RESUMO
Establishing a functional circulatory system is required for post-implantation development during murine embryogenesis. Previous studies in loss-of-function mouse models showed that FOXO1, a Forkhead family transcription factor, is required for yolk sac (YS) vascular remodeling and survival beyond embryonic day (E) 11. Here, we demonstrate that at E8.25, loss of Foxo1 in Tie2-cre expressing cells resulted in increased sprouty 2 (Spry2) and Spry4 expression, reduced arterial gene expression and reduced Kdr (also known as Vegfr2 and Flk1) transcripts without affecting overall endothelial cell identity, survival or proliferation. Using a Dll4-BAC-nlacZ reporter line, we found that one of the earliest expressed arterial genes, delta like 4, is significantly reduced in Foxo1 mutant YS without being substantially affected in the embryo proper. We show that FOXO1 binds directly to previously identified Spry2 gene regulatory elements (GREs) and newly identified, evolutionarily conserved Spry4 GREs to repress their expression. Furthermore, overexpression of Spry4 in transient transgenic embryos largely recapitulates the reduced expression of arterial genes seen in conditional Foxo1 mutants. Together, these data reveal a novel role for FOXO1 as a key transcriptional repressor regulating both pre-flow arterial specification and subsequent vessel remodeling within the murine YS.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Remodelação Vascular , Saco Vitelino , Animais , Artérias , Embrião de Mamíferos/metabolismo , Células Endoteliais/metabolismo , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Camundongos , Remodelação Vascular/genética , Saco Vitelino/metabolismoRESUMO
As with all glial cells, the major role of retinal Müller glia (MG) is to provide essential neuronal support. However, the MG of some non-mammalian species have the additional ability to generate new retinal neurons capable of sight restoration. Unfortunately, mammalian MG do not possess this ability. However, if we could understand the reasons why, we may be able to devise strategies to confer regenerative potential. The recent discovery that the Hippo signaling pathway acts as an intrinsic block to mammalian MG proliferation, along with reports of adeno-associated virus (AAV)-based MG reprogramming and functional photoreceptor differentiation, may indicate a watershed moment in the field of mammalian retinal regeneration. However, as researchers delve deeper into the cellular and molecular mechanisms, and further refine MG reprogramming strategies, we should recall past misinterpretations of data in this field and proceed with caution. Here, we provide a summary of these emerging data and a discussion of technical concerns specific to AAV-mediated reprogramming experiments that must be addressed in order for the field to move forward.
Assuntos
Proliferação de Células , Técnicas de Reprogramação Celular , Reprogramação Celular , Células Ependimogliais/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Regeneração , Animais , Dependovirus , Vetores Genéticos , HumanosRESUMO
Combined methylmalonic acidemia and homocystinuria, cblC type, is the most common inherited disorder of cobalamin metabolism and is characterized by severe fetal developmental defects primarily impacting the central nervous system, hematopoietic system, and heart. CblC was previously shown to be due to mutations in the MMACHC gene, which encodes a protein thought to function in intracellular cobalamin trafficking and biosynthesis of adenosylcobalamin (AdoCbl) and methylcobalamin (MeCbl). These coenzymes are required for the production of succinyl-CoA and methionine, respectively. However, it is currently unclear whether additional roles for MMACHC exist outside of cobalamin metabolism. Furthermore, due to a lack of sufficient animal models, the exact pathophysiology of cblC remains unknown. Here, we report the generation and characterization of two new mouse models to study the role of MMACHC in vivo. CRISPR/Cas9 genome editing was used to develop a Mmachc floxed allele (Mmachcflox/flox), which we validated as a conditional null. For a gain-of-function approach, we generated a transgenic mouse line that over-expresses functional Mmachc (Mmachc-OE+/tg) capable of rescuing Mmachc homozygous mutant lethality. Surprisingly, our data also suggest that these mice may exhibit a partially penetrant maternal-effect rescue, which might have implications for in utero therapeutic interventions to treat cblC. Both the Mmachcflox/flox and Mmachc-OE+/tg mouse models will be valuable resources for understanding the biological roles of MMACHC in a variety of tissue contexts and allow for deeper understanding of the pathophysiology of cblC.
