Excessive Dpp signaling induces cardial apoptosis through dTAK1 and dJNK during late embryogenesis of Drosophila

<p>Abstract</p> <p>Background</p> <p>To identify genes involved in the heart development of <it>Drosophila</it>, we found that embryos lacking <it>raw </it>function exhibited cardial phenotypes. <it>raw </it>was initially identified as a dorsal open group gene. The dorsal open phenotype was demonstrated to be resulted from the aberrant expression of <it>decapentaplegic </it>(<it>dpp</it>), a member of the tumor growth factor beta (TGF-β), signaling pathway. Despite the role of <it>dpp </it>in pattering cardioblasts during early embryogenesis of <it>Drosophila </it>have been demonstrated, how mutation in <it>raw </it>and/or excessive <it>dpp </it>signaling involves in the differentiating heart of <it>Drosophila </it>has not been fully elaborated at late stages.</p> <p>Results</p> <p>We show that <it>raw </it>mutation produced a mild overspecification of cardial cells at stage 14, but these overproduced cells were mostly eliminated in late mutant embryos due to apoptosis. Aberrant <it>dpp </it>signaling is likely to contribute to the cardial phenotype found in <it>raw </it>mutants, because expression of <it>dpp </it>or constitutively activated <it>thickven </it>(<it>tkv^CA</it>), the type I receptor of Dpp, induced a <it>raw</it>-like phenotype. Additionally, we show that <it>dpp </it>induced non-autonomous apoptosis through TGFβ activated kinase 1 (<it>TAK1</it>), because mis-expression of a dominant negative form of <it>Drosophila TAK1 </it>(<it>dTAK1^DN</it>) was able to suppress cell death in <it>raw </it>mutants or embryos overexpressing <it>dpp</it>. Importantly, we demonstrated that <it>dpp </it>induce its own expression through <it>dTAK1</it>, which also leads to the hyperactivation of <it>Drosophila </it>JNK (DJNK). The hyperactivated DJNK was attributed to be the cause of Dpp/DTAK1-induced apoptosis because overexpression of a dominant negative DJNK, <it>basket </it>(<it>bsk^DN</it>), suppressed cell death induced by Dpp or DTAK1. Moreover, targeted overexpression of the anti-apoptotic P35 protein, or a dominant negative proapoptotic P53 (P53^DN) protein blocked Dpp/DTAK1-induced apoptosis, and rescued heart cells under the <it>raw </it>mutation background.</p> <p>Conclusions</p> <p>We find that ectopic Dpp led to DJNK-dependent cardial apoptosis through the non-canonical TGF-β pathway during late embryogenesis of <it>Drosophila</it>. This certainly will increase our understanding of the pathogenesis of cardiomyopathy, because haemodynamic overload can up-regulate TGF-β and death of cardiomyocytes is observed in virtually every myocardial disease. Thus, our study may provide possible medical intervention for human cardiomyopathy.</p

Sequence and developmental expression of amphioxus AmphiNk2–1 : insights into the evolutionary origin of the vertebrate thyroid gland and forebrain

 We characterized an amphioxus NK-2 homeobox gene ( AmphiNk2–1 ), a homologue of vertebrate Nkx2–1 , which is involved in the development of the central nervous system and thyroid gland. At the early neurula stage of amphioxus, AmphiNk2–1 expression is first detected medially in the neural plate. By the mid-neurula stage, expression is localized ventrally in the nerve cord and also begins in the endoderm. During the late neurula stage, the ventral neural expression becomes transiently segmented posteriorly and is then down-regulated except in the cerebral vesicle at the anterior end of the central nervous system. Within the cerebral vesicle AmphiNk2–1 is expressed in a broad ventral domain, probably comprising both the floor plate and basal plate regions; this pattern is comparable to Nkx2–1 expression in the mouse diencephalon. In the anterior part of the gut, expression becomes intense in the endostyle (the right wall of the pharynx), which is the presumed homologue of the vertebrate thyroid gland. More posteriorly, there is transitory expression in the midgut and hindgut. In sum, the present results help to support homologies (1) between the amphioxus endostyle and the vertebrate thyroid gland and (2) between the amphioxus cerebral vesicle and the vertebrate diencephalic forebrain.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42247/1/427-209-4-254_92090254.pd

