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OBJECTIVE: Mitochondrial proteostasis is critical for cellular function. The molecular chaperone HSP60 is essential for cell function and dysregulation of HSP60 expression has been implicated in cancer and diabetes. The few reported patients carrying HSP60 gene variants show neurodevelopmental delay and brain hypomyelination. Hsp60 interacts with more than 260 mitochondrial proteins but the mitochondrial proteins and functions affected by HSP60 deficiency are poorly characterized. METHODS: We studied two model systems for HSP60 deficiency: (1) engineered HEK cells carrying an inducible dominant negative HSP60 mutant protein, (2) zebrafish HSP60 knockout larvae. Both systems were analyzed by RNASeq, proteomics, and targeted metabolomics, and several functional assays relevant for the respective model. In addition, skin fibroblasts from patients with disease-associated HSP60 variants were analyzed by proteomics. RESULTS: We show that HSP60 deficiency leads to a differentially downregulated mitochondrial matrix proteome, transcriptional activation of stress responses, and dysregulated cholesterol biosynthesis. This leads to lipid accumulation in zebrafish knockout larvae. CONCLUSIONS: Our data provide a compendium of the effects of HSP60 deficiency on the mitochondrial matrix proteome. We show that HSP60 is a master regulator and modulator of mitochondrial functions and metabolic pathways. HSP60 dysfunction also affects cellular metabolism and disrupts the integrated stress response. The effect on cholesterol synthesis explains the effect of HSP60 dysfunction on myelination observed in patients carrying genetic variants of HSP60.
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Zebrafish have become a widely used vertebrate model in physiology and reliable measures of their metabolic rate are needed. We have developed a 3D-printed respirometer and swim tunnel system and used it for obtaining accurate measurement of standard metabolic rate (SMR) and maximal, aerobic metabolic rate (MMR) in zebrafish under rest and maximal exercise, respectively. We compared a slow (stepwise) protocol to a fast (continuous) protocol for determining MMR. The fast protocol yielded slightly (but not significantly) higher oxygen consumption rates than the slow protocol and the data, in contrast to the slow protocol, followed a normal distribution. These findings point to the fast protocol as a fast and reliable method for obtaining accurate values of MMR in zebrafish. We make the 3D drawings for printing the system available to researchers, to help streamline the field of metabolic research in zebrafish and other smaller fish species.
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Consumo de Oxigênio , Impressão Tridimensional , Natação , Peixe-Zebra , Animais , Metabolismo Energético , Metabolismo BasalRESUMO
Motor neuron (MN) development and nerve regeneration requires orchestrated action of a vast number of molecules. Here, we identify SorCS2 as a progranulin (PGRN) receptor that is required for MN diversification and axon outgrowth in zebrafish and mice. In zebrafish, SorCS2 knockdown also affects neuromuscular junction morphology and fish motility. In mice, SorCS2 and PGRN are co-expressed by newborn MNs from embryonic day 9.5 until adulthood. Using cell-fate tracing and nerve segmentation, we find that SorCS2 deficiency perturbs cell-fate decisions of brachial MNs accompanied by innervation deficits of posterior nerves. Additionally, adult SorCS2 knockout mice display slower motor nerve regeneration. Interestingly, primitive macrophages express high levels of PGRN, and their interaction with SorCS2-positive motor axon is required during axon pathfinding. We further show that SorCS2 binds PGRN to control its secretion, signaling, and conversion into granulins. We propose that PGRN-SorCS2 signaling controls MN development and regeneration in vertebrates.
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Peptídeos e Proteínas de Sinalização Intercelular , Peixe-Zebra , Camundongos , Animais , Progranulinas , Peixe-Zebra/metabolismo , Neurônios Motores/metabolismo , Granulinas , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismo , Receptores de Superfície Celular/metabolismoRESUMO
Previous studies using myoglobin (Mb) knockout mice and knockdown zebrafish have presented conflicting results about in vivo phenotypes resulting from the loss of this conserved and highly expressed protein, and therefore a new well-characterized knockout model is warranted. We here describe the generation of three distinct zebrafish mb knockout lines using the CRISPR/Cas system. None of the three lines exhibited any morphological phenotypes, changes in length, or lethality during embryonic and larval development. The adult homozygous knockout mb(Auzf13.2) zebrafish line were absent of Mb protein, had an almost complete degradation of mb mRNA, and showed no changes in viability, length, or heart size. Furthermore, transcriptomic analysis of adult heart tissue showed that mb knockout did not cause altered expression of other genes. Lastly, no off-targeting was observed in 36 screened loci. In conclusion, we have generated three mb knockout lines with indistinguishable phenotypes during embryonic and larval development and validated one of these lines, mb(Auzf13.2), to have no signs of genetic compensation or off-target effects in the adult heart. These findings suggests that the mb(Auzf13.2) shows promise as a candidate for investigating the biological role of Mb in zebrafish.