Assuntos
Homocistinúria , Oxirredutases , Deficiência de Vitamina B 12/congênito , Animais , Modelos Animais de Doenças , Homocistinúria/genética , Homocistinúria/metabolismo , Homocistinúria/patologia , Homocistinúria/fisiopatologia , Camundongos , Camundongos Transgênicos , Oxirredutases/genética , Oxirredutases/metabolismo , Deficiência de Vitamina B 12/genética , Deficiência de Vitamina B 12/metabolismo , Deficiência de Vitamina B 12/patologia , Deficiência de Vitamina B 12/fisiopatologiaRESUMO
Macrophages are well characterized as immune cells. However, in recent years, a multitude of non-immune functions have emerged many of which play essential roles in a variety of developmental processes (Wynn et al., 2013; DeFalco et al., 2014). In adult animals, macrophages are derived from circulating monocytes originating in the bone marrow, but much of the tissue-resident population arise from erythro-myeloid progenitors (EMPs) in the extra-embryonic yolk sac, appearing around the same time as primitive erythroblasts (Schulz et al., 2012; Kierdorf et al., 2013; McGrath et al., 2015; Gomez Perdiguero et al., 2015; Mass et al., 2016). Of particular interest to our group, macrophages have been shown to act as pro-angiogenic regulators during development (Wynn et al., 2013; DeFalco et al., 2014; Hsu et al., 2015), but there is still much to learn about these early cells. The goal of the present study was to isolate and expand progenitors of yolk-sac-derived Embryonic Macrophages (EMs) in vitro to generate a new platform for mechanistic studies of EM differentiation. To accomplish this goal, we isolated pure (>98%) EGFP+ populations by flow cytometry from embryonic day 9.5 (E9.5) Csf1r-EGFP+/tg mice, then evaluated the angiogenic potential of EMs relative to Bone Marrow-Derived Macrophages (BMDMs). We found that EMs expressed more pro-angiogenic and less pro-inflammatory macrophage markers than BMDMs. EMs also promoted more endothelial cell (EC) cord formation in vitro, as compared to BMDMs in a manner that required direct cell-to-cell contact. Importantly, EMs preferentially matured into microglia when co-cultured with mouse Neural Stem/Progenitor Cells (NSPCs). In conclusion, we have established a protocol to isolate and propagate EMs in vitro, have further defined specialized properties of yolk-sac-derived macrophages, and have identified EM-EC and EM-NSPC interactions as key inducers of EC tube formation and microglial cell maturation, respectively.
Assuntos
Células Precursoras Eritroides/fisiologia , Macrófagos/fisiologia , Células Progenitoras Mieloides/fisiologia , Animais , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Técnicas de Cocultura/métodos , Citometria de Fluxo/métodos , Células-Tronco Hematopoéticas/fisiologia , Macrófagos/citologia , Camundongos/embriologia , Fenótipo , Saco Vitelino/citologiaRESUMO
Mitochondria are incredibly dynamic organelles that undergo continuous fission and fusion events to control morphology, which profoundly impacts cell physiology including cell cycle progression. This is highlighted by the fact that most major human neurodegenerative diseases are due to specific disruptions in mitochondrial fission or fusion machinery and null alleles of these genes result in embryonic lethality. To gain a better understanding of the pathophysiology of such disorders, tools for the in vivo assessment of mitochondrial dynamics are required. It would be particularly advantageous to simultaneously image mitochondrial fission-fusion coincident with cell cycle progression. To that end, we have generated a new transgenic reporter mouse, called mito::mKate2 that ubiquitously expresses a mitochondria localized far-red mKate2 fluorescent protein. Here we show that mito::mKate2 mice are viable and fertile and that mKate2 fluorescence can be spectrally separated from the previously developed Fucci cell cycle reporters. By crossing mito::mKate2 mice to the ROSA26R-mTmG dual fluorescent Cre reporter line, we also demonstrate the potential utility of mito::mKate2 for genetic mosaic analysis of mitochondrial phenotypes.
