RESUMO
Cannabis has demonstrated anticonvulsant properties, and about thirty percent of epileptic patients do not have satisfactory seizure management with standard treatment and could potentially benefit from cannabis-based intervention. Here, we report the use of cannabinoids to treat pentylenetetrazol (PTZ)-induced convulsions in a zebrafish model, their effect on gene expression, and a simple assay for assessing their uptake in zebrafish tissues. Using an optimized behavioral assay, we show that cannabidiol (CBD) and cannabichromene (CBC) and cannabinol (CBN) are effective at reducing seizures at low doses, with little evidence of sedation, and our novel HPLC assay indicates that CBC is effective with the lowest accumulation in larval tissues. All cannabinoids tested were effective at higher concentrations. Pharmacological manipulation of potential receptors demonstrates that Gpr55 partially mediates the anticonvulsant effects of CBD. Treatment of zebrafish larvae with endocannabinoids, such as 2-arachidonoylglycerol (2-AG) and anandamide (AEA), altered larvae movement, and the expression of genes that regulate their metabolism was affected by phytocannabinoid treatment, highlighting the possibility that changes to endocannabinoid levels may represent one facet of the anticonvulsant effect of phytocannabinoids.
Assuntos
Canabidiol , Cannabis , Humanos , Animais , Endocanabinoides , Peixe-Zebra , Anticonvulsivantes/farmacologia , Convulsões/induzido quimicamente , Convulsões/tratamento farmacológico , Convulsões/genética , Agonistas de Receptores de Canabinoides , Canabidiol/farmacologia , Canabinol , Expressão GênicaRESUMO
Bone diseases such as otosclerosis (conductive hearing loss) and osteoporosis (low bone mineral density) can result from the abnormal expression of genes that regulate cartilage and bone development. The forkhead box transcription factor FOXL1 has been identified as the causative gene in a family with autosomal dominant otosclerosis and has been reported as a candidate gene in GWAS meta-analyses for osteoporosis. This potentially indicates a novel role for foxl1 in chondrogenesis, osteogenesis, and bone remodelling. We created a foxl1 mutant zebrafish strain as a model for otosclerosis and osteoporosis and examined jaw bones that are homologous to the mammalian middle ear bones, and mineralization of the axial skeleton. We demonstrate that foxl1 regulates the expression of collagen genes such as collagen type 1 alpha 1a and collagen type 11 alpha 2, and results in a delay in jawbone mineralization, while the axial skeleton remains unchanged. foxl1 may also act with other forkhead genes such as foxc1a, as loss of foxl1 in a foxc1a mutant background increases the severity of jaw calcification phenotypes when compared to each mutant alone. Our zebrafish model demonstrates atypical cartilage formation and mineralization in the zebrafish craniofacial skeleton in foxl1 mutants and demonstrates that aberrant collagen expression may underlie the development of otosclerosis.
Assuntos
Osteoporose , Otosclerose , Animais , Biomarcadores/metabolismo , Cartilagem , Mamíferos , Mutação , Otosclerose/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
Sequencing exomes/genomes have been successful for identifying recessive genes; however, discovery of dominant genes including deafness genes (DFNA) remains challenging. We report a new DFNA gene, ATP11A, in a Newfoundland family with a variable form of bilateral sensorineural hearing loss (SNHL). Genome-wide SNP genotyping linked SNHL to DFNA33 (LOD = 4.77), a locus on 13q34 previously mapped in a German family with variable SNHL. Whole-genome sequencing identified 51 unremarkable positional variants on 13q34. Continuous clinical ascertainment identified several key recombination events and reduced the disease interval to 769 kb, excluding all but one variant. ATP11A (NC_000013.11: chr13:113534963G>A) is a novel variant predicted to be a cryptic donor splice site. RNA studies verified in silico predictions, revealing the retention of 153 bp of intron in the 3' UTR of several ATP11A isoforms. Two unresolved families from Israel were subsequently identified with a similar, variable form of SNHL and a novel duplication (NM_032189.3:c.3322_3327+2dupGTCCAGGT) in exon 28 of ATP11A extended exon 28 by 8 bp, leading to a frameshift and premature stop codon (p.Asn1110Valfs43Ter). ATP11A is a type of P4-ATPase that transports (flip) phospholipids from the outer to inner leaflet of cell membranes to maintain asymmetry. Haploinsufficiency of ATP11A, the phospholipid flippase that specially transports phosphatidylserine (PS) and phosphatidylethanolamine (PE), could leave cells with PS/PE at the extracellular side vulnerable to phagocytic degradation. Given that surface PS can be pharmaceutically targeted, hearing loss due to ATP11A could potentially be treated. It is also likely that ATP11A is the gene underlying DFNA33.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Surdez , Perda Auditiva Neurossensorial , Perda Auditiva , Humanos , Regiões 3' não Traduzidas , Transportadores de Cassetes de Ligação de ATP/genética , Surdez/genética , Perda Auditiva/genética , Perda Auditiva Neurossensorial/genética , Mutação , Linhagem , Fosfolipídeos/metabolismo , Sítios de Splice de RNARESUMO
