Developmental Genes Research Articles

Protective role of ghrelin against 6PPD-quinone-induced neurotoxicity in zebrafish larvae (Danio rerio) via the GHSR pathway

The toxicity mechanisms of N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine quinone (6PPD-Q), an antioxidant derivative of 6PPD via ozone reaction commonly used in rubber and tire industries, were investigated in zebrafish larvae with concentrations ranging from 0 to 50 μg/L. Despite normal hatchability, 6PPD-Q exposure led to reduced body length and swimming distance in 120 hours post-fertilization (hpf) larvae. At the highest concentration (50 μg/L), 6PPD-Q significantly impaired dopaminergic neuron development and neurotransmitter levels, including dopamine, 5-hydroxytryptamine, and glutamate. Transcriptome profiling unveiled perturbations in growth and developmental gene expression, such as upregulation of runx2a, runx2b, and ghrl (ghrelin and obestatin prepropeptide), and downregulation of stat1b, auto1, and cidea. Notably, anamorelin, a growth hormone secretagogue receptor (GHSR) agonist, recovered the behavioral deficits induced by 6PPD-Q, implying a neuroprotective role of ghrelin possibly mediated via the ghrelin/GHSR pathway. Collectively, our findings indicate that ghrelin upregulation may counteract 6PPD-Q toxicity in zebrafish larvae, shedding light on potential therapeutic avenues for mitigating the adverse effects of this antioxidant byproduct.

Chromatin assembly factor subunit CHAF1A as a monogenic cause for oculo-auriculo-vertebral spectrum.

Oculo-auriculo-vertebral spectrum (OAVS) is characterized by abnormal development of the 1st and 2nd branchial arches. Despite arguments against a monogenic condition, a few genes have been involved in a minority of cases. We now report heterozygous, presumably loss-of function variants in the CHAF1A gene in 8 individuals, including 3 members of the same family. Four cases fulfill stringent diagnostic criteria for OAVS, including asymmetric ear dysplasia, preauricular tags, mandibular asymmetry +/- vertebral malformations. Two patients also presented with kidney malformations. CHAF1A encodes a subunit of CAF-1 (chromatin assembly factor-1), a heterotrimeric protein complex responsible for the deposition of newly synthesized histones H3-H4 onto the newly synthetized DNA strand during replication. The identification of loss-of-unction variants in CHAF1A is consistent with the hypothesis of early developmental genes dysregulation driving OAVS and other associations recently lumped under the acronym Recurrent Constellations of Embryonic Malformations (RCEM).

A high-throughput gene expression analysis software tool for developmental time series and gene signature analysis of human cardiomyocyte differentiation

Publicly available high-throughput gene expression data enable the investigation of biological processes by the scientific community. Although several bioinformatics tools offer methodologies for basic differential gene expression analysis, difficulties arise in the analysis of multiple sample groups comprising a developmental time series, especially when the identification and classification of unique gene expression patterns is the primary goal of the study. Data analysis using these tools requires programming experience, which limits the accessibility of these tools to the broader community. To streamline developmental time-series investigations, we created the Developmental Gene Expression Analysis (devGEA) tool. This environment can be implemented locally or via web browsers to expedite differential gene expression analysis. This tool provides gene signature determination methods that can classify differentially expressed genes based on their correlation with gene expression patterns. devGEA was used to characterize cardiac development gene expression signatures from high-throughput RNA-seq datasets profiling small-molecule directed cardiomyocyte differentiation of human pluripotent stem cell lines (hiPSCs). After pre-processing, discrete gene expression criteria-based expected changes were used to classify the genes into developmental signatures. Several cardiomyocyte differentiation markers and candidate cardiac genes representing different developmental signatures were experimentally validated using the GIBCOTM hiPSC line. This method was then compared to a gene signature correlation approach that classified expressed genes based on their degree of similarity with key cardiac developmental signatures representing the stages of cardiomyocyte differentiation. Therefore, devGEA provides a robust workflow for investigator-driven analysis of developmental time series, allowing for the identification of differentially expressed genes and gene signatures for extensive experimental investigation. We also introduced a method for classifying genes by their correlation with genes or developmental patterns of interest. Our correlation-based method takes advantage of a priori knowledge of an experiment and is conceptually simpler than unsupervised clustering approaches.

