RESUMEN
The liver is the largest solid organ in the body, yet it remains incompletely characterized. Here we present a spatial proteogenomic atlas of the healthy and obese human and murine liver combining single-cell CITE-seq, single-nuclei sequencing, spatial transcriptomics, and spatial proteomics. By integrating these multi-omic datasets, we provide validated strategies to reliably discriminate and localize all hepatic cells, including a population of lipid-associated macrophages (LAMs) at the bile ducts. We then align this atlas across seven species, revealing the conserved program of bona fide Kupffer cells and LAMs. We also uncover the respective spatially resolved cellular niches of these macrophages and the microenvironmental circuits driving their unique transcriptomic identities. We demonstrate that LAMs are induced by local lipid exposure, leading to their induction in steatotic regions of the murine and human liver, while Kupffer cell development crucially depends on their cross-talk with hepatic stellate cells via the evolutionarily conserved ALK1-BMP9/10 axis.
Asunto(s)
Evolución Biológica , Hepatocitos/metabolismo , Macrófagos/metabolismo , Proteogenómica , Animales , Núcleo Celular/metabolismo , Hígado Graso/genética , Hígado Graso/patología , Homeostasis , Humanos , Macrófagos del Hígado/metabolismo , Antígenos Comunes de Leucocito/metabolismo , Lípidos/química , Hígado/metabolismo , Linfocitos/metabolismo , Ratones Endogámicos C57BL , Modelos Biológicos , Células Mieloides/metabolismo , Obesidad/patología , Proteoma/metabolismo , Transducción de Señal , Transcriptoma/genéticaRESUMEN
Bi-allelic mutations in the gene coding for human trans-membrane anterior-posterior transformation protein 1 (TAPT1) result in a broad phenotypic spectrum, ranging from syndromic disease with severe skeletal and congenital abnormalities to isolated early-onset cataract. We present here the first patient with a frameshift mutation in the TAPT1 gene, resulting in both bilateral early-onset cataract and skeletal abnormalities, in addition to several dysmorphic features, in this way further expanding the phenotypic spectrum associated with TAPT1 mutations. A tapt1a/tapt1b double knock-out (KO) zebrafish model generated by CRISPR/Cas9 gene editing revealed an early larval phenotype with eye malformations, loss of vision, increased photokinetics and hyperpigmentation, without visible skeletal involvement. Ultrastructural analysis of the eyes showed a smaller condensed lens, loss of integrity of the lens capsule with formation of a secondary lens and hyperplasia of the cells in the ganglion and inner plexiform layers of the retina. Transcriptomic analysis pointed to an impaired lens development with aberrant expression of many of the crystallin and other lens-specific genes. Furthermore, the phototransduction and visual perception pathways were found to be significantly disturbed. Differences in light perception are likely the cause of the increased dark photokinetics and generalized hyperpigmentation observed in this zebrafish model. In conclusion, this study validates TAPT1 as a new gene for early-onset cataract and sheds light on its ultrastructural and molecular characteristics.
Asunto(s)
Catarata , Cristalino , Animales , Humanos , Catarata/genética , Cristalino/metabolismo , Mutación , Retina/metabolismo , Pez Cebra/genética , Proteínas de la Membrana/metabolismoRESUMEN
Arterial tortuosity syndrome (ATS) is a recessively inherited connective tissue disorder, mainly characterized by tortuosity and aneurysm formation of the major arteries. ATS is caused by loss-of-function mutations in SLC2A10, encoding the facilitative glucose transporter GLUT10. Former studies implicated GLUT10 in the transport of dehydroascorbic acid, the oxidized form of ascorbic acid (AA). Mouse models carrying homozygous Slc2a10 missense mutations did not recapitulate the human phenotype. Since mice, in contrast to humans, are able to intracellularly synthesize AA, we generated a novel ATS mouse model, deficient for Slc2a10 as well as Gulo, which encodes for L-gulonolactone oxidase, an enzyme catalyzing the final step in AA biosynthesis in mouse. Gulo;Slc2a10 double knock-out mice showed mild phenotypic anomalies, which were absent in single knock-out controls. While Gulo;Slc2a10 double knock-out mice did not fully phenocopy human ATS, histological and immunocytochemical analysis revealed compromised extracellular matrix formation. Transforming growth factor beta signaling remained unaltered, while mitochondrial function was compromised in smooth muscle cells derived from Gulo;Slc2a10 double knock-out mice. Altogether, our data add evidence that ATS is an ascorbate compartmentalization disorder, but additional factors underlying the observed phenotype in humans remain to be determined.
