RESUMEN
Histone deacetylases (HDACs) are key posttranslational modulators of the proteome. We show that expression of histone deacetylase 6 ( hdac6 ) is dynamic and appears in a tissue specific manner throughout embryonic development of the frog Xenopus laevis . Interestingly, hdac6 transcripts often associate with ciliated tissues, like the left-right organizer at neurula stage or the pronephros. In the embryonic skin, Hdac6 protein localizes to the cilia base, suggesting a functional link.
RESUMEN
The vertebrate left-right axis is specified during embryogenesis by a transient organ: the left-right organizer (LRO). Species including fish, amphibians, rodents and humans deploy motile cilia in the LRO to break bilateral symmetry, while reptiles, birds, even-toed mammals and cetaceans are believed to have LROs without motile cilia. We searched for genes whose loss during vertebrate evolution follows this pattern and identified five genes encoding extracellular proteins, including a putative protease with hitherto unknown functions that we named ciliated left-right organizer metallopeptide (CIROP). Here, we show that CIROP is specifically expressed in ciliated LROs. In zebrafish and Xenopus, CIROP is required solely on the left side, downstream of the leftward flow, but upstream of DAND5, the first asymmetrically expressed gene. We further ascertained 21 human patients with loss-of-function CIROP mutations presenting with recessive situs anomalies. Our findings posit the existence of an ancestral genetic module that has twice disappeared during vertebrate evolution but remains essential for distinguishing left from right in humans.
Asunto(s)
Evolución Biológica , Tipificación del Cuerpo , Redes Reguladoras de Genes , Metaloproteasas , Animales , Humanos , Tipificación del Cuerpo/genética , Tipificación del Cuerpo/fisiología , Cilios/genética , Mutación con Pérdida de Función , Metaloproteasas/genética , Metaloproteasas/fisiología , Proteínas/genética , Proteínas/fisiología , Vertebrados/genéticaRESUMEN
Rotating cilia at the vertebrate left-right organizer (LRO) generate an asymmetric leftward flow, which is sensed by cells at the left LRO margin. Ciliary activity of the calcium channel Pkd2 is crucial for flow sensing. How this flow signal is further processed and relayed to the laterality-determining Nodal cascade in the left lateral plate mesoderm (LPM) is largely unknown. We previously showed that flow down-regulates mRNA expression of the Nodal inhibitor Dand5 in left sensory cells. De-repression of the co-expressed Nodal, complexed with the TGFß growth factor Gdf3, drives LPM Nodal cascade induction. Here, we show that post-transcriptional repression of dand5 is a central process in symmetry breaking of Xenopus, zebrafish and mouse. The RNA binding protein Bicc1 was identified as a post-transcriptional regulator of dand5 and gdf3 via their 3'-UTRs. Two distinct Bicc1 functions on dand5 mRNA were observed at pre- and post-flow stages, affecting mRNA stability or flow induced translational inhibition, respectively. To repress dand5, Bicc1 co-operates with Dicer1, placing both proteins in the process of flow sensing. Intriguingly, Bicc1 mediated translational repression of a dand5 3'-UTR mRNA reporter was responsive to pkd2, suggesting that a flow induced Pkd2 signal triggers Bicc1 mediated dand5 inhibition during symmetry breakage.
Asunto(s)
Tipificación del Cuerpo/genética , Regulación del Desarrollo de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular/genética , Proteínas de Unión al ARN/genética , Ribonucleasa III/genética , Xenopus laevis/genética , Pez Cebra/genética , Regiones no Traducidas 3'/genética , Animales , Desarrollo Embrionario/genética , Ratones , Estabilidad del ARN/genética , Xenopus laevis/embriología , Pez Cebra/embriologíaRESUMEN
Cilia are protrusions of the cell surface and composed of hundreds of proteins many of which are evolutionary and functionally well conserved. In cells assembling motile cilia the expression of numerous ciliary components is under the control of the transcription factor FOXJ1. Here, we analyse the evolutionary conserved FOXJ1 target CFAP161 in Xenopus and mouse. In both species Cfap161 expression correlates with the presence of motile cilia and depends on FOXJ1. Tagged CFAP161 localises to the basal bodies of multiciliated cells of the Xenopus larval epidermis, and in mice CFAP161 protein localises to the axoneme. Surprisingly, disruption of the Cfap161 gene in both species did not lead to motile cilia-related phenotypes, which contrasts with the conserved expression in cells carrying motile cilia and high sequence conservation. In mice mutation of Cfap161 stabilised the mutant mRNA making genetic compensation triggered by mRNA decay unlikely. However, genes related to microtubules and cilia, microtubule motor activity and inner dyneins were dysregulated, which might buffer the Cfap161 mutation.