Otosclerosis is a bone disorder of the otic capsule and common form of late-onset hearing impairment. Considered a complex disease, little is known about its pathogenesis. Over the past 20 years, ten autosomal dominant loci (OTSC1-10) have been mapped but no genes identified. Herein, we map a new OTSC locus to a 9.96 Mb region within the FOX gene cluster on 16q24.1 and identify a 15 bp coding deletion in Forkhead Box L1 co-segregating with otosclerosis in a Caucasian family. Pre-operative phenotype ranges from moderate to severe hearing loss to profound sensorineural loss requiring a cochlear implant. Mutant FOXL1 is both transcribed and translated and correctly locates to the cell nucleus. However, the deletion of 5 residues in the C-terminus of mutant FOXL1 causes a complete loss of transcriptional activity due to loss of secondary (alpha helix) structure. FOXL1 (rs764026385) was identified in a second unrelated case on a shared background. We conclude that FOXL1 (rs764026385) is pathogenic and causes autosomal dominant otosclerosis and propose a key inhibitory role for wildtype Foxl1 in bone remodelling in the otic capsule. New insights into the molecular pathology of otosclerosis from this study provide molecular targets for non-invasive therapeutic interventions.
Assuntos
Otosclerose , Fatores de Transcrição Forkhead/genética , Humanos , Otosclerose/genéticaRESUMO
Axenfeld-Rieger syndrome (ARS) encompasses a group of developmental disorders that affect the anterior segment of the eye, as well as systemic developmental defects in some patients. Malformation of the ocular anterior segment often leads to secondary glaucoma, while some patients also present with cardiovascular malformations, craniofacial and dental abnormalities and additional periumbilical skin. Genes that encode two transcription factors, FOXC1 and PITX2, account for almost half of known cases, while the genetic lesions in the remaining cases remain unresolved. Given the genetic similarity between zebrafish and humans, as well as robust antisense inhibition and gene editing technologies available for use in these animals, loss of function zebrafish models for ARS have been created and shed light on the mechanism(s) whereby mutations in these two transcription factors cause such a wide array of developmental phenotypes. This review summarizes the published phenotypes in zebrafish foxc1 and pitx2 loss of function models and discusses possible mechanisms that may be used to target pharmaceutical development and therapeutic interventions.
Assuntos
Segmento Anterior do Olho/anormalidades , Anormalidades do Olho/genética , Oftalmopatias Hereditárias/genética , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Segmento Anterior do Olho/patologia , Anormalidades do Olho/patologia , Oftalmopatias Hereditárias/patologia , Fatores de Transcrição Forkhead/genética , Humanos , Fatores de Transcrição/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
The onset of circulation in a developing embryo requires intact blood vessels to prevent hemorrhage. The development of endothelial cells, and their subsequent recruitment of perivascular mural cells are important processes to establish and maintain vascular integrity. These processes are genetically controlled during development, and mutations that affect endothelial cell specification, pattern formation, or maturation through the addition of mural cells can result in early developmental hemorrhage. We created a strong loss of function allele of the zebrafish GDP-mannose 4,6 dehydratase (gmds) gene that is required for the de novo synthesis of GDP-fucose, and homozygous embryos display cerebral hemorrhages. Our data demonstrate that gmds mutants have early defects in vascular patterning with ectopic branches observed at time of hemorrhage. Subsequently, defects in the number of mural cells that line the vasculature are observed. Moreover, activation of Notch signaling rescued hemorrhage phenotypes in gmds mutants, highlighting a potential downstream pathway that requires protein fucosylation for vascular integrity. Finally, supplementation with fucose can rescue hemorrhage frequency in gmds mutants, demonstrating that synthesis of GDP-fucose via an alternative (salvage) pathway may provide an avenue toward therapeutic correction of phenotypes observed due to defects in de novo GDP-fucose synthesis. Together, these data are consistent with a novel role for the de novo and salvage protein fucosylation pathways in regulating vascular integrity through a Notch dependent mechanism.
