RESUMEN
The receptor tyrosine kinase ROR2 mediates noncanonical WNT5A signaling to orchestrate tissue morphogenetic processes, and dysfunction of the pathway causes Robinow syndrome, brachydactyly B, and metastatic diseases. The domain(s) and mechanisms required for ROR2 function, however, remain unclear. We solved the crystal structure of the extracellular cysteine-rich (CRD) and Kringle (Kr) domains of ROR2 and found that, unlike other CRDs, the ROR2 CRD lacks the signature hydrophobic pocket that binds lipids/lipid-modified proteins, such as WNTs, suggesting a novel mechanism of ligand reception. Functionally, we showed that the ROR2 CRD, but not other domains, is required and minimally sufficient to promote WNT5A signaling, and Robinow mutations in the CRD and the adjacent Kr impair ROR2 secretion and function. Moreover, using function-activating and -perturbing antibodies against the Frizzled (FZ) family of WNT receptors, we demonstrate the involvement of FZ in WNT5A-ROR signaling. Thus, ROR2 acts via its CRD to potentiate the function of a receptor super-complex that includes FZ to transduce WNT5A signals.
Asunto(s)
Receptores Huérfanos Similares al Receptor Tirosina Quinasa , Vía de Señalización Wnt , Animales , Humanos , Ratones , Cristalografía por Rayos X , Conformación Proteica , Dominios Proteicos , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/química , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/genética , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Proteínas Wnt/metabolismo , Proteínas Wnt/genética , Proteína Wnt-5a/metabolismo , Proteína Wnt-5a/genéticaRESUMEN
Detailed binding experiments reveal new insights into the Norrin/Wnt signaling pathway that helps to control vascularization in the retina.
Asunto(s)
Proteínas del Ojo , Proteínas del Tejido Nervioso , Retina , Vía de Señalización Wnt , Proteínas del Ojo/metabolismo , Proteínas del Ojo/genética , Humanos , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Retina/metabolismo , Animales , Unión ProteicaRESUMEN
Deciphering non-canonical WNT signaling has proven to be both fascinating and challenging. Discovered almost 30 years ago, non-canonical WNT ligands signal independently of the transcriptional co-activator ß-catenin to regulate a wide range of morphogenetic processes during development. The molecular and cellular mechanisms that underlie non-canonical WNT function, however, remain nebulous. Recent results from various model systems have converged to define a core non-canonical WNT pathway consisting of the prototypic non-canonical WNT ligand, WNT5A, the receptor tyrosine kinase ROR, the seven transmembrane receptor Frizzled and the cytoplasmic scaffold protein Dishevelled. Importantly, mutations in each of these signaling components cause Robinow syndrome, a congenital disorder characterized by profound tissue morphogenetic abnormalities. Moreover, dysregulation of the pathway has also been linked to cancer metastasis. As new knowledge concerning the WNT5A-ROR pathway continues to grow, modeling these mutations will likely provide crucial insights into both the physiological regulation of the pathway and the etiology of WNT5A-ROR-driven diseases.
Asunto(s)
Receptores Huérfanos Similares al Receptor Tirosina Quinasa , Proteínas Wnt , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/genética , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Proteína Wnt-5a/genética , Proteína Wnt-5a/metabolismo , Transducción de Señal/fisiología , Morfogénesis , Vía de Señalización WntRESUMEN
Wnt5a-Ror signaling is a conserved pathway that regulates morphogenetic processes during vertebrate development [R. T. Moon et al, Development 119, 97-111 (1993); I. Oishi et al, Genes Cells 8, 645-654 (2003)], but its downstream signaling events remain poorly understood. Through a large-scale proteomic screen in mouse embryonic fibroblasts, we identified the E3 ubiquitin ligase Pdzrn3 as a regulatory target of the Wnt5a-Ror pathway. Upon pathway activation, Pdzrn3 is degraded in a ß-catenin-independent, ubiquitin-proteasome system-dependent manner. We developed a flow cytometry-based reporter to monitor Pdzrn3 abundance and delineated a signaling cascade involving Frizzled, Dishevelled, Casein kinase 1, and Glycogen synthase kinase 3 that regulates Pdzrn3 stability. Epistatically, Pdzrn3 is regulated independently of Kif26b, another Wnt5a-Ror effector. Wnt5a-dependent degradation of Pdzrn3 requires phosphorylation of three conserved amino acids within its C-terminal LNX3H domain [M. Flynn, O. Saha, P. Young, BMC Evol. Biol. 11, 235 (2011)], which acts as a bona fide Wnt5a-responsive element. Importantly, this phospho-dependent degradation is essential for Wnt5a-Ror modulation of cell migration. Collectively, this work establishes a Wnt5a-Ror cell morphogenetic cascade involving Pdzrn3 phosphorylation and degradation.