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Mioglobina , Peixe-Zebra , Animais , Camundongos , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Mioglobina/genética , Mioglobina/metabolismo , Proteínas de Peixe-Zebra/genética , Sistemas CRISPR-Cas , Fenótipo , Técnicas de Inativação de GenesRESUMO
The genetic architecture of the QT interval, defined as the period from onset of depolarisation to completion of repolarisation of the ventricular myocardium, is incompletely understood. Only a minor part of the QT interval variation in the general population has been linked to autosomal variant loci. Altered X chromosome dosage in humans, as seen in sex chromosome aneuploidies such as Turner syndrome (TS) and Klinefelter syndrome (KS), is associated with altered QTc interval (heart rate corrected QT), indicating that genes, located in the pseudoautosomal region 1 of the X and Y chromosomes may contribute to QT interval variation. We investigate the dosage effect of the pseudoautosomal gene SLC25A6, encoding the membrane ADP/ATP translocase 3 in the inner mitochondrial membrane, on QTc interval duration. To this end we used human participants and in vivo zebrafish models. Analyses in humans, based on 44 patients with KS, 44 patients with TS, 59 male and 22 females, revealed a significant negative correlation between SLC25A6 expression level and QTc interval duration. Similarly, downregulation of slc25a6 in zebrafish increased QTc interval duration with pharmacological inhibition of KATP channels restoring the systolic duration, whereas overexpression of SLC25A6 shortened QTc, which was normalized by pharmacological activation of KATP channels. Our study demonstrate an inverse relationship between SLC25A6 dosage and QTc interval indicating that SLC25A6 contributes to QT interval variation.
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Síndrome de Klinefelter , Síndrome do QT Longo , Síndrome de Turner , Animais , Feminino , Humanos , Masculino , Trifosfato de Adenosina , Eletrocardiografia , Síndrome do QT Longo/genética , Cromossomo X , Peixe-Zebra/genética , Translocador 3 do Nucleotídeo AdeninaRESUMO
BACKGROUND: A variant in the SLC4A3 anion exchanger has been identified as a novel cause of short QT syndrome (SQTS), but the clinical importance of SLC4A3 as a cause of SQTS or sudden cardiac death remains unknown. OBJECTIVE: The purpose of this study was to investigate the prevalence of potential disease-causing variants in SQTS patients using gene panels including SLC4A3. METHODS: In this multicenter study, genetic testing was performed in 34 index patients with SQTS. The pathogenicity of novel SLC4A3variants was validated in a zebrafish embryo heart model. RESULTS: Potentially disease-causing variants were identified in 9 (26%) patients and were mainly (15%) located in SLC4A3: 4 patients heterozygous for novel nonsynonymous SLC4A3 variants-p.Arg600Cys, p.Arg621Trp, p.Glu852Asp, and p.Arg952His-and 1 patient with the known p.Arg370His variant. In other SQTS genes, potentially disease-causing variants were less frequent (2× in KCNQ1, 1× in KCNJ2, and CACNA1C each). SLC4A3 variant carriers (n = 5) had a similar heart rate but shorter QT and J point to T wave peak intervals than did noncarriers (n = 29). Knockdown of slc4a3 in zebrafish resulted in shortened heart rate-corrected QT intervals (calculated using the Bazett formula) that could be rescued by overexpression of the native human SLC4A3-encoded protein (AE3), but neither by the mutated AE3 variants p.Arg600Cys, p.Arg621Trp, p.Glu852Asp nor by p.Arg952His, suggesting pathogenicity of these variants. Dysfunction in slc4a3/AE3 was associated with alkaline cytosol and shortened action potential of cardiomyocytes. CONCLUSION: In about a quarter of patients with SQTS, a potentially disease-causing variant can be identified. Nonsynonymous variants in SLC4A3 represent the most common cause of SQTS, underscoring the importance of including SLC4A3 in the genetic screening of patients with SQTS or sudden cardiac death.