Asunto(s)
Arterias/anomalías , Deficiencia de Ácido Ascórbico/genética , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Inestabilidad de la Articulación/genética , L-Gulonolactona Oxidasa/genética , Enfermedades Cutáneas Genéticas/genética , Malformaciones Vasculares/genética , Animales , Arterias/metabolismo , Arterias/patología , Ácido Ascórbico/biosíntesis , Ácido Ascórbico/genética , Deficiencia de Ácido Ascórbico/metabolismo , Deficiencia de Ácido Ascórbico/patología , Modelos Animales de Enfermedad , Homocigoto , Humanos , Inestabilidad de la Articulación/metabolismo , Inestabilidad de la Articulación/patología , Ratones , Ratones Noqueados , Mitocondrias/genética , Mitocondrias/metabolismo , Mitocondrias/patología , Respiración/genética , Transducción de Señal/genética , Enfermedades Cutáneas Genéticas/metabolismo , Enfermedades Cutáneas Genéticas/patología , Malformaciones Vasculares/metabolismo , Malformaciones Vasculares/patologíaRESUMEN
The type I collagenopathies are a group of heterogeneous connective tissue disorders, that are caused by mutations in the genes encoding type I collagen and include specific forms of osteogenesis imperfecta (OI) and the Ehlers-Danlos syndrome (EDS). These disorders present with a broad disease spectrum and large clinical variability of which the underlying genetic basis is still poorly understood. In this study, we systematically analyzed skeletal phenotypes in a large set of zebrafish, with diverse mutations in the genes encoding type I collagen, representing different genetic forms of human OI, and a zebrafish model resembling human EDS, which harbors a number of soft connective tissues defects, typical of EDS. Furthermore, we provide insight into how zebrafish and human type I collagen are compositionally and functionally related, which is relevant in the interpretation of human type I collagen-related disease models. Our studies reveal a high degree of intergenotype variability in phenotypic expressivity that closely correlates with associated OI severity. Furthermore, we demonstrate the potential for select mutations to give rise to phenotypic variability, mirroring the clinical variability associated with human disease pathology. Therefore, our work suggests the future potential for zebrafish to aid in identifying unknown genetic modifiers and mechanisms underlying the phenotypic variability in OI and related disorders. This will improve diagnostic strategies and enable the discovery of new targetable pathways for pharmacological intervention.
Asunto(s)
Colágeno Tipo I , Modelos Animales de Enfermedad , Síndrome de Ehlers-Danlos , Osteogénesis Imperfecta , Pez Cebra , Animales , Animales Modificados Genéticamente , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Síndrome de Ehlers-Danlos/genética , Síndrome de Ehlers-Danlos/metabolismo , Síndrome de Ehlers-Danlos/patología , Humanos , Osteogénesis Imperfecta/genética , Osteogénesis Imperfecta/metabolismo , Osteogénesis Imperfecta/patología , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
One of the most frequently applied techniques in zebrafish (Danio rerio) research is the visualisation or manipulation of specific cell populations using transgenic reporter lines. The generation of these transgenic zebrafish, displaying cell- or tissue-specific expression of frequently used fluorophores such as Green Fluorescent Protein (GFP) or mCherry, is relatively easy using modern techniques. Fluorophores with different emission wavelengths and driven by different promoters can be monitored simultaneously in the same animal. Photoconvertible fluorescent proteins (pcFPs) are different from these standard fluorophores because their emission spectrum is changed when exposed to UV light, a process called photoconversion. Here, the benefits and versatility of using pcFPs for both single and dual fluorochrome imaging in zebrafish skeletal research in a previously generated osx:Kaede transgenic line are illustrated. In this line, Kaede, which is expressed under control of the osterix, otherwise known as sp7, promoter thereby labelling immature osteoblasts, can switch from green to red fluorescence upon irradiation with UV light. First, this study demonstrates that osx:Kaede exhibits an expression pattern similar to a previously described osx:nuGFP transgenic line in both larval and adult stages, hereby validating the use of this line for the imaging of immature osteoblasts. More in-depth experiments highlight different applications for osx:Kaede, such as lineage tracing and its combined use with in vivo skeletal staining and other transgenic backgrounds. Mineral staining in combination with osx:Kaede confirms osteoblast-independent mineralisation of the notochord. Osteoblast lineage tracing reveals migration and dedifferentiation of scleroblasts during fin regeneration. Finally, this study shows that combining two transgenics, osx:Kaede and osc:GFP, with similar emission wavelengths is possible when using a pcFP such as Kaede.
Asunto(s)
Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Imagen Óptica , Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente , Colorantes Fluorescentes/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
BACKGROUND: Transgenic zebrafish lines with the expression of a fluorescent reporter under the control of a cell-type specific promoter, enable transcriptome analysis of FACS sorted cell populations. RNA quality and yield are key determinant factors for accurate expression profiling. Limited cell number and FACS induced cellular stress make RNA isolation of sorted zebrafish cells a delicate process. We aimed to optimize a workflow to extract sufficient amounts of high-quality RNA from a limited number of FACS sorted cells from Tg(fli1a:GFP) zebrafish embryos, which can be used for accurate gene expression analysis. RESULTS: We evaluated two suitable RNA isolation kits (the RNAqueous micro and the RNeasy plus micro kit) and determined that sorting cells directly into lysis buffer is a critical step for success. For low cell numbers, this ensures direct cell lysis, protects RNA from degradation and results in a higher RNA quality and yield. We showed that this works well up to 0.5× dilution of the lysis buffer with sorted cells. In our sort settings, this corresponded to 30,000 and 75,000 cells for the RNAqueous micro kit and RNeasy plus micro kit respectively. Sorting more cells dilutes the lysis buffer too much and requires the use of a collection buffer. We also demonstrated that an additional genomic DNA removal step after RNA isolation is required to completely clear the RNA from any contaminating genomic DNA. For cDNA synthesis and library preparation, we combined SmartSeq v4 full length cDNA library amplification, Nextera XT tagmentation and sample barcoding. Using this workflow, we were able to generate highly reproducible RNA sequencing results. CONCLUSIONS: The presented optimized workflow enables to generate high quality RNA and allows accurate transcriptome profiling of small populations of sorted zebrafish cells.
Asunto(s)
Citometría de Flujo , ARN/genética , ARN/aislamiento & purificación , Análisis de Secuencia de ARN , Pez Cebra/genética , Animales , Recuento de Células , Poli A/genética , Control de CalidadRESUMEN
The evolutionarily conserved transmembrane anterior posterior transformation 1 protein, encoded by TAPT1, is involved in murine axial skeletal patterning, but its cellular function remains unknown. Our study demonstrates that TAPT1 mutations underlie a complex congenital syndrome, showing clinical overlap between lethal skeletal dysplasias and ciliopathies. This syndrome is characterized by fetal lethality, severe hypomineralization of the entire skeleton and intra-uterine fractures, and multiple congenital developmental anomalies affecting the brain, lungs, and kidneys. We establish that wild-type TAPT1 localizes to the centrosome and/or ciliary basal body, whereas defective TAPT1 mislocalizes to the cytoplasm and disrupts Golgi morphology and trafficking and normal primary cilium formation. Knockdown of tapt1b in zebrafish induces severe craniofacial cartilage malformations and delayed ossification, which is shown to be associated with aberrant differentiation of cranial neural crest cells.
Asunto(s)
Cilios/genética , Trastornos de la Motilidad Ciliar/genética , Anomalías Craneofaciales/genética , Proteínas de la Membrana/genética , Mutación/genética , Osificación Heterotópica/genética , Osteocondrodisplasias/genética , Secuencia de Aminoácidos , Animales , Tipificación del Cuerpo , Diferenciación Celular , Movimiento Celular , Cilios/metabolismo , Cilios/patología , Embrión no Mamífero/anomalías , Femenino , Regulación del Desarrollo de la Expresión Génica , Humanos , Hibridación in Situ , Masculino , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Cresta Neural/citología , Cresta Neural/metabolismo , Linaje , Transporte de Proteínas , Homología de Secuencia de Aminoácido , Transducción de Señal , Pez Cebra/embriología , Pez Cebra/genéticaRESUMEN
Recently, we identified in two individuals with intellectual disability (ID) different de novo mutations in DEAF1, which encodes a transcription factor with an important role in embryonic development. To ascertain whether these mutations in DEAF1 are causative for the ID phenotype, we performed targeted resequencing of DEAF1 in an additional cohort of over 2,300 individuals with unexplained ID and identified two additional individuals with de novo mutations in this gene. All four individuals had severe ID with severely affected speech development, and three showed severe behavioral problems. DEAF1 is highly expressed in the CNS, especially during early embryonic development. All four mutations were missense mutations affecting the SAND domain of DEAF1. Altered DEAF1 harboring any of the four amino acid changes showed impaired transcriptional regulation of the DEAF1 promoter. Moreover, behavioral studies in mice with a conditional knockout of Deaf1 in the brain showed memory deficits and increased anxiety-like behavior. Our results demonstrate that mutations in DEAF1 cause ID and behavioral problems, most likely as a result of impaired transcriptional regulation by DEAF1.
