Conservation Of Gene Function Research Articles

Zebrafish: A Versatile Animal Model in Biomedical Research

Zebrafish (Danio rerio) has emerged as a powerful animal model in biomedical research, offering a unique combination of genetic tractability, rapid development, and conserved gene function with humans. This review highlights the significance of zebrafish in understanding various human diseases, including neurodegenerative disorders, metabolic disorders, cancer, and more. The zebrafish genome, with over 70% similarity to the human genome, allows for the modelling of human diseases with high fidelity. The ease of genetic manipulation, high fecundity, and low maintenance costs make zebrafish an attractive model for large-scale genetic screens and drug discovery. Zebrafish models of neurodegenerative diseases, such as Parkinson's disease, Alzheimer's disease, and Huntington's disease, have been developed, providing insights into disease mechanisms and potential therapeutic targets. Similarly, zebrafish models of metabolic disorders, including diabetes, dyslipidaemia, and atherosclerosis, have been established, enabling the study of disease pathogenesis and prevention strategies.The zebrafish has also been used to model various types of cancer, including leukaemia, melanoma, and pancreatic cancer, facilitating the discovery of novel oncogenes and tumor suppressors. The transparent nature of zebrafish embryos and larvae allows for in vivo imaging of cancer cells, enabling the study of cancer cell behavior and response to therapy. Furthermore, zebrafish has been used to investigate brain-to-organ communication, cell transplantation, and cell-cell interactions, providing insights into the complex interactions between different tissues and organs. The development of novel tools, such as CRISPR/Cas9 genome editing and single-cell RNA sequencing, has further expanded the capabilities of zebrafish research.

Adaptive Evolution of the OAS Gene Family Provides New Insights into the Antiviral Ability of Laurasiatherian Mammals.

Many mammals risk damage from virus invasion due to frequent environmental changes. The oligoadenylate synthesis (OAS) gene family, which is an important component of the immune system, provides an essential response to the antiviral activities of interferons by regulating immune signal pathways. However, little is known about the evolutionary characteristics of OASs in Laurasiatherian mammals. Here, we examined the evolution of the OAS genes in 64 mammals to explore the accompanying molecular mechanisms of the antiviral ability of Laurasiatherian mammals living in different environments. We found that OAS2 and OAS3 were found to be pseudogenes in Odontoceti species. This may be related to the fact that they live in water. Some Antilopinae, Caprinae, and Cervidae species lacked the OASL gene, which may be related to their habitats being at higher altitudes. The OASs had a high number of positive selection sites in Cetartiodactyla, which drove the expression of strong antiviral ability. The OAS gene family evolved in Laurasiatherian mammals at different rates and was highly correlated with the species' antiviral ability. The gene evolution rate in Cetartiodactyla was significantly higher than that in the other orders. Compared to other species of the Carnivora family, the higher selection pressure on the OAS gene and the absence of positive selection sites in Canidae may be responsible for its weak resistance to rabies virus. The OAS gene family was relatively conserved during evolution. Conserved genes are able to provide better maintenance of gene function. The rate of gene evolution and the number of positively selected sites combine to influence the resistance of a species to viruses. The positive selection sites demonstrate the adaptive evolution of the OAS gene family to the environment. Adaptive evolution combined with conserved gene function improves resistance to viruses. Our findings offer insights into the molecular and functional evolution of the antiviral ability of Laurasian mammals.

Ancient origin and conserved gene function in terpene pheromone and defense evolution of stink bugs and hemipteran insects

Insects use diverse arrays of small molecules such as metabolites of the large class of terpenes for intra- and inter-specific communication and defense. These molecules are synthesized by specialized metabolic pathways; however, the origin of enzymes involved in terpene biosynthesis and their evolution in insect genomes is still poorly understood. We addressed this question by investigating the evolution of isoprenyl diphosphate synthase (IDS)-like genes with terpene synthase (TPS) function in the family of stink bugs (Pentatomidae) within the large order of piercing–sucking Hemipteran insects. Stink bugs include species of global pest status, many of which emit structurally related 15-carbon sesquiterpenes as sex or aggregation pheromones. We provide evidence for the emergence of IDS-type TPS enzymes at the onset of pentatomid evolution over 100 million years ago, coinciding with the evolution of flowering plants. Stink bugs of different geographical origin maintain small IDS-type families with genes of conserved TPS function, which stands in contrast to the diversification of TPS genes in plants. Expanded gene mining and phylogenetic analysis in other hemipteran insects further provides evidence for an ancient emergence of IDS-like genes under presumed selection for terpene-mediated chemical interactions, and this process occurred independently from a similar evolution of IDS-type TPS genes in beetles. Our findings further suggest differences in TPS diversification in insects and plants in conjunction with different modes of gene functionalization in chemical interactions.