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Eletrocardiografia , Peixe-Zebra , Animais , Humanos , Arritmias Cardíacas , Morte Súbita Cardíaca/prevenção & controle , Eletrocardiografia/métodosRESUMO
Superoxide dismutase 3 (SOD3) is an extracellular protein with the capacity to convert superoxide into hydrogen peroxide, an important secondary messenger in redox regulation. To investigate the utility of zebrafish in functional studies of SOD3 and its relevance for redox regulation, we have characterized the zebrafish orthologues; Sod3a and Sod3b. Our analyses show that both recombinant Sod3a and Sod3b express SOD activity, however, only Sod3b is able to bind heparin. Furthermore, RT-PCR analyses reveal that sod3a and sod3b are expressed in zebrafish embryos and are present primarily in separate organs in adult zebrafish, suggesting distinct functions in vivo. Surprisingly, both RT-PCR and whole mount in situ hybridization showed specific expression of sod3b in skeletal tissue. To further investigate this observation, we compared femoral bone obtained from wild-type and SOD3-/- mice to determine whether a functional difference was apparent in healthy adult mice. Here we report, that bone from SOD3-/- mice is less mineralized and characterized by significant reduction of cortical and trabecular thickness in addition to reduced mechanical strength. These analyses show that SOD3 plays a hitherto unappreciated role in bone development and homeostasis.
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Superóxido Dismutase , Peixe-Zebra , Animais , Osso e Ossos/metabolismo , Homeostase , Camundongos , Camundongos Knockout , Oxirredução , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
Nanoparticles can acquire a biomolecular corona with a species-specific biological identity. However, "non-self" incompatibility of recipient biological systems is often not considered, for example, when rodents are used as a model organism for preclinical studies of biomolecule-inspired nanomedicines. Using zebrafish embryos as an emerging model for nanobioimaging, here we unravel the in vivo fate of intravenously injected 70 nm SiO2 nanoparticles with a protein corona preformed from fetal bovine serum (FBS), representing a non-self biological identity. Strikingly rapid sequestration and endolysosomal acidification of nanoparticles with the preformed FBS corona were observed in scavenger endothelial cells within minutes after injection. This led to loss of blood vessel integrity and to inflammatory activation of macrophages over the course of several hours. As unmodified nanoparticles or the equivalent dose of FBS proteins alone failed to induce the observed pathophysiology, this signifies how the corona enriched with a differential repertoire of proteins can determine the fate of the nanoparticles in vivo. Our findings thus reveal the adverse outcome triggered by incompatible protein coronas and indicate a potential pitfall in the use of mismatched species combinations during nanomedicine development.
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Nanopartículas , Coroa de Proteína , Animais , Células Endoteliais , Dióxido de Silício , Peixe-ZebraRESUMO
Human patients carrying PAPP-A2 inactivating mutations have low bone mineral density. The underlying mechanisms for this reduced calcification are poorly understood. Using a zebrafish model, we report that Papp-aa regulates bone calcification by promoting Ca2+-transporting epithelial cell (ionocyte) quiescence-proliferation transition. Ionocytes, which are normally quiescent, re-enter the cell cycle under low [Ca2+] stress. Genetic deletion of Papp-aa, but not the closely related Papp-ab, abolished ionocyte proliferation and reduced calcified bone mass. Loss of Papp-aa expression or activity resulted in diminished IGF1 receptor-Akt-Tor signaling in ionocytes. Under low Ca2+ stress, Papp-aa cleaved Igfbp5a. Under normal conditions, however, Papp-aa proteinase activity was suppressed and IGFs were sequestered in the IGF/Igfbp complex. Pharmacological disruption of the IGF/Igfbp complex or adding free IGF1 activated IGF signaling and promoted ionocyte proliferation. These findings suggest that Papp-aa-mediated local Igfbp5a cleavage functions as a [Ca2+]-regulated molecular switch linking IGF signaling to bone calcification by stimulating epithelial cell quiescence-proliferation transition under low Ca2+ stress.
