RESUMEN
During development, lymph node (LN) initiation is coordinated by lymphoid tissue organizer (LTo) cells that attract lymphoid tissue inducer (LTi) cells at strategic positions within the embryo. The identity and function of LTo cells during the initial attraction of LTi cells remain poorly understood. Using lineage tracing, we demonstrated that a subset of Osr1-expressing cells was mesenchymal LTo progenitors. By investigating the heterogeneity of Osr1+ cells, we uncovered distinct mesenchymal LTo signatures at diverse anatomical locations, identifying a common progenitor of mesenchymal LTos and LN-associated adipose tissue. Osr1 was essential for LN initiation, driving the commitment of mesenchymal LTo cells independent of neural retinoic acid, and for LN-associated lymphatic vasculature assembly. The combined action of chemokines CXCL13 and CCL21 was required for LN initiation. Our results redefine the role and identity of mesenchymal organizer cells and unify current views by proposing a model of cooperative cell function in LN initiation.
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Organogénesis , Factores de Transcripción , Diferenciación Celular , Ganglios Linfáticos , Tejido LinfoideRESUMEN
The mechanism of pattern formation during limb muscle development remains poorly understood. The canonical view holds that naïve limb muscle progenitor cells (MPCs) invade a pre-established pattern of muscle connective tissue, thereby forming individual muscles. Here, we show that early murine embryonic limb MPCs highly accumulate pSMAD1/5/9, demonstrating active signaling of bone morphogenetic proteins (BMP) in these cells. Overexpression of inhibitory human SMAD6 (huSMAD6) in limb MPCs abrogated BMP signaling, impaired their migration and proliferation, and accelerated myogenic lineage progression. Fewer primary myofibers developed, causing an aberrant proximodistal muscle pattern. Patterning was not disturbed when huSMAD6 was overexpressed in differentiated muscle, implying that the proximodistal muscle pattern depends on BMP-mediated expansion of MPCs before their differentiation. We show that limb MPCs differentially express Hox genes, and Hox-expressing MPCs displayed active BMP signaling. huSMAD6 overexpression caused loss of HOXA11 in early limb MPCs. In conclusion, our data show that BMP signaling controls expansion of embryonic limb MPCs as a prerequisite for establishing the proximodistal muscle pattern, a process that involves expression of Hox genes.
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Proteínas Morfogenéticas Óseas , Músculo Esquelético , Animales , Humanos , Ratones , Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación Celular/fisiología , Genes Homeobox , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Proteína smad6/metabolismoRESUMEN
Coordinated development of muscles, tendons, and their attachment sites ensures emergence of functional musculoskeletal units that are adapted to diverse anatomical demands among different species. How these different tissues are patterned and functionally assembled during embryogenesis is poorly understood. Here, we investigated the morphogenesis of extraocular muscles (EOMs), an evolutionary conserved cranial muscle group that is crucial for the coordinated movement of the eyeballs and for visual acuity. By means of lineage analysis, we redefined the cellular origins of periocular connective tissues interacting with the EOMs, which do not arise exclusively from neural crest mesenchyme as previously thought. Using 3D imaging approaches, we established an integrative blueprint for the EOM functional unit. By doing so, we identified a developmental time window in which individual EOMs emerge from a unique muscle anlage and establish insertions in the sclera, which sets these muscles apart from classical muscle-to-bone type of insertions. Further, we demonstrate that the eyeballs are a source of diffusible all-trans retinoic acid (ATRA) that allow their targeting by the EOMs in a temporal and dose-dependent manner. Using genetically modified mice and inhibitor treatments, we find that endogenous local variations in the concentration of retinoids contribute to the establishment of tendon condensations and attachment sites that precede the initiation of muscle patterning. Collectively, our results highlight how global and site-specific programs are deployed for the assembly of muscle functional units with precise definition of muscle shapes and topographical wiring of their tendon attachments.