Ancient developmental genes underlie evolutionary novelties in walking fish

A critical question in biology is how new traits evolve, but studying this in wild animals remains challenging. Here, we probe the genetic basis of trait gain in sea robin fish, which have evolved specialized leg-like appendages for locomotion and digging along the ocean floor. We use genome sequencing, transcriptional profiling, and interspecific hybrid analysis to explore the molecular and developmental basis of leg formation. We identified the ancient, conserved transcription factor tbx3a as a major determinant of sensory leg development. Genome editing confirms that tbx3a is required for normal leg formation in sea robins, and for formation of enlarged central nervous system lobes, sensory papillae, and adult digging behavior. Our study establishes sea robins as a model organism for studying the evolution of major trait gain and illustrates how ancient developmental control genes can underlie novel organ formation.

Common variants increase risk for congenital diaphragmatic hernia within the context of de novo variants

Congenital diaphragmatic hernia (CDH) is a severe congenital anomaly often accompanied by other structural anomalies and/or neurobehavioral manifestations. Rare de novo protein-coding variants and copy-number variations contribute to CDH in the population. However, most individuals with CDH remain genetically undiagnosed. Here, we perform integrated de novo and common-variant analyses using 1,469 CDH individuals, including 1,064 child-parent trios and 6,133 ancestry-matched, unaffected controls for the genome-wide association study. We identify candidate CDH variants in 15 genes, including eight novel genes, through deleterious de novo variants. We further identify two genomic loci contributing to CDH risk through common variants with similar effect sizes among Europeans and Latinx. Both loci are in putative transcriptional regulatory regions of developmental patterning genes. Estimated heritability in common variants is ∼19%. Strikingly, there is no significant difference in estimated polygenic risk scores between isolated and complex CDH or between individuals harboring deleterious de novo variants and individuals without these variants. The data support a polygenic model as part of the CDH genetic architecture.

Evolutionary Innovations in Conserved Regulatory Elements Associate With Developmental Genes in Mammals.

Transcriptional enhancers orchestrate cell type- and time point-specific gene expression programs. Genetic variation within enhancer sequences is an important contributor to phenotypic variation including evolutionary adaptations and human disease. Certain genes and pathways may be more prone to regulatory evolution than others, with different patterns across diverse organisms, but whether such patterns exist has not been investigated at a sufficient scale. To address this question, we identified signatures of accelerated sequence evolution in conserved enhancer elements throughout the mammalian phylogeny at an unprecedented scale. While different genes and pathways were enriched for regulatory evolution in different parts of the tree, we found a striking overall pattern of pleiotropic genes involved in gene regulatory and developmental processes being enriched for accelerated enhancer evolution. These genes were connected to more enhancers than other genes, which was the basis for having an increased amount of sequence acceleration over all their enhancers combined. We provide evidence that sequence acceleration is associated with turnover of regulatory function. Detailed study of one acceleration event in an enhancer of HES1 revealed that sequence evolution led to a new activity domain in the developing limb that emerged concurrently with the evolution of digit reduction in hoofed mammals. Our results provide evidence that enhancer evolution has been a frequent contributor to regulatory innovation at conserved developmental signaling genes in mammals.

Mono-allelic epigenetic regulation of bi-directional silencing of RNA Polymerase II polycistronic transcription initiation in Trypanosoma brucei.

The single-cell parasite Trypanosoma brucei causes lethal diseases in both humans and livestock. T. brucei undergoes multiple developmental changes to adapt in different environments during its digenetic life cycle. With protein-coding genes organized as polycistronic transcription and apparent absence of promoter-mediated regulation of transcription initiation, it is believed that developmental gene regulation in trypanosomes is essentially post-transcriptional. In this study, we found reversible Pol II transcriptional silencing of two adjacent polycistronic gene arrays that correlates with the novel DNA base J modification of the shared promoter region. Our findings support epigenetic regulation of Pol II transcription initiation as a viable mechanism of gene expression control in T. brucei . This has implications for our understanding how trypanosomes utilize polycistronic genome organization to regulate gene expression during its life cycle.