Asunto(s)
Proteómica , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Vía de Señalización Wnt , Proteína Wnt-5a/metabolismo , Animales , Movimiento Celular , Ratones , Fosforilación , Complejo de la Endopetidasa Proteasomal/metabolismo , Dominios Proteicos , Proteolisis , Reproducibilidad de los Resultados , Ubiquitina/metabolismoRESUMEN
WNT5a is a mainly "non-canonical" WNT ligand whose dysregulation is observed in lung diseases such as idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD) and asthma. Germline deletion of Wnt5a disrupts embryonic lung development. However, the temporal-specific function of WNT5a remains unknown. In this study, we generated a conditional loss-of-function mouse model (Wnt5aCAG) and examined the specific role of Wnt5a during the saccular and alveolar phases of lung development. The lack of Wnt5a in the saccular phase blocked distal airway expansion and attenuated differentiation of endothelial and alveolar epithelial type I (AT1) cells and myofibroblasts. Postnatal Wnt5a inactivation disrupted alveologenesis, producing a phenotype resembling human bronchopulmonary dysplasia (BPD). Mutant lungs showed hypoalveolization, but endothelial and epithelial differentiation was unaffected. The major impact of Wnt5a inactivation on alveologenesis was on myofibroblast differentiation and migration, with reduced expression of key regulatory genes. These findings were validated in vitro using isolated lung fibroblasts. Conditional inactivation of the WNT5a receptors Ror1 and Ror2 in alveolar myofibroblasts recapitulated the Wnt5aCAG phenotype, demonstrating that myofibroblast defects are the major cause of arrested alveologenesis in Wnt5aCAG lungs. Finally, we show that WNT5a is reduced in human BPD lung samples, indicating the clinical relevance and potential role for WNT5a in pathogenesis of BPD.
Asunto(s)
Organogénesis , Alveolos Pulmonares/embriología , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Transducción de Señal , Proteína Wnt-5a/metabolismo , Animales , Animales Recién Nacidos , Diferenciación Celular , Movimiento Celular , Células Cultivadas , Células Endoteliales/citología , Regulación del Desarrollo de la Expresión Génica , Humanos , Recién Nacido , Ratones , Modelos Biológicos , Miofibroblastos/citologíaRESUMEN
Adult neurogenesis in the hippocampus may represent a form of plasticity in brain functions including mood, learning and memory. However, mechanisms underlying neural stem/progenitor cells (NSPCs) proliferation are not well understood. We found that Agrin, a factor critical for neuromuscular junction formation, is elevated in the hippocampus of mice that are stimulated by enriched environment (EE). Genetic deletion of the Agrn gene in excitatory neurons decreases NSPCs proliferation and increases depressive-like behavior. Low-density lipoprotein receptor-related protein 4 (Lrp4), a receptor for Agrin, is expressed in hippocampal NSPCs and its mutation blocked basal as well as EE-induced NSPCs proliferation and maturation of newborn neurons. Finally, we show that Lrp4 interacts with and activates receptor tyrosine kinase-like orphan receptor 2 (Ror2); and Ror2 mutation impairs NSPCs proliferation. Together, these observations identify a role of Agrin-Lrp4-Ror2 signaling for adult neurogenesis, uncovering previously unexpected functions of Agrin and Lrp4 in the brain.