Assuntos
Cruzamentos Genéticos , Genes Reporter , Camundongos Transgênicos , Dinâmica Mitocondrial/genética , Animais , Ciclo Celular/genética , Feminino , Imunofluorescência , Expressão Gênica , Vetores Genéticos , Proteínas Luminescentes/genética , Masculino , Camundongos , Mosaicismo , Fenótipo , Proteína Vermelha FluorescenteRESUMO
Programmed capillary regression and remodeling are essential developmental processes. However, the cellular and molecular mechanisms that regulate vessel regression are only the beginning to be understood. Here, using in vivo, dynamic, confocal imaging of mouse transgenic reporters as well as static confocal and electron microscopy, we studied the embryonic development and postnatal regression of the transient mouse pupillary membrane (PM) vasculature. This approach allowed us to directly observe the precise temporal sequence of cellular events preceding and during the elimination of the PM from the mouse eye. Imaging of Tcf/Lef-H2B::GFP Wnt-reporter mice uncovered that, unlike the hyaloid vasculature of the posterior eye, a PM endothelial cell (EC) Wnt/ß-catenin response is unlikely to be part of the regression mechanism. Live imaging of EC and macrophage dynamics revealed highly active Csf1r-GFP+ macrophages making direct contact with the Flk1-myr::mCherry+ vessel surface and with membrane protrusions or filopodia extending from the ECs. Flk1-myr::mCherry+ EC membrane particles were observed on and around ECs as well as within macrophages. Electron microscopy studies confirmed that they were in phagosomes within macrophages, indicating that the macrophages engulfed the membrane particles. Interestingly, EC plasma membrane uptake by PM macrophages did not correlate with apoptosis and was found shortly after vessel formation at mid-gestation stages in the embryo; long before vessel regression begins during postnatal development. Additionally, genetic ablation of macrophages showed that EC membrane particles were still shed in the absence of macrophages suggesting that macrophages do not induce the formation or release of EC microparticles. These studies have uncovered a novel event during programmed capillary regression in which resident macrophages scavenge endothelial cell microparticles released from the PM vessels. This finding suggests that there may be an initial disruption in vessel homeostasis embryonically as the PM forms that may underlie its ultimate regression postnatally.
Assuntos
Micropartículas Derivadas de Células/imunologia , Olho/irrigação sanguínea , Macrófagos/imunologia , Fagocitose/imunologia , Pupila/fisiologia , Animais , Capilares , Membrana Celular/fisiologia , Extensões da Superfície Celular/metabolismo , Células Endoteliais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMO
The present study examined the consequences of eliminating horizontal cells from the outer retina during embryogenesis upon the organization and assembly of the outer plexiform layer (OPL). Retinal horizontal cells exhibit a migration defect in Lim1-conditional knock-out (Lim1-CKO) mice and become mispositioned in the inner retina before birth, redirecting their dendrites into the inner plexiform layer. The resultant (mature) OPL, developing in the absence of horizontal cells, shows a retraction of rod spherules into the outer nuclear layer and a sprouting of rod bipolar cell dendrites to reach ectopic ribbon-protein puncta. Cone pedicles and the dendrites of type 7 cone bipolar cells retain their characteristic stratification and colocalization within the collapsed OPL, although both are atrophic and the spatial distribution of the pedicles is disrupted. Developmental analysis of Lim1-CKO retina reveals that components of the rod and cone pathways initially co-assemble within their normal strata in the OPL, indicating that horizontal cells are not required for the correct targeting of photoreceptor terminals or bipolar cell dendrites. As the rod spherules begin to retract during the second postnatal week, rod bipolar cells initially show no signs of ectopic growth, sprouting only subsequently and continuing to do so well after the eighth postnatal week. These results demonstrate the critical yet distinctive roles for horizontal cells on the rod and cone pathways and highlight a unique and as-yet-unrecognized maintenance function of an inhibitory interneuron that is not required for the initial targeting and co-stratification of other components in the circuit.
Assuntos
Plasticidade Neuronal/fisiologia , Células Horizontais da Retina/fisiologia , Neurônios Retinianos/fisiologia , Vias Visuais/crescimento & desenvolvimento , Animais , Feminino , Camundongos , Camundongos Transgênicos , Sinapses/metabolismo , Vias Visuais/fisiologiaRESUMO
Neuronal populations display conspicuous variability in their size among individuals, but the genetic sources of this variation are largely undefined. We demonstrate a large and highly heritable variation in neuron number within the mouse retina, affecting a critical population of interneurons, the horizontal cells. Variation in the size of this population maps to the distal end of chromosome (Chr) 13, a region homologous to human Chr 5q11.1-11.2. This region contains two genes known to modulate retinal cell number. Using conditional knock-out mice, we demonstrate that one of these genes, the LIM homeodomain gene Islet-1 (Isl1), plays a role in regulating horizontal cell number. Genetic differences in Isl1 expression are high during the period of horizontal cell production, and cis-regulation of Isl1 expression within the retina is demonstrated directly. We identify a single nucleotide polymorphism in the 5' UTR of Isl1 that creates an E-box sequence as a candidate causal variant contributing to this variation in horizontal cell number.
