RESUMEN
The polyglutamine (polyQ) diseases are a group of nine neurodegenerative diseases caused by the expansion of a polyQ tract that results in protein aggregation. Unlike other model organisms, Dictyostelium discoideum is a proteostatic outlier, naturally encoding long polyQ tracts yet resistant to polyQ aggregation. Here we identify serine-rich chaperone protein 1 (SRCP1) as a molecular chaperone that is necessary and sufficient to suppress polyQ aggregation. SRCP1 inhibits aggregation of polyQ-expanded proteins, allowing for their degradation via the proteasome, where SRCP1 is also degraded. SRCP1's C-terminal domain is essential for its activity in cells, and peptides that mimic this domain suppress polyQ aggregation in vitro. Together our results identify a novel type of molecular chaperone and reveal how nature has dealt with the problem of polyQ aggregation.
Asunto(s)
Chaperonas Moleculares/metabolismo , Péptidos/metabolismo , Línea Celular , Dictyostelium/metabolismo , Células HEK293 , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Serina/metabolismo , Ubiquitina/metabolismoRESUMEN
BACKGROUND: The goal of this study was to identify and characterize cell-cell interactions that facilitate endothelial tip cell fusion downstream of BMP (bone morphogenic protein)-mediated venous plexus formation. METHODS: High resolution and time-lapse imaging of transgenic reporter lines and loss-of-function studies were carried out to study the involvement of mesenchymal stromal cells during venous angiogenesis. RESULTS: BMP-responsive stromal cells facilitate timely and precise fusion of venous tip cells during developmental angiogenesis. CONCLUSIONS: Stromal cells are required for anastomosis of venous tip cells in the embryonic caudal hematopoietic tissue.
Asunto(s)
Proteínas Morfogenéticas Óseas , Células Madre Mesenquimatosas , Animales , Fusión Celular , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Animales Modificados Genéticamente , Comunicación Celular , Células del Estroma/metabolismoRESUMEN
Progenitor cell nuclei in the rapidly expanding epithelium of the embryonic vertebrate central nervous system undergo a process called interkinetic nuclear migration (IKNM). Movements of IKNM are generally believed to involve smooth migration of nuclei from apical to basal and back during the G1 and G2 phases of the cell cycle, respectively. Yet, this has not been formally demonstrated, nor have the molecular mechanisms that drive IKNM been identified. Using time-lapse confocal microscopy to observe nuclear movements in zebrafish retinal neuroepithelial cells, we show that, except for brief apical nuclear translocations preceding mitosis, IKNM is stochastic rather than smooth and directed. We also show that IKNM is driven largely by actomyosin-dependent forces as it still occurs when the microtubule cytoskeleton is compromised but is blocked when MyosinII activity is inhibited.
Asunto(s)
Actomiosina/metabolismo , Núcleo Celular/metabolismo , Retina/citología , Pez Cebra/embriología , Animales , Complejo Dinactina , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Células Neuroepiteliales/citología , Células Neuroepiteliales/metabolismo , Retina/embriología , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
The development of the biliary system is a complex yet poorly understood process, with relevance to multiple diseases, including biliary atresia, choledochal cysts and gallbladder agenesis. We present here a crucial role for Hippo-Yap/Taz signaling in this context. Analysis of sav1 mutant zebrafish revealed dysplastic morphology and expansion of both intrahepatic and extrahepatic biliary cells, and ultimately larval lethality. Biliary dysgenesis, but not larval lethality, is driven primarily by Yap signaling. Re-expression of Sav1 protein in sav1-/- hepatocytes is able to overcome these initial deficits and allows sav1-/- fish to survive, suggesting cell non-autonomous signaling from hepatocytes. Examination of sav1-/- rescued adults reveals loss of gallbladder and formation of dysplastic cell masses expressing biliary markers, suggesting roles for Hippo signaling in extrahepatic biliary carcinomas. Deletion of stk3 revealed that the phenotypes observed in sav1 mutant fish function primarily through canonical Hippo signaling and supports a role for phosphatase PP2A, but also suggests Sav1 has functions in addition to facilitating Stk3 activity. Overall, this study defines a role for Hippo-Yap signaling in the maintenance of both intra- and extrahepatic biliary ducts.
