todarodes pacificus, also harbored dicyemids. Chromidinid ciliates were found only in decapods (squid and cuttlefish). One dicyemid species was found in branchial heart appendages of Rossia pacifica. Dicyemids and chromidinids occasionally occurred simultaneously in Euprymna morsei, Sepia kobiensis, S. peterseni, and T. pacificus. The small-sized cephalopod species, Idiosepius paradoxus and Octopus parvus, harbored no parasites. Comparative histology revealed that the external surface of renal organs varies morphologically in various cephalopod species. The small-sized cephalopod species have a simple external surface. In contrast, the medium- to large-sized cephalopod species have a complex external surface. In the medium- to large-sized cephalopod species, their juveniles have a simple external surface of the renal organs. The external surface subsequently becomes complicated as they grow. Dicyemids and chromidinids attach their heads to epithelia or insert their heads into folds of renal appendages, pancreatic appendages, and branchial heart appendages. The rugged and convoluted external surface provides a foothold for dicyemids and chromidinids with a conical head. They apparently do not harm these tissues of their host cephalopods.

A G-to-T transversion at the splice acceptor site of dystrophin exon 14 shows multiple splicing outcomes that are not exemplified by transition mutations.

Mutations at splicing consensus sequences have been shown to induce splicing errors such as exon skipping or cryptic splice site activation. Here, we identified eight splicing products caused by a G-to-T transversion mutation at the splice acceptor site of exon 14 of the dystrophin gene (c.1603-1G>T). Unexpectedly, the most abundant product showed skipping of the two consecutive exons 14 and 15, and exon 14 skipping was observed as the second most abundant product. To examine the cause of this splicing multiplicity, minigenes containing dystrophin exons 14 and 15 with their flanking introns were constructed and subjected to in vitro splicing. Minigenes with the wild-type sequence or a G>A transition at position c.1603-1 produced only the mature mRNA. On the other hand, the minigenes with a G>T or G>C transversion mutation produced multiple splicing products. A time-course analysis of the in vitro splicing revealed that splicing of the middle intron, intron 14, was the first step in transcript maturation for all four minigene constructs. The identity of the mutant nucleotide, but not its position, is a factor leading to multiple splicing outcomes. Our results suggest that exon skipping therapy for Duchenne's muscular dystrophy should be carefully monitored for their splicing outcomes.

Chemical treatment enhances skipping of a mutated exon in the dystrophin gene.

Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease caused by a loss of the dystrophin protein. Control of dystrophin mRNA splicing to convert severe DMD to a milder phenotype is attracting much attention. Here we report a dystrophinopathy patient who has a point mutation in exon 31 of the dystrophin gene. Although the mutation generates a stop codon, a small amount of internally deleted, but functional, dystrophin protein is produced in the patient cells. An analysis of the mRNA reveals that the mutation promotes exon skipping and restores the open reading frame of dystrophin. Presumably, the mutation disrupts an exonic splicing enhancer and creates an exonic splicing silencer. Therefore, we searched for small chemicals that enhance exon skipping, and found that TG003 promotes the skipping of exon 31 in the endogenous dystrophin gene in a dose-dependent manner and increases the production of the dystrophin protein in the patient's cells.

The Hippo signaling pathway components Lats and Yap pattern Tead4 activity to distinguish mouse trophectoderm from inner cell mass.

Outside cells of the preimplantation mouse embryo form the trophectoderm (TE), a process requiring the transcription factor Tead4. Here, we show that transcriptionally active Tead4 can induce Cdx2 and other trophoblast genes in parallel in embryonic stem cells. In embryos, the Tead4 coactivator protein Yap localizes to nuclei of outside cells, and modulation of Tead4 or Yap activity leads to changes in Cdx2 expression. In inside cells, Yap is phosphorylated and cytoplasmic, and this involves the Hippo signaling pathway component Lats. We propose that active Tead4 promotes TE development in outside cells, whereas Tead4 activity is suppressed in inside cells by cell contact- and Lats-mediated inhibition of nuclear Yap localization. Thus, differential signaling between inside and outside cell populations leads to changes in cell fate specification during TE formation.

Mammalian Tead proteins regulate cell proliferation and contact inhibition as transcriptional mediators of Hippo signaling.