Asunto(s)
Agrina/metabolismo , Hipocampo/crecimiento & desarrollo , Proteínas Relacionadas con Receptor de LDL/metabolismo , Células-Madre Neurales/fisiología , Neurogénesis , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Transducción de Señal , Agrina/deficiencia , Animales , Proliferación Celular , Técnicas de Inactivación de Genes , Proteínas Relacionadas con Receptor de LDL/deficiencia , Ratones , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/deficienciaRESUMEN
Multiple vertebrate embryonic structures such as organ primordia are composed of confluent cells. Although mechanisms that shape tissue sheets are increasingly understood, those which shape a volume of cells remain obscure. Here we show that 3D mesenchymal cell intercalations are essential to shape the mandibular arch of the mouse embryo. Using a genetically encoded vinculin tension sensor that we knock-in to the mouse genome, we show that cortical force oscillations promote these intercalations. Genetic loss- and gain-of-function approaches show that Wnt5a functions as a spatial cue to coordinate cell polarity and cytoskeletal oscillation. These processes diminish tissue rigidity and help cells to overcome the energy barrier to intercalation. YAP/TAZ and PIEZO1 serve as downstream effectors of Wnt5a-mediated actomyosin polarity and cytosolic calcium transients that orient and drive mesenchymal cell intercalations. These findings advance our understanding of how developmental pathways regulate biophysical properties and forces to shape a solid organ primordium.
Asunto(s)
Polaridad Celular , Citoesqueleto/fisiología , Mandíbula/embriología , Mandíbula/fisiología , Proteína Wnt-5a/fisiología , Citoesqueleto de Actina , Actomiosina/metabolismo , Animales , Calcio/metabolismo , Ciclo Celular , Citosol/metabolismo , Elasticidad , Células Epiteliales/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Mutación , Oscilometría , Transducción de Señal , Estrés Mecánico , Vinculina/metabolismo , ViscosidadRESUMEN
Our understanding of the cellular mechanisms governing carcinoma invasiveness and metastasis has evolved dramatically over the last several years. The previous emphasis on the epithelial-mesenchymal transition as a driver of the migratory properties of single cells has expanded with the observation that carcinoma cells often invade and migrate collectively as adherent groups. Moreover, recent analyses suggest that circulating tumor cells within the vasculature often exist as multicellular clusters and that clusters more efficiently seed metastatic lesions than single circulating tumor cells. While these observations point to a key role for collective cell migration in carcinoma metastasis, the molecular mechanisms driving collective tumor cell migration remain to be discerned. Wnt/PCP (planar cell polarity) signaling, one of the noncanonical Wnt signaling pathways, mediates collective migratory events such as convergent extension during developmental processes. Wnt/PCP signaling components are frequently dysregulated in solid tumors, and aberrant pathway activation contributes to tumor cell migratory properties. Here we summarize key studies that address the mechanisms by which Wnt/PCP signaling mediate collective cell migration in developmental and tumor contexts. We emphasize Wnt/PCP component localization within migrating cells and discuss how component asymmetry may govern the spatiotemporal control of downstream cytoskeletal effectors to promote collective cell motility.
Asunto(s)
Carcinoma/patología , Movimiento Celular , Polaridad Celular , Transición Epitelial-Mesenquimal , Proteínas Wnt/metabolismo , Animales , Carcinoma/metabolismo , Humanos , Metástasis de la NeoplasiaRESUMEN
Mutations in the human receptor tyrosine kinase ROR2 are associated with Robinow syndrome (RRS) and brachydactyly type B1. Amongst others, the shortened limb phenotype associated with RRS is recapitulated in Ror2-/- mutant mice. In contrast, Ror1-/- mutant mice are viable and show no limb phenotype. Ror1-/- ;Ror2-/- double mutants are embryonic lethal, whereas double mutants containing a hypomorphic Ror1 allele (Ror1hyp ) survive up to birth and display a more severe shortened limb phenotype. Both orphan receptors have been shown to act as possible Wnt coreceptors and to mediate the Wnt5a signal. Here, we analyzed genetic interactions between the Wnt ligand, Wnt9a, and Ror2 or Ror1, as Wnt9a has also been implicated in skeletal development. Wnt9a-/- single mutants display a mild shortening of the long bones, whereas these are severely shortened in Ror2-/- mutants. Ror2-/- ;Wnt9a-/- double mutants displayed even more severely shortened long bones, and intermediate phenotypes were observed in compound Ror2;Wnt9a mutants. Long bones were also shorter in Ror1hyp/hyp ;Wnt9a-/- double mutants. In addition, Ror1hyp/hyp ;Wnt9a-/- double mutants displayed a secondary palate cleft phenotype, which was not present in the respective single mutants. Interestingly, 50% of compound mutant pups heterozygous for Ror2 and homozygous mutant for Ror1 also developed a secondary palate cleft phenotype.
