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Purpose: Myopia, or nearsightedness, is the most common form of refractive error and is increasing in prevalence. While significant efforts have been made to identify genetic variants that predispose individuals to myopia, these variants are believed to account for only a small portion of the myopia prevalence, leading to a feedback theory of emmetropization, which depends on the active perception of environmental visual cues. Consequently, there has been renewed interest in studying myopia in the context of light perception, beginning with the opsin family of G-protein coupled receptors (GPCRs). Refractive phenotypes have been characterized in every opsin signaling pathway studied, leaving only Opsin 3 (OPN3), the most widely expressed and blue-light sensing noncanonical opsin, to be investigated for function in the eye and refraction. Methods: Opn3 expression was assessed in various ocular tissues using an Opn3eGFP reporter. Weekly refractive development in Opn3 retinal and germline mutants from 3 to 9 weeks of age was measured using an infrared photorefractor and spectral domain optical coherence tomography (SD-OCT). Susceptibility to lens-induced myopia was then assessed using skull-mounted goggles with a -30 diopter experimental and a 0 diopter control lens. Mouse eye biometry was similarly tracked from 3 to 6 weeks. A myopia gene expression signature was assessed 24 h after lens induction for germline mutants to further assess myopia-induced changes. Results: Opn3 was found to be expressed in a subset of retinal ganglion cells and a limited number of choroidal cells. Based on an assessment of Opn3 mutants, the OPN3 germline, but not retina conditional Opn3 knockout, exhibits a refractive myopia phenotype, which manifests in decreased lens thickness, shallower aqueous compartment depth, and shorter axial length, atypical of traditional axial myopias. Despite the short axial length, Opn3 null eyes demonstrate normal axial elongation in response to myopia induction and mild changes in choroidal thinning and myopic shift, suggesting that susceptibility to lens-induced myopia is largely unchanged. Additionally, the Opn3 null retinal gene expression signature in response to induced myopia after 24 h is distinct, with opposing Ctgf, Cx43, and Egr1 polarity compared to controls. Conclusions: The data suggest that an OPN3 expression domain outside the retina can control lens shape and thus the refractive performance of the eye. Prior to this study, the role of Opn3 in the eye had not been investigated. This work adds OPN3 to the list of opsin family GPCRs that are implicated in emmetropization and myopia. Further, the work to exclude retinal OPN3 as the contributing domain in this refractive phenotype is unique and suggests a distinct mechanism when compared to other opsins.
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Miopía , Errores de Refracción , Animales , Ratones , Miopía/genética , Refracción Ocular , Retina , Opsinas/genética , Opsinas de BastonesRESUMEN
Neurons in the hypothalamic preoptic area (POA) regulate multiple homeostatic processes, including thermoregulation and sleep, by sensing afferent input and modulating sympathetic nervous system output. The POA has an autonomous circadian clock and may also receive circadian signals indirectly from the suprachiasmatic nucleus. We have previously defined a subset of neurons in the POA termed QPLOT neurons that are identified by the expression of molecular markers (Qrfp, Ptger3, LepR, Opn5, Tacr3) that suggest receptivity to multiple stimuli. Because Ptger3, Opn5, and Tacr3 encode G-protein coupled receptors (GPCRs), we hypothesized that elucidating the G-protein signaling in these neurons is essential to understanding the interplay of inputs in the regulation of metabolism. Here, we describe how the stimulatory Gs-alpha subunit (Gnas) in QPLOT neurons regulates metabolism in mice. We analyzed Opn5cre; Gnasfl/fl mice using indirect calorimetry at ambient temperatures of 22°C (a historical standard), 10°C (a cold challenge), and 28°C (thermoneutrality) to assess the ability of QPLOT neurons to regulate metabolism. We observed a marked decrease in nocturnal locomotion of Opn5cre; Gnasfl/fl mice at both 28°C and 22°C, but no overall differences in energy expenditure, respiratory exchange, or food and water consumption. To analyze daily rhythmic patterns of metabolism, we assessed circadian parameters including amplitude, phase, and MESOR. Loss-of-function GNAS in QPLOT neurons resulted in several subtle rhythmic changes in multiple metabolic parameters. We observed that Opn5cre; Gnasfl/fl mice show a higher rhythm-adjusted mean energy expenditure at 22°C and 10°C, and an exaggerated respiratory exchange shift with temperature. At 28°C, Opn5cre; Gnasfl/fl mice have a significant delay in the phase of energy expenditure and respiratory exchange. Rhythmic analysis also showed limited increases in rhythm-adjusted means of food and water intake at 22°C and 28°C. Together, these data advance our understanding of Gαs-signaling in preoptic QPLOT neurons in regulating daily patterns of metabolism.