Asunto(s)
Cilios/metabolismo , Factores de Transcripción Forkhead/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animales , Axonema/metabolismo , Cuerpos Basales/metabolismo , Células Epidérmicas/metabolismo , Epidermis/metabolismo , Femenino , Masculino , Ratones , Microtúbulos/metabolismoRESUMEN
Cilia are complex cellular protrusions consisting of hundreds of proteins. Defects in ciliary structure and function, many of which have not been characterised molecularly, cause ciliopathies: a heterogeneous group of human syndromes. Here, we report on the FOXJ1 target gene Cfap206, orthologues of which so far have only been studied in Chlamydomonas and Tetrahymena In mouse and Xenopus, Cfap206 was co-expressed with and dependent on Foxj1 CFAP206 protein localised to the basal body and to the axoneme of motile cilia. In Xenopus crispant larvae, the ciliary beat frequency of skin multiciliated cells was enhanced and bead transport across the epidermal mucociliary epithelium was reduced. Likewise, Cfap206 knockout mice revealed ciliary phenotypes. Electron tomography of immotile knockout mouse sperm flagella indicated a role in radial spoke formation reminiscent of FAP206 function in Tetrahymena Male infertility, hydrocephalus and impaired mucociliary clearance of the airways in the absence of laterality defects in Cfap206 mutant mice suggests that Cfap206 may represent a candidate for the subgroup of human primary ciliary dyskinesias caused by radial spoke defects.
Asunto(s)
Encéfalo/embriología , Encéfalo/metabolismo , Proteínas del Citoesqueleto/metabolismo , Factores de Transcripción Forkhead/metabolismo , Pulmón/metabolismo , Depuración Mucociliar , Motilidad Espermática , Animales , Axonema/metabolismo , Cuerpos Basales/metabolismo , Cilios/metabolismo , Proteínas del Citoesqueleto/química , Desarrollo Embrionario , Células Epiteliales/metabolismo , Fluorescencia , Hidrocefalia/patología , Infertilidad Masculina/patología , Masculino , Ratones Noqueados , Moco/metabolismo , Mutación/genética , Transporte de Proteínas , Espermatozoides/metabolismo , Espermatozoides/ultraestructura , Xenopus laevis/embriología , Xenopus laevis/metabolismoRESUMEN
Neural crest cells (NCCs) are a unique embryonic cell population that initially reside at the dorsal neural tube but later migrate in the embryo and differentiate into multiple types of derivatives. To acquire motility, NCCs undergo epithelial-to-mesenchymal transition and invade the surrounding extracellular matrix (ECM). Matrix metalloproteases (MMPs) are a large family of proteases which regulate migration of various embryonic and adult cells via ECM remodeling. The gelatinase's subgroup of MMPs is the most studied one due to its key role in metastasis. As it is composed of only two proteases, MMP2 and MMP9, it is important to understand whether each is indispensable or redundant in its biological function. Here we explored the role of the gelatinases in executing NCC migration, by determining whether MMP2 and/or MMP9 regulate migration across species in singular, combined, or redundant manners. Chick and mouse embryos were utilized to compare expression and activity of both MMPs using genetic and pharmacological approaches in multiple in vivo and ex vivo assays. Both MMPs were found to be expressed and active in mouse and chick NCCs. Inhibition of each MMP was sufficient to prevent NCC migration in both species. Yet, NCC migration was maintained in MMP2-/- or MMP9-/- mouse mutants due to compensation between the gelatinases, but reciprocal pharmacological inhibition in each mutant prevented NCC migration. This study reveals for the first time that both gelatinases are expressed in avian and mammalian NCCs, and demonstrates their fundamental and conserved role in promoting embryonic cell migration.