Spinocerebellar ataxia type 8 larger triplet expansion alters histone modification and induces RNA foci

<p>Abstract</p> <p>Background</p> <p>Spinocerebellar ataxia type 8 (SCA8) involves the expression of an expanded CTG/CAG combined repeats (CR) from opposite strands producing CUG expansion transcripts (ataxin 8 opposite strand, ATXN8OS) and a polyglutamine expansion protein (ataxin 8, ATXN8). The pathogenesis of SCA8 is complex and the spectrum of clinical presentations is broad.</p> <p>Results</p> <p>Using stably induced cell models expressing 0, 23, 88 and 157 CR, we study the role of ATXN8OS transcripts in SCA8 pathogenesis. In the absence of doxycycline, the stable ATXN8OS CR cell lines exhibit low levels of ATXN8OS expression and a repeat length-related increase in staurosporine sensitivity and in the number of annexin positive cells. A repeat length-dependent repression of ATXN8OS expression was also notable. Addition of doxycycline leads to 25~50 times more ATXN8OS RNA expression with a repeat length-dependent increase in fold of ATXN8OS RNA induction. ChIP-PCR assay using anti-dimethyl-histone H3-K9 and anti-acetyl-histone H3-K14 antibodies revealed increased H3-K9 dimethylation and reduced H3-K14 acetylation around the ATXN8OS cDNA gene in 157 CR line. The repeat length-dependent increase in induction fold is probably due to the increased RNA stability as demonstrated by monitoring ATXN8OS RNA decay in cells treated with the transcriptional inhibitor, actinomycin D. In cells stably expressing ATXN8OS, RNA FISH experiments further revealed ribonuclear foci formation in cells carrying expanded 88 and 157 CR.</p> <p>Conclusion</p> <p>The present study demonstrates that the expanded CUG-repeat tracts are toxic to human cells and may affect ATXN8OS RNA expression and stability through epigenetic and post-transcriptional mechanisms.</p

Deactivation of TBP contributes to SCA17 pathogenesis

Spinocerebellar ataxia type 17 (SCA17) is an autosomal dominant cerebellar ataxia caused by the expansion of polyglutamine (polyQ) within the TATA box-binding protein (TBP). Previous studies have shown that polyQexpanded TBP forms neurotoxic aggregates and alters downstream genes. However, how expanded polyQ tracts affect the function of TBP and the link between dysfunctional TBP and SCA17 is not clearly understood. In this study, we generated novel Drosophila models for SCA17 that recapitulate pathological features such as aggregate formation, mobility defects and premature death. In addition to forming neurotoxic aggregates, we determined that polyQ-expanded TBP reduces its own intrinsic DNA-binding and transcription abilities. Dysfunctional TBP also disrupts normal TBP function. Furthermore, heterozygous dTbp amorph mutant flies exhibited SCA17-like phenotypes and flies expressing polyQ-expanded TBP exhibited enhanced retinal degeneration, suggesting that loss of TBP function may contribute to SCA17 pathogenesis. We further determined that the downregulation of TBP activity enhances retinal degeneration in SCA3 and Huntington&apos;s disease fly models, indicating that the deactivation of TBP is likely to play a common role in polyQ-induced neurodegeneration

Nationwide Surveillance of Influenza during the Pandemic (2009–10) and Post-Pandemic (2010–11) Periods in Taiwan

INTRODUCTION: Although WHO declared the world moving into the post-pandemic period on August 10, 2010, influenza A(H1N1) 2009 virus continued to circulate globally. Its impact was expected to continue during the 2010-11 influenza season. This study describes the nationwide surveillance findings of the pandemic and post-pandemic influenza periods in Taiwan and assesses the impact of influenza A(H1N1) 2009 during the post-pandemic period. METHODS: The Influenza Laboratory Surveillance Network consisted of 12 contract laboratories for collecting and testing samples with acute respiratory tract infections. Surveillance of emergency room visits and outpatient department visits for influenza-like illness (ILI) were conducted using the Real-Time Outbreak and Disease Surveillance system and the National Health Insurance program data, respectively. Hospitalized cases with severe complications and deaths were reported to the National Notifiable Disease Surveillance System. RESULTS: During the 2009-10 influenza season, pandemic A(H1N1) 2009 was the predominant circulating strain and caused 44 deaths. However, the 2010-11 influenza season began with A(H3N2) being the predominant circulating strain, changing to A(H1N1) 2009 in December 2010. Emergency room and outpatient department ILI surveillance displayed similar trends. By March 31, 2011, there were 1,751 cases of influenza with severe complications; 50.1% reported underlying diseases. Of the reported cases, 128 deaths were associated with influenza. Among these, 93 (72.6%) were influenza A(H1N1) 2009 and 30 (23.4%) A(H3N2). Compared to the pandemic period, during the immediate post-pandemic period, increased number of hospitalizations and deaths were observed, and the patients were consistently older. CONCLUSIONS: Reemergence of influenza A(H1N1) 2009 during the 2010-11 influenza season had an intense activity with age distribution shift. To further mitigate the impact of future influenza epidemics, Taiwan must continue its multifaceted influenza surveillance systems, remain flexible with antiviral use policies, and revise the vaccine policies to include the population most at risk