Glial regenerative response in the imaginal discs of Drosophila melanogaster.

Glial regenerative response in the imaginal discs of Drosophila melanogaster.

Ancient Origin and Conserved Gene Function in Terpene Pheromone and Defense Evolution of Stink Bugs and Hemipteran Insects

Ancient Origin and Conserved Gene Function in Terpene Pheromone and Defense Evolution of Stink Bugs and Hemipteran Insects

The use of CRISPR to generate a whole-gene humanized MAPT and the examination of P301L and G272V clinical variants, along with the creation of deletion null alleles of ptl-1, pgrn-1 and alfa-1 loci.

To study important genes involved in Frontotemporal Dementia ( MAPT , GRN and C9orf72 ), we created deletion alleles in the three orthologous genes ( ptl-1 , pgrn-1 , and alfa-1 ). Simultaneously, we replaced the C. elegans ptl-1 gene with the predicted orthologous human MAPT gene, often called whole-gene humanization, which allows direct assessment of conserved gene function, as well as the opportunity to examine consequences of clinical disease-associated patient variations. Each gene was manipulated using a different selection strategy, including a novel strategy using an unc-18 mutation rescue technique. Clinical MAPT ALS/FTD missense variants G272V and P301L were successfully inserted in hMAPT . Neither ptl-1 loss or clinical variants caused neuronal defects in young adult or aged C. elegans , based on examination of glutamatergic phasmid neurons. Yet, we noted decreased survival to day 9 in the P301L hMAPT strain, compared to control strains. Based on these results, we comment on strategies for humanization, including the importance of confirming C. elegans gene predictions and identifying loss of function defects for each gene before embarking on humanization, and we report the creation of strains and a new gene-editing selection strategy that will be useful for future studies.

The Use of TnSeq to Identify Essential Alphaproteobacterial Genes Reveals Operational Variability in Conserved Developmental and Cell Cycle Systems.

A powerful method for examining genetic fitness and function on a large scale is to couple saturating transposon mutagenesis with high-throughput sequencing (TnSeq). By mapping where transposon insertions can be tolerated in a genome, it is possible to analyze the fitness of every gene in a genome simultaneously under a given growth condition. While this technique can describe genes as essential or nonessential under those growth conditions, sufficient mutagenesis and sequencing depth can provide more subtle differences in fitness. In this paper, TnSeq was used to analyze gene fitness of two Alphaproteobacteria from different environments: the freshwater oligotroph Brevundimonas subvibrioides (Caulobacterales) and the soil plant pathogen Agrobacterium tumefaciens (Rhizobiales) for the purpose of comparing conservation of gene function.

A Bioinformatics Pipeline for Investigating Molecular Evolution and Gene Expression using RNA-seq.

Distilling and reporting large datasets, such as whole genome or transcriptome data, is often a daunting task. One way to break down results is to focus on one or more gene families that are significant to the organism and study. In this protocol, we outline bioinformatic steps to generate a phylogeny and to quantify the expression of genes of interest. Phylogenetic trees can give insight into how genes are evolving within and between species as well as reveal orthology. These results can be enhanced using RNA-seq data to compare the expression of these genes in different individuals or tissues. Studies of molecular evolution and expression can reveal modes of evolution and conservation of gene function between species. The characterization of a gene family can serve as a springboard for future studies and can highlight an important gene family in a new genome or transcriptome paper.

Simple, efficient and open-source CRISPR/Cas9 strategy for multi-site genome editing in Populus tremula × alba.