Asunto(s)
Discapacidad Intelectual/genética , Trastornos Mentales/genética , Proteínas Nucleares/genética , Trastornos del Habla/genética , Secuencia de Aminoácidos , Animales , Niño , Estudios de Cohortes , Análisis Mutacional de ADN , Proteínas de Unión al ADN , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Mutación , Estructura Terciaria de Proteína/genética , Factores de TranscripciónRESUMEN
GLUT10 belongs to a family of transporters that catalyze the uptake of sugars/polyols by facilitated diffusion. Loss-of-function mutations in the SLC2A10 gene encoding GLUT10 are responsible for arterial tortuosity syndrome (ATS). Since subcellular distribution of the transporter is dubious, we aimed to clarify the localization of GLUT10. In silico GLUT10 localization prediction suggested its presence in the endoplasmic reticulum (ER). Immunoblotting showed the presence of GLUT10 protein in the microsomal, but not in mitochondrial fractions of human fibroblasts and liver tissue. An even cytosolic distribution with an intense perinuclear decoration of GLUT10 was demonstrated by immunofluorescence in human fibroblasts, whilst mitochondrial markers revealed a fully different decoration pattern. GLUT10 decoration was fully absent in fibroblasts from three ATS patients. Expression of exogenous, tagged GLUT10 in fibroblasts from an ATS patient revealed a strict co-localization with the ER marker protein disulfide isomerase (PDI). The results demonstrate that GLUT10 is present in the ER.
Asunto(s)
Arterias/anomalías , Retículo Endoplásmico/metabolismo , Fibroblastos/metabolismo , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Inestabilidad de la Articulación/metabolismo , Enfermedades Cutáneas Genéticas/metabolismo , Malformaciones Vasculares/metabolismo , Arterias/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Espacio Intracelular/metabolismo , Inestabilidad de la Articulación/genética , Microsomas/metabolismo , Unión Proteica , Transporte de Proteínas , Enfermedades Cutáneas Genéticas/genética , Malformaciones Vasculares/genéticaRESUMEN
Proteoglycans are important components of cell plasma membranes and extracellular matrices of connective tissues. They consist of glycosaminoglycan chains attached to a core protein via a tetrasaccharide linkage, whereby the addition of the third residue is catalyzed by galactosyltransferase II (ß3GalT6), encoded by B3GALT6. Homozygosity mapping and candidate gene sequence analysis in three independent families, presenting a severe autosomal-recessive connective tissue disorder characterized by skin fragility, delayed wound healing, joint hyperlaxity and contractures, muscle hypotonia, intellectual disability, and a spondyloepimetaphyseal dysplasia with bone fragility and severe kyphoscoliosis, identified biallelic B3GALT6 mutations, including homozygous missense mutations in family 1 (c.619G>C [p.Asp207His]) and family 3 (c.649G>A [p.Gly217Ser]) and compound heterozygous mutations in family 2 (c.323_344del [p.Ala108Glyfs(∗)163], c.619G>C [p.Asp207His]). The phenotype overlaps with several recessive Ehlers-Danlos variants and spondyloepimetaphyseal dysplasia with joint hyperlaxity. Affected individuals' fibroblasts exhibited a large decrease in ability to prime glycosaminoglycan synthesis together with impaired glycanation of the small chondroitin/dermatan sulfate proteoglycan decorin, confirming ß3GalT6 loss of function. Dermal electron microcopy disclosed abnormalities in collagen fibril organization, in line with the important regulatory role of decorin in this process. A strong reduction in heparan sulfate level was also observed, indicating that ß3GalT6 deficiency alters synthesis of both main types of glycosaminoglycans. In vitro wound healing assay revealed a significant delay in fibroblasts from two index individuals, pointing to a role for glycosaminoglycan defect in impaired wound repair in vivo. Our study emphasizes a crucial role for ß3GalT6 in multiple major developmental and pathophysiological processes.
