RESUMO
Vsx2 is a transcription factor essential for retinal proliferation and bipolar cell differentiation, but the molecular mechanisms underlying its developmental roles are unclear. Here, we have profiled VSX2 genomic occupancy during mouse retinogenesis, revealing extensive retinal genetic programs associated with VSX2 during development. VSX2 binds and transactivates its enhancer in association with the transcription factor PAX6. Mice harboring deletions in the Vsx2 regulatory landscape exhibit specific abnormalities in retinal proliferation and in bipolar cell differentiation. In one of those deletions, a complete loss of bipolar cells is associated with a bias towards photoreceptor production. VSX2 occupies cis-regulatory elements nearby genes associated with photoreceptor differentiation and homeostasis in the adult mouse and human retina, including a conserved region nearby Prdm1, a factor implicated in the specification of rod photoreceptors and suppression of bipolar cell fate. VSX2 interacts with the transcription factor OTX2 and can act to suppress OTX2-dependent enhancer transactivation of the Prdm1 enhancer. Taken together, our analyses indicate that Vsx2 expression can be temporally and spatially uncoupled at the enhancer level, and they illuminate important mechanistic insights into how VSX2 is engaged with gene regulatory networks that are essential for retinal proliferation and cell fate acquisition.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio , Adulto , Animais , Diferenciação Celular/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Retina/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Visual information is transmitted from the eye to the brain along the optic nerve, a structure composed of retinal ganglion cell (RGC) axons. The optic nerve is highly vulnerable to damage in neurodegenerative diseases, such as glaucoma, and there are currently no FDA-approved drugs or therapies to protect RGCs from death. Zebrafish possess remarkable neuroprotective and regenerative abilities. Here, utilizing an optic nerve transection (ONT) injury and an RNA-seq-based approach, we identify genes and pathways active in RGCs that may modulate their survival. Through pharmacological perturbation, we demonstrate that Jak/Stat pathway activity is required for RGC survival after ONT. Furthermore, we show that immune responses directly contribute to RGC death after ONT; macrophages/microglia are recruited to the retina and blocking neuroinflammation or depleting these cells after ONT rescues survival of RGCs. Taken together, these data support a model in which crosstalk between macrophages/microglia and RGCs, mediated by Jak/Stat pathway activity, regulates RGC survival after optic nerve injury.
Assuntos
Imunidade Inata , Janus Quinases/imunologia , Traumatismos do Nervo Óptico/imunologia , Células Ganglionares da Retina/imunologia , Fatores de Transcrição STAT/imunologia , Transdução de Sinais/imunologia , Proteínas de Peixe-Zebra/imunologia , Peixe-Zebra/imunologia , Animais , Animais Geneticamente Modificados , Feminino , Janus Quinases/genética , Masculino , Traumatismos do Nervo Óptico/genética , Fatores de Transcrição STAT/genética , Transdução de Sinais/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
The retinal pigment epithelium (RPE) plays numerous critical roles in maintaining vision and this is underscored by the prevalence of degenerative blinding diseases like age-related macular degeneration (AMD), in which visual impairment is caused by progressive loss of RPE cells. In contrast to mammals, zebrafish possess the ability to intrinsically regenerate a functional RPE layer after severe injury. The molecular underpinnings of this regenerative process remain largely unknown yet hold tremendous potential for developing treatment strategies to stimulate endogenous regeneration in the human eye. In this study, we demonstrate that the mTOR pathway is activated in RPE cells post-genetic ablation. Pharmacological and genetic inhibition of mTOR activity impaired RPE regeneration, while mTOR activation enhanced RPE recovery post-injury, demonstrating that mTOR activity is essential for RPE regeneration in zebrafish. RNA-seq of RPE isolated from mTOR-inhibited larvae identified a number of genes and pathways dependent on mTOR activity at early and late stages of regeneration; amongst these were components of the immune system, which is emerging as a key regulator of regenerative responses across various tissue and model systems. Our results identify crosstalk between macrophages/microglia and the RPE, wherein mTOR activity is required for recruitment of macrophages/microglia to the RPE injury site. Macrophages/microglia then reinforce mTOR activity in regenerating RPE cells. Interestingly, the function of macrophages/microglia in maintaining mTOR activity in the RPE appeared to be inflammation-independent. Taken together, these data identify mTOR activity as a key regulator of RPE regeneration and link the mTOR pathway to immune responses in facilitating RPE regeneration.
