Multiple Model Organisms Research Articles

SLC16A8 is a causal contributor to age-related macular degeneration risk

Age-related macular degeneration (AMD), a complex neurodegenerative disease, is a leading cause of visual impairment worldwide with a strong genetic component. Genetic studies have identified several loci, but few causal genes with functional characterization. Here we highlight multiple lines of evidence which show a causal role in AMD for SLC16A8, which encodes MCT3, a retinal pigment epithelium (RPE) specific lactate transporter. First, in an unbiased, genome-wide analysis of rare coding variants we show multiple SLC16A8 rare variants are associated with AMD risk, corroborating previous borderline significant reports from AMD rare variant studies. Second, we report a novel SLC16A8 mutation in a three-generation family with early onset macular degeneration. Finally, mis-expression in multiple model organisms shows functional and anatomic retinal consequences. This study highlights the important role for SLC16A8 and lactate regulation towards outer retina/RPE health and highlights a potential new therapeutic opportunity for the treatment of AMD.

Identification of a novel MYO1D variant associated with laterality defects, congenital heart diseases, and sperm defects in humans.

The establishment of left-right asymmetry is a fundamental process in animal development. Interference with this process leads to a range of disorders collectively known as laterality defects, which manifest as abnormal arrangements of visceral organs. Among patients with laterality defects, congenital heart diseases (CHD) are prevalent. Through multiple model organisms, extant research has established that myosin-Id (MYO1D) deficiency causes laterality defects. This study investigated over a hundred cases and identified a novel biallelic variant of MYO1D (NM_015194: c.1531G>A; p.D511N) in a consanguineous family with complex CHD and laterality defects. Further examination of the proband revealed asthenoteratozoospermia and shortened sperm. Afterward, the effects of the D511N variant and another known MYO1D variant (NM_015194: c.2293C>T; p.P765S) were assessed. The assessment showed that both enhance the interaction with β-actin and SPAG6. Overall, this study revealed the genetic heterogeneity of this rare disease and found that MYO1D variants are correlated with laterality defects and CHD in humans. Furthermore, this research established a connection between sperm defects and MYO1D variants. It offers guidance for exploring infertility and reproductive health concerns. The findings provide a critical basis for advancing personalized medicine and genetic counseling.

The Function, Regulation, and Mechanism of Protein Turnover in Circadian Systems in Neurospora and Other Species.

Circadian clocks drive a large array of physiological and behavioral activities. At the molecular level, circadian clocks are composed of positive and negative elements that form core oscillators generating the basic circadian rhythms. Over the course of the circadian period, circadian negative proteins undergo progressive hyperphosphorylation and eventually degrade, and their stability is finely controlled by complex post-translational pathways, including protein modifications, genetic codon preference, protein-protein interactions, chaperon-dependent conformation maintenance, degradation, etc. The effects of phosphorylation on the stability of circadian clock proteins are crucial for precisely determining protein function and turnover, and it has been proposed that the phosphorylation of core circadian clock proteins is tightly correlated with the circadian period. Nonetheless, recent studies have challenged this view. In this review, we summarize the research progress regarding the function, regulation, and mechanism of protein stability in the circadian clock systems of multiple model organisms, with an emphasis on Neurospora crassa, in which circadian mechanisms have been extensively investigated. Elucidation of the highly complex and dynamic regulation of protein stability in circadian clock networks would greatly benefit the integrated understanding of the function, regulation, and mechanism of protein stability in a wide spectrum of other biological processes.

