RESUMO
G-quadruplex (G4), a non-canonical higher-order structure formed by guanine-rich nucleic acid sequences, affects various genetic events in cis, including replication, transcription and translation. Whereas up-regulation of innate immune/interferon-stimulated genes (ISGs) is implicated in cancer progression, G4-forming oligonucleotides that mimic telomeric repeat-containing RNA suppress ISG induction in three-dimensional (3D) culture of cancer cells. However, it is unclear how G4 suppresses ISG expression in trans. In this study, we found that G4 binding to splicing factor 3B subunit 2 (SF3B2) down-regulated STAT1 phosphorylation and ISG expression in 3D-cultured cancer cells. Liquid chromatography-tandem mass spectrometry analysis identified SF3B2 as a G4-binding protein. Either G4-forming oligonucleotides or SF3B2 knockdown suppressed ISG induction, whereas Phen-DC3, a G4-stabilizing compound, reversed the inhibitory effect of G4-forming oligonucleotides on ISG induction. Phen-DC3 inhibited SF3B2 binding to G4 in vitro. SF3B2-mediated ISG induction appeared to occur independently of RNA splicing because SF3B2 knockdown did not affect pre-mRNA splicing under the experimental conditions, and pharmacological inhibition of splicing by pladienolide B did not repress ISG induction. These observations suggest that G4 disrupts the ability of SF3B2 to induce ISGs in cancer. We propose a new mode for gene regulation, which employs G4 as an inhibitory trans-element.
Assuntos
Quadruplex G , Regulação da Expressão Gênica , Imunidade Inata/genética , Ácidos Nucleicos/metabolismo , Fatores de Processamento de RNA/metabolismo , Linhagem Celular Tumoral , Citocinas/genética , Citocinas/metabolismo , Compostos de Anéis Fundidos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Ontologia Genética , Humanos , Imunidade Inata/efeitos dos fármacos , Ligantes , Modelos Biológicos , Oligonucleotídeos/metabolismo , Ligação Proteica/efeitos dos fármacos , Splicing de RNA/efeitos dos fármacos , Splicing de RNA/genética , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMO
Telomere-binding protein TRF2 protects the linear chromosome ends, telomeres, from being recognized as damaged DNA. TRF2 also regulates gene expression outside telomeres, but the detailed mechanism has not been fully understood. Mukherjee and colleagues have employed ChIP-Seq and biochemical analyses to identify G-quadruplexes at gene promoters across the genome as nontelomeric TRF2-binding sites. TRF2 occupancy on such target sites leads to epigenetic gene repression, implicating TRF2-G-quadruplex interaction as a sophisticated regulator of gene expression.
Assuntos
Quadruplex G , Proteína 2 de Ligação a Repetições Teloméricas/genética , Animais , Sítios de Ligação , Dano ao DNA , TelômeroRESUMO
Loss of telomere protection occurs during physiological cell senescence and ageing, due to attrition of telomeric repeats and insufficient retention of the telomere-binding factor TRF2. Subsequently formed telomere fusions trigger rampant genomic instability leading to cell death or tumorigenesis. Mechanistically, telomere fusions require either the classical non-homologous end-joining (C-NHEJ) pathway dependent on Ku70/80 and LIG4, or the alternative non-homologous end-joining (A-NHEJ), which relies on PARP1 and LIG3. Here, we show that the tumour suppressor BRCA1, together with its interacting partner CtIP, both acting in end resection, also promotes end-joining of uncapped telomeres. BRCA1 and CtIP do not function in the ATM-dependent telomere damage signalling, nor in telomere overhang removal, which are critical for telomere fusions by C-NHEJ. Instead, BRCA1 and CtIP act in the same pathway as LIG3 to promote joining of de-protected telomeres by A-NHEJ. Our work therefore ascribes novel roles for BRCA1 and CtIP in end-processing and fusion reactions at uncapped telomeres, underlining the complexity of DNA repair pathways that act at chromosome ends lacking protective structures. Moreover, A-NHEJ provides a mechanism of previously unanticipated significance in telomere dysfunction-induced genome instability.