Although the CRISPR/Cas9 system has been successfully used for crop breeding, its application remains limited in forest trees. Here, we describe an efficient gene editing strategy for hybrid poplar, (Populus tremula × alba INRA clone 717-1B4) based on the Golden Gate MoClo cloning. To test the system efficiency for generating single gene mutants, two single guide RNAs (sgRNAs) were designed and incorporated into the MoClo Tool Kit level 2 binary vector with the Cas9 expression cassette to mutate the SHORT ROOT (SHR) gene. Moreover, we also tested its efficiency for introducing mutations in two genes simultaneously by expressing one sgRNA targeting a single site of the YUC4 gene and the other sgRNA targeting the PLT1 gene. For a robust evaluation of the approach, we repeated the strategy to target the LBD12 and LBD4 genes simultaneously, using an independent construct. We generated hairy roots by Agrobacterium rhizogenes-mediated leaf transformation. Sequencing results confirmed the CRISPR/Cas9-mediated mutation in the targeted sites of PtaSHR. Biallelic and homozygous knockout mutations were detected. A deletion spanning both target sites and small insertions/deletions were the most common mutations. Out of the 22 SHR alleles sequenced, 21 were mutated. The phenotype’s characterization showed that transgenic roots with biallelic mutations for the SHR gene lacked a defined endodermal single cell layer, suggesting a conserved gene function similar to its homolog in Arabidopsis Arabidopsis thaliana (L.) Heynh. Sequencing results also revealed the high efficiency of the system for generating double mutants. Biallelic mutations for both genes in the yuc4/plt1 and lbd12/lbd4 roots were detected in three (yuc4/plt1) and two (lbd12/lbd4) out of four transgenic roots evaluated. A small deletion or a single nucleotide insertion at the single target site was the most common mutations. This CRISPR/Cas9 strategy arises as a rapid, simple and standardized gene-editing tool to evaluate the gene role in essential developmental programs such as radial cell differentiation of poplar roots.

Conserved pleiotropy of an ancient plant homeobox gene uncovered by cis-regulatory dissection

Divergence of gene function is a hallmark of evolution, but assessing functional divergence over deep time is not trivial. The few alleles available for cross-species studies often fail to expose the entire functional spectrum of genes, potentially obscuring deeply conserved pleiotropic roles. Here, we explore the functional divergence of WUSCHEL HOMEOBOX9 (WOX9), suggested to have species-specific roles in embryo and inflorescence development. Using a cis-regulatory editing drive system, we generate a comprehensive allelic series in tomato, which revealed hidden pleiotropic roles for WOX9. Analysis of accessible chromatin and conserved cis-regulatory sequences identifies the regions responsible for this pleiotropic activity, the functions of which are conserved in groundcherry, a tomato relative. Mimicking these alleles in Arabidopsis, distantly related to tomato and groundcherry, reveals new inflorescence phenotypes, exposing a deeply conserved pleiotropy. We suggest that targeted cis-regulatory mutations can uncover conserved gene functions and reduce undesirable effects in crop improvement.

Functional characterization of retinoid X receptor with an emphasis on the mediation of organotin poisoning in the Pacific oyster (Crassostrea gigas).

Marine mollusks suffer harmful effects due to environmental organotin compounds such as tributyltin (TBT) and triphenyltin (TPT). It is known that gastropod imposex caused by organotins is mediated by a key nuclear receptor, retinoid X receptor (RXR). The organotin-mediated toxic effects on oysters grown in seawater include a thicker shell, incomplete growth, disrupted development and a high rate of mortality. However, few studies have been conducted to determine the role of RXR in the toxic effects of organotins on bivalves. Here, we cloned an RXR homolog (CgRXR) from the Pacific oyster (Crassostrea gigas) and characterized its molecular function. Expression of the CgRXR RNA transcripts was assessed in whole developmental stages and tissues, with the highest expression detected in the blastula and mantle, respectively. The subcellular localization experiment confirmed that CgRXR protein was expressed in the nucleus exclusively as a nuclear receptor. Electrophoretic mobility shift assay indicated that CgRXR could bind to the DNA motifs DR0-DR5. The dual-luciferase reporter assay demonstrated that the transcriptional activity of CgRXR was activated by conserved ligands (9-cis retinoic acid and cis-4,7,10,13,16,19-docosahexanoic acid) and endocrine-disrupting chemicals (TBT and TPT). These results revealed the conserved gene function involved in protein localization, ligand binding and heterodimer formation with thyroid hormone receptor. However, the DNA binding properties of CgRXR differed from those of other invertebrate and vertebrate RXRs. CgRXR had the highest expression level in the blastula and mantle, and the disrupted development or shell malformation induced by organotins suggested a possible correlation of CgRXR with shell formation in bivalves. The results indicated the potential involvement of CgRXR in the toxic effects of organotins (TBT and TPT) through signaling pathway disruption. Functional characterization of CgRXR will help us better understand the endocrinology of bivalves.