Efficient CRISPR/Cas9-mediated genome editing in the European corn borer, Ostrinia nubilalis.

The European corn borer (Ostrinia nubilalis) is an agricultural pest and burgeoning model for research on speciation, seasonal adaptation and insect resistance management. Although previous work in O. nubilalis has identified genes associated with differences in life cycle, reproduction, and resistance to Bt toxins, the general lack of a robust gene-editing protocol for O. nubilalis has been a barrier to functional validation of candidate genes. Here, we demonstrate an efficient and practical methodology for heritable gene mutagenesis in O. nubilalis using the CRISPR/Cas9 genome editing system. Precise loss-of-function (LOF) mutations were generated at two circadian clock genes, period (per) and pigment-dispersing factor receptor (pdfr), and a developmental gene, prothoracicotropic hormone (ptth). Precluding the need for a visible genetic marker, gene-editing efficiency remained high across different single guide RNAs (sgRNA) and germline transmission of mutations to F1 offspring approached 100%. When single or dual sgRNAs were injected at a high concentration, gene-specific phenotypic differences in behaviour and development were identified in F0 mutants. Specifically, F0 gene mutants demonstrated that PER, but not PDFR, is essential for normal timing of eclosion. PTTH F0 mutants were significantly heavier and exhibited a higher incidence of diapause. This work will accelerate future studies of gene function in O. nubilalis and facilitate the development of similar screens in other Lepidopteran and non-model insects.

Regional patterns of human cortex development correlate with underlying neurobiology

Human brain morphology undergoes complex changes over the lifespan. Despite recent progress in tracking brain development via normative models, current knowledge of underlying biological mechanisms is highly limited. We demonstrate that human cortical thickness development and aging trajectories unfold along patterns of molecular and cellular brain organization, traceable from population-level to individual developmental trajectories. During childhood and adolescence, cortex-wide spatial distributions of dopaminergic receptors, inhibitory neurons, glial cell populations, and brain-metabolic features explain up to 50% of the variance associated with a lifespan model of regional cortical thickness trajectories. In contrast, modeled cortical thickness change patterns during adulthood are best explained by cholinergic and glutamatergic neurotransmitter receptor and transporter distributions. These relationships are supported by developmental gene expression trajectories and translate to individual longitudinal data from over 8000 adolescents, explaining up to 59% of developmental change at cohort- and 18% at single-subject level. Integrating neurobiological brain atlases with normative modeling and population neuroimaging provides a biologically meaningful path to understand brain development and aging in living humans.

De Novo Transcriptome Profiling of Mustard Aphid (Lipaphis erysimi) and Differential Expression of Transcripts Associated with Feeding and Non-Feeding Conditions and Developmental Stages.

Lipaphis erysimi is a specialist aphid of the Indian subcontinent that causes significant yield losses in oilseed Brassicas. Several aphid genes have been used as preferred targets in RNAi-based transgenic plants for aphid resistance. In order to enhance the repertoire of potential target genes for aphid control and to identify the genes associated with aphid feeding and development, we performed a two-way comparative study of differential gene expression profiles between (i) feeding and non-feeding adults and (ii) adult and nymph developmental stages of L. erysimi. De novo RNA-seq of aphids using Illumina technology generated a final transcriptome comprising 52,652 transcripts. Potential transcripts for host selection, detoxification, salivary proteins and effectors, molecular chaperones and developmental genes were identified. Differential gene expression studies identified variations in the expression of 1502 transcripts between feeding and non-feeding adults and 906 transcripts between nymphs and adults. These data were used to identify novel target genes for RNAi-based aphid control and facilitate further studies on the molecular basis of aphid feeding and development.

A bHLH transcription factor AaMYC2-type positively regulates glandular trichome density and artemisinin biosynthesis in Artemisia annua.