Assuntos
Degeneração Macular , Epitélio Pigmentado da Retina , Animais , Degeneração Macular/genética , Degeneração Macular/metabolismo , Mamíferos/metabolismo , Regeneração/genética , Epitélio Pigmentado da Retina/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
Molecular insights into the selective vulnerability of retinal ganglion cells (RGCs) in optic neuropathies and after ocular trauma can lead to the development of novel therapeutic strategies aimed at preserving RGCs. However, little is known about what molecular contexts determine RGC susceptibility. In this study, we show the molecular mechanisms underlying the regional differential vulnerability of RGCs after optic nerve injury. We identified RGCs in the mouse peripheral ventrotemporal (VT) retina as the earliest population of RGCs susceptible to optic nerve injury. Mechanistically, the serotonin transporter (SERT) is upregulated on VT axons after injury. Utilizing SERT-deficient mice, loss of SERT attenuated VT RGC death and led to robust retinal axon regeneration. Integrin ß3, a factor mediating SERT-induced functions in other systems, is also upregulated in RGCs and axons after injury, and loss of integrin ß3 led to VT RGC protection and axon regeneration. Finally, RNA sequencing analyses revealed that loss of SERT significantly altered molecular signatures in the VT retina after optic nerve injury, including expression of the transmembrane protein, Gpnmb. GPNMB is rapidly downregulated in wild-type, but not SERT- or integrin ß3-deficient VT RGCs after injury, and maintaining expression of GPNMB in RGCs via AAV2 viruses even after injury promoted VT RGC survival and axon regeneration. Taken together, our findings demonstrate that the SERT-integrin ß3-GPNMB molecular axis mediates selective RGC vulnerability and axon regeneration after optic nerve injury.
Assuntos
Axônios , Regeneração Nervosa , Doenças do Sistema Nervoso/metabolismo , Células Ganglionares da Retina/citologia , Proteínas da Membrana Plasmática de Transporte de Serotonina/fisiologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Doenças do Sistema Nervoso/patologiaRESUMO
Loss of the retinal pigment epithelium (RPE) because of dysfunction or disease can lead to blindness in humans. Harnessing the intrinsic ability of the RPE to self-repair is an attractive therapeutic strategy; however, mammalian RPE is limited in its regenerative capacity. Zebrafish possess tremendous intrinsic regenerative potential in ocular tissues, including the RPE, but little is known about the mechanisms driving RPE regeneration. Here, utilizing transgenic and mutant zebrafish lines, pharmacological manipulations, transcriptomics, and imaging analyses, we identified elements of the immune response as critical mediators of intrinsic RPE regeneration. After genetic ablation, the RPE express immune-related genes, including leukocyte recruitment factors such as interleukin 34 We demonstrate that macrophage/microglia cells are responsive to RPE damage and that their function is required for the timely progression of the regenerative response. These data identify the molecular and cellular underpinnings of RPE regeneration and hold significant potential for translational approaches aimed toward promoting a pro-regenerative environment in mammalian RPE.
