Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 11 de 11
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Nat Aging ; 3(12): 1544-1560, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37957359

RESUMO

Late-life-initiated dietary interventions show limited efficacy in extending longevity or mitigating frailty, yet the underlying causes remain unclear. Here we studied the age-related fasting response of the short-lived killifish Nothobranchius furzeri. Transcriptomic analysis uncovered the existence of a fasting-like transcriptional program in the adipose tissue of old fish that overrides the feeding response, setting the tissue in persistent metabolic quiescence. The fasting-refeeding cycle triggers an inverse oscillatory expression of genes encoding the AMP-activated protein kinase (AMPK) regulatory subunits Prkag1 (γ1) and Prkag2 (γ2) in young individuals. Aging blunts such regulation, resulting in reduced Prkag1 expression. Transgenic fish with sustained AMPKγ1 countered the fasting-like transcriptional program, exhibiting a more youthful feeding and fasting response in older age, improved metabolic health and longevity. Accordingly, Prkag1 expression declines with age in human tissues and is associated with multimorbidity and multidimensional frailty risk. Thus, selective activation of AMPKγ1 prevents metabolic quiescence and preserves healthy aging in vertebrates, offering potential avenues for intervention.


Assuntos
Fragilidade , Longevidade , Animais , Humanos , Longevidade/genética , Proteínas Quinases Ativadas por AMP/genética , Envelhecimento/genética , Peixes/metabolismo
2.
Front Cell Dev Biol ; 11: 959611, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37020464

RESUMO

Introduction: Deciphering the biological and physical requirements for the outset of multicellularity is limited to few experimental models. The early embryonic development of annual killifish represents an almost unique opportunity to investigate de novo cellular aggregation in a vertebrate model. As an adaptation to seasonal drought, annual killifish employs a unique developmental pattern in which embryogenesis occurs only after undifferentiated embryonic cells have completed epiboly and dispersed in low density on the egg surface. Therefore, the first stage of embryogenesis requires the congregation of embryonic cells at one pole of the egg to form a single aggregate that later gives rise to the embryo proper. This unique process presents an opportunity to dissect the self-organizing principles involved in early organization of embryonic stem cells. Indeed, the physical and biological processes required to form the aggregate of embryonic cells are currently unknown. Methods: Here, we developed an in silico, agent-based biophysical model that allows testing how cell-specific and environmental properties could determine the aggregation dynamics of early Killifish embryogenesis. In a forward engineering approach, we then proceeded to test two hypotheses for cell aggregation (cell-autonomous and a simple taxis model) as a proof of concept of modeling feasibility. In a first approach (cell autonomous system), we considered how intrinsic biophysical properties of the cells such as motility, polarity, density, and the interplay between cell adhesion and contact inhibition of locomotion drive cell aggregation into self-organized clusters. Second, we included guidance of cell migration through a simple taxis mechanism to resemble the activity of an organizing center found in several developmental models. Results: Our numerical simulations showed that random migration combined with low cell-cell adhesion is sufficient to maintain cells in dispersion and that aggregation can indeed arise spontaneously under a limited set of conditions, but, without environmental guidance, the dynamics and resulting structures do not recapitulate in vivo observations. Discussion: Thus, an environmental guidance cue seems to be required for correct execution of early aggregation in early killifish development. However, the nature of this cue (e.g., chemical or mechanical) can only be determined experimentally. Our model provides a predictive tool that could be used to better characterize the process and, importantly, to design informed experimental strategies.

3.
Cold Spring Harb Protoc ; 2023(11): pdb.prot107886, 2023 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-37100471

RESUMO

The ability to perform in vitro fertilization, together with sperm cryopreservation, greatly facilitates the long-term laboratory maintenance of wild-type and transgenic model organisms and helps prevent genetic drift. It is also useful in cases where reproduction may be compromised. In this protocol, we present a method for in vitro fertilization of the African Turquoise killifish Nothobranchius furzeri that is compatible with the use of fresh or cryopreserved sperm.