Assuntos
Embrião de Mamíferos/metabolismo , Proteínas de Homeodomínio/genética , Retina/metabolismo , Regiões 5' não Traduzidas/genética , Animais , Contagem de Células , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Feminino , Imunofluorescência , Folistatina/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas com Homeodomínio LIM , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout , Camundongos Transgênicos , Polimorfismo de Nucleotídeo Único , Gravidez , Regiões Promotoras Genéticas/genética , Locos de Características Quantitativas/genética , Retina/citologia , Retina/embriologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Fatores de TranscriçãoRESUMO
ß2-glycoprotein I (ß2-Gp1) is a cardiolipin-binding plasma glycoprotein. It is evolutionarily conserved from invertebrates, and cardiolipin-bound ß2-Gp1 is a major target of antiphospholipid antibodies seen in autoimmune disorders. Cardiolipin is almost exclusively present in mitochondria, and mitochondria are present in circulating blood. We show that ß2-Gp1 binds to cell-free mitochondria (CFM) in the circulation and promotes its phagocytosis by macrophages at physiological plasma concentrations. Exogenous CFM had a short circulation time of less than 10 minutes in mice. Following infusion of CFM, ß2-Gp1-deficient mice had significantly higher levels of transfused mitochondria at 5 minutes (9.9 ± 6.4 pg/ml versus 4.0 ± 2.3 pg/ml in wildtype, p = 0.01) and at 10 minutes (3.0 ± 3.6 pg/ml versus 1.0 ± 0.06 pg/ml in wild-type, p = 0.033, n = 10). In addition, the splenic macrophages had less phagocytosed CFM in ß2-Gp1-deficient mice (24.4 ± 2.72% versus 35.6 ± 3.5 in wild-type, p = 0.001, n = 5). A patient with abnormal ß2-Gp1, unable to bind cardiolipin, has increased CFM in blood (5.09 pg/ml versus 1.26 ± 1.35 in normal) and his plasma induced less phagocytosis of CFM by macrophages (47.3 ± 1.6% versus 54.3 ± 1.3, p = 0.01) compared to normal plasma. These results show the evolutionarily conserved ß2-Gp1 is one of the mediators of the clearance of CFM in circulation.
Assuntos
Síndrome Antifosfolipídica , Cardiolipinas , Humanos , Animais , Camundongos , beta 2-Glicoproteína I , Cardiolipinas/metabolismo , Anticorpos Antifosfolipídeos , Macrófagos/metabolismo , FagocitoseRESUMO
Unidirectional airflow in the avian lung enables gas exchange during both inhalation and exhalation. The underlying developmental process and how it deviates from that of the bidirectional mammalian lung are poorly understood. Sampling key developmental stages with multiscale 3D imaging and single-cell transcriptomics, we delineate morphogenic, molecular, and cellular features that accommodate the unidirectional airflow in the chicken lung. Primary termini of hyper-elongated branches are eliminated via proximal-short and distal-long fusions, forming parabronchi. Neoform termini extend radially through parabronchial smooth muscle to form gas-exchanging alveoli. Supporting this radial alveologenesis, branch stalks halt their proximalization, defined by SOX9-SOX2 transition, and become SOX9 low parabronchi. Primary and secondary vascular plexi interface with primary and neoform termini, respectively. Single-cell and Stereo-seq spatial transcriptomics reveal a third, chicken-specific alveolar cell type expressing KRT14, hereby named luminal cells. Luminal, alveolar type 2, and alveolar type 1 cells sequentially occupy concentric zones radiating from the parabronchial lumen. Our study explores the evolutionary space of lung diversification and lays the foundation for functional analysis of species-specific genetic determinants.
