RESUMO
Three-dimensional (3D) microscaffolds for cell biology have shown their potential in mimicking physiological environments and simulating complex multicellular constructs. However, controlling the localization of cells precisely on microfabricated structures is still complex and usually limited to two-dimensional assays. Indeed, the implementation of an efficient method to selectively target different cell types to specific regions of a 3D microscaffold would represent a decisive step toward cell-by-cell assembly of complex cellular arrangements. Here, we use two-photon lithography (2PL) to fabricate 3D microarchitectures with functional photoresists. UV-mediated click reactions are used to functionalize their surfaces with single-stranded DNA oligonucleotides, using sequential repetition to decorate different scaffold regions with individual DNA addresses. Various immortalized cell lines and stem cells modified by grafting complementary oligonucleotides onto the phospholipid membranes can then be immobilized onto complementary regions of the 3D structures by selective hybridization. This allows controlled cocultures to be established with spatially separated arrays of eukaryotic cells in 3D.
RESUMO
The vertebrate sense of smell employs four main receptor families for detection of odors, among them the V1R/ORA family, which is unusually small and highly conserved in teleost fish. Zebrafish possess just seven ORA receptors, enabling a comprehensive analysis of the expression patterns of the entire family. The olfactory organ of zebrafish is representative for teleosts, cup-shaped, with lamella covered with sensory epithelium protruding into the cup from a median raphe. We have performed quantitative in situ hybridization on complete series of horizontal cryostat sections of adult zebrafish olfactory organ, and have analysed the location of ora-expressing cells in three dimensions, radial diameter, laminar height, and height-within-the-organ. We report broadly overlapping, but distinctly different distributions for all ora genes, even for ora3a and ora3b, the most recent gene duplication. Preferred positions in different dimensions are independent of each other. This spatial logic is very similar to previous reports for the much larger families of odorant receptor (or) and V2R-related olfC genes in zebrafish. Preferred positions for ora genes tend to be more central and more apical than those we observed for these other two families, consistent with expression in non-canonical sensory neuron types.
Assuntos
Neurônios Receptores Olfatórios , Receptores Odorantes , Animais , Mucosa Olfatória/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Seleção Genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Mitochondria are essential organelles because of their function in energy conservation. Here, we show an involvement of mitochondria in phytochrome-dependent light sensing in fungi. Phytochrome photoreceptors are found in plants, bacteria, and fungi and contain a linear, heme-derived tetrapyrrole as chromophore. Linearization of heme requires heme oxygenases (HOs) which reside inside chloroplasts in planta. Despite the poor degree of conservation of HOs, we identified two candidates in the fungus Alternaria alternata. Deletion of either one phenocopied phytochrome deletion. The two enzymes had a cooperative effect and physically interacted with phytochrome, suggesting metabolon formation. The metabolon was attached to the surface of mitochondria with a C-terminal anchor (CTA) sequence in HoxA. The CTA was necessary and sufficient for mitochondrial targeting. The affinity of phytochrome apoprotein to HoxA was 57,000-fold higher than the affinity of the holoprotein, suggesting a "kiss-and-go" mechanism for chromophore loading and a function of mitochondria as assembly platforms for functional phytochrome. Hence, two alternative approaches for chromophore biosynthesis and insertion into phytochrome evolved in plants and fungi.