Asunto(s)
Sistema Biliar/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Pez Cebra/metabolismo , Aciltransferasas , Animales , Animales Modificados Genéticamente/crecimiento & desarrollo , Animales Modificados Genéticamente/metabolismo , Sistema Biliar/anatomía & histología , Sistema Biliar/crecimiento & desarrollo , Sistemas CRISPR-Cas/genética , Hidrolasas de Éster Carboxílico/metabolismo , Vesícula Biliar/anatomía & histología , Vesícula Biliar/crecimiento & desarrollo , Vesícula Biliar/metabolismo , Larva/crecimiento & desarrollo , Larva/metabolismo , Hígado/anatomía & histología , Hígado/metabolismo , Fenotipo , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Serina-Treonina Quinasa 3 , Transducción de Señal , Transactivadores/genética , Factores de Transcripción/genética , Proteínas Señalizadoras YAP , Pez Cebra/crecimiento & desarrollo , Pez Cebra/metabolismo , Proteínas de Pez Cebra/deficiencia , Proteínas de Pez Cebra/genéticaRESUMEN
Adherens junction remodeling regulated by apical polarity proteins constitutes a major driving force for tissue morphogenesis, although the precise mechanism remains inconclusive. Here, we report that, in zebrafish, the Crumbs complex component MPP5a interacts with small GTPase Rab11 in Golgi to transport cadherin and Crumbs components synergistically to the apical domain, thus establishing apical epithelial polarity and adherens junctions. In contrast, Par complex recruited by MPP5a is incapable of interacting with Rab11 but might assemble cytoskeleton to facilitate cadherin exocytosis. In accordance, dysfunction of MPP5a induces an invasive migration of epithelial cells. This adherens junction remodeling pattern is frequently observed in zebrafish lens epithelial cells and neuroepithelial cells. The data identify an unrecognized MPP5a-Rab11 complex and describe its essential role in guiding apical polarization and zonula adherens formation in epithelial cells.
Asunto(s)
Uniones Adherentes/metabolismo , Movimiento Celular/fisiología , Polaridad Celular/fisiología , Guanilato Ciclasa/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Proteínas de Unión al GTP rab/metabolismo , Uniones Adherentes/genética , Animales , Cadherinas/genética , Cadherinas/metabolismo , Células Epiteliales , Aparato de Golgi/genética , Aparato de Golgi/metabolismo , Guanilato Ciclasa/genética , Transporte de Proteínas/fisiología , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteínas de Unión al GTP rab/genéticaRESUMEN
The Hippo-Yap pathway regulates multiple cellular processes in response to mechanical and other stimuli. In Drosophila, the polarity protein Lethal (2) giant larvae [L(2)gl], negatively regulates Hippo-mediated transcriptional output. However, in vertebrates, little is known about its homolog Llgl1. Here, we define a novel role for vertebrate Llgl1 in regulating Yap stability in cardiomyocytes, which impacts heart development. In contrast to the role of Drosophila L(2)gl, Llgl1 depletion in cultured rat cardiomyocytes decreased Yap protein levels and blunted target gene transcription without affecting Yap transcript abundance. Llgl1 depletion in zebrafish resulted in larger and dysmorphic cardiomyocytes, pericardial effusion, impaired blood flow and aberrant valvulogenesis. Cardiomyocyte Yap protein levels were decreased in llgl1 morphants, whereas Notch, which is regulated by hemodynamic forces and participates in valvulogenesis, was more broadly activated. Consistent with the role of Llgl1 in regulating Yap stability, cardiomyocyte-specific overexpression of Yap in Llgl1-depleted embryos ameliorated pericardial effusion and restored blood flow velocity. Altogether, our data reveal that vertebrate Llgl1 is crucial for Yap stability in cardiomyocytes and its absence impairs cardiac development.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Corazón/embriología , Miocitos Cardíacos/metabolismo , Transactivadores/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Proteínas de Ciclo Celular/genética , Estabilidad Proteica , Transactivadores/genética , Proteínas Señalizadoras YAP , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
The different cell types in the central nervous system develop from a common pool of progenitor cells. The nuclei of progenitors move between the apical and basal surfaces of the neuroepithelium in phase with their cell cycle, a process termed interkinetic nuclear migration (INM). In the retina of zebrafish mikre oko (mok) mutants, in which the motor protein Dynactin-1 is disrupted, interkinetic nuclei migrate more rapidly and deeply to the basal side and more slowly to the apical side. We found that Notch signaling is predominantly activated on the apical side in both mutants and wild-type. Mutant progenitors are, thus, less exposed to Notch and exit the cell cycle prematurely. This leads to an overproduction of early-born retinal ganglion cells (RGCs) at the expense of later-born interneurons and glia. Our data indicate that the function of INM is to balance the exposure of progenitor nuclei to neurogenic versus proliferative signals.