Assuntos
Fundulidae , Animais , Masculino , Sêmen , Laboratórios , Fertilização in vitro , Envelhecimento
4.
Cold Spring Harb Protoc ; 2023(11): pdb.prot107885, 2023 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-36921999

RESUMO

Sperm cryopreservation is an essential method for the genetic preservation and long-term storage of wild-type and transgenic animal stocks. In addition, it allows for the synchronization of gamete availability and the transport and sharing of lines between different laboratories. Here, we describe a protocol developed in our laboratory for the extraction and cryopreservation of killifish (Nothobranchius furzeri) sperm.


Assuntos
Fundulidae , Masculino , Animais , Sêmen , Animais Geneticamente Modificados , Criopreservação , Envelhecimento
5.
Cold Spring Harb Protoc ; 2023(8): 107745, 2023 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-36828575

RESUMO

Blood withdrawal is a common procedure performed on laboratory animals to monitor key processes and indicators of fish health and physiology, such as hematopoiesis, hemostasis, and lipid and glucose metabolism. Moreover, the ability to extract blood with minimal invasiveness and without sacrificing animals enables repeated sampling, allowing both longitudinal studies of individual animals, as well as reducing the number of experimental animals needed in a study. The African turquoise killifish is an emerging animal model that is progressively being adopted worldwide for aging studies because of its naturally short life span. However, because of the small body size of this species, nonlethal blood collection is a challenging procedure. Here we present a detailed protocol enabling repeated blood sampling from the same individual fish. This method, if correctly executed, is minimally invasive and does not cause any lasting damage. The protocol has been tested on animals spanning from 6 to 24 wk of age and the amount of blood that could be extracted varied from 0.5 to 8 µL, greatly depending on specimen age, sex, and size. This volume is sufficient to perform analyses such as blood glucose measurement, blood cell counts, or histological stains on blood smears.


Assuntos
Ciprinodontiformes , Fundulidae , Animais , Fundulidae/fisiologia , Ciprinodontiformes/fisiologia , Envelhecimento , Longevidade
6.
Cold Spring Harb Protoc ; 2023(8): 107884, 2023 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-36828576

RESUMO

Aging is associated with an increase in body fat mass and a concomitant decrease in lean mass and bone density in mammals. Body adiposity can also be redistributed with age, resulting in abdominal fat accumulation and subcutaneous fat reduction. In addition, specific variation in fat distribution is considered to be a risk factor for a number of age-related metabolic disorders. Micro computed tomography (micro-CT) is a nondestructive high-resolution imaging method that uses planar X-ray images captured at various angles around a sample of interest to yield a three-dimensional array of radiodensity values, which can then be used to computationally extract the adipose volume in situ using its innate contrast properties. This method was successfully used to study adipose tissue dynamics in rodents and more recently in zebrafish. The naturally short-lived African turquoise killifish is an emerging model organism to study the biology of aging. Like mammals, killifish also have different fat deposits (visceral and subcutaneous), making them a suitable model to study age-related changes in fat mass and distribution. However, procedures allowing precise quantification of fat content and distribution are missing in this species. Here, we provide an optimized protocol to measure and quantify fat distribution in turquoise killifish by micro-CT scan analysis and show the applicability of the method in young and old animals of both sexes.


Assuntos
Fundulidae , Masculino , Animais , Feminino , Microtomografia por Raio-X/métodos , Peixe-Zebra , Tecido Adiposo/diagnóstico por imagem , Mamíferos
7.
Sci Rep ; 11(1): 17145, 2021 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-34433853