Assuntos
Cegueira/genética , Imunidade/genética , Interleucinas/genética , Regeneração/genética , Proteínas de Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Cegueira/parasitologia , Cegueira/terapia , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Microglia/metabolismo , Microglia/patologia , Mutação/genética , Epitélio Pigmentado da Retina/crescimento & desenvolvimento , Epitélio Pigmentado da Retina/patologia , Transcriptoma/genética , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
BACKGROUND: Pathogenic variants in human MAB21L2 result in microphthalmia, anophthalmia, and coloboma. The exact molecular function of MAB21L2 is currently unknown. We conducted a series of yeast two-hybrid (Y2H) experiments to determine protein interactomes of normal human and zebrafish MAB21L2/mab21l2 as well as human disease-associated variant MAB21L2-p.(Arg51Gly) using human adult retina and zebrafish embryo libraries. RESULTS: These screens identified klhl31, tnpo1, TNPO2/tnpo2, KLC2/klc2, and SPTBN1/sptbn1 as co-factors of MAB21L2/mab21l2. Several factors, including hspa8 and hspa5, were found to interact with MAB21L2-p.Arg51Gly but not wild-type MAB21L2/mab21l2 in Y2H screens. Further analyses via 1-by-1 Y2H assays, co-immunoprecipitation, and mass spectrometry revealed that both normal and variant MAB21L2 interact with HSPA5 and HSPA8. In situ hybridization detected co-expression of hspa5 and hspa8 with mab21l2 during eye development in zebrafish. Examination of zebrafish mutant hspa8hi138Tg identified reduced hspa8 expression associated with severe ocular developmental defects, including small eye, coloboma, and anterior segment dysgenesis. To investigate the effects of hspa8 deficiency on the mab21l2Arg51_Phe52del allele, corresponding zebrafish double mutants were generated and found to be more severely affected than single mutant lines. CONCLUSION: This study identifies heat shock proteins as interacting partners of MAB21L2/mab21l2 and suggests a role for this interaction in vertebrate eye development.
Assuntos
Coloboma , Anormalidades do Olho , Adulto , Animais , Humanos , Coloboma/patologia , Olho , Proteínas do Olho/metabolismo , Proteínas de Choque Térmico HSC70/genética , Peptídeos e Proteínas de Sinalização Intracelular , Retina/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
The zebrafish retina possesses tremendous regenerative potential. Müller glia underlie retinal regeneration through their ability to reprogram and generate multipotent neuronal progenitors that re-differentiate into lost neurons. Many factors required for Müller glia reprogramming and proliferation have been identified; however, we know little about the epigenetic and transcriptional regulation of these genes during regeneration. Here, we determined whether transcriptional regulation by members of the Bromodomain (Brd) family is required for Müller glia-dependent retinal regeneration. Our data demonstrate that three brd genes were expressed in Müller glia upon injury. brd2a and brd2b were expressed in all Müller glia and brd4 was expressed only in reprogramming Müller glia. Utilizing (+)-JQ1, a pharmacological inhibitor of Brd function, we demonstrate that transcriptional regulation by Brds plays a critical role in Müller glia reprogramming and regeneration. (+)-JQ1 treatment prevented cell cycle re-entry of Müller glia and the generation of neurogenic progenitors. Modulating the (+)-JQ1 exposure window, we identified the first 48 h post-injury as the time-period during which Müller glia reprogramming occurs. (+)-JQ1 treatments after 48 h post-injury had no effect on the re-differentiation of UV cones, indicating that Brd function is required only for Müller glia reprogramming and not subsequent specification/differentiation events. Brd inhibition also prevented the expression of reprogramming genes like ascl1a and lepb in Müller glia, but not effector genes like mmp9, nor did it affect microglial recruitment after injury. These results demonstrate that transcriptional regulation by Brds plays a critical role during Müller glia-dependent retinal regeneration in zebrafish.
RESUMO
For centuries, the eye has fascinated scientists and philosophers alike, and as a result the visual system has always been at the forefront of integrating cutting-edge technology in research. We are again at a turning point at which technical advances have expanded the range of organisms we can study developmentally and deepened what we can learn. In this new era, we are finally able to understand eye development in animals across the phylogenetic tree. In this Review, we highlight six areas in comparative visual system development that address questions that are important for understanding the developmental basis of evolutionary change. We focus on the opportunities now available to biologists to study the developmental genetics, cell biology and morphogenesis that underlie the incredible variation of visual organs found across the Metazoa. Although decades of important work focused on gene expression has suggested homologies and potential evolutionary relationships between the eyes of diverse animals, it is time for developmental biologists to move away from this reductive approach. We now have the opportunity to celebrate the differences and diversity in visual organs found across animal development, and to learn what it can teach us about the fundamental principles of biological systems and how they are built.
