African Turquoise Killifish Research Articles

A short dasatinib and quercetin treatment is sufficient to reinstate potent adult neuroregenesis in the aged killifish

The young African turquoise killifish has a high regenerative capacity, but loses it with advancing age, adopting several aspects of the limited form of mammalian regeneration. We deployed a proteomic strategy to identify pathways that underpin the loss of regenerative power caused by aging. Cellular senescence stood out as a potential brake on successful neurorepair. We applied the senolytic cocktail Dasatinib and Quercetin (D + Q) to test clearance of chronic senescent cells from the aged killifish central nervous system (CNS) as well as rebooting the neurogenic output. Our results show that the entire aged killifish telencephalon holds a very high senescent cell burden, including the parenchyma and the neurogenic niches, which could be diminished by a short-term, late-onset D + Q treatment. Reactive proliferation of non-glial progenitors increased substantially and lead to restorative neurogenesis after traumatic brain injury. Our results provide a cellular mechanism for age-related regeneration resilience and a proof-of-concept of a potential therapy to revive the neurogenic potential in an already aged or diseased CNS.

Rapid and precise genome engineering in a naturally short-lived vertebrate.

The African turquoise killifish is a powerful vertebrate system to study complex phenotypes at scale, including aging and age-related disease. Here, we develop a rapid and precise CRISPR/Cas9-mediated knock-in approach in the killifish. We show its efficient application to precisely insert fluorescent reporters of different sizes at various genomic loci in order to drive cell-type- and tissue-specific expression. This knock-in method should allow the establishment of humanized disease models and the development of cell-type-specific molecular probes for studying complex vertebrate biology.

In Vitro Fertilization of the African Turquoise Killifish Nothobranchius furzeri.

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.

Chromatin Accessibility Profiling and Data Analysis Using ATAC-seq in Nothobranchius furzeri.

The state of genome-wide chromatin accessibility in cells, tissues, or organisms can be investigated with a technique called assay for transposase-accessible chromatin using sequencing (ATAC-seq). ATAC-seq is a powerful approach for profiling the epigenomic landscape of cells using very low input materials. Analysis of chromatin accessibility data allows for prediction of gene expression and identification of regulatory elements such as potential enhancers and specific transcription-factor binding sites. Here, we describe an optimized ATAC-seq protocol for the preparation of isolated nuclei and subsequent next-generation sequencing from whole embryos and tissues of the African turquoise killifish (Nothobranchius furzeri). Importantly, we provide an overview of a pipeline for processing and analyzing ATAC-seq data from killifish.

Visually Driven Behavior Assays to Study Functionality and Recovery after Damage of the Aged African Turquoise Killifish Visual System.

Loss of vision is a prominent feature of aging and vision is considered by many to be the most valuable sense to be lost. In our graying society, we are increasingly challenged by age-related deterioration of the central nervous system (CNS), as well as by age-associated neurodegenerative diseases and brain injuries, all often affecting the visual system and thus its performance. Here, we describe two visually driven behavior assays to evaluate visual performance upon aging or CNS damage in the fast-aging killifish. The first test, the optokinetic response (OKR), measures the reflexive eye movement triggered by motion in the visual field and allows assessment of visual acuity. The second assay, the dorsal light reflex (DLR), evaluates the swimming angle based on input of light coming from above. The OKR can be used to study the effect of aging on visual acuity as well as visual improvement and recovery after rejuvenation therapy or visual system injury or disease, whereas the DLR is best used to assess functional repair after a unilateral optic nerve crush.

Morphological Analysis of Aging Hallmarks in the Central Nervous System of the Fast-Aging African Turquoise Killifish.

As the number of elderly individuals is increasing in modern society, the need for a relevant gerontology model is higher than ever before. Aging can be defined by specific cellular hallmarks, described by López-Otín and colleagues, who provided a map which can be used to scavenge the aging tissue environment. As revealing the presence of individual hallmarks does not necessarily indicate aging, here we provide different (immuno)histochemical approaches that can be used to investigate several aging hallmarks-namely, genomic damage, mitochondrial dysfunction/oxidative stress, cellular senescence, stem cell exhaustion, and altered intercellular communication-in the killifish retina, optic tectum, and/or telencephalon at a morphological level. In combination with molecular and biochemical analysis of these aging hallmarks, this protocol offers the opportunity to fully characterize the aged killifish central nervous system.

