RESUMEN
Angioblasts that form the major axial blood vessels of the dorsal aorta and cardinal vein migrate toward the embryonic midline from distant lateral positions. Little is known about what controls the precise timing of angioblast migration and their final destination at the midline. Using zebrafish, we found that midline angioblast migration requires neighboring tissue rearrangements generated by somite morphogenesis. The somitic shape changes cause the adjacent notochord to separate from the underlying endoderm, creating a ventral midline cavity that provides a physical space for the angioblasts to migrate into. The anterior to posterior progression of midline angioblast migration is facilitated by retinoic acid-induced anterior to posterior somite maturation and the subsequent progressive opening of the ventral midline cavity. Our work demonstrates a critical role for somite morphogenesis in organizing surrounding tissues to facilitate notochord positioning and angioblast migration, which is ultimately responsible for creating a functional cardiovascular system.
Asunto(s)
Embrión no Mamífero/irrigación sanguínea , Desarrollo Embrionario/fisiología , Neovascularización Fisiológica/fisiología , Somitos/fisiología , Animales , Animales Modificados Genéticamente , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Retinoides/farmacología , Tretinoina/metabolismo , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , p-Aminoazobenceno/análogos & derivados , p-Aminoazobenceno/farmacologíaRESUMEN
Somitogenesis, the segmentation of the antero-posterior axis in vertebrates, is thought to result from the interactions between a genetic oscillator and a posterior-moving determination wavefront. The segment (somite) size is set by the product of the oscillator period and the velocity of the determination wavefront. Surprisingly, while the segmentation period can vary by a factor three between 20 °C and 32 °C, the somite size is constant. How this temperature independence is achieved is a mystery that we address in this study. Using RT-qPCR we show that the endogenous fgf8 mRNA concentration decreases during somitogenesis and correlates with the exponent of the shrinking pre-somitic mesoderm (PSM) size. As the temperature decreases, the dynamics of fgf8 and many other gene transcripts, as well as the segmentation frequency and the PSM shortening and tail growth rates slows down as T-Tc (with Tc = 14.4 °C). This behavior characteristic of a system near a critical point may account for the temperature independence of somitogenesis in zebrafish.
Asunto(s)
Embrión no Mamífero/metabolismo , Desarrollo Embrionario/fisiología , Factor 8 de Crecimiento de Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Animales , Desarrollo Embrionario/genética , Factor 8 de Crecimiento de Fibroblastos/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Pez Cebra , p-Aminoazobenceno/análogos & derivados , p-Aminoazobenceno/farmacologíaRESUMEN
BACKGROUND: Recent evidence in cancer research, developed the notion that malignant tumors consist of different subpopulations of cells, one of them, known as cancer stem cells, being attributed many important properties such as enhanced tumorigenicity, proliferation potential and profound multidrug resistance to chemotherapy. Several key stem cells markers were identified in colon cancer. In our study we focused on the aldehyde dehydrogenase type 1 (ALDH1) expression in colon cancer-derived cell lines HT-29/eGFP, HCT-116/eGFP and LS-180/eGFP, and its role in the chemoresistance and tumorigenic potential. METHODS: The effect of pharmacological inhibition of ALDH activity by diethylaminobenzaldehyde (DEAB) and also effect of molecular inhibition by specific siRNA was evaluated in vitro in cultures of human colorectal cell lines. The expression level of different isoenzymes of aldehyde dehydrogenase was determined using qPCR. Changes in cell biology were evaluated by expression analysis, western blot and apoptosis assay. The efficiency of cytotoxic treatment in the presence of different chemotherapeutic drugs was analyzed by fluorimetric assay. Tumorigenicity of cells with specific ALDH1A1 siRNA was tested in xenograft model in vivo. RESULTS: Treatment by DEAB partially sensitized the tested cell lines to chemotherapeutics. Subsequently the molecular inhibition of specific isoforms of ALDH by ALDH1A1 or ALDH1A3 siRNA led to sensitizing of cell lines HT-29/eGFP, HCT-116/eGFP to capecitabine and 5-FU. On the model of athymic mice we observed the effect of molecular inhibition of ALDH1A1 in HT-29/eGFP cells by siRNA. We observed inhibition of proliferation of subcutaneous xenografts in comparison to control cells. CONCLUSION: This research, verifies the significance of the ALDH1A isoforms in multidrug resistance of human colorectal cancer cells and its potential as a cancer stem cell marker. This provides the basis for the development of new approaches regarding the treatment of patients with colorectal adenocarcinoma and potentially the treatment of other tumor malignancies.