Asunto(s)
Fisura del Paladar/genética , Epistasis Genética , Deformidades Congénitas de las Extremidades/genética , Mutación , Osteogénesis/genética , Proteínas Wnt/genética , Animales , Ratones , Ratones Endogámicos C57BL , Fenotipo , Receptores Huérfanos Similares al Receptor Tirosina QuinasaRESUMEN
Domestic dog breeds exhibit remarkable morphological variations that result from centuries of artificial selection and breeding. Identifying the genetic changes that contribute to these variations could provide critical insights into the molecular basis of tissue and organismal morphogenesis. Bulldogs, French Bulldogs and Boston Terriers share many morphological and disease-predisposition traits, including brachycephalic skull morphology, widely set eyes and short stature. Unlike other brachycephalic dogs, these breeds also exhibit vertebral malformations that result in a truncated, kinked tail (screw tail). Whole genome sequencing of 100 dogs from 21 breeds identified 12.4 million bi-allelic variants that met inclusion criteria. Whole Genome Association of these variants with the breed defining phenotype of screw tail was performed using 10 cases and 84 controls and identified a frameshift mutation in the WNT pathway gene DISHEVELLED 2 (DVL2) (Chr5: 32195043_32195044del, p = 4.37 X 10-37) as the most strongly associated variant in the canine genome. This DVL2 variant was fixed in Bulldogs and French Bulldogs and had a high allele frequency (0.94) in Boston Terriers. The DVL2 variant segregated with thoracic and caudal vertebral column malformations in a recessive manner with incomplete and variable penetrance for thoracic vertebral malformations between different breeds. Importantly, analogous frameshift mutations in the human DVL1 and DVL3 genes cause Robinow syndrome, a congenital disorder characterized by similar craniofacial, limb and vertebral malformations. Analysis of the canine DVL2 variant protein showed that its ability to undergo WNT-induced phosphorylation is reduced, suggesting that altered WNT signaling may contribute to the Robinow-like syndrome in the screwtail breeds.
Asunto(s)
Anomalías Craneofaciales/veterinaria , Proteínas Dishevelled/genética , Enfermedades de los Perros/genética , Perros/genética , Enanismo/veterinaria , Deformidades Congénitas de las Extremidades/veterinaria , Anomalías Urogenitales/veterinaria , Secuencia de Aminoácidos , Animales , Anomalías Craneofaciales/genética , Anomalías Craneofaciales/metabolismo , Proteínas Dishevelled/metabolismo , Enfermedades de los Perros/metabolismo , Perros/anatomía & histología , Perros/clasificación , Enanismo/genética , Enanismo/metabolismo , Femenino , Mutación del Sistema de Lectura , Variación Genética , Estudio de Asociación del Genoma Completo , Humanos , Deformidades Congénitas de las Extremidades/genética , Deformidades Congénitas de las Extremidades/metabolismo , Masculino , Compuestos de Organosilicio , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Cola (estructura animal)/anatomía & histología , Anomalías Urogenitales/genética , Anomalías Urogenitales/metabolismo , Vía de Señalización Wnt/genéticaRESUMEN
KIF26B is a member of the kinesin superfamily with evolutionarily conserved functions in controlling aspects of embryogenesis, including the development of the nervous system, though its function is incompletely understood. We describe an infant with progressive microcephaly, pontocerebellar hypoplasia, and arthrogryposis secondary to the involvement of anterior horn cells and ventral (motor) nerves. We performed whole exome sequencing on the trio and identified a de novo KIF26B missense variant, p.Gly546Ser, in the proband. This variant alters a highly conserved amino acid residue that is part of the phosphate-binding loop motif and motor-like domain and is deemed pathogenic by several in silico methods. Functional analysis of the variant protein in cultured cells revealed a reduction in the KIF26B protein's ability to promote cell adhesion, a defect that potentially contributes to its pathogenicity. Overall, KIF26B may play a critical role in the brain development and, when mutated, cause pontocerebellar hypoplasia with arthrogryposis.