Asunto(s)
Núcleo Celular/metabolismo , Células Neuroepiteliales/citología , Organogénesis , Retina/embriología , Animales , Tipificación del Cuerpo , Ciclo Celular , Diferenciación Celular , Complejo Dinactina , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Asociadas a Microtúbulos/genética , Mutación , Células Neuroepiteliales/metabolismo , Receptores Notch/metabolismo , Retina/citología , Células Ganglionares de la Retina/metabolismo , Células Madre/citología , Células Madre/metabolismo , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
The response of the adult mammalian heart to injury such as myocardial infarction has long been described as primarily fibrotic scarring and adverse remodeling with little to no regeneration of cardiomyocytes. Emerging studies have challenged this paradigm by demonstrating that, indeed, adult mammalian cardiomyocytes are capable of completing cytokinesis albeit at levels vastly insufficient to compensate for the loss of functional cardiomyocytes following ischemic injury. Thus, there is great interest in identifying mechanisms to guide adult cardiomyocyte cell cycle re-entry and facilitate endogenous heart regeneration. The Hippo signaling pathway is a core kinase cascade that functions to suppress the transcriptional co-activators Yap and Taz by phosphorylation and therefore cytoplasmic retention or phospho-degradation. This pathway has recently sparked interest in the field of cardiac regeneration as inhibition of Hippo kinase signaling or overdriving the transcriptional co-activator, Yap, significantly promotes proliferation of terminally differentiated adult mammalian cardiomyocytes and can restore function in failing mouse hearts. Thus, the Hippo pathway is an attractive therapeutic target for promoting cardiomyocyte renewal and cardiac regeneration. Although the core kinases and transcriptional activators of the Hippo pathway have been studied extensively over the last twenty years, the regulatory inputs of this pathway, particularly in vertebrates, are poorly understood. Recent studies have elucidated several upstream regulatory inputs to the Hippo pathway in adult mammalian cardiomyocytes that influence cell proliferation and heart regeneration. Considering upstream inputs to the Hippo pathway are thought to be context and cell type specific, targeting these various components could serve as a therapeutic approach for refining Hippo-Yap signaling in the heart. Here, we provide an overview of the emerging regulatory inputs to the Hippo pathway as they relate to mammalian cardiomyocytes and heart regeneration.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Corazón/fisiología , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Regeneración , Transducción de Señal , Factores de Transcripción/metabolismo , Vía de Señalización Hippo , HumanosRESUMEN
The retinal pigment epithelium (RPE) is a specialized monolayer of pigmented cells within the eye that is critical for maintaining visual system function. Diseases affecting the RPE have dire consequences for vision, and the most prevalent of these is atrophic (dry) age-related macular degeneration (AMD), which is thought to result from RPE dysfunction and degeneration. An intriguing possibility for treating RPE degenerative diseases like atrophic AMD is the stimulation of endogenous RPE regeneration; however, very little is known about the mechanisms driving successful RPE regeneration in vivo. Here, we developed a zebrafish transgenic model (rpe65a:nfsB-eGFP) that enabled ablation of large swathes of mature RPE. RPE ablation resulted in rapid RPE degeneration, as well as degeneration of Bruch's membrane and underlying photoreceptors. Using this model, we demonstrate for the first time that zebrafish are capable of regenerating a functional RPE monolayer after RPE ablation. Regenerated RPE cells first appear at the periphery of the RPE, and regeneration proceeds in a peripheral-to-central fashion. RPE ablation elicits a robust proliferative response in the remaining RPE. Subsequently, proliferative cells move into the injury site and differentiate into RPE. BrdU incorporation assays demonstrate that the regenerated RPE is likely derived from remaining peripheral RPE cells. Pharmacological disruption using IWR-1, a Wnt signaling antagonist, significantly reduces cell proliferation in the RPE and impairs overall RPE recovery. These data demonstrate that the zebrafish RPE possesses a robust capacity for regeneration and highlight a potential mechanism through which endogenous RPE regenerate in vivo.