Asunto(s)
Aldehído Deshidrogenasa/antagonistas & inhibidores , Neoplasias Colorrectales/patología , Resistencia a Antineoplásicos/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , p-Aminoazobenceno/análogos & derivados , Familia de Aldehído Deshidrogenasa 1 , Animales , Línea Celular Tumoral , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Humanos , Ratones , Células Madre Neoplásicas/efectos de los fármacos , Retinal-Deshidrogenasa , Ensayos Antitumor por Modelo de Xenoinjerto , p-Aminoazobenceno/farmacologíaRESUMEN
Respiratory motoneuron pools must provide rhythmic inspiratory drive that is robust and reliable, yet dynamic enough to respond to respiratory challenges. One form of plasticity that is hypothesized to contribute to motor output stability by sensing and responding to inadequate respiratory neural activity is inactivity-induced phrenic motor facilitation (iPMF), an increase in inspiratory output triggered by a reduction in phrenic synaptic inputs. Evidence suggests that mechanisms giving rise to iPMF differ depending on the pattern of reduced respiratory neural activity (i.e., neural apnea). A prolonged neural apnea elicits iPMF via a spinal TNF-α-induced increase in atypical PKC activity, but little is known regarding mechanisms that elicit iPMF following intermittent neural apnea. We tested the hypothesis that iPMF triggered by intermittent neural apnea requires retinoic acid and protein synthesis. Phrenic nerve activity was recorded in urethane-anesthetized and -ventilated rats treated intrathecally with an inhibitor of retinoic acid synthesis (4-diethlyaminobenzaldehyde, DEAB), a protein synthesis inhibitor (emetine), or vehicle (artificial cerebrospinal fluid) before intermittent (5 episodes, ~1.25 min each) or prolonged (30 min) neural apnea. Both DEAB and emetine abolished iPMF elicited by intermittent neural apnea but had no effect on iPMF elicited by a prolonged neural apnea. Thus different patterns of reduced respiratory neural activity elicit phenotypically similar iPMF via distinct spinal mechanisms. Understanding mechanisms that allow respiratory motoneurons to dynamically tune their output may have important implications in the context of respiratory control disorders that involve varied patterns of reduced respiratory neural activity, such as central sleep apnea and spinal cord injury.NEW & NOTEWORTHY We identify spinal retinoic acid and protein synthesis as critical components in the cellular cascade whereby repetitive reductions in respiratory neural activity elicit rebound increases in phrenic inspiratory activity.
Asunto(s)
Apnea/fisiopatología , Emetina/farmacología , Neuronas Motoras/fisiología , Nervio Frénico/fisiología , Inhibidores de la Síntesis de la Proteína/farmacología , Tretinoina/metabolismo , Animales , Apnea/metabolismo , Masculino , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/metabolismo , Nervio Frénico/efectos de los fármacos , Nervio Frénico/metabolismo , Proteína Quinasa C/metabolismo , Ratas , Ratas Sprague-Dawley , Factor de Necrosis Tumoral alfa/metabolismo , p-Aminoazobenceno/análogos & derivados , p-Aminoazobenceno/farmacologíaRESUMEN
Recent studies have revealed that aldehyde dehydrogenase (ALDH) is a candidate marker for thyroid cancer stem cells, although its activity is flexible. The goal of this study is to clarify the functional significance of ALDH enzymatic activity on thyroid cancer stem cells properties in anaplastic thyroid cancer cell lines. In vitro sphere formation assay was used to judge the stemness of 4 anaplastic thyroid cancer cell lines (FRO, ACT1, 8505C, and KTC3). Two well-known ALDH inhibitors, N,N-diethylaminobenzaldehyde (DEAB) and disulfiram (DS), were first used. DEAB (50 µM) almost completely suppressed ALDH activity without affecting cell proliferation or spherogenicity. Lack of effect of ALDH suppression on spherogenicity was confirmed using shRNA for ALDH1A3, an ALDH isozyme predominantly expressed in anaplastic thyroid cancer cell lines. In contrast, an ALDH2 inhibitor DS (1 µM) inhibited spherogenicity but did not inhibit ALDH1A3 activity. Based on the recent article from another group reporting the importance of sonic hedgehog (Shh) signaling in ALDH activity and spherogenicity in thyroid cancer, the effects of the Shh inhibitor cyclopamine were also studied. Like DS, cyclopamine (1 µM) decreased spherogenicity but not ALDH activity. Finally, exogenous expression of ALDH1A3 in otherwise ALDH- TPC1 cells (a papillary thyroid cancer cell line) revealed no effect on spherogenicity. In conclusion, we here show no functional role for ALDH activity in thyroid thyroid cancer stem cells properties. That is, ALDH activity and spherogenicity are clearly dissociable. Further understanding of thyroid cancer stem cells biology in thyroid cancers remains necessary for the future development of thyroid thyroid cancer stem cells-targeted therapies.
