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1.
PLoS Genet ; 15(6): e1008213, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31199790

RESUMO

The neural crest (NC) is a vertebrate-specific cell type that contributes to a wide range of different tissues across all three germ layers. The gene regulatory network (GRN) responsible for the formation of neural crest is conserved across vertebrates. Central to the induction of the NC GRN are AP-2 and SoxE transcription factors. NC induction robustness is ensured through the ability of some of these transcription factors to compensate loss of function of gene family members. However the gene regulatory events underlying compensation are poorly understood. We have used gene knockout and RNA sequencing strategies to dissect NC induction and compensation in zebrafish. We genetically ablate the NC using double mutants of tfap2a;tfap2c or remove specific subsets of the NC with sox10 and mitfa knockouts and characterise genome-wide gene expression levels across multiple time points. We find that compensation through a single wild-type allele of tfap2c is capable of maintaining early NC induction and differentiation in the absence of tfap2a function, but many target genes have abnormal expression levels and therefore show sensitivity to the reduced tfap2 dosage. This separation of morphological and molecular phenotypes identifies a core set of genes required for early NC development. We also identify the 15 somites stage as the peak of the molecular phenotype which strongly diminishes at 24 hpf even as the morphological phenotype becomes more apparent. Using gene knockouts, we associate previously uncharacterised genes with pigment cell development and establish a role for maternal Hippo signalling in melanocyte differentiation. This work extends and refines the NC GRN while also uncovering the transcriptional basis of genetic compensation via paralogues.


Assuntos
Desenvolvimento Embrionário/genética , Crista Neural/crescimento & desenvolvimento , Fatores de Transcrição SOXE/genética , Fator de Transcrição AP-2/genética , Proteínas de Peixe-Zebra/genética , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento/genética , Redes Reguladoras de Genes/genética , Melanócitos/metabolismo , Fator de Transcrição Associado à Microftalmia/genética , Crista Neural/metabolismo , Pigmentação/genética , Proteínas Serina-Treonina Quinases/genética , Serina-Treonina Quinase 3 , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento
2.
PLoS Genet ; 13(8): e1006959, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28806732

RESUMO

KDM2A is a histone demethylase associated with transcriptional silencing, however very little is known about its in vivo role in development and disease. Here we demonstrate that loss of the orthologue kdm2aa in zebrafish causes widespread transcriptional disruption and leads to spontaneous melanomas at a high frequency. Fish homozygous for two independent premature stop codon alleles show reduced growth and survival, a strong male sex bias, and homozygous females exhibit a progressive oogenesis defect. kdm2aa mutant fish also develop melanomas from early adulthood onwards which are independent from mutations in braf and other common oncogenes and tumour suppressors as revealed by deep whole exome sequencing. In addition to effects on translation and DNA replication gene expression, high-replicate RNA-seq in morphologically normal individuals demonstrates a stable regulatory response of epigenetic modifiers and the specific de-repression of a group of zinc finger genes residing in constitutive heterochromatin. Together our data reveal a complex role for Kdm2aa in regulating normal mRNA levels and carcinogenesis. These findings establish kdm2aa mutants as the first single gene knockout model of melanoma biology.


Assuntos
Histona Desmetilases com o Domínio Jumonji/genética , Melanoma/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Replicação do DNA , Modelos Animais de Doenças , Epigênese Genética , Exoma , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Masculino , Mutação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de RNA , Peixe-Zebra/embriologia
3.
BMC Genomics ; 16: 578, 2015 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-26238335

RESUMO

BACKGROUND: We present a genome-wide messenger RNA (mRNA) sequencing technique that converts small amounts of RNA from many samples into molecular phenotypes. It encompasses all steps from sample preparation to sequence analysis and is applicable to baseline profiling or perturbation measurements. RESULTS: Multiplex sequencing of transcript 3' ends identifies differential transcript abundance independent of gene annotation. We show that increasing biological replicate number while maintaining the total amount of sequencing identifies more differentially abundant transcripts. CONCLUSIONS: This method can be implemented on polyadenylated RNA from any organism with an annotated reference genome and in any laboratory with access to Illumina sequencing.