Assuntos
Proteína BRCA1/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Reparo do DNA por Junção de Extremidades/fisiologia , Telômero/metabolismo , Animais , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Dano ao DNA , DNA Ligase Dependente de ATP , DNA Ligases/genética , DNA Ligases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Humanos , Autoantígeno Ku , Camundongos , Camundongos Knockout , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose , Telômero/genética , Proteínas de XenopusRESUMO
Mammalian telomeres repress DNA-damage activation at natural chromosome ends by recruiting specific inhibitors of the DNA-damage machinery that form a protective complex termed shelterin. Within this complex, TRF2 (also known as TERF2) has a crucial role in end protection through the suppression of ATM activation and the formation of end-to-end chromosome fusions. Here we address the molecular properties of TRF2 that are both necessary and sufficient to protect chromosome ends in mouse embryonic fibroblasts. Our data support a two-step mechanism for TRF2-mediated end protection. First, the dimerization domain of TRF2 is required to inhibit ATM activation, the key initial step involved in the activation of a DNA-damage response (DDR). Next, TRF2 independently suppresses the propagation of DNA-damage signalling downstream of ATM activation. This novel modulation of the DDR at telomeres occurs at the level of the E3 ubiquitin ligase RNF168 (ref. 3). Inhibition of RNF168 at telomeres involves the deubiquitinating enzyme BRCC3 and the ubiquitin ligase UBR5, and is sufficient to suppress chromosome end-to-end fusions. This two-step mechanism for TRF2-mediated end protection helps to explain the apparent paradox of frequent localization of DDR proteins at functional telomeres without concurrent induction of detrimental DNA-repair activities.
Assuntos
Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos de Mamíferos/genética , Cromossomos de Mamíferos/metabolismo , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Enzimas Desubiquitinantes , Endopeptidases/deficiência , Endopeptidases/metabolismo , Ativação Enzimática , Camundongos , Multimerização Proteica , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico , Transdução de Sinais , Telômero/genética , Telômero/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/química , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/metabolismoRESUMO
This paper investigates the applicability of 1-µm-band mode-detection optical time domain reflectometry (OTDR), which detects microbending in single-mode fibers with high sensitivity by conducting measurements in the two-mode region of the fibers under test and observing the second-order mode of the backscattered light. To demonstrate its feasibility, we evaluate microbending losses as determined by the technique, and comparisons are made with conventional OTDR operating at the wavelength of 1650 nm. In addition, we discuss two potential applications of the technique. One is microbend sensing of fibers and cables for detecting microbending losses that are too small to be found with conventional OTDR. The other is health monitoring of installed fiber cables. This enables us to detect the growth in microbending losses at an earlier stage. Proof of concept is demonstrated experimentally.
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Glioblastoma (GBM) is an invariably fatal brain tumor in which a small subpopulation of self-renewable glioma stem cells (GSCs) contributes to tumor propagation and relapse. Targeting GSCs could therefore have a significant clinical impact for GBM. Telomestatin is a naturally-occurring compound that preferentially impairs GSC growth by perturbing transcription and inducing a DNA damage response. Telomestatin stabilizes G-quadruplexes (G4s), which are guanine-rich four-strand nucleic acid structures observed in vitro and in vivo. However, the mechanism underlying the GSC-selective nature of the DNA damage response remains unknown. Here we demonstrate that GSCs are more susceptible to telomestatin-induced telomere dysfunction and replication stress when compared with GSC-derived non-stem glioma cells (NSGCs). Telomestatin induced dissociation of the telomere-capping protein TRF2 from telomeres, leading to telomeric DNA damage in GSCs-but not in NSGCs. BIBR1532, a telomerase catalytic inhibitor, did not preferentially inhibit GSC growth, suggesting that telomestatin promotes telomere dysfunction in a telomerase-independent manner. GSCs and NSGCs had comparable levels of G4s in their nuclei, and both responded to telomestatin with phosphorylation of RPA2 at Ser33-a hallmark of replication stress. However, activation of the checkpoint kinase Chk1, induction of a DNA damage response, and subsequent growth inhibition occurred only in telomestatin-treated GSCs. These observations suggest that telomestatin impairs GSC growth through removal of TRF2 from telomeres and potent activation of the replication stress response pathway. Therefore, a novel G4-directed therapeutic strategy could specifically target cancer stem cells in GBM.