Asunto(s)
Células Madre Neoplásicas/enzimología , Carcinoma Anaplásico de Tiroides/enzimología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Disulfiram/farmacología , Humanos , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Carcinoma Anaplásico de Tiroides/patología , p-Aminoazobenceno/análogos & derivados , p-Aminoazobenceno/farmacologíaRESUMEN
The aim of this study was the characterization of transcriptional regulatory pathways mediated by retinoic acid (RA) in Senegalese sole larvae. For this purpose, pre-metamorphic larvae were treated with a low concentration of DEAB, an inhibitor of RALDH enzyme, until the end of metamorphosis. No differences in growth, eye migration or survival were observed. Nevertheless, gene expression analysis revealed a total of 20 transcripts differentially expressed during larval development and only six related with DEAB treatments directly involved in RA metabolism and actions (rdh10a, aldh1a2, crbp1, igf2r, rarg and cyp26a1) to adapt to a low-RA environment. In a second experiment, post-metamorphic larvae were exposed to the all-trans RA (atRA) observing an opposite regulation for those genes involved in RA synthesis and degradation (rdh10a, aldh1a2, crbp1 and cyp26a1) as well as other related with thyroid- (dio2) and IGF-axes (igfbp1, igf2r and igfbp5) to balance RA levels. In a third experiment, DEAB-pretreated post-metamorphic larvae were exposed to atRA and TTNPB (a specific RAR agonist). Both drugs down-regulated rdh10a and aldh1a2 and up-regulated cyp26a1 expression demonstrating their important role in RA homeostasis. Moreover, five retinoic receptors that mediate RA actions, the thyroid receptor thrb, and five IGF binding proteins changed differentially their expression. Overall, this study demonstrates that exogenous RA modulates the expression of some genes involved in the RA synthesis, degradation and cellular transport through RAR-mediated regulatory pathways establishing a negative feedback regulatory mechanism necessary to balance endogenous RA levels and gradients.
Asunto(s)
Peces Planos/genética , Peces Planos/metabolismo , Regulación de la Expresión Génica , Larva/genética , Larva/metabolismo , Tretinoina/metabolismo , Animales , Benzoatos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Larva/crecimiento & desarrollo , Metamorfosis Biológica/efectos de los fármacos , Metamorfosis Biológica/genética , Receptores de Ácido Retinoico/agonistas , Receptores de Ácido Retinoico/genética , Receptores de Ácido Retinoico/metabolismo , Retinoides/farmacología , p-Aminoazobenceno/análogos & derivados , p-Aminoazobenceno/farmacologíaRESUMEN
The effect of sulfanilamides (soluble streptocid as an example) on changing of the electrophysical properties (EP) of microbial cells of Escherichia coli XL-1, BL-Ril, Pseudomonasputida C-11 and BA-11 was studied. It was shown that significant changes in the orientation spectra (OS) of the cell suspensions incubated at various concentrations of the sulfanilamide resulted in changing of the electrooptic (EO) signal of the cell suspension at the first five frequencies of the orientation electric field (10-1000 Hz) with the use of soluble streptocid in a concentration of 0.3 mcg/ml. The dynamics of the drug effect on the microbial cells demonstrated a decrease of the EO signal value 5 minutes after the exposure by -59% vs. the control (the cells not exposed to the drug). During the following exposure the EO signal value practically did not change (within 5%). The changes of the OS of the cell suspensions exposed to soluble streptocid significantly differed for the susceptible and resistant strains. Determination of the activity of sulfanilamides by electrooptic analysis of microbial cell suspensions was considered possible. Changing of the microbial suspencion OS under the effect of sulfanilamides can be used as a test on the microbial cell susceptibility to drugs.
Asunto(s)
Escherichia coli/efectos de los fármacos , Pruebas de Sensibilidad Microbiana/métodos , Pseudomonas putida/efectos de los fármacos , Sulfanilamidas/farmacología , p-Aminoazobenceno/análogos & derivados , Impedancia Eléctrica , Escherichia coli/crecimiento & desarrollo , Pruebas de Sensibilidad Microbiana/instrumentación , Dispositivos Ópticos , Pseudomonas putida/crecimiento & desarrollo , Solubilidad , p-Aminoazobenceno/farmacologíaRESUMEN
Apolipoproteins, the major components of lipoproteins, play physiological roles in lipoprotein metabolism. Contrary to the well-documented effects on plasma lipid, little is known about the function and regulation of Apolipoproteins during embryonic development. Here we have shown that apolipoprotein C1 (apoc1) gene is highly expressed in the yolk syncytial layer, a structure implicated in embryonic and larval nutrition. The apoc1 transcripts are also observed in the deep cell layer at the ventral and lateral region during gastrulation, and in the tail paraxial mesoderm during somitogenesis. By whole-mount in situ hybridization and quantified RT-RCR, we further demonstrate that apoc1 expression is induced by bone morphogenetic proteins (BMPs) signaling, while retinoic acid (RA) signaling suppresses the expression of BMP ligands and inhibits the BMP effect in this process.