RESUMO
Combined methylmalonic acidemia and homocystinuria (cblC) is the most common inborn error of intracellular cobalamin metabolism and due to mutations in Methylmalonic Aciduria type C and Homocystinuria (MMACHC). Recently, mutations in the transcriptional regulators HCFC1 and RONIN (THAP11) were shown to result in cellular phenocopies of cblC. Since HCFC1/RONIN jointly regulate MMACHC, patients with mutations in these factors suffer from reduced MMACHC expression and exhibit a cblC-like disease. However, additional de-regulated genes and the resulting pathophysiology is unknown. Therefore, we have generated mouse models of this disease. In addition to exhibiting loss of Mmachc, metabolic perturbations, and developmental defects previously observed in cblC, we uncovered reduced expression of target genes that encode ribosome protein subunits. We also identified specific phenotypes that we ascribe to deregulation of ribosome biogenesis impacting normal translation during development. These findings identify HCFC1/RONIN as transcriptional regulators of ribosome biogenesis during development and their mutation results in complex syndromes exhibiting aspects of both cblC and ribosomopathies.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Homocistinúria/genética , Fator C1 de Célula Hospedeira/genética , Oxirredutases/genética , Proteínas Repressoras/genética , Ribossomos/genética , Deficiência de Vitamina B 12/genética , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/patologia , Animais , Modelos Animais de Doenças , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Homocistinúria/metabolismo , Homocistinúria/patologia , Fator C1 de Célula Hospedeira/deficiência , Humanos , Masculino , Camundongos , Camundongos Knockout , Mutação , Biogênese de Organelas , Oxirredutases/deficiência , Biossíntese de Proteínas , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Repressoras/deficiência , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Ribossomos/patologia , Vitamina B 12/metabolismo , Deficiência de Vitamina B 12/metabolismo , Deficiência de Vitamina B 12/patologiaRESUMO
OBJECTIVE: Classical measures of vessel morphology, including diameter and density, are employed to study microvasculature in endothelial membrane labeled mice. These measurements prove sufficient for some studies; however, they are less well suited for quantifying changes in microcirculatory networks lacking hierarchical structure. We demonstrate that automated multifractal analysis and lacunarity may be used with classical methods to quantify microvascular morphology. METHODS: Using multifractal analysis and lacunarity, we present an automated extraction tool with a processing pipeline to characterize 2D representations of 3D microvasculature. We apply our analysis on four tissues and the hyaloid vasculature during remodeling. RESULTS: We found that the vessel networks analyzed have multifractal geometries and that kidney microvasculature has the largest fractal dimension and the lowest lacunarity compared to microvasculature networks in the cortex, skin, and thigh muscle. Also, we found that, during hyaloid remodeling, there were differences in multifractal spectra reflecting the functional transition from a space filling vasculature which nurtures the lens to a less dense vasculature as it regresses, permitting unobstructed vision. CONCLUSION: Multifractal analysis and lacunarity are valuable additions to classical measures of vascular morphology and will have utility in future studies of normal, developing, and pathological tissues.
Assuntos
Microvasos/anatomia & histologia , Modelos Anatômicos , Modelos Cardiovasculares , Algoritmos , Animais , Córtex Cerebral/irrigação sanguínea , Fractais , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional/estatística & dados numéricos , Rim/irrigação sanguínea , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Músculos/irrigação sanguínea , Vasos Retinianos/anatomia & histologia , Pele/irrigação sanguínea , SoftwareRESUMO
Cone photoreceptors detect light and are responsible for color vision. These cells display a distinct polarized morphology where nuclei are precisely aligned in the apical retina. However, little is known about the mechanisms involved in cone nuclear positioning or the impact of this organization on retina function. We show that the serine/threonine kinase LKB1 and one of its substrates, AMPK, regulate cone nuclear positioning. In the absence of either molecule, cone nuclei are misplaced along the axon, resulting in altered nuclear lamination. LKB1 is required specifically in cones to mediate this process, and disruptions in nuclear alignment result in reduced cone function. Together, these results identify molecular determinants of cone nuclear position and indicate that cone nuclear position alignment enables proper visual function.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Visão Ocular/fisiologia , Animais , CamundongosRESUMO
The highly vascularized mouse eye is an excellent model system in which to elucidate the molecular genetic basis of blood vessel development and disease. However, the analysis of ocular vessel defects has traditionally been derived from fixed tissue, which fails to account for dynamic events such as blood flow and cell migration. To overcome the limitations of static analysis, tremendous advances in imaging technology and fluorescent protein reporter mouse lines now enable the direct visualization of developing cells in vivo. Here, we demonstrate that the Flk1-myr::mCherry transgenic mouse is an extremely useful live reporter with broad applicability to retinal, hyaloid, and choroid vascular research.