Assuntos
Morfogênese/fisiologia , Retina/citologia , Retina/metabolismo , Animais , Evolução Biológica , FilogeniaRESUMO
A crucial step in eye development is the closure of the choroid fissure (CF), a transient structure in the ventral optic cup through which vasculature enters the eye and ganglion cell axons exit. Although many factors have been identified that function during CF closure, the molecular and cellular mechanisms mediating this process remain poorly understood. Failure of CF closure results in colobomas. Recently, MITF was shown to be mutated in a subset of individuals with colobomas, but how MITF functions during CF closure is unknown. To address this issue, zebrafish with mutations in mitfa and tfec, two members of the Mitf family of transcription factors, were analyzed and their functions during CF closure determined. mitfa;tfec mutants possess severe colobomas and our data demonstrate that Mitf activity is required within cranial neural crest cells (cNCCs) during CF closure. In the absence of Mitf function, cNCC migration and localization in the optic cup are perturbed. These data shed light on the cellular mechanisms underlying colobomas in individuals with MITF mutations and identify a novel role for Mitf function in cNCCs during CF closure.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Corioide/citologia , Corioide/embriologia , Fator de Transcrição Associado à Microftalmia/metabolismo , Crista Neural/citologia , Crânio/citologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Coloboma/patologia , Embrião de Mamíferos/citologia , Humanos , Mutação/genética , Crista Neural/metabolismo , Epitélio Pigmentado da Retina/embriologiaRESUMO
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.
Assuntos
Degeneração Macular/genética , Regeneração/genética , Epitélio Pigmentado da Retina/crescimento & desenvolvimento , cis-trans-Isomerases/genética , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Apoptose/genética , Lâmina Basilar da Corioide/crescimento & desenvolvimento , Lâmina Basilar da Corioide/metabolismo , Diferenciação Celular/genética , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/genética , Humanos , Imidas/administração & dosagem , Larva/genética , Larva/crescimento & desenvolvimento , Degeneração Macular/patologia , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/patologia , Quinolinas/administração & dosagem , Retina/crescimento & desenvolvimento , Retina/patologia , Epitélio Pigmentado da Retina/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
BACKGROUND: Since the COVID-19 pandemic of 2020, there has been a marked rise in the use of telemedicine to evaluate patients after total knee arthroplasty (TKA). The purpose of our study was to assess a novel stem with an embedded sensor that can remotely and objectively monitor a patient's mobility after TKA. METHODS: A single anatomically designed knee system was implanted in concert with an interconnected tibial stem extension containing 3D accelerometers, 3D gyroscopes, a power source, and a telemetry transmission capability in 3 cadaveric pelvis to toe specimens. The legs were moved by hand to preset tibial positions at full knee extension, midflexion, flexion, and back to midflexion and extension for a total of 16 trials across 6 knees. RESULTS: Sensor data were successfully transmitted with good quality of signal to an external base station. Good correlation to the range of motion of the tibia was found (mean error 0.1 degrees; root mean square error 3.8 degrees). The signal from the heel drop tests suggests the sensor could detect heel strike during activities of daily living in vivo and the potential for additional signal processing to analyze vibratory and motion patterns detected by the sensors. A frequency domain analysis of a properly cemented and poorly cemented implant during the heel drop test suggests a difference in accelerometer signal in these implant states. CONCLUSION: The results confirm signals generated from an embedded TKA sensor can transmit through bone and cement, providing accurate range of motion data and may be capable of detecting changes in prosthesis fixation remotely.