Asunto(s)
Degeneración Macular/genética , Regeneración/genética , Epitelio Pigmentado de la Retina/crecimiento & desarrollo , cis-trans-Isomerasas/genética , Animales , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/crecimiento & desarrollo , Apoptosis/genética , Lámina Basal de la Coroides/crecimiento & desarrollo , Lámina Basal de la Coroides/metabolismo , Diferenciación Celular/genética , Modelos Animales de Enfermedad , Proteínas Fluorescentes Verdes/genética , Humanos , Imidas/administración & dosificación , Larva/genética , Larva/crecimiento & desarrollo , Degeneración Macular/patología , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/patología , Quinolinas/administración & dosificación , Retina/crecimiento & desarrollo , Retina/patología , Epitelio Pigmentado de la Retina/metabolismo , Vía de Señalización Wnt/efectos de los fármacos , Pez Cebra/genética , Pez Cebra/crecimiento & desarrolloRESUMEN
Balancing the rate of differentiation and proliferation in developing tissues is essential to produce organs of robust size and composition. Although many molecular regulators have been established, how these connect to physical and geometrical aspects of tissue architecture is poorly understood. Here, using high-resolution timelapse imaging, we find that changes to cell geometry associated with dense tissue packing play a significant role in regulating differentiation rate in the zebrafish neural tube. Specifically, progenitors that are displaced away from the apical surface due to crowding, tend to differentiate in a Notch-dependent manner. Using simulations we show that interplay between progenitor density, cell shape and changes in differentiation rate could naturally result in negative-feedback control on progenitor cell number. Given these results, we suggest a model whereby differentiation rate is regulated by density dependent effects on cell geometry to: (1) correct variability in cell number; and (2) balance the rates of proliferation and differentiation over development to 'fill' the available space.
Asunto(s)
Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Células-Madre Neurales/metabolismo , Tubo Neural/embriología , Neurogénesis/fisiología , Pez Cebra/embriología , Animales , Células-Madre Neurales/citología , Tubo Neural/citología , Receptores Notch/genética , Receptores Notch/metabolismo , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Vertebrates have a unique 3D body shape in which correct tissue and organ shape and alignment are essential for function. For example, vision requires the lens to be centred in the eye cup which must in turn be correctly positioned in the head. Tissue morphogenesis depends on force generation, force transmission through the tissue, and response of tissues and extracellular matrix to force. Although a century ago D'Arcy Thompson postulated that terrestrial animal body shapes are conditioned by gravity, there has been no animal model directly demonstrating how the aforementioned mechano-morphogenetic processes are coordinated to generate a body shape that withstands gravity. Here we report a unique medaka fish (Oryzias latipes) mutant, hirame (hir), which is sensitive to deformation by gravity. hir embryos display a markedly flattened body caused by mutation of YAP, a nuclear executor of Hippo signalling that regulates organ size. We show that actomyosin-mediated tissue tension is reduced in hir embryos, leading to tissue flattening and tissue misalignment, both of which contribute to body flattening. By analysing YAP function in 3D spheroids of human cells, we identify the Rho GTPase activating protein ARHGAP18 as an effector of YAP in controlling tissue tension. Together, these findings reveal a previously unrecognised function of YAP in regulating tissue shape and alignment required for proper 3D body shape. Understanding this morphogenetic function of YAP could facilitate the use of embryonic stem cells to generate complex organs requiring correct alignment of multiple tissues.