Assuntos
Proteínas Fúngicas/biossíntese , Mitocôndrias/metabolismo , Fitocromo/biossíntese , Alternaria , Proteínas Fúngicas/genética , Heme/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Fitocromo/genética , Transporte ProteicoRESUMO
BACKGROUND: The sense of smell is unrivaled in terms of molecular complexity of its input channels. Even zebrafish, a model vertebrate system in many research fields including olfaction, possesses several hundred different olfactory receptor genes, organized in four different gene families. For one of these families, the initially discovered odorant receptors proper, segregation of expression into distinct spatial subdomains within a common sensory surface has been observed both in teleost fish and in mammals. However, for the remaining three families, little to nothing was known about their spatial coding logic. Here we wished to investigate, whether the principle of spatial segregation observed for odorant receptors extends to another olfactory receptor family, the V2R-related OlfC genes. Furthermore we thought to examine, how expression of OlfC genes is integrated into expression zones of odorant receptor genes, which in fish share a single sensory surface with OlfC genes. RESULTS: To select representative genes, we performed a comprehensive phylogenetic study of the zebrafish OlfC family, which identified a novel OlfC gene, reduced the number of pseudogenes to 1, and brought the total family size to 60 intact OlfC receptors. We analyzed the spatial pattern of OlfC-expressing cells for seven representative receptors in three dimensions (height within the epithelial layer, horizontal distance from the center of the olfactory organ, and height within the olfactory organ). We report non-random distributions of labeled neurons for all OlfC genes analysed. Distributions for sparsely expressed OlfC genes are significantly different from each other in nearly all cases, broad overlap notwithstanding. For two of the three coordinates analyzed, OlfC expression zones are intercalated with those of odorant receptor zones, whereas in the third dimension some segregation is observed. CONCLUSION: Our results show that V2R-related OlfC genes follow the same spatial logic of expression as odorant receptors and their expression zones intermingle with those of odorant receptor genes. Thus, distinctly different expression zones for individual receptor genes constitute a general feature shared by teleost and tetrapod V2R/OlfC and odorant receptor families alike.
Assuntos
Perfilação da Expressão Gênica , Receptores Odorantes/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Olfato/genética , Peixe-Zebra/fisiologiaRESUMO
Genetic hardwiring during brain development provides computational architectures for innate neuronal processing. Thus, the paradigmatic chick retinotectal projection, due to its neighborhood preserving, topographic organization, establishes millions of parallel channels for incremental visual field analysis. Retinal axons receive targeting information from quantitative guidance cue gradients. Surprisingly, novel adaptation assays demonstrate that retinal growth cones robustly adapt towards ephrin-A/EphA forward and reverse signals, which provide the major mapping cues. Computational modeling suggests that topographic accuracy and adaptability, though seemingly incompatible, could be reconciled by a novel mechanism of coupled adaptation of signaling channels. Experimentally, we find such 'co-adaptation' in retinal growth cones specifically for ephrin-A/EphA signaling. Co-adaptation involves trafficking of unliganded sensors between the surface membrane and recycling endosomes, and is presumably triggered by changes in the lipid composition of membrane microdomains. We propose that co-adaptative desensitization eventually relies on guidance sensor translocation into cis-signaling endosomes to outbalance repulsive trans-signaling.
Assuntos
Orientação de Axônios , Efrinas/metabolismo , Cones de Crescimento/fisiologia , Receptores da Família Eph/metabolismo , Retina/embriologia , Células Ganglionares da Retina/fisiologia , Animais , Embrião de Galinha , Simulação por Computador , Endossomos/metabolismo , Proteínas de Membrana/metabolismoRESUMO
Axonal projections between two populations of neurons, which preserve neighborhood relationships, are called topographic. They are ubiquitous in the brain. The development of the retinotectal projection, mapping the retinal output onto the roof of the midbrain, has been studied for decades as a model system. The rigid precision of normal retinotopic mapping has prompted the chemoaffinity hypothesis, positing axonal targeting to be based on fixed biochemical affinities between fibers and targets. In addition, however, abundant evidence has been gathered mainly in the 1970s and 80s that the mapping can adjust to variegated targets with stunning flexibility demonstrating the extraordinary robustness of the guidance process. The identification of ephrins and Eph-receptors as the underlying molecular cues has mostly been interpreted as supporting the fiber-target chemoaffinity hypothesis, while the evidence on mapping robustness has largely been neglected. By having a fresh look on the old data, we expound that they indicate, in addition to fiber-target chemoaffinity, the existence of a second autonomous guidance influence, which we call fiber-fiber chemoaffinity. Classical in vitro observations suggest both influences be composed of opposing monofunctional guidance activities. Based on the molecular evidence, we propose that those might be ephrin/Eph forward and reverse signaling, not only in fiber-target but also in fiber-fiber interactions. In fact, computational models based on this assumption can reconcile the seemingly conflicting findings on rigid and flexible topographic mapping. Supporting the suggested parsimonious and powerful mechanism, they contribute to an understanding of the evolutionary success of robust topographic mass wiring of axons.
Assuntos
Axônios/fisiologia , Simulação por Computador , Modelos Neurológicos , Transdução de Sinais/fisiologia , Animais , Mapeamento Encefálico/métodos , Humanos , Receptores da Família Eph/metabolismoRESUMO
Stripe assays are frequently used for studying binary growth decisions of cells and axons towards surface-bound molecules in vitro. In particular in the fields of neurodevelopment and axon guidance, stripe assays have become a routine tool. Several variants of the stripe assay have been developed since its introduction by Bonhoeffer and colleagues in 1987 (Development 101:685-696, 1987). In all variants, however, the principle is the generation of a structured binary growth substrate, consisting of two sets of cues, arranged in alternating stripes. There are two major classes of stripe assays, mainly distinguished by the source material used for stripe pattern manufacturing: membrane stripe assays, where the stripe patterns are generated with membrane fractions isolated from tissue or cells, and stripe assays with purified proteins, also called modified stripe assays. In this chapter we describe in detail the classical membrane stripe assay, the commonly used modified stripe assay employing purified proteins, and a novel stripe assay for high-affinity interacting proteins, like receptor/ligand pairs.
Assuntos
Axônios/metabolismo , Bioensaio/métodos , Animais , Centrifugação , Células HEK293 , Humanos , Ligantes , Membranas , Camundongos , Proteínas/metabolismo , Receptores de Superfície Celular/metabolismo , Sacarose , TransfecçãoRESUMO
The retinotectal projection, which topographically maps retinal axons onto the tectum of the midbrain, is an ideal model system with which to investigate the molecular genetics of embryonic brain wiring. Corroborating Sperry's seminal hypothesis, ephrin/Eph counter-gradients on both retina and tectum were found to represent matching chemospecificity markers. Intriguingly, however, it has never been possible to reconstitute topographically appropriate fiber growth in vitro with these cues. Moreover, experimentally derived molecular mechanisms have failed to provide explanations as to why the mapping adapts to grossly diverse targets in some experiments, while displaying strict point-to-point specificity in others. In vitro, ephrin-A/EphA forward, as well as reverse, signaling mediate differential repulsion to retinal fibers, instead of providing topographic guidance. We argue that those responses are indicative of ephrin-A and EphA being members of a guidance system that requires two counteracting cues per axis. Experimentally, we demonstrate by introducing novel double-cue stripe assays that the simultaneous presence of both cues indeed suffices to elicit topographically appropriate guidance. The peculiar mechanism, which uses forward and reverse signaling through a single receptor/ligand combination, entails fiber/fiber interactions. We therefore propose to extend Sperry's model to include ephrin-A/EphA-based fiber/fiber chemospecificity, eventually out-competing fiber/target interactions. By computational simulation, we show that our model is consistent with stripe assay results. More importantly, however, it not only accounts for classical in vivo evidence of point-to-point and adaptive topographic mapping, but also for the map duplication found in retinal EphA knock-in mice. Nonetheless, it is based on a single constraint of topographic growth cone navigation: the balancing of ephrin-A/EphA forward and reverse signaling.
Assuntos
Efrinas/metabolismo , Cones de Crescimento/fisiologia , Modelos Biológicos , Receptores da Família Eph/metabolismo , Retina/embriologia , Transdução de Sinais/fisiologia , Teto do Mesencéfalo/embriologia , Animais , Mapeamento Encefálico/métodos , Simulação por Computador , Camundongos , Vias Visuais/embriologiaRESUMO
Chondroitin sulfate (CS) carrying proteoglycans (PGs) are widely expressed in the nervous system, and there is increasing evidence that they regulate developmental mechanisms like neurite outgrowth, axonal guidance and neuronal migration. Moreover, they can also act indirectly by organizing and/or modulating growth factors and guidance molecules. We found that chondroitin-4-sulfate is coexpressed with semaphorin 3A (Sema 3A) in the striatal mantle zone (SMZ), a nontarget region of neuropilin (Nrp)-1-expressing cortical interneurons flanking their migratory route in the subpallium. Using in vitro assays, we showed that CS PGs exert a repulsive effect on cortical interneurons, independently of Sema 3A, due to the CS side chains. We further showed that extracellular Sema 3A binds to CS. Disrupting Sema 3A-Nrp-1 signaling led migrating medial ganglionic eminence neurons to inappropriately invade the SMZ and even more so after removal of the CS side chains. Moreover, we found that soluble Sema 3A enhances the CS-induced repulsion in vitro. We concluded that CS acts as a repellent for cortical interneurons and that, in addition, CS restricts secreted Sema 3A within SMZ. Thus, both molecules act in concert to repel cortical interneurons from the SMZ during tangential migration toward the cerebral cortex.
Assuntos
Movimento Celular/fisiologia , Córtex Cerebral/citologia , Sulfatos de Condroitina/metabolismo , Corpo Estriado/metabolismo , Interneurônios/fisiologia , Semaforina-3A/metabolismo , Animais , Movimento Celular/efeitos dos fármacos , Condroitina ABC Liase/farmacologia , Proteoglicanas de Sulfatos de Condroitina/genética , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Fluorescência Verde/genética , Interneurônios/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal/métodos , Neuropilina-1/metabolismo , Técnicas de Cultura de Órgãos , Semaforina-3A/genéticaRESUMO
Cadherins are superfamily of Ca2+-dependent transmembrane glycoproteins with more than 100 members. They play a role in a wide variety of developmental mechanisms, including cell proliferation, cell differentiation, cell-cell recognition, neurite outgrowth and synaptogenesis. We cloned 16 novel members of the classic cadherin and delta-protocadherin subgroups from ferret brain. Their expression patterns were investigated by in situ hybridization in the developing primary visual cortex (V1) of the ferret. Fifteen out of the 16 cadherins are expressed in a spatiotemporally restricted fashion throughout development. Each layer of V1 can be characterized by the combinatorial expression of a subset of cadherins at any given developmental stage. A few cadherins are expressed by subsets of neurons in specific layers or by neurons dispersed throughout all cortical layers. Generally, the expression of protocadherins is more widespread, whereas that of classic cadherins is more restricted to specific layers. At the V1/V2 boundary, changes in layer-specific cadherin expression are observed. In conclusion, our results suggest that cadherins provide a code of potentially adhesive cues for layer formation in ferret V1. The persistence of expression in the adult suggests a functional role also in the mature cortex.
Assuntos
Caderinas/biossíntese , Córtex Visual/crescimento & desenvolvimento , Animais , Mapeamento Encefálico , Caderinas/genética , Clonagem Molecular , Primers do DNA , DNA Complementar/biossíntese , DNA Complementar/genética , Feminino , Furões , Fluoresceína-5-Isotiocianato/análogos & derivados , Hibridização In Situ , Hibridização in Situ Fluorescente , Gravidez , RNA/biossíntese , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tiramina/análogos & derivadosRESUMO
Cortical interneurons are born in the germinative zones of the ganglionic eminences in the subpallium, and migrate tangentially in spatially and temporally well-defined corridors into the neocortex. Because ephrin-A5 is expressed in the ventricular zone (VZ) of the ganglionic eminences at these developmental stages, we examined the possible effects of this molecule on interneuron migration. Double-immunocytochemistry of dissociated neurons from the medial ganglionic eminences (MGE) revealed that calbindin-positive cells express the EphA4-receptor. In situ, EphA4 is strongly expressed in the subventricular zone of the ganglionic eminences. Using different in vitro assays, we found that ephrin-A5 acts as a repellent cue for MGE neurons. We then examined interneuron migration in slice overlay experiments, where MGE-derived explants from enhanced green fluorescent protein-expressing transgenic mice were homotopically grafted into host slices from wild-type littermate embryos. In these in vitro preparations, interneurons recapitulated in vivo cell migration in several respects. However, interneurons in brain slices also migrated in the VZ of the ganglionic eminences, a region that is strictly avoided in vivo. In situ hybridizations revealed that ephrin-A5 became downregulated in the VZ in vitro. When recombinant ephrin-A5-Fc was added to the slices, it preferentially bound to the VZ, and migrating MGE neurons avoided the VZ as in vivo. The restoration of the normal migration pathway in slices required ephrin-A5 clustering and signalling of Src family kinases. Together, these experiments suggest that ephrin-A5 acts as an inhibitory flank that contributes to define the pathway of migrating interneurons.
Assuntos
Movimento Celular/fisiologia , Efrina-A5/metabolismo , Interneurônios/fisiologia , Animais , Encéfalo/anatomia & histologia , Encéfalo/metabolismo , Técnicas de Cocultura , Sinais (Psicologia) , Efrina-A5/genética , Feminino , Fibroblastos/citologia , Fibroblastos/fisiologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Interneurônios/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células NIH 3T3 , Receptor EphA4/metabolismoRESUMO
The Eph receptor tyrosine kinases and their membrane-bound ligands, the ephrins, are involved in a variety of developmental processes such as axonal guidance, cell migration, cell adhesion, proliferation, and differentiation. In addition to repulsive effects, ephrins can also induce attractive responses. Up to now, little was known about the underlying signaling mechanisms that regulate attractive versus repulsive effects. In this study, we show that ephrin-A5 enhances the motility of cortical neurons that is dependent on the activity of Src-family kinases (SFKs). Ephrin-A5 further changes the adhesive properties of neurons by inducing the formation of cell aggregates. Using the stripe assay, we found that the motogenic effect of ephrin-A5 is the result of repulsive ephrin-A interactions. Blocking SFK function leads to a conversion of repulsion into adhesion, suggesting that SFKs can act as a biological switch for the response of EphA receptors. Finally, we discovered a ligand-induced release of membrane particles containing EphA receptors, suggesting membrane ripping as a novel mechanism to overcome the "ephrin paradox" of repulsion after high-affinity receptor-ligand binding.
Assuntos
Efrina-A5/fisiologia , Neurônios/enzimologia , Quinases da Família src/fisiologia , Animais , Agregação Celular/fisiologia , Movimento Celular/fisiologia , Humanos , Camundongos , Células NIH 3T3 , Neurônios/citologia , Neurônios/metabolismo , Receptor EphA5/antagonistas & inibidores , Receptor EphA5/metabolismo , Receptor EphA5/fisiologia , Quinases da Família src/antagonistas & inibidoresRESUMO
The complex task of wiring up the brain during embryonic development is achieved by a multitude of guidance signals acting in complex combinations to drive growing axons to their proper targets. The somatosensory system provides an extensively studied model system featuring many universal mechanisms of neural development. In rodents, it constitutes an important model to study how precise topographic connections are achieved. Recent evidence suggests that the Eph/ephrin family of guidance molecules is of pivotal importance for the development of the somatosensory system. Members of Eph/ephrin family are thought to be involved in the global presorting of thalamic axons projecting to the cortex, in labeling specific cortical areas for innervation, in providing topographic cues within the target area, and in distinguishing cortical layers for intracortical wiring. The Eph/ephrin system also seems to contribute to the formation of specific corticothalamic feedback projections. So far, the functions of only a few members of the Eph/ephrin family have been examined, but expression analysis indicates complex combinatorial effects of these signaling molecules. Understanding the Eph/ephrin wiring code is expected to yield new insights into the development and plasticity of brain circuits involved in higher functions.
Assuntos
Axônios/fisiologia , Efrinas/metabolismo , Córtex Somatossensorial/anatomia & histologia , Tálamo/anatomia & histologia , Animais , Humanos , Vias Neurais , Córtex Somatossensorial/metabolismo , Tálamo/metabolismoRESUMO
Olfactory receptor neurons select a single odourant receptor gene for expression out of a large gene family. The mechanisms of this extreme selectivity are largely unknown. We have determined in detail the developmental expression dynamics of a representative subset of the zebrafish odourant receptor repertoire, using in situ hybridization analysis. We have thus generated a dataset, which allows us to test hypotheses of odourant receptor gene regulation. The receptors chosen belong to four different groups with respect to ontogenetic onset of expression (onset groups). Statistical analysis of the data supports a model in which the final choice of an individual odourant receptor gene occurs stochastically from within a group of genes sharing a deterministically defined onset of expression. Genomic mapping revealed a pronounced correlation of onset of expression with genomic neighbourhood. During a protracted juvenile developmental period individual regulatory influences seem to modify the expression of odourant receptor genes, a notable example being a transient decrease in expressivity of two odourant receptor genes.