Artemisinin-based combinational therapies (ACTs) constitute the first line of malaria treatment. However, due to its trichome-specific biosynthesis, low concentration, and poor understanding of regulatory mechanisms involved in artemisinin biosynthesis and trichome development, it becomes very difficult to meet the increased demand for ACTs. Here, we have reported that a bHLH transcription factor, AaMYC2-type, plays an important role in regulating GST development and artemisinin biosynthesis in Artemisia annua. AaMYC2-type encodes a protein that is transcriptionally active and localised to the nucleus. It is prominently expressed in aerial parts like leaves, stems, inflorescence and least expressed in roots. AaMYC2-type expression is significantly increased under different hormonal treatments. In transgenic overexpression lines, AaMYC2-type OE, a significant increase in the expression of trichome development and artemisinin biosynthesis genes was observed. While in knockdown lines, Aamyc2-type, expression of trichome development and artemisinin biosynthesis genes were significantly reduced. Yeast one-hybrid assay clearly shows that the AaMYC2-type directly binds to the E-boxes in the promoter regions of ADS and CYP71AVI. The SEM microscopy depicted the number of trichomes elevated from 11 mm-2 in AaMYC2-type OE lines to 6.1 mm-2 in Aamyc2-type. The final effect of the alteration in biosynthetic and trichome developmental genes was observed in the accumulation of artemisinin. In the AaMYC2-type OE, the artemisinin content was 12 mg g-1DW, which was reduced to 3.2 mg g-1DW in the Aamyc2-type. Altogether, the above findings suggest that the AaMYC2-type play a dual regulating role in controlling both trichome developmental and artemisinin biosynthetic genes.

Design the fusion double-strand RNAs to control two global sap-sucking pests

RNA interference (RNAi) is an effective pest management strategy through silencing the crucial genes in target organisms. However, the effectiveness of targeting a single gene is often limited by the silencing efficiency due to tissue or developmental stage-specific gene expression. Moreover, multiple pests often infest the same crop simultaneously under current ecological conditions. Therefore, a combined strategy of “targeting multiple genes” and “controlling multiple pests” is expected to yield better management results. In this study, homologous genes from two globally sap-sucking pests, the peach aphid (Myzus persicae) and the whitefly (Bemisia tabaci), were screened on a genome-wide scale. Subsequently, RNAi bioassays showed silencing the genes (MpAbd-A, MpH3, MpRpL27a, and MpScr) exhibited high mortalities in both species, which were further selected for designing fusion dsRNAs. These fusion dsRNAs resulted in higher mortalities in both pests than single gene silencing and posed a minimal off-target risk to the predator ladybeetle (Propylaea japonica) based on the sequence analysis. Finally, the tobacco plants expressing the fusion dsRNAs through virus-induced gene silencing (VIGS) technology enhanced the resistance to both pests. In conclusion, this study proposes a novel RNAi-based approach for managing two sap-sucking pests simultaneously.

Splicing is dynamically regulated during limb development

Modifications to highly conserved developmental gene regulatory networks are thought to underlie morphological diversification in evolution and contribute to human congenital malformations. Relationships between gene expression and morphology have been extensively investigated in the limb, where most of the evidence for alterations to gene regulation in development consists of pre-transcriptional mechanisms that affect expression levels, such as epigenetic alterations to regulatory sequences and changes to cis-regulatory elements. Here we report evidence that alternative splicing (AS), a post-transcriptional process that modifies and diversifies mRNA transcripts, is dynamic during limb development in two mammalian species. We evaluated AS patterns in mouse (Mus musculus) and opossum (Monodelphis domestica) across the three key limb developmental stages: the ridge, bud, and paddle. Our data show that splicing patterns are dynamic over developmental time and suggest differences between the two mammalian taxa. Additionally, multiple key limb development genes, including Fgf8, are differentially spliced across the three stages in both species, with expression levels of the conserved splice variants, Fgf8a and Fgf8b, changing across developmental time. Our data demonstrates that AS is a critical mediator of mRNA diversity in limb development and provides an additional mechanism for evolutionary tweaking of gene dosage.

Strong Activation of ID1, ID2, and ID3 Genes Is Coupled with the Formation of Vasculogenic Mimicry Phenotype in Melanoma Cells.