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Regulación de la Temperatura Corporal , Hipotálamo , Animales , Ratones , Regulación de la Temperatura Corporal/fisiología , Ritmo Circadiano/fisiología , Metabolismo Energético , Homeostasis , Hipotálamo/metabolismo , Proteínas de la Membrana/metabolismo , Neuronas/metabolismo , Opsinas/metabolismo , TemperaturaRESUMEN
We live on a planet that is bathed in daily and seasonal sunlight cycles. In this context, terrestrial life forms have evolved mechanisms that directly harness light energy (plants) or decode light information for adaptive advantage. In animals, the main light sensors are a family of G protein-coupled receptors called opsins. Opsin function is best described for the visual sense. However, most animals also use opsins for extraocular light sensing for seasonal behavior and camouflage. While it has long been believed that mammals do not have an extraocular light sensing capacity, recent evidence suggests otherwise. Notably, encephalopsin (OPN3) and neuropsin (OPN5) are both known to mediate extraocular light sensing in mice. Examples of this mediation include photoentrainment of circadian clocks in skin (by OPN5) and acute light-dependent regulation of metabolic pathways (by OPN3 and OPN5). This review summarizes current findings in the expanding field of extraocular photoreception and their relevance for human physiology.
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Opsinas , Opsinas de Bastones , Ratones , Humanos , Animales , Opsinas/fisiología , Piel/metabolismo , Mamíferos , Proteínas de la Membrana/metabolismoRESUMEN
Activating non-inherited mutations in the guanine nucleotide-binding protein G(q) subunit alpha (GNAQ) gene family have been identified in childhood vascular tumors. Patients experience extensive disfigurement, chronic pain and severe complications including a potentially lethal coagulopathy termed Kasabach-Merritt phenomenon. Animal models for this class of vascular tumors do not exist. This has severely hindered the discovery of the molecular consequences of GNAQ mutations in the vasculature and, in turn, the preclinical development of effective targeted therapies. Here we report a mouse model expressing hyperactive mutant GNAQ in endothelial cells. Mutant mice develop vascular and coagulopathy phenotypes similar to those seen in patients. Mechanistically, by transcriptomic analysis we demonstrate increased mitogen activated protein kinase signaling in the mutant endothelial cells. Targeting of this pathway with Trametinib suppresses the tumor growth by reducing vascular cell proliferation and permeability. Trametinib also prevents the development of coagulopathy and improves mouse survival.
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Melanoma , Neoplasias de la Úvea , Neoplasias Vasculares , Animales , Ratones , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Células Endoteliales/metabolismo , Apoptosis , Melanoma/genética , Neoplasias de la Úvea/genética , Mutación , Modelos Animales de Enfermedad , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Línea Celular TumoralRESUMEN
Opsin photoreceptors outside of the central nervous system have been shown to mediate smooth muscle photorelaxation in several organs. We hypothesized that opsin receptor activation in the colon would have a similar effect and influence colonic motility. We detected Opsin 3 (OPN3) protein expression in the colonic wall and demonstrated that OPN3 was present in enteric neurons in the muscularis propria of the murine colon. Precontracted murine colon segments demonstrated blue light (BL) -mediated relaxation ex vivo. This photorelaxation was wavelength specific and was increased with the administration of the chromophore 9-cis retinal and a G protein receptor kinase 2 (GRK2) inhibitor. Light-mediated relaxation of the colon was not inhibited by L-NAME or tetrodotoxin (TTX). Furthermore, BL exposure in the presence of 9-cis retinal decreased the frequency of colonic migrating motor complexes (CMMC) in spontaneously contracting mouse colons ex vivo. These results demonstrate for the first time a receptor-mediated photorelaxation of colonic smooth muscle and implicate opsins as possible new targets in the treatment of spasmodic gastrointestinal dysmotility.