Assuntos
Células Endoteliais/metabolismo , Hidroliases/metabolismo , Receptores Notch/metabolismo , Animais , Padronização Corporal/genética , Diferenciação Celular/genética , Movimento Celular/genética , Fucose/metabolismo , Glicosilação , Guanosina Difosfato Fucose/metabolismo , Hemorragia/genética , Hemorragia/prevenção & controle , Hidroliases/genética , Mutação com Perda de Função/genética , Mutação , Fenótipo , Receptores Notch/fisiologia , Transdução de Sinais , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
Precise spatiotemporal expression of the Nodal-Lefty-Pitx2 cascade in the lateral plate mesoderm establishes the left-right axis, which provides vital cues for correct organ formation and function. Mutations of one cascade constituent PITX2 and, separately, the Forkhead transcription factor FOXC1 independently cause a multi-system disorder known as Axenfeld-Rieger syndrome (ARS). Since cardiac involvement is an established ARS phenotype and because disrupted left-right patterning can cause congenital heart defects, we investigated in zebrafish whether foxc1 contributes to organ laterality or situs. We demonstrate that CRISPR/Cas9-generated foxc1a and foxc1b mutants exhibit abnormal cardiac looping and that the prevalence of cardiac situs defects is increased in foxc1a-/-; foxc1b-/- homozygotes. Similarly, double homozygotes exhibit isomerism of the liver and pancreas, which are key features of abnormal gut situs. Placement of the asymmetric visceral organs relative to the midline was also perturbed by mRNA overexpression of foxc1a and foxc1b. In addition, an analysis of the left-right patterning components, identified in the lateral plate mesoderm of foxc1 mutants, reduced or abolished the expression of the NODAL antagonist lefty2. Together, these data reveal a novel contribution from foxc1 to left-right patterning, demonstrating that this role is sensitive to foxc1 gene dosage, and provide a plausible mechanism for the incidence of congenital heart defects in Axenfeld-Rieger syndrome patients.
Assuntos
Segmento Anterior do Olho/anormalidades , Anormalidades do Olho/diagnóstico , Anormalidades do Olho/etiologia , Oftalmopatias Hereditárias/diagnóstico , Oftalmopatias Hereditárias/etiologia , Fatores de Transcrição Forkhead/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Fenótipo , Alelos , Animais , Biologia Computacional/métodos , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Estudos de Associação Genética/métodos , Genótipo , Humanos , Mesoderma/embriologia , Mesoderma/metabolismo , Mutação , Peixe-ZebraRESUMO
Cellular membrane asymmetry is a hallmark characteristic of all eukaryotic cells. The balance of phospholipid composition within the cytoplasmic inner leaflet and the extracellular outer leaflet of the plasma membrane (PM) maintains cellular function and vitality. The proper exposure of particular phospholipids is necessary to maintain cellular signalling, controlled apoptosis, and vesicle transportation among other roles. Phospholipid asymmetry is coordinated by P4-type phospholipid transferases (flippases or ATPases). ATP11A, ATP11B, and ATP11C belong to class VI of the P4-flippase family (vertebrates) and are responsible for the movement of phosphatidylserine (PS) from the outer leaflet to the inner leaflet of the PM. To date, there is a lack of knowledge of the tissue specific expression of these three flippases on a whole-organism level in a vertebrate system. Here we have determined the spatial-temporal expression profiles of each gene in a zebrafish model using in situ hybridization and performed comparative phylogenetic analyses with other vertebrates. Our data reveals sequence similarity between vertebrate flippases and specific synteny of zebrafish and human chromosomes. Both atp11b and atp11c are maternally expressed in zebrafish, while zygotic expression analysis demonstrates tissue and temporal specificity for all three genes. atp11a is expressed in the neural crest cells as well as in the developing eye and ear, while atp11b is expressed early in the ventricular epithelial lining and later in the ear. atp11c is expressed in the anterior most rhombomeres of the hindbrain, pharyngeal arches, and liver. Our expression data suggests that each of the three flippases are integral for the development of specific tissues, and aberrant function of either could lead to visual, hearing, neural, or liver dysfunction.