Asunto(s)
Anomalías Múltiples/genética , Síndrome de Ehlers-Danlos/genética , Galactosiltransferasas/genética , Glicosaminoglicanos/biosíntesis , Anomalías Múltiples/diagnóstico por imagen , Anomalías Múltiples/metabolismo , Adulto , Secuencia de Aminoácidos , Secuencia de Bases , Niño , Consanguinidad , Síndrome de Ehlers-Danlos/diagnóstico por imagen , Síndrome de Ehlers-Danlos/metabolismo , Femenino , Estudios de Asociación Genética , Pleiotropía Genética , Humanos , Lactante , Masculino , Datos de Secuencia Molecular , Mutación Missense , Linaje , Radiografía , Análisis de Secuencia de ADN , Cicatrización de Heridas/genéticaRESUMEN
PURPOSE: Submicroscopic deletions of chromosome band 2p25.3 are associated with intellectual disability and/or central obesity. Although MYT1L is believed to be a critical gene responsible for intellectual disability, so far no unequivocal data have confirmed this hypothesis. METHODS: In this study we evaluated a cohort of 22 patients (15 sporadic patients and two families) with a 2p25.3 aberration to further refine the clinical phenotype and to delineate the role of MYT1L in intellectual disability and obesity. In addition, myt1l spatiotemporal expression in zebrafish embryos was analyzed by quantitative polymerase chain reaction and whole-mount in situ hybridization. RESULTS: Complete MYT1L deletion, intragenic deletion, or duplication was observed in all sporadic patients, in addition to two patients with a de novo point mutation in MYT1L. The familial cases comprise a 6-Mb deletion in a father and his three children and a 5' MYT1L overlapping duplication in a father and his two children. Expression analysis in zebrafish embryos shows specific myt1l expression in the developing brain. CONCLUSION: Our data strongly strengthen the hypothesis that MYT1L is the causal gene for the observed syndromal intellectual disability. Moreover, because 17 patients present with obesity/overweight, haploinsufficiency of MYT1L might predispose to weight problems with childhood onset.Genet Med 17 6, 460-466.
Asunto(s)
Deleción Cromosómica , Cromosomas Humanos Par 2 , Discapacidad Intelectual/genética , Proteínas del Tejido Nervioso/genética , Obesidad/genética , Factores de Transcripción/genética , Adolescente , Adulto , Animales , Niño , Preescolar , Mapeo Cromosómico , Estudios de Cohortes , Facies , Femenino , Duplicación de Gen , Expresión Génica , Estudios de Asociación Genética , Humanos , Masculino , Persona de Mediana Edad , Mutación Puntual , Adulto Joven , Pez CebraRESUMEN
Arterial tortuosity syndrome (ATS) is an autosomal recessive disorder characterized by tortuosity, elongation, stenosis and aneurysm formation in the major arteries owing to disruption of elastic fibers in the medial layer of the arterial wall. Previously, we used homozygosity mapping to map a candidate locus in a 4.1-Mb region on chromosome 20q13.1 (ref. 2). Here, we narrowed the candidate region to 1.2 Mb containing seven genes. Mutations in one of these genes, SLC2A10, encoding the facilitative glucose transporter GLUT10, were identified in six ATS families. GLUT10 deficiency is associated with upregulation of the TGFbeta pathway in the arterial wall, a finding also observed in Loeys-Dietz syndrome, in which aortic aneurysms associate with arterial tortuosity. The identification of a glucose transporter gene responsible for altered arterial morphogenesis is notable in light of the previously suggested link between GLUT10 and type 2 diabetes. Our data could provide new insight on the mechanisms causing microangiopathic changes associated with diabetes and suggest that therapeutic compounds intervening with TGFbeta signaling represent a new treatment strategy.