Assuntos
Artroplastia do Joelho , COVID-19 , Prótese do Joelho , Atividades Cotidianas , Artroplastia do Joelho/efeitos adversos , Fenômenos Biomecânicos , COVID-19/prevenção & controle , Cadáver , Estudos de Viabilidade , Humanos , Articulação do Joelho/cirurgia , Monitorização Fisiológica , Pandemias , Amplitude de Movimento Articular , Tíbia/cirurgiaRESUMO
Lens abnormalities are a major cause of reduced vision and blindness. One mechanism that can lead to reduced lens transparency, i.e. cataract, is abnormal behavior of lens epithelial cells (LECs), the precursors of the transparent lens fiber cells. Here we describe a zebrafish mutation causing the embryonic lens epithelium to generate cellular masses comprising partially differentiated lens fiber cells. We identify the mutant gene as plod3, which encodes for Lysyl hydroxylase 3 (Lh3), an enzyme essential for modification of collagens, including Collagen IV, a main component of the lens capsule. We show that plod3-deficient lenses have abnormal lens epithelium from an early developmental stage, as well as abnormal lens capsules. Subsequently, upregulation of TGFß signaling takes place, which drives the formation of lens epithelial cellular masses. We identify a similar phenotype in Collagen IVα5-deficient embryos, suggesting a key role for the defective lens capsule in the pathogenesis. We propose that plod3 and col4a5 mutant zebrafish can serve as useful models for better understanding the biology of LECs during embryonic development and in formation of lens epithelium-derived cataract.
Assuntos
Glicosiltransferases/genética , Cápsula do Cristalino/embriologia , Cápsula do Cristalino/metabolismo , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/genética , Proteínas de Peixe-Zebra/genética , Actinas/genética , Actinas/metabolismo , Animais , Catarata/genética , Diferenciação Celular/fisiologia , Desenvolvimento Embrionário , Células Epiteliais/patologia , Epitélio/patologia , Glicosiltransferases/metabolismo , Cristalino/embriologia , Fenótipo , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
Opacification of the ocular lens, termed cataract, is a common cause of blindness. To become transparent, lens fiber cells undergo degradation of their organelles, including their nuclei, presenting a fundamental question: does signaling/transcription sufficiently explain differentiation of cells progressing toward compromised transcriptional potential? We report that a conserved RNA-binding protein Celf1 post-transcriptionally controls key genes to regulate lens fiber cell differentiation. Celf1-targeted knockout mice and celf1-knockdown zebrafish and Xenopus morphants have severe eye defects/cataract. Celf1 spatiotemporally down-regulates the cyclin-dependent kinase (Cdk) inhibitor p27Kip1 by interacting with its 5' UTR and mediating translation inhibition. Celf1 deficiency causes ectopic up-regulation of p21Cip1. Further, Celf1 directly binds to the mRNA of the nuclease Dnase2b to maintain its high levels. Together these events are necessary for Cdk1-mediated lamin A/C phosphorylation to initiate nuclear envelope breakdown and DNA degradation in fiber cells. Moreover, Celf1 controls alternative splicing of the membrane-organization factor beta-spectrin and regulates F-actin-crosslinking factor Actn2 mRNA levels, thereby controlling fiber cell morphology. Thus, we illustrate new Celf1-regulated molecular mechanisms in lens development, suggesting that post-transcriptional regulatory RNA-binding proteins have evolved conserved functions to control vertebrate oculogenesis.