Asunto(s)
Tamaño Corporal/genética , Proteínas de Peces/metabolismo , Morfogénesis/genética , Oryzias/anatomía & histología , Oryzias/embriología , Actomiosina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Embrión no Mamífero/anatomía & histología , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Proteínas de Peces/genética , Proteínas Activadoras de GTPasa/metabolismo , Genes Esenciales/genética , Gravitación , Humanos , Mutación/genética , Tamaño de los Órganos/genética , Oryzias/genética , Fenotipo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Esferoides Celulares/citología , Esferoides Celulares/metabolismoRESUMEN
The PITX2 (paired-like homeodomain 2) gene encodes a bicoid-like homeodomain transcription factor linked with several human disorders. The main associated congenital phenotype is Axenfeld-Rieger syndrome, type 1, an autosomal dominant condition characterized by variable defects in the anterior segment of the eye, an increased risk of glaucoma, craniofacial dysmorphism and dental and umbilical anomalies; in addition to this, one report implicated PITX2 in ring dermoid of the cornea and a few others described cardiac phenotypes. We report three novel PITX2 mutations-c.271C > T, p.(Arg91Trp); c.259T > C, p.(Phe87Leu); and c.356delA, p.(Gln119Argfs*36)-identified in independent families with typical Axenfeld-Rieger syndrome characteristics and some unusual features such as corneal guttata, Wolf-Parkinson-White syndrome, and hyperextensibility. To gain further insight into the diverse roles of PITX2/pitx2 in vertebrate development, we generated various genetic lesions in the pitx2 gene via TALEN-mediated genome editing. Affected homozygous zebrafish demonstrated congenital defects consistent with the range of PITX2-associated human phenotypes: abnormal development of the cornea, iris and iridocorneal angle; corneal dermoids; and craniofacial dysmorphism. In addition, via comparison of pitx2M64* and wild-type embryonic ocular transcriptomes we defined molecular changes associated with pitx2 deficiency, thereby implicating processes potentially underlying disease pathology. This analysis identified numerous affected factors including several members of the Wnt pathway and collagen types I and V gene families. These data further support the link between PITX2 and the WNT pathway and suggest a new role in regulation of collagen gene expression during development.
Asunto(s)
Segmento Anterior del Ojo/anomalías , Anomalías del Ojo/genética , Enfermedades Hereditarias del Ojo/genética , Glaucoma/genética , Proteínas de Homeodominio/genética , Factores de Transcripción/genética , Síndrome de Wolff-Parkinson-White/genética , Proteínas de Pez Cebra/genética , Animales , Segmento Anterior del Ojo/fisiopatología , Colágeno Tipo I/genética , Colágeno Tipo V/genética , Anomalías del Ojo/fisiopatología , Enfermedades Hereditarias del Ojo/fisiopatología , Edición Génica , Regulación del Desarrollo de la Expresión Génica , Glaucoma/fisiopatología , Humanos , Mutación , Linaje , Factores de Transcripción/deficiencia , Vía de Señalización Wnt , Pez Cebra/genética , Proteínas de Pez Cebra/deficiencia , Proteína del Homeodomínio PITX2RESUMEN
Background Interpreting genetic variants is one of the greatest challenges impeding analysis of rapidly increasing volumes of genomic data from patients. For example, SHROOM3 is an associated risk gene for CKD, yet causative mechanism(s) of SHROOM3 allele(s) are unknown.Methods We used our analytic pipeline that integrates genetic, computational, biochemical, CRISPR/Cas9 editing, molecular, and physiologic data to characterize coding and noncoding variants to study the human SHROOM3 risk locus for CKD.Results We identified a novel SHROOM3 transcriptional start site, which results in a shorter isoform lacking the PDZ domain and is regulated by a common noncoding sequence variant associated with CKD (rs17319721, allele frequency: 0.35). This variant disrupted allele binding to the transcription factor TCF7L2 in podocyte cell nuclear extracts and altered transcription levels of SHROOM3 in cultured cells, potentially through the loss of repressive looping between rs17319721 and the novel start site. Although common variant mechanisms are of high utility, sequencing is beginning to identify rare variants involved in disease; therefore, we used our biophysical tools to analyze an average of 112,849 individual human genome sequences for rare SHROOM3 missense variants, revealing 35 high-effect variants. The high-effect alleles include a coding variant (P1244L) previously associated with CKD (P=0.01, odds ratio=7.95; 95% CI, 1.53 to 41.46) that we find to be present in East Asian individuals at an allele frequency of 0.0027. We determined that P1244L attenuates the interaction of SHROOM3 with 14-3-3, suggesting alterations to the Hippo pathway, a known mediator of CKD.Conclusions These data demonstrate multiple new SHROOM3-dependent genetic/molecular mechanisms that likely affect CKD.