Asunto(s)
Movimiento Celular , Embrión de Mamíferos/fisiología , Metaloproteinasa 2 de la Matriz/fisiología , Metaloproteinasa 9 de la Matriz/fisiología , Cresta Neural/fisiología , Animales , Pollos , Embrión de Mamíferos/citología , Matriz Extracelular/fisiología , Femenino , Masculino , Ratones , Ratones Noqueados , Cresta Neural/citologíaRESUMEN
Malfunctions of motile cilia cause a variety of developmental defects and diseases in humans and animal model organisms. Defects include impaired mucociliary clearance of the airways, sperm immotility, hydrocephalus and organ laterality. Here, we characterize the evolutionary conserved Cfap43 gene by loss-of-function experiments in the mouse and the frog Xenopus laevis. Cfap43 is expressed in tissues carrying motile cilia and acts as a target gene of the transcription factor FOXJ1, which is essential for the induction of motile ciliogenesis. We show that CFAP43, a protein of unknown biochemical function, localizes to the ciliary axoneme. CFAP43 is involved in the regulation of the beating frequency of tracheal cilia and loss of CFAP43 causes severe mucus accumulation in the nasal cavity. Likewise, morphant and crispant frog embryos revealed impaired function of motile cilia of the larval epidermis, a model for airway mucociliary epithelia. CFAP43 participates in the formation of flagellar axonemes during spermatogenesis as mice mutant for Cfap43 display male infertility, consistent with observations in male sterile patients. In addition, mice mutant for Cfap43 display early onset hydrocephalus. Together, these results confirm the role of CFAP43 in the male reproductive tract and pinpoint additional functions in airway epithelia mucus clearance and brain development.
Asunto(s)
Cilios/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Proteínas del Citoesqueleto/genética , Células Epidérmicas/metabolismo , Factores de Transcripción Forkhead/metabolismo , Hidrocefalia/genética , Infertilidad Masculina/genética , Masculino , Ratones , Ratones Noqueados , Cola del Espermatozoide/metabolismo , Espermatogénesis/genética , Espermatozoides/metabolismo , Tráquea/citología , Proteínas de Xenopus/genética , Xenopus laevisRESUMEN
Animal asymmetries are widespread, from lobster claws to human handedness. Controlled by the left-sided Nodal signaling cascade, asymmetric morphogenesis and placement of vertebrate organs (heart, gut, etc.) are executed during embryogenesis. Fish, amphibians and mammals use a ciliated epithelium to break bilateral symmetry and induce the Nodal cascade. Cilia tilt and polarize to the posterior cell pole, such that clockwise rotation causes a leftward flow at the cell surface. Recent progress in Xenopus showed that mechanical strain drives cilia lengthening and polarization. Studying mutant alleles causing human organ situs defects and following novel EvoDevo approaches, new genes were discovered and functionally characterized in the frog, facilitated by a unique set of experimental tools.
Asunto(s)
Tipificación del Cuerpo/genética , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Xenopus laevis/genética , Animales , Cilios/genética , Cilios/metabolismo , Epitelio/embriología , Epitelio/metabolismo , Evolución Molecular , Estrés Mecánico , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologíaRESUMEN
Organ left-right (LR) asymmetry is a conserved vertebrate feature, which is regulated by left-sided activation of Nodal signaling. Nodal asymmetry is established by a leftward fluid-flow generated at the ciliated LR organizer (LRO). Although the role of fibroblast growth factor (FGF) signaling pathways during mesoderm development is conserved, diverging results from different model organisms suggest a non-conserved function in LR asymmetry. Here, we demonstrate that FGF is required during gastrulation in a dual function at consecutive stages of Xenopus embryonic development. In the early gastrula, FGF is necessary for LRO precursor induction, acting in parallel with FGF-mediated mesoderm induction. During late gastrulation, the FGF/Ca2+-branch is required for specification of the flow-sensing lateral LRO cells, a function related to FGF-mediated mesoderm morphogenesis. This second function in addition requires input from the calcium channel Polycystin-2. Thus, analogous to mesoderm development, FGF activity is required in a dual role for laterality specification; namely, for generating and sensing leftward flow. Moreover, our findings in Xenopus demonstrate that FGF functions in LR development share more conserved features across vertebrate species than previously anticipated.