Asunto(s)
Arterias/patología , Proteínas Facilitadoras del Transporte de la Glucosa/fisiología , Mutación , Neovascularización Patológica/genética , Enfermedades Vasculares/genética , Secuencia de Aminoácidos , Mapeo Cromosómico , Cromosomas Humanos Par 20 , Femenino , Proteínas Facilitadoras del Transporte de la Glucosa/química , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Humanos , Masculino , Datos de Secuencia Molecular , Linaje , Homología de Secuencia de AminoácidoRESUMEN
Growth factor signaling results in dramatic phenotypic changes in cells, which require commensurate alterations in cellular metabolism. Mutations in SLC2A10/GLUT10, a member of the facilitative glucose transporter family, are associated with altered transforming growth factor-ß (TGFß) signaling in patients with arterial tortuosity syndrome (ATS). The objective of this work was to test whether SLC2A10/GLUT10 can serve as a link between TGFß-related transcriptional regulation and metabolism during development. In zebrafish embryos, knockdown of slc2a10 using antisense morpholino oligonucleotide injection caused a wavy notochord and cardiovascular abnormalities with a reduced heart rate and blood flow, which was coupled with an incomplete and irregular vascular patterning. This was phenocopied by treatment with a small-molecule inhibitor of TGFß receptor (tgfbr1/alk5). Array hybridization showed that the changes at the transcriptome level caused by the two treatments were highly correlated, revealing that a reduced tgfbr1 signaling is a key feature of ATS in early zebrafish development. Interestingly, a large proportion of the genes, which were specifically dysregulated after glut10 depletion gene and not by tgfbr1 inhibition, play a major role in mitochondrial function. Consistent with these results, slc2a10 morphants showed decreased respiration and reduced TGFß reporter gene activity. Finally, co-injection of antisense morpholinos targeting slc2a10 and smad7 (a TGFß inhibitor) resulted in a partial rescue of smad7 morphant phenotypes, suggesting scl2a10/glut10 functions downstream of smads. Taken together, glut10 is essential for cardiovascular development by facilitating both mitochondrial respiration and TGFß signaling.
Asunto(s)
Anomalías Cardiovasculares/etiología , Proteínas Facilitadoras del Transporte de la Glucosa/fisiología , Mitocondrias/metabolismo , Notocorda/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Anomalías Cardiovasculares/metabolismo , Anomalías Cardiovasculares/patología , Luciferasas/metabolismo , Mitocondrias/patología , Datos de Secuencia Molecular , Morfolinos/farmacología , Mutación/genética , Notocorda/patología , Fenotipo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal , Transcriptoma , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Pez Cebra/crecimiento & desarrolloRESUMEN
Cross-species genome comparisons have revealed a substantial number of ultraconserved non-coding elements (UCNEs). Several of these elements have proved to be essential tissue- and cell type-specific cis-regulators of developmental gene expression. Here, we characterize a set of UCNEs as candidate CREs (cCREs) during retinal development and evaluate the contribution of their genomic variation to rare eye diseases, for which pathogenic non-coding variants are emerging. Integration of bulk and single-cell retinal multi-omics data reveals 594 genes under potential cis-regulatory control of UCNEs, of which 45 are implicated in rare eye disease. Mining of candidate cis-regulatory UCNEs in WGS data derived from the rare eye disease cohort of Genomics England reveals 178 ultrarare variants within 84 UCNEs associated with 29 disease genes. Overall, we provide a comprehensive annotation of ultraconserved non-coding regions acting as cCREs during retinal development which can be targets of non-coding variation underlying rare eye diseases.
Asunto(s)
Oftalmopatías , Multiómica , Humanos , Retina/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Genoma , Oftalmopatías/genética , Oftalmopatías/metabolismoRESUMEN
Osteopoikilosis, Buschke-Ollendorff syndrome (BOS) and melorheostosis are disorders characterized by increased bone density. The occurrence of one or more of these phenotypes in the same individual or family suggests that these entities might be allelic. We collected data from three families in which affected individuals had osteopoikilosis with or without manifestations of BOS or melorheostosis. A genome-wide linkage analysis in these families, followed by the identification of a microdeletion in an unrelated individual with these diseases, allowed us to map the gene that is mutated in osteopoikilosis. All the affected individuals that we investigated were heterozygous with respect to a loss-of-function mutation in LEMD3 (also called MAN1), which encodes an inner nuclear membrane protein. A somatic mutation in the second allele of LEMD3 could not be identified in fibroblasts from affected skin of an individual with BOS and an individual with melorheostosis. XMAN1, the Xenopus laevis ortholog, antagonizes BMP signaling during embryogenesis. In this study, LEMD3 interacted with BMP and activin-TGFbeta receptor-activated Smads and antagonized both signaling pathways in human cells.