Assuntos
Dano ao DNA , Replicação do DNA/genética , Quadruplex G , Glioblastoma/genética , Células-Tronco Neoplásicas/fisiologia , Telômero/genética , Linhagem Celular Tumoral , Humanos , Homeostase do Telômero/genéticaRESUMO
Active vitamin D is a second line treatment followed by bisphosphonate in the Japanese guideline of glucocorticoid-induced osteoporosis (GIO) . Although it shows relatively mild but significant effect on BMD and fracture prevention, it is utilized as combination therapy with bisphosphonate. Vitamin D is recommended for GIO with gastrointestinal disease and hepatobiliary disorders. Vitamin D may be the first line treatment for younger GIO patients.
Assuntos
Conservadores da Densidade Óssea/uso terapêutico , Osso e Ossos/efeitos dos fármacos , Glucocorticoides/efeitos adversos , Osteoporose/tratamento farmacológico , Vitamina D/uso terapêutico , Animais , Densidade Óssea/efeitos dos fármacos , Osso e Ossos/metabolismo , Humanos , Osteoporose/induzido quimicamente , Vitamina D/metabolismoRESUMO
INTRODUCTION: Semi-extended tibial nailing techniques include the extra-articular technique (EAT) and the patellar eversion technique (PET). These approaches differ regarding the exposure of the patellar retinaculum and the size of the surgical field. This study compared the postoperative alignment and intramedullary nailing entry points between the EAT and PET for tibial fractures. PATIENTS AND METHODS: A total of 54 patients (aged ≥18 years) who had undergone intramedullary nailing by the EAT (n = 29) or PET (n = 25) for a tibial shaft fracture were evaluated. The intramedullary nailing entry point and postoperative alignment were measured, and the 1-year postoperative follow-up results were compared. RESULTS: For the EAT and PET, the intramedullary nailing entry point was located at a mean distance of 4.04 mm medial to the optimal entry point and 0.27 mm lateral to the optimal entry point, respectively. The mean angular deformation observed in anteroposterior radiographs following surgery using the EAT and PET were 2.49° and 0.32° valgus, respectively. CONCLUSION: The intramedullary nailing entry point affected postoperative alignment. Intramedullary nailing may result in malalignment while performing the EAT due to the interference of the patella at the time of nailing.
Assuntos
Fixação Intramedular de Fraturas , Fraturas da Tíbia , Adolescente , Adulto , Pinos Ortopédicos , Fixação Intramedular de Fraturas/métodos , Humanos , Patela/diagnóstico por imagem , Patela/cirurgia , Estudos Retrospectivos , Fraturas da Tíbia/diagnóstico por imagem , Fraturas da Tíbia/cirurgiaRESUMO
A novel technology for simultaneous WDM signal monitoring is presented based on ultrafast field sampling. Its flexibility regarding channel allocation and/or channel bandwidth is unique and attractive, as is its ultrafast nature. Two- and four-channel WDM signals, with the total bandwidth of 800 GHz, are successfully discriminated and observed with the proposed scheme.
RESUMO
The potential of mesenchymal stem cells (MSCs) to differentiate into nonmesodermal cells such as pancreatic beta cells has been reported. New cell-based therapy using MSCs for diabetes mellitus is anticipated as an alternative treatment option to insulin injection or islet transplantation in both human and veterinary medicine. Several protocols were reported for differentiation of MSCs into insulin-producing cells (IPCs), but no studies have reported IPCs generated from canine MSCs. The purpose of this study was to generate IPCs from canine adipose tissue-derived MSCs (AT-MSCs) in vitro and to investigate the effects of IPC transplantation on diabetic mice in vivo. Culturing AT-MSCs with the differentiation protocol under a two-dimensional culture system did not produce IPCs. However, spheroid-like small clusters consisting of canine AT-MSCs and human recombinant peptide µ-pieces developed under a three-dimensional (3D) culture system were successfully differentiated into IPCs. The generated IPCs under 3D culture condition were stained with dithizone and anti-insulin antibody. Canine IPCs also showed gene expression typical for pancreatic beta cells and increased insulin secretion in response to glucose stimulation. The blood glucose levels in streptozotocin-induced diabetic mice were decreased after injection with the supernatant of canine IPCs, but the hyperglycemic states of diabetic mice were not improved after transplanting IPCs subcutaneously or intramesenterically. The histological examination showed that the transplanted small clusters of IPCs were successfully engrafted to the mice and included cells positive for insulin by immunofluorescence. Several factors, such as the transplanted cell number, the origin of AT-MSCs, and the differentiation protocol, were considered potential reasons for the inability to improve the hyperglycemic state after IPC transplantation. These findings suggest that canine AT-MSCs can be differentiated into IPCs under a 3D culture system and IPC transplantation may be a new treatment option for dogs with diabetes mellitus.