Assuntos
Estudos de Associação Genética , Estudo de Associação Genômica Ampla , Sequenciamento de Nucleotídeos em Larga Escala , Tipagem Molecular , RNA Mensageiro/genética , Análise de Sequência de RNA , Animais , Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , Biblioteca Gênica , Estudos de Associação Genética/métodos , Estudo de Associação Genômica Ampla/métodos , Tipagem Molecular/métodos , Mutação , Peixe-Zebra
4.
Dis Model Mech ; 17(1)2024 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-38131137

RESUMO

Shigella flexneri is a human-adapted pathovar of Escherichia coli that can invade the intestinal epithelium, causing inflammation and bacillary dysentery. Although an important human pathogen, the host response to S. flexneri has not been fully described. Zebrafish larvae represent a valuable model for studying human infections in vivo. Here, we use a Shigella-zebrafish infection model to generate mRNA expression profiles of host response to Shigella infection at the whole-animal level. Immune response-related processes dominate the signature of early Shigella infection (6 h post-infection). Consistent with its clearance from the host, the signature of late Shigella infection (24 h post-infection) is significantly changed, and only a small set of immune-related genes remain differentially expressed, including acod1 and gpr84. Using mutant lines generated by ENU, CRISPR mutagenesis and F0 crispants, we show that acod1- and gpr84-deficient larvae are more susceptible to Shigella infection. Together, these results highlight the power of zebrafish to model infection by bacterial pathogens and reveal the mRNA expression of the early (acutely infected) and late (clearing) host response to Shigella infection.


Assuntos
Disenteria Bacilar , Animais , Humanos , Disenteria Bacilar/genética , Shigella flexneri/genética , Shigella flexneri/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/microbiologia , Inflamação/microbiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
5.
Nat Genet ; 56(3): 395-407, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38429495

RESUMO

In digenic inheritance, pathogenic variants in two genes must be inherited together to cause disease. Only very few examples of digenic inheritance have been described in the neuromuscular disease field. Here we show that predicted deleterious variants in SRPK3, encoding the X-linked serine/argenine protein kinase 3, lead to a progressive early onset skeletal muscle myopathy only when in combination with heterozygous variants in the TTN gene. The co-occurrence of predicted deleterious SRPK3/TTN variants was not seen among 76,702 healthy male individuals, and statistical modeling strongly supported digenic inheritance as the best-fitting model. Furthermore, double-mutant zebrafish (srpk3-/-; ttn.1+/-) replicated the myopathic phenotype and showed myofibrillar disorganization. Transcriptome data suggest that the interaction of srpk3 and ttn.1 in zebrafish occurs at a post-transcriptional level. We propose that digenic inheritance of deleterious changes impacting both the protein kinase SRPK3 and the giant muscle protein titin causes a skeletal myopathy and might serve as a model for other genetic diseases.


Assuntos
Doenças Musculares , Peixe-Zebra , Animais , Humanos , Masculino , Conectina/genética , Conectina/metabolismo , Músculo Esquelético , Doenças Musculares/genética , Doenças Musculares/metabolismo , Doenças Musculares/patologia , Mutação , Peixe-Zebra/genética
6.
Neurosci Biobehav Rev ; 135: 104559, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35124155

RESUMO

Psychiatric disorders represent a significant burden in our societies. Despite the convincing evidence pointing at gene and gene-environment interaction contributions, the role of genetics in the etiology of psychiatric disease is still poorly understood. Forward genetic screens in animal models have helped elucidate causal links. Here we discuss the application of mutagenesis-based forward genetic approaches in common animal model species: two invertebrates, nematodes (Caenorhabditis elegans) and fruit flies (Drosophila sp.); and two vertebrates, zebrafish (Danio rerio) and mice (Mus musculus), in relation to psychiatric disease. We also discuss the use of large scale genomic studies in human populations. Despite the advances using data from human populations, animal models coupled with next-generation sequencing strategies are still needed. Although with its own limitations, zebrafish possess characteristics that make them especially well-suited to forward genetic studies exploring the etiology of psychiatric disorders.