Competition and evolutionary selection among core regulatory motifs in gene expression control

Gene products that are beneficial in one environment may become burdensome in another, prompting the emergence of diverse regulatory schemes that carry their own bioenergetic cost. By ensuring that regulators are only expressed when needed, we demonstrate that autoregulation generally offers an advantage in an environment combining mutation and time-varying selection. Whether positive or negative feedback emerges as dominant depends primarily on the demand for the target gene product, typically to ensure that the detrimental impact of inevitable mutations is minimized. While self-repression of the regulator curbs the spread of these loss-of-function mutations, self-activation instead facilitates their propagation. By analyzing the transcription network of multiple model organisms, we reveal that reduced bioenergetic cost may contribute to the preferential selection of autoregulation among transcription factors. Our results not only uncover how seemingly equivalent regulatory motifs have fundamentally different impact on population structure, growth dynamics, and evolutionary outcomes, but they can also be leveraged to promote the design of evolutionarily robust synthetic gene circuits.

Insulin signaling in development.

Nutrient intake is obligatory for animal growth and development, but nutrients alone are not sufficient. Indeed, insulin and homologous hormones are required for normal growth even in the presence of nutrients. These hormones communicate nutrient status between organs, allowing animals to coordinate growth and metabolism with nutrient supply. Insulin and related hormones, such as insulin-like growth factors and insulin-like peptides, play important roles in development and metabolism, with defects in insulin production and signaling leading to hyperglycemia and diabetes. Here, we describe the insulin hormone family and the signal transduction pathways activated by these hormones. We highlight the roles of insulin signaling in coordinating maternal and fetal metabolism and growth during pregnancy, and we describe how secretion of insulin is regulated at different life stages. Additionally, we discuss the roles of insulin signaling in cell growth, stem cell proliferation and cell differentiation. We provide examples of the role of insulin in development across multiple model organisms: Caenorhabditis elegans, Drosophila, zebrafish, mouse and human.

Evolutionarily Conserved Role of Thioredoxin Systems in Determining Longevity.

Thioredoxin and thioredoxin reductase are evolutionarily conserved antioxidant enzymes that protect organisms from oxidative stress. These proteins also play roles in redox signaling and can act as a redox-independent cellular chaperone. In most organisms, there is a cytoplasmic and mitochondrial thioredoxin system. A number of studies have examined the role of thioredoxin and thioredoxin reductase in determining longevity. Disruption of either thioredoxin or thioredoxin reductase is sufficient to shorten lifespan in model organisms including yeast, worms, flies and mice, thereby indicating conservation across species. Similarly, increasing the expression of thioredoxin or thioredoxin reductase can extend longevity in multiple model organisms. In humans, there is an association between a specific genetic variant of thioredoxin reductase and lifespan. Overall, the cytoplasmic and mitochondrial thioredoxin systems are both important for longevity.

Bioelectricity in Developmental Patterning and Size Control: Evidence and Genetically Encoded Tools in the Zebrafish Model.

Developmental patterning is essential for regulating cellular events such as axial patterning, segmentation, tissue formation, and organ size determination during embryogenesis. Understanding the patterning mechanisms remains a central challenge and fundamental interest in developmental biology. Ion-channel-regulated bioelectric signals have emerged as a player of the patterning mechanism, which may interact with morphogens. Evidence from multiple model organisms reveals the roles of bioelectricity in embryonic development, regeneration, and cancers. The Zebrafish model is the second most used vertebrate model, next to the mouse model. The zebrafish model has great potential for elucidating the functions of bioelectricity due to many advantages such as external development, transparent early embryogenesis, and tractable genetics. Here, we review genetic evidence from zebrafish mutants with fin-size and pigment changes related to ion channels and bioelectricity. In addition, we review the cell membrane voltage reporting and chemogenetic tools that have already been used or have great potential to be implemented in zebrafish models. Finally, new perspectives and opportunities for bioelectricity research with zebrafish are discussed.

Use of Frogs as a Model to Study the Etiology of HLHS.

A frog is a classical model organism used to uncover processes and regulations of early vertebrate development, including heart development. Recently, we showed that a frog also represents a useful model to study a rare human congenital heart disease, hypoplastic left heart syndrome. In this review, we first summarized the cellular events and molecular regulations of vertebrate heart development, and the benefit of using a frog model to study congenital heart diseases. Next, we described the challenges in elucidating the etiology of hypoplastic left heart syndrome and discussed how a frog model may contribute to our understanding of the molecular and cellular bases of the disease. We concluded that a frog model offers its unique advantage in uncovering the cellular mechanisms of hypoplastic left heart syndrome; however, combining multiple model organisms, including frogs, is needed to gain a comprehensive understanding of the disease.