Role of the CCAAT-Binding Protein NFY in SCA17 Pathogenesis

Spinocerebellar ataxia 17 (SCA17) is caused by expansion of the polyglutamine (polyQ) tract in human TATA-box binding protein (TBP) that is ubiquitously expressed in both central nervous system and peripheral tissues. The spectrum of SCA17 clinical presentation is broad. The precise pathogenic mechanism in SCA17 remains unclear. Previously proteomics study using a cellular model of SCA17 has revealed reduced expression of heat shock 70 kDa protein 5 (HSPA5) and heat shock 70 kDa protein 8 (HSPA8), suggesting that impaired protein folding may contribute to the cell dysfunction of SCA17 (Lee et al., 2009). In lymphoblastoid cells, HSPA5 and HSPA8 expression levels in cells with mutant TBP were also significantly lower than that of the control cells (Chen et al., 2010). As nuclear transcription factor Y (NFY) has been reported to regulate HSPA5 transcription, we focused on if NFY activity and HSPA5 expression in SCA17 cells are altered. Here, we show that TBP interacts with NFY subunit A (NFYA) in HEK-293 cells and NFYA incorporated into mutant TBP aggregates. In both HEK-293 and SH-SY5Y cells expressing TBP/Q61∼79, the level of soluble NFYA was significantly reduced. In vitro binding assay revealed that the interaction between TBP and NFYA is direct. HSPA5 luciferase reporter assay and endogenous HSPA5 expression analysis in NFYA cDNA and siRNA transfection cells further clarified the important role of NFYA in regulating HSPA5 transcription. In SCA17 cells, HSPA5 promoter activity was activated as a compensatory response before aggregate formation. NFYA dysfunction was indicated in SCA17 cells as HSPA5 promoter activity reduced along with TBP aggregate formation. Because essential roles of HSPA5 in protection from neuronal apoptosis have been shown in a mouse model, NFYA could be a target of mutant TBP in SCA17

New Variants and Age Shift to High Fatality Groups Contribute to Severe Successive Waves in the 2009 Influenza Pandemic in Taiwan

Past influenza pandemics have been characterized by the signature feature of multiple waves. However, the reasons for multiple waves in a pandemic are not understood. Successive waves in the 2009 influenza pandemic, with a sharp increase in hospitalized and fatal cases, occurred in Taiwan during the winter of 2010. In this study, we sought to discover possible contributors to the multiple waves in this influenza pandemic. We conducted a large-scale analysis of 4703 isolates in an unbiased manner to monitor the emergence, dominance and replacement of various variants. Based on the data from influenza surveillance and epidemic curves of each variant clade, we defined virologically and temporally distinct waves of the 2009 pandemic in Taiwan from May 2009 to April 2011 as waves 1 and 2, an interwave period and wave 3. Except for wave 3, each wave was dominated by one distinct variant. In wave 3, three variants emerged and co-circulated, and formed distinct phylogenetic clades, based on the hemagglutinin (HA) genes and other segments. The severity of influenza was represented as the case fatality ratio (CFR) in the hospitalized cases. The CFRs in waves 1 and 2, the interwave period and wave 3 were 6.4%, 5.1%, 15.2% and 9.8%, respectively. The results highlight the association of virus evolution and variable influenza severity. Further analysis revealed that the major affected groups were shifted in the waves to older individuals, who had higher age-specific CFRs. The successive pandemic waves create challenges for the strategic preparedness of health authorities and make the pandemic uncertain and variable. Our findings indicate that the emergence of new variants and age shift to high fatality groups might contribute potentially to the occurrence of successive severe pandemic waves and offer insights into the adjustment of national responses to mitigate influenza pandemics