Assuntos
Transtornos Mentais , Peixe-Zebra , Animais , Caenorhabditis elegans/genética , Modelos Animais de Doenças , Drosophila , Humanos , Transtornos Mentais/genética , Camundongos , Modelos Animais , Peixe-Zebra/genética
7.
Dis Model Mech ; 15(6)2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35514229

RESUMO

Manganese neurotoxicity is a hallmark of hypermanganesemia with dystonia 2, an inherited manganese transporter defect caused by mutations in SLC39A14. To identify novel potential targets of manganese neurotoxicity, we performed transcriptome analysis of slc39a14-/- mutant zebrafish that were exposed to MnCl2. Differentially expressed genes mapped to the central nervous system and eye, and pathway analysis suggested that Ca2+ dyshomeostasis and activation of the unfolded protein response are key features of manganese neurotoxicity. Consistent with this interpretation, MnCl2 exposure led to decreased whole-animal Ca2+ levels, locomotor defects and changes in neuronal activity within the telencephalon and optic tectum. In accordance with reduced tectal activity, slc39a14-/- zebrafish showed changes in visual phototransduction gene expression, absence of visual background adaptation and a diminished optokinetic reflex. Finally, numerous differentially expressed genes in mutant larvae normalised upon MnCl2 treatment indicating that, in addition to neurotoxicity, manganese deficiency is present either subcellularly or in specific cells or tissues. Overall, we assembled a comprehensive set of genes that mediate manganese-systemic responses and found a highly correlated and modulated network associated with Ca2+ dyshomeostasis and cellular stress. This article has an associated First Person interview with the first author of the paper.


Assuntos
Proteínas de Transporte de Cátions , Distonia , Animais , Cálcio/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Distonia/genética , Íons/metabolismo , Manganês/metabolismo , Manganês/toxicidade , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
8.
Front Psychiatry ; 12: 795175, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-35082702

RESUMO

Developmental consequences of prenatal drug exposure have been reported in many human cohorts and animal studies. The long-lasting impact on the offspring-including motor and cognitive impairments, cranial and cardiac anomalies and increased prevalence of ADHD-is a socioeconomic burden worldwide. Identifying the molecular changes leading to developmental consequences could help ameliorate the deficits and limit the impact. In this study, we have used zebrafish, a well-established behavioral and genetic model with conserved drug response and reward pathways, to identify changes in behavior and cellular pathways in response to developmental exposure to amphetamine, nicotine or oxycodone. In the presence of the drug, exposed animals showed altered behavior, consistent with effects seen in mammalian systems, including impaired locomotion and altered habituation to acoustic startle. Differences in responses seen following acute and chronic exposure suggest adaptation to the presence of the drug. Transcriptomic analysis of exposed larvae revealed differential expression of numerous genes and alterations in many pathways, including those related to cell death, immunity and circadian rhythm regulation. Differential expression of circadian rhythm genes did not correlate with behavioral changes in the larvae, however, two of the circadian genes, arntl2 and per2, were also differentially expressed at later stages of development, suggesting a long-lasting impact of developmental exposures on circadian gene expression. The immediate-early genes, egr1, egr4, fosab, and junbb, which are associated with synaptic plasticity, were downregulated by all three drugs and in situ hybridization showed that the expression for all four genes was reduced across all neuroanatomical regions, including brain regions implicated in reward processing, addiction and other psychiatric conditions. We anticipate that these early changes in gene expression in response to drug exposure are likely to contribute to the consequences of prenatal exposure and their discovery might pave the way to therapeutic intervention to ameliorate the long-lasting deficits.

9.
Elife ; 82019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30777146

RESUMO

The vertebrate eye originates from the eye field, a domain of cells specified by a small number of transcription factors. In this study, we show that Tcf7l1a is one such transcription factor that acts cell-autonomously to specify the eye field in zebrafish. Despite the much-reduced eye field in tcf7l1a mutants, these fish develop normal eyes revealing a striking ability of the eye to recover from a severe early phenotype. This robustness is not mediated through genetic compensation at neural plate stage; instead, the smaller optic vesicle of tcf7l1a mutants shows delayed neurogenesis and continues to grow until it achieves approximately normal size. Although the developing eye is robust to the lack of Tcf7l1a function, it is sensitised to the effects of additional mutations. In support of this, a forward genetic screen identified mutations in hesx1, cct5 and gdf6a, which give synthetically enhanced eye specification or growth phenotypes when in combination with the tcf7l1a mutation.