Asunto(s)
Factores de Crecimiento de Fibroblastos/metabolismo , Animales , Tipificación del Cuerpo/genética , Tipificación del Cuerpo/fisiología , Cilios/metabolismo , Factores de Crecimiento de Fibroblastos/genética , Factores de Transcripción Forkhead/metabolismo , Gástrula/metabolismo , Gastrulación/fisiología , Mesodermo/metabolismo , Transducción de Señal/fisiología , Canales Catiónicos TRPP/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMEN
Joubert syndrome (JS) is a congenital autosomal-recessive or-in rare cases-X-linked inherited disease. The diagnostic hallmark of the so-called molar tooth sign describes the morphological manifestation of the mid- and hind-brain in axial brain scans. Affected individuals show delayed development, intellectual disability, ataxia, hyperpnea, sleep apnea, abnormal eye, and tongue movements as well as hypotonia. At the cellular level, JS is associated with the compromised biogenesis of sensory cilia, which identifies JS as a member of the large group of ciliopathies. Here we report on the identification of novel compound heterozygous variants (p.Y503C and p.Q485*) in the centrosomal gene PIBF1 in a patient with JS via trio whole exome sequencing. We have studied the underlying disease mechanism in the frog Xenopus, which offers fast assessment of cilia functions in a number of embryological contexts. Morpholino oligomer (MO) mediated knockdown of the orthologous Xenopus pibf1 gene resulted in defective mucociliary clearance in the larval epidermis, due to reduced cilia numbers and motility on multiciliated cells. To functionally assess patient alleles, mutations were analyzed in the larval skin: the p.Q485* nonsense mutation resulted in a disturbed localization of PIBF1 to the ciliary base. This mutant failed to rescue the ciliation phenotype following knockdown of endogenous pibf1. In contrast, the missense variant p.Y503C resulted in attenuated rescue capacity compared to the wild type allele. Based on these results, we conclude that in the case of this patient, JS is the result of a pathogenic combination of an amorphic and a hypomorphic PIBF1 allele. Our study underscores the versatility of the Xenopus model to study ciliopathies such as JS in a rapid and cost-effective manner, which should render this animal model attractive for future studies of human ciliopathies.
RESUMEN
Nodal signaling controls asymmetric organ placement during vertebrate embryogenesis. Nodal is induced by a leftward fluid flow at the ciliated left-right organizer (LRO). The mechanism of flow sensing, however, has remained elusive. pkd2 encodes the calcium channel Polycystin-2, which is required for kidney development and laterality, and may act in flow perception. Here, we have studied the role of Polycystin-2 in Xenopus and show that pkd2 is indispensable for left-right (LR) asymmetry. Knockdown of pkd2 prevented left-asymmetric nodal cascade induction in the lateral plate mesoderm. Defects were due to failure of LRO specification, morphogenesis, and, consequently, absence of leftward flow. Polycystin-2 synergizes with the unconventional nodal-type signaling molecule Xnr3 to induce the LRO precursor tissue before gastrulation, upstream of symmetry breakage. Our data uncover an unknown function of pkd2 in LR axis formation, which we propose represents an ancient role of Polycystin-2 during LRO induction in lower vertebrates.
RESUMEN
The transcription factor FOXJ1 is essential for the formation of motile cilia throughout the animal kingdom. Target genes therefore likely constitute an important part of the motile cilia program. Here, we report on the analysis of one of these targets, Fam183b, in Xenopus and mice. Fam183b encodes a protein with unknown function which is conserved from the green algae Chlamydomonas to humans. Fam183b is expressed in tissues harbouring motile cilia in both mouse and frog embryos. FAM183b protein localises to basal bodies of cilia in mIMCD3 cells and of multiciliated cells of the frog larval epidermis. In addition, FAM183b interacts with NUP93, which also localises to basal bodies. During frog embryogenesis, Fam183b was dispensable for laterality specification and brain development, but required for ciliogenesis and motility of epidermal multiciliated cells and nephrostomes, i.e. the embryonic kidney. Surprisingly, mice homozygous for a null allele did not display any defects indicative of disrupted motile ciliary function. The lack of a cilia phenotype in mouse and the limited requirements in frog contrast with high sequence conservation and the correlation of gene expression with the presence of motile cilia. This finding may be explained through compensatory mechanisms at sites where no defects were observed in our FAM183b-loss-of-function studies.