The Optic Nerve Crush Injury Paradigm in African Turquoise Killifish to Study Axonal Regeneration in an Aged Environment.

In our graying world population, we are increasingly facing brain injuries and age-associated neurodegenerative diseases, which are often characterized by axonal pathology. Here, we propose the killifish visual/retinotectal system as a model for investigating central nervous system repair, more specifically axonal regeneration, in an aging context. We first describe an optic nerve crush (ONC) injury paradigm in killifish to induce and study both de- and regeneration of retinal ganglion cells (RGCs) and their axons. Subsequently, we summarize several methods for mapping different steps of the regenerative process-namely, axonal regrowth and synapse reformation-using retro- and anterograde tracing methods, (immuno)histochemistry, and morphometrical analyses.

Preparation of High-Viability Single-Cell Suspensions from African Turquoise Killifish Brain Tissue.

Studying the brain at the single-cell level has become increasingly popular in recent years. This, however, remains challenging, especially in emerging model organisms. To carry out single-cell sequencing, the preparation of a high-viability single-cell suspension is critical. In this protocol, we describe how to prepare a high-viability single-cell suspension starting from brain tissue of the African turquoise killifish (Nothobranchius furzeri). The protocol consists of dissection, enzymatic and mechanical dissociation of the brain tissue, and debris removal. The protocol described here has been successfully used for 10× Genomics single-cell sequencing of the telencephalon of adult killifish, which requires a cell viability of at least 70%. In addition to single-cell sequencing experiments, the single-cell suspension generated can be used for other applications, including cell culture and flow cytometry.

Molecular and Biochemical Analyses of Aging Hallmarks in the Central Nervous System of the Fast-Aging African Turquoise Killifish.

As modern society is graying, aging research and biogerontology models, in which the aging process can be studied, are becoming increasingly important. A proper aging model can be defined as one that displays many of the aging hallmarks. Here, we provide two different practical approaches-namely, real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting-that can be used to investigate cellular senescence (RT-qPCR for p21 and p27), altered intercellular communication/inflammaging (RT-qPCR for il-10, sirt-1, il-6, il-1b, il-8, and tnf), and oxidative stress (western blotting for 4-HNE) in the killifish central nervous system, and, more specifically, in the retina, optic tectum, and telencephalon. These molecular and biochemical analyses are a first step in confirming the aging characteristics but should preferably be combined with morphological analyses.

Life Span Assessment in the African Turquoise Killifish Nothobranchius furzeri.

The African turquoise killifish (Nothobranchius furzeri) is the shortest-lived vertebrate bred in captivity, with a median life span of 4-6 mo. Within its short life span, the killifish recapitulates critical aspects of human aging, including neurodegeneration and increased frailty. Developing standardized protocols for life span assessment in killifish is critical for identifying environmental and genetic factors that impact vertebrate life span. A standardized life span protocol should have low variability and high reproducibility, and it should enable comparison of life spans between laboratories. Here, we report our standardized protocol for measuring life span in the African turquoise killifish.

Husbandry of the African Turquoise Killifish Nothobranchius furzeri.

The African turquoise killifish (Nothobranchius furzeri) is an extremely short-lived vertebrate that has emerged as a powerful model organism for several research areas, including aging and embryonic diapause, which is the temporary suspension of embryonic development. The killifish research community is expanding and developing new solutions to improve the tractability of the killifish as a model system. Starting a killifish colony from scratch can present numerous challenges. In this protocol, we aim to highlight critical elements in building and maintaining a killifish colony. This protocol should help laboratories start a killifish colony and standardize aspects of killifish husbandry.

Breeding and Reproduction of the African Turquoise Killifish Nothobranchius furzeri.

The successful breeding and reproduction of the African turquoise killifish Nothobranchius furzeri in a controlled laboratory setting are required to establish this fish species as a model system for studying vertebrate development and aging. Here, we describe a protocol to care for and hatch African turquoise killifish embryos, raise the juvenile fish to adulthood, and breed this species using sand as the breeding bedding. We also provide suggestions for generating a large quantity of good-quality embryos.

Micro-CT Analysis of Fat in the Killifish Nothobranchius furzeri.

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.