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Músculos Oculomotores/embriología , Músculos Oculomotores/crecimiento & desarrollo , Tretinoina/metabolismo , Animales , Tejido Conectivo/fisiología , Desarrollo Embrionario , Ojo , Imagenología Tridimensional/métodos , Ratones/embriología , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Morfogénesis , Transducción de Señal , Tendones/fisiología , Tretinoina/fisiologíaRESUMEN
For decades, limb development has been a paradigm of three-dimensional patterning. Moreover, as the limb muscles and the other tissues of the limb's musculoskeletal system arise from distinct developmental sources, it has been a prime example of integrative morphogenesis and cross-tissue communication. As the limbs grow, all components of the musculoskeletal system (muscles, tendons, connective tissue, nerves) coordinate their growth and differentiation, ultimately giving rise to a functional unit capable of executing elaborate movement. While the molecular mechanisms governing global three-dimensional patterning and formation of the skeletal structures of the limbs has been a matter of intense research, patterning of the soft tissues is less understood. Here, we review the development of limb muscles with an emphasis on their interaction with other tissue types and the instructive roles these tissues play. Furthermore, we discuss the role of adult correlates of these embryonic accessory tissues in muscle regeneration.
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Extremidades/embriología , Desarrollo de Músculos , Músculo Esquelético/embriología , Regeneración , Animales , Diferenciación Celular , Humanos , Músculo Esquelético/citología , Músculo Esquelético/metabolismoRESUMEN
PURPOSE: We aimed to identify the underlying genetic cause for a novel form of distal arthrogryposis. METHODS: Rare variant family-based genomics, exome sequencing, and disease-specific panel sequencing were used to detect ADAMTS15 variants in affected individuals. Adamts15 expression was analyzed at the single-cell level during murine embryogenesis. Expression patterns were characterized using in situ hybridization and RNAscope. RESULTS: We identified homozygous rare variant alleles of ADAMTS15 in 5 affected individuals from 4 unrelated consanguineous families presenting with congenital flexion contractures of the interphalangeal joints and hypoplastic or absent palmar creases. Radiographic investigations showed physiological interphalangeal joint morphology. Additional features included knee, Achilles tendon, and toe contractures, spinal stiffness, scoliosis, and orthodontic abnormalities. Analysis of mouse whole-embryo single-cell sequencing data revealed a tightly regulated Adamts15 expression in the limb mesenchyme between embryonic stages E11.5 and E15.0. A perimuscular and peritendinous expression was evident in in situ hybridization in the developing mouse limb. In accordance, RNAscope analysis detected a significant coexpression with Osr1, but not with markers for skeletal muscle or joint formation. CONCLUSION: In aggregate, our findings provide evidence that rare biallelic recessive trait variants in ADAMTS15 cause a novel autosomal recessive connective tissue disorder, resulting in a distal arthrogryposis syndrome.
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Artrogriposis , Contractura , Proteínas ADAMTS , Animales , Artrogriposis/genética , Consanguinidad , Contractura/genética , Homocigoto , Humanos , Ratones , Mutación , Linaje , FenotipoRESUMEN
Connective tissues support organs and play crucial roles in development, homeostasis and fibrosis, yet our understanding of their formation is still limited. To gain insight into the molecular mechanisms of connective tissue specification, we selected five zinc-finger transcription factors - OSR1, OSR2, EGR1, KLF2 and KLF4 - based on their expression patterns and/or known involvement in connective tissue subtype differentiation. RNA-seq and ChIP-seq profiling of chick limb micromass cultures revealed a set of common genes regulated by all five transcription factors, which we describe as a connective tissue core expression set. This common core was enriched with genes associated with axon guidance and myofibroblast signature, including fibrosis-related genes. In addition, each transcription factor regulated a specific set of signalling molecules and extracellular matrix components. This suggests a concept whereby local molecular niches can be created by the expression of specific transcription factors impinging on the specification of local microenvironments. The regulatory network established here identifies common and distinct molecular signatures of limb connective tissue subtypes, provides novel insight into the signalling pathways governing connective tissue specification, and serves as a resource for connective tissue development.