Asunto(s)
Cinesinas/genética , Atrofias Olivopontocerebelosas/genética , Atrofias Musculares Espinales de la Infancia/diagnóstico , Atrofias Musculares Espinales de la Infancia/genética , Secuencia de Aminoácidos , Animales , Encéfalo/anomalías , Encéfalo/diagnóstico por imagen , Adhesión Celular , Modelos Animales de Enfermedad , Expresión Génica , Humanos , Cinesinas/química , Imagen por Resonancia Magnética/métodos , Ratones , Modelos Moleculares , Conformación Proteica , Secuenciación del ExomaRESUMEN
Noncanonical Wnt signaling functions independently of the ß-catenin pathway to control diverse developmental processes, and dysfunction of the pathway contributes to a number of human pathological conditions, including birth defects and metastatic cancer. Progress in the field, however, has been hampered by the scarcity of functional assays for measuring noncanonical Wnt signaling activity. We recently described the Wnt5a-Ror-Kif26b (WRK) reporter assay, which directly monitors a post-transcriptional regulatory event in noncanonical Wnt signaling. In this protocol, we describe the generation of the stable GFP-Kif26b reporter cell line and a quantitative reporter assay for detecting and measuring Wnt5a signaling activities in live cells via flow cytometry.
RESUMEN
Noncanonical WNT pathways function independently of the ß-catenin transcriptional co-activator to regulate diverse morphogenetic and pathogenic processes. Recent studies showed that noncanonical WNTs, such as WNT5A, can signal the degradation of several downstream effectors, thereby modulating these effectors' cellular activities. The protein domain(s) that mediates the WNT5A-dependent degradation response, however, has not been identified. By coupling protein mutagenesis experiments with a flow cytometry-based degradation reporter assay, we have defined a protein domain in the kinesin superfamily protein KIF26B that is essential for WNT5A-dependent degradation. We found that a human disease-causing KIF26B mutation located at a conserved amino acid within this domain compromises the ability of WNT5A to induce KIF26B degradation. Using pharmacological perturbation, we further uncovered a role of glycogen synthase kinase 3 (GSK3) in WNT5A regulation of KIF26B degradation. Lastly, based on the identification of the WNT5A-responsive domain, we developed a new reporter system that allows for efficient profiling of WNT5A-KIF26B signaling activity in both somatic and stem cells. In conclusion, our study identifies a new protein domain that mediates WNT5A-dependent degradation of KIF26B and provides a new tool for functional characterization of noncanonical WNT5A signaling in cells.
RESUMEN
Wnt5a-Ror signaling constitutes a developmental pathway crucial for embryonic tissue morphogenesis, reproduction and adult tissue regeneration, yet the molecular mechanisms by which the Wnt5a-Ror pathway mediates these processes are largely unknown. Using a proteomic screen, we identify the kinesin superfamily protein Kif26b as a downstream target of the Wnt5a-Ror pathway. Wnt5a-Ror, through a process independent of the canonical Wnt/ß-catenin-dependent pathway, regulates the cellular stability of Kif26b by inducing its degradation via the ubiquitin-proteasome system. Through this mechanism, Kif26b modulates the migratory behavior of cultured mesenchymal cells in a Wnt5a-dependent manner. Genetic perturbation of Kif26b function in vivo caused embryonic axis malformations and depletion of primordial germ cells in the developing gonad, two phenotypes characteristic of disrupted Wnt5a-Ror signaling. These findings indicate that Kif26b links Wnt5a-Ror signaling to the control of morphogenetic cell and tissue behaviors in vertebrates and reveal a new role for regulated proteolysis in noncanonical Wnt5a-Ror signal transduction.