Assuntos
Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Animais , Membrana Celular/enzimologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Hibridização In Situ , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Modelos Animais , Fosfatidilserinas/metabolismo , Fosfolipídeos/metabolismo , Filogenia , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Vascular smooth muscle of the head derives from neural crest, but developmental mechanisms and early transcriptional drivers of the vSMC lineage are not well characterized. We find that in early development, the transcription factor foxc1b is expressed in mesenchymal cells that associate with the vascular endothelium. Using timelapse imaging, we observe that foxc1b expressing mesenchymal cells differentiate into acta2 expressing vascular mural cells. We show that in zebrafish, while foxc1b is co-expressed in acta2 positive smooth muscle cells that associate with large diameter vessels, it is not co-expressed in capillaries where pdgfrß positive pericytes are located. In addition to being an early marker of the lineage, foxc1 is essential for vSMC differentiation; we find that foxc1 loss of function mutants have defective vSMC differentiation and that early genetic ablation of foxc1b or acta2 expressing populations blocks vSMC differentiation. Furthermore, foxc1 is expressed upstream of acta2 and is required for acta2 expression in vSMCs. Using RNA-Seq we determine an enriched intersectional gene expression profile using dual expression of foxc1b and acta2 to identify novel vSMC markers. Taken together, our data suggests that foxc1 is a marker of vSMCs and plays a critical functional role in promoting their differentiation.
Assuntos
Diferenciação Celular , Embrião não Mamífero/citologia , Fatores de Transcrição Forkhead/metabolismo , Cabeça/irrigação sanguínea , Cabeça/embriologia , Músculo Liso Vascular/citologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Encéfalo/embriologia , Encéfalo/metabolismo , Diferenciação Celular/genética , Embrião não Mamífero/metabolismo , Endotélio/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/metabolismo , Miócitos de Músculo Liso/metabolismo , Pericitos/metabolismo , Transcriptoma/genética , Regulação para Cima , Peixe-Zebra/genéticaRESUMO
Mutation of FOXC1 causes Axenfeld-Rieger Syndrome (ARS) with early onset or congenital glaucoma. We assessed retinal ganglion cell (RGC) number in zebrafish due to CRISPR-mediated mutation and antisense inhibition of two-forkhead box transcription factors, foxc1a and foxc1b. These genes represent duplicated homologues of human FOXC1. Using a CRISPR induced null mutation in foxc1b, in combination with antisense inhibition of foxc1a, we demonstrate reduced cell number in the retinal ganglion cell layer of developing zebrafish eyes. As early as 5â¯days post fertilization (dpf), fewer RGCs are found in foxc1b homozygous mutants injected with foxc1a morpholinos, and a thinner optic nerve results. Our data illustrates that foxc1 is required for the expression of atonal homolog 7 (atoh7), a gene that is necessary for RGC differentiation. As markers of differentiated RGCs (pou4f2) are downregulated in foxc1b-/- mutants injected with foxc1a morpholinos and no cell death is observed, our results are consistent with defects in the differentiation of RGCs leading to reduced cell number, as opposed to increased cell death of RGCs or off targets effects of morpholino injection. Our zebrafish model demonstrates that aberrant regulation of RGC number could act in concert with other known glaucoma risk factors to influence the development of congenital and early onset glaucoma due to FOXC1 mutation.
Assuntos
Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Mutação , Nervo Óptico/embriologia , Células Ganglionares da Retina/patologia , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Animais , Axônios/patologia , Contagem de Células , Morte Celular , Diferenciação Celular , Proteínas de Ligação a DNA/genética , Embrião não Mamífero , Inativação Gênica/efeitos dos fármacos , Glaucoma/embriologia , Glaucoma/genética , Hibridização In Situ , Morfolinos/farmacologia , Reação em Cadeia da Polimerase , TransfecçãoRESUMO
Patients with cerebral small-vessel disease (CSVD) exhibit perturbed end-artery function and have an increased risk for stroke and age-related cognitive decline. Here, we used targeted genome-wide association (GWA) analysis and defined a CSVD locus adjacent to the forkhead transcription factor FOXC1. Moreover, we determined that the linked SNPs influence FOXC1 transcript levels and demonstrated that patients as young as 1 year of age with altered FOXC1 function exhibit CSVD. MRI analysis of patients with missense and nonsense mutations as well as FOXC1-encompassing segmental duplication and deletion revealed white matter hyperintensities, dilated perivascular spaces, and lacunar infarction. In a zebrafish model, overexpression or morpholino-induced suppression of foxc1 induced cerebral hemorrhage. Inhibition of foxc1 perturbed platelet-derived growth factor (Pdgf) signaling, impairing neural crest migration and the recruitment of mural cells, which are essential for vascular stability. GWA analysis also linked the FOXC1-interacting transcription factor PITX2 to CSVD, and both patients with PITX2 mutations and murine Pitx2-/- mutants displayed brain vascular phenotypes. Together, these results extend the genetic etiology of stroke and demonstrate an increasing developmental basis for human cerebrovascular disease.