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Diferenciación Celular/genética , Pollos/metabolismo , Tejido Conectivo/metabolismo , Factores de Transcripción/metabolismo , Animales , Pollos/genética , Clonación Molecular , Extremidades , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Inmunohistoquímica , Hibridación in Situ , Morfogénesis/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ARN , Transducción de Señal , Dedos de Zinc/genéticaRESUMEN
Transcriptional signal cointegrators associate with transcription factors or nuclear receptors and coregulate tissue-specific gene transcription. We report on recessive loss-of-function mutations in two genes (TRIP4 and ASCC1) that encode subunits of the nuclear activating signal cointegrator 1 (ASC-1) complex. We used autozygosity mapping and whole-exome sequencing to search for pathogenic mutations in four families. Affected individuals presented with prenatal-onset spinal muscular atrophy (SMA), multiple congenital contractures (arthrogryposis multiplex congenita), respiratory distress, and congenital bone fractures. We identified homozygous and compound-heterozygous nonsense and frameshift TRIP4 and ASCC1 mutations that led to a truncation or the entire absence of the respective proteins and cosegregated with the disease phenotype. Trip4 and Ascc1 have identical expression patterns in 17.5-day-old mouse embryos with high expression levels in the spinal cord, brain, paraspinal ganglia, thyroid, and submandibular glands. Antisense morpholino-mediated knockdown of either trip4 or ascc1 in zebrafish disrupted the highly patterned and coordinated process of α-motoneuron outgrowth and formation of myotomes and neuromuscular junctions and led to a swimming defect in the larvae. Immunoprecipitation of the ASC-1 complex consistently copurified cysteine and glycine rich protein 1 (CSRP1), a transcriptional cofactor, which is known to be involved in spinal cord regeneration upon injury in adult zebrafish. ASCC1 mutant fibroblasts downregulated genes associated with neurogenesis, neuronal migration, and pathfinding (SERPINF1, DAB1, SEMA3D, SEMA3A), as well as with bone development (TNFRSF11B, RASSF2, STC1). Our findings indicate that the dysfunction of a transcriptional coactivator complex can result in a clinical syndrome affecting the neuromuscular system.
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Fracturas Óseas/genética , Regulación del Desarrollo de la Expresión Génica , Atrofia Muscular Espinal/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Artrogriposis/diagnóstico , Artrogriposis/genética , Proteínas Portadoras/genética , Células Cultivadas , Fibroblastos/citología , Fibroblastos/metabolismo , Fracturas Óseas/diagnóstico , Perfilación de la Expresión Génica , Homocigoto , Humanos , Proteínas con Dominio LIM/genética , Ratones , Datos de Secuencia Molecular , Atrofia Muscular Espinal/diagnóstico , Mutación , Proteínas Nucleares/genética , Linaje , Fenotipo , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
Chordin-Like 1 (CHRDL1) mutations cause non-syndromic X-linked megalocornea (XMC) characterized by enlarged anterior eye segments. Mosaic corneal degeneration, presenile cataract and secondary glaucoma are associated with XMC. Beside that CHRDL1 encodes Ventroptin, a secreted bone morphogenetic protein (BMP) antagonist, the molecular mechanism of XMC is not well understood yet. In a family with broad phenotypic variability of XMC, we identified the novel CHRDL1 frameshift mutation c.807_808delTC [p.H270Wfs*22] presumably causing CHRDL1 loss of function. Using Xenopus laevis as model organism, we demonstrate that chrdl1 is specifically expressed in the ocular tissue at late developmental stages. The chrdl1 knockdown directly resembles the human XMC phenotype and confirms CHRDL1 deficiency to cause XMC. Interestingly, secondary to this bmp4 is down-regulated in the Xenopus eyes. Moreover, phospho-SMAD1/5 is altered and BMP receptor 1A is reduced in a XMC patient. Together, we classify these observations as negative-feedback regulation due to the deficient BMP antagonism in XMC. As CHRDL1 is preferentially expressed in the limbal stem cell niche of adult human cornea, we assume that CHRDL1 plays a key role in cornea homeostasis. In conclusion, we provide novel insights into the molecular mechanism of XMC as well as into the specific role of CHRDL1 during cornea organogenesis, among others by the establishment of the first XMC in vivo model. We show that unravelling monogenic cornea disorders like XMC-with presumably disturbed cornea growth and differentiation-contribute to the identification of potential limbal stem cell niche factors that are promising targets for regenerative therapies of corneal injuries.