Asunto(s)
Cinesinas/metabolismo , Transducción de Señal , Proteína Wnt-5a/metabolismo , Animales , Línea Celular , Desarrollo Embrionario/fisiología , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Humanos , Cinesinas/genética , Ratones , Ratones Endogámicos C57BL , Morfogénesis/efectos de los fármacos , Proteómica , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/genética , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Vía de Señalización Wnt , Proteína Wnt-5a/farmacología , beta Catenina/metabolismoRESUMEN
The Ror family receptor tyrosine kinases, Ror1 and Ror2, play important roles in regulating developmental morphogenesis and tissue- and organogenesis, but their roles in tissue regeneration in adult animals remain largely unknown. In this study, we examined the expression and function of Ror1 and Ror2 during skeletal muscle regeneration. Using an in vivo skeletal muscle injury model, we show that expression of Ror1 and Ror2 in skeletal muscles is induced transiently by the inflammatory cytokines, TNF-α and IL-1ß, after injury and that inhibition of TNF-α and IL-1ß by neutralizing antibodies suppresses expression of Ror1 and Ror2 in injured muscles. Importantly, expression of Ror1, but not Ror2, was induced primarily in Pax7-positive satellite cells (SCs) after muscle injury, and administration of neutralizing antibodies decreased the proportion of Pax7-positive proliferative SCs after muscle injury. We also found that stimulation of a mouse myogenic cell line, C2C12 cells, with TNF-α or IL-1ß induced expression of Ror1 via NF-κB activation and that suppressed expression of Ror1 inhibited their proliferative responses in SCs. Intriguingly, SC-specific depletion of Ror1 decreased the number of Pax7-positive SCs after muscle injury. Collectively, these findings indicate for the first time that Ror1 has a critical role in regulating SC proliferation during skeletal muscle regeneration. We conclude that Ror1 might be a suitable target in the development of diagnostic and therapeutic approaches to manage muscular disorders.
Asunto(s)
Músculo Esquelético/lesiones , Músculo Esquelético/fisiología , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Regeneración , Células Satélite del Músculo Esquelético/citología , Animales , Línea Celular , Proliferación Celular , Regulación Enzimológica de la Expresión Génica , Interleucina-1beta/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Desarrollo de Músculos , FN-kappa B/metabolismo , Factor de Transcripción PAX7/metabolismo , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/genética , Células Satélite del Músculo Esquelético/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Blastocyst implantation is a complex process requiring coordination of a dynamic sequence of embryo-uterine interactions. Blood vessels enter the uterus from the mesometrium, demarcating the uterus into mesometrial (M) and antimesometrial (AM) domains. Implantation occurs along the uterine longitudinal axis within specialized implantation chambers (crypts) that originate within the evaginations directed from the primary lumen toward the AM domain. The morphological orientation of crypts in rodent uteri was recognized more than a century ago, but the mechanism remained unknown. Here we provide evidence that planar cell polarity (PCP) signaling orchestrates directed epithelial evaginations to form crypts for implantation in mice. Uterine deletion of Vang-like protein 2, but not Vang-like protein 1, conferred aberrant PCP signaling, misdirected epithelial evaginations, defective crypt formation, and blastocyst attachment, leading to severely compromised pregnancy outcomes. The study reveals a previously unrecognized role for PCP in executing spatial cues for crypt formation and implantation. Because PCP is an evolutionarily conserved phenomenon, our study is likely to inspire implantation studies of this signaling pathway in humans and other species.
Asunto(s)
Polaridad Celular/fisiología , Implantación del Embrión/fisiología , Útero/fisiología , Animales , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/fisiología , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Comunicación Celular/fisiología , Proteínas Dishevelled/fisiología , Epitelio/anatomía & histología , Epitelio/fisiología , Femenino , Masculino , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Embarazo , Resultado del Embarazo , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/deficiencia , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/genética , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/fisiología , Transducción de Señal/fisiología , Útero/anatomía & histología , Proteína Wnt-5a/deficiencia , Proteína Wnt-5a/genética , Proteína Wnt-5a/fisiologíaRESUMEN
Cell segregation is the process by which cells self-organize to establish developmental boundaries, an essential step in tissue formation. Cell segregation is a common outcome of Eph/ephrin signaling, but the mechanisms remain unclear. In craniofrontonasal syndrome, X-linked mosaicism for ephrin-B1 expression has been hypothesized to lead to aberrant Eph/ephrin-mediated cell segregation. Here, we use mouse genetics to exploit mosaicism to study cell segregation in the mammalian embryo and integrate live-cell imaging to examine the underlying cellular and molecular mechanisms. Our data demonstrate that dramatic ephrin-B1-mediated cell segregation occurs in the early neuroepithelium. In contrast to the paradigm that repulsive bidirectional signaling drives cell segregation, unidirectional EphB kinase signaling leads to cell sorting by the Rho kinase-dependent generation of a cortical actin differential between ephrin-B1- and EphB-expressing cells. These results define mechanisms of Eph/ephrin-mediated cell segregation, implicating unidirectional regulation of cortical actomyosin contractility as a key effector of this fundamental process.