Asunto(s)
Proteínas de Microfilamentos/genética , Insuficiencia Renal Crónica/genética , Alelos , Animales , Núcleo Celular , Frecuencia de los Genes , Sitios Genéticos , Células HEK293 , Humanos , Ratones , Mutación Missense , Podocitos , Isoformas de Proteínas/genética , Proteína 2 Similar al Factor de Transcripción 7/genética , Transcripción Genética , Pez CebraRESUMEN
Zebrafish morphants of osm-3/kif17, a kinesin-2 family member and intraflagellar transport motor, have photoreceptor outer segments that are dramatically reduced in number and size. However, two genetic mutant lines, osm-3/kif17sa0119 and osm-3/kif17sa18340, reportedly lack any observable morphological outer segment defects. In this work, we use TALENs to generate an independent allele, osm-3/kif17mw405, and show that both osm-3/kif17sa0119 and osm-3/kif17mw405 have an outer segment developmental delay in both size and density that is fully recovered by 6 days post-fertilization. Additionally, we use CRISPRs to generate cos2/kif7mw406, a mutation in the kinesin-4 family member cos2/kif7 that has been implicated in controlling ciliary architecture and Hedgehog signaling to test whether it may be functioning redundantly with osm-3/kif17. We show that cos2/kif7mw406 has an outer segment developmental delay similar to the osm-3/kif17 mutants. Using a three-dimensional mathematical model of outer segments, we show that while cos2/kif7mw406 and osm-3/kif17mw405 outer segments are smaller throughout the first 6 days of development, the volumetric rates of outer segment morphogenesis are not different among wild-type, cos2/kif7mw406, and osm-3/kif17mw405 after 60hpf. Instead, our model suggests that cos2/kif7mw406 and osm-3/kif17mw405 impact outer segment morphogenesis through upstream events that that are different for each motor. In the case of cos2/kif7mw406 mutants, we show that early defects in Hedgehog signaling lead to a general, non-photoreceptor-specific delay of retinal neurogenesis, which in turn causes the secondary phenotype of delayed outer segment morphogenesis. In contrast, the osm-3/kif17mw405 outer segment morphogenesis delays are linked specifically to initial disc morphogenesis of photoreceptors rather than an upstream event. Further, we show that osm-3/kif17 mutant mice also exhibit a similarly delayed outer segment development, suggesting a role for osm-3/kif17 in early outer segment development that is conserved across species. In conclusion, we show that both osm-3/kif17 and cos2/kif7 have comparable outer segment developmental delays, although through independent mechanisms.
Asunto(s)
Cinesinas/metabolismo , Morfogénesis , Segmento Externo de las Células Fotorreceptoras Retinianas/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Secuencia de Bases , Sistemas CRISPR-Cas/genética , Cilios/efectos de los fármacos , Cilios/metabolismo , Edición Génica , Proteínas Hedgehog/metabolismo , Ratones Endogámicos C57BL , Modelos Biológicos , Morfogénesis/efectos de los fármacos , Morfolinos/farmacología , Mutación/genética , Neurogénesis/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Temperatura , Nucleasas de los Efectores Tipo Activadores de la TranscripciónRESUMEN
BACKGROUND: KIF17, a kinesin-2 motor that functions in intraflagellar transport, can regulate the onset of photoreceptor outer segment development. However, the function of KIF17 in a mature photoreceptor remains unclear. Additionally, the ciliary localization of KIF17 is regulated by a C-terminal consensus sequence (KRKK) that is immediately adjacent to a conserved residue (mouse S1029/zebrafish S815) previously shown to be phosphorylated by CaMKII. Yet, whether this phosphorylation can regulate the localization, and thus function, of KIF17 in ciliary photoreceptors remains unknown. RESULTS: Using transgenic expression in zebrafish photoreceptors, we show that phospho-mimetic KIF17 has enhanced localization along the cone outer segment. Importantly, expression of phospho-mimetic KIF17 is associated with greatly enhanced turnover of the photoreceptor outer segment through disc shedding in a cell-autonomous manner, while genetic mutants of kif17 in zebrafish and mice have diminished disc shedding. Lastly, cone expression of constitutively active tCaMKII leads to a kif17-dependent increase in disc shedding. CONCLUSIONS: Taken together, our data support a model in which phosphorylation of KIF17 promotes its photoreceptor outer segment localization and disc shedding, a process essential for photoreceptor maintenance and homeostasis. While disc shedding has been predominantly studied in the context of the mechanisms underlying phagocytosis of outer segments by the retinal pigment epithelium, this work implicates photoreceptor-derived signaling in the underlying mechanisms of disc shedding.