Gene expression patterns are very sensitive to external influences and are reflected in phenotypic changes. It was previously described that transferring melanoma cells from a plastic surface to Matrigel led to formation of de novo vascular networks-vasculogenic mimicry-that are characteristic to a stemness phenotype in aggressive tumors. Up to now there was no detailed data about the gene signature accompanying this process. Here, we show that this transfer shortly led to extremely strong epigenetic changes in gene expression in the melanoma cells. We observed that on Matrigel numerous genes controlling ribosome biogenesis were upregulated. However, most of the activated genes were inhibitors of the differentiation genes (ID1, ID2, and ID3). At the same time, the genes that control differentiation were downregulated. Both the upregulated and the downregulated genes are simultaneously targeted by different transcription factors shaping sets of co-expressed genes. The specific group of downregulated genes shaping contacts with rDNA genes are also associated with the H3K27me3 mark and with numerous lincRNAs and miRNAs. We conclude that the stemness phenotype of melanoma cells is due to the downregulation of developmental genes and formation of dedifferentiated cells.

Cooperative insulation of regulatory domains by CTCF-dependent physical insulation and promoter competition

The specificity of gene expression during development requires the insulation of regulatory domains to avoid inappropriate enhancer-gene interactions. In vertebrates, this insulator function is mostly attributed to clusters of CTCF sites located at topologically associating domain (TAD) boundaries. However, TAD boundaries allow some physical crosstalk across regulatory domains, which is at odds with the specific and precise expression of developmental genes. Here we show that developmental genes and nearby clusters of CTCF sites cooperatively foster the robust insulation of regulatory domains. By genetically dissecting a couple of representative loci in mouse embryonic stem cells, we show that CTCF sites prevent undesirable enhancer-gene contacts (i.e. physical insulation), while developmental genes preferentially contribute to regulatory insulation through non-structural mechanisms involving promoter competition rather than enhancer blocking. Overall, our work provides important insights into the insulation of regulatory domains, which in turn might help interpreting the pathological consequences of certain structural variants.

A PRE loop at the dac locus acts as a topological chromatin structure that restricts and specifies enhancer-promoter communication.

Three-dimensional (3D) genome folding has a fundamental role in the regulation of developmental genes by facilitating or constraining chromatin interactions between cis-regulatory elements (CREs). Polycomb response elements (PREs) are a specific kind of CRE involved in the memory of transcriptional states in Drosophila melanogaster. PREs act as nucleation sites for Polycomb group (PcG) proteins, which deposit the repressive histone mark H3K27me3, leading to the formation of a class of topologically associating domain (TAD) called a Polycomb domain. PREs can establish looping contacts that stabilize the gene repression of key developmental genes during development. However, the mechanism by which PRE loops fine-tune gene expression is unknown. Using clustered regularly interspaced short palindromic repeats and Cas9 genome engineering, we specifically perturbed PRE contacts or enhancer function and used complementary approaches including 4C-seq, Hi-C and Hi-M to analyze how chromatin architecture perturbation affects gene expression. Our results suggest that the PRE loop at the dac gene locus acts as a constitutive 3D chromatin scaffold during Drosophila development that forms independently of gene expression states and has a versatile function; it restricts enhancer-promoter communication and contributes to enhancer specificity.

Comparative Transcriptome Analysis Reveals Differential Cutaneous Gene Expression in the Color Variation of Two Ornamental Discus, Red Melon and Red Cover.

Red Melon (RM) and Red Cover (RC) discus (Symphysodon spp.) are ornamental fish varieties that were selectively bred from the wild parental lineages of the brown discus S. aquafaciatus over many generations, resulting in distinct cutaneous patterns from juveniles to adults. To better understand the underlying mechanisms, skin samples were collected from juveniles aged 60 days and adults aged 1 year from RM and RC for investigations. Microscopic observation detected xanthophores and erythrophores in all samples, except RC juveniles with no erythrophores. Melanophores were presented only in RC. The comparative analysis revealed that genes involved in pteridine synthesis (gch1 and zgc:153031), one-carbon metabolism (aldh1l2 and zgc153031), and lipid metabolism (apoda and klf1) were differentially expressed in RM juveniles, which may be associated with the development of erythrophores and xanthophores. The temporal inhibition of melanophore differentiation and development was observed in RM juveniles, coupled with elevated expression of notum2 and sost, two antagonist genes in Wnt-signaling, suggesting their roles in melanophore development. Distinct pigment pattern between RM and RC since the juvenile stage may be driven by the differential expression of multiple axial developmental genes, including GATA, ankyrin, and mitotic spindle orientation proteins. This is the first report to describe the differential growth of cutaneous pigments and the molecular processes involved in red discus. The results provided valuable insights into pigment pattern differences in an interesting ornamental fish model.