RESUMO

Over the last decade, the African turquoise killifish, Nothobranchius furzeri, has emerged as an important model system for the study of vertebrate biology and ageing. Propagation of laboratory inbred strains of Nothobranchius furzeri, such as GRZ, however, can pose challenges due to the short window of fertility, the efforts and space requirements involved in continuous strain maintenance, and the risks of further inbreeding. The current method for long term strain preservation relies on arrest of embryos in diapause. To create an alternative for long term maintenance, we developed a robust protocol to cryopreserve and revive sperm for in vitro fertilization (IVF). We tested a variety of extender and activator buffers for sperm IVF, as well as cryoprotectants to achieve practical long-term storage and fertilization conditions tailored to this species. Our protocol enabled sperm to be preserved in a cryogenic condition for months and to be revived with an average of 40% viability upon thawing. Thawed sperm were able to fertilize nearly the same number of eggs as natural fertilization, with an average of ~ 25% and peaks of ~ 55% fertilization. This technical advance will greatly facilitate the use of N. furzeri as a model organism.


Assuntos
Criopreservação/métodos , Fertilização in vitro/métodos , Fundulidae/fisiologia , Preservação do Sêmen/métodos , Animais , Feminino , Masculino
8.
Evodevo ; 10: 29, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31728179

RESUMO

BACKGROUND: Annual killifishes are adapted to surviving and reproducing over alternating dry and wet seasons. During the dry season, all adults die and desiccation-resistant embryos remain encased in dry mud for months or years in a state of diapause where their development is halted in anticipation of the months that have to elapse before their habitats are flooded again. Embryonic development of annual killifishes deviates from canonical teleost development. Epiblast cells disperse during epiboly, and a "dispersed phase" precedes gastrulation. In addition, annual fish have the ability to enter diapause and block embryonic development at the dispersed phase (diapause I), mid-somitogenesis (diapause II) and the final phase of development (diapause III). Developmental transitions associated with diapause entry and exit can be linked with cell cycle events. Here we set to image this transition in living embryos. RESULTS: To visibly explore cell cycle dynamics during killifish development in depth, we created a stable transgenic line in Nothobranchius furzeri that expresses two fluorescent reporters, one for the G1 phase and one for the S/G2 phases of the cell cycle, respectively (Fluorescent Ubiquitination-based Cell Cycle Indicator, FUCCI). Using this tool, we observed that, during epiboly, epiblast cells progressively become quiescent and exit the cell cycle. All embryos transit through a phase where dispersed cells migrate, without showing any mitotic activity, possibly blocked in the G1 phase (diapause I). Thereafter, exit from diapause I is synchronous and cells enter directly into the S phase without transiting through G1. The developmental trajectories of embryos entering diapause and of those that continue to develop are different. In particular, embryos entering diapause have reduced growth along the medio-lateral axis. Finally, exit from diapause II is synchronous for all cells and is characterized by a burst of mitotic activity and growth along the medio-lateral axis such that, by the end of this phase, the morphology of the embryos is identical to that of direct-developing embryos. CONCLUSIONS: Our study reveals surprising levels of coordination of cellular dynamics during diapause and provides a reference framework for further developmental analyses of this remarkable developmental quiescent state.

9.
BMC Biol ; 15(1): 9, 2017 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-28193224

RESUMO

BACKGROUND: A widespread modulation of gene expression occurs in the aging brain, but little is known as to the upstream drivers of these changes. MicroRNAs emerged as fine regulators of gene expression in many biological contexts and they are modulated by age. MicroRNAs may therefore be part of the upstream drivers of the global gene expression modulation correlated with aging and aging-related phenotypes. RESULTS: Here, we show that microRNA-29 (miR-29) is induced during aging in short-lived turquoise killifish brain and genetic antagonism of its function induces a gene-expression signature typical of aging. Mechanicistically, we identified Ireb2 (a master gene for intracellular iron delivery that encodes for IRP2 protein), as a novel miR-29 target. MiR-29 is induced by iron loading and, in turn, it reduces IRP2 expression in vivo, therefore limiting intracellular iron delivery in neurons. Genetically modified fish with neuro-specific miR-29 deficiency exhibit increased levels of IRP2 and transferrin receptor, increased iron content, and oxidative stress. CONCLUSIONS: Our results demonstrate that age-dependent miR-29 upregulation is an adaptive mechanism that counteracts the expression of some aging-related phenotypes and its anti-aging activity is primarily exerted by regulating intracellular iron homeostasis limiting excessive iron-exposure in neurons.


