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1.
Nucleic Acids Res ; 52(10): 5804-5824, 2024 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-38676942

RESUMO

MicroRNAs (miRNAs) that share identical or near-identical sequences constitute miRNA families and are predicted to act redundantly. Yet recent evidence suggests that members of the same miRNA family with high sequence similarity might have different roles and that this functional divergence might be rooted in their precursors' sequence. Current knock-down strategies such as antisense oligonucleotides (ASOs) or miRNA sponges cannot distinguish between identical or near identical miRNAs originating from different precursors to allow exploring unique functions of these miRNAs. We here develop a novel strategy based on short 2'-OMe/LNA-modified oligonucleotides to selectively target specific precursor molecules and ablate the production of individual members of miRNA families in vitro and in vivo. Leveraging the highly conserved Xenopus miR-181a family as proof-of-concept, we demonstrate that 2'-OMe/LNA-ASOs targeting the apical region of pre-miRNAs achieve precursor-selective inhibition of mature miRNA-5p production. Furthermore, we extend the applicability of our approach to the human miR-16 family, illustrating its universality in targeting precursors generating identical miRNAs. Overall, our strategy enables efficient manipulation of miRNA expression, offering a powerful tool to dissect the functions of identical or highly similar miRNAs derived from different precursors within miRNA families.


Assuntos
MicroRNAs , Oligonucleotídeos , MicroRNAs/genética , MicroRNAs/metabolismo , Animais , Humanos , Oligonucleotídeos/química , Oligonucleotídeos/genética , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Precursores de RNA/metabolismo , Precursores de RNA/genética , Precursores de RNA/química , Xenopus/genética
2.
EMBO J ; 39(6): e102513, 2020 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-32073171

RESUMO

Various species of non-coding RNAs (ncRNAs) are enriched in specific subcellular compartments, but the mechanisms orchestrating their localization and their local functions remain largely unknown. We investigated both aspects using the elongating retinal ganglion cell axon and its tip, the growth cone, as models. We reveal that specific endogenous precursor microRNAs (pre-miRNAs) are actively trafficked to distal axons by hitchhiking primarily on late endosomes/lysosomes. Upon exposure to the axon guidance cue semaphorin 3A (Sema3A), pre-miRNAs are processed specifically within axons into newly generated miRNAs, one of which, in turn, silences the basal translation of tubulin beta 3 class III (TUBB3), but not amyloid beta precursor protein (APP). At the organismal level, these mature miRNAs are required for growth cone steering and a fully functional visual system. Overall, our results uncover a novel mode of ncRNA transport from one cytosolic compartment to another within polarized cells. They also reveal that newly generated miRNAs are critical components of a ncRNA-based signaling pathway that transduces environmental signals into the structural remodeling of subcellular compartments.


Assuntos
MicroRNAs/genética , RNA não Traduzido/genética , Transdução de Sinais , Animais , Axônios/fisiologia , Transporte Biológico , Endossomos/metabolismo , Feminino , Cones de Crescimento/fisiologia , Camundongos Endogâmicos C57BL , Precursores de RNA/genética , Células Ganglionares da Retina/fisiologia , Xenopus laevis
3.
RNA ; 28(3): 433-446, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34949721

RESUMO

Detection of nucleic acids within subcellular compartments is key to understanding their function. Determining the intracellular distribution of nucleic acids requires quantitative retention and estimation of their association with different organelles by immunofluorescence microscopy. This is particularly important for the delivery of nucleic acid therapeutics, which depends on endocytic uptake and endosomal escape. However, the current protocols fail to preserve the majority of exogenously delivered nucleic acids in the cytoplasm. To solve this problem, by monitoring Cy5-labeled mRNA delivered to primary human adipocytes via lipid nanoparticles (LNP), we optimized cell fixation, permeabilization, and immunostaining of a number of organelle markers, achieving quantitative retention of mRNA and allowing visualization of levels that escape detection using conventional procedures. The optimized protocol proved effective on exogenously delivered siRNA, miRNA, as well as endogenous miRNA. Our protocol is compatible with RNA probes of single molecule fluorescence in situ hybridization (smFISH) and molecular beacon, thus demonstrating that it is broadly applicable to study a variety of nucleic acids in cultured cells.