Assuntos
Proteínas CELF1/fisiologia , Núcleo Celular/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/genética , Endodesoxirribonucleases/genética , Cristalino/crescimento & desenvolvimento , Proteínas de Ligação a RNA/fisiologia , Proteínas de Xenopus/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Linhagem Celular , Regulação da Expressão Gênica , Cristalino/citologia , Cristalino/metabolismo , Camundongos , Xenopus laevis , Peixe-ZebraRESUMO
DNA hydroxymethylation has recently been shown to play critical roles in regulating gene expression and terminal differentiation events in a variety of developmental contexts. However, little is known about its function during eye development. Methylcytosine dioxygenases of the Tet family convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), an epigenetic mark thought to serve as a precursor for DNA demethylation and as a stable mark in neurons. Here, we report a requirement for Tet activity during zebrafish retinal neurogenesis. In tet2-/-;tet3-/- mutants, retinal neurons are specified but most fail to terminally differentiate. While differentiation of the first born retinal neurons, the retinal ganglion cells (RGCs), is less affected in tet2-/-;tet3-/- mutants than other retinal cell types, the majority of RGCs do not undergo terminal morphogenesis and form axons. Moreover, the few photoreceptors that differentiate in tet2-/-;tet3-/- mutants fail to form outer segments, suggesting that Tet function is also required for terminal morphogenesis of differentiated retinal neurons. Mosaic analyses revealed a surprising cell non-autonomous requirement for tet2 and tet3 activity in facilitating retinal neurogenesis. Through a combination of candidate gene analysis, transcriptomics and pharmacological manipulations, we identified the Notch and Wnt pathways as cell-extrinsic pathways regulated by tet2 and tet3 activity during RGC differentiation and morphogenesis. Transcriptome analyses also revealed the ectopic expression of non-retinal genes in tet2-/-;tet3-/- mutant retinae, and this correlated with locus-specific reduction in 5hmC. These data provide the first evidence that Tet-dependent regulation of 5hmC formation is critical for retinal neurogenesis, and highlight an additional layer of complexity in the progression from retinal progenitor cell to differentiated retinal neuron during development of the vertebrate retina.
Assuntos
Metilação de DNA/genética , Dioxigenases/genética , Retina/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genética , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animais , Axônios/metabolismo , Diferenciação Celular/genética , Neurogênese/genética , Neurônios/metabolismo , Receptores Notch/genética , Retina/metabolismo , Transcriptoma/genética , Via de Sinalização Wnt/genética , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
BACKGROUND: Mutations in MAB21L2 result in severe ocular defects including microphthalmia, anophthalmia, coloboma, microcornea, and cataracts. The molecular and cellular underpinnings of these defects are unknown, as is the normal cellular function of MAB21L2. Zebrafish mab21l2 au10 mutants possess ocular defects resembling those in humans with MAB21L2 mutations, providing an excellent model to characterize mab21l2 functions during eye development. RESULTS: mab21l2 -/- mutants possessed a host of ocular defects including microphthalmia and colobomas as well as small, disorganized lenses and cornea dysgenesis. Decreased proliferation, increased cell death, and defects in marker gene expression were detected in the lens. Cell death in the optic stalk was elevated in mab21l2 -/- mutants and the basement membrane between the edges of the choroid fissure failed to break down. Neuronal differentiation in the retina was normal, however. mab21l2 -/- mutant corneas were disorganized, possessed an increased number of cells, some of which proliferated ectopically, and failed to differentiate the corneal stroma. CONCLUSIONS: mab21l2 function is required for morphogenesis and cell survival in the lens and optic cup, and basement membrane breakdown in the choroid fissure. mab21l2 function also regulates proliferation in the lens and cornea; in its absence, the lens is small and mispatterned, and corneal morphogenesis and patterning are also disrupted.