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Calcificação Fisiológica/fisiologia , Células Epiteliais/metabolismo , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Metaloendopeptidases/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Proliferação de Células/fisiologia , Peixe-ZebraRESUMO
Despite the common knowledge that the reticuloendothelial system is largely responsible for blood clearance of systemically administered nanoparticles, the sequestration mechanism remains a "black box". Using transgenic zebrafish embryos with cell type-specific fluorescent reporters and fluorescently labeled model nanoparticles (70 nm SiO2), we here demonstrate simultaneous three-color in vivo imaging of intravenously injected nanoparticles, macrophages, and scavenger endothelial cells (SECs). The trafficking processes were further revealed at ultrastructural resolution by transmission electron microscopy. We also find, using a correlative light-electron microscopy approach, that macrophages rapidly sequester nanoparticles via membrane adhesion and endocytosis (including macropinocytosis) within minutes after injection. In contrast, SECs trap single nanoparticles via scavenger receptor-mediated endocytosis, resulting in gradual sequestration with a time scale of hours. Inhibition of the scavenger receptors prevented SECs from accumulating nanoparticles but enhanced uptake in macrophages, indicating the competitive nature of nanoparticle clearance in vivo. To directly quantify the relative contributions of the two cell types to overall nanoparticle sequestration, the differential sequestration kinetics was studied within the first 30 min post-injection. This revealed a much higher and increasing relative contribution of SECs, as they by far outnumber macrophages in zebrafish embryos, suggesting the importance of the macrophage:SECs ratio in a given tissue. Further characterizing macrophages on their efficiency in nanoparticle clearance, we show that inflammatory stimuli diminish the uptake of nanoparticles per cell. Our study demonstrates the strength of transgenic zebrafish embryos for intravital real-time and ultrastructural imaging of nanomaterials that may provide mechanistic insights into nanoparticle clearance in rodent models and humans.
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Células Endoteliais/química , Macrófagos/química , Nanopartículas/metabolismo , Dióxido de Silício/metabolismo , Animais , Células Endoteliais/metabolismo , Cinética , Macrófagos/metabolismo , Nanopartículas/química , Tamanho da Partícula , Dióxido de Silício/química , Propriedades de Superfície , Fatores de Tempo , Peixe-Zebra/embriologiaRESUMO
Normal brain development depends on tight temporal and spatial regulation of connections between cells. Mutations in L1cam, a member of the immunoglobulin (Ig) superfamily that mediate cell-cell contacts through homo- and heterophilic interactions, are associated with several developmental abnormalities of the nervous system, including mental retardation, limb spasticity, hydrocephalus, and corpus callosum aplasia. L1cam has been reported to be shed from the cell surface, but the significance of this during different phases of brain development is unknown. We here show that ADAM10-mediated shedding of L1cam is regulated by its fibronectin type III (FNIII) domains. Specifically, the third FNIII domain is important for maintaining a conformation where access to a membrane proximal cleavage site is restricted. To define the role of ADAM10/17/BACE1-mediated shedding of L1cam during brain development, we used a zebrafish model system. Knockdown of the zebrafish, l1camb, caused hydrocephalus, defects in axonal outgrowth, and myelination abnormalities. Rescue experiments with proteinase-resistant and soluble L1cam variants showed that proteolytic cleavage is not required for normal axonal outgrowth and development of the ventricular system. In contrast, metalloproteinase-mediated shedding is required for efficient myelination, and only specific fragments are able to mediate this stimulatory function of the shedded L1cam.
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Encéfalo/crescimento & desenvolvimento , Hidrocefalia/genética , Molécula L1 de Adesão de Célula Nervosa/genética , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Proteínas ADAM/metabolismo , Animais , Ácido Aspártico Endopeptidases/metabolismo , Encéfalo/metabolismo , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Hidrocefalia/metabolismo , Molécula L1 de Adesão de Célula Nervosa/química , Proteólise , Peixe-Zebra , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Formation of functional myelin sheaths within the central nervous system depends on expression of myelin basic protein (MBP). Following process extension and wrapping around axonal segments, this highly basic protein is required for compaction of the multi-layered membrane sheath produced by oligodendrocytes. MBP is hypothesized to be targeted to the membrane sheath by mRNA transport and local translation, which ensures that its expression is temporally and spatially restricted. The mechanistic details of how this might be regulated are still largely unknown, in particular because a model system that allows this process to be studied in vivo is lacking. We here show that the expression of the zebrafish MBP orthologs, mbpa and mbpb, is developmentally regulated, and that expression of specific mbpa isoforms is restricted to the peripheral nervous system. By analysis of transgenic zebrafish, which express a fluorescent reporter protein specifically in myelinating oligodendrocytes, we demonstrate that both mbpa and mbpb include a 3'UTR sequence, by which mRNA transport and translation is regulated in vivo. Further functional analysis suggests that: (1) the 3'UTRs delay the onset of protein expression; and that (2) several regulatory elements contribute to targeting of the mbp mRNA to the myelin sheath. Finally, we show that a pharmacological compound known to enhance neuronal activity stimulates the translation of Mbp in zebrafish in a 3'UTR-dependent manner. A similar effect was obtained following stimulation with a TrkB receptor agonist, and cell-based assays further confirmed that the receptor ligand, BDNF, in combination with other signals reversed the inhibitory effect of the 3'UTR on translation.