Characteristics and functions of DNA N(6)-methyladenine in embryonic chicken muscle development

DNA N(6)-methyladenine (DNA-6mA) is a new epigenetic mark in eukaryotes, the distribution and functions of which in genomic DNA remain unknown. Although recent studies have suggested that 6mA is present in multiple model organisms and is dynamically regulated during development, the genomic features of 6mA in avian species have yet to be elucidated. 6mA immunoprecipitation sequencing approach was used to analysis the distribution and function of 6mA in the muscle genomic DNA during embryonic chicken development. 6mA immunoprecipitation sequencing was combined with transcriptomic sequencing to reveal the role of 6mA in the regulation of gene expression and to explore possible pathways by which 6mA is involved in muscle development. We here provide evidence that 6mA modification exists widely throughout the chicken genome, and show preliminary data regarding genome-wide distribution of this epigenetic mark. Gene expression was shown to be inhibited by 6mA modification in promoter regions. In addition, the promoters of some genes related to development were modified by 6mA, indicating that 6mA may be involved in embryonic chicken development. Furthermore, 6mA may participate in muscle development and immune function by regulating HSPB8 and OASL expression. Our study improves our understanding of the distribution and function of 6mA modification in higher organisms and provide new information about differences between mammals and other vertebrates. These findings demonstrate an epigenetic role for 6mA in gene expression and potential involvement in chicken muscle development. Furthermore, the results suggest a potential epigenetic role for 6mA in avian embryonic development.

Prime editing in chicken fibroblasts and primordial germ cells.

CRISPR/Cas9-based genome editing technologies are revolutionizing developmental biology. One of the advanced CRISPR-based techniques is prime editing (PE), which enables precise gene modification in multiple model organisms. However, there has been no report of taking advantage of the PE system for gene editing in primordial germ cells (PGCs) thus far. In the current study, we describe a method to apply PE to the genome of chicken fibroblasts and PGCs. By combining PE with a transposon-mediated genomic integration, drug selection, and the single-cell culture method, we successfully generated prime-edited chicken fibroblasts and PGCs. The chicken PGC is widely used as an experimental model to study germ cell formation and as a vector for gene transfer to produce transgenic chickens. Such experimental models are useful in the developmental biology field and as potential bioreactors to produce pharmaceutical and nutritious proteins. Thus, the method presented here will provide not only a powerful tool to investigate gene function in germ cell development but also a basis for generating prime-edited transgenic birds.

TEDD: a database of temporal gene expression patterns during multiple developmental periods in human and model organisms.

Characterization of the specific expression and chromatin profiles of genes enables understanding how they contribute to tissue/organ development and the mechanisms leading to diseases. Whilst the number of single-cell sequencing studies is increasing dramatically; however, data mining and reanalysis remains challenging. Herein, we systematically curated the up-to-date and most comprehensive datasets of sequencing data originating from 2760 bulk samples and over 5.1 million single-cells from multiple developmental periods from humans and multiple model organisms. With unified and systematic analysis, we profiled the gene expression and chromatin accessibility among 481 cell-types, 79 tissue-types and 92 timepoints, and pinpointed cells with the co-expression of target genes. We also enabled the detection of gene(s) with a temporal and cell-type specific expression profile that is similar to or distinct from that of a target gene. Additionally, we illustrated the potential upstream and downstream gene-gene regulation interactions, particularly under the same biological process(es) or KEGG pathway(s). Thus, TEDD (Temporal Expression during Development Database), a value-added database with a user-friendly interface, not only enables researchers to identify cell-type/tissue-type specific and temporal gene expression and chromatin profiles but also facilitates the association of genes with undefined biological functions in development and diseases. The database URL is https://TEDD.obg.cuhk.edu.hk/.