Assuntos
Anormalidades do Olho/genética , Proteínas do Olho/genética , Olho/crescimento & desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Peixe-Zebra/genética , Animais , Córnea , Desenvolvimento Embrionário , Olho/embriologia , Cristalino , Morfogênese , Proteínas Mutantes/genética , Peixe-Zebra/anatomia & histologia , Peixe-Zebra/embriologiaRESUMO
Defects in choroid fissure (CF) formation and closure lead to coloboma, a major cause of childhood blindness. Despite genetic advances, the cellular defects underlying coloboma remain poorly elucidated due to our limited understanding of normal CF morphogenesis. We address this deficit by conducting high-resolution spatio-temporal analyses of CF formation and closure in the chick, mouse and fish. We show that a small ventral midline invagination initiates CF formation in the medial-proximal optic cup, subsequently extending it dorsally toward the lens, and proximally into the optic stalk. Unlike previously supposed, the optic disc does not form solely as a result of this invagination. Morphogenetic events that alter the shape of the proximal optic cup also direct clusters of outer layer and optic stalk cells to form dorsal optic disc. A cross-species comparison suggests that CF closure can be accomplished by breaking down basement membranes (BM) along the CF margins, and by establishing BM continuity along the dorsal and ventral surfaces of the CF. CF closure is subsequently accomplished via two distinct mechanisms: tissue fusion or the intercalation of various tissues into the inter-CF space. We identify several novel cell behaviors that underlie CF fusion, many of which involve remodeling of the retinal epithelium. In addition to BM disruption, these include NCAD downregulation along the SOX2+ retinal CF margin, and the protrusion or movement of partially polarized retinal cells into the inter-CF space to mediate fusion. Proximally, the inter-CF space does not fuse or narrow and is instead loosely packed with migrating SOX2+/PAX2+/Vimentin+ astrocytes until it is closed by the outgoing optic nerve. Taken together, our results highlight distinct proximal-distal differences in CF morphogenesis and closure and establish detailed cellular models that can be utilized for understanding the genetic bases of coloboma.
Assuntos
Corioide/embriologia , Coloboma/embriologia , Coloboma/fisiopatologia , Animais , Embrião de Galinha , Corioide/fisiologia , Coloboma/genética , Olho/embriologia , Camundongos/embriologia , Morfogênese/fisiologia , Disco Óptico/embriologia , Retina/embriologia , Análise Espaço-Temporal , Peixe-Zebra/embriologiaRESUMO
Photoreception is a ubiquitous sensory ability found across the Metazoa, and photoreceptive organs are intricate and diverse in their structure. Although the morphology of the compound eye in Drosophila and the single-chambered eye in vertebrates have elaborated independently, the amount of conservation within the 'eye' gene regulatory network remains controversial, with few taxa studied. To better understand the evolution of photoreceptive organs, we established the cephalopod Doryteuthis pealeii as a lophotrochozoan model for eye development. Utilizing histological, transcriptomic and molecular assays, we characterize eye formation in Doryteuthis pealeii Through lineage tracing and gene expression analyses, we demonstrate that cells expressing Pax and Six genes incorporate into the lens, cornea and iris, and the eye placode is the sole source of retinal tissue. Functional assays demonstrate that Notch signaling is required for photoreceptor cell differentiation and retinal organization. This comparative approach places the canon of eye research in traditional models into perspective, highlighting complexity as a result of both conserved and convergent mechanisms.
Assuntos
Cefalópodes/embriologia , Cefalópodes/metabolismo , Olho/embriologia , Olho/metabolismo , Células Fotorreceptoras/citologia , Células Fotorreceptoras/metabolismo , Animais , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Morfogênese/genética , Morfogênese/fisiologiaRESUMO
A critical aspect of vertebrate eye development is closure of the choroid fissure (CF). Defects in CF closure result in colobomas, which are a significant cause of childhood blindness worldwide. Despite the growing number of mutated loci associated with colobomas, we have a limited understanding of the cell biological underpinnings of CF closure. Here, we utilize the zebrafish embryo to identify key phases of CF closure and regulators of the process. Utilizing Laminin-111 as a marker for the basement membrane (BM) lining the CF, we determine the spatial and temporal patterns of BM breakdown in the CF, a prerequisite for CF closure. Similarly, utilizing a combination of in vivo time-lapse imaging, ß-catenin immunohistochemistry and F-actin staining, we determine that tissue fusion, which serves to close the fissure, follows BM breakdown closely. Periocular mesenchyme (POM)-derived endothelial cells, which migrate through the CF to give rise to the hyaloid vasculature, possess distinct actin foci that correlate with regions of BM breakdown. Disruption of talin1, which encodes a regulator of the actin cytoskeleton, results in colobomas and these correlate with structural defects in the hyaloid vasculature and defects in BM breakdown. cloche mutants, which entirely lack a hyaloid vasculature, also possess defects in BM breakdown in the CF. Taken together, these data support a model in which the hyaloid vasculature and/or the POM-derived endothelial cells that give rise to the hyaloid vasculature contribute to BM breakdown during CF closure.