Comparative Studies on Gene Expression of Rice and Wheat in Response to Fungal Infection

Cereals are the foremost necessary foods for growing population of human. Wheat (Triticum aestivum) is the first important and strategic cereal crop for the majority of world’s populations. It is the most necessary staple food. Although rice (oryzae sativa) has the second place because of planted area but it serves as the most important food source for Asian countries. Wheat and rice production may be severely restricted by biotic and abiotic constraints. Disease is the major biotic stress in several regions.Comparative sequence analysis is a powerful tool to study homologous gene families, define conserved gene functions between orthologs, and identify lineage- and species-specific genes. Most annotations of newly sequenced genomes are based on similarity with sequences for which functional information is available. Apart from conserved sequences, inter-species differences provide important clues about evolutionary history and species-specific adaptations. In our study, two RNA-sequencing data sets of resistant variety of wheat (Triticum aestivum L.,) after infection with leaf rust fungus, Puccinia triticina and resistant variety of rice (Oryza sativa L.,) after infection with blast fungus, Magnaporthe oryzae were compared. 31768 up-regulated genes in wheat and 3902 up-regulated genes in rice were filtered according to fold change more than 3 and removing variants, 250 upregulated genes of wheat and rice were aligned and phylogenetic tree was generated. The result of phylogenetic tree showed close relationship between ten aligned gene pairs of wheat and rice. Two pairs of aligned gene pairs were selected randomly, super family of these pairs were obtained, the result showed that each aligned pair of proteins shared the same protein family and the same annotation and all pairs participate in plant defense pathways. Then, the gene expression of the two pairs were validated by Real-time PCR after infecting wheat with Puccinia triticina and rice with Magnaporthe grisea. Each aligned pair of the two pairs shared the same manner of expression with few exceptions in rice.

Expression of new genes in vertebrate tooth development and p63 signaling.

p63 is an evolutionarily ancient transcription factor essential to vertebrate tooth development. Our recent gene expression screen comparing wild-type and "toothless" p63-/- mouse embryos implicated in tooth development several new genes that we hypothesized act downstream of p63 in dental epithelium, where p63 is also expressed. Via in situ hybridization and immunohistochemistry, we probed mouse embryos (embryonic days 10.5-14.5) and spotted gar fish embryos (14 days postfertilization) for these newly linked genes, Cbln1, Cldn23, Fermt1, Krt15, Pltp and Prss8, which were expressed in mouse and gar dental epithelium. Loss of p63 altered expression levels but not domains. Expression was comparable between murine upper and lower tooth organs, implying conserved gene functions in maxillary and mandibular dentitions. Our meta-analysis of gene expression databases supported that these genes act within a p63-driven gene regulatory network important to tooth development in mammals and more evolutionary ancient vertebrates (fish, amphibians). Cbln1, Cldn23, Fermt1, Krt15, Pltp, and Prss8 were expressed in mouse and fish dental epithelium at placode, bud, and/or cap stages. We theorize that these genes participate in cell-cell adhesion, cell polarity, and extracellular matrix signaling to support dental epithelium integrity, folding, and epithelial-mesenchymal cross talk during tooth development.

CRISPR-Cas9 human gene replacement and phenomic characterization in Caenorhabditis elegans to understand the functional conservation of human genes and decipher variants of uncertain significance.