Asunto(s)
Cilios/fisiología , Proteínas del Citoesqueleto/metabolismo , Células Epidérmicas/fisiología , Locomoción , Animales , Cuerpos Basales/química , Proteínas del Citoesqueleto/genética , Factores de Transcripción Forkhead/metabolismo , Técnicas de Inactivación de Genes , Ratones , Ratones Noqueados , Xenopus , Proteínas de Xenopus/metabolismoRESUMEN
The function of normal and defective candidate genes for human genetic diseases, which are rapidly being identified in large numbers by human geneticists and the biomedical community at large, will be best studied in relevant and predictive model organisms that allow high-speed verification, analysis of underlying developmental, cellular and molecular mechanisms, and establishment of disease models to test therapeutic options. We describe and discuss the pros and cons of the frog Xenopus, which has been extensively used to uncover developmental mechanisms in the past, but which is being underutilized as a biomedical model. We argue that Xenopus complements the more commonly used mouse and zebrafish as a time- and cost-efficient animal model to study human disease alleles and mechanisms.
Asunto(s)
Anomalías Congénitas/genética , Modelos Animales de Enfermedad , Enfermedades Genéticas Congénitas/genética , Xenopus laevis/genética , Alelos , Animales , Trastornos de la Motilidad Ciliar/embriología , Trastornos de la Motilidad Ciliar/genética , Trastornos de la Motilidad Ciliar/fisiopatología , Ciliopatías/embriología , Ciliopatías/genética , Ciliopatías/fisiopatología , Anomalías Congénitas/embriología , Anomalías Congénitas/fisiopatología , Enfermedades Genéticas Congénitas/embriología , Enfermedades Genéticas Congénitas/fisiopatología , Cardiopatías Congénitas/embriología , Cardiopatías Congénitas/genética , Cardiopatías Congénitas/fisiopatología , Humanos , Mutación , Xenopus laevis/embriología , Xenopus laevis/fisiologíaRESUMEN
Left-right organizers require motile and polarized cilia to break symmetry. In this issue of Developmental Cell, Chien et al. (2018) demonstrate that gastrulation-derived mechanical strain of the precursor tissue orients cilia and is required for cilia lengthening and motility.
Asunto(s)
Tipificación del Cuerpo , Cilios , GastrulaciónRESUMEN
Symmetry is appealing, be it in architecture, art or facial expression, where symmetry is a key feature to finding someone attractive or not. Yet, asymmetries are widespread in nature, not as an erroneous deviation from the norm but as a way to adapt to the prevailing environmental conditions at a time. Asymmetries in many cases are actively selected for: they might well have increased the evolutionary fitness of a species. Even many single-celled organisms are built asymmetrically, such as the pear-shaped ciliate Paramecium, which may depend on its asymmetry to navigate towards the oxygen-richer surface of turbid waters, at least based on modeling. Everybody knows the lobster with its asymmetric pair of claws, the large crusher usually on the left and the smaller cutter on the right. Snail shells coil asymmetrically, as do the organs they house. Organ asymmetries are found throughout the animal kingdom, referring to asymmetric positioning, asymmetric morphology or both, with the vertebrate heart being an example for the latter. Functional asymmetries, such as that of the human brain with its localization of the language center in one hemisphere, add to the complexity of organ asymmetries and presumably played a decisive role for sociocultural evolution. The evolutionary origin of organ asymmetries may have been a longer than body length gut, which allows efficient retrieval of nutrients, and the need to stow a long gut in the body cavity in an orderly manner that ensures optimal functioning. Vertebrate organ asymmetries (situs solitus) are quite sophisticated: in humans, the apex of the asymmetrically built heart points to the left; the lung in turn, due to space restrictions, has fewer lobes on the left than on the right side (two versus three in humans), stomach and spleen are found on the left, the liver on the right, and small and large intestine coil in a chiral manner (Figure 1A). In very rare cases (1:10,000), the organ situs is inverted (situs inversus), while heterotaxia refers to another rare situation (about 1:1,000), in which subsets of organs show normal or aberrant positioning or morphology (Figure 1B). Individuals with situs solitus or situs inversus are healthy, whereas heterotaxia presents severe congenital malformations. Many human syndromes are known in which patients suffer from laterality defects, such as Katagener syndrome, in which the organ situs is inverted in one half of patients and males are sterile. Snail shells and vertebrate organs are examples of biased asymmetries with on average only one inversion in every 10,000 cases. Other asymmetries such as the coiling of the tails of piglets occur randomly with a 50:50 distribution. This primer exclusively deals with organ asymmetries in the animal kingdom, specifically with the mechanisms that ensure the development of biased asymmetries during embryogenesis.