Assuntos
Corioide/embriologia , Artéria Oftálmica/embriologia , Animais , Membrana Basal/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Corioide/irrigação sanguínea , Corioide/ultraestrutura , Coloboma/embriologia , Coloboma/genética , Mesoderma/fisiologia , Microinjeções , RNA Mensageiro/genética , Talina/deficiência , Talina/genética , Talina/fisiologia , Imagem com Lapso de Tempo , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologiaRESUMO
BACKGROUND: Rho GTPases are small monomeric G-proteins that play key roles in many cellular processes. Due to Rho GTPases' widespread expression and broad functions, analyses of their function during late development require tissue-specific modulation of activity. The GAL4/UAS system provides an excellent tool for investigating the function of Rho GTPases in vivo. With this in mind, we created a transgenic tool kit enabling spatial and temporal modulation of Rho GTPase activity in zebrafish. RESULTS: Transgenic constructs were assembled driving dominant-negative, constitutively active, and wild-type versions of Cdc42, RhoA, and Rac1 under 10XUAS control. The self-cleaving viral peptide F2A was utilized to allow bicistronic expression of a fluorescent reporter and Rho GTPase. Global heat shock of hsp70l:gal4(+) transgenic embryos confirmed GAL4-specific construct expression. Western blot analysis indicated myc-tagged Rho GTPases were expressed only in the presence of GAL4. Construct expression was confined to proper cells when combined with pou4f3:gal4 or ptf1a:gal4. Finally, transgene expression resulted in reproducible defects in lens formation, indicating that the transgenes are functional in vivo. CONCLUSIONS: We generated and validated 10 transgenic lines, creating a versatile tool kit for the temporal-spatial modulation of Cdc42, RhoA, and Rac1 activity in vivo. These lines will enable systematic analysis of Rho GTPase function in any tissue of interest. Developmental Dynamics 245:844-853, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Ligação a DNA/genética , Fatores de Transcrição/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Two vascular networks nourish the embryonic eye as it develops - the hyaloid vasculature, located at the anterior of the eye between the retina and lens, and the choroidal vasculature, located at the posterior of the eye, surrounding the optic cup. Little is known about hyaloid development and morphogenesis, however. To begin to identify the morphogenetic underpinnings of hyaloid formation, we utilized in vivo time-lapse confocal imaging to characterize morphogenesis of the zebrafish hyaloid through 5 days post fertilization (dpf). Our data segregate hyaloid formation into three distinct morphogenetic stages: Stage I: arrival of hyaloid cells at the lens and formation of the hyaloid loop; Stage II: formation of a branched hyaloid network; Stage III: refinement of the hyaloid network. Utilizing fixed and dissected tissues, distinct Stage II and Stage III aspects of hyaloid formation were quantified over time. Combining in vivo imaging with microangiography, we demonstrate that the hyaloid system becomes fully enclosed by 5dpf. To begin to identify the molecular and cellular mechanisms underlying hyaloid morphogenesis, we identified a recessive mutation in the mab21l2 gene, and in a subset of mab21l2 mutants the lens does not form. Utilizing these "lens-less" mutants, we determined whether the lens was required for hyaloid morphogenesis. Our data demonstrate that the lens is not required for Stage I of hyaloid formation; however, Stages II and III of hyaloid formation are disrupted in the absence of a lens, supporting a role for the lens in hyaloid maturation and maintenance. Taken together, this study provides a foundation on which the cellular, molecular and embryologic mechanisms underlying hyaloid morphogenesis can be elucidated.