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During development of the central nervous system not all axons are myelinated, and axons may have distinct myelination patterns. Furthermore, the number of myelin sheaths formed by each oligodendrocyte is highly variable. However, our current knowledge about the axo-glia communication that regulates the formation of myelin sheaths spatially and temporally is limited. By using axon-mimicking microfibers and a zebrafish model system, we show that axonal ephrin-A1 inhibits myelination. Ephrin-A1 interacts with EphA4 to activate the ephexin1-RhoA-Rock-myosin 2 signaling cascade and causes inhibition of oligodendrocyte process extension. Both in myelinating co-cultures and in zebrafish larvae, activation of EphA4 decreases myelination, whereas myelination is increased by inhibition of EphA4 signaling at different levels of the pathway, or by receptor knockdown. Mechanistically, the enhanced myelination is a result of a higher number of myelin sheaths formed by each oligodendrocyte, not an increased number of mature cells. Thus, we have identified EphA4 and ephrin-A1 as novel negative regulators of myelination. Our data suggest that activation of an EphA4-RhoA pathway in oligodendrocytes by axonal ephrin-A1 inhibits stable axo-glia interaction required for generating a myelin sheath.
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Sistema Nervoso Central/crescimento & desenvolvimento , Sistema Nervoso Central/metabolismo , Efrina-A1/metabolismo , Bainha de Mielina/metabolismo , Receptor EphA4/metabolismo , Animais , Animais Geneticamente Modificados , Axônios/metabolismo , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/metabolismo , Técnicas de Cocultura , Ratos , Transdução de Sinais , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Patients with short QT syndrome (SQTS) may present with syncope, ventricular fibrillation or sudden cardiac death. Six SQTS susceptibility genes, encoding cation channels, explain <25% of SQTS cases. Here we identify a missense mutation in the anion exchanger (AE3)-encoding SLC4A3 gene in two unrelated families with SQTS. The mutation causes reduced surface expression of AE3 and reduced membrane bicarbonate transport. Slc4a3 knockdown in zebrafish causes increased cardiac pHi, short QTc, and reduced systolic duration, which is rescued by wildtype but not mutated SLC4A3. Mechanistic analyses suggest that an increase in pHi and decrease in [Cl-]i shortened the action potential duration. However, other mechanisms may also play a role. Altered anion transport represents a mechanism for development of arrhythmia and may provide new therapeutic possibilities.
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Antiporters/genética , Arritmias Cardíacas/genética , Mutação com Perda de Função , Potenciais de Ação/genética , Animais , Antiporters/deficiência , Antiporters/fisiologia , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/fisiopatologia , Membrana Celular/fisiologia , Antiportadores de Cloreto-Bicarbonato/deficiência , Antiportadores de Cloreto-Bicarbonato/genética , Antiportadores de Cloreto-Bicarbonato/fisiologia , Modelos Animais de Doenças , Eletrocardiografia , Feminino , Técnicas de Silenciamento de Genes , Predisposição Genética para Doença , Células HEK293 , Coração/fisiopatologia , Heterozigoto , Humanos , Concentração de Íons de Hidrogênio , Masculino , Mutação de Sentido Incorreto , Linhagem , Sequenciamento do Exoma , Peixe-Zebra/genética , Peixe-Zebra/fisiologia , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologiaRESUMO
Congenital heart defects are a major cause of perinatal mortality and morbidity, affecting >1% of all live births in the Western world, yet a large fraction of such defects have an unknown etiology. Recent studies demonstrated surprising dual roles for immune-related molecules and their effector mechanisms during fetal development and adult homeostasis. In this article, we describe the function of an endogenous complement inhibitor, mannan-binding lectin (MBL)-associated protein (MAp)44, in regulating the composition of a serine protease-pattern recognition receptor complex, MBL-associated serine protease (MASP)-3/collectin-L1/K1 hetero-oligomer, which impacts cardiac neural crest cell migration. We used knockdown and rescue strategies in zebrafish, a model allowing visualization and assessment of heart function, even in the presence of severe functional defects. Knockdown of embryonic expression of MAp44 caused impaired cardiogenesis, lowered heart rate, and decreased cardiac output. These defects were associated with aberrant neural crest cell behavior. We found that MAp44 competed with MASP-3 for pattern recognition molecule interaction, and knockdown of endogenous MAp44 expression could be rescued by overexpression of wild-type MAp44. Our observations provide evidence that immune molecules are centrally involved in the orchestration of cardiac tissue development.