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The telomeres are nucleoprotein complexes essential for maintaining the genomic integrity of linear chromosomes. Six telomere localizing proteins form a complex named "shelterin/telosome" to cooperatively regulate telomere length and protect chromosomal ends from DNA damage and repair responses. Mouse embryonic stem (ES) cells lacking TRF1, a shelterin component, exhibit a high-incidence of broken or lost telomere FISH signals, supporting a critical role for TRF1 in telomere maintenance. We demonstrate that these abnormal telomere structures are not caused by the inability of TRF1-deficient cells to recruit TIN2 but are due to a specific role for TRF1 at telomeres. Furthermore, we provide evidence that the mTRF1 TRF homology (TRFH) domain is crucial for this abnormal telomere FISH phenotype. These novel findings suggest that the TRFH domain is crucial not only for dimerization of TRF1 and TIN2-telomere recruitment, but also telomere stabilization.
Assuntos
Telômero/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo , Animais , Galinhas , Instabilidade Cromossômica , Dano ao DNA , Dimerização , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Humanos , Hibridização in Situ Fluorescente , Camundongos , Fenótipo , Mutação Puntual , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/químicaRESUMO
This paper proposes a novel detection technique for wavelength-division multiplexing (WDM) signals that uses the ultrafast field sampling approach. The proposed technique simultaneously samples the total field of the WDM signal with no wavelength-demultiplexing; its electrical post-processing provides a filtering function in the digital domain. As a result, the individual fields of the WDM channels, including mutual phase relationship, can be jointly reconstructed. As a preliminary demonstration, the simultaneous monitoring of a WDM signal composed of two channels with independent polarization states, is successfully performed using a dual-channel field sampling system with polarization diversity. This demonstration can be expanded to the detection of a WDM signal with N-channels by using an N-channel field sampling system. It is also experimentally-verified that the reconstructed fields preserve the mutual phase relationship of the original fields. This 'field sensitive' WDM detection may open new possibilities for advanced WDM monitoring systems and even the electrical compensation of linear and/or non-linear inter-channel cross-talk in digital coherent detection systems.
Assuntos
Tecnologia de Fibra Óptica/instrumentação , Dispositivos Ópticos , Processamento de Sinais Assistido por Computador/instrumentação , Telecomunicações/instrumentação , Desenho Assistido por Computador , Desenho de Equipamento , Análise de Falha de Equipamento , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Telomeres, the protective structures of chromosome ends are gradually shortened by each cell division, eventually leading to senescence or apoptosis. Cancer cells maintain the telomere length for unlimited growth by telomerase reactivation or a recombination-based mechanism. Recent genome-wide analyses have unveiled genetic and epigenetic alterations of the telomere maintenance machinery in cancer. While telomerase inhibition reveals that longer telomeres are more advantageous for cell survival, cancer cells often have paradoxically shorter telomeres compared with those found in the normal tissues. In this review, we summarize the latest knowledge about telomere length alterations in cancer and revisit its rationality. Finally, we discuss the potential utility of telomere length as a prognostic biomarker.
Assuntos
Neoplasias/genética , Encurtamento do Telômero/genética , Animais , Epigênese Genética , Humanos , Mutação/genética , Regiões Promotoras Genéticas/genética , Telomerase/genética , Telomerase/metabolismoRESUMO
This study was designed to examine whether or not phospholipid is contained in the secretory granules of the rat palatine gland acinar cells, and if present, to examine the movements of phospholipid in the secretory granules during postnatal development. The palatine glands of male Wistar rats aged 0 to 56 days were used. Acid-hematin staining showed a few positive acinar cells with a faint reaction in the acini on day 0, numerous positive cells with an intense reaction on day 7, a weakening reaction in the cells on day 14, and almost no reactivity on day 35 and after. In contrast, alcian blue staining showed acinar cells with a weak reaction on day 7, a gradual increase in the reaction from day 14, and the presence of many cells with an intense reaction on day 28 and after. Electron probe microanalysis (EPMA) revealed a higher density of phosphorus in samples on day 7 than on day 56. These findings suggest that developing rat palatine gland acinar cells contain phospholipid in the secretory granules, being particularly more conspicuous around postnatal day 7, but that the amount of phospholipid decreases as the cells change to mature mucous cells.