A sensitive and specific genetically-encoded potassium ion biosensor for in vivo applications across the tree of life.

Potassium ion (K+) plays a critical role as an essential electrolyte in all biological systems. Genetically-encoded fluorescent K+ biosensors are promising tools to further improve our understanding of K+-dependent processes under normal and pathological conditions. Here, we report the crystal structure of a previously reported genetically-encoded fluorescent K+ biosensor, GINKO1, in the K+-bound state. Using structure-guided optimization and directed evolution, we have engineered an improved K+ biosensor, designated GINKO2, with higher sensitivity and specificity. We have demonstrated the utility of GINKO2 for in vivo detection and imaging of K+ dynamics in multiple model organisms, including bacteria, plants, and mice.

Simple to Complex: The Role of Actin and Microtubules in Mitochondrial Dynamics in Amoeba, Yeast, and Mammalian Cells.

Mitochondria are complex organelles that provide energy for the cell in the form of adenosine triphosphate (ATP) and have very specific structures. For most organisms, this is a reticular or tubular mitochondrial network, while others have singular oval-shaped organelles. Nonetheless, maintenance of this structure is dependent on the mitochondrial dynamics, fission, fusion, and motility. Recently, studies have shown that the cytoskeleton has a significant role in the regulation of mitochondrial dynamics. In this review, we focus on microtubules and actin filaments and look at what is currently known about the cytoskeleton’s role in mitochondrial dynamics in complex models like mammals and yeast, as well as what is known in the simple model system, Dictyostelium discoideum. Understanding how the cytoskeleton is involved in mitochondrial dynamics increases our understanding of mitochondrial disease, especially neurodegenerative diseases. Increases in fission, loss of fusion, and fragmented mitochondria are seen in several neurodegenerative diseases such as Parkinson’s, Alzheimer’s, and Huntington’s disease. There is no known cure for these diseases, but new therapeutic strategies using drugs to alter mitochondrial fusion and fission activity are being considered. The future of these therapeutic studies is dependent on an in-depth understanding of the mechanisms of mitochondrial dynamics. Understanding the cytoskeleton’s role in dynamics in multiple model organisms will further our understanding of these mechanisms and could potentially uncover new therapeutic targets for these neurodegenerative diseases.

Wear and tear of the intestinal visceral musculature by intrinsic and extrinsic factors.

The gut visceral musculature plays essential roles in not only moving substances through the lumen but also maintaining the function and physiology of the gut. Although the development of the visceral musculature has been studied in multiple model organisms, how it degenerates is poorly understood. Here, we employ the Drosophila midgut as a model to demonstrate that the visceral musculature is disrupted by intrinsic and extrinsic factors, such as aging, feeding, chemical-induced tissue damage, and oncogenic transformation in the epithelium. Notably, we define four prominent visceral musculature disruption phenotypes, which we refer as "sprout," "discontinuity," "furcation," and "crossover" of the longitudinal muscle. Given that the occurrence of these phenotypes is increased during aging and under various stresses, we propose that these phenotypes can be used as quantitative readouts of deterioration of the visceral musculature. Intriguingly, administration of a tissue-damaging chemical dextran sulfate sodium (DSS) induced similar visceral musculature disruption phenotypes in zebrafish larvae, indicating that ingestion of a tissue-damaging chemical can disrupt the visceral musculature in a vertebrate as well. Our study provides insights into the deterioration of the gut visceral musculature and lays a groundwork for investigating the underlying mechanisms in Drosophila as well as other animals.