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Recently, we characterized blue light-mediated relaxation (photorelaxation) of airway smooth muscle (ASM) and implicated the involvement of opsin 3 (OPN3), an atypical opsin. In the present study, we characterized the cellular signaling mechanisms of photorelaxation. We confirmed the functional role of OPN3 in blue light photorelaxation using trachea from OPN3 null mice (maximal relaxation 52 ± 13% compared with wild-type mice 90 ± 4.3%, P < 0.05). We then demonstrated colocalization of OPN3 and Gαs using co-IP and proximity ligation assays in primary human ASM cells, which was further supported by an increase in cAMP in mouse trachea treated with blue light compared with dark controls (23 ± 3.6 vs. 14 ± 2.6 pmol cAMP/ring, P < 0.05). Downstream PKA (protein kinase A) involvement was shown by inhibiting photorelaxation using Rp-cAMPS (P < 0.0001). Moreover, we observed converging mechanisms of desensitization by chronic ß2-agonist exposure in mouse trachea and correlated this finding with colocalization of OPN3 and GRK2 (G protein receptor kinase) in primary human ASM cells. Finally, an overexpression model of OPN1LW (a red light photoreceptor in the same opsin family) in human ASM cells showed an increase in intracellular cAMP levels following red light exposure compared with nontransfected cells (48 ± 13 vs. 13 ± 2.1 pmol cAMP/mg protein, P < 0.01), suggesting a conserved photorelaxation mechanism for wavelengths of light that are more tissue penetrant. Together, these results demonstrate that blue light photorelaxation in ASM is mediated by the OPN3 receptor interacting with Gαs, which increases cAMP levels, activating PKA and modulated by GRK2.
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Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Relajación Muscular/fisiología , Músculo Liso/metabolismo , Miocitos del Músculo Liso/metabolismo , Opsinas de Bastones/metabolismo , Tráquea/metabolismo , Animales , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Opsinas/metabolismo , Transducción de Señal/fisiologíaRESUMEN
The opsin family of G-protein-coupled receptors are used as light detectors in animals. Opsin 5 (also known as neuropsin or OPN5) is a highly conserved opsin that is sensitive to visible violet light1,2. In mice, OPN5 is a known photoreceptor in the retina3 and skin4 but is also expressed in the hypothalamic preoptic area (POA)5. Here we describe a light-sensing pathway in which POA neurons that express Opn5 regulate thermogenesis in brown adipose tissue (BAT). We show that Opn5 is expressed in glutamatergic warm-sensing POA neurons that receive synaptic input from several thermoregulatory nuclei. We further show that Opn5 POA neurons project to BAT and decrease its activity under chemogenetic stimulation. Opn5-null mice show overactive BAT, increased body temperature, and exaggerated thermogenesis when cold-challenged. Moreover, violet photostimulation during cold exposure acutely suppresses BAT temperature in wild-type mice but not in Opn5-null mice. Direct measurements of intracellular cAMP ex vivo show that Opn5 POA neurons increase cAMP when stimulated with violet light. This analysis thus identifies a violet light-sensitive deep brain photoreceptor that normally suppresses BAT thermogenesis.