Assuntos
Doenças de Pequenos Vasos Cerebrais/genética , Fatores de Transcrição Forkhead/genética , Proteínas de Homeodomínio/genética , Fatores de Transcrição/genética , Animais , Hemorragia Cerebral/genética , Códon sem Sentido , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Leucoencefalopatias/genética , Desequilíbrio de Ligação , Mutação de Sentido Incorreto , Fator de Crescimento Derivado de Plaquetas/fisiologia , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Transdução de Sinais , Peixe-Zebra , Proteína Homeobox PITX2RESUMO
PURPOSE: To characterize the molecular mechanisms underlying retinal apoptosis induced by loss of Gdf6, a TGFß ligand. METHODS: The role of Gdf6 in regulating apoptosis was studied using a zebrafish gdf6a(-/-) mutant, which encodes a truncated, nonfunctional protein. To investigate whether intrinsic or extrinsic apoptotic mechanisms were involved, morpholino antisense oligonucleotides targeting baxa, baxb, and p53 were employed. Caspase-3 immunohistochemistry (IHC) was performed to assay apoptosis. Pharmacologic inhibition (using SB203580) and IHC were used to investigate the role of p38 mitogen activated protein (MAP) kinase activation in gdf6a(-/-)-induced apoptosis. To assess the role of Gdf6a in transcriptional regulation of TGFß signal transducers, in situ hybridization (ISH) was performed using probes to smad1, 5, 7, and 8. RESULTS: Results indicate maximal ocular apoptosis occurs 28 hours post fertilization (hpf) in gdf6a(-/-) mutants that is mediated independently of p53 by intrinsic mechanisms involving Bax proteins. Also, gdf6a(-/-) mutants exhibit markedly increased p38 MAP kinase activation that can be inhibited to significantly reduce retinal apoptosis. A reduction in retinal smad1 expression was also noted in gdf6a(-/-) mutants. CONCLUSIONS: gdf6a(-/-)-induced apoptosis is characterized by the involvement of intrinsic apoptotic pathways, p38 MAP kinases, and dysregulated smad expression. Modulation of key mediators can inhibit retinal apoptosis offering potential avenues of therapy. However, the efficacy of pharmacomodulation in improvement of visual function needs to be further examined.
Assuntos
Apoptose/fisiologia , Fator 6 de Diferenciação de Crescimento/fisiologia , Retina/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Caspase 3/metabolismo , Fator 6 de Diferenciação de Crescimento/genética , Imuno-Histoquímica , Retina/metabolismo , Transdução de Sinais/fisiologia , Proteína Smad1/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologiaRESUMO
PURPOSE: Vision is critically dependent on ocular size, which is regulated by environmental and genetic factors. Mutation of human Growth and Differentiation Factor 6 (GDF6) or zebrafish gdf6a results in a spectrum of small eye phenotypes (microphthalmia, anophthalmia, and coloboma). However, current models do not explain their etiology fully. As such, analyses of apoptosis and cell cycle regulation were undertaken in a zebrafish gdf6a mutant. METHODS: Microarray analysis was performed at 2 days after fertilization to uncover novel gdf6a-dependent cell cycle regulators. Altered expression of Gdf6a targets was confirmed by in situ hybridization, and resulting changes in cell proliferation were assessed by phosphohistone H3 immunohistochemistry. Analysis of apoptosis was evaluated through activated Caspase 3 immunohistochemistry and chemical inhibitors of cell death. RESULTS: Reduced numbers of retinal progenitor cells are observed at 24 hours post fertilization (hpf), resulting in microphthalmic eyes in gdf6a(-/-) embryos. At 28 hpf, a wave of apoptosis occurs; however, apoptosis inhibition does not rescue eye size, indicating a limited contribution. Mutants display altered proliferation and expression levels of cell cycle regulators, including members of the forkhead box i (foxi) transcription factor family expressed in the ciliary marginal zone. Notably, inhibition of foxi2 in gdf6a(-/-) embryos further reduces eye size. CONCLUSIONS: These data support a model whereby the gdf6a(-/-)-induced microphthalmia is based on early regulation of retinal progenitor cell number, and later by regulation of proliferation in the ciliary marginal zone. Foxi genes represent downstream effectors of Gdf6a function in the CMZ required for eye size determination.