Pan-cancer analyses of synonymous mutations based on tissue-specific codon optimality

Codon optimality has been demonstrated to be an important determinant of mRNA stability and expression levels in multiple model organisms and human cell lines. However, tissue-specific codon optimality has not been developed to investigate how codon optimality is usually perturbed by somatic synonymous mutations in human cancers. Here, we determined tissue-specific codon optimality in 29 human tissues based on mRNA expression data from the Genotype-Tissue Expression project. We found that optimal codons were associated with differentiation, whereas non-optimal codons were correlated with proliferation. Furthermore, codons biased toward differentiation displayed greater tissue specificity in codon optimality, and the tissue specificity of codon optimality was primarily present in amino acids with high degeneracy of the genetic code. By applying tissue-specific codon optimality to somatic synonymous mutations in 8532 tumor samples across 24 cancer types and to those in 416 normal cells across six human tissues, we found that synonymous mutations frequently increased optimal codons in tumor cells and cancer-related genes (e.g., genes involved in cell cycle). Furthermore, an elevated frequency of optimal codon gain was found to promote tumor cell proliferation in three cancer types characterized by DNA damage repair deficiency and could act as a prognostic biomarker for patients with triple-negative breast cancer. In summary, this study profiled tissue-specific codon optimality in human tissues, revealed alterations in codon optimality caused by synonymous mutations in human cancers, and highlighted the non-negligible role of optimal codon gain in tumorigenesis and therapeutics.

Dogs lacking Apolipoprotein E show advanced atherosclerosis leading to apparent clinical complications.

Atherosclerotic cardiovascular disease resulting from dysregulated lipid metabolism is the leading cause of morbidity and mortality worldwide. Apolipoprotein E (ApoE) plays a critical role in cholesterol metabolism. Knockouts in lipid-metabolizing proteins including ApoE in multiple model organisms such as mice and rats exhibiting elevated levels of cholesterol have been widely used for dissecting the pathology of atherosclerosis, but few of these animal models exhibit advanced atherosclerotic plaques leading to ischemia-induced clinical symptoms, limiting their use for translational studies. Here we report hypercholesterolemia and severe atherosclerosis characterized by stenosis and occlusion of arteries, together with clinical manifestations of stroke and gangrene, in ApoE knockout dogs generated by CRISPR/Cas9 and cloned by somatic cell nuclear transfer technologies. Importantly, the hypercholesterolemia and atherosclerotic complications in F0 mutants are recapitulated in their offspring. As the ApoE-associated atherosclerosis and clinical manifestations in mutant dogs are more similar to that in human patients compared with those in other animal models, these mutant dogs will be invaluable in developing and evaluating new therapies, including endovascular procedures, against atherosclerosis and related disorders.

Folding Mitochondrial-Mediated Cytosolic Proteostasis Into the Mitochondrial Unfolded Protein Response.

Several studies reported that mitochondrial stress induces cytosolic proteostasis. How mitochondrial stress activates proteostasis in the cytosol remains unclear. However, the cross-talk between the mitochondria and cytosolic proteostasis has far reaching implications for treatment of proteopathies including neurodegenerative diseases. This possibility appears within reach since selected drugs have begun to emerge as being able to stimulate mitochondrial-mediated cytosolic proteostasis. In this review, we focus on studies describing how mitochondrial stress activates proteostasis in the cytosol across multiple model organisms. A model is proposed linking mitochondrial-mediated regulation of cytosolic translation, folding capacity, ubiquitination, and proteasome degradation and autophagy as a multi layered control of cytosolic proteostasis that overlaps with the integrated stress response (ISR) and the mitochondrial unfolded protein response (UPRmt). By analogy to the conductor in an orchestra managing multiple instrumental sections into a dynamically integrated musical piece, the cross-talk between these signaling cascades places the mitochondria as a major conductor of cellular integrity.