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Color , Luz , Proteínas de la Membrana/metabolismo , Neuronas/metabolismo , Neuronas/efectos de la radiación , Opsinas/metabolismo , Área Preóptica/citología , Termogénesis/efectos de la radiación , Tejido Adiposo Pardo/inervación , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Pardo/efectos de la radiación , Animales , Temperatura Corporal , Frío , AMP Cíclico/metabolismo , Femenino , Masculino , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Opsinas/deficiencia , Opsinas/genética , Termogénesis/genéticaRESUMEN
Almost all life forms can detect and decode light information for adaptive advantage. Examples include the visual system, in which photoreceptor signals are processed into virtual images, and the circadian system, in which light entrains a physiological clock. Here we describe a light response pathway in mice that employs encephalopsin (OPN3, a 480 nm, blue-light-responsive opsin) to regulate the function of adipocytes. Germline null and adipocyte-specific conditional null mice show a light- and Opn3-dependent deficit in thermogenesis and become hypothermic upon cold exposure. We show that stimulating mouse adipocytes with blue light enhances the lipolysis response and, in particular, phosphorylation of hormone-sensitive lipase. This response is Opn3 dependent. These data establish a key mechanism in which light-dependent, local regulation of the lipolysis response in white adipocytes regulates energy metabolism.
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Adipocitos Marrones/metabolismo , Adipocitos Marrones/efectos de la radiación , Adipocitos Blancos/metabolismo , Adipocitos Blancos/efectos de la radiación , Luz , Opsinas de Bastones/metabolismo , Termogénesis/efectos de la radiación , Animales , Frío , Metabolismo Energético/efectos de la radiación , Perfilación de la Expresión Génica , Lipólisis/efectos de la radiación , Ratones Endogámicos C57BL , Fenotipo , Fotones , Termogénesis/genéticaRESUMEN
Nearly all mammalian tissues have functional, autonomous circadian clocks, which free-run with non-24 h periods and must be synchronized (entrained) to the 24 h day. This entrainment mechanism is thought to be hierarchical, with photic input to the retina entraining the master circadian clock in the suprachiasmatic nuclei (SCN) and the SCN in turn synchronizing peripheral tissues via endocrine mechanisms. Here, we assess the function of a population of melanocyte precursor cells in hair and vibrissal follicles that express the photopigment neuropsin (OPN5). Organotypic cultures of murine outer ear and vibrissal skin entrain to a light-dark cycle ex vivo, requiring cis-retinal chromophore and Opn5 gene function. Short-wavelength light strongly phase shifts skin circadian rhythms ex vivo via an Opn5-dependent mechanism. In vivo, the normal amplitude of Period mRNA expression in outer ear skin is dependent on both the light-dark cycle and Opn5 function. In Opn4-/-; Pde6brd1/rd1 mice that cannot behaviorally entrain to light-dark cycles, the phase of skin-clock gene expression remains synchronized to the light-dark cycle, even as other peripheral clocks remain phase-locked to the free-running behavioral rhythm. Taken together, these results demonstrate the presence of a direct photic circadian entrainment pathway and direct light-response elements for clock genes in murine skin, similar to pathways previously described for invertebrates and certain non-mammalian vertebrates.
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Relojes Circadianos/genética , Ritmo Circadiano/fisiología , Luz , Proteínas de la Membrana/genética , Opsinas/genética , Fotoperiodo , Animales , Relojes Circadianos/efectos de la radiación , Ritmo Circadiano/efectos de la radiación , Femenino , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Opsinas/metabolismo , Distribución Aleatoria , PielRESUMEN
During mouse postnatal eye development, the embryonic hyaloid vascular network regresses from the vitreous as an adaption for high-acuity vision. This process occurs with precisely controlled timing. Here, we show that opsin 5 (OPN5; also known as neuropsin)-dependent retinal light responses regulate vascular development in the postnatal eye. In Opn5-null mice, hyaloid vessels regress precociously. We demonstrate that 380-nm light stimulation via OPN5 and VGAT (the vesicular GABA/glycine transporter) in retinal ganglion cells enhances the activity of inner retinal DAT (also known as SLC6A3; a dopamine reuptake transporter) and thus suppresses vitreal dopamine. In turn, dopamine acts directly on hyaloid vascular endothelial cells to suppress the activity of vascular endothelial growth factor receptor 2 (VEGFR2) and promote hyaloid vessel regression. With OPN5 loss of function, the vitreous dopamine level is elevated and results in premature hyaloid regression. These investigations identify violet light as a developmental timing cue that, via an OPN5-dopamine pathway, regulates optic axis clearance in preparation for visual function.