Assuntos
Olho/irrigação sanguínea , Vasos Retinianos/embriologia , Vasos Retinianos/patologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Animais , Olho/patologia , Imunofluorescência , Técnicas In Vitro , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Reação em Cadeia da PolimeraseRESUMO
Live fluorescent microscopy of whole-mount rodent retinal explants has proved to be extremely valuable for understanding dynamic events during retinogenesis. However, to obtain three-dimensional images with high-quality axial resolution, investigators are restricted to specific areas of the retina and require microscopes, such as two photon, with a higher level of depth penetrance. As an alternative, we report a retinal live-imaging protocol using slice cultures that are suitable for capturing discrete cellular events during retinal development and differentiation. This is a significant improvement upon current methods, as it is more amenable to a wider array of imaging systems and does not compromise resolution of retinal cross-sectional area.
Assuntos
Microscopia de Fluorescência/métodos , Retina/citologia , Animais , Diferenciação Celular/fisiologia , Testes Diagnósticos de Rotina/métodos , Imageamento Tridimensional/métodos , Camundongos , Técnicas de Cultura de Órgãos/métodosRESUMO
SIGNIFICANCE: The retina is critical for vision, and several diseases may alter its biomechanical properties. However, assessing the biomechanical properties of the retina nondestructively is a challenge due to its fragile nature and location within the eye globe. Advancements in Brillouin spectroscopy have provided the means for nondestructive investigations of retina biomechanical properties. AIM: We assessed the biomechanical properties of mouse retinas using Brillouin microscopy noninvasively and showed the potential of Brillouin microscopy to differentiate the type and layers of retinas based on stiffness. APPROACH: We used Brillouin microscopy to quantify stiffness of fresh and paraformaldehyde (PFA)-fixed retinas. As further proof-of-concept, we demonstrated a change in the stiffness of a retina with N-methyl-D-aspartate (NMDA)-induced damage, compared to an undamaged sample. RESULTS: We found that the retina layers with higher cell body density had higher Brillouin modulus compared to less cell-dense layers. We have also demonstrated that PFA-fixed retina samples were stiffer compared with fresh samples. Further, NMDA-induced neurotoxicity leads to retinal ganglion cell (RGC) death and reactive gliosis, increasing the stiffness of the RGC layer. CONCLUSION: Brillouin microscopy can be used to characterize the stiffness distribution of the layers of the retina and can be used to differentiate tissue at different conditions based on biomechanical properties.
Assuntos
Microscopia , Retina , Animais , Camundongos , N-Metilaspartato , Células Ganglionares da Retina , Visão OcularRESUMO
In response to retinal damage, the Müller glial cells (MGs) of the zebrafish retina have the ability to undergo a cellular reprogramming event in which they enter the cell cycle and divide asymmetrically, thereby producing multipotent retinal progenitors capable of regenerating lost retinal neurons. However, mammalian MGs do not exhibit such a proliferative and regenerative ability. Here, we identify Hippo pathway-mediated repression of the transcription cofactor YAP as a core regulatory mechanism that normally blocks mammalian MG proliferation and cellular reprogramming. MG-specific deletion of Hippo pathway components Lats1 and Lats2, as well as transgenic expression of a Hippo non-responsive form of YAP (YAP5SA), resulted in dramatic Cyclin D1 upregulation, loss of adult MG identity, and attainment of a highly proliferative, progenitor-like cellular state. Our results reveal that mammalian MGs may have latent regenerative capacity that can be stimulated by repressing Hippo signaling.
Assuntos
Reprogramação Celular , Células Ependimogliais/citologia , Células Ependimogliais/enzimologia , Mamíferos/metabolismo , Neuroglia/citologia , Neuroglia/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Retina/citologia , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Ciclina D1/metabolismo , Ciclina D3/metabolismo , Via de Sinalização Hippo , Camundongos , Células-Tronco/metabolismo , Proteínas de Sinalização YAPRESUMO
Although much is known about the transcriptional regulation that coordinates retinal cell fate determination, very little is known about the developmental processes that establish the characteristic laminar architecture of the retina, in particular, the specification of neuronal positioning. The LIM class homeodomain transcription factor Lim1 (Lhx1) is expressed in postmitotic, differentiating, and mature retinal horizontal cells. We show that conditional ablation of Lim1 results in the ectopic localization of horizontal cells to inner aspects of the inner nuclear layer, among the retinal amacrine cells. The ectopic cells maintain a molecular phenotype consistent with horizontal cell identity; however, these neurons adopt a unique morphology more reminiscent of amacrine cells, including a dendritic arbor positioned within the inner plexiform layer. All other retinal cell populations appear unaltered. Our data suggest a model whereby Lim1 lies downstream of horizontal cell fate determination factors and functions cell autonomously to instruct differentiating horizontal cells to the appropriate laminar position in the developing retina. This study is the first to describe a cell type-specific genetic program that is essential for targeting a discrete retinal neuron population to the proper lamina.