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Enfermedades Hereditarias del Ojo/genética , Proteínas del Ojo/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Proteínas del Tejido Nervioso/genética , Adolescente , Animales , Secuencia de Bases , Proteína Morfogenética Ósea 4/genética , Proteína Morfogenética Ósea 4/metabolismo , Córnea/patología , Análisis Mutacional de ADN , Femenino , Mutación del Sistema de Lectura , Expresión Génica , Estudios de Asociación Genética , Humanos , Masculino , Linaje , Transducción de Señal , Xenopus laevisRESUMEN
Skeletal and heart muscle-specific variant of the alpha subunit of nascent polypeptide associated complex (skNAC) is exclusively found in striated muscle cells. Its function, however, is largely unknown. Previous reports could demonstrate that skNAC binds to Smyd1 (SET and MYND domain containing protein 1). The facts that (a) SET domains have histone methyltransferase activity, and (b) MYND domains are known recruiters of histone deacetylases (HDACs), implicate the skNAC-Smyd1 complex in transcriptional control. To study potential target genes, we carried out cDNA microarray analysis on differentiating C2C12 myoblasts in which expression of the skNAC gene had been knocked down. We found and confirmed a series of targets, specifically genes encoding regulators of inflammation, cellular metabolism, and cell migration. Mechanistically, as shown by Western blot, ELISA, and ChIP analysis at selected promoter regions, transcriptional control by skNAC-Smyd1 appears to be exerted at least in part by affecting a series of histone modifications, specifically H3K4 di- and trimethylation and potentially also histone acetylation. Taken together, our data suggest that the skNAC-Smyd1 complex is involved in transcriptional regulation both via the control of histone methylation and histone (de)acetylation.
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Proteínas de Unión al ADN/genética , Histonas/metabolismo , Chaperonas Moleculares/genética , Proteínas Musculares/genética , Factores de Transcripción/genética , Transcripción Genética/genética , Acetilación , Animales , Diferenciación Celular , Línea Celular , Movimiento Celular/genética , Metabolismo Energético/genética , Regulación de la Expresión Génica , Histona Desacetilasas/metabolismo , Inflamación/genética , Metilación , Ratones , Músculo Esquelético/metabolismo , Mioblastos Cardíacos/citología , Mioblastos Esqueléticos/citología , Miocardio/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas/genética , Interferencia de ARN , ARN Interferente Pequeño , Succinato Deshidrogenasa/metabolismo , Factores de Intercambio de Guanina Nucleótido ras/biosíntesisRESUMEN
The morphology of bones is genetically determined, but the molecular mechanisms that control shape, size and the overall gestalt of bones remain unclear. We previously showed that metacarpals in the synpolydactyly homolog (spdh) mouse, which carries a mutation in Hoxd13 similar to the human condition synpolydactyly (SPD), were transformed to carpal-like bones with cuboid shape that lack cortical bone and a perichondrium and are surrounded by a joint surface. Here we provide evidence that spdh metacarpal growth plates have a defect in cell polarization with a random instead of linear orientation. In parallel prospective perichondral cells failed to adopt the characteristic flattened cell shape. We observed a similar cell polarity defect in metacarpals of Wnt5a(-/-) mice. Wnt5a and the closely related Wnt5b were downregulated in spdh handplates, and HOXD13 induced expression of both genes in vitro. Concomitant we observed mislocalization of core planar cell polarity (PCP) components DVL2 and PRICKLE1 in spdh metacarpals indicating a defect in the WNT/PCP pathway. Conversely the WNT/ß-CATENIN pathway, a hallmark of joint cells lining carpal bones, was upregulated in the perichondral region. Finally, providing spdh limb explant cultures with cells expressing either HOXD13 or WNT5A led to a non-cell autonomous partial rescue of cell polarity the perichondral region and restored the expression of perichondral markers. This study provides a so far unrecognized link between HOX proteins and cell polarity in the perichondrium and the growth plate, a failure of which leads to transformation of metacarpals to carpal-like structures.
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Cartílago/embriología , Placa de Crecimiento/embriología , Proteínas de Homeodominio/metabolismo , Huesos del Metacarpo/embriología , Factores de Transcripción/metabolismo , Proteínas Wnt/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Cartílago/metabolismo , Polaridad Celular , Células Cultivadas , Proteínas Dishevelled , Placa de Crecimiento/metabolismo , Proteínas de Homeodominio/genética , Humanos , Proteínas con Dominio LIM/metabolismo , Huesos del Metacarpo/metabolismo , Ratones , Ratones Noqueados , Morfogénesis/genética , Fosfoproteínas/metabolismo , Receptores de Fenciclidina/metabolismo , Sindactilia/genética , Factores de Transcripción/genética , Proteínas Wnt/genética , Proteína Wnt-5a , beta Catenina/metabolismoRESUMEN
BACKGROUND: Components of the limb musculoskeletal system have distinct mesoderm origins. Limb skeletal muscles originate from somites, while the skeleton and attachments (tendons and connective tissues) derive from limb lateral plate. Despite distinct mesoderm origins, the development of muscle, skeleton and attachments is highly coordinated both spatially and temporally to ensure complete function of the musculoskeletal system. A system to study molecular interactions between somitic-derived tissues (muscles) and lateral-plate-derived tissues (skeletal components and attachments) during limb development is missing. RESULTS: We designed a gene delivery system in chick embryos with the ultimate aim to study the interactions between the components of the musculoskeletal system during limb development. We combined the Tol2 genomic integration system with the viral T2A system and developed new vectors that lead to stable and bicistronic expression of two proteins at comparable levels in chick cells. Combined with limb somite and lateral plate electroporation techniques, two fluorescent reporter proteins were co-expressed in stoichiometric proportion in the muscle lineage (somitic-derived) or in skeleton and their attachments (lateral-plate-derived). In addition, we designed three vectors with different promoters to target muscle cells at different steps of the differentiation process. CONCLUSION: Limb somite electroporation technique using vectors containing these different promoters allowed us to target all myogenic cells, myoblasts or differentiated muscle cells. These stable and promoter-specific vectors lead to bicistronic expression either in somitic-derived myogenic cells or lateral plate-derived cells, depending on the electroporation sites and open new avenues to study the interactions between myogenic cells and tendon or connective tissue cells during limb development.
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Huesos/embriología , Extremidades/embriología , Esbozos de los Miembros/embriología , Músculo Esquelético/embriología , Somitos/embriología , Actinas/genética , Animales , Embrión de Pollo , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/genética , Electroporación , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes/genética , Desarrollo de Músculos/fisiología , Cadenas Ligeras de Miosina/genética , Organogénesis/genética , Organogénesis/fisiología , Regiones Promotoras Genéticas/genéticaRESUMEN
Brachydactyly type A1 (BDA1) was the first recorded disorder of the autosomal dominant Mendelian trait in humans, characterized by shortened or absent middle phalanges in digits. It is associated with heterozygous missense mutations in indian hedgehog (IHH). Hedgehog proteins are important morphogens for a wide range of developmental processes. The capacity and range of signalling is thought to be regulated by its interaction with the receptor PTCH1 and antagonist HIP1. Here we show that a BDA1 mutation (E95K) in Ihh impairs the interaction of IHH with PTCH1 and HIP1. This is consistent with a recent paper showing that BDA1 mutations cluster in a calcium-binding site essential for the interaction with its receptor and cell-surface partners. Furthermore, we show that in a mouse model that recapitulates the E95K mutation, there is a change in the potency and range of signalling. The mice have digit abnormalities consistent with the human disorder.
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Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Deformidades Congénitas de las Extremidades/genética , Deformidades Congénitas de las Extremidades/metabolismo , Mutación/genética , Transducción de Señal , Animales , Pollos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Receptores Patched , Receptor Patched-1 , Unión Proteica , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismoRESUMEN
Indian hedgehog (IHH) is a secreted signaling molecule of the hedgehog family known to play important roles in the regulation of chondrocyte differentiation, cortical bone formation, and the development of joints. Here, we describe that copy-number variations of the IHH locus involving conserved noncoding elements (CNEs) are associated with syndactyly and craniosynostosis. These CNEs are able to drive reporter gene expression in a pattern highly similar to wild-type Ihh expression. We postulate that the observed duplications lead to a misexpression and/or overexpression of IHH and by this affect the complex regulatory signaling network during digit and skull development.
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Craneosinostosis/genética , Variaciones en el Número de Copia de ADN , Duplicación de Gen , Sitios Genéticos , Proteínas Hedgehog/genética , Sindactilia/genética , Animales , Secuencia Conservada/genética , Femenino , Dedos/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Masculino , Ratones , Ratones Transgénicos , Mutación , Cráneo/crecimiento & desarrolloRESUMEN
Proteoglycans are a major component of extracellular matrix and contribute to normal embryonic and postnatal development by ensuring tissue stability and signaling functions. We studied five patients with recessive joint dislocations and congenital heart defects, including bicuspid aortic valve (BAV) and aortic root dilatation. We identified linkage to chromosome 11 and detected a mutation (c.830G>A, p.Arg277Gln) in B3GAT3, the gene coding for glucuronosyltransferase-I (GlcAT-I). The enzyme catalyzes an initial step in the synthesis of glycosaminoglycan side chains of proteoglycans. Patients' cells as well as recombinant mutant protein showed reduced glucuronyltransferase activity. Patient fibroblasts demonstrated decreased levels of dermatan sulfate, chondroitin sulfate, and heparan sulfate proteoglycans, indicating that the defect in linker synthesis affected all three lines of O-glycanated proteoglycans. Further studies demonstrated that GlcAT-I resides in the cis and cis-medial Golgi apparatus and is expressed in the affected tissues, i.e., heart, aorta, and bone. The study shows that reduced GlcAT-I activity impairs skeletal as well as heart development and results in variable combinations of heart malformations, including mitral valve prolapse, ventricular septal defect, and bicuspid aortic valve. The described family constitutes a syndrome characterized by heart defects and joint dislocations resulting from altered initiation of proteoglycan synthesis (Larsen-like syndrome, B3GAT3 type).