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Dopamina/metabolismo , Ojo/irrigación sanguínea , Luz , Proteínas de la Membrana/metabolismo , Opsinas/metabolismo , Animales , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/antagonistas & inhibidores , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/química , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Endotelio Vascular/metabolismo , Ojo/enzimología , Ojo/crecimiento & desarrollo , Ojo/metabolismo , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Opsinas/genética , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/efectos de la radiación , Treonina/metabolismo , Proteínas del Transporte Vesicular de Aminoácidos Inhibidores/fisiología , Cuerpo Vítreo/metabolismoRESUMEN
Normal development requires tight regulation of cell proliferation and cell death. Here, we have investigated these control mechanisms in the hyaloid vessels, a temporary vascular network in the mammalian eye that requires a Wnt/ß-catenin response for scheduled regression. We investigated whether the hyaloid Wnt response was linked to the oncogene Myc, and the cyclin-dependent kinase inhibitor CDKN1A (P21), both established regulators of cell cycle progression and cell death. Our analysis showed that the Wnt pathway co-receptors LRP5 and LRP6 have overlapping activities that mediate the Wnt/ß-catenin signaling in hyaloid vascular endothelial cells (VECs). We also showed that both Myc and Cdkn1a are downstream of the Wnt response and are required for hyaloid regression but for different reasons. Conditional deletion of Myc in VECs suppressed both proliferation and cell death. By contrast, conditional deletion of Cdkn1a resulted in VEC overproliferation that countered the effects of cell death on regression. When combined with analysis of MYC and CDKN1A protein levels, this analysis suggests that a Wnt/ß-catenin and MYC-CDKN1A pathway regulates scheduled hyaloid vessel regression.
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Apoptosis/fisiología , Proliferación Celular/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Endotelio Vascular/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , beta Catenina/metabolismo , Animales , Línea Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Células Endoteliales/metabolismo , Endotelio Vascular/citología , Ojo/irrigación sanguínea , Células HEK293 , Humanos , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Ratones , Ratones Noqueados , Proteínas Proto-Oncogénicas c-myc/genética , Vía de Señalización Wnt/fisiologíaRESUMEN
Formation of neural and sensory progenitors in the inner ear requires Sox2 in mammals, and in other species is thought to rely on both Sox2 and Sox3. How Sox2 and/or Sox3 promote different fates is poorly understood. Our mutant analysis in zebrafish showed that sox2 is uniquely required for sensory development while sox3 is uniquely required for neurogenesis. Moderate misexpression of sox2 during placodal stages led to development of otic vesicles with expanded sensory and reduced neurogenic domains. However, high-level misexpression of sox2 or sox3 expanded both sensory and neurogenic domains to fill the medial and lateral halves of the otic vesicle, respectively. Disruption of medial factor pax2a eliminated the ability of sox2/3 misexpression to expand sensory but not neurogenic domains. Additionally, mild misexpression of fgf8 during placodal development was sufficient to specifically expand the zone of prosensory competence. Later, cross-repression between atoh1a and neurog1 helps maintain the sensory-neural boundary, but unlike mouse this does not require Notch activity. Together, these data show that sox2 and sox3 exhibit intrinsic differences in promoting sensory vs. neural competence, but at high levels these factors can mimic each other to enhance both states. Regional cofactors like pax2a and fgf8 also modify sox2/3 functions.
