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1.
Int J Mol Sci ; 22(14)2021 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-34299109

RESUMO

The domestic goat (Capra aegagrus hircus), a mammalian species with high genetic merit for production of milk and meat, can be a tremendously valuable tool for transgenic research. This research is focused on the production and multiplication of genetically engineered or genome-edited cloned specimens by applying somatic cell nuclear transfer (SCNT), which is a dynamically developing assisted reproductive technology (ART). The efficiency of generating the SCNT-derived embryos, conceptuses, and progeny in goats was found to be determined by a variety of factors controlling the biological, molecular, and epigenetic events. On the one hand, the pivotal objective of our paper was to demonstrate the progress and the state-of-the-art achievements related to the innovative and highly efficient solutions used for the creation of transgenic cloned does and bucks. On the other hand, this review seeks to highlight not only current goals and obstacles but also future challenges to be faced by the approaches applied to propagate genetically modified SCNT-derived goats for the purposes of pharmacology, biomedicine, nutritional biotechnology, the agri-food industry, and modern livestock breeding.


Assuntos
Animais Geneticamente Modificados/genética , Clonagem de Organismos/veterinária , Embrião de Mamíferos/citologia , Engenharia Genética/veterinária , Técnicas de Transferência Nuclear/veterinária , Animais , Animais Geneticamente Modificados/crescimento & desenvolvimento , Cabras
2.
Toxicol Lett ; 350: 71-80, 2021 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-34252508

RESUMO

Sanguinarine, derived from the root of Sanguinaria canadensis, have multiple biological activities, such as antimicrobial, insecticidal, antitumor, anti-inflammatory and anti-angiogenesis effect, but little is known about its toxicity on normal embryonic development. Here, we study the developmental toxicity using zebrafish model. Notably, sanguinarine caused a significant increase of the malformation rate and decrease of hatching rates and body length of zebrafish embryos. Sanguinarine also impaired the normal development of heart, liver and nerve system of zebrafish embryos. Further, the ROS level and MDA concentrations were remarkably increased, while the activity of T-SOD was decreased. In addition, obvious increase of apoptosis were observed by AO staining or TUNEL assay. Further studies showed that the oxidative stress-, apoptosis-related genes were changed, while genes of nrf2 and wnt pathways were inhibited by sangunarine. To sum up, our study will be helpful to understand the adverse effect of sanguinarine on embryonic development and the underlying molecular mechanism.


Assuntos
Apoptose/efeitos dos fármacos , Benzofenantridinas/toxicidade , Isoquinolinas/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Peixe-Zebra/embriologia , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados/crescimento & desenvolvimento , Embrião não Mamífero/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Variação Genética , Genótipo , Modelos Animais , Raízes de Plantas/química , Raízes de Plantas/toxicidade , Sanguinaria/química , Sanguinaria/toxicidade
3.
Int J Biol Macromol ; 184: 721-730, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-34174306

RESUMO

Aquaporin (AQP) transport solutes across cell membranes in both unicellular and multicellular organisms. In this study, the aquaporin CsPrip was identified in Chilo suppressalis, an important pest of rice. CsPrip was comprised of two variants, CsPrip_v1 and CsPrip_v2; the former variant was <103 bp was shorter than the latter, although both exhibited the same open reading frame (ORF). Transmembrane topology and protein structure analyses showed that CsPrip retained the conserved features of water-selective insect AQPs, including six transmembrane domains, two conserved hydrophobic asparagine-proline-alanine motifs and the aromatic/arginine constriction region. Expression in Xenopus oocytes revealed that CsPrip preferentially transported water and urea instead of trehalose and glycerol. The CsPrip transcript was expressed in multiple organs and tissues of C. suppressalis larvae and was most abundant in the hindgut and Malpighian tubules. CsPrip transcription was highest in male adults and was relatively stable throughout development. CsPrip expression in larvae was significantly altered by thermal stress, and relative humidity levels impacted CsPrip transcription in 3rd and 5th instar larvae. This study confirms that the aquaporin CsPrip performs multiple critical functions in maintaining water equilibrium in C. suppressalis.