Assuntos
Tonsila Palatina/crescimento & desenvolvimento , Tonsila Palatina/metabolismo , Fosfolipídeos/metabolismo , Ratos Wistar/crescimento & desenvolvimento , Vesículas Secretórias/metabolismo , Animais , Microanálise por Sonda Eletrônica , Histocitoquímica , Masculino , Microscopia Eletrônica de Transmissão , Tonsila Palatina/citologia , Fósforo/metabolismo , Ratos , Saliva/metabolismo , Vesículas Secretórias/ultraestruturaRESUMO
Tankyrase, a member of the poly(ADP-ribose) polymerase (PARP) family, regulates various intracellular responses, such as telomere maintenance, Wnt/ß-catenin signaling and cell cycle progression through its interactions with multiple target proteins. Tankyrase contains a long stretch of 24 ankyrin repeats that are further divided into five subdomains, called ANK repeat clusters (ARCs). Each ARC works as an independent ligand-binding unit, which implicates tankyrase as a platform for multiple protein-protein interactions. Furthermore, tankyrase distributes to various intracellular loci, suggesting potential distinct but yet unidentified physiological functions. To explore the novel functions of tankyrase, we performed liquid chromatography-mass spectrometry analysis and identified the BRE-BRCC36-MERIT40 complex, a regulator of homologous recombination, as tankyrase-binding proteins. Among the complex components, MERIT40 was directly associated with tankyrase via a tankyrase-binding consensus motif, as previously reported. In X-ray-irradiated non-small cell lung cancer cells, tankyrase localized to DNA double-stranded break sites in a MERIT40-dependent manner. MERIT40 knockdown increased the cell sensitivity to X-ray, whereas the wild-type, but not the tankyrase-unbound mutant, MERIT40 rescued the phenotype of the knockdown cells. Tankyrase inhibitors, such as G007-LK and XAV939, increased the cellular sensitivity to X-ray irradiation and anticancer drugs that induce DNA double-stranded breaks. These observations suggest that tankyrase plays a role in the DNA damage repair response and implicates a potential therapeutic utility of tankyrase inhibitors in combination treatments with DNA-damaging anticancer drugs.
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During exercise, tidal volume initially contributes to ventilatory responses more than respiratory frequency, and respiratory frequency then increases rapidly while tidal volume stabilizes. Dyspnea intensity is also known to increase in a threshold-like manner. We tested the possibility that the threshold of tachypneic breathing is equal to that of dyspnea perception during cycle ergometer exercise (n = 27). Dyspnea intensity was scored by a visual analog scale. Thresholds were expressed as values of pulmonary O2 uptake at each breakpoint. Dyspnea intensity and respiratory frequency started increasing rapidly once the intensity of stimuli exceeded a threshold level. The thresholds for dyspnea intensity and for occurrence of tachypnea were significantly correlated. An intraclass correlation coefficient of 0.71 and narrow limits of agreement on the Bland-Altman plot indicated a good agreement between these thresholds. These results suggest that the start of tachypneic breathing coincides with the threshold for dyspnea intensity during cycle ergometer exercise.