Reassortment and Mutations Associated with Emergence and Spread of Oseltamivir-Resistant Seasonal Influenza A/H1N1 Viruses in 2005–2009

A dramatic increase in the frequency of the H275Y mutation in the neuraminidase (NA), conferring resistance to oseltamivir, has been detected in human seasonal influenza A/H1N1 viruses since the influenza season of 2007–2008. The resistant viruses emerged in the ratio of 14.3% and quickly reached 100% in Taiwan from September to December 2008. To explore the mechanisms responsible for emergence and spread of the resistant viruses, we analyzed the complete genome sequences of 25 viruses collected during 2005–2009 in Taiwan, which were chosen from various clade viruses, 1, 2A, 2B-1, 2B-2, 2C-1 and 2C-2 by the classification of hemagglutinin (HA) sequences. Our data revealed that the dominant variant, clade 2B-1, in the 2007–2008 influenza emerged through an intra-subtype 4+4 reassortment between clade 1 and 2 viruses. The dominant variant acquired additional substitutions, including A206T in HA, H275Y and D354G in NA, L30R and H41P in PB1-F2, and V411I and P453S in basic polymerase 2 (PB2) proteins and subsequently caused the 2008–2009 influenza epidemic in Taiwan, accompanying the widespread oseltamivir-resistant viruses. We also characterized another 3+5 reassortant virus which became double resistant to oseltamivir and amantadine. Comparison of oseltamivir-resistant influenza A/H1N1 viruses belonging to various clades in our study highlighted that both reassortment and mutations were associated with emergence and spread of these viruses and the specific mutation, H275Y, conferring to antiviral resistance, was acquired in a hitch-hiking mechanism during the viral evolutionary processes

Cell-type specification during heart development.

Compared to early heart morphogenesis, the specification of various heart cell types is less well understood. In tinman mutants all the different heart cells are absent, suggesting that tin is essential for heart development and the diversification of different cardiac cells. It is believed that tin might interact with various genes that are expressed in all or a subset of heart precursors to specify different heart cell fates. Based on this rationale, a novel gene, apontic (apt), which is expressed in the mesoderm and heart cells, has been cloned. apt encodes a protein that contains opa repeats between a hydrophobic domain and a stretch of asparagine (N). Phenotypic analysis reveals that apt mutants exhibit minor abnormalities in the heart of Drosophila. The heart beat rate is also greatly decreased in apt mutant embryos, suggesting that apt also plays a physiological role in Drosophila. Zinc finger homeodomain gene-1 (zfh-1) is expressed in the mesoderm and later in cardial as well as pericardial cells. This expression profile of zfh-1 has made it a potential candidate to be involved in some aspects of cardiac diversification. The EPCs are selectively missing in zfh-1 mutant embryos at late stages, suggesting that zfh-1 is essential for the proper development of EPCs. even-skipped (eve) itself has also been shown to be essential for even-skipped expressing pericardial cell (EPC) development. As in zfh-1 mutant embryos, removal of the function of eve causes the absence of the EPCs. This result suggests that both zfh-1 and eve act in the same genetic pathway to specify the EPC cell fate. An enhancer that is sufficient for mesodermal eve expression has been identified, and has been shown to be bound by zfh-1 protein in vitro. This result demonstrates that eve is a direct target of zfh-1 in the formation of EPCs. To examine the role of zfh-1 in the cardiogenesis of vertebrates I have identified a mouse zfh-1 homolog (mzfh-1) with a high degree of similarity in the organization of DNA binding domains and amino acids between Drosophila and mouse. The expression pattern of mzfh-1 resembles that of zfh-1. The missing EPCs can be restored in zfh-1 mutant embryos when mzfh-1 is over-expressed. This result demonstrates that zfh-1 and mzfh-1 are functionally interchangeable, and thus the function of zfh-1 is likely to be evolutionary conserved.Ph.D.Biological SciencesCellular biologyGeneticsMolecular biologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/131549/2/9910002.pd