ABSTRACTOur ability to sequence genomes has vastly surpassed our ability to interpret the genetic variation we discover. This presents a major challenge in the clinical setting, where the recent application of whole-exome and whole-genome sequencing has uncovered thousands of genetic variants of uncertain significance. Here, we present a strategy for targeted human gene replacement and phenomic characterization, based on CRISPR-Cas9 genome engineering in the genetic model organism Caenorhabditis elegans, that will facilitate assessment of the functional conservation of human genes and structure-function analysis of disease-associated variants with unprecedented precision. We validate our strategy by demonstrating that direct single-copy replacement of the C. elegans ortholog (daf-18) with the critical human disease-associated gene phosphatase and tensin homolog (PTEN) is sufficient to rescue multiple phenotypic abnormalities caused by complete deletion of daf-18, including complex chemosensory and mechanosensory impairments. In addition, we used our strategy to generate animals harboring a single copy of the known pathogenic lipid phosphatase inactive PTEN variant (PTEN-G129E), and showed that our automated in vivo phenotypic assays could accurately and efficiently classify this missense variant as loss of function. The integrated nature of the human transgenes allows for analysis of both homozygous and heterozygous variants and greatly facilitates high-throughput precision medicine drug screens. By combining genome engineering with rapid and automated phenotypic characterization, our strategy streamlines the identification of novel conserved gene functions in complex sensory and learning phenotypes that can be used as in vivo functional assays to decipher variants of uncertain significance.

Advancing epilepsy treatment through personalized genetic zebrafish models.

With an increase in the number of disease causing genetic mutations identified from epilepsy cohorts, zebrafish are proving to be an attractive vertebrate model for functional analysis of these allele variants. Not only do zebrafish have conserved gene functions, but larvae harboring mutations in identified human epileptic genes show spontaneous seizure activity and mimic the convulsive behavioral movements observed in humans. With zebrafish being compatible with medium to high-throughput screening, they are also proving to be a unique and powerful system for early preclinical drug screening, including novel target identification, pharmacology, and toxicology. Additionally, with recent advances in genomic engineering technologies, it is now possible to study the precise pathophysiology of patient-specific gene mutations in zebrafish. The following sections highlight how the unique attributes of zebrafish, in combination with genetic modifications, are continuing to transform our understanding of epilepsy and help identify personalized therapeutics for specific patient cohorts.

GO2TR: a gene ontology-based workflow to generate target regions for target enrichment experiments

Although new sequencing technology has enabled biologists to examine genome-wide variation in their taxa of interest, new experimental design, analysis, and cost hurdles impede many researchers from using the benefits of next-generation sequencing. Here we present a workflow to help researchers identify genomic intervals associated with a desired gene function by using gene ontology terms to filter an annotated genome. Resulting intervals comprise a target region that can then be used to design complementary sequences or baits for capturing desired targets in a target enrichment experiment. We show that target regions produced by our workflow and Ensembl Genebuild share intervals but nonetheless differ, and then demonstrate our workflow’s utility for target enrichment experiments involving non-model organisms. Using available turtle genomes and bait sequences designed to capture a workflow-generated target region, in silico analysis predicts between 48 and 86 % of baits will bind to complementary sequences in genomes across the Order Testudines (turtles and tortoises). We then use these bait sequences in an actual target enrichment experiment and show that bait performance falls within the range predicted by in silico analysis. We show that by selecting a reference genome related as closely as possible to taxa of interest and focusing on important and likely conserved gene functions, users can acquire valuable genomic data from non-model organisms.

The iBeetle large-scale RNAi screen reveals gene functions for insect development and physiology.

Genetic screens are powerful tools to identify the genes required for a given biological process. However, for technical reasons, comprehensive screens have been restricted to very few model organisms. Therefore, although deep sequencing is revealing the genes of ever more insect species, the functional studies predominantly focus on candidate genes previously identified in Drosophila, which is biasing research towards conserved gene functions. RNAi screens in other organisms promise to reduce this bias. Here we present the results of the iBeetle screen, a large-scale, unbiased RNAi screen in the red flour beetle, Tribolium castaneum, which identifies gene functions in embryonic and postembryonic development, physiology and cell biology. The utility of Tribolium as a screening platform is demonstrated by the identification of genes involved in insect epithelial adhesion. This work transcends the restrictions of the candidate gene approach and opens fields of research not accessible in Drosophila.

CSGM Designer: a platform for designing cross-species intron-spanning genic markers linked with genome information of legumes.