Assuntos
Apoptose/fisiologia , Proteínas Morfogenéticas Ósseas/fisiologia , Proliferação de Células , Microftalmia/patologia , Animais , Ciclo Celular/genética , Modelos Animais de Doenças , Embrião não Mamífero , Olho/embriologia , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Fator 6 de Diferenciação de Crescimento/metabolismo , Imuno-Histoquímica , Análise em Microsséries , Microftalmia/genética , Microftalmia/fisiopatologia , Células-Tronco/patologia , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/metabolismoRESUMO
Retinal dystrophies are predominantly caused by mutations affecting the visual phototransduction system and cilia, with few genes identified that function to maintain photoreceptor survival. We reasoned that growth factors involved with early embryonic retinal development would represent excellent candidates for such diseases. Here we show that mutations in the transforming growth factor-ß (TGF-ß) ligand Growth Differentiation Factor 6, which specifies the dorso-ventral retinal axis, contribute to Leber congenital amaurosis. Furthermore, deficiency of gdf6 results in photoreceptor degeneration, so demonstrating a connection between Gdf6 signaling and photoreceptor survival. In addition, in both murine and zebrafish mutant models, we observe retinal apoptosis, a characteristic feature of human retinal dystrophies. Treatment of gdf6-deficient zebrafish embryos with a novel aminopropyl carbazole, P7C3, rescued the retinal apoptosis without evidence of toxicity. These findings implicate for the first time perturbed TGF-ß signaling in the genesis of retinal dystrophies, support the study of related morphogenetic genes for comparable roles in retinal disease and may offer additional therapeutic opportunities for genetically heterogeneous disorders presently only treatable with gene therapy.
Assuntos
Sobrevivência Celular , Fator 6 de Diferenciação de Crescimento/genética , Amaurose Congênita de Leber/genética , Retinose Pigmentar/genética , Sequência de Aminoácidos , Animais , Apoptose , Análise Mutacional de DNA , Estudos de Associação Genética , Fator 6 de Diferenciação de Crescimento/fisiologia , Humanos , Amaurose Congênita de Leber/patologia , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Linhagem , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/fisiologia , Retina/patologia , Retinose Pigmentar/patologia , Peixe-ZebraRESUMO
In this review, we highlight recent literature concerning the signaling mechanisms underlying the development of two neural birth defects, holoprosencephaly and coloboma. Holoprosencephaly, the most common forebrain defect, occurs when the cerebral hemispheres fail to separate and is typically associated with mispatterning of embryonic midline tissue. Coloboma results when the choroid fissure in the eye fails to close. It is clear that Sonic hedgehog (Shh) signaling regulates both forebrain and eye development, with defects in Shh, or components of the Shh signaling cascade leading to the generation of both birth defects. In addition, other intercellular signaling pathways are known factors in the incidence of holoprosencephaly and coloboma. This review will outline recent advances in our understanding of forebrain and eye embryonic pattern formation, with a focus on zebrafish studies of Shh and retinoic acid pathways. Given the clear overlap in the mechanisms that generate both diseases, we propose that holoprosencephaly and coloboma can represent mild and severe aspects of single phenotypic spectrum resulting from aberrant forebrain development. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases.