Asunto(s)
Melorreostosis/genética , Proteínas de la Membrana/genética , Mutación , Proteínas Nucleares/genética , Osteopoiquilosis/genética , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Humanos Par 12 , Proteínas de Unión al ADN , Femenino , Haplotipos , Humanos , Masculino , Datos de Secuencia Molecular , Nevo/genética , Linaje , SíndromeRESUMEN
Osteogenesis imperfecta (OI) is a family of rare heritable skeletal disorders associated with dominant mutations in the collagen type I encoding genes and recessive defects in proteins involved in collagen type I synthesis and processing and in osteoblast differentiation and activity. Historically, it was believed that the OI bone phenotype was only caused by abnormal collagen type I fibrils in the extracellular matrix, but more recently it became clear that the altered bone cell homeostasis, due to mutant collagen retention, plays a relevant role in modulating disease severity in most of the OI forms and it is correlated to impaired bone cell differentiation. Despite in vitro evidence, in vivo data are missing. To better understand the physiopathology of OI, we used two zebrafish models: Chihuahua (Chi/+), carrying a dominant p.G736D substitution in the α1 chain of collagen type I, and the recessive p3h1-/-, lacking prolyl 3-hydroxylase (P3h1) enzyme. Both models share the delay of collagen type I folding, resulting in its overmodification and partial intracellular retention. The regeneration of the bony caudal fin of Chi/+ and p3h1-/- was employed to investigate the impact of abnormal collagen synthesis on bone cell differentiation. Reduced regenerative ability was evident in both models, but it was associated to impaired osteoblast differentiation and osteoblastogenesis/adipogenesis switch only in Chi/+. On the contrary, reduced osteoclast number and activity were found in both models during regeneration. The dominant OI model showed a more detrimental effect in the extracellular matrix organization. Interestingly, the chemical chaperone 4-phenylbutyrate (4-PBA), known to reduce cellular stress and increase collagen secretion, improved bone formation only in p3h1-/- by favoring caudal fin growth without affecting bone cell markers expression. Taken together, our in vivo data proved the negative impact of structurally abnormal collagen type I on bone formation but revealed a gene mutation-specific effect on bone cell differentiation and matrix organization in OI. These, together with the distinct ability to respond to the chaperone treatment, underline the need for precision medicine approaches to properly treat the disease.
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Colágeno Tipo I , Osteogénesis Imperfecta , Animales , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Osteogénesis Imperfecta/genética , Osteogénesis Imperfecta/metabolismo , Osteogénesis/genética , Pez Cebra/genética , Pez Cebra/metabolismo , Colágeno/metabolismo , Chaperonas Moleculares/genética , Mutación , Diferenciación CelularRESUMEN
Introduction: Trimeric intracellular potassium channels TRIC-A and -B are endoplasmic reticulum (ER) integral membrane proteins, involved in the regulation of calcium release mediated by ryanodine (RyRs) and inositol 1,4,5-trisphosphate (IP3Rs) receptors, respectively. While TRIC-A is mainly expressed in excitable cells, TRIC-B is ubiquitously distributed at moderate level. TRIC-B deficiency causes a dysregulation of calcium flux from the ER, which impacts on multiple collagen specific chaperones and modifying enzymatic activity, leading to a rare form of osteogenesis imperfecta (OI Type XIV). The relevance of TRIC-B on cell homeostasis and the molecular mechanism behind the disease are still unknown. Results: In this study, we exploited zebrafish to elucidate the role of TRIC-B in skeletal tissue. We demonstrated, for the first time, that tmem38a and tmem38b genes encoding Tric-a and -b, respectively are expressed at early developmental stages in zebrafish, but only the latter has a maternal expression. Two zebrafish mutants for tmem38b were generated by CRISPR/Cas9, one carrying an out of frame mutation introducing a premature stop codon (tmem38b-/- ) and one with an in frame deletion that removes the highly conserved KEV domain (tmem38bΔ120-7/Δ120-7 ). In both models collagen type I is under-modified and partially intracellularly retained in the endoplasmic reticulum, as described in individuals affected by OI type XIV. Tmem38b-/- showed a mild skeletal phenotype at the late larval and juvenile stages of development whereas tmem38bΔ120-7/Δ120-7 bone outcome was limited to a reduced vertebral length at 21 dpf. A caudal fin regeneration study pointed towards impaired activity of osteoblasts and osteoclasts associated with mineralization impairment. Discussion: Our data support the requirement of Tric-b during early development and for bone cell differentiation.