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Coração/embriologia , Serina Proteases Associadas a Proteína de Ligação a Manose/metabolismo , Animais , Técnicas de Silenciamento de Genes , Humanos , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
Calmodulin (CaM) is the central mediator of intracellular Ca(2+) signalling in cardiomyocytes, where it conveys the intricate Ca(2+) transients to the proteins controlling cardiac contraction. We recently linked two separate mutations in CaM (N53I and N97S) to dominantly inherited catecholaminergic polymorphic ventricular tachycardia (CPVT), an arrhythmic disorder in which exercise or acute emotion can lead to syncope and sudden cardiac death. Given the ubiquitous presence of CaM in all eukaryote cells, it is particular intriguing that carriers of either mutation show no additional symptoms. Here, we investigated the effects of the CaM CPVT mutations in a zebrafish animal model. Three-day-old embryos injected with either CaM mRNA showed no detectable pathologies or developmental abnormalities. However, embryos injected with CPVT CaM mRNA displayed increased heart rate compared to wild-type CaM mRNA under ß-adrenergic stimulation, demonstrating a conserved dominant cardiac specific effect between zebrafish and human carriers of these mutations. Motivated by the highly similar physiological phenotypes, we compared the effects of the N53I and N97S mutations on the biophysical and functional properties of CaM. Surprisingly, the mutations have opposing effects on CaM C-lobe Ca(2+) binding affinity and kinetics, and changes to the CaM N-lobe Ca(2+) binding are minor and specific to the N53I mutation. Furthermore, both mutations induce differential perturbations to structure and stability towards unfolding. Our results suggest different molecular disease mechanisms for the CPVT (N53I and N97S mutations) and strongly support that cardiac contraction is the physiological process most sensitive to CaM integrity.
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Calmodulina/química , Calmodulina/metabolismo , Taquicardia Ventricular/metabolismo , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/metabolismo , Animais , Sinalização do Cálcio/genética , Sinalização do Cálcio/fisiologia , Calmodulina/genética , Mutação , Dobramento de Proteína , Taquicardia Ventricular/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Pregnancy-associated plasma protein A2 (PAPP-A2, also known as pappalysin-2) is a large metalloproteinase that is known to be required for normal postnatal growth and bone development in mice. We here report the detection of zebrafish papp-a2 mRNA in the chordamesoderm, notochord and lower jaw of zebrafish (Danio rerio) embryos, and that papp-a2-knockdown embryos display broadened axial mesoderm, notochord bends and severely reduced cranial cartilages. Genetic data link these phenotypes to insulin-like growth factor (Igf)-binding protein-3 (Igfbp-3) and bone morphogenetic protein (Bmp) signaling, and biochemical analysis show specific Igfbp-3 proteolysis by Papp-a2, implicating Papp-a2 in the modulation of Bmp signaling by Igfbp-3 proteolysis. Knockdown of papp-a2 additionally resulted in angiogenesis defects, strikingly similar to previous observations in embryos with mutations in components of the Notch system. Accordingly, we find that Notch signaling is modulated by Papp-a2 in vivo, and, furthermore, that human PAPP-A2 is capable of modulating Notch signaling independently of its proteolytic activity in cell culture. Based on these results, we conclude that Papp-a2 modulates Bmp and Notch signaling by independent mechanisms in zebrafish embryos. In conclusion, these data link pappalysin function in zebrafish to two different signaling pathways outside the IGF system.