Asunto(s)
Cinesinas/metabolismo , Segmento Externo de las Células Fotorreceptoras Retinianas/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Línea Celular , Cilios/metabolismo , Humanos , Cinesinas/química , Ratones Endogámicos C57BL , Mutación/genética , Fagosomas/metabolismo , Fagosomas/ultraestructura , Fosforilación , Células Fotorreceptoras Retinianas Conos/metabolismo , Células Fotorreceptoras Retinianas Conos/ultraestructura , Proteínas de Pez Cebra/químicaRESUMEN
Genome-wide association studies (GWAS) identify regions of the genome correlated with disease risk but are restricted in their ability to identify the underlying causative mechanism(s). Thus, GWAS are useful "roadmaps" that require functional analysis to establish the genetic and mechanistic structure of a particular locus. Unfortunately, direct functional testing in humans is limited, demonstrating the need for complementary approaches. Here we used an integrated approach combining zebrafish, rat, and human data to interrogate the function of an established GWAS locus (SHROOM3) lacking prior functional support for chronic kidney disease (CKD). Congenic mapping and sequence analysis in rats suggested Shroom3 was a strong positional candidate gene. Transferring a 6.1-Mb region containing the wild-type Shroom3 gene significantly improved the kidney glomerular function in FHH (fawn-hooded hypertensive) rat. The wild-type Shroom3 allele, but not the FHH Shroom3 allele, rescued glomerular defects induced by knockdown of endogenous shroom3 in zebrafish, suggesting that the FHH Shroom3 allele is defective and likely contributes to renal injury in the FHH rat. We also show for the first time that variants disrupting the actin-binding domain of SHROOM3 may cause podocyte effacement and impairment of the glomerular filtration barrier.
Asunto(s)
Barrera de Filtración Glomerular/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas de Pez Cebra/metabolismo , Alelos , Secuencia de Aminoácidos , Animales , Animales Congénicos , Animales Modificados Genéticamente , Clonación Molecular , Exones , Femenino , Sitios Genéticos , Variación Genética , Estudio de Asociación del Genoma Completo , Humanos , Enfermedades Renales/genética , Masculino , Proteínas de Microfilamentos/genética , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Plásmidos/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Análisis de Secuencia de ADN , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
The optic vesicle comprises a pool of bi-potential progenitor cells from which the retinal pigment epithelium (RPE) and neural retina fates segregate during ocular morphogenesis. Several transcription factors and signaling pathways have been shown to be important for RPE maintenance and differentiation, but an understanding of the initial fate specification and determination of this ocular cell type is lacking. We show that Yap/Taz-Tead activity is necessary and sufficient for optic vesicle progenitors to adopt RPE identity in zebrafish. A Tead-responsive transgene is expressed within the domain of the optic cup from which RPE arises, and Yap immunoreactivity localizes to the nuclei of prospective RPE cells. yap (yap1) mutants lack a subset of RPE cells and/or exhibit coloboma. Loss of RPE in yap mutants is exacerbated in combination with taz (wwtr1) mutant alleles such that, when Yap and Taz are both absent, optic vesicle progenitor cells completely lose their ability to form RPE. The mechanism of Yap-dependent RPE cell type determination is reliant on both nuclear localization of Yap and interaction with a Tead co-factor. In contrast to loss of Yap and Taz, overexpression of either protein within optic vesicle progenitors leads to ectopic pigmentation in a dosage-dependent manner. Overall, this study identifies Yap and Taz as key early regulators of RPE genesis and provides a mechanistic framework for understanding the congenital ocular defects of Sveinsson's chorioretinal atrophy and congenital retinal coloboma.
Asunto(s)
Linaje de la Célula , Proteínas de Unión al ADN/metabolismo , Células Epiteliales/citología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Epitelio Pigmentado de la Retina/citología , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Alelos , Animales , Apoptosis/genética , Núcleo Celular/metabolismo , Proliferación Celular , Coloboma/patología , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Células HEK293 , Humanos , Morfogénesis/genética , Mutación , Fenotipo , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Epitelio Pigmentado de la Retina/trasplante , Transducción de Señal/genética , Factores de Transcripción de Dominio TEA , Transactivadores/genética , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ , Transgenes , Regulación hacia Arriba , Proteínas Señalizadoras YAP , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
By analysing the cellular and subcellular events that occur in the centre of the developing zebrafish neural rod, we have uncovered a novel mechanism of cell polarisation during lumen formation. Cells from each side of the neural rod interdigitate across the tissue midline. This is necessary for localisation of apical junctional proteins to the region where cells intersect the tissue midline. Cells assemble a mirror-symmetric microtubule cytoskeleton around the tissue midline, which is necessary for the trafficking of proteins required for normal lumen formation, such as partitioning defective 3 and Rab11a to this point. This occurs in advance and is independent of the midline cell division that has been shown to have a powerful role in lumen organisation. To our knowledge, this is the first example of the initiation of apical polarisation part way along the length of a cell, rather than at a cell extremity. Although the midline division is not necessary for apical polarisation, it confers a morphogenetic advantage by efficiently eliminating cellular processes that would otherwise bridge the developing lumen.