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Glucuronosiltransferasa/genética , Cardiopatías Congénitas/patología , Proteoglicanos/biosíntesis , Adolescente , Secuencia de Aminoácidos , Válvula Aórtica/patología , Estudios de Casos y Controles , Niño , Sulfatos de Condroitina/análisis , Cromosomas Humanos Par 11/genética , Consanguinidad , Dermatán Sulfato/análisis , Electroforesis en Gel de Poliacrilamida , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente , Proteoglicanos de Heparán Sulfato/análisis , Humanos , Immunoblotting , Masculino , Válvula Mitral/patología , Modelos Moleculares , Datos de Secuencia Molecular , LinajeRESUMEN
Facial branchiomotor neurons (FBMs) of vertebrates typically develop in rhombomere 4 (r4), and in mammals and several other vertebrate taxa, migrate caudally into r6 and subsequently laterally and ventrally to the pial surface. How similar or dissimilar these migratory processes between species are at a molecular level remains unclear. In zebrafish and mouse, mutations in certain PCP genes disrupt normal caudal migration of FBMs. Zebrafish prickle1a (prickle-like 1a) and prickle1b, two orthologs of Prickle1, act non-cell-autonomously and cell-autonomously, respectively, to regulate FBM migration. Here, we show that, in Prickle1 (C251X/C251X) mice which have reduced Prickle1 expression, the caudal migration of FBMs is affected. Most FBM neurons do not migrate caudally along the floor plate. However, some neurons perform limited caudal migration such that the neurons eventually lie near the pial surface from r4 to anterior r6. FBMs in Prickle1 (C251X/C251X) mice survive until P0 and form an ectopic nucleus dorsal to the olivo-cochlear efferents of r4. Ror2, which modifies the PCP pathway in other systems, is expressed by the migrating mouse FBMs, but is not required for FBM caudal migration. Our results suggest that, in mice, Prickle1 is part of a molecular mechanism that regulates FBM caudal migration and separates the FBM and the olivo-cochlear efferents. This defective caudal migration of FBMs in Prickle1C251X mutants resembles Vangl2 mutant defects. In contrast to other developing systems that show similar defects in Prickle1, Wnt5a and Ror2, the latter two only have limited or no effect on FBM caudal migration.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Movimiento Celular , Cara/inervación , Proteínas con Dominio LIM/metabolismo , Neuronas Motoras/citología , Neuronas Motoras/metabolismo , Animales , Núcleo Celular/metabolismo , Polaridad Celular , Supervivencia Celular , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Ratones , Ratones Mutantes , Mutación/genética , Neuronas Eferentes/citología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismoRESUMEN
BACKGROUND: Metacarpal 4-5 fusion (MF4; MIM %309630) is a rare congenital malformation of the hand characterised by the partial or complete fusion of the fourth and fifth metacarpals. The anomaly occurs as an isolated trait or part of a genetic syndrome. METHODS: To search for disease-causing mutation, whole exome sequencing (WES) was performed on samples from a single trio. Before WES, molecular screening including gene sequencing and array comparative genomic hybridisation was applied. Validation of WES and segregation studies were done using routine Sanger sequencing. RESULTS: Exome sequencing detected a nonsense mutation (c.C535T; p.R179X) in exon 3 of the FGF16 gene, which maps to chromosome Xq21.1. Mutational screening of the FGF16 gene performed in an unrelated proband of different ethnicity showed another nonsense mutation in exon 3 (c.C470A; p.S157X). CONCLUSIONS: This study shows that truncating mutations of FGF16 are associated with X-linked recessive metacarpal 4-5 fusion. The study provides evidence for the involvement of FGF16 in the fine tuning of the human skeleton of the hand.
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Codón sin Sentido , Exoma , Factores de Crecimiento de Fibroblastos/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Deformidades Congénitas de la Mano/genética , Huesos del Metacarpo/anomalías , Niño , Embrión de Mamíferos , Femenino , Humanos , Masculino , Especificidad de Órganos , Análisis de Secuencia de ADNRESUMEN
Brachydactyly type E (BDE), shortened metacarpals, metatarsals, cone-shaped epiphyses, and short stature commonly occurs as a sole phenotype. Parathyroid hormone-like protein (PTHrP) has been shown to be responsible in all forms to date, either directly or indirectly. We used linkage and then whole genome sequencing in a small pedigree, to elucidate BDE and identified a truncated disintegrin-and-metalloproteinase-19 (ADAM19) allele in all affected family members, but not in nonaffected persons. Since we had shown earlier that the extracellular domain of the parathyroid hormone receptor (PTHR1) is subject to an unidentified metalloproteinase cleavage, we tested the hypothesis that ADAM19 is a sheddase for PTHR1. WT ADAM19 cleaved PTHR1, while mutated ADAM-19 did not. We mapped the cleavage site that we verified with mass spectrometry between amino acids 64-65. ADAM-19 cleavage increased Gq and decreased Gs activation. Moreover, perturbed PTHR1 cleavage by ADAM19 increased ß-arrestin2 recruitment, while cAMP accumulation was not altered. We suggest that ADAM19 serves as a regulatory element for PTHR1 and could be responsible for BDE. This sheddase may affect other PTHrP or PTH-related functions.