Asunto(s)
Apolipoproteína C-I/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Tretinoina/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Animales , Yema de Huevo/metabolismo , Embrión no Mamífero , Femenino , Gastrulación , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Tretinoina/farmacología , p-Aminoazobenceno/análogos & derivados , p-Aminoazobenceno/farmacologíaRESUMEN
The vitamin A metabolite, retinoic acid, is important for memory formation and hippocampal synaptic plasticity in vertebrate species. In our studies in the mollusc Lymnaea stagnalis, we have shown that retinoic acid plays a role in memory formation following operant conditioning of the aerial respiratory behaviour. Inhibition of either retinaldehyde dehydrogenase (RALDH) or the retinoid receptors prevents long-term memory (LTM) formation, whereas synthetic retinoid receptor agonists promote memory formation by converting intermediate-term memory (ITM) into LTM. In this study, animals were exposed to constant darkness in order to test whether light-sensitive retinoic acid would promote memory formation. However, we found that exposure to constant darkness alone (in the absence of retinoic acid) enhanced memory formation. To determine whether the memory-promoting effects of darkness could override the memory-inhibiting effects of the retinoid signaling inhibitors, we exposed snails to RALDH inhibitors or a retinoid receptor antagonist in constant darkness. We found that darkness overcame the inhibitory effects of RALDH inhibition, but did not overcome the inhibitory effects of the retinoid receptor antagonist. We also tested whether constant darkness and training affected the mRNA levels of the retinoid metabolic enzymes RALDH and Cyp26, or the mRNA levels of the retinoid receptors, but found no significant effect. Overall, these data demonstrate an interaction between environmental light conditions and the retinoid signaling pathway, which influence long-term memory formation in a mollusc.
Asunto(s)
Condicionamiento Operante/fisiología , Oscuridad , Memoria a Largo Plazo/fisiología , Transducción de Señal/fisiología , Monoterpenos Acíclicos , Animales , Conducta Animal/efectos de los fármacos , Conducta Animal/fisiología , Benzoatos/farmacología , Compuestos de Bifenilo/farmacología , Condicionamiento Operante/efectos de los fármacos , Ambiente , Lymnaea , Memoria a Largo Plazo/efectos de los fármacos , Monoterpenos/farmacología , Retinal-Deshidrogenasa/antagonistas & inhibidores , Receptores X Retinoide/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , p-Aminoazobenceno/análogos & derivados , p-Aminoazobenceno/farmacologíaRESUMEN
Retinoic acid, a metabolite of vitamin A, is proposed to play an important role in vertebrate learning and memory, as well as hippocampal-dependent synaptic plasticity. However, it has not yet been determined whether retinoic acid plays a similar role in learning and memory in invertebrates. In this study, we report that retinoid signaling in the mollusc Lymnaea stagnalis, is required for long-term memory formation following operant conditioning of its aerial respiratory behaviour. Animals were exposed to inhibitors of the RALDH enzyme (which synthesizes retinoic acid), or various retinoid receptor antagonists. Following exposure to these inhibitors, neither learning nor intermediate-term memory (lasting 2 h) was affected, but long-term memory formation (tested at either 24 or 72 h) was inhibited. We next demonstrated that various retinoid receptor agonists promoted long-term memory formation. Using a training paradigm shown only to produce intermediate-term memory (lasting 2 h, but not 24 h) we found that exposure of animals to synthetic retinoids promoted memory formation that lasted up to 30 h. These findings suggest that the role of retinoids in memory formation is ancient in origin, and that retinoid signaling is also important for the formation of implicit memories, in addition to its previously demonstrated role in hippocampal-dependent memories.
Asunto(s)
Condicionamiento Operante/efectos de los fármacos , Memoria a Largo Plazo/efectos de los fármacos , Receptores de Ácido Retinoico/antagonistas & inhibidores , Retinoides/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Monoterpenos Acíclicos , Animales , Conducta Animal/efectos de los fármacos , Dibenzazepinas/farmacología , Lymnaea/efectos de los fármacos , Monoterpenos/farmacología , p-Aminoazobenceno/análogos & derivados , p-Aminoazobenceno/farmacologíaRESUMEN
Aldehyde dehydrogenases (ALDHs), enzymes responsible for detoxification and retinoic acid biosynthesis, are considered a potent functional stem cell marker of normal and malignant cells in many tissues. To date, however, there are no available data on ALDH distributions and functions in oral mucosa. This study aims to clarify the levels and types of ALDH expression using immunohistochemistry with accompanying mRNA expression as well as an ALDEFLUOR assay, and to assess phenotypic and histological changes after manipulation of the ALDH activity of oral keratinocytes to increase the potency of a tissue-engineered oral mucosa by a specific ALDH inhibitor, diethylaminobenzaldehyde (DEAB), together with small interfering RNA of ALDH1A3 and ALDH3A1. Results showed the mRNA and cytoplasmic protein expression of ALDH1A3 and ALDH3A1 to be mostly localized in the upper suprabasal layer although no ALDH1A1 immunoreaction was detected throughout the epithelium. Oral keratinocytes with high ALDH activity exhibited a profile of differentiating cells. By pharmacological inhibition, the phenotypic analysis revealed the proliferating cell-population shifting to a more quiescent state compared with untreated cells. Furthermore, a well-structured epithelial layer showing a normal differentiation pattern and a decrease in Ki-67 immunopositive basal cells was developed by DEAB incubation, suggesting a slower turnover rate efficient to maintain undifferentiated cells. Histological findings of a regenerated oral epithelium by ALDH1A3 siRNA were similar to those when treated with DEAB while ALDH3A1 siRNA eradicated the epithelial regenerative capacity. These observations suggest the effects of phenotypic and morphological alterations by DEAB on oral keratinocytes are mainly consequent to the inhibition of ALDH1A3 activity.