Asunto(s)
Actomiosina/metabolismo , Diferenciación Celular , Efrina-B1/metabolismo , Células Neuroepiteliales/citología , Receptores de la Familia Eph/metabolismo , Transducción de Señal , Actinas/metabolismo , Animales , Recuento de Células , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Ratones , Modelos Biológicos , Células Neuroepiteliales/metabolismo , Quinasas Asociadas a rho/metabolismoRESUMEN
Mouse ovarian germ cells enter meiosis in a wave that propagates from anterior to posterior, but little is known about contribution of germ cells to initiation or propagation of meiosis. In a Ror2 mutant with diminished germ cell number and migration, we find that overall timing of meiotic initiation is delayed at the population level. We use chemotherapeutic depletion to exclude a profoundly reduced number of germ cells as a cause for meiotic delay. We rule out sex reversal or failure to specify somatic support cells as contributors to the meiotic phenotype. Instead, we find that anomalies in the distribution of germ cells as well as gonad shape in mutants contribute to aberrant initiation of meiosis. Our analysis supports a model of meiotic initiation via diffusible signal(s), excludes a role for germ cells in commencing the meiotic wave and furnishes the first phenotypic demonstration of the wave of meiotic entry. Finally, our studies underscore the importance of considering germ cell migration defects while studying meiosis to discern secondary effects resulting from positioning versus primary meiotic entry phenotypes.
Asunto(s)
Células Germinativas/metabolismo , Gónadas/patología , Meiosis/genética , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/genética , Animales , Recuento de Células , Movimiento Celular/genética , Forma de la Célula/genética , Femenino , Células Germinativas/crecimiento & desarrollo , Células Germinativas/patología , Gónadas/crecimiento & desarrollo , Ratones , Mutación , Ovario/crecimiento & desarrollo , Ovario/metabolismo , Ovario/patología , Transducción de Señal/genéticaRESUMEN
The two cortical hemispheres of the mammalian forebrain are interconnected by major white matter tracts, including the corpus callosum (CC) and the posterior branch of the anterior commissure (ACp), that bridge the telencephalic midline. We show here that the intracellular signaling domains of the EphB1 and EphB2 receptors are critical for formation of both the ACp and CC. We observe partial and complete agenesis of the corpus callosum, as well as highly penetrant ACp misprojection phenotypes in truncated EphB1/2 mice that lack intracellular signaling domains. Consistent with the roles for these receptors in formation of the CC and ACp, we detect expression of these receptors in multiple brain regions associated with the formation of these forebrain structures. Taken together, our findings suggest that a combination of forward and reverse EphB1/2 receptor-mediated signaling contribute to ACp and CC axon guidance.
Asunto(s)
Comisura Anterior Cerebral/embriología , Comisura Anterior Cerebral/metabolismo , Cuerpo Calloso/embriología , Cuerpo Calloso/metabolismo , Receptor EphB1/metabolismo , Receptor EphB2/metabolismo , Animales , Comisura Anterior Cerebral/citología , Axones/metabolismo , Movimiento Celular/fisiología , Cuerpo Calloso/citología , Técnicas de Sustitución del Gen , Técnicas de Inactivación de Genes , Inmunohistoquímica , Espacio Intracelular , Ratones Transgénicos , Técnicas de Trazados de Vías Neuroanatómicas , Dominios Proteicos , Receptor EphB1/genética , Receptor EphB2/genética , Transducción de SeñalRESUMEN
Embryo homing and implantation occur within a crypt (implantation chamber) at the antimesometrial (AM) pole along the uterus. The mechanism by which this is achieved is not known. Here, we show that villi-like epithelial projections from the main uterine lumen toward the AM pole at regularly spaced intervals that form crypts for embryo implantation were disrupted in mice with uterine loss or gain of function of Wnt5a, or loss of function of both Ror1 and Ror2. This disruption of Wnt5a-ROR signaling resulted in disorderly epithelial projections, crypt formation, embryo spacing, and impaired implantation. These early disturbances under abnormal Wnt5a-ROR signaling were reflected in adverse late pregnancy events, including defective decidualization and placentation, ultimately leading to compromised pregnancy outcomes. This study presents deeper insight regarding the formation of organized epithelial projections for crypt formation and embryo implantation for pregnancy success.