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Cartilagem/enzimologia , Neovascularização Fisiológica , Proteína Plasmática A Associada à Gravidez/metabolismo , Crânio/enzimologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Cartilagem/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Técnicas de Silenciamento de Genes , Genótipo , Células HEK293 , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Proteína 4 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Dados de Sequência Molecular , Fenótipo , Proteína Plasmática A Associada à Gravidez/genética , RNA Mensageiro/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Transdução de Sinais , Crânio/embriologia , Fatores de Tempo , Transfecção , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Synapsin 1 (SYN1) is a phosphoprotein involved in nerve signal transmission. The porcine SYN1 promoter orthologue was cloned and characterized to provide a means of expressing a transgene specifically in neurons. The nucleotide sequence of the promoter displayed a high degree of conservation of elements responsible for neuron-specific expression. Expression analysis of SYN1 demonstrated presence of transcript during embryonic development. Analysis of GFP expression in transgenic zebrafish embryos suggests that the pig SYN1 promoter directs expression in neuronal cells. Thus, the SYN1 promoter is a good candidate for use in the generation of pig models of human neurodegenerative disorders.
RESUMO
Naâº/Kâº-ATPase maintains electrochemical gradients of Na⺠and K⺠essential for a variety of cellular functions including neuronal activity. The α-subunit of the Naâº/Kâº-ATPase exists in four different isoforms (α1-α4) encoded by different genes. With a view to future use of pig as an animal model in studies of human diseases caused by Naâº/Kâº-ATPase mutations, we have determined the porcine coding sequences of the α1-α3 genes, ATP1A1, ATP1A2, and ATP1A3, their chromosomal localization, and expression patterns. Our ATP1A1 sequence accords with the sequences from several species at five positions where the amino acid residue of the previously published porcine ATP1A1 sequence differs. These corrections include replacement of glutamine 841 with arginine. Analysis of the functional consequences of substitution of the arginine revealed its importance for Na⺠binding, which can be explained by interaction of the arginine with the C-terminus, stabilizing one of the Na⺠sites. Quantitative real-time PCR expression analyses of porcine ATP1A1, ATP1A2, and ATP1A3 mRNA showed that all three transcripts are expressed in the embryonic brain as early as 60 days of gestation. Expression of α3 is confined to neuronal tissue. Generally, the expression patterns of ATP1A1, ATP1A2, and ATP1A3 transcripts were found similar to their human counterparts, except for lack of α3 expression in porcine heart. These expression patterns were confirmed at the protein level. We also report the sequence of the porcine ATP1A3 promoter, which was found to be closely homologous to its human counterpart. The function and specificity of the porcine ATP1A3 promoter was analyzed in transgenic zebrafish, demonstrating that it is active and drives expression in embryonic brain and spinal cord. The results of the present study provide a sound basis for employing the ATP1A3 promoter in attempts to generate transgenic porcine models of neurological diseases caused by ATP1A3 mutations.
Assuntos
ATPase Trocadora de Sódio-Potássio/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Chlorocebus aethiops , Mapeamento Cromossômico , Clonagem Molecular , Metilação de DNA , Genes Reporter , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Especificidade de Órgãos , Regiões Promotoras Genéticas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , ATPase Trocadora de Sódio-Potássio/química , Sus scrofa , Peixe-ZebraRESUMO
Pregnancy-associated plasma protein-A (PAPP-A) is a large metalloproteinase specifically cleaving insulin-like growth factor (IGF) binding proteins, causing increased IGF bioavailability and, hence, local regulation of IGF receptor activation. We have identified two highly conserved zebrafish homologs of the human PAPP-A gene. Expression of zebrafish Papp-a, one of the two paralogs, begins during gastrulation and persists throughout the first week of development, and analyses demonstrate highly conserved patterns of expression between adult zebrafish, humans, and mice. We show that the specific knockdown of zebrafish papp-a limits the developmental rate beginning during gastrulation without affecting the normal patterning of the embryo. This phenotype is different from those resulting from deficiency of Igf receptor or ligand in zebrafish, suggesting a function of Papp-a outside of the Igf system. Biochemical analysis of recombinant zebrafish Papp-a demonstrates conservation of proteolytic activity, specificity, and the intrinsic regulatory mechanism. However, in vitro transcribed mRNA, which encodes a proteolytically inactive Papp-a mutant, recues the papp-a knockdown phenotype as efficiently as wild-type Papp-a. Thus, the developmental phenotype of papp-a knockdown is not a consequence of lacking Papp-a proteolytic activity. We conclude that Papp-a possesses biological functions independent of its proteolytic activity. Our data represent the first evidence for a non-proteolytic function of PAPP-A.