Regulation of organ size is important for development and tissue homeostasis. In Drosophila, Hippo signaling controls organ size by regulating the activity of a TEAD transcription factor, Scalloped, through modulation of its co-activator protein Yki. Here, we show that mouse Tead proteins regulate cell proliferation by mediating Hippo signaling. In NIH3T3 cells, cell density and Hippo signaling regulated the activity of endogenous Tead proteins by modulating nuclear localization of a Yki homolog, Yap1, and the resulting change in Tead activity altered cell proliferation. Tead2-VP16 mimicked Yap1 overexpression, including increased cell proliferation, reduced cell death, promotion of EMT, lack of cell contact inhibition and promotion of tumor formation. Growth-promoting activities of various Yap1 mutants correlated with their Tead-co-activator activities. Tead2-VP16 and Yap1 regulated largely overlapping sets of genes. However, only a few of the Tead/Yap1-regulated genes in NIH3T3 cells were affected in Tead1(-/-);Tead2(-/-) or Yap1(-/-) embryos. Most of the previously identified Yap1-regulated genes were not affected in NIH3T3 cells or mutant mice. In embryos, levels of nuclear Yap1 and Tead1 varied depending on cell type. Strong nuclear accumulation of Yap1 and Tead1 were seen in myocardium, correlating with requirements of Tead1 for proliferation. However, their distribution did not always correlate with proliferation. Taken together, mammalian Tead proteins regulate cell proliferation and contact inhibition as a transcriptional mediator of Hippo signaling, but the mechanisms by which Tead/Yap1 regulate cell proliferation differ depending on the cell type, and Tead, Yap1 and Hippo signaling may play multiple roles in mouse embryos.

Differentiation of autonomic neurons by BMP-independent mechanisms.

A number of signaling molecules and transcription factors play important roles in the development of the autonomic nervous system. Here, we show that mouse trunk neural crest cells can differentiate into autonomic neurons expressing mammalian achaete-scute homolog 1 (mash1), Phox2b, tyrosine hydroxylase, and/or dopamine-beta-hydroxylase in the absence of bone morphogenetic protein (BMP)-4. The expression of mash1 and Phox2b is induced even in the presence of noggin or chordin, which are inhibitors of BMP signaling. Whereas these autonomic neurons do not express c-ret, the receptor for glial-cell-line-derived neurotrophic factor (GDNF), GDNF promotes the differentiation of c-ret-positive autonomic neurons in the presence of noggin. Autonomic neurogenesis is completely prevented by fibroblast growth factor (FGF)-2 treatment or by activation of Notch signaling. Furthermore, the suppression of Phox2b expression by FGF-2 can be recovered by treatment with Notch-1 small interfering RNA. Our data suggest that BMP-independent mechanisms promote the differentiation of autonomic neurons, and that FGF-2 suppresses autonomic neurogenesis by means of the activation of Notch signaling.

Tead4 is required for specification of trophectoderm in pre-implantation mouse embryos.

During pre-implantation mouse development, embryos form blastocysts with establishment of the first two cell lineages: the trophectoderm (TE) which gives rise to the placenta, and the inner cell mass (ICM) which will form the embryo proper. Differentiation of TE is regulated by the transcription factor Caudal-related homeobox 2 (Cdx2), but the mechanisms which act upstream of Cdx2 expression remain unknown. Here we show that the TEA domain family transcription factor, Tead4, is required for TE development. Tead1, Tead2 and Tead4 were expressed in pre-implantation embryos, and at least Tead1 and Tead4 were expressed widely in both TE and ICM lineages. Tead4-/- embryos died at pre-implantation stages without forming the blastocoel. The mutant embryos continued cell proliferation, and adherens junction and cell polarity were not significantly affected. In Tead4-/- embryos, Cdx2 was weakly expressed at the morula stage but was not expressed in later stages. None of the TE specific genes, including Eomes and a Cdx2 independent gene, Fgfr2, was detected in Tead4-/- embryos. Instead, the ICM specific transcription factors, Oct3/4 and Nanog, were expressed in all the blastomeres. Tead4-/- embryos also failed to differentiate trophoblast giant cells when they were cultured in vitro. ES cells with normal in vitro differentiation abilities were established from Tead4-/- embryos. These results suggest that Tead4 has a distinct role from Tead1 and Tead2 in trophectoderm specification of pre-implantation embryos, and that Tead4 is an early transcription factor required for specification and development of the trophectoderm lineage, which includes expression of Cdx2.