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Regulación del Desarrollo de la Expresión Génica/fisiología , Células Ciliadas Auditivas Internas/metabolismo , Neurogénesis/fisiología , Factores de Transcripción SOX/biosíntesis , Proteínas de Pez Cebra/biosíntesis , Pez Cebra/embriología , Animales , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Células Ciliadas Auditivas Internas/citología , Factor de Transcripción PAX2/genética , Factor de Transcripción PAX2/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Factores de Transcripción SOX/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Resident tissue myeloid cells play a role in many aspects of physiology including development of the vascular systems. In the blood vasculature, myeloid cells use VEGFC to promote angiogenesis and can use Wnt ligands to control vascular branching and to promote vascular regression. Here we show that myeloid cells also regulate development of the dermal lymphatic vasculature using Wnt ligands. Using myeloid-specific deletion of the WNT transporter Wntless we show that myeloid Wnt ligands are active at two distinct stages of development of the dermal lymphatics. As lymphatic progenitors are emigrating from the cardinal vein and intersomitic vessels, myeloid Wnt ligands regulate both their numbers and migration distance. Later in lymphatic development, myeloid Wnt ligands regulate proliferation of lymphatic endothelial cells (LEC) and thus control lymphatic vessel caliber. Myeloid-specific deletion of WNT co-receptor Lrp5 or Wnt5a gain-of-function also produce elevated caliber in dermal lymphatic capillaries. These data thus suggest that myeloid cells produce Wnt ligands to regulate lymphatic development and use Wnt pathway co-receptors to regulate the balance of Wnt ligand activity during the macrophage-LEC interaction.
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Dermis/metabolismo , Células Endoteliales/metabolismo , Linfangiogénesis/fisiología , Vasos Linfáticos/metabolismo , Células Mieloides/metabolismo , Proteínas Wnt/metabolismo , Animales , Proliferación Celular/fisiología , Ratones , Vía de Señalización WntRESUMEN
The molecular circadian clocks in the mammalian retina are locally synchronized by environmental light cycles independent of the suprachiasmatic nuclei (SCN) in the brain. Unexpectedly, this entrainment does not require rods, cones, or melanopsin (OPN4), possibly suggesting the involvement of another retinal photopigment. Here, we show that the ex vivo mouse retinal rhythm is most sensitive to short-wavelength light but that this photoentrainment requires neither the short-wavelength-sensitive cone pigment [S-pigment or cone opsin (OPN1SW)] nor encephalopsin (OPN3). However, retinas lacking neuropsin (OPN5) fail to photoentrain, even though other visual functions appear largely normal. Initial evidence suggests that OPN5 is expressed in select retinal ganglion cells. Remarkably, the mouse corneal circadian rhythm is also photoentrainable ex vivo, and this photoentrainment likewise requires OPN5. Our findings reveal a light-sensing function for mammalian OPN5, until now an orphan opsin.
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Córnea/fisiología , Proteínas de la Membrana/fisiología , Opsinas/fisiología , Retina/fisiología , Núcleo Supraquiasmático/fisiología , Animales , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Opsinas/genética , Rayos UltravioletaRESUMEN
The belly spot and tail (Bst(+/-)) mouse phenotype is caused by mutations of the ribosomal protein L24 (Rpl24). Among various phenotypes in Bst(+/-) mice, the most interesting are its retinal abnormalities, consisting of delayed closure of choroid fissures, decreased ganglion cells and subretinal vascularization. We further characterized the Bst(+/-) mouse and investigated the underlying molecular mechanisms to assess the feasibility of using this strain as a model for stem cell therapy of retinal degenerative diseases due to retinal ganglion cell (RGC) loss. We found that, although RGCs are significantly reduced in retinal ganglion cell layer in Bst(+/-) mouse, melanopsin(+) RGCs, also called ipRGCs, appear to be unchanged. Pupillary light reflex was completely absent in Bst(+/-) mice but they had a normal circadian rhythm. In order to examine the pathological abnormalities in Bst(+/-) mice, we performed electron microscopy in RGC and found that mitochondria morphology was deformed, having irregular borders and lacking cristae. The complex activities of the mitochondrial electron transport chain were significantly decreased. Finally, for subretinal vascularization, we also found that angiogenesis is delayed in Bst(+/-) associated with delayed hyaloid regression. Characterization of Bst(+/-) retina suggests that the Bst(+/-) mouse strain could be a useful murine model. It might be used to explore further the pathogenesis and strategy of treatment of retinal degenerative diseases by employing stem cell technology.