Assuntos
Coloboma/etiologia , Regulação da Expressão Gênica no Desenvolvimento , Holoprosencefalia/etiologia , Prosencéfalo/anormalidades , Prosencéfalo/metabolismo , Transdução de Sinais , Animais , Coloboma/metabolismo , Coloboma/patologia , Modelos Animais de Doenças , Holoprosencefalia/metabolismo , Holoprosencefalia/patologia , Humanos , Prosencéfalo/embriologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
BACKGROUND: During visual system development, multiple signalling pathways cooperate to specify axial polarity within the retina and optic tectum. This information is required for the topographic mapping of retinal ganglion cell axons on the tectum. Meis1 is a TALE-class homeodomain transcription factor known to specify anterior-posterior identity in the hindbrain, but its role in visual system patterning has not been investigated. RESULTS: meis1 is expressed in both the presumptive retina and tectum. An analysis of retinal patterning reveals that Meis1 is required to correctly specify both dorsal-ventral and nasal-temporal identity in the zebrafish retina. Meis1-knockdown results in a loss of smad1 expression and an upregulation in follistatin expression, thereby causing lower levels of Bmp signalling and a partial ventralization of the retina. Additionally, Meis1-deficient embryos exhibit ectopic Fgf signalling in the developing retina and a corresponding loss of temporal identity. Meis1 also positively regulates ephrin gene expression in the tectum. Consistent with these patterning phenotypes, a knockdown of Meis1 ultimately results in retinotectal mapping defects. CONCLUSIONS: In this work we describe a novel role for Meis1 in regulating Bmp signalling and in specifying temporal identity in the retina. By patterning both the retina and tectum, Meis1 plays an important role in establishing the retinotectal map and organizing the visual system.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Retina/embriologia , Colículos Superiores/embriologia , Aminoácidos , Animais , Animais Geneticamente Modificados , Apolipoproteína A-II/genética , Apolipoproteína A-II/metabolismo , Padronização Corporal/genética , Carbocianinas , Embrião não Mamífero , Perfilação da Expressão Gênica/métodos , Fator 6 de Diferenciação de Crescimento/química , Fator 6 de Diferenciação de Crescimento/genética , Proteínas de Homeodomínio/química , Mutação/genética , Proteína Meis1 , Proteínas de Neoplasias/química , Oligodesoxirribonucleotídeos Antissenso/farmacologia , RNA Mensageiro/farmacologia , Retina/citologia , Retina/efeitos dos fármacos , Células Ganglionares da Retina/fisiologia , Transdução de Sinais/genética , Proteína Smad5/química , Proteína Smad5/genética , Colículos Superiores/citologia , Colículos Superiores/efeitos dos fármacos , Peixe-Zebra , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Ocular mal-development results in heterogeneous and frequently visually disabling phenotypes that include coloboma and microphthalmia. Due to the contribution of bone morphogenetic proteins to such processes, the function of the paralogue Growth Differentiation Factor 3 was investigated. Multiple mis-sense variants were identified in patients with ocular and/or skeletal (Klippel-Feil) anomalies including one individual with heterozygous alterations in GDF3 and GDF6. These variants were characterized, individually and in combination, through integrated biochemical and zebrafish model organism analyses, demonstrating appreciable effects with western blot analyses, luciferase based reporter assays and antisense morpholino inhibition. Notably, inhibition of the zebrafish co-orthologue of GDF3 accurately recapitulates patient phenotypes. By demonstrating the pleiotropic effects of GDF3 mutation, these results extend the contribution of perturbed BMP signaling to human disease and potentially implicate multi-allelic inheritance of BMP variants in developmental disorders.
Assuntos
Anormalidades do Olho/genética , Fator 3 de Diferenciação de Crescimento/genética , Músculo Esquelético/anormalidades , Mutação , Sequência de Aminoácidos , Animais , Linhagem Celular , Anormalidades do Olho/metabolismo , Feminino , Fator 3 de Diferenciação de Crescimento/química , Fator 3 de Diferenciação de Crescimento/metabolismo , Humanos , Masculino , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Linhagem , Alinhamento de Sequência , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Dorsal-ventral patterning of the vertebrate retina is essential for accurate topographic mapping of retinal ganglion cell (RGC) axons to visual processing centers. Bone morphogenetic protein (Bmp) growth factors regulate dorsal retinal identity in vertebrate models, but the developmental timing of this signaling and the relative roles of individual Bmps remain unclear. In this study, we investigate the functions of two zebrafish Bmps, Gdf6a and Bmp4, during initiation of dorsal retinal identity, and subsequently during lens differentiation. Knockdown of zebrafish Gdf6a blocks initiation of retinal Smad phosphorylation and dorsal marker expression, while knockdown of Bmp4 produces no discernable retinal phenotype. These data, combined with analyses of embryos ectopically expressing Bmps, demonstrate that Gdf6a is necessary and sufficient for initiation of dorsal retinal identity. We note a profound expansion of ventral retinal identity in gdf6a morphants, demonstrating that dorsal BMP signaling antagonizes ventral marker expression. Finally, we demonstrate a role for Gdf6a in non-neural ocular tissues. Knockdown of Gdf6a leads to defects in lens-specific gene expression, and when combined with Bmp signaling inhibitors, disrupts lens fiber cell differentiation. Taken together, these data indicate that Gdf6a initiates dorsal retinal patterning independent of Bmp4, and regulates lens differentiation.