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Braquidactilia , Proteína Relacionada con la Hormona Paratiroidea , Humanos , Proteína Relacionada con la Hormona Paratiroidea/genética , Braquidactilia/genética , Receptor de Hormona Paratiroídea Tipo 1/genética , Receptor de Hormona Paratiroídea Tipo 1/metabolismo , Metaloproteasas , Proteínas ADAMRESUMEN
Patients affected by neurofibromatosis type 1 (NF1) frequently show muscle weakness with unknown etiology. Here we show that, in mice, Neurofibromin 1 (Nf1) is not required in muscle fibers, but specifically in early postnatal myogenic progenitors (MPs), where Nf1 loss led to cell cycle exit and differentiation blockade, depleting the MP pool resulting in reduced myonuclear accretion as well as reduced muscle stem cell numbers. This was caused by precocious induction of stem cell quiescence coupled to metabolic reprogramming of MPs impinging on glycolytic shutdown, which was conserved in muscle fibers. We show that a Mek/Erk/NOS pathway hypersensitizes Nf1-deficient MPs to Notch signaling, consequently, early postnatal Notch pathway inhibition ameliorated premature quiescence, metabolic reprogramming and muscle growth. This reveals an unexpected role of Ras/Mek/Erk signaling supporting postnatal MP quiescence in concert with Notch signaling, which is controlled by Nf1 safeguarding coordinated muscle growth and muscle stem cell pool establishment. Furthermore, our data suggest transmission of metabolic reprogramming across cellular differentiation, affecting fiber metabolism and function in NF1.
Asunto(s)
Neurofibromatosis 1 , Neurofibromina 1 , Ratones , Humanos , Animales , Neurofibromina 1/genética , Neurofibromina 1/metabolismo , Neurofibromatosis 1/genética , Neurofibromatosis 1/metabolismo , Transducción de Señal/fisiología , Sistema de Señalización de MAP Quinasas , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismoRESUMEN
Neurofibromatosis type 1 (NF1) is a multi-system disease caused by mutations in the NF1 gene encoding a Ras-GAP protein, neurofibromin, which negatively regulates Ras signaling. Besides neuroectodermal malformations and tumors, the skeletal system is often affected (e.g. scoliosis and long bone dysplasia) demonstrating the importance of neurofibromin for development and maintenance of the musculoskeletal system. Here, we focus on the role of neurofibromin in skeletal muscle development. Nf1 gene inactivation in the early limb bud mesenchyme using Prx1-cre (Nf1(Prx1)) resulted in muscle dystrophy characterized by fibrosis, reduced number of muscle fibers and reduced muscle force. This was caused by an early defect in myogenesis affecting the terminal differentiation of myoblasts between E12.5 and E14.5. In parallel, the muscle connective tissue cells exhibited increased proliferation at E14.5 and an increase in the amount of connective tissue as early as E16.5. These changes were accompanied by excessive mitogen-activated protein kinase pathway activation. Satellite cells isolated from Nf1(Prx1) mice showed normal self-renewal, but their differentiation was impaired as indicated by diminished myotube formation. Our results demonstrate a requirement of neurofibromin for muscle formation and maintenance. This previously unrecognized function of neurofibromin may contribute to the musculoskeletal problems in NF1 patients.