Asunto(s)
Aldehído Oxidorreductasas/metabolismo , Inhibidores Enzimáticos/farmacología , Silenciador del Gen , Queratinocitos/enzimología , Mucosa Bucal/enzimología , ARN Interferente Pequeño/genética , p-Aminoazobenceno/análogos & derivados , Adulto , Aldehído Deshidrogenasa/antagonistas & inhibidores , Aldehído Deshidrogenasa/genética , Aldehído Deshidrogenasa/metabolismo , Aldehído Oxidorreductasas/antagonistas & inhibidores , Aldehído Oxidorreductasas/genética , Proliferación Celular/efectos de los fármacos , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Inmunohistoquímica/métodos , Queratinocitos/patología , Antígeno Ki-67/metabolismo , Masculino , Mucosa Bucal/patología , ARN Mensajero/metabolismo , Regeneración/efectos de los fármacos , p-Aminoazobenceno/farmacologíaRESUMEN
Vitamin A deficiency causes impaired vision and blindness in millions of children around the world. Previous studies in zebrafish have demonstrated that retinoic acid (RA), the acid form of vitamin A, plays a vital role in early eye development. The objective of this study was to describe the effects of early RA deficiency by treating zebrafish with diethylaminobenzaldehyde (DEAB), a potent inhibitor of the enzyme retinaldehyde dehydrogenase (RALDH) that converts retinal to RA. Zebrafish embryos were treated for 2 h beginning at 9 h postfertilization. Gross morphology and retinal development were examined at regular intervals for 5 days after treatment. The optokinetic reflex (OKR) test, visual background adaptation (VBA) test, and the electroretinogram (ERG) were performed to assess visual function and behavior. Early treatment of zebrafish embryos with 100 µM DEAB (9 h) resulted in reduced eye size, and this microphthalmia persisted through larval development. Retinal histology revealed that DEAB eyes had significant developmental abnormalities but had relatively normal retinal lamination by 5.5 days postfertilization. However, the fish showed neither an OKR nor a VBA response. Further, the retina did not respond to light as measured by the ERG. We conclude that early deficiency of RA during eye development causes microphthalmia as well as other visual defects, and that timing of the RA deficiency is critical to the developmental outcome.
Asunto(s)
Microftalmía/etiología , Tretinoina/fisiología , Deficiencia de Vitamina A/complicaciones , Pez Cebra/fisiología , Adaptación Ocular/efectos de los fármacos , Adaptación Ocular/fisiología , Animales , Conducta Animal/fisiología , Electrorretinografía/efectos de los fármacos , Embrión no Mamífero/patología , Ojo/patología , Larva , Microftalmía/fisiopatología , Nistagmo Optoquinético/efectos de los fármacos , Nistagmo Optoquinético/fisiología , Fenotipo , Reflejo/efectos de los fármacos , Tretinoina/antagonistas & inhibidores , Tretinoina/metabolismo , Deficiencia de Vitamina A/inducido químicamente , Deficiencia de Vitamina A/fisiopatología , p-Aminoazobenceno/análogos & derivados , p-Aminoazobenceno/farmacologíaRESUMEN
Retinoic acid (RA) is known to regulate definitive myelopoiesis but its role in vertebrate primitive myelopoiesis remains unclear. Here we report that zebrafish primitive myelopoiesis is restricted by RA in a dose dependent manner mainly before 11 hpf (hours post fertilization) when anterior hemangioblasts are initiated to form. RA treatment significantly reduces expressions of anterior hemangioblast markers scl, lmo2, gata2 and etsrp in the rostral end of ALPM (anterior lateral plate mesoderm) of the embryos. The result indicates that RA restricts primitive myelopoiesis by suppressing formation of anterior hemangioblasts. Analyses of ALPM formation suggest that the defective primitive myelopoiesis resulting from RA treatment before late gastrulation may be secondary to global loss of cells for ALPM fate whereas the developmental defect resulting from RA treatment during 10-11 hpf should be due to ALPM patterning shift. Overexpressions of scl and lmo2 partially rescue the block of primitive myelopoiesis in the embryos treated with 250 nM RA during 10-11 hpf, suggesting RA acts upstream of scl to control primitive myelopoiesis. However, the RA treatment blocks the increased primitive myelopoiesis caused by overexpressing gata4/6 whereas the abolished primitive myelopoiesis in gata4/5/6 depleted embryos is well rescued by 4-diethylamino-benzaldehyde, a retinal dehydrogenase inhibitor, or partially rescued by knocking down aldh1a2, the major retinal dehydrogenase gene that is responsible for RA synthesis during early development. Consistently, overexpressing gata4/6 inhibits aldh1a2 expression whereas depleting gata4/5/6 increases aldh1a2 expression. The results reveal that RA signaling acts downstream of gata4/5/6 to control primitive myelopoiesis. But, 4-diethylamino-benzaldehyde fails to rescue the defective primitive myelopoiesis in either cloche embryos or lycat morphants. Taken together, our results demonstrate that RA signaling restricts zebrafish primitive myelopoiesis through acting downstream of gata4/5/6, upstream of, or parallel to, cloche, and upstream of scl.