BackgroundGenetic markers are tools that can facilitate molecular breeding, even in species lacking genomic resources. An important class of genetic markers is those based on orthologous genes, because they can guide hypotheses about conserved gene function, a situation that is well documented for a number of agronomic traits. For under-studied species a key bottleneck in gene-based marker development is the need to develop molecular tools (e.g., oligonucleotide primers) that reliably access genes with orthology to the genomes of well-characterized reference species.ResultsHere we report an efficient platform for the design of cross-species gene-derived markers in legumes. The automated platform, named CSGM Designer (URL: http://tgil.donga.ac.kr/CSGMdesigner), facilitates rapid and systematic design of cross-species genic markers. The underlying database is composed of genome data from five legume species whose genomes are substantially characterized. Use of CSGM is enhanced by graphical displays of query results, which we describe as “circular viewer” and “search-within-results” functions. CSGM provides a virtual PCR representation (eHT-PCR) that predicts the specificity of each primer pair simultaneously in multiple genomes. CSGM Designer output was experimentally validated for the amplification of orthologous genes using 16 genotypes representing 12 crop and model legume species, distributed among the galegoid and phaseoloid clades. Successful cross-species amplification was obtained for 85.3% of PCR primer combinations.ConclusionCSGM Designer spans the divide between well-characterized crop and model legume species and their less well-characterized relatives. The outcome is PCR primers that target highly conserved genes for polymorphism discovery, enabling functional inferences and ultimately facilitating trait-associated molecular breeding.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-015-0074-6) contains supplementary material, which is available to authorized users.

Patterns and Polarity in Floral Meristem and Floral Organ Initiation

The initial event in plant floral organogenesis is bract specification, followed by floral meristem (FM) initiation in bract axils, but initiation signals and the interplay between both lateral organs remain unelucidated. Floral organs are initiated on the flanks of the outgrowing FM and the enormous diversity in floral morphology throughout the plant kingdom reflects variations in organ position, meristy and ontogeny. Classical models of floral development have focused on Arabidopsis, which has mostly actinomorphic flowers, and Antirrhinum, which exhibits zygomorphy, although neither species is typical or representative of angiosperm flower diversity. Although the ABCE model defines a centripetal model of organ identity establishment in different whorls, the characterization of floral organ initiation in many species has relied on their morphological appearance, due to a lack of founder cell-specific markers. Recent progress in early Arabidopsis floral development using histology, molecular markers and mutants has led to refinements of existing floral organ initiation paradigms. In Arabidopsis, sepals initiate unidirectionally, in a temporal window characterized by the absence of CLAVATA3 and WUSCHEL stem cell markers and are partly dependent on PRESSED FLOWER function, whereas initiation of inner-whorl organs occurs centripetally. Arabidopsis mutants reveal that the FM is highly polarized along an ab-/adaxial axis and a comparison of floral development in Arabidopsis and Antirrhinum suggests that heterochrony of conserved gene functions has been evolutionarily adaptive.This review discusses current views on FM and organ specification signals, the gene regulatory networks that underlie floral meristem polarity, and analogies between the development of floral and leaf primordia as lateral organs. Alternative stem-cell proliferation mechanisms and the bifurcation of founder cell populations can help to explain the diversity in floral diversity throughout the plant kingdom and underpin comparative evolutionary biology and macroevolution. An analysis of plants with divergent body plans at the level of organ specification is urgently needed.

Chapter Three - New Insight into the Regulation of Floral Morphogenesis

The beauty and complexity of flowers have held the fascination of scientists for centuries, from Linnaeus, to Goethe, to Darwin, through to the present. During the past decade, enormous progress has been made in understanding the molecular regulation of flower morphogenesis. It seems likely that there are both highly conserved aspects to flower development in addition to significant differences in developmental patterning that can contribute to the unique morphologies of different species. Furthermore, floral development is attractive in that several key genes regulating fundamental processes have been identified. Crucial functional studies of floral organ identity genes in diverse taxa are allowing the real insight into the conservation of gene function, while findings on the genetic control of organ elaboration open up new avenues for investigation. These fundamentals of floral organ differentiation and growth are therefore an ideal subject for comparative analyses of flower development, which will lead to a better understanding of molecular mechanisms that control flower morphogenesis.