Noninvasive Ejaculate Collection from the African Turquoise Killifish Nothobranchius furzeri.

Ejaculate collection from live animals is a fundamental technique used to perform repeated measures on the same individual-for instance, to test the effect of different treatments/conditions on the same animal or to conduct longitudinal studies. Moreover, collecting the ejaculate allows one to perform in vitro fertilizations (IVFs) or sperm cryopreservation from valuable individuals (e.g., mutant, transgenic animals) without the need to kill the animals. We developed a noninvasive protocol based on a stripping procedure for collecting the ejaculate from live males of the African turquoise killifish (Nothobranchius furzeri). The proposed procedure allows the retrieval of mature sperm along with the seminal fluid. The ejaculate can be used within 1 h of the collection to perform in vitro fertilizations and/or sperm quality measurements. Contamination by mucus, water, urine, or feces can activate the sperm and/or negatively affect sperm quality, so special care is needed to avoid contamination and thus prolong sperm viability. Since in small fish the amount of ejaculate collected is limited to a few microliters, in the present protocol we also provide recommendations on housing conditions to maximize the probability to obtain ejaculate samples.

Sequencing the Immunoglobulin Heavy-Chain Locus (IgH) in Turquoise Killifish (Nothobranchius furzeri).

B cells undergo a process of somatic recombination leading to the synthesis of a staggering diversity of transmembrane and secreted antibodies. This process dates back to the evolution of jawed vertebrates and is pivotal to the origin of lymphocyte-based adaptive immune responses. Here, we optimized a sequencing-based protocol to characterize B-cell-specific IgH transcripts from bulk RNA in the African turquoise killifish, an emerging model organism characterized by naturally short life span and by a vast range of age-dependent dysfunctions.

Dynamic regulation of gonadal transposon control across the lifespan of the naturally short-lived African turquoise killifish.

Although germline cells are considered to be functionally "immortal," both the germline and supporting somatic cells in the gonad within an organism experience aging. With increased age at parenthood, the age-related decline in reproductive success has become an important biological issue for an aging population. However, molecular mechanisms underlying reproductive aging across sexes in vertebrates remain poorly understood. To decipher molecular drivers of vertebrate gonadal aging across sexes, we perform longitudinal characterization of the gonadal transcriptome throughout the lifespan in the naturally short-lived African turquoise killifish (Nothobranchius furzeri). By combining mRNA-seq and small RNA-seq from 26 individuals, we characterize the aging gonads of young-adult, middle-aged, and old female and male fish. We analyze changes in transcriptional patterns of genes, transposable elements (TEs), and piRNAs. We find that testes seem to undergo only marginal changes during aging. In contrast, in middle-aged ovaries, the time point associated with peak female fertility in this strain, PIWI pathway components are transiently down-regulated, TE transcription is elevated, and piRNA levels generally decrease, suggesting that egg quality may already be declining at middle-age. Furthermore, we show that piRNA ping-pong biogenesis declines steadily with age in ovaries, whereas it is maintained in aging testes. To our knowledge, this data set represents the most comprehensive transcriptomic data set for vertebrate gonadal aging. This resource also highlights important pathways that are regulated during reproductive aging in either ovaries or testes, which could ultimately be leveraged to help restore aspects of youthful reproductive function.

The fast‐aging killifish: A unique vertebrate model to identify molecular targets for neuroreparative strategies in the aged retina