Assuntos
Fator 6 de Diferenciação de Crescimento/fisiologia , Cristalino/embriologia , Retina/embriologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Padronização Corporal/fisiologia , Proteína Morfogenética Óssea 4/biossíntese , Proteína Morfogenética Óssea 4/genética , Embrião não Mamífero/fisiologia , Técnicas de Silenciamento de Genes , Fator 6 de Diferenciação de Crescimento/biossíntese , Fator 6 de Diferenciação de Crescimento/genética , Cristalino/metabolismo , Retina/metabolismo , Transdução de Sinais/fisiologia , Proteínas Smad/antagonistas & inibidores , Proteínas Smad/fisiologia , Proteínas com Domínio T/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/biossíntese , Proteínas de Peixe-Zebra/genéticaRESUMO
Proteins of the bone morphogenetic protein (BMP) family are known to have a role in ocular and skeletal development; however, because of their widespread expression and functional redundancy, less progress has been made identifying the roles of individual BMPs in human disease. We identified seven heterozygous mutations in growth differentiation factor 6 (GDF6), a member of the BMP family, in patients with both ocular and vertebral anomalies, characterized their effects with a SOX9-reporter assay and western analysis, and demonstrated comparable phenotypes in model organisms with reduced Gdf6 function. We observed a spectrum of ocular and skeletal anomalies in morphant zebrafish, the latter encompassing defective tail formation and altered expression of somite markers noggin1 and noggin2. Gdf6(+/-) mice exhibited variable ocular phenotypes compatible with phenotypes observed in patients and zebrafish. Key differences evident between patients and animal models included pleiotropic effects, variable expressivity and incomplete penetrance. These data establish the important role of this determinant in ocular and vertebral development, demonstrate the complex genetic inheritance of these phenotypes, and further understanding of BMP function and its contributions to human disease.
Assuntos
Anormalidades Múltiplas/genética , Anormalidades Múltiplas/patologia , Fator 6 de Diferenciação de Crescimento/genética , Penetrância , Sequência de Aminoácidos , Animais , Análise Mutacional de DNA , Genes Reporter , Fator 6 de Diferenciação de Crescimento/química , Humanos , Camundongos , Modelos Animais , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Mutação/genética , Oligonucleotídeos Antissenso/farmacologia , Peixe-Zebra , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genéticaRESUMO
BACKGROUND: Obesity is caused by the excessive accumulation of adipose tissue as a result of a chronic energy surplus. Little is known regarding the molecular mechanisms involved in the response to an energy surplus in human adipose tissue at the genomic level. OBJECTIVE: The objective was to investigate changes in the transcriptome of abdominal subcutaneous adipose tissue after a positive energy challenge induced by overfeeding in both lean and obese subjects to identify novel obesity candidate genes. DESIGN: A total of 26 men were recruited and classified on the basis of percentage body fat (measured by dual-energy X-ray absorptiometry) as lean (<20%) or obese (>25%) to participate in the baseline comparison. Sixteen men participated in the overfeeding study (8 lean and 8 obese). Adipose tissue biopsy samples were collected from all subjects at the subumbilical region. Global gene expression profiles were determined at baseline and after a 7-d hypercaloric diet at 40% above normal energy requirements by using whole human genome DNA microarrays. RESULTS: Overfeeding induced differential expression in 45 genes. Six genes displayed a significant interaction effect between adiposity status and overfeeding treatment, including transferrin (TF), stearoyl-CoA desaturase (SCD), transaldolase 1 (TALDO1), cathepsin C (CTSC), insulin receptor substrate 2 (IRS2), and pyruvate dehydrogenase kinase, isozyme 4 (PDK4). Overfeeding resulted in changes in expression of these genes in lean subjects, whereas no significant changes were evident in obese subjects. CONCLUSIONS: Differential expression of these 6 genes may represent a protective mechanism at the molecular level in lean subjects in response to an energy surplus. These genes represent valuable candidates for downstream studies related to obesity.