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Retina/patología , Retina/fisiopatología , Animales , Inmunohistoquímica , Ratones , Ratones Mutantes , Mitocondrias/metabolismo , Neovascularización Fisiológica , Consumo de Oxígeno , Fenotipo , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/patología , Vasos Retinianos/metabolismo , Vasos Retinianos/patología , Opsinas de Bastones/metabolismo , Factor de Transcripción Brn-3A/metabolismoRESUMEN
Angiogenesis defines the process in which new vessels grow from existing vessels. Using the mouse retina as a model system, we show that cysteine-rich motor neuron 1 (Crim1), a type I transmembrane protein, is highly expressed in angiogenic endothelial cells. Conditional deletion of the Crim1 gene in vascular endothelial cells (VECs) causes delayed vessel expansion and reduced vessel density. Based on known Vegfa binding by Crim1 and Crim1 expression in retinal vasculature, where angiogenesis is known to be Vegfa dependent, we tested the hypothesis that Crim1 is involved in the regulation of Vegfa signaling. Consistent with this hypothesis, we showed that VEC-specific conditional compound heterozygotes for Crim1 and Vegfa exhibit a phenotype that is more severe than each single heterozygote and indistinguishable from that of the conditional homozygotes. We further showed that human CRIM1 knockdown in cultured VECs results in diminished phosphorylation of VEGFR2, but only when VECs are required to rely on an autocrine source of VEGFA. The effect of CRIM1 knockdown on reducing VEGFR2 phosphorylation was enhanced when VEGFA was also knocked down. Finally, an anti-VEGFA antibody did not enhance the effect of CRIM1 knockdown in reducing VEGFR2 phosphorylation caused by autocrine signaling, but VEGFR2 phosphorylation was completely suppressed by SU5416, a small-molecule VEGFR2 kinase inhibitor. These data are consistent with a model in which Crim1 enhances the autocrine signaling activity of Vegfa in VECs at least in part via Vegfr2.
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Receptores de Proteínas Morfogenéticas Óseas/metabolismo , Vasos Retinianos/crecimiento & desarrollo , Vasos Retinianos/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Alelos , Animales , Comunicación Autocrina , Receptores de Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Receptores de Proteínas Morfogenéticas Óseas/genética , Proliferación Celular , Células Endoteliales/metabolismo , Técnicas de Silenciamiento del Gen , Heterocigoto , Homocigoto , Células Endoteliales de la Vena Umbilical Humana , Humanos , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Mutantes , Ratones Transgénicos , Neovascularización Fisiológica , Pericitos/metabolismo , Fenotipo , Fosforilación , ARN Interferente Pequeño/genética , Vasos Retinianos/embriología , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Neuroblasts of the statoacoustic ganglion (SAG) initially form in the floor of the otic vesicle during a relatively brief developmental window. They soon delaminate and undergo a protracted phase of proliferation and migration (transit-amplification). Neuroblasts eventually differentiate and extend processes bi-directionally to synapse with hair cells in the inner ear and various targets in the hindbrain. Our studies in zebrafish have shown that Fgf signaling controls multiple phases of this complex developmental process. Moderate levels of Fgf in a gradient emanating from the nascent utricular macula specify SAG neuroblasts in laterally adjacent otic epithelium. At a later stage, differentiating SAG neurons express Fgf5, which serves two functions: First, as SAG neurons accumulate, increasing levels of Fgf exceed an upper threshold that terminates the initial phase of neuroblast specification. Second, elevated Fgf delays differentiation of transit-amplifying cells, balancing the rate of progenitor renewal with neuronal differentiation. Laser-ablation of mature SAG neurons abolishes feedback-inhibition and causes precocious neuronal differentiation. Similar effects are obtained by Fgf5-knockdown or global impairment of Fgf signaling, whereas Fgf misexpression has the opposite effect. Thus Fgf signaling renders SAG development self-regulating, ensuring steady production of an appropriate number of neurons as the larva grows.