Asunto(s)
Mielopoyesis , Transducción de Señal , Tretinoina/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Aciltransferasas/metabolismo , Aldehído Deshidrogenasa/metabolismo , Animales , Tipificación del Cuerpo/efectos de los fármacos , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/metabolismo , Fertilización/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Mesodermo/efectos de los fármacos , Mesodermo/metabolismo , Mielopoyesis/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Proteínas de Pez Cebra/metabolismo , p-Aminoazobenceno/análogos & derivados , p-Aminoazobenceno/farmacologíaRESUMEN
Depression of the peripheral blood platelet count during acute infection is a hallmark of dengue. This thrombocytopenia has been attributed, in part, to an insufficient level of platelet production by megakaryocytes that reside in the bone marrow (BM). Interestingly, it was observed that dengue patients experience BM suppression at the onset of fever. However, few studies focus on the interaction between dengue virus (DENV) and megakaryocytes and how this interaction can lead to a reduction in platelets. In the studies reported herein, BM cells from normal healthy rhesus monkeys (RM) and humans were utilized to identify the cell lineage(s) that were capable of supporting virus infection and replication. A number of techniques were employed in efforts to address this issue. These included the use of viral RNA quantification, nonstructural protein and infectivity assays, phenotypic studies utilizing immunohistochemical staining, anti-differentiation DEAB treatment, and electron microscopy. Cumulative results from these studies revealed that cells in the BM were indeed highly permissive for DENV infection, with human BM having higher levels of viral production compared to RM. DENV-like particles were predominantly observed in multi-nucleated cells that expressed CD61+. These data suggest that megakaryocytes are likely the predominant cell type infected by DENV in BM, which provides one explanation for the thrombocytopenia and the dysfunctional platelets characteristic of dengue virus infection.
Asunto(s)
Células de la Médula Ósea/virología , Médula Ósea/virología , Linaje de la Célula/fisiología , Dengue/virología , Integrina beta3/metabolismo , Animales , Médula Ósea/metabolismo , Células de la Médula Ósea/metabolismo , Ensayo de Unidades Formadoras de Colonias , Dengue/metabolismo , Virus del Dengue , Humanos , Macaca mulatta , Megacariocitos/metabolismo , Megacariocitos/virología , Trombocitopenia/metabolismo , Trombocitopenia/virología , p-Aminoazobenceno/análogos & derivados , p-Aminoazobenceno/farmacologíaRESUMEN
Pancreatic ß-cells are an essential source of insulin and their destruction because of autoimmunity causes type I diabetes. We conducted a chemical screen to identify compounds that would induce the differentiation of insulin-producing ß-cells in vivo. To do this screen, we brought together the use of transgenic zebrafish as a model of ß-cell differentiation, a unique multiwell plate that allows easy visualization of lateral views of swimming larval fish and a library of clinical drugs. We identified six hits that can induce precocious differentiation of secondary islets in larval zebrafish. Three of these six hits were known drugs with a considerable background of published data on mechanism of action. Using pharmacological approaches, we have identified and characterized two unique pathways in ß-cell differentiation in the zebrafish, including down-regulation of GTP production and retinoic acid biosynthesis.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Descubrimiento de Drogas/métodos , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Preparaciones Farmacéuticas/metabolismo , Acetanilidas/farmacología , Animales , Animales Modificados Genéticamente , Ácidos Cafeicos/farmacología , Línea Celular Tumoral , Proliferación Celular , Cartilla de ADN/genética , Dimetilsulfóxido , Relación Dosis-Respuesta a Droga , Epirizol/farmacología , Técnica del Anticuerpo Fluorescente , Proteínas Fluorescentes Verdes , Guanosina Trifosfato/biosíntesis , Proteína HMGB1/metabolismo , Larva/efectos de los fármacos , Microscopía Confocal , Ácido Micofenólico/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Ácidos Sulfanílicos/farmacología , Tretinoina/metabolismo , Pez Cebra , p-Aminoazobenceno/análogos & derivados , p-Aminoazobenceno/farmacologíaRESUMEN
In mouse, Hedgehog (Hh) signalling is required for most ventral spinal neurons to form. Here, we analyse the spinal cord phenotype of zebrafish maternal-zygotic smoothened (MZsmo) mutants that completely lack Hh signalling. We find that most V3 domain cells and motoneurons are lost, whereas medial floorplate still develops normally and V2, V1 and V0v cells form in normal numbers. This phenotype resembles that of mice that lack both Hh signalling and Gli repressor activity. Ventral spinal cord progenitor domain transcription factors are not expressed at 24 hpf in zebrafish MZsmo mutants. However, pMN, p2 and p1 domain markers are expressed at early somitogenesis stages in these mutants. This suggests that Gli repressor activity does not extend into zebrafish ventral spinal cord at these stages, even in the absence of Hh signalling. Consistent with this, ectopic expression of Gli3R represses ventral progenitor domain expression at these early stages and knocking down Gli repressor activity rescues later expression. We investigated whether retinoic acid (RA) signalling specifies ventral spinal neurons in the absence of Hh signalling. The results suggest that RA is required for the correct number of many different spinal neurons to form. This is probably mediated, in part, by an effect on cell proliferation. However, V0v, V1 and V2 cells are still present, even in the absence of both Hh and RA signalling. We demonstrate that Gli1 has a Hh-independent role in specifying most of the remaining motoneurons and V3 domain cells in embryos that lack Hh signalling, but removal of Gli1 activity does not affect more dorsal neurons.