Assuntos
Olho/crescimento & desenvolvimento , Morfogênese , Proteína 1 Semelhante ao Fator 7 de Transcrição/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Animais , Proliferação de Células , Embrião não Mamífero/metabolismo , Olho/patologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Loci Gênicos , Cinética , Masculino , Mutação/genética , Placa Neural/embriologia , Neurogênese , Penetrância , Fenótipo , Prosencéfalo/embriologia , Proteína 1 Semelhante ao Fator 7 de Transcrição/genética , Regulação para Cima/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Zigoto/metabolismo
10.
Nat Commun ; 10(1): 2792, 2019 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-31243271

RESUMO

The Deciphering the Mechanisms of Developmental Disorders programme has analysed the morphological and molecular phenotypes of embryonic and perinatal lethal mouse mutant lines in order to investigate the causes of embryonic lethality. Here we show that individual whole-embryo RNA-seq of 73 mouse mutant lines (>1000 transcriptomes) identifies transcriptional events underlying embryonic lethality and associates previously uncharacterised genes with specific pathways and tissues. For example, our data suggest that Hmgxb3 is involved in DNA-damage repair and cell-cycle regulation. Further, we separate embryonic delay signatures from mutant line-specific transcriptional changes by developing a baseline mRNA expression catalogue of wild-type mice during early embryogenesis (4-36 somites). Analysis of transcription outside coding sequence identifies deregulation of repetitive elements in Morc2a mutants and a gene involved in gene-specific splicing. Collectively, this work provides a large scale resource to further our understanding of early embryonic developmental disorders.


Assuntos
Embrião de Mamíferos/metabolismo , Análise de Sequência de RNA , Transcrição Gênica , Animais , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Mutação , Transcriptoma
11.
Wellcome Open Res ; 2: 77, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29568807

RESUMO

BACKGROUND: Mutations in proteins involved in telomere maintenance lead to a range of human diseases, including dyskeratosis congenita, idiopathic pulmonary fibrosis and cancer. Telomerase functions to add telomeric repeats back onto the ends of chromosomes, however non-canonical roles of components of telomerase have recently been suggested. METHODS: Here we use a zebrafish telomerase mutant which harbours a nonsense mutation in tert to investigate the adult phenotypes of fish derived from heterozygous parents of different ages. Furthermore we use whole genome sequencing data to estimate average telomere lengths. RESULTS: We show that homozygous offspring from older heterozygotes exhibit signs of body wasting at a younger age than those of younger parents, and that offspring of older heterozygous parents weigh less irrespective of genotype. We also demonstrate that tert homozygous mutant fish have a male sex bias, and that clutches from older parents also have a male sex bias in the heterozygous and wild-type populations. Telomere length analysis reveals that the telomeres of younger heterozygous parents are shorter than those of older heterozygous parents. CONCLUSIONS: These data indicate that the phenotypes observed in offspring from older parents cannot be explained by telomere length. Instead we propose that Tert functions outside of telomere length maintenance in an age-dependent manner to influence the adult phenotypes of the next generation.

12.
Elife ; 62017 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-29144233

RESUMO

We have produced an mRNA expression time course of zebrafish development across 18 time points from 1 cell to 5 days post-fertilisation sampling individual and pools of embryos. Using poly(A) pulldown stranded RNA-seq and a 3' end transcript counting method we characterise temporal expression profiles of 23,642 genes. We identify temporal and functional transcript co-variance that associates 5024 unnamed genes with distinct developmental time points. Specifically, a class of over 100 previously uncharacterised zinc finger domain containing genes, located on the long arm of chromosome 4, is expressed in a sharp peak during zygotic genome activation. In addition, the data reveal new genes and transcripts, differential use of exons and previously unidentified 3' ends across development, new primary microRNAs and temporal divergence of gene paralogues generated in the teleost genome duplication. To make this dataset a useful baseline reference, the data can be browsed and downloaded at Expression Atlas and Ensembl.


Assuntos
Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , RNA Mensageiro/biossíntese , Peixe-Zebra/embriologia , Animais , Perfilação da Expressão Gênica , Análise de Sequência de RNA , Fatores de Tempo
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