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The interplay between neural progenitors and stem cells (NPSCs), and their extracellular matrix (ECM) is a crucial regulatory mechanism that determines their behavior. Nonetheless, how the ECM dictates the state of NPSCs remains elusive. The hindbrain is valuable to examine this relationship, as cells in the ventricular surface of hindbrain boundaries (HBs), which arise between any two neighboring rhombomeres, express the NPSC marker Sox2, while being surrounded with the membrane-bound ECM molecule chondroitin sulphate proteoglycan (CSPG), in chick and mouse embryos. CSPG expression was used to isolate HB Sox2+ cells for RNA-sequencing, revealing their distinguished molecular properties as typical NPSCs, which express known and newly identified genes relating to stem cells, cancer, the matrisome and cell cycle. In contrast, the CSPG- non-HB cells, displayed clear neural-differentiation transcriptome. To address whether CSPG is significant for hindbrain development, its expression was manipulated in vivo and in vitro. CSPG manipulations shifted the stem versus differentiation state of HB cells, evident by their behavior and altered gene expression. These results provide further understanding of the uniqueness of hindbrain boundaries as repetitive pools of NPSCs in-between the rapidly growing rhombomeres, which rely on their microenvironment to maintain their undifferentiated state during development.
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Células-Madre Neurales , Proteoglicanos , Ratones , Animales , Proteoglicanos/metabolismo , Sulfatos de Condroitina , Proteoglicanos Tipo Condroitín Sulfato , Matriz Extracelular/metabolismo , Rombencéfalo/metabolismo , Células-Madre Neurales/metabolismoRESUMEN
BACKGROUND: Compartment boundaries are an essential developmental mechanism throughout evolution, designated to act as organizing centers and to regulate and localize differently fated cells. The hindbrain serves as a fascinating example for this phenomenon as its early development is devoted to the formation of repetitive rhombomeres and their well-defined boundaries in all vertebrates. Yet, the actual role of hindbrain boundaries remains unresolved, especially in amniotes. RESULTS: Here, we report that hindbrain boundaries in the chick embryo consist of a subset of cells expressing the key neural stem cell (NSC) gene Sox2. These cells co-express other neural progenitor markers such as Transitin (the avian Nestin), GFAP, Pax6 and chondroitin sulfate proteoglycan. The majority of the Sox2(+) cells that reside within the boundary core are slow-dividing, whereas nearer to and within rhombomeres Sox2(+) cells are largely proliferating. In vivo analyses and cell tracing experiments revealed the contribution of boundary Sox2(+) cells to neurons in a ventricular-to-mantle manner within the boundaries, as well as their lateral contribution to proliferating Sox2(+) cells in rhombomeres. The generation of boundary-derived neurospheres from hindbrain cultures confirmed the typical NSC behavior of boundary cells as a multipotent and self-renewing Sox2(+) cell population. Inhibition of Sox2 in boundaries led to enhanced and aberrant neural differentiation together with inhibition in cell-proliferation, whereas Sox2 mis-expression attenuated neurogenesis, confirming its significant function in hindbrain neuronal organization. CONCLUSIONS: Data obtained in this study deciphers a novel role of hindbrain boundaries as repetitive pools of neural stem/progenitor cells, which provide proliferating progenitors and differentiating neurons in a Sox2-dependent regulation.
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Tipificación del Cuerpo , Células-Madre Neurales/citología , Rombencéfalo/citología , Factores de Transcripción SOXB1/metabolismo , Animales , Biomarcadores/metabolismo , Diferenciación Celular , Movimiento Celular , Proliferación Celular , Autorrenovación de las Células , Embrión de Pollo , Modelos Biológicos , Células-Madre Neurales/metabolismo , Rombencéfalo/embriología , Esferoides Celulares/citología , Factores de TiempoRESUMEN
Rearrangement of the actin cytoskeleton is controlled by RhoGTPases which are activated by RhoGEFs. We identified homozygosity for Arg204Trp mutation in the Rho guanidine exchange factor (RhoGEF) PLEKHG2 gene in five patients with profound mental retardation, dystonia, postnatal microcephaly, and distinct neuroimaging pattern. The activity of the mutant PLEKHG2 was significantly decreased, both in basal state and when Gßγ- or lysophosphatidic acid (LPA)-stimulated. SDF1a-stimulated actin polymerization was significantly impaired in patient cells, and this abnormality was duplicated in control cells when PLEKHG2 expression was downregulated. These results underscore the role of PLEKHG2 in actin polymerization and delineate the clinical and radiological findings in PLEKHG2 deficiency.
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Actinas/metabolismo , Distonía/genética , Factores de Intercambio de Guanina Nucleótido/genética , Microcefalia/genética , Árabes , Consanguinidad , Distonía/complicaciones , Familia , Femenino , Células HEK293 , Humanos , Masculino , Microcefalia/complicaciones , Medio Oriente , Mutación Missense , Linaje , Multimerización de Proteína/genéticaRESUMEN
BACKGROUND: Hereditary spastic paraparesis (HSP) (syn. Hereditary spastic paraplegia, SPG) are a group of genetic disorders characterised by spasticity of the lower limbs due to pyramidal tract dysfunction. Nearly 60 disease loci have been identified, which include mutations in two genes (KIF5A and KIF1A) that encode motor proteins of the kinesin superfamily. Here we report a novel genetic defect in KIF1C of patients with spastic paraparesis and cerebellar dysfunction in two consanguineous families of Palestinian and Moroccan ancestry. METHODS AND RESULTS: We performed autozygosity mapping in a Palestinian and classic linkage analysis in a Moroccan family and found a locus on chromosome 17 that had previously been associated with spastic ataxia type 2 (SPAX2, OMIM %611302). Whole-exome sequencing revealed two homozygous mutations in KIF1C that were absent among controls: a nonsense mutation (c.2191C>T, p.Arg731*) that segregated with the disease phenotype in the Palestinian kindred resulted in the entire absence of KIF1C protein from the patient's fibroblasts, and a missense variant (c.505C>T, p.Arg169Trp) affecting a conserved amino acid of the motor domain that was found in the Moroccan kindred. CONCLUSIONS: Kinesin genes encode a family of cargo/motor proteins and are known to cause HSP if mutated. Here we identified nonsense and missense mutations in a further member of this protein family. The KIF1C mutation is associated with a HSP subtype (SPAX2/SAX2) that combines spastic paraplegia and weakness with cerebellar dysfunction.
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Enfermedades Cerebelosas/genética , Cinesinas/genética , Paraparesia Espástica/genética , Adolescente , Secuencia de Aminoácidos , Secuencia de Bases , Niño , Preescolar , Consanguinidad , Análisis Mutacional de ADN , Femenino , Estudios de Asociación Genética , Ligamiento Genético , Células HEK293 , Humanos , Lactante , Masculino , Datos de Secuencia Molecular , Linaje , Polimorfismo de Nucleótido Simple , Adulto JovenRESUMEN
PURPOSE: To determine the molecular basis of familial, autosomal-recessive, non-obstructive azoospermia in a consanguineous Iranian Jewish family. METHODS: We investigated the genetic cause of non-obstructive azoospermia in two affected siblings from a consanguineous family. Homozygosity mapping in the DNA samples of the patients and their normospermic brother was followed by exome analysis of one of the patients. Other family members were genotyped for the mutation by Sanger sequencing. The mutation effect was demonstrated by immunostaining of the patients' testicular tissue. RESULTS: The two patients were homozygous for a splice site mutation in SYCE1 which resulted in retention of intron three in the cDNA and premature stop codon. SYCE1 encodes a Synaptonemal Complex protein which plays an essential role during meiosis. Immunostaining of patient's testicular tissue with anti-Syce1 antibody revealed an undetectable level of Syce1. Histological examination of the patients' tissue disclosed immature-stages spermatocytes without mature forms, indicating maturation arrest. CONCLUSION: The significance of most synaptonemal complex proteins was previously demonstrated in a mutant mouse model. The present report underscores the importance of synaptonemal complex proteins in spermatogenenesis in humans. Our new approach, combining homozygosity mapping and exome sequencing, resulted in one of the first reports of an autosomal-recessive form of NOA.
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Azoospermia/genética , Codón sin Sentido , Proteínas Nucleares/genética , Consanguinidad , Proteínas de Unión al ADN , Homocigoto , Humanos , Masculino , Mutación , Proteínas Nucleares/química , Linaje , Sitios de Empalme de ARN/genéticaRESUMEN
In 4 infants with a new lethal autonomic sensory neuropathy with clinical features similar to familial dysautonomia as well as contractures, we identified a deleterious mutation in the DST gene, using homozygosity mapping followed by exome sequencing. DST encodes dystonin, a cytoskeleton linker protein, and the mutation results in an unstable transcript. Interestingly, dystonin is significantly more abundant in cells of familial dysautonomia patients with IKBKAP (I-κ-B kinase complex-associated protein) mutation compared to fibroblasts of controls, suggesting that upregulation of dystonin is responsible for the milder course in familial dysautonomia. Homozygosity mapping followed by exome sequencing is a successful approach to identify mutated genes in rare monogenic disorders.
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Proteínas Portadoras/genética , Proteínas del Citoesqueleto/genética , Neuropatías Hereditarias Sensoriales y Autónomas/genética , Mutación , Proteínas del Tejido Nervioso/genética , Secuencia de Bases , Preescolar , Mapeo Cromosómico , Análisis Mutacional de ADN , Distonina , Femenino , Predisposición Genética a la Enfermedad , Neuropatías Hereditarias Sensoriales y Autónomas/patología , Humanos , Lactante , Judíos , Masculino , Microtúbulos/genética , Microtúbulos/patología , Datos de Secuencia Molecular , LinajeRESUMEN
BACKGROUND: Significant advancements in understanding the molecular pathophysiology of laterality determination were recently made. However, there are large gaps in our knowledge of the initial processes that lead to laterality defects, such as heterotaxy syndrome (HS, also known as situs ambiguous) and situs inversus totalis (SIT). The former refers to abnormal distribution of visceral organs, and the latter refers to a complete laterality inversion of both abdominal and thoracic viscera. METHODS: In order to identify a mutated gene in SIT and HS patients, the authors performed homozygosity mapping in a consanguineous family with laterality disorders identified in two siblings. RESULTS: A homozygous deleterious mutation in the CCDC11 gene was identified in the patients. The mutation resulted in an abnormally smaller protein in the patient's skin fibroblasts. The parents and five healthy siblings were heterozygous for the mutation, which was not present in 112 anonymous controls. CONCLUSIONS: Few genes have been associated with both SIT and HS, usually accompanied by other abnormalities. The authors suggest that CCDC11 is associated with autosomal recessive laterality defects of diverse phenotype resulting in SIT in one individual family member who is otherwise healthy, and in complex laterality anomalies (HS) in another member. This report underscores the importance of CCDC11 in laterality determination.
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Genes Recesivos , Síndrome de Heterotaxia/genética , Mutación , Situs Inversus/genética , Adolescente , Secuencia de Aminoácidos , Secuencia de Bases , Western Blotting , Mapeo Cromosómico , Consanguinidad , Fibroblastos , Humanos , Masculino , Datos de Secuencia Molecular , Linaje , ARN/genética , ARN/aislamiento & purificación , Análisis de Secuencia de ADNRESUMEN
The IGF2BP family of RNA binding proteins consists of three paralogs that regulate intracellular RNA localization, RNA stability, and translational control. Although IGF2BP1 and 3 are oncofetal proteins, IGF2BP2 expression is maintained in many tissues, including the heart, into adulthood. IGF2BP2 is upregulated in cardiomyocytes during cardiac stress and remodeling and returns to normal levels in recovering hearts. We wondered whether IGF2BP2 might play an adaptive role during cardiac stress and recovery. Enhanced expression of an IGF2BP2 transgene in a conditional, inducible mouse line leads to dilated cardiomyopathy (DCM) and death within 3-4 weeks in newborn or adult hearts. Downregulation of the transgene after 2 weeks, however, rescues these mice, with complete recovery by 12 weeks. Hearts overexpressing IGF2BP2 downregulate sarcomeric and mitochondrial proteins and have fragmented mitochondria and elongated, thinner sarcomeres. IGF2BP2 is also upregulated in DCM or myocardial infarction patients. These results suggest that IGF2BP2 may be an attractive target for therapeutic intervention in cardiomyopathies.
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Cardiomiopatías , Cardiomiopatía Dilatada , Adulto , Animales , Humanos , Ratones , Cardiomiopatías/metabolismo , Cardiomiopatía Dilatada/genética , Miocitos Cardíacos/metabolismo , ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Avian blastoderm can enter into diapause when kept at low temperatures and successfully resume development (SRD) when re-incubated in body temperature. These abilities, which are largely affected by the temperature and duration of the diapause, are poorly understood at the cellular and molecular level. To determine how temperature affects embryonic morphology during diapause, high-resolution episcopic microscopy (HREM) analysis was utilized. While blastoderms diapausing at 12 °C for 28 days presented typical cytoarchitecture, similar to non-diapaused embryos, at 18 °C, much thicker blastoderms with higher cell number were observed. RNAseq was conducted to discover the genes underlying these phenotypes, revealing differentially expressed cell cycle regulatory genes. Among them, WEE1, a negative regulator of G2/M transition, was highly expressed at 12 °C compared to 18 °C. This finding suggested that cells at 12 °C are arrested at the G2/M phase, as supported by bromodeoxyuridine incorporation (BrdU) assay and phospho-histone H3 (pH 3) immunostaining. Inhibition of WEE1 during diapause at 12 °C resulted in cell cycle progression beyond the G2/M and augmented tissue volume, resembling the morphology of 18 °C-diapaused embryos. These findings suggest that diapause at low temperatures leads to WEE1 upregulation, which arrests the cell cycle at the G2/M phase, promoting the perseverance of embryonic cytoarchitecture and future SRD. In contrast, WEE1 is not upregulated during diapause at higher temperature, leading to continuous proliferation and maladaptive morphology associated with poor survivability. Combining HREM-based analysis with RNAseq and molecular manipulations, we present a novel mechanism that regulates the ability of diapaused avian embryos to maintain their cytoarchitecture via cell cycle arrest, which enables their SRD.
RESUMEN
The avian embryo has a remarkable ability that allows it to suspend its development during blastulation for a long time at low temperatures, and to resume normal development when incubated. This ability is used by poultry hatcheries to store eggs prior to incubation. We have previously found that this ability correlates with the temperature during storage; embryos recover much better following prolonged storage at 12°C rather than at 18°C. However, the molecular and cellular mechanisms underlying these differences are poorly understood. To successfully resume development following storage, the embryo has to shift from the blastulation phase to gastrulation. Several genes are known to partake in the blastulation-to-gastrulation transition under normal conditions, such as the pluripotency-related genes Inhibitor of DNA Binding 2 (ID2) and NANOG that are expressed during blastulation, and the gastrulation-regulating genes NODAL and Brachyury (TBXT). However, their expression and activity following storage is unknown. To elucidate the molecular mechanisms that initiate the ability to successfully transit from blastulation to gastrulation following storage, embryos were stored for 28 days at 12°C or 18°C, and were assessed either prior to incubation, 12, or 18 h of incubation at 37.8°C. Immediately following storage at 18°C group showed remarkable impaired morphology compared to the blastoderm of the 12°C group and of non-stored control embryos. Concurrently with these, expression of ID2 and NANOG was maintained following storage at 12°C similar to the control group, but was significantly reduced upon storage at 18°C. Nevertheless, when the 18°C-stored embryos were incubated, the morphology and the reduced genes were reverted to resemble those of the 12°C group. At variance, key gastrulation genes, NODAL and its downstream effector Brachyury (TBXT), which were similarly expressed in the control and the 12°C group, were not restored in the 18°C embryos following incubation. Notably, ectopic administration of Activin rescued NODAL and TBXT expression in the 18°C group, indicating that these embryos maintain the potential to initiate. Collectively, this study suggests a temperature-dependent mechanisms that direct the transition from blastulation to gastrulation. These mechanisms promote a successful developmental resumption following prolonged storage at low temperatures.
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Peripheral and intraspinal feedback is required to shape and update the output of spinal networks that execute motor behavior. We report that lumbar dI2 spinal interneurons in chicks receive synaptic input from afferents and premotor neurons. These interneurons innervate contralateral premotor networks in the lumbar and brachial spinal cord, and their ascending projections innervate the cerebellum. These findings suggest that dI2 neurons function as interneurons in local lumbar circuits, are involved in lumbo-brachial coupling, and that part of them deliver peripheral and intraspinal feedback to the cerebellum. Silencing of dI2 neurons leads to destabilized stepping in posthatching day 8 hatchlings, with occasional collapses, variable step profiles, and a wide-base walking gait, suggesting that dI2 neurons may contribute to the stabilization of the bipedal gait.
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Marcha/fisiología , Interneuronas/fisiología , Médula Espinal , Animales , Embrión de Pollo , Pollos , Región Lumbosacra , Médula Espinal/citología , Médula Espinal/fisiología , Tractos Espinocerebelares/citología , Tractos Espinocerebelares/fisiología , Sinapsis/fisiologíaRESUMEN
Carbamazepine (CBZ) is an anticonvulsant drug used for epilepsy and other disorders. Prescription of CBZ during pregnancy increases the risk for congenital malformations. CBZ is ubiquitous in effluents and persistent during wastewater treatment. Thus, it is re-introduced into agricultural ecosystems upon irrigation with reclaimed wastewater. People consuming produce irrigated with reclaimed wastewater were found to be exposed to CBZ. However, environmental concentrations of CBZ (µgL-1) are magnitudes lower than its therapeutic levels (µgml-1), raising the question of whether and how environmental levels of CBZ affect embryonic development. The chick embryo is a powerful and highly sensitive amniotic model system that enables to assess environmental contaminants in the living organism. Since the chick embryonic development is highly similar to mammalians, yet, it develops in an egg, toxic effects can be directly analyzed in a well-controlled system without maternal influences. This research utilized the chick embryo to test whether CBZ is embryo-toxic by using morphological, cellular, molecular and imaging strategies. Three key embryonic stages were monitored: after blastulation (st.1HH), gastrulation/neurulation (st.8HH) and organogenesis (st.15HH). Here we demonstrate that environmental relevant concentrations of CBZ impair morphogenesis in a dose- and stage- dependent manner. Effects on gastrulation, neural tube closure, differentiation and proliferation were exhibited in early stages by exposing embryos to CBZ dose as low as 0.1µgL-1. Quantification of developmental progression revealed a significant difference in the total score obtained by CBZ-treated embryos compared to controls (up to 5-fold difference, p<0.05). Yet, defects were unnoticed as embryos passed gastrulation/neurulation. This study provides the first evidence for teratogenic effect of environmental-relevant concentrations of CBZ in amniotic embryos that impair early but not late stages of development. These findings call for in-depth risk analysis to ensure that the environmental presence of CBZ and other drugs is not causing irreversible ecological and public-health damages.
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Anticonvulsivantes/toxicidad , Carbamazepina/toxicidad , Animales , Anticonvulsivantes/química , Carbamazepina/química , Embrión de Pollo , Femenino , Defectos del Tubo Neural/inducido químicamente , Embarazo , Prueba de Estudio Conceptual , Aguas Residuales/análisisRESUMEN
The study of plant anatomy, which can be traced back to the seventeenth century, advanced hand in hand with light microscopy technology and relies on traditional histologic techniques, which are based on serial two-dimensional (2D) sections. However, these valuable techniques lack spatial arrangement of the tissue and hence provide only partial information. A new technique of whole-mount three-dimensional (3D) imaging termed high-resolution episcopic microscopy (HREM) can overcome this obstacle and generate a 3D model of the specimen at a near-histological resolution. Here, we describe the application of HREM technique in plants by analyzing two plant developmental processes in woody plants: oil secretory cavity development in citrus fruit and adventitious root formation in persimmon rootstock cuttings. HREM 3D models of citrus fruit peel showed that oil cavities were initiated schizogenously during the early stages of fruitlet development. Citrus secretory cavity formation, shape, volume, and distribution were analyzed, and new insights are presented. HREM 3D model comparison of persimmon rootstock clones, which differ in their rooting ability, revealed that difficult-to-root clones failed to develop adventitious roots due to their inability to initiate root primordia.
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Leaf axil patterning occurs concomitantly with leaf development and takes place at the boundary zone which demarcates the initiating leaf primordium from the shoot apical meristem. Subsequent growth and differentiation result in establishment of the axillary meristem and abscission zone (AZ) along the proximal-distal axis of the leaf axil, yet the molecular mechanisms that regulate these events are poorly understood. We studied the role of the tomato BLADE ON PETIOLE (SlBOP) boundary gene family on the development of the leaf axil using BOP-silenced plants as well as BOP-mutated lines. We show that silencing of the tomato SlBOP gene family affects patterning of the leaf axil along the proximal-distal axis, manifested by dispositioning of the AM and abnormal development of the adjacent tissue resulting in lack of a functional leaf AZ. Dissection of the role of each of the three tomato SlBOPs by analysis of single, double and triple null-mutants demonstrated that SlBOP2 is the dominant gene in leaf axil patterning, but does not rule out involvement of SlBOP1 and SlBOP3 in correct AM positioning. We further studied the potential role of TERMINATING FLOWER (TMF), a transcription factor which was previously shown to interact with SlBOPs, in leaf axil patterning using TMF mutant tomato lines. The results suggest that similar to SlBOP2, TMF is involved in leaf axil proximal-distal patterning and AZ development.
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Cripto-1 (TDGF1) is a multifunctional signaling factor that stimulates cellular effects, including proliferation, migration, survival, epithelial-to-mesenchymal transition, and angiogenesis, to regulate embryogenesis, tissue homeostasis, and tumorigenesis. Those cell behaviors are also associated with implantation of the embryo into the uterine wall, and this led us to investigate the role of embryo-derived Cripto in embryo attachment and implantation. In this study, we show that Cripto and its signaling mediator GRP78 are uniquely localized to embryo implantation sites. We knocked down Cripto expression specifically in trophoblast cells and found that this resulted in a corresponding decrease in the levels of its downstream signaling mediators, phosphorylated (phospho-)SMAD2, phospho-SRC, phospho-extracellular signal-regulated kinase, and phospho-AKT, which are also known mediators of embryo implantation. We then transplanted Cripto knockdown and control embryos into uteri of pseudopregnant female mice and found that embryos with Cripto-depleted trophoblast cells had dramatically impaired capacity to attach to the uterine wall when compared with controls. This loss of appropriate embryo attachment following Cripto knockdown in trophoblast cells was associated with abnormally enlarged implantation sites that were almost completely devoid of microvessels. A role for Cripto in embryo implantation was further supported by our demonstration that attachment of trophoblast-derived spheroids to endometrial cells in vitro was stimulated by Cripto treatment and diminished by treatment with either of two mechanistically distinct Cripto blocking agents. Collectively, our findings identify Cripto as a novel and critical embryo attachment factor and suggest that modulation of Cripto signaling may have significant therapeutic potential for the treatment of infertility and other related disorders.
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Implantación del Embrión/fisiología , Endometrio/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Trofoblastos/metabolismo , Animales , Línea Celular , Endometrio/irrigación sanguínea , Chaperón BiP del Retículo Endoplásmico , Factor de Crecimiento Epidérmico/genética , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Glicoproteínas de Membrana/genética , Ratones , Proteínas de Neoplasias/genética , Transducción de Señal/fisiologíaRESUMEN
Axial determination occurs during early stages of embryogenesis. Flaws in laterality patterning result in abnormal positioning of visceral organs, as manifested in heterotaxy syndrome, or complete left-right inversion as in situs inversus totalis. These malformations are often associated with ciliopathies, as seen in primary ciliary dyskinesia. We have recently described a novel mutation in the Coiled-Coil Domain-Containing 11 (CCDC11) gene associated with laterality disorders in a consanguineous family of Arab-Muslim origin with two affected siblings presenting with diverse phenotypes, one with heterotaxy syndrome and the other with non-primary ciliary dyskinesia situs inversus totalis. This study further characterizes the roles of CCDC11 and the implications of the identified mutation on left-right axial patterning in patient-derived cells and in the frog embryo as a model organism. We analyzed patient-derived cells and manipulated Ccdc11 levels in Xenopus laevis frog embryos. Cilia length in patient cells was longer than in controls, and CCDC11 was localized to the centriole and the actin cytoskeleton. Mutated truncated protein accumulated and was also localized to the centriole and actin cytoskeleton. In frog embryos, Ccdc11 was regulated downstream of FoxJ1, and overexpression of the full-length or truncated protein, or downregulation of the gene resulted in severe disruption of embryonic left-right axial patterning. Taken together, our initial description of the deleterious mutation in CCDC11 in patients, the current results and more recent supportive studies highlight the important role of CCDC11 in axial patterning.
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Tipificación del Cuerpo , Mapeo Encefálico , Proteínas del Citoesqueleto/fisiología , Fibroblastos/metabolismo , Síndrome de Heterotaxia/genética , Proteínas de Xenopus/fisiología , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animales , Proteínas del Citoesqueleto/genética , Citoesqueleto/metabolismo , Modelos Animales de Enfermedad , Embrión no Mamífero/metabolismo , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Humanos , Islamismo , Mutación , Fenotipo , Proteínas de Xenopus/genética , Xenopus laevis/embriología , Pez Cebra/genéticaRESUMEN
The somite and its intermediate derivatives, sclerotome and dermomyotome (DM), are composed of distinct subdomains based on lineage analysis and gene expression patterns. This sets the grounds for elucidating the mechanisms underlying differential cell specification and morphogenesis. By examining the in vivo roles of N-cadherin on discrete domains of the somitic epithelium at various times, our recent studies highlight the existence of a regional and temporal heterogeneity in cellular responsiveness. As examples of this assortment, we document a coupling between asymmetric cell division and fate segregation in the DM sheet, sequential effects of N-cadherin-mediated adhesion on early myogenic specification compared to later myofiber patterning, and a differential behavior of pioneer myoblasts compared to later myogenic waves.
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Aves/embriología , Tipificación del Cuerpo/fisiología , Cadherinas/metabolismo , División Celular/fisiología , Linaje de la Célula/fisiología , Músculos/embriología , Somitos/fisiología , Animales , Adhesión Celular/fisiologíaRESUMEN
For muscles to function, myofibers have to stretch and anchor at the myotendinous junction (MTJ), a region rich in extracellular matrix (ECM). Integrin signaling is required for MTJ formation, and mutations affecting the cascade lead to muscular dystrophies in mice and humans. Underlying mechanisms for integrin activation at the MTJ and ECM modifications regulating its signaling are unclear. We show that lysyl oxidase-like 3 (LoxL3) is a key regulator of integrin signaling that ensures localized control of the cascade. In LoxL3 mutants, myofibers anchor prematurely or overshoot to adjacent somites, and are loose and lack tension. We find that LoxL3 complexes with and directly oxidizes Fibronectin (FN), an ECM scaffold protein and integrin ligand enriched at the MTJ. We identify a mechanism whereby localized LoxL3 secretion from myofiber termini oxidizes FN, enabling enhanced integrin activation at the tips of myofibers and ensuring correct positioning and anchoring of myofibers along the MTJ.
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Aminoácido Oxidorreductasas/metabolismo , Adhesión Celular/fisiología , Fibronectinas/metabolismo , Integrinas/metabolismo , Músculos/metabolismo , Animales , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Ratones , Miofibrillas/metabolismo , Oxidación-Reducción , Somitos/metabolismo , Tendones/metabolismoRESUMEN
More than 20 years after characterization of the key regulator of mammalian energy balance, leptin, we identified the leptin (LEP) genes of chicken (Gallus gallus) and duck (Anas platyrhynchos). The extreme guanine-cytosine content (â¼70%), the location in a genomic region with low-complexity repetitive and palindromic sequence elements, the relatively low sequence conservation, and low level of expression have hampered the identification of these genes until now. In vitro-expressed chicken and duck leptins specifically activated signaling through the chicken leptin receptor in cell culture. In situ hybridization demonstrated expression of LEP mRNA in granular and Purkinje cells of the cerebellum, anterior pituitary, and in embryonic limb buds, somites, and branchial arches, suggesting roles in adult brain control of energy balance and during embryonic development. The expression patterns of LEP and the leptin receptor (LEPR) were explored in chicken, duck, and quail (Coturnix japonica) using RNA-sequencing experiments available in the Short Read Archive and by quantitative RT-PCR. In adipose tissue, LEP and LEPR were scarcely transcribed, and the expression level was not correlated to adiposity. Our identification of the leptin genes in chicken and duck genomes resolves a long lasting controversy regarding the existence of leptin genes in these species. This identification was confirmed by sequence and structural similarity, conserved exon-intron boundaries, detection in numerous genomic, and transcriptomic datasets and characterization by PCR, quantitative RT-PCR, in situ hybridization, and bioassays. Our results point to an autocrine/paracrine mode of action for bird leptin instead of being a circulating hormone as in mammals.
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Comunicación Autocrina/genética , Leptina/genética , Comunicación Paracrina/genética , ARN Mensajero/metabolismo , Receptores de Leptina/genética , Tejido Adiposo/metabolismo , Animales , Región Branquial/metabolismo , Cerebelo/metabolismo , Pollos , Coturnix , Patos , Sistema Endocrino , Femenino , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Humanos , Hibridación in Situ , Riñón/metabolismo , Leptina/metabolismo , Leptina/fisiología , Esbozos de los Miembros/metabolismo , Masculino , Miocardio/metabolismo , Ovario/metabolismo , Adenohipófisis/metabolismo , Células de Purkinje/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Leptina/metabolismo , Somitos/metabolismo , Testículo/metabolismo , TranscriptomaRESUMEN
The exosome is a multi-protein complex, required for the degradation of AU-rich element (ARE) containing messenger RNAs (mRNAs). EXOSC8 is an essential protein of the exosome core, as its depletion causes a severe growth defect in yeast. Here we show that homozygous missense mutations in EXOSC8 cause progressive and lethal neurological disease in 22 infants from three independent pedigrees. Affected individuals have cerebellar and corpus callosum hypoplasia, abnormal myelination of the central nervous system or spinal motor neuron disease. Experimental downregulation of EXOSC8 in human oligodendroglia cells and in zebrafish induce a specific increase in ARE mRNAs encoding myelin proteins, showing that the imbalanced supply of myelin proteins causes the disruption of myelin, and explaining the clinical presentation. These findings show the central role of the exosomal pathway in neurodegenerative disease.