Asunto(s)
Diferenciación Celular/fisiología , Proteínas Hedgehog/metabolismo , Neuronas/fisiología , Transducción de Señal/fisiología , Médula Espinal/citología , Tretinoina/metabolismo , Pez Cebra/embriología , Animales , Inmunohistoquímica , Hibridación in Situ , Morfolinos/genética , Proteínas Oncogénicas/metabolismo , Receptores Acoplados a Proteínas G/genética , Transducción de Señal/efectos de los fármacos , Receptor Smoothened , Médula Espinal/embriología , Transactivadores/metabolismo , Alcaloides de Veratrum/farmacología , Proteínas de Pez Cebra/genética , Proteína con Dedos de Zinc GLI1 , p-Aminoazobenceno/análogos & derivados , p-Aminoazobenceno/farmacologíaRESUMEN
The lateral line system of teleosts has recently become a model system to study patterning and morphogenesis. However, its embryonic origins are still not well understood. In zebrafish, the posterior lateral line (PLL) system is formed in two waves, one that generates the embryonic line of seven to eight neuromasts and 20 afferent neurons and a second one that generates three additional lines during larval development. The embryonic line originates from a postotic placode that produces both a migrating sensory primordium and afferent neurons. Nothing is known about the origin and innervation of the larval lines. Here we show that a "secondary" placode can be detected at 24 h postfertilization (hpf), shortly after the primary placode has given rise to the embryonic primordium and ganglion. The secondary placode generates two additional sensory primordia, primD and primII, as well as afferent neurons. The primary and secondary placodes require retinoic acid signaling at the same stage of late gastrulation, suggesting that they share a common origin. Neither primary nor secondary neurons show intrinsic specificity for neuromasts derived from their own placode, but the sequence of neuromast deposition ensures that neuromasts are primarily innervated by neurons derived from the cognate placode. The delayed formation of secondary afferent neurons accounts for the capability of the fish to form a new PLL ganglion after ablation of the embryonic ganglion at 24 hpf.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Sistema de la Línea Lateral/citología , Sistema de la Línea Lateral/embriología , Neuronas/fisiología , Pez Cebra/fisiología , Técnicas de Ablación/métodos , Aminoácidos/metabolismo , Animales , Animales Modificados Genéticamente , Tipificación del Cuerpo/fisiología , Diferenciación Celular , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Embrión no Mamífero , Ganglios Sensoriales/citología , Ganglios Sensoriales/embriología , Proteínas Fluorescentes Verdes/genética , Larva/crecimiento & desarrollo , Neuronas/clasificación , Transducción de Señal/fisiología , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , p-Aminoazobenceno/análogos & derivados , p-Aminoazobenceno/farmacologíaRESUMEN
BACKGROUND: Over-expression of aldehyde dehydrogenase and other stem cell markers is characteristic of cells with tumorigenic potential in NOD/SCID mice. Most of these studies have focused on metastatic cells in bone marrow and on solid tumors. There are no studies on correlation of marker expression with ALDH1 expression in cells from human peripheral blood apheresis (HPC-A) samples. METHODS: HPC-A samples from 44 patients were incubated with Aldefluor with or without the presence of aldehyde dehydrogenase inhibitor DEAB. Cells with high aldehyde dehydrogenase expression (ALDH1(bright)) were analyzed for stem/progenitor markers CD34, CD90, CD117, and CD133. Electronic volume measured by Coulter principal in a Quanta flow analyzer was correlated with ALDH1 and marker expression. RESULTS: In ALDH1(bright)/SSC(low) cells, 0.13% of the cells had CD34(+) expression and three distinct populations were seen. Expression of CD90 was dim and the frequency of ALDH1(bright)/SSC(low)/CD90(dim) cells amongst the nonlineage depleted samples was 0.04%. CD117(dim-bright) expression was seen in 0.17% of the samples. Three distinct populations of cells with CD133 expression were seen in ALDH1(bright)/SSC(low) nonlineage depleted cells with a frequency of 0.28%. The ALDH1(bright)/CD90(dim) cells had the smallest mean electronic volume of 264.9 microm(3) when compared with cells with CD34(bright) expression (270.2 microm(3)) and ALDH1(dim)/CD90(dim) cells (223 microm(3)). CONCLUSIONS: ALDH1(bright)/SSC(low) cells show heterogeneity in expression of the four stem cell markers studied. The CD90 cells in both the ALDH1(bright) and ALDH1(dim) populations had the smallest mean electronic volume when compared with similar cells with CD117 expression.