KMT2A oncoproteins induce epigenetic resistance to targeted therapies.

Chromosomal translocations involving the Lysine-Methyl-Transferase-2A ( KMT2A ) locus generate potent oncogenic fusion proteins (oncoproteins) that disrupt regulation of developmental gene expression. By profiling the oncoprotein-target sites of 36 broadly representative KMT2A -rearranged leukemia samples, including three samples that underwent a lymphoid-to-myeloid lineage-switching event in response to therapy, we find the genomic enrichment of the oncoprotein is highly variable between samples and subject to dynamic regulation. At high levels of expression, the oncoproteins preferentially activate either an acute lymphoblastic leukemia (ALL) program, enriched for pro-B-cell genes, or an acute myeloid leukemia (AML) program, enriched for hematopoietic-stem-cell genes. The fusion-partner-specific-binding patterns over these gene sets are highly correlated with the prevalence of each mutation in ALL versus AML. In lineage-switching samples the oncoprotein levels are reduced and the oncoproteins preferentially activate granulocyte-monocyte progenitor (GMP) genes. In a sample that lineage switched during treatment with the menin inhibitor revumenib, the oncoprotein and menin are reduced to undetectable levels, but ENL, a transcriptional cofactor of the oncoprotein, persists on numerous oncoprotein-target loci, including genes in the GMP-like lineage-switching program. We propose KMT2A oncoproteins promote lineage-switching events through dynamic chromatin binding and can induce epigenetic lesions, marked by ENL, that support resistance to targeted therapies.

The ribonucleoprotein complex factor Ybx1 stabilizes the maternal mRNA of the <i>ssx2ip</i> gene encoding the centrosome maturation protein in <i>Xenopus laevis</i> embryogenesis

Our study investigates the mechanisms that regulate early developmental gene expression in Xenopus laevis frog embryos. Our previous study demonstrated that maternal mRNAs of two developmentally significant genes, the nuclear retinoic acid receptor rxrg and the pluripotency factor pou5f3, form complexes with ribonucleoprotein complex Ybx1. Based on the results of the present study, we determined and demonstrated that the stability of the maternal mRNA ssx2ip, which encodes a conserved protein, also called Msd1 or ADIP, which is involved in centrosome maturation, is dependent on Ybx1. This research shows that Ybx1 forms a ribonucleoprotein complex with ssx2ip mRNA, which is mediated by its cold shock domain (CSD). This study confirms our hypothesis of Ybx1 selectively binding to maternal transcripts. It opens up new opportunities to study new mechanisms of gene expression regulation at the earliest stages of development by searching for possible cis-motifs for recognition by trans-regulators such as Ybx1.

Regional patterns of human cortex development correlate with underlying neurobiology.

Human brain morphology undergoes complex changes over the lifespan. Despite recent progress in tracking brain development via normative models, current knowledge of underlying biological mechanisms is highly limited. We demonstrate that human cortical thickness development and aging trajectories unfold along patterns of molecular and cellular brain organization, traceable from population-level to individual developmental trajectories. During childhood and adolescence, cortex-wide spatial distributions of dopaminergic receptors, inhibitory neurons, glial cell populations, and brain-metabolic features explain up to 50% of variance associated with a lifespan model of regional cortical thickness trajectories. In contrast, modeled cortical thickness change patterns during adulthood are best explained by cholinergic and glutamatergic neurotransmitter receptor and transporter distributions. These relationships are supported by developmental gene expression trajectories and translate to individual longitudinal data from over 8,000 adolescents, explaining up to 59% of developmental change at cohort- and 18% at single-subject level. Integrating neurobiological brain atlases with normative modeling and population neuroimaging provides a biologically meaningful path to understand brain development and aging in living humans.