Asunto(s)
Evolución Biológica , Lateralidad Funcional , Morfogénesis , Vertebrados/fisiología , Animales , Vertebrados/anatomía & histología , Vertebrados/clasificaciónRESUMEN
Anatomical and functional asymmetries are widespread in the animal kingdom [1, 2]. In vertebrates, many visceral organs are asymmetrically placed [3]. In snails, shells and inner organs coil asymmetrically, and in Drosophila, genitalia and hindgut undergo a chiral rotation during development. The evolutionary origin of these asymmetries remains an open question [1]. Nodal signaling is widely used [4], and many, but not all, vertebrates use cilia for symmetry breaking [5]. In Drosophila, which lacks both cilia and Nodal, the unconventional myosin ID (myo1d) gene controls dextral rotation of chiral organs [6, 7]. Here, we studied the role of myo1d in left-right (LR) axis formation in Xenopus. Morpholino oligomer-mediated myo1d downregulation affected organ placement in >50% of morphant tadpoles. Induction of the left-asymmetric Nodal cascade was aberrant in >70% of cases. Expression of the flow-target gene dand5 was compromised, as was flow itself, due to shorter, fewer, and non-polarized cilia at the LR organizer. Additional phenotypes pinpointed Wnt/planar cell polarity signaling and suggested that myo1d, like in Drosophila [8], acted in the context of the planar cell polarity pathway. Indeed, convergent extension of gastrula explant cultures was inhibited in myo1d morphants, and the ATF2 reporter gene for non-canonical Wnt signaling was downregulated. Finally, genetic interference experiments demonstrated a functional interaction between the core planar cell polarity signaling gene vangl2 and myo1d in LR axis formation. Thus, our data identified myo1d as a common denominator of arthropod and chordate asymmetry, in agreement with a monophyletic origin of animal asymmetry.
Asunto(s)
Tipificación del Cuerpo/genética , Embrión no Mamífero/embriología , Regulación del Desarrollo de la Expresión Génica/genética , Morfogénesis/genética , Miosinas/genética , Proteínas de Xenopus/genética , Xenopus laevis/embriología , Animales , Polaridad Celular/genética , Gástrula/embriología , Miosinas/metabolismo , Proteínas de Xenopus/metabolismoRESUMEN
ARL13B encodes for the ADP-ribosylation factor-like 13B GTPase, which is required for normal cilia structure and Sonic hedgehog (Shh) signaling. Disruptions in cilia structure or function lead to a class of human disorders called ciliopathies. Joubert syndrome is characterized by a wide spectrum of symptoms, including a variable degree of intellectual disability, ataxia, and ocular abnormalities. Here we report a novel homozygous missense variant c.[223G>A] (p.(Gly75Arg) in the ARL13B gene, which was identified by whole-exome sequencing of a trio from a consanguineous family with multiple-affected individuals suffering from intellectual disability, ataxia, ocular defects, and epilepsy. The same variant was also identified in a second family. We saw a striking difference in the severity of ataxia between affected male and female individuals in both families. Both ARL13B and ARL13B-c.[223G>A] (p.(Gly75Arg) expression rescued the cilia length and Shh defects displayed by Arl13b hennin (null) cells, indicating that the variant did not disrupt either ARL13B function. In contrast, ARL13B-c.[223G>A] (p.(Gly75Arg) displayed a marked loss of ARL3 guanine nucleotide-exchange factor activity, with retention of its GTPase activities, highlighting the correlation between its loss of function as an ARL3 guanine nucleotide-exchange factor and Joubert syndrome.