Asunto(s)
Aldehído Deshidrogenasa/biosíntesis , Antígenos CD/biosíntesis , Eliminación de Componentes Sanguíneos , Citometría de Flujo/métodos , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/metabolismo , Aldehído Deshidrogenasa/antagonistas & inhibidores , Aldehído Deshidrogenasa/inmunología , Antígenos CD/inmunología , Biomarcadores/sangre , Células Madre Hematopoyéticas/enzimología , Humanos , Sensibilidad y Especificidad , p-Aminoazobenceno/análogos & derivados , p-Aminoazobenceno/farmacologíaRESUMEN
Hematopoietic stem cells (HSCs) are enriched for aldehyde dehydrogenase (ALDH) activity and ALDH is a selectable marker for human HSCs. However, the function of ALDH in HSC biology is not well understood. We sought to determine the function of ALDH in regulating HSC fate. Pharmacologic inhibition of ALDH with diethylaminobenzaldehyde (DEAB) impeded the differentiation of murine CD34(-)c-kit(+)Sca-1(+)lineage(-) (34(-)KSL) HSCs in culture and facilitated a ninefold expansion of cells capable of radioprotecting lethally irradiated mice compared to input 34(-)KSL cells. Treatment of bone marrow (BM) 34(-)KSL cells with DEAB caused a fourfold increase in 4-week competitive repopulating units, verifying the amplification of short-term HSCs (ST-HSCs) in response to ALDH inhibition. Targeted siRNA of ALDH1a1 in BM HSCs caused a comparable expansion of radioprotective progenitor cells in culture compared to DEAB treatment, confirming that ALDH1a1 was the target of DEAB inhibition. The addition of all trans retinoic acid blocked DEAB-mediated expansion of ST-HSCs in culture, suggesting that ALDH1a1 regulates HSC differentiation via augmentation of retinoid signaling. Pharmacologic inhibition of ALDH has therapeutic potential as a means to amplify ST-HSCs for transplantation purposes.
Asunto(s)
Aldehído Deshidrogenasa/antagonistas & inhibidores , Proliferación Celular/efectos de los fármacos , Citoprotección/fisiología , Inhibidores Enzimáticos/farmacología , Células Madre Hematopoyéticas/enzimología , Trasplante de Células Madre/métodos , Aldehído Deshidrogenasa/genética , Aldehído Deshidrogenasa/metabolismo , Familia de Aldehído Deshidrogenasa 1 , Animales , Antineoplásicos/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , División Celular/efectos de los fármacos , División Celular/fisiología , Células Cultivadas , Citoprotección/efectos de los fármacos , Inhibidores Enzimáticos/uso terapéutico , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de la radiación , Humanos , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , ARN Interferente Pequeño/genética , Radiación Ionizante , Retinal-Deshidrogenasa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Tretinoina/metabolismo , Tretinoina/farmacología , p-Aminoazobenceno/análogos & derivados , p-Aminoazobenceno/farmacología , p-Aminoazobenceno/uso terapéuticoRESUMEN
We have isolated and characterized a novel zebrafish pancreas mutant. Mutant embryos lack expression of isl1 and sst in the endocrine pancreas, but retain isl1 expression in the CNS. Non-endocrine endodermal gene expression is less affected in the mutant, with varying degrees of residual expression observed for pdx1, carbA, hhex, prox1, sid4, transferrin and ifabp. In addition, mutant embryos display a swollen pericardium and lack fin buds. Genetic mapping revealed a mutation resulting in a glycine to arginine change in the catalytic domain of the aldh1a2 gene, which is required for the production of retinoic acid from vitamin A. Comparison of our mutant (aldh1a2(um22)) to neckless (aldh1a2(i26)), a previously identified aldh1a2 mutant, revealed similarities in residual endodermal gene expression. In contrast, treatment with DEAB (diethylaminobenzaldehyde), a competitive reversible inhibitor of Aldh enzymes, produces a more severe phenotype with complete loss of endodermal gene expression, indicating that a source of Aldh activity persists in both mutants. We find that mRNA from the aldh1a2(um22) mutant allele is inactive, indicating that it represents a null allele. Instead, the residual Aldh activity is likely due to maternal aldh1a2, since we find that translation-blocking, but not splice-blocking, aldh1a2 morpholinos produce a phenotype similar to DEAB treatment. We conclude that Aldh1a2 is the primary Aldh acting during pancreas development and that maternal Aldh1a2 activity persists in aldh1a2(um22) and aldh1a2(i26) mutant embryos.