Assuntos
Aquaporinas/genética , Aquaporinas/metabolismo , Lepidópteros/metabolismo , Oryza/parasitologia , Processamento Alternativo , Animais , Animais Geneticamente Modificados/crescimento & desenvolvimento , Aquaporinas/química , Feminino , Regulação da Expressão Gênica , Proteínas de Insetos/química , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Lepidópteros/genética , Masculino , Modelos Moleculares , Especificidade de Órgãos , Conformação Proteica , Domínios Proteicos , Caracteres Sexuais , Ureia/metabolismo , Água/metabolismo , Xenopus/genética , Xenopus/crescimento & desenvolvimento
4.
Methods Mol Biol ; 2272: 281-318, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34009621

RESUMO

5-methylcytosine (5mC) is a gene-regulatory mark associated with transcriptional repression. 5mC can be erased through the catalytic action of Ten-eleven translocation (TET) methylcytosine dioxygenases (TET1, TET2, TET3), which oxidize 5mC resulting in its removal from the genome. In vertebrates, TET enzymes facilitate DNA demethylation of regulatory regions linked to genes involved in developmental processes. Consequently, TET ablation leads to severe morphological defects and developmental arrest. Here we describe a system that can facilitate the study of relationships between TET enzymes, 5mC, and embryo development. We provide detailed descriptions for the generation of F0 zebrafish tet1/2/3 knockouts using CRISPR/Cas9 technology and elaborate on the strategies to assess the impact of TET loss by reduced representation bisulfite sequencing (RRBS).


Assuntos
Animais Geneticamente Modificados/metabolismo , Metilação de DNA , Dioxigenases/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Dioxigenases/genética , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genética
5.
FASEB J ; 35(5): e21537, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33817834

RESUMO

Cannabidiol (CBD), a phytocannabinoid from the Cannabis sativa plant, exhibits a broad spectrum of potential therapeutic properties for neurodegenerative diseases. An accumulation of amyloid-ß (Aß) protein is one of the most important neuropathology in neurodegenerative diseases like Alzheimer's disease (AD). Data on the effect of CBD on the amelioration of Aß-induced neurite degeneration and its consequences of life and health spans is sparse. This study aimed to investigate the effects of CBD on neurite outgrowth in cells and lifespan and health span in Caenorhabditis elegans (C. elegans). In human SH-SY5Y neuronal cells, CBD prevented neurite lesion induced by Aß1-42 and increased the expression of fatty acid amide hydrolase (FAAH) and cannabinoid receptor 1 (CB1R). Furthermore, CBD both protected the reduction of dendritic spine density and rescued the activity of synaptic Ca2+ /calmodulin-dependent protein kinase II (CaMKII) from Aß1-42 toxicity in primary hippocampal neurons. In C. elegans, we used the transgenic CL2355 strain of C. elegans, which expresses the human Aß peptide throughout the nervous system and found that CBD treatment extended lifespan and improved health span. The neuroprotective effect of CBD was further explored by observing the dopaminergic neurons using transgenic dat-1: GFP strains using the confocal microscope. This study shows that CBD prevents the neurite degeneration induced by Aß, by a mechanism involving CB1R activation, and extends lifespan and improves health span in Aß-overexpressing worms. Our findings support the potential therapeutic approach of CBD for the treatment of AD patients.