AbstractThe rise in life expectancy in our modern societies concurs with an increasing prevalence of age‐associated neurodegenerative disorders and central nervous system (CNS) trauma. Despite many research attempts trying to unravel aging processes and their role in functional decline, the mechanisms underlying healthy CNS aging remain elusive. Moreover, the reparative approaches proposed to combat loss of neurons due to damage or (age‐associated) disease are failing. One of the reasons might be that the currently used animal models often lack the capability to properly recapitulate the aged environment in which these pathologies manifest and the repair processes need to occur. We introduce the African turquoise killifish as an opportune model to study CNS aging as well as neuroreparative strategies. Indeed, while sharing adult neuroregenerative potential with other teleost fish like zebrafish, the short‐lived killifish displays many of the human aging hallmarks, making it a promising biogerontology model.As vision decline manifests during human aging and in many neurodegenerative diseases, we performed a comprehensive characterization of the aged killifish visual system, comprising the retina, optic projections and optic tectum. Immunohistochemical, biochemical and molecular analyses revealed the presence of many aging traits in this system, including oxidative stress, genomic damage, cellular senescence, altered cellular communication, reduced neurogenic potential and even neurodegeneration. Specifically, microglia changed to a pro‐inflammatory secretion profile upon aging and macroglia became gliotic in the aging killifish visual system. Altogether, these age‐related changes led to a diminished visual performance in old killifish.Using an optic nerve injury model, we further disclosed an age‐related decline in functional circuit restoration, with different phases of the repair process being affected depending on the age. While young adult killifish were able to functionally recover after injury, old fish did not regain vision at all. Here we observed for the first time in a regenerative teleost fish model, a glial scar that formed a mechanical barrier physically preventing axonal regrowth. Moreover, in the optic tectum the inflammatory response shifted from acute to chronic upon aging. Macroglia, which showed a transient gliotic response after injury in young adult fish, displayed a permanent response in old animals. Overall, as in mammals, both a reduced intrinsic growth potential and a non‐supportive cellular environment, orchestrated by activated micro‐ and macroglia, were found to underlie the declined repair potential in aged killifish.To decode the cellular and molecular framework that defines visual system aging and the failed regenerative response in old fish, we are currently performing single‐cell RNA sequencing of the young adult and aged killifish retina. We envision our bioinformatic approaches to result in innovative druggable targets for future development of rejuvenative/curative therapies in the aged mammalian retina.In summary, our study highlights the importance of a neuron–glia crosstalk in health, disease and repair. It puts forward the strengths of the killifish as a vertebrate model with mammalian‐like characteristics upon aging, that offers unique opportunities to identify rejuvenating and neuroreparative strategies ensuring healthy aging.

An automated feeding system for the African killifish reveals the impact of diet on lifespan and allows scalable assessment of associative learning.

The African turquoise killifish is an exciting new vertebrate model for aging studies. A significant challenge for any model organism is the control over its diet in space and time. To address this challenge, we created an automated and networked fish feeding system. Our automated feeder is designed to be open-source, easily transferable, and built from widely available components. Compared to manual feeding, our automated system is highly precise and flexible. As a proof of concept for the feeding flexibility of these automated feeders, we define a favorable regimen for growth and fertility for the African killifish and a dietary restriction regimen where both feeding time and quantity are reduced. We show that this dietary restriction regimen extends lifespan in males (but not in females) and impacts the transcriptomes of killifish livers in a sex-specific manner. Moreover, combining our automated feeding system with a video camera, we establish a quantitative associative learning assay to provide an integrative measure of cognitive performance for the killifish. The ability to precisely control food delivery in the killifish opens new areas to assess lifespan and cognitive behavior dynamics and to screen for dietary interventions and drugs in a scalable manner previously impossible with traditional vertebrate model organisms.

CRISPR-Cas9 Genome Editing in Nothobranchius furzeri for Gene Knockout and Knock-In.

The African turquoise killifish Nothobranchius furzeri has recently gained interest as an emerging vertebrate model system for the study of aging, owing to its naturally short life span and generation time. Here, we provide a step-by-step guide for effective genome engineering using the CRISPR-Cas9 system to generate loss-of-function (i.e., knockout) alleles and for precise editing (i.e., knock-in) of short sequences into the genome. Using this approach, a new stable line can be created within several months. The killifish's tough chorion, rapid growth, and short life span are considered in this protocol and account for the key deviations from similar protocols in other fish models.

Molecular Sexing of Nothobranchius furzeri Embryos and Larvae.

Differences between the sexes are of increasing interest in basic and applied research with regard to development, behavior, aging, and diseases. Although the African turquoise killifish Nothobranchius furzeri, a model for aging research well known for its remarkably short life span, develops strong sexual dimorphism in adulthood, there is no visible indicator of its sex in embryonic and juvenile stages. To address this issue, we developed a molecular sexing assay exploiting two large sequence polymorphisms in the minimal sex-determining region (SDR) of N. furzeri These polymorphisms are sequence deletions on the Y chromosome that involve the lack or truncation of one or multiple microsatellites. The simple polymerase chain reaction (PCR) readout of the assays described here allows the sexing of N. furzeri embryos and larvae in a medium- to high-throughput and cost-efficient manner.