Assuntos
Imunofluorescência/métodos , Hibridização in Situ Fluorescente/métodos , RNA/metabolismo , Células Cultivadas , Fixadores/química , Corantes Fluorescentes/química , Células HeLa , Humanos , Nanopartículas/química , RNA/química , Processamento Pós-Transcricional do RNA , Transporte de RNA
4.
Int J Mol Sci ; 21(22)2020 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-33218218

RESUMO

During neuronal circuit formation, axons progressively develop into a presynaptic compartment aided by extracellular signals. Axons display a remarkably high degree of autonomy supported in part by a local translation machinery that permits the subcellular production of proteins required for their development. Here, we review the latest findings showing that microRNAs (miRNAs) are critical regulators of this machinery, orchestrating the spatiotemporal regulation of local translation in response to cues. We first survey the current efforts toward unraveling the axonal miRNA repertoire through miRNA profiling, and we reveal the presence of a putative axonal miRNA signature. We also provide an overview of the molecular underpinnings of miRNA action. Our review of the available experimental evidence delineates two broad paradigms: cue-induced relief of miRNA-mediated inhibition, leading to bursts of protein translation, and cue-induced miRNA activation, which results in reduced protein production. Overall, this review highlights how a decade of intense investigation has led to a new appreciation of miRNAs as key elements of the local translation regulatory network controlling axon development.


Assuntos
Axônios/metabolismo , MicroRNAs/genética , Neurônios/metabolismo , Biossíntese de Proteínas/genética , Animais , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
5.
Semin Cell Dev Biol ; 24(3): 146-55, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23219835

RESUMO

Since the discovery of the first microRNA (miRNA) almost 20 years ago, insight into their functional role has gradually been accumulating. This class of non-coding RNAs has recently been implicated as key molecular regulators in the biology of most eukaryotic cells, contributing to the physiology of various systems including immune, cardiovascular, nervous systems and also to the pathophysiology of cancers. Interestingly, Semaphorins, a class of evolutionarily conserved signalling molecules, are acknowledged to play major roles in these systems also. This, combined with the fact that Semaphorin signalling requires tight spatiotemporal regulation, a hallmark of miRNA expression, suggests that miRNAs could be crucial regulators of Semaphorin function. Here, we review evidence suggesting that Semaphorin signalling is regulated by miRNAs in various systems in health and disease. In particular, we focus on neural circuit formation, including axon guidance, where Semaphorin function was first discovered.


Assuntos
MicroRNAs/genética , Sistema Nervoso/metabolismo , Semaforinas/genética , Animais , Movimento Celular , Humanos , Sistema Nervoso/citologia , Plasticidade Neuronal , Semaforinas/metabolismo , Transdução de Sinais
6.
Gen Comp Endocrinol ; 205: 55-61, 2014 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-24746676

RESUMO

While growth hormone (GH) is obligatory for postnatal growth, it is not required for a number of growth-without-GH syndromes, such as early embryonic or fetal growth. Instead, these syndromes are thought to be dependent upon local growth factors, rather than pituitary GH. The GH gene is, however, also expressed in many extrapituitary tissues, particularly during early development and extrapituitary GH may be one of the local growth factors responsible for embryonic or fetal growth. Moreover, as the expression of the GH receptor (GHR) gene mirrors that of GH in extrapituitary tissues the actions of GH in early development are likely to be mediated by local autocrine or paracrine mechanisms, especially as extrapituitary GH expression occurs prior to the ontogeny of pituitary somatotrophs or the appearance of GH in the circulation. The extrapituