RESUMO
During development, the central nervous system (CNS) vasculature grows to precisely meet the metabolic demands of neurons and glia. In addition, the vast majority of the CNS vasculature acquires a unique set of molecular and cellular properties-collectively referred to as the blood-brain barrier-that minimize passive diffusion of molecules between the blood and the CNS parenchyma. Both of these processes are controlled by signals emanating from neurons and glia. In this review, we describe the nature and mechanisms-of-action of these signals, with an emphasis on vascular endothelial growth factor (VEGF) and beta-catenin (canonical Wnt) signaling, the two best-understood systems that regulate CNS vascular development. We highlight foundational discoveries, interactions between different signaling systems, the integration of genetic and cell biological studies, advances that are of clinical relevance, and questions for future research.
Assuntos
Fator A de Crescimento do Endotélio Vascular , Via de Sinalização Wnt , Barreira Hematoencefálica/metabolismo , Sistema Nervoso Central , Fator A de Crescimento do Endotélio Vascular/metabolismo , Via de Sinalização Wnt/fisiologiaRESUMO
Norrin/Frizzled4 (Fz4) signaling activates the canonical Wnt pathway to control retinal vascular development. Using genetically engineered mice, we show that precocious Norrin production leads to premature retinal vascular invasion and delayed Norrin production leads to characteristic defects in intraretinal vascular architecture. In genetic mosaics, wild-type endothelial cells (ECs) instruct neighboring Fz4(-/-) ECs to produce an architecturally normal mosaic vasculature, a cell nonautonomous effect. However, over the ensuing weeks, Fz4(-/-) ECs are selectively eliminated from the mosaic vasculature, implying the existence of a quality control program that targets defective ECs. In the adult retina and cerebellum, gain or loss of Norrin/Fz4 signaling results in a cell-autonomous gain or loss, respectively, of blood retina barrier and blood brain barrier function, indicating an ongoing requirement for Frizzled signaling in barrier maintenance and substantial plasticity in mature CNS vascular structure.
Assuntos
Barreira Hematoencefálica , Barreira Hematorretiniana , Células Endoteliais/metabolismo , Proteínas do Olho/metabolismo , Receptores Frizzled/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Retina/metabolismo , Transdução de Sinais , Animais , Movimento Celular , Proliferação de Células , Células Endoteliais/citologia , Proteínas do Olho/genética , Receptores Frizzled/genética , Humanos , Camundongos , Mosaicismo , Proteínas do Tecido Nervoso/genética , Plasticidade Neuronal , Retina/citologia , Retina/embriologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The 2023 Lasker-DeBakey Clinical Medical Research Award is being presented to James Fujimoto, David Huang, and Eric Swanson for their invention and development of optical coherence tomography (OCT), an imaging technology that uses light to visualize microscopic structures within tissues such as the retina. OCT has dramatically changed the practice of ophthalmology and improved the lives of millions of people. It also has great potential in a wide range of other medical fields.
Assuntos
Distinções e Prêmios , Pesquisa Biomédica , Humanos , Tomografia de Coerência Óptica , Retina/diagnóstico por imagemRESUMO
Diverse extracellular proteins negatively regulate WNT signaling. One such regulator is adenomatosis polyposis coli down-regulated 1 (APCDD1), a conserved single-span transmembrane protein. In response to WNT signaling in a variety of tissues, APCDD1 transcripts are highly up-regulated. We have determined the three-dimensional structure of the extracellular domain of APCDD1, and this structure reveals an unusual architecture consisting of two closely apposed ß-barrel domains (ABD1 and ABD2). ABD2, but not ABD1, has a large hydrophobic pocket that accommodates a bound lipid. The APCDD1 ECD can also bind to WNT7A, presumably via its covalently bound palmitoleate, a modification that is common to all WNTs and is essential for signaling. This work suggests that APCDD1 functions as a negative feedback regulator by titrating WNT ligands at the surface of responding cells.
Assuntos
Polipose Adenomatosa do Colo , Proteínas de Membrana , Humanos , Proteínas de Membrana/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Via de Sinalização Wnt , Lipídeos , beta Catenina/genética , beta Catenina/metabolismoRESUMO
The lacrimal gland is of central interest in ophthalmology both as the source of the aqueous component of tear fluid and as the site of autoimmune pathology in the context of Sjogren's syndrome (SjS). To provide a foundational description of mouse lacrimal gland cell types and their patterns of gene expression, we have analyzed single-cell transcriptomes from wild-type (Balb/c) mice and from two genetically based SjS models, MRL/lpr and NOD (nonobese diabetic).H2b, and defined the localization of multiple cell-type-specific protein and mRNA markers. This analysis has uncovered a previously undescribed cell type, Car6+ cells, which are located at the junction of the acini and the connecting ducts. More than a dozen secreted polypeptides that are likely to be components of tear fluid are expressed by acinar cells and show pronounced sex differences in expression. Additional examples of gene expression heterogeneity within a single cell type were identified, including a gradient of Claudin4 along the length of the ductal system and cell-to-cell heterogeneity in transcription factor expression within acinar and myoepithelial cells. The patterns of expression of channels, transporters, and pumps in acinar, Car6+, and ductal cells make strong predictions regarding the mechanisms of water and electrolyte secretion. In MRL/lpr and NOD.H2b lacrimal glands, distinctive changes in parenchymal gene expression and in immune cell subsets reveal widespread interferon responses, a T cell-dominated infiltrate in the MRL/lpr model, and a mixed B cell and T cell infiltrate in the NOD.H2b model.
Assuntos
Aparelho Lacrimal , Síndrome de Sjogren , Feminino , Camundongos , Masculino , Animais , Síndrome de Sjogren/metabolismo , Aparelho Lacrimal/metabolismo , Camundongos Endogâmicos MRL lpr , Camundongos Endogâmicos NOD , Camundongos Endogâmicos BALB C , Modelos Animais de DoençasRESUMO
In many organs, small openings across capillary endothelial cells (ECs) allow the diffusion of low-molecular weight compounds and small proteins between the blood and tissue spaces. These openings contain a diaphragm composed of radially arranged fibers, and current evidence suggests that a single-span type II transmembrane protein, plasmalemma vesicle-associated protein-1 (PLVAP), constitutes these fibers. Here, we present the three-dimensional crystal structure of an 89-amino acid segment of the PLVAP extracellular domain (ECD) and show that it adopts a parallel dimeric alpha-helical coiled-coil configuration with five interchain disulfide bonds. The structure was solved using single-wavelength anomalous diffraction from sulfur-containing residues (sulfur SAD) to generate phase information. Biochemical and circular dichroism (CD) experiments show that a second PLVAP ECD segment also has a parallel dimeric alpha-helical configuration-presumably a coiled coil-held together with interchain disulfide bonds. Overall, ~2/3 of the ~390 amino acids within the PLVAP ECD adopt a helical configuration, as determined by CD. We also determined the sequence and epitope of MECA-32, an anti-PLVAP antibody. Taken together, these data lend strong support to the model of capillary diaphragms formulated by Tse and Stan in which approximately ten PLVAP dimers are arranged within each 60- to 80-nm-diameter opening like the spokes of a bicycle wheel. Passage of molecules through the wedge-shaped pores is presumably determined both by the length of PLVAP-i.e., the long dimension of the pore-and by the chemical properties of amino acid side chains and N-linked glycans on the solvent-accessible faces of PLVAP.
Assuntos
Diafragma , Células Endoteliais , Diafragma/metabolismo , Células Endoteliais/metabolismo , Proteínas de Transporte/metabolismo , Endotélio Vascular/metabolismo , Dissulfetos/metabolismo , Dicroísmo CircularRESUMO
Over the past 3 decades, a diverse collection of small protein domains have been used as scaffolds to generate general purpose protein-binding reagents using a variety of protein display and enrichment technologies. To expand the repertoire of scaffolds and protein surfaces that might serve this purpose, we have explored the utility of (i) a pair of anti-parallel alpha-helices in a small highly disulfide-bonded 4-helix bundle, the CC4 domain from reversion-inducing Cysteine-rich Protein with Kazal Motifs and (ii) a concave beta-sheet surface and two adjacent loops in the human FN3 domain, the scaffold for the widely used monobody platform. Using M13 phage display and next generation sequencing, we observe that, in both systems, libraries of â¼30 million variants contain binding proteins with affinities in the low µM range for baits corresponding to the extracellular domains of multiple mammalian proteins. CC4- and FN3-based binding proteins were fused to the N- and/or C-termini of Fc domains and used for immunostaining of transfected cells. Additionally, FN3-based binding proteins were inserted into VP1 of AAV to direct AAV infection to cells expressing a defined surface receptor. Finally, FN3-based binding proteins were inserted into the Pvc13 tail fiber protein of an extracellular contractile injection system particle to direct protein cargo delivery to cells expressing a defined surface receptor. These experiments support the utility of CC4 helices B and C and of FN3 beta-strands C, D, and F together with adjacent loops CD and FG as surfaces for engineering general purpose protein-binding reagents.
Assuntos
Biblioteca de Peptídeos , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Animais , Humanos , Bacteriófago M13 , Técnicas de Visualização da Superfície Celular , Células HEK293 , Ligação ProteicaRESUMO
In central nervous system vascular endothelial cells, signaling via the partially redundant ligands WNT7A and WNT7B requires two co-activator proteins, GPR124 and RECK. WNT7A and RECK have been shown previously to play a role in limb development, but the mechanism of RECK action in this context is unknown. The roles of WNT7B and GPR124 in limb development have not been investigated. Using combinations of conventional and/or conditional loss-of-function alleles for mouse Wnt7a, Wnt7b, Gpr124 and Reck, including a Reck allele that codes for a protein that is specifically defective in WNT7A/WNT7B signaling, we show that reductions in ligand and/or co-activator function synergize to cause reduced and dysmorphic limb bone growth. Two additional limb phenotypes - loss of distal Lmx1b expression and ectopic growth of nail-like structures - occur with reduced Wnt7a/Wnt7b gene copy number and, respectively, with Reck mutations and with combined Reck and Gpr124 mutations. A third limb phenotype - bleeding into a digit - occurs with the most severe combinations of Wnt7a/Wnt7b, Reck and Gpr124 mutations. These data imply that the WNT7A/WNT7B-FRIZZLED-LRP5/LRP6-GPR124-RECK signaling system functions as an integral unit in limb development.
Assuntos
Embrião de Mamíferos/metabolismo , Extremidades/embriologia , Proteínas Ligadas por GPI/metabolismo , Receptores Acoplados a Proteínas G , Proteínas Wnt , Animais , Sistema Nervoso Central/metabolismo , Células Endoteliais/metabolismo , Ligantes , Camundongos , Proteínas Proto-Oncogênicas/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMO
Disorders of vascular structure and function play a central role in a wide variety of CNS diseases. Mutations in the Frizzled-4 (Fz4) receptor, Lrp5 coreceptor, or Norrin ligand cause retinal hypovascularization, but the mechanisms by which Norrin/Fz4/Lrp signaling controls vascular development have not been defined. Using mouse genetic and cell culture models, we show that loss of Fz4 signaling in endothelial cells causes defective vascular growth, which leads to chronic but reversible silencing of retinal neurons. Loss of Fz4 in all endothelial cells disrupts the blood brain barrier in the cerebellum, whereas excessive Fz4 signaling disrupts embryonic angiogenesis. Sox17, a transcription factor that is upregulated by Norrin/Fz4/Lrp signaling, plays a central role in inducing the angiogenic program controlled by Norrin/Fz4/Lrp. These experiments establish a cellular basis for retinal hypovascularization diseases due to insufficient Frizzled signaling, and they suggest a broader role for Frizzled signaling in vascular growth, remodeling, maintenance, and disease.
Assuntos
Células Endoteliais/metabolismo , Proteínas do Olho/metabolismo , Receptores Frizzled/metabolismo , Proteínas Relacionadas a Receptor de LDL/metabolismo , Neovascularização Fisiológica , Proteínas do Tecido Nervoso/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Neurônios Retinianos/metabolismo , Transdução de Sinais , Animais , Cerebelo/metabolismo , Receptores Frizzled/genética , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Camundongos , Receptores Acoplados a Proteínas G/genética , Retina/citologia , Retina/metabolismo , Proteínas Wnt/metabolismoRESUMO
Five small protein domains, the CC-domains, at the N terminus of the RECK protein, play essential roles in signaling by WNT7A and WNT7B in the context of central nervous system angiogenesis and blood-brain barrier formation and maintenance. We have determined the structure of CC domain 4 (CC4) at 1.65-Å resolution and find that it folds into a compact four-helix bundle with three disulfide bonds. The CC4 structure, together with homology modeling of CC1, reveals the surface locations of critical residues that were shown in previous mutagenesis studies to mediate GPR124 binding and WNT7A/WNT7B recognition and signaling. Surprisingly, sequence and structural homology searches reveal no other cell-surface or secreted domains in vertebrates that resemble the CC domain, a pattern that is in striking contrast to other ancient and similarly sized domains, such as Epidermal Growth Factor, Fibronectin Type 3, Immunoglobulin, and Thrombospondin type 1 domains, which are collectively present in hundreds of proteins.
Assuntos
Evolução Molecular , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/metabolismo , Sequência de Aminoácidos , Animais , Caenorhabditis elegans , Proteínas Ligadas por GPI/genética , Humanos , Camundongos , Domínios Proteicos , Alinhamento de SequênciaRESUMO
During mitosis, Kif11, a kinesin motor protein, promotes bipolar spindle formation and chromosome movement, and during interphase, Kif11 mediates diverse trafficking processes in the cytoplasm. In humans, inactivating mutations in KIF11 are associated with (1) retinal hypovascularization with or without microcephaly and (2) multi-organ syndromes characterized by variable combinations of lymphedema, chorioretinal dysplasia, microcephaly and/or mental retardation. To explore the pathogenic basis of KIF11-associated retinal vascular disease, we generated a Kif11 conditional knockout (CKO) mouse and investigated the consequences of early postnatal inactivation of Kif11 in vascular endothelial cells (ECs). The principal finding is that postnatal EC-specific loss of Kif11 leads to severely stunted growth of the retinal vasculature, mildly stunted growth of the cerebellar vasculature and little or no effect on the vasculature elsewhere in the central nervous system (CNS). Thus, in mice, Kif11 function in early postnatal CNS ECs is most significant in the two CNS regions-the retina and cerebellum-that exhibit the most rapid rate of postnatal growth, which may sensitize ECs to impaired mitotic spindle function. Several lines of evidence indicate that these phenotypes are not caused by reduced beta-catenin signaling in ECs, despite the close resemblance of the Kif11 CKO phenotype to that caused by EC-specific reductions in beta-catenin signaling. Based on prior work, defective beta-catenin signaling had been the only known mechanism responsible for monogenic human disorders of retinal hypovascularization. The present study implies that retinal hypovascularization can arise from a second and mechanistically distinct cause.
Assuntos
Vitreorretinopatias Exsudativas Familiares/genética , Deficiência Intelectual/genética , Cinesinas/genética , Degeneração Retiniana/genética , Animais , Análise Mutacional de DNA , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Vitreorretinopatias Exsudativas Familiares/patologia , Humanos , Deficiência Intelectual/patologia , Camundongos , Mutação/genética , Linhagem , Fenótipo , Degeneração Retiniana/patologiaRESUMO
Autoimmune uveoretinitis is a significant cause of visual loss, and mouse models offer unique opportunities to study its disease mechanisms. Aire-/- mice fail to express self-antigens in the thymus, exhibit reduced central tolerance, and develop a spontaneous, chronic, and progressive uveoretinitis. Using single-cell RNA sequencing (scRNA-seq), we characterized wild-type and Aire-/- retinas to define, in a comprehensive and unbiased manner, the cell populations and gene expression patterns associated with disease. Based on scRNA-seq, immunostaining, and in situ hybridization, we infer that 1) the dominant effector response in Aire-/- retinas is Th1-driven, 2) a subset of monocytes convert to either a macrophage/microglia state or a dendritic cell state, 3) the development of tertiary lymphoid structures constitutes part of the Aire-/- retinal phenotype, 4) all major resident retinal cell types respond to interferon gamma (IFNG) by changing their patterns of gene expression, and 5) Muller glia up-regulate specific genes in response to IFN gamma and may act as antigen-presenting cells.
RESUMO
The mammalian CNS is capable of tolerating chronic hypoxia, but cell type-specific responses to this stress have not been systematically characterized. In the Norrin KO (NdpKO ) mouse, a model of familial exudative vitreoretinopathy (FEVR), developmental hypovascularization of the retina produces chronic hypoxia of inner nuclear-layer (INL) neurons and Muller glia. We used single-cell RNA sequencing, untargeted metabolomics, and metabolite labeling from 13C-glucose to compare WT and NdpKO retinas. In NdpKO retinas, we observe gene expression responses consistent with hypoxia in Muller glia and retinal neurons, and we find a metabolic shift that combines reduced flux through the TCA cycle with increased synthesis of serine, glycine, and glutathione. We also used single-cell RNA sequencing to compare the responses of individual cell types in NdpKO retinas with those in the hypoxic cerebral cortex of mice that were housed for 1 week in a reduced oxygen environment (7.5% oxygen). In the hypoxic cerebral cortex, glial transcriptome responses most closely resemble the response of Muller glia in the NdpKO retina. In both retina and brain, vascular endothelial cells activate a previously dormant tip cell gene expression program, which likely underlies the adaptive neoangiogenic response to chronic hypoxia. These analyses of retina and brain transcriptomes at single-cell resolution reveal both shared and cell type-specific changes in gene expression in response to chronic hypoxia, implying both shared and distinct cell type-specific physiologic responses.
Assuntos
Hipóxia/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Vitreorretinopatias Exsudativas Familiares/genética , Vitreorretinopatias Exsudativas Familiares/fisiopatologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/metabolismo , Retina/metabolismo , Retina/fisiologia , Neurônios Retinianos/metabolismo , Vasos Retinianos/metabolismo , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodosRESUMO
ß-Catenin signaling controls the development and maintenance of the blood-brain barrier (BBB) and the blood-retina barrier (BRB), but the division of labor and degree of redundancy between the two principal ligand-receptor systems-the Norrin and Wnt7a/Wnt7b systems-are incompletely defined. Here, we present a loss-of-function genetic analysis of postnatal BBB and BRB maintenance in mice that shows striking threshold and partial redundancy effects. In particular, the combined loss of Wnt7a and Norrin or Wnt7a and Frizzled4 (Fz4) leads to anatomically localized BBB defects that are far more severe than observed with loss of Wnt7a, Norrin, or Fz4 alone. In the cerebellum, selective loss of Wnt7a in glia combined with ubiquitous loss of Norrin recapitulates the phenotype observed with ubiquitous loss of both Wnt7a and Norrin, implying that glia are the source of Wnt7a in the cerebellum. Tspan12, a coactivator of Norrin signaling in the retina, is also active in BBB maintenance but is less potent than Norrin, consistent with a model in which Tspan12 enhances the amplitude of the Norrin signal in vascular endothelial cells. Finally, in the context of a partially impaired Norrin system, the retina reveals a small contribution to BRB development from the Wnt7a/Wnt7b system. Taken together, these experiments define the extent of CNS region-specific cooperation for several components of the Norrin and Wnt7a/Wnt7b systems, and they reveal substantial regional heterogeneity in the extent to which partially redundant ligands, receptors, and coactivators maintain the BBB and BRB.
Assuntos
Barreira Hematoencefálica/crescimento & desenvolvimento , Barreira Hematoencefálica/fisiologia , Barreira Hematorretiniana/crescimento & desenvolvimento , Barreira Hematorretiniana/fisiologia , Proteínas do Olho/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Wnt/fisiologia , Animais , Barreira Hematoencefálica/citologia , Barreira Hematorretiniana/citologia , Técnicas de Cultura de Células , Proteínas do Olho/genética , Receptores Frizzled/deficiência , Receptores Frizzled/genética , Receptores Frizzled/fisiologia , Camundongos , Camundongos Knockout , Modelos Biológicos , Modelos Neurológicos , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais , Tetraspaninas/deficiência , Tetraspaninas/genética , Tetraspaninas/fisiologia , Proteínas Wnt/deficiência , Proteínas Wnt/genética , beta Catenina/fisiologiaRESUMO
Astrotactins are vertebrate-specific membrane proteins implicated in neuron-glia interactions during central nervous system development and in hair follicle polarity during skin development. By studying epitope-tagged derivatives of mouse astrotactin-2 (Astn2) produced in transfected cells, we determined that the amino and carboxyl termini reside in the extracellular space and are initially linked by two transmembrane segments and a single cytoplasmic domain. We further show that Astn2 undergoes proteolytic cleavage in the second transmembrane domain (TM2) and that a disulfide bond holds the resulting two fragments together. Recombinant Astn1 also undergoes TM2 cleavage, as does Astn2 isolated from mouse cerebellum. Astn2 intramembrane proteolysis is insensitive to replacement of TM2 by the transmembrane domain of CD74 or by 21 alanines. However, replacement of TM2 by the transmembrane domain of CD4, the asialoglycoprotein receptor, or the transferrin receptor eliminates intramembrane proteolysis, as does leucine substitution of residues that overlap or are immediately upstream of the cleavage site. Replacement of the transmembrane domain of CD74 or the asialoglycoprotein receptor with Astn2 TM2 leads to the appearance of a carboxyl-terminal fragment consistent with intramembrane proteolysis. These experiments define a highly unusual transmembrane topology for the astrotactins, reveal intramembrane proteolysis as a feature of astrotactin maturation, and constrain the substrate sequences that are permissive for cleavage of one type 2 transmembrane segment.
Assuntos
Membrana Celular/metabolismo , Glicoproteínas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Membrana Celular/ultraestrutura , Cerebelo/metabolismo , Chlorocebus aethiops , Glicoproteínas/análise , Células HEK293 , Humanos , Camundongos , Proteínas do Tecido Nervoso/análise , Domínios Proteicos , Multimerização Proteica , ProteóliseRESUMO
Peropsin is a non-visual opsin in both vertebrate and invertebrate species. In mammals, peropsin is present in the apical microvilli of retinal pigment epithelial (RPE) cells. These structures interdigitate with the outer segments of rod and cone photoreceptor cells. RPE cells play critical roles in the maintenance of photoreceptors, including the recycling of visual chromophore for the opsin visual pigments. Here, we sought to identify the function of peropsin in the mouse eye. To this end, we generated mice with a null mutation in the peropsin gene (Rrh). These mice exhibited normal retinal histology, normal morphology of outer segments and RPE cells, and no evidence of photoreceptor degeneration. Biochemically, Rrh-/- mice had â¼2-fold higher vitamin A (all-trans-retinol (all-trans-ROL)) in the neural retina following a photobleach and 5-fold lower retinyl esters in the RPE. This phenotype was similar to those reported in mice that lack interphotoreceptor retinoid-binding protein (IRBP) or cellular retinol-binding protein, suggesting that peropsin plays a role in the movement of all-trans-ROL from photoreceptors to the RPE. We compared the phenotypes in mice lacking both peropsin and IRBP with those of mice lacking peropsin or IRBP alone and found that the retinoid phenotype was similarly severe in each of these knock-out mice. We conclude that peropsin controls all-trans-ROL movement from the retina to the RPE or may regulate all-trans-ROL storage within the RPE. We propose that peropsin affects light-dependent regulation of all-trans-ROL uptake from photoreceptors into RPE cells through an as yet undefined mechanism.
Assuntos
Rodopsina/metabolismo , Vitamina A/fisiologia , Animais , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Camundongos , Camundongos Knockout , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Pigmentos da Retina/metabolismo , Retinaldeído/metabolismo , Retinoides/metabolismo , Proteínas de Ligação ao Retinol/genética , Proteínas de Ligação ao Retinol/metabolismo , Proteínas Celulares de Ligação ao Retinol/metabolismo , Rodopsina/genética , Rodopsina/fisiologia , Opsinas de Bastonetes/metabolismo , Vitamina A/metabolismoAssuntos
Demografia , Crescimento Demográfico , África , África do Norte , Países em Desenvolvimento , Egito , Alimentos , Dinâmica PopulacionalRESUMO
Planar cell polarity (PCP) signaling controls the global orientation of surface structures, such as hairs and bristles, in both vertebrates and invertebrates. In Frizzled6(-/-) (Fz6(-/-)) mice, hair follicle orientations on the head and back are nearly random at birth, but reorient during early postnatal development to eventually generate a nearly parallel anterior-to-posterior array. We report the identification of a naturally occurring exon 5 deletion in Astrotactin2 (Astn2) that acts as a recessive genetic modifier of the Fz6(-/-) hair patterning phenotype. A genetically engineered Astn2 exon 5 deletion recapitulates the modifier phenotype. In Fz6(-/-);Astn2(ex5del/del) mice, hair orientation on the back is subtly biased from posterior-to-anterior, leading to a 180-degree orientation reversal in mature mice. These experiments suggest that Astn2, an endosomal membrane protein, modulates PCP signaling.
Assuntos
Receptores Frizzled/genética , Glicoproteínas/genética , Folículo Piloso/crescimento & desenvolvimento , Proteínas do Tecido Nervoso/genética , Animais , Padronização Corporal/genética , Polaridade Celular , Glicoproteínas/fisiologia , Camundongos , Proteínas do Tecido Nervoso/fisiologia , Transdução de SinaisRESUMO
In mammals, hair follicles cover most of the body surface and exhibit precise and stereotyped orientations relative to the body axes. Follicle orientation is controlled by the planar cell polarity (PCP; or, more generally, tissue polarity) system, as determined by the follicle mis-orientation phenotypes observed in mice with PCP gene mutations. The present study uses conditional knockout alleles of the PCP genes Frizzled6 (Fz6), Vangl1, and Vangl2, together with a series of Cre drivers to interrogate the spatio-temporal domains of PCP gene action in the developing mouse epidermis required for follicle orientation. Fz6 is required starting between embryonic day (E)11.5 and E12.5. Eliminating Fz6 in either the anterior or the posterior halves of the embryo or in either the feet or the torso leads to follicle mis-orientation phenotypes that are limited to the territories associated with Fz6 loss, implying either that PCP signaling is required for communicating polarity information on a local but not a global scale, or that there are multiple independent sources of global polarity information. Eliminating Fz6 in most hair follicle cells or in the inter-follicular epidermis at E15.5 suggests that PCP signaling in developing follicles is not required to maintain their orientation. The asymmetric arrangement of Merkel cells around the base of each guard hair follicle dependents on Fz6 expression in the epidermis but not in differentiating Merkel cells. These experiments constrain current models of PCP signaling and the flow of polarity information in mammalian skin.
Assuntos
Polaridade Celular , Receptores Frizzled/química , Receptores Frizzled/metabolismo , Folículo Piloso/citologia , Folículo Piloso/embriologia , Animais , Padronização Corporal , Proteínas de Transporte/metabolismo , Embrião de Mamíferos/metabolismo , Genes Reporter , Integrases/metabolismo , Proteínas de Membrana/metabolismo , Células de Merkel/citologia , Células de Merkel/metabolismo , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismo , Especificidade de Órgãos , Estrutura Terciária de Proteína , Transdução de Sinais , Fatores de TempoRESUMO
The dorsal surface of the mouse tongue is covered by ~7000 papillae, asymmetric epithelial protrusions that are precisely oriented to create a stereotyped macroscopic pattern. Within the context of this large-scale pattern, neighboring papillae exhibit a high degree of local order that minimizes the differences in their orientations. We show here that the orientations of lingual papillae are under the control of the core planar cell polarity (PCP) genes Vangl1, Vangl2, and Celsr1. Using K14-Cre and Nkx2.5-Cre to induce conditional knockout of Vangl1 and/or Vangl2 in the tongue epithelium, we observe more severe disruptions to local order among papillae with inactivation of larger numbers of Vangl genes, a greater role for Vangl2 than Vangl1, and a more severe phenotype with the Vangl2 Looptail (Lp) allele than the Vangl2 null allele, consistent with a dominant negative mode of action of the Vangl2Lp allele. Interestingly, Celsr1-/- tongues show disruption of both local and global order, with many papillae in the anterior tongue showing a reversed orientation. To quantify each of these phenotypes, we have developed and applied three procedures for sampling the orientations of papillae and assessing the degree of order on different spatial scales. The experiments reported here establish the dorsal surface of the mouse tongue as a favorable system for studying PCP control of epithelial patterning.