Asunto(s)
Oído Interno , Factor 5 de Crecimiento de Fibroblastos , Neuronas , Pez Cebra , Animales , Diferenciación Celular , Oído Interno/crecimiento & desarrollo , Oído Interno/inervación , Oído Interno/metabolismo , Epitelio/metabolismo , Factor 5 de Crecimiento de Fibroblastos/genética , Factor 5 de Crecimiento de Fibroblastos/metabolismo , Ganglión/metabolismo , Regulación del Desarrollo de la Expresión Génica , Larva/crecimiento & desarrollo , Larva/metabolismo , Neurogénesis , Neuronas/citología , Neuronas/metabolismo , Transducción de Señal , Pez Cebra/crecimiento & desarrollo , Pez Cebra/metabolismoRESUMEN
Atoh1 is required for differentiation of sensory hair cells in the vertebrate inner ear. Moreover, misexpression of Atoh1 is sufficient to establish ectopic sensory epithelia, making Atoh1 a good candidate for gene therapy to restore hearing. However, competence to form sensory epithelia appears to be limited to discrete regions of the inner ear. To better understand the developmental factors influencing sensory-competence, we examined the effects of misexpressing atoh1a in zebrafish embryos under various developmental conditions. Activation of a heat shock-inducible transgene, hs:atoh1a, resulted in ectopic expression of early markers of sensory development within 2h, and mature hair cells marked by brn3c:GFP began to accumulate 9h after heat shock. The ability of atoh1a to induce ectopic sensory epithelia was maximal when activated during placodal or early otic vesicle stages but declined rapidly thereafter. At no stage was atoh1a sufficient to induce sensory development in dorsal or lateral non-sensory regions of the otic vesicle. However, co-misexpression of atoh1a with fgf3, fgf8 or sox2, genes normally acting in the same gene network with atoh1a, stimulated sensory development in all regions of the otic vesicle. Thus, expression of fgf3, fgf8 or sox2 strongly enhances competence to respond to Atoh1.
Asunto(s)
Oído Interno/fisiología , Factores de Crecimiento de Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Redes Reguladoras de Genes/genética , Células Ciliadas Auditivas/fisiología , Factores de Transcripción SOX/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Oído Interno/embriología , Perfilación de la Expresión Génica , Redes Reguladoras de Genes/fisiología , Proteínas Fluorescentes Verdes/metabolismo , Hibridación in Situ , Factores de Transcripción/genética , Transgenes/genética , Proteínas de Pez Cebra/genéticaRESUMEN
The zebrafish otic vesicle initially forms with only two sensory epithelia, the utricular and saccular maculae, which primarily mediate vestibular and auditory function, respectively. Here, we test the role of pax5, which is preferentially expressed in the utricular macula. Morpholino knockdown of pax5 disrupts vestibular function but not hearing. Neurons of the statoacoustic ganglion (SAG) develop normally. Utricular hair cells appear to form normally but a variable number subsequently undergo apoptosis and are extruded from the otic vesicle. Dendrites of the SAG persist in the utricle but become disorganized after hair cell loss. Hair cells in the saccule develop and survive normally. Otic expression of pax5 requires pax2a and fgf3, mutations in which cause vestibular defects, albeit by distinct mechanisms. Thus, pax5 works in conjunction with fgf3 and pax2a to establish and/or maintain the utricular macula and is essential for vestibular function.