BMP and FGF-2 regulate neurogenin-2 expression and the differentiation of sensory neurons and glia.

We have examined the effects of signaling molecules and Notch signaling on the mechanisms regulating neurogenin (ngn)-2 expression. This ngn-2 is a transcription factor that is essential for the specification of early differentiating sensory neurons in the dorsal root ganglia. In the presence of bone morphogenetic protein (BMP), anti-ngn-2-positive cells appeared in mouse trunk neural crest cell cultures, and they expressed Brn3, indicating that ngn-2-expressing cells are sensory neurons. These cells did not differentiate after fibroblast growth factor (FGF)-2 treatment or after Notch activation. The suppression of ngn-2 expression by FGF-2 was recovered by treatment with a Notch signaling inhibitor. Thus, FGF-2 may prevent ngn-2 expression through Notch activation. Whereas BMP-4 inhibited glial differentiation, FGF-2 promoted gliogenesis by means of Notch activation. Our data suggest that BMP and FGF-2 act as positive and negative regulators in ngn-2 expression, respectively, and that these signaling molecules regulate the differentiation of sensory neurons and glia.

Induction of neurogenin-1 expression by sonic hedgehog: Its role in development of trigeminal sensory neurons.

We have examined the roles of signaling molecules in the mechanisms underlying the induction of neurogenin (ngn)-1 expression. ngn-1 is a basic helix-loop-helix (bHLH) transcription factor, which is essential for the specification of trigeminal sensory neurons. Semiquantitative reverse transcriptase-polymerase chain reaction using cranial explants in organ cultures showed that sonic hedgehog (Shh) promotes ngn-1 expression. This promoting activity was not observed in other signaling molecules examined. The promotion of ngn-1 expression by Shh, furthermore, was inhibited by cyclopamine, a specific inhibitor of Shh signaling. Shh did not affect the expression of ngn-2, a bHLH transcription factor that plays an important role in the specification of epibranchial placode-derived sensory neurons. The expression levels of ngn-1 and ngn-2 decreased after fibroblast growth factor-2 treatment. These results suggest that Shh induces ngn-1 expression specifically and that expression of ngn-1 and ngn-2 is regulated by different mechanisms. The induction of ngn-1 expression by Shh suggests that this signaling molecule participates in the specification of trigeminal sensory neurons. We therefore examined the effect of Shh on the development of these neurons. Immunostaining using anti-ngn-1 demonstrated that Shh promotes ngn-1 expression in trigeminal neural crest cells. Trigeminal neural crest cells are derived from the posterior mesencephalon and the most-anterior rhombencephalon, and they contain a subset of precursors of trigeminal sensory neurons. Moreover, a subpopulation of trigeminal neural crest cells expressed the Shh receptor Patched. The number of cells that express Brn3a, a POU-domain transcription factor that plays an important role in differentiation of sensory neurons, also increased with Shh treatment. Our data suggest that Shh signaling is involved in the specification of trigeminal sensory neurons through the induction of ngn-1 expression. Furthermore, Shh promotes the differentiation of neural crest cells into trigeminal sensory neurons.

The significance of N-linked glycosylation in pig endogenous retrovirus infectivity.

The significance of the envelope glycoprotein in the transmission of pig endogenous retrovirus (PERV) to human cells was investigated. Pig endothelial cells (PEC) were transduced with the LacZ gene by a pseudotype infection and then infected with PERV subtype B. Culture supernatants of the infected PEC previously incubated with several types of drugs were inoculated into HEK293 cells. The inoculated cells were then stained and the number of LacZ-positive foci was counted. PERV from tunicamycin treated PEC was not transmitted to human cells, indicating the importance of N-linked sugars in this process. Moreover, while inhibition of the terminal alpha-glucose residues from the precursor N-glycan by castanospermine and 1-deoxynojirimycin attenuated PERV infectivity, the mannosidase inhibitors, 1-deoxymannojirimycin and swainsonine, upregulated the infectivity. In addition, treatment with alpha-mannosidase and incubation with concanavalin A completely abrogated the transmission of PERV to HEK293. These data imply that the high-mannose type of N-glycan plays a key role in PERV infectivity.