Assuntos
Proteínas de Homeodomínio/fisiologia , Células Horizontais da Retina/embriologia , Células Horizontais da Retina/metabolismo , Sequência de Aminoácidos , Animais , Diferenciação Celular/genética , Movimento Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Proteínas com Homeodomínio LIM , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Dados de Sequência Molecular , Células Horizontais da Retina/citologia , Fatores de TranscriçãoRESUMO
Retinal neurons extend their dendritic fields to achieve a degree of dendritic overlap with homotypic neighbors that is cell-type specific. How these neurons regulate their dendritic growth is unclear. The dendritic field of a retinal horizontal cell varies inversely with horizontal cell density across different strains of mice, suggesting that proximity to neighboring cells regulates dendritic growth. To test this directly, we have employed the Cre-loxP conditional gene targeting strategy to achieve inactivation of Lim1 function in developing horizontal cells. Through this approach, Lim1 function was prevented within a subset of horizontal cells that in turn fail to migrate to the horizontal cell layer and differentiate normally. For those remaining horizontal cells with Lim1 intact (about half of the normal population in these mice), we show that they spread themselves out tangentially and differentiate a dendritic morphology that is essentially normal but for the fact that it has nearly doubled in area. Such larger horizontal cells, sampling from an area of retina containing twice their normal afferent number, differentiate a dendritic field with nearly double the number of higher order branches and terminal clusters. These results demonstrate directly that positioning and dendritic growth are regulated by interactions with homotypic neighbors, whereas afferents instruct the differentiation of dendritic patterning.
Assuntos
Comunicação Celular/fisiologia , Forma Celular/fisiologia , Dendritos/fisiologia , Células Horizontais da Retina/citologia , Células Horizontais da Retina/crescimento & desenvolvimento , Animais , Contagem de Células/métodos , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Proteínas com Homeodomínio LIM , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fatores de TranscriçãoRESUMO
BACKGROUND: Ex vivo, whole-mount explant culture of the rodent retina has proved to be a valuable approach for studying retinal development. In a limited number of recent studies, this method has been coupled to live fluorescent microscopy with the goal of directly observing dynamic cellular events. However, retinal tissue thickness imposes significant technical limitations. To obtain 3-dimensional images with high quality axial resolution, investigators are restricted to specific areas of the retina and require microscopes, such as 2-photon, with a higher level of depth penetrance. Here, we report a retinal live imaging method that is more amenable to a wider array of imaging systems and does not compromise resolution of retinal cross-sectional area. RESULTS: Mouse retinal slice cultures were prepared and standard, inverted confocal microscopy was used to generate movies with high quality resolution of retinal cross-sections. To illustrate the ability of this method to capture discrete, physiologically relevant events during retinal development, we imaged the dynamics of the Fucci cell cycle reporter in both wild type and Cyclin D1 mutant retinal progenitor cells (RPCs) undergoing interkinetic nuclear migration (INM). Like previously reported for the zebrafish, mouse RPCs in G1 phase migrated stochastically and exhibited overall basal drift during development. In contrast, mouse RPCs in G2 phase displayed directed, apical migration toward the ventricular zone prior to mitosis. We also determined that Cyclin D1 knockout RPCs in G2 exhibited a slower apical velocity as compared to wild type. These data are consistent with previous IdU/BrdU window labeling experiments on Cyclin D1 knockout RPCs indicating an elongated cell cycle. Finally, to illustrate the ability to monitor retinal neuron differentiation, we imaged early postnatal horizontal cells (HCs). Time lapse movies uncovered specific HC neurite dynamics consistent with previously published data showing an instructive role for transient vertical neurites in HC mosaic formation. CONCLUSIONS: We have detailed a straightforward method to image mouse retinal slice culture preparations that, due to its relative ease, extends live retinal imaging capabilities to a more diverse group of scientists. We have also shown that, by using a slice technique, we can achieve excellent lateral resolution, which is advantageous for capturing intracellular dynamics and overall cell movements during retinal development and differentiation.