Assuntos
Envelhecimento/genética , Ferro/metabolismo , Peixes Listrados/crescimento & desenvolvimento , Peixes Listrados/genética , MicroRNAs/metabolismo , Neurônios/metabolismo , Animais , Sequência de Bases , Encéfalo/metabolismo , Homeostase/genética , Sobrecarga de Ferro/genética , Sobrecarga de Ferro/metabolismo , Proteína 2 Reguladora do Ferro/genética , Proteína 2 Reguladora do Ferro/metabolismo , MicroRNAs/genética , Modelos Biológicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulação para Cima/genética , Peixe-Zebra/genética
10.
Evodevo ; 5: 32, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25276337

RESUMO

BACKGROUND: Annual killifishes inhabit temporary ponds and their embryos survive the dry season encased in the mud by entering diapause, a process that arrests embryonic development during hostile conditions. Annual killifishes are present within three clades distributed in Africa (one East and one West of the Dahomey gap) and South America. Within each of these phylogenetic clades, a non-annual clade is sister taxon to a annual clade and therefore represent an example of convergent evolution. Early cleavage of teleost embryos is characterized by a very fast cell cycle (15-30 minutes) and lack of G1 and G2 phases. Here, we decided to investigate rates of early cleavage in annual killifishes. In addition, we specifically tested whether also annual killifish embryos lack G1 and G2 phases. RESULTS: We used time lapse brightfield microscopy to investigate cell division kinetics during the first developmental stages of annual- and non-annual species belonging to the three different phylogenetic clades. Annual killifishes of all three clades showed cleavage times significantly longer when compared to their non-annual sister taxa (average 35 min vs. average 75 min). Using FUCCI fluorescent imaging of the cell cycle after microinjection in the annual species Nothobranchius furzeri, we demonstrate that the first 5 division are synchronous and do not show a G1 phase. Cell cycle synchronization is lost after the 5th cleavage division. CONCLUSIONS: Our results show, for the first time, that cell cycle rate during cleavage, a trait thought to be rather evolutionary conserved can undergo convergent evolutionary change in response to variations in life-history.

11.
PLoS One ; 7(11): e50536, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23226307

RESUMO

tbx5, a member of the T-box gene family, encodes one of the key transcription factors mediating vertebrate heart development. Tbx5 function in heart development appears to be exquisitely sensitive to gene dosage, since both haploinsufficiency and gene duplication generate the cardiac abnormalities associated with Holt-Oram syndrome (HOS), a highly penetrant autosomal dominant disease characterized by congenital heart defects of varying severity and upper limb malformation. It is suggested that tight integration of microRNAs and transcription factors into the cardiac genetic circuitry provides a rich and robust array of regulatory interactions to control cardiac gene expression. Based on these considerations, we performed an in silico screening to identify microRNAs embedded in genes highly sensitive to Tbx5 dosage. Among the identified microRNAs, we focused our attention on miR-218-1 that, together with its host gene, slit2, is involved in heart development. We found correlated expression of tbx5 and miR-218 during cardiomyocyte differentiation of mouse P19CL6 cells. In zebrafish embryos, we show that both Tbx5 and miR-218 dysregulation have a severe impact on heart development, affecting early heart morphogenesis. Interestingly, down-regulation of miR-218 is able to rescue the heart defects generated by tbx5 over-expression supporting the notion that miR-218 is a crucial mediator of Tbx5 in heart development and suggesting its possible involvement in the onset of heart malformations.


Assuntos
Coração/crescimento & desenvolvimento , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas com Domínio T/genética , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/genética , Animais , Diferenciação Celular/genética , Linhagem Celular , Movimento Celular/genética , Regulação para Baixo/genética , Expressão Gênica , Humanos , Camundongos , Miócitos Cardíacos/citologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...