Asunto(s)
Comunicación Celular , Microtúbulos/metabolismo , Tubo Neural/embriología , Neurulación , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Tipificación del Cuerpo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , División Celular , Movimiento Celular , Polaridad Celular , Embrión no Mamífero/citología , Embrión no Mamífero/embriología , Proteínas Fluorescentes Verdes/química , Sustancias Luminiscentes/química , Microtúbulos/genética , Mutación , Tubo Neural/citología , Nocodazol/farmacología , Transporte de Proteínas/efectos de los fármacos , Proteínas Recombinantes de Fusión , Moduladores de Tubulina/farmacología , Pez Cebra/anatomía & histología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Zebrafish (Danio rerio) provide many advantages as a model organism for studying ocular disease and development, and there is great interest in the ability to non-invasively assess their photoreceptor mosaic. Despite recent applications of scanning light ophthalmoscopy, fundus photography, and gonioscopy to in vivo imaging of the adult zebrafish eye, current techniques either lack accurate scaling information (limiting quantitative analyses) or require euthanizing the fish (precluding longitudinal analyses). Here we describe improved methods for imaging the adult zebrafish retina using spectral domain optical coherence tomography (OCT). Transgenic fli1:eGFP zebrafish were imaged using the Bioptigen Envisu R2200 broadband source OCT with a 12-mm telecentric probe to measure axial length and a mouse retina probe to acquire retinal volume scans subtending 1.2 × 1.2 mm nominally. En face summed volume projections were generated from the volume scans using custom software that allows the user to create contours tailored to specific retinal layer(s) of interest. Following imaging, the eyes were dissected for ex vivo fluorescence microscopy, and measurements of blood vessel branch points were compared to those made from the en face OCT images to determine the OCT lateral scale as a function of axial length. Using this scaling model, we imaged the photoreceptor layer of five wild-type zebrafish and quantified the density and packing geometry of the UV cone submosaic. Our in vivo cone density measurements agreed with measurements from previously published histology values. The method presented here allows accurate, quantitative assessment of cone structure in vivo and will be useful for longitudinal studies of the zebrafish cone mosaics.
Asunto(s)
Células Fotorreceptoras Retinianas Conos/citología , Tomografía de Coherencia Óptica , Pez Cebra/anatomía & histología , Animales , Animales Modificados Genéticamente , Proteínas Fluorescentes Verdes/genética , Microscopía Fluorescente , Pez Cebra/genéticaRESUMEN
To gain insights into the cellular mechanisms of neurogenesis, we analyzed retinal neuroepithelia deficient for Llgl1, a protein implicated in apicobasal cell polarity, asymmetric cell division, cell shape and cell cycle exit. We found that vertebrate retinal neuroepithelia deficient for Llgl1 retained overt apicobasal polarity, but had expanded apical domains. Llgl1 retinal progenitors also had increased Notch activity and reduced rates of neurogenesis. Blocking Notch function by depleting Rbpj restored normal neurogenesis. Experimental expansion of the apical domain, through inhibition of Shroom3, also increased Notch activity and reduced neurogenesis. Significantly, in wild-type retina, neurogenic retinal progenitors had smaller apical domains compared with proliferative neuroepithelia. As nuclear position during interkinetic nuclear migration (IKNM) has been previously linked with cell cycle exit, we analyzed this phenomenon in cells depleted of Llgl1. We found that although IKNM was normal, the relationship between nuclear position and neurogenesis was shifted away from the apical surface, consistent with increased pro-proliferative and/or anti-neurogenic signals associated with the apical domain. These data, in conjunction with other findings, suggest that, in retinal neuroepithelia, the size of the apical domain modulates the strength of polarized signals that influence neurogenesis.