Asunto(s)
Factores de Ribosilacion-ADP/genética , Anomalías Múltiples/genética , Cerebelo/anomalías , Anomalías del Ojo/genética , Enfermedades Renales Quísticas/genética , Mutación con Pérdida de Función , Retina/anomalías , Factores de Ribosilacion-ADP/metabolismo , Anomalías Múltiples/diagnóstico , Adolescente , Adulto , Animales , Línea Celular Tumoral , Células Cultivadas , Niño , Anomalías del Ojo/diagnóstico , Femenino , Guanosina Trifosfato/metabolismo , Homocigoto , Humanos , Enfermedades Renales Quísticas/diagnóstico , Masculino , Ratones , Mutación Missense , LinajeRESUMEN
Goosecoid (Gsc) expression marks the primary embryonic organizer in vertebrates and beyond. While functions have been assigned during later embryogenesis, the role of Gsc in the organizer has remained enigmatic. Using conditional gain-of-function approaches in Xenopus and mouse to maintain Gsc expression in the organizer and along the axial midline, neural tube closure defects (NTDs) arose and dorsal extension was compromised. Both phenotypes represent convergent extension (CE) defects, arising from impaired Wnt/planar cell polarity (PCP) signaling. Dvl2 recruitment to the cell membrane was inhibited by Gsc in Xenopus animal cap assays and key Wnt/PCP factors (RhoA, Vangl2, Prickle, Wnt11) rescued Gsc-mediated NTDs. Re-evaluation of endogenous Gsc functions in MO-mediated gene knockdown frog and knockout mouse embryos unearthed PCP/CE-related phenotypes as well, including cartilage defects in Xenopus and misalignment of inner ear hair cells in mouse. Our results assign a novel function to Gsc as an inhibitor of Wnt/PCP-mediated CE. We propose that in the organizer Gsc represses CE as well: Gsc-expressing prechordal cells, which leave the organizer first, migrate and do not undergo CE like the Gsc-negative notochordal cells, which subsequently emerge from the organizer. In this model, Gsc provides a switch between cell migration and CE, i.e. cell intercalation.
Asunto(s)
Proteína Goosecoide/metabolismo , Organizadores Embrionarios/metabolismo , Proteínas Wnt/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animales , Polaridad Celular , Proteínas Dishevelled/metabolismo , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/patología , Embrión no Mamífero/metabolismo , Embrión no Mamífero/patología , Desarrollo Embrionario , Genes Reporteros , Proteína Goosecoide/deficiencia , Proteína Goosecoide/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Electrónica de Rastreo , Transducción de Señal , Proteínas de Xenopus/genéticaRESUMEN
Conjoined twins fused at the thorax display an enigmatic left-right defect: although left twins are normal, laterality is disturbed in one-half of right twins [1-3]. Molecularly, this randomization corresponds to a lack of asymmetric Nodal cascade induction in right twins [4]. We studied leftward flow [5, 6] at the left-right organizer (LRO) [7, 8] in thoracopagus twins in Xenopus, which displayed a duplicated, fused, and ciliated LRO. Cilia were motile and produced a leftward flow from the right LRO margin of the right to the left margin of the left twin. Motility was required for correct laterality in left twins, as knockdown of dynein motor dnah9 prevented Nodal cascade induction. Nodal was rescued by parallel knockdown of the inhibitor dand5 [9, 10] on the left side of the left twin. Lack of Nodal induction in the right twin, despite the presence of flow, was due to insufficient suppression of dand5. Knockdown of dand5 at the center of the fused LRO resulted in asymmetric Nodal cascade induction in the right twin as well. Manipulation of leftward flow and dand5 in a targeted and sided manner induced the Nodal cascade in a predictable manner, in the left twin, the right one, both, or neither. Laterality in conjoined twins thus was determined by cilia-driven leftward fluid flow like in single embryos, which solves a century-old riddle, as the phenomenon was already studied by some of the founders of experimental embryology, including Dareste [11], Fol and Warynsky [12], and Spemann and Falkenberg [13] (reviewed in [14]).