Asunto(s)
Canales Iónicos , Osteogénesis Imperfecta , Proteínas de Pez Cebra , Pez Cebra , Animales , Huesos/metabolismo , Calcio/metabolismo , Diferenciación Celular/genética , Canales Iónicos/genética , Osteogénesis Imperfecta/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genéticaRESUMEN
SNARE proteins comprise a conserved protein family responsible for catalyzing membrane fusion during vesicle traffic. Syntaxin18 (STX18) is a poorly characterized endoplasmic reticulum (ER)-resident t-SNARE. Recently, together with TANGO1 and SLY1, its involvement was shown in ER to Golgi transport of collagen II during chondrogenesis. We report a fetus with a severe osteochondrodysplasia in whom we identified a homozygous substitution of the highly conserved p.Arg10 to Pro of STX18. CRISPR/Cas9-mediated Stx18 deficiency in zebrafish reveals a crucial role for Stx18 in cartilage and bone development. Furthermore, increased expression of multiple components of the Stx18 SNARE complex and of COPI and COPII proteins suggests that Stx18 deficiency impairs antero- and retrograde vesicular transport in the crispant stx18 zebrafish. Taken together, our studies highlight a new candidate gene for a recessive form of osteochondrodysplasia, thereby possibly broadening the SNAREopathy phenotypic spectrum and opening new doors toward future research avenues. © 2023 American Society for Bone and Mineral Research (ASBMR).
Asunto(s)
Osteocondrodisplasias , Pez Cebra , Animales , Humanos , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Osteocondrodisplasias/metabolismo , Aparato de Golgi/metabolismo , Cartílago/metabolismo , Desarrollo Óseo , Transporte de ProteínasRESUMEN
Pseudoxanthoma elasticum (PXE) is a hereditary ectopic calcification disorder affecting the skin, eyes, and blood vessels. Recently, the DNA damage response (DDR), in particular PARP1, was shown to be involved in aberrant mineralization, raising the hypothesis that excessive DDR/PARP1 signaling also contributes to PXE pathogenesis. Using dermal fibroblasts of patients with PXE and healthy controls, (lesional) skin tissue, and abcc6aâ/â zebrafish, we performed expression analysis of DDR/PARP1 targets with QRT-PCR, western blot, immunohistochemistry, and enzyme activity assays before and after treatment with the PARP1 inhibitor minocycline. PARP1 and the ATMâp21âp53 axis was found to be significantly increased in PXE. In addition, PARP1 downstream targets IL-6, signal transducer and activator of transcription 1/3, TET1, and RUNX2 were upregulated, whereas the RUNX2 antagonist microRNA-204 was decreased. In PXE fibroblasts, DDR/PARP1 signaling increased with advancing ectopic calcification. Minocycline treatment attenuated DDR/PARP1 overexpression and reduced aberrant mineralization in PXE fibroblasts and abcc6aâ/â zebrafish. In summary, we showed the involvement of excessive DDR/PARP1 signaling in PXE pathophysiology, identifying a signal transducer and activator of transcriptionâdriven cascade resulting in increased expression of the epigenetic modifier TET1 and procalcifying transcription factor RUNX2. Minocycline attenuated this deleterious molecular mechanism and reduced ectopic calcification both in vitro and in vivo, fueling the exciting prospect of a therapeutic compound for PXE.
Asunto(s)
MicroARNs , Seudoxantoma Elástico , Transportadoras de Casetes de Unión a ATP/genética , Animales , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Daño del ADN , Humanos , Minociclina/farmacología , Minociclina/uso terapéutico , Oxigenasas de Función Mixta/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Proto-Oncogénicas/metabolismo , Seudoxantoma Elástico/tratamiento farmacológico , Seudoxantoma Elástico/genética , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genéticaRESUMEN
The popularity of zebrafish in both basic biological and biomedical research has led to an increased need for understanding their behavior. Locomotor behavior is an important outcome of different factors, such as specific genotypes or external stimuli that influence the nervous and musculoskeletal system. Locomotion can be studied by forced swimming in a swim tunnel, a device capable of generating a laminar water flow at different speeds in a chamber where zebrafish can be placed. However, commercially available swim tunnels are relatively expensive and in-house built systems are mostly presented without clear building instructions or proper validation procedures. In this study, we developed an alternative, cheap (<250 euro), and user-friendly, but customizable benchtop swim tunnel, called the "Zebrafish exercise-tunnel" (ZE-Tunnel). Detailed step-by-step instructions on how to construct the tunnel components, including the frame, mechanical, and electric components are given. The ZE-Tunnel was reliably used to exercise fish for prolonged periods and its performance was successfully validated by replicating previously published experiments on critical speed testing in zebrafish. Finally, implementation of behavioral video analysis using freely available motion-tracking software showed differences in swimming dynamics in the Chihuahua skeletal zebrafish mutant.