Assuntos
Dispneia/fisiopatologia , Exercício Físico/fisiologia , Percepção/fisiologia , Taxa Respiratória/fisiologia , Taquipneia/fisiopatologia , Teste de Esforço , Humanos , Masculino , Consumo de Oxigênio/fisiologia , Ventilação Pulmonar/fisiologia , Volume de Ventilação Pulmonar/fisiologia , Adulto JovemRESUMO
OBJECTIVES: Both nocturnal hypertension (HT) and systemic inflammation underlying rheumatoid arthritis (RA) have been shown to be independent predictors of cardiovascular disease (CVD), although little is known on the relationship between nocturnal blood pressure (BP) and disease activity in RA patients. METHODS: We performed 24-hour ambulatory BP monitoring (ABPM) in 71 RA patients to examine the relationship of nocturnal fall in BP and RA disease activity based on a disease activity score of 28 joint counts with C-reactive protein (CRP, 28-joint disease activity score (DAS28)-CRP). Among them, 25 RA patients whose consent obtained were reexamined by ABPM to assess the improvement of nocturnal fall in BP after RA therapeutic intervention. RESULTS: The mean DAS28-CRP level was 4.8±1.6 in 71 RA patients. The mean nocturnal fall in BP was 5.6±8.9%. DAS28-CRP was associated significantly and independently in a negative manner with the nocturnal fall in BP (ß = -0.388, P = 0.004). In 25 RA patients, DAS28-CRP improved from 5.4±1.1 to 3.5±0.8 (P < 0.0001) and the nocturnal fall in BP increased significantly from 4.5±9.2% to 10.6±5.8% (P = 0.002) with the significant decrease of nighttime systolic BP (SBP) from 121.2±22.5mm Hg to 112.5±18.8mm Hg (P = 0.02) in spite of no change in daytime BP after 4 weeks of RA treatment. CONCLUSIONS: The present study observed that higher RA activity was associated with lower nocturnal fall in BP, but not daytime BP, in RA patients.
Assuntos
Artrite Reumatoide/epidemiologia , Ritmo Circadiano , Hipertensão/epidemiologia , Idoso , Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/imunologia , Artrite Reumatoide/fisiopatologia , Monitorização Ambulatorial da Pressão Arterial , Proteína C-Reativa/imunologia , Doenças Cardiovasculares/epidemiologia , Feminino , Humanos , Hipertensão/fisiopatologia , Masculino , Pessoa de Meia-IdadeRESUMO
The planarian brain has an inverted-U shaped structure with functional regionalization. To investigate how each region in the brain connects to each other, we traced neural projections by microinjection of fluorescence dye tracers. We found that external light and olfactory/taste signals received in the head region are conveyed in the main lobes (sponge region) of the brain. Chemosensory neurons distributed in the lateral branches project to the peripheral region of the sponge and visual neurons project to the medial region of the sponge. Parts of the sensory neurons project directly to the corresponding sensory neurons on the opposite side of the brain. However, all of the dye labeled brain neurons in the left and right lobes connect to each other via commissural neurons in the central region of the sponge. In addition to these observations, we detected regional differences in the planarian visual neurons. Posterior visual neurons have ipsilateral projection, but anterior visual neurons project to the contralateral side of the brain. A pair of longitudinal ventral nerve cords (VNC) connect to the brain on the ventral side, suggesting that they transmit signals which are integrated and processed in the brain. We also detected the direct connection of neurons in the brain and those of the pharynx, even though most pharynx neurons connect to VNC neurons. Here, we report for the first time on neural connections in the planarian central nervous system after overcoming technical difficulties specific to flatworms.
Assuntos
Neurônios/ultraestrutura , Planárias/anatomia & histologia , Animais , Encéfalo/anatomia & histologia , Encéfalo/ultraestrutura , Corantes FluorescentesRESUMO
When exposed to light, planarians display a distinctive light avoidance behavior known as negative phototaxis. Such behavior is temporarily suppressed when animals are decapitated, and it is restored once the animals regenerate their heads. Head regeneration and the simple but reproducible phototactic response of planarians provides an opportunity to study the association between neuronal differentiation and the establishment of behavior in a simple, experimentally tractable metazoan. We have devised a phototaxis assay system to analyze light response recovery during head regeneration and determined that light evasion is markedly re-established 5 days after amputation. Immunohistological and in situ hybridization studies indicate that the photoreceptors and optic nerve connections to the brain begin by the fourth day of cephalic regeneration. To experimentally manipulate the light response recovery, we performed gene knockdown analysis using RNA interference (RNAi) on two genes (1020HH and eye53) previously reported to be expressed at 5 days after amputation and in the dorso-medial region of the brain (where the optic nerves project). Although RNAi failed to produce morphological defects in either the brain or the visual neurons, the recovery of the phototactic response normally observed in 5-day regenerates was significantly suppressed. The data suggest that 1020HH and eye53 may be involved in the functional recovery and maintenance of the visual system, and that the phototaxis assay presented here can be used to reliably quantify the negative phototactic behavior of planarians.