An economical and highly adaptable optogenetics system for individual and population-level manipulation of Caenorhabditis elegans

BackgroundOptogenetics allows the experimental manipulation of excitable cells by a light stimulus without the need for technically challenging and invasive procedures. The high degree of spatial, temporal, and intensity control that can be achieved with a light stimulus, combined with cell type-specific expression of light-sensitive ion channels, enables highly specific and precise stimulation of excitable cells. Optogenetic tools have therefore revolutionized the study of neuronal circuits in a number of models, including Caenorhabditis elegans. Despite the existence of several optogenetic systems that allow spatial and temporal photoactivation of light-sensitive actuators in C. elegans, their high costs and low flexibility have limited wide access to optogenetics. Here, we developed an inexpensive, easy-to-build, modular, and adjustable optogenetics device for use on different microscopes and worm trackers, which we called the OptoArm.ResultsThe OptoArm allows for single- and multiple-worm illumination and is adaptable in terms of light intensity, lighting profiles, and light color. We demonstrate OptoArm’s power in a population-based multi-parameter study on the contributions of motor circuit cells to age-related motility decline. We found that individual components of the neuromuscular system display different rates of age-dependent deterioration. The functional decline of cholinergic neurons mirrors motor decline, while GABAergic neurons and muscle cells are relatively age-resilient, suggesting that rate-limiting cells exist and determine neuronal circuit ageing.ConclusionWe have assembled an economical, reliable, and highly adaptable optogenetics system which can be deployed to address diverse biological questions. We provide a detailed description of the construction as well as technical and biological validation of our set-up. Importantly, use of the OptoArm is not limited to C. elegans and may benefit studies in multiple model organisms, making optogenetics more accessible to the broader research community.

Hsp90 and its mitochondrial homologue TRAP-1 independently regulate hypoxia adaptations in Caenorhabditis elegans

Mitochondrial adaptations to various environmental cues contribute to cellular and organismal adaptations across multiple model organisms. Due to increased complexity, a direct connection between mitochondrial integrity and oxygen fluctuations, and survival fitness was not demonstrated. Here, using C. elegans as a model system, we studied the role of HIF-1, Hsp90, and TRAP-1 in mitochondrial adaptations during chemical hypoxia. We show that Hsp90mt (Hsp90 mutant) but not HIF-1mt (HIF-1 mutant) affects hypoxia adaptation in nematodes. TRAP-1KD (TRAP-1 knockdown) interfered with the survival and fecundity of worms. Compared to Hsp90mt, TRAP-1KD has induced a significant decrease in mitochondrial integrity and oxygen consumption rate. The complex I inhibitor rotenone did not affect ATP levels in Hsp90mt worms. However, ATP levels were decreased in TRAP-1KD worms under similar conditions. The glucose restriction has reduced, and glucose supplementation has increased the survival rate in Hsp90mt worms. Neither glucose restriction nor glucose supplementation has significantly affected the survival of TRAP-1KD worms in response to hypoxia. However, TRAP-1 inhibition using a nanocarrier drug has dramatically reduced the survival rate in response to hypoxia. Our results suggest that Hsp90 and TRAP-1 independently regulate hypoxia adaptations and metabolic plasticity in C. elegans. Considering the emerging roles of TRAP-1 in altered energy metabolism and cellular adaptations, our findings gain importance.

Genetic signature of human longevity in PKC and NF-κB signaling.

Gene variants associated with longevity are also associated with protection against cognitive decline, dementia and Alzheimer's disease, suggesting that common physiologic pathways act at the interface of longevity and cognitive function. To test the hypothesis that variants in genes implicated in cognitive function may promote exceptional longevity, we performed a comprehensive 3‐stage study to identify functional longevity‐associated variants in ~700 candidate genes in up to 450 centenarians and 500 controls by target capture sequencing analysis. We found an enrichment of longevity‐associated genes in the nPKC and NF‐κB signaling pathways by gene‐based association analyses. Functional analysis of the top three gene variants (NFKBIA, CLU, PRKCH) suggests that non‐coding variants modulate the expression of cognate genes, thereby reducing signaling through the nPKC and NF‐κB. This matches genetic studies in multiple model organisms, suggesting that the evolutionary conservation of reduced PKC and NF‐κB signaling pathways in exceptional longevity may include humans.