Assuntos
Peptídeos beta-Amiloides/toxicidade , Caenorhabditis elegans/crescimento & desenvolvimento , Canabidiol/farmacologia , Longevidade , Neuroblastoma/tratamento farmacológico , Crescimento Neuronal , Receptor CB1 de Canabinoide/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Anticonvulsivantes/farmacologia , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Neurônios/citologia , Neurônios/metabolismo , Fármacos Neuroprotetores , Fosforilação , Receptor CB1 de Canabinoide/genética , Fator de Transcrição STAT3/genética
6.
Sci Rep ; 11(1): 671, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436662

RESUMO

We developed a novel reporter transgenic zebrafish model called MITO-Luc/GFP zebrafish in which GFP and luciferase expression are under the control of the master regulator of proliferation NF-Y. In MITO-Luc/GFP zebrafish it is possible to visualize cell proliferation in vivo by fluorescence and bioluminescence. In this animal model, GFP and luciferase expression occur in early living embryos, becoming tissue specific in juvenile and adult zebrafish. By in vitro and ex vivo experiments we demonstrate that luciferase activity in adult animals occurs in intestine, kidney and gonads, where detectable proliferating cells are located. Further, by time lapse experiments in live embryos, we observed a wave of GFP positive cells following fin clip. In adult zebrafish, in addition to a bright bioluminescence signal on the regenerating tail, an early unexpected signal coming from the kidney occurs indicating not only a fin cell proliferation, but also a systemic response to tissue damage. Finally, we observed that luciferase activity was inhibited by anti-proliferative interventions, i.e. 5FU, cell cycle inhibitors and X-Rays. In conclusion, MITO-Luc/GFP zebrafish is a novel animal model that may be crucial to assess the spatial and temporal evolution of cell proliferation in vivo.


Assuntos
Animais Geneticamente Modificados/crescimento & desenvolvimento , Proliferação de Células , Evolução Molecular , Proteínas de Fluorescência Verde/metabolismo , Luciferases/metabolismo , Análise Espaço-Temporal , Peixe-Zebra/crescimento & desenvolvimento , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Proteínas de Fluorescência Verde/genética , Luciferases/genética , Regeneração , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
7.
BMC Biol ; 19(1): 11, 2021 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-33478487

RESUMO

BACKGROUND: Thermal plasticity is pivotal for evolution in changing climates and in mediating resilience to its potentially negative effects. The efficacy to respond to environmental change depends on underlying mechanisms. DNA methylation induced by DNA methyltransferase 3 enzymes in the germline or during early embryonic development may be correlated with responses to environmental change. This developmental plasticity can interact with reversible acclimation within adult organisms, which would increase the speed of response and could alleviate potential mismatches between parental or early embryonic environments and those experienced at later life stages. Our aim was to determine whether there is a causative relationship between DNMT3 enzyme and developmental thermal plasticity and whether either or both interact with short-term acclimation to alter fitness and thermal responses in zebrafish (Danio rerio). RESULTS: We developed a novel DNMT3a knock-out model to show that sequential knock-out of DNA methyltransferase 3a isoforms (DNMT3aa-/- and DNMT3aa-/-ab-/-) additively decreased survival and increased deformities when cold developmental temperatures in zebrafish offspring mismatched warm temperatures experienced by parents. Interestingly, short-term cold acclimation of parents before breeding rescued DNMT3a knock-out offspring by restoring survival at cold temperatures. DNMT3a knock-out genotype interacted with developmental temperatures to modify thermal performance curves in offspring, where at least one DNMT3a isoform was necessary to buffer locomotion from increasing temperatures. The thermal sensitivity of citrate synthase activity, an indicator of mitochondrial density, was less severely affected by DNMT3a knock-out, but there was nonetheless a significant interaction between genotype and developmental temperatures. CONCLUSIONS: Our results show that DNMT3a regulates developmental thermal plasticity and that the phenotypic effects of different DNMT3a isoforms are additive. However, DNMT3a interacts with other mechanisms, such as histone (de)acetylation, induced during short-term acclimation to buffer phenotypes from environmental change. Interactions between these mechanisms make phenotypic compensation for climate change more efficient and make it less likely that thermal plasticity incurs a cost resulting from environmental mismatches.


Assuntos
Adaptação Fisiológica/genética , Mudança Climática , DNA (Citosina-5-)-Metiltransferases/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Animais Geneticamente Modificados/fisiologia , DNA (Citosina-5-)-Metiltransferases/metabolismo , Feminino , Masculino , Fenótipo , Termotolerância/genética , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/metabolismo
8.
Bioorg Chem ; 107: 104624, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33465669

RESUMO

Two novel monoterpenoid indole alkaloids (MIAs), gelsechizines A-B (1-2), along with four known ones (3-6) were isolated from the fruits of Gelsemium elegans. Compound 1 features a new carbon skeleton with two additional carbon atoms forming a 4-methylpyridine unit. Their structures with absolute configurations were elucidated by NMR, MS, X-ray diffraction and electronic circular dichroism (ECD) calculations. Compounds 1-3 showed significant anti-inflammatory effects in vivo and in vitro, which may be related to the inhibition of the trecruitment of neutrophils and macrophages as well as the secretion of TNF-α and IL-6. Preliminary structure-activity relationship analysis revealed that the ß-N-acrylate moiety plays an important role in the anti-inflammatory effect.


Assuntos
Anti-Inflamatórios/farmacologia , Gelsemium/química , Macrófagos/efeitos dos fármacos , Alcaloides de Triptamina e Secologanina/química , Animais , Animais Geneticamente Modificados/crescimento & desenvolvimento , Animais Geneticamente Modificados/metabolismo , Anti-Inflamatórios/química , Anti-Inflamatórios/isolamento & purificação , Frutas/química , Frutas/metabolismo , Gelsemium/metabolismo , Interleucina-6/metabolismo , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Larva/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/metabolismo , Espectroscopia de Ressonância Magnética , Camundongos , Conformação Molecular , Neutrófilos/citologia , Neutrófilos/patologia , Células RAW 264.7 , Alcaloides de Triptamina e Secologanina/isolamento & purificação , Alcaloides de Triptamina e Secologanina/farmacologia , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo
9.
Gene ; 771: 145366, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33346099

RESUMO

The Drosophila melanogaster ß2-tubulin gene (Dm-ß2t) controls the function of microtubules in the testis and sperm, and has been evaluated for use in biocontrol strategies based on the sterile insect technique, including sexing and the induction of male sterility. The spotted-wing Drosophila (Drosophila suzukii) is native to eastern Asia but has spread globally as an invasive pest of fruit crops, so biocontrol strategies are urgently required for this species. We therefore isolated the ß2tubulin ortholog Ds-ß2t from the USA laboratory strain of D. suzukii and confirmed the presence of functional motifs by aligning orthologs from multiple insects. The developmental expression profile of Ds-ß2t was determined by RT-PCR using gene-specific primers and was similar to that of Dm-ß2t. We then isolated the Ds-ß2t promoter and used it to generate transgenic strains expressing a testis-specific fluorescent protein starting from the thirdinstar larvae. Efficient sexing was achieved based on fluorescence detection, and the transgenic males showed a similar survival rate to wild-type males. Fluorescence imaging and PCR were also used to confirm the insemination of wild-type females by transgenic males. We therefore confirm that D. suzukii strains expressing fluorescent markers under the control of the Ds-ß2t promoter can be used for sexing and the confirmation of mating, and we discuss the wider potential of the Ds-ß2t promoter in the context of genetic control strategies for D. suzukii.


Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/crescimento & desenvolvimento , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Animais , Animais Geneticamente Modificados/crescimento & desenvolvimento , Animais Geneticamente Modificados/metabolismo , Drosophila/genética , Drosophila/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Inseminação , Espécies Introduzidas , Masculino , Imagem Óptica , Regiões Promotoras Genéticas , Reprodução , Processos de Determinação Sexual
10.
Front Endocrinol (Lausanne) ; 11: 578885, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33133021

RESUMO

In vertebrate germ cell differentiation, gonadal somatic cells and germ cells are closely related. By analyzing this relationship, it has recently been reported in mammals that primordial germ cells (PGCs), induced from pluripotent stem cells and germline stem cells, can differentiate into functional gametes when co-cultured in vitro with fetal gonadal somatic cells. In some fish species, differentiation into functional sperm by reaggregation or co-culture of gonadal somatic cells and germ cells has also been reported; however, the relationship between gonadal somatic cells and germ cells in these species is not well-understood. Here, we report the transcriptional regulation of Müllerian inhibiting substance (MIS) and the establishment of a gonadal somatic cell line using mis-GFP transgenic fish, in medaka (Oryzias latipes)-a fish model which offers many advantages for molecular genetics. MIS is a glycoprotein belonging to the transforming growth factor ß superfamily. In medaka, mis mRNA is expressed in gonadal somatic cells of both sexes before sex differentiation, and MIS regulates the proliferation of germ cells during this period. Using luciferase assays, we found that steroidogenic factor 1 (SF1) and liver receptor homolog 1 (LRH1) activate medaka mis gene transcription, probably by binding to the mis promoter. We also report that mis-GFP transgenic medaka emit GFP fluorescence specific to gonadal somatic cells in the gonads. By fusing Sertoli cells from transgenic medaka with a cell line derived from medaka hepatoma cancer, we produced a hybridoma cell line that expresses gonadal somatic cell-specific markers, including Sertoli and Leydig cell markers. Moreover, embryonic PGCs co-cultured with the established hybridoma, as feeder cells, proliferated and formed significant colonies after 1 week. PGCs cultured for 3 weeks expressed a germ cell marker dnd, as well as the meiotic markers sycp1 and sycp3. Thus, we here provide the first evidence in teleosts that we have successfully established a gonadal somatic cell-derived hybridoma that can induce both the proliferation and meiosis of germ cells.


Assuntos
Animais Geneticamente Modificados/metabolismo , Hormônio Antimülleriano/metabolismo , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica , Células Germinativas/metabolismo , Gônadas/metabolismo , Oryzias/metabolismo , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Hormônio Antimülleriano/genética , Diferenciação Celular , Células Cultivadas , Proteínas de Peixes/genética , Células Germinativas/citologia , Gônadas/citologia , Oryzias/genética , Oryzias/crescimento & desenvolvimento
13.
Zebrafish ; 17(6): 373-381, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33112719

RESUMO

The melanocortin system is a key structure in the regulation of energy balance. Overexpression of inverse agonists, agouti-signaling protein (ASIP), and agouti-related protein (AGRP) results in increased food intake, linear growth, and body weight. ASIP regulates dorsal-ventral pigment polarity through melanocortin 1 receptor (MC1R) and overexpression induces obesity in mice by binding to central MC4R. Asip1 overexpression in transgenic zebrafish (asip1-Tg) enhances growth, yet experiments show fish overexpressing Asip1 do not develop obesity even under severe feeding regimes. Asip1-Tg fish do not need to eat more to grow larger and faster; thus, increased food efficiency can be observed. In addition, asip1-Tg fish reared at high density are able to grow far more than wild-type (WT) fish reared at low density, although asip1-Tg fish seem to be more sensitive to crowding stress than WT fish, thus making the melanocortin system a target for sustainable aquaculture, especially as the U.S. Food and Drug Association has recently approved transgenic fish trading.


Assuntos
Proteína Agouti Sinalizadora/genética , Dieta , Expressão Gênica , Obesidade/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteína Agouti Sinalizadora/metabolismo , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Aglomeração , Estresse Fisiológico , Peixe-Zebra/genética
14.
PLoS Genet ; 16(10): e1009102, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33104696

RESUMO

Ryanodine receptors (RyR) are essential regulators of cellular calcium homeostasis and signaling. Vertebrate genomes contain multiple RyR gene isoforms, expressed in different tissues and executing different functions. In contrast, invertebrate genomes contain a single RyR-encoding gene and it has long been proposed that different transcripts generated by alternative splicing may diversify their functions. Here, we analyze the expression and function of alternative exons in the C. elegans RyR gene unc-68. We show that specific isoform subsets are created via alternative promoters and via alternative splicing in unc-68 Divergent Region 2 (DR2), which actually corresponds to a region of high sequence variability across vertebrate isoforms. The expression of specific unc-68 alternative exons is enriched in different tissues, such as in body wall muscle, neurons and pharyngeal muscle. In order to infer the function of specific alternative promoters and alternative exons of unc-68, we selectively deleted them by CRISPR/Cas9 genome editing. We evaluated pharyngeal function, as well as locomotor function in swimming and crawling with high-content computer-assisted postural and behavioral analysis. Our data provide a comprehensive map of the pleiotropic impact of isoform-specific mutations and highlight that tissue-specific unc-68 isoforms fulfill distinct functions. As a whole, our work clarifies how the C. elegans single RyR gene unc-68 can fulfill multiple tasks through tissue-specific isoforms, and provide a solid foundation to further develop C. elegans as a model to study RyR channel functions and malfunctions.


Assuntos
Processamento Alternativo/genética , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Contração Muscular/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Animais , Animais Geneticamente Modificados/crescimento & desenvolvimento , Caenorhabditis elegans/crescimento & desenvolvimento , Sinalização do Cálcio/genética , Modelos Animais de Doenças , Éxons , Humanos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Mutação/genética , Especificidade de Órgãos/genética , Isoformas de Proteínas/genética , Rianodina/metabolismo
15.
Curr Biol ; 30(17): 3277-3292.e5, 2020 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-32649909

RESUMO

The musculoskeletal system is a striking example of how cell identity and position is coordinated across multiple tissues to ensure function. However, it is unclear upon tissue loss, such as complete loss of cells of a central musculoskeletal connecting tendon, whether neighboring tissues harbor progenitors capable of mediating regeneration. Here, using a zebrafish model, we genetically ablate all embryonic tendon cells and find complete regeneration of tendon structure and pattern. We identify two regenerative progenitor populations, sox10+ perichondrial cells surrounding cartilage and nkx2.5+ cells surrounding muscle. Surprisingly, laser ablation of sox10+ cells, but not nkx2.5+ cells, increases tendon progenitor number in the perichondrium, suggesting a mechanism to regulate attachment location. We find BMP signaling is active in regenerating progenitor cells and is necessary and sufficient for generating new scxa+ cells. Our work shows that muscle and cartilage connective tissues harbor progenitor cells capable of fully regenerating tendons, and this process is regulated by BMP signaling.


Assuntos
Animais Geneticamente Modificados/crescimento & desenvolvimento , Proteínas Morfogenéticas Ósseas/metabolismo , Sistema Musculoesquelético/metabolismo , Regeneração , Células-Tronco/citologia , Tendões/citologia , Peixe-Zebra/crescimento & desenvolvimento , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Proteínas Morfogenéticas Ósseas/genética , Diferenciação Celular , Células-Tronco/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
16.
Insect Mol Biol ; 29(5): 452-465, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32654295

RESUMO

The silkworm is an economically important insect producing plentiful silk fibre in the silk gland. In this study, we reported a cross-talk between the fat body, silk gland and midgut through a glycine-serine biosynthetic pathway in the silkworm. Amino acid sequence and functional domains of glycine transporter gene BmGT1-L were mapped. Our results indicated that BmGT1-L was specifically expressed in the midgut microvilli and persistently expressed during the feeding stages. RNA interference of BmGT1-L activated glycine biosynthesis, and BmGT1-L overexpression facilitated serine biosynthesis in the BmN4-SID1 cell. In addition, silkworms after FibH gene knock-out or silk gland extirpation showed markedly decreased BmGT1-L transcripts in the midgut and disturbed glycine-serine biosynthesis as silk yield decreased. Finally, BmGT1-L ectopic expression in the posterior silk gland promoted glycine biosynthesis, and enhanced silk yield via increasing fibroin synthesis. These results suggested that cross-talk between tissues can be used for enhancing silk yield in the silkworm.


Assuntos
Bombyx/metabolismo , Expressão Ectópica do Gene , Proteínas de Insetos/genética , Seda/biossíntese , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Animais Geneticamente Modificados/metabolismo , Bombyx/genética , Bombyx/crescimento & desenvolvimento , Glândulas Exócrinas/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Tegumento Comum/fisiologia , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Alinhamento de Sequência , Seda/genética
17.
PLoS One ; 15(7): e0235433, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32726316

RESUMO

ADP-ribosylhydrolase-like 1 (Adprhl1) is a pseudoenzyme expressed in the developing heart myocardium of all vertebrates. In the amphibian Xenopus laevis, knockdown of the two cardiac Adprhl1 protein species (40 and 23 kDa) causes failure of chamber outgrowth but this has only been demonstrated using antisense morpholinos that interfere with RNA-splicing. Transgenic production of 40 kDa Adprhl1 provides only part rescue of these defects. CRISPR/Cas9 technology now enables targeted mutation of the adprhl1 gene in G0-generation embryos with routine cleavage of all alleles. Testing multiple gRNAs distributed across the locus reveals exonic locations that encode critical amino acids for Adprhl1 function. The gRNA recording the highest frequency of a specific ventricle outgrowth phenotype directs Cas9 cleavage of an exon 6 sequence, where microhomology mediated end-joining biases subsequent DNA repairs towards three small in-frame deletions. Mutant alleles encode discrete loss of 1, 3 or 4 amino acids from a di-arginine (Arg271-Arg272) containing peptide loop at the centre of the ancestral ADP-ribosylhydrolase site. Thus despite lacking catalytic activity, it is the modified (adenosine-ribose) substrate binding cleft of Adprhl1 that fulfils an essential role during heart formation. Mutation results in striking loss of myofibril assembly in ventricle cardiomyocytes. The defects suggest Adprhl1 participation from the earliest stage of cardiac myofibrillogenesis and are consistent with previous MO results and Adprhl1 protein localization to actin filament Z-disc boundaries. A single nucleotide change to the gRNA sequence renders it inactive. Mice lacking Adprhl1 exons 3-4 are normal but production of the smaller ADPRHL1 species is unaffected, providing further evidence that cardiac activity is concentrated at the C-terminal protein portion.


Assuntos
Ventrículos do Coração/crescimento & desenvolvimento , Coração/crescimento & desenvolvimento , Desenvolvimento Muscular/genética , N-Glicosil Hidrolases/genética , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Catálise , Domínio Catalítico/genética , Coração/fisiopatologia , Ventrículos do Coração/patologia , Humanos , Camundongos , Camundongos Knockout , Morfolinos/genética , Oligodesoxirribonucleotídeos Antissenso/genética , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Organogênese/genética , Xenopus laevis/genética , Xenopus laevis/crescimento & desenvolvimento
18.
Development ; 147(14)2020 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-32586974

RESUMO

Long-term survival of an animal species depends on development being robust to environmental variations and climate changes. We used C. elegans to study how mechanisms that sense environmental changes trigger adaptive responses that ensure animals develop properly. In water, the nervous system induces an adaptive response that reinforces vulval development through an unknown backup signal for vulval induction. This response involves the heterotrimeric G-protein EGL-30//Gαq acting in motor neurons. It also requires body-wall muscle, which is excited by EGL-30-stimulated synaptic transmission, suggesting a behavioral function of neurons induces backup signal production from muscle. We now report that increased acetylcholine during liquid growth activates an EGL-30-Rho pathway, distinct from the synaptic transmission pathway, that increases Wnt production from motor neurons. We also provide evidence that this neuronal Wnt contributes to EGL-30-stimulated vulval development, with muscle producing a parallel developmental signal. As diverse sensory modalities stimulate motor neurons via acetylcholine, this mechanism enables broad sensory perception to enhance Wnt-dependent development. Thus, sensory perception improves animal fitness by activating distinct neuronal functions that trigger adaptive changes in both behavior and developmental processes.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Neurônios/metabolismo , Proteínas Wnt/metabolismo , Acetilcolina/metabolismo , Citoesqueleto de Actina , Animais , Animais Geneticamente Modificados/crescimento & desenvolvimento , Animais Geneticamente Modificados/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/genética , Fator de Crescimento Epidérmico/metabolismo , Feminino , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Genótipo , Mutagênese , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais , Transmissão Sináptica/genética , Vulva/crescimento & desenvolvimento , Vulva/metabolismo , Proteínas Wnt/genética
19.
Mutat Res ; 821: 111713, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32593030

RESUMO

The present study investigated the effect of four carvacrol ethers (propyl-, butyl, octyl- and benzyl) on the viability, level of dominant lethal mutations of Drosophila melanogaster and their influence on the multiplication of the nuclear genome in salivary gland cells. The fertility and viability of fruit flies were assessed after oral administration (0.05 % to culture medium) and inhalation exposure (5 mg per 1 cm2 of polyvinyl alcohol film) of compounds 1‒4 and initial carvacrol. The influence of terpenoid and its ethers on the degree of chromosomes polyteny in salivary gland cells of D. melanogaster larvae has been revealed. Among all tested compounds, carvacrol exhibited the most significant impact on frequency of dominant lethal mutations, fecundity and insect survival when inhaled or adding to the culture medium. Oral administration of ethers 1‒4 was found to decrease the average level of chromosome polyteny degree (366 C-500 C) while pure carvacrol adding to culture medium had the opposite effect (763 C) compared to control (695 C). The possible mechanism of action for carvacrol and its ethers is discussed.


Assuntos
Animais Geneticamente Modificados/crescimento & desenvolvimento , Duplicação Cromossômica/efeitos dos fármacos , Cromossomos/efeitos dos fármacos , Cimenos/toxicidade , Drosophila melanogaster/efeitos dos fármacos , Éteres/toxicidade , Mutação , Animais , Animais Geneticamente Modificados/genética , Núcleo Celular , Dano ao DNA , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Genótipo , Masculino
20.
Development ; 147(12)2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32467235

RESUMO

The transcription factor Stat3 is required for proliferation and pluripotency of embryonic stem cells; we have prepared and characterized fluorescent Stat3-reporter zebrafish based on repeats of minimal responsive elements. These transgenic lines mimic in vivo Stat3 expression patterns and are responsive to exogenous Stat3; notably, fluorescence is inhibited by both stat3 knockout and IL6/Jak/STAT inhibitors. At larval stages, Stat3 reporter activity correlates with proliferating regions of the brain, haematopoietic tissue and intestine. In the adult gut, the reporter is active in sparse proliferating cells, located at the base of intestinal folds, expressing the stemness marker sox9b and having the morphology of mammalian crypt base columnar cells; noteworthy, zebrafish stat3 mutants show defects in intestinal folding. Stat3 reporter activity in the gut is abolished with mutation of T cell factor 4 (Tcf7l2), the intestinal mediator of Wnt/ß-catenin-dependent transcription. The Wnt/ß-catenin dependence of Stat3 activity in the gut is confirmed by abrupt expansion of Stat3-positive cells in intestinal adenomas of apc heterozygotes. Our findings indicate that Jak/Stat3 signalling is needed for intestinal stem cell maintenance and possibly crucial in controlling Wnt/ß-catenin-dependent colorectal cancer cell proliferation.


Assuntos
Mucosa Intestinal/metabolismo , Fator de Transcrição STAT3/metabolismo , Via de Sinalização Wnt , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados/crescimento & desenvolvimento , Animais Geneticamente Modificados/metabolismo , Intestinos/crescimento & desenvolvimento , Intestinos/fisiologia , Janus Quinase 1 , Larva/crescimento & desenvolvimento , Larva/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Fatores de Transcrição SOX9/metabolismo , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/deficiência , Fator de Transcrição STAT3/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/deficiência , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Transcrição Genética/efeitos dos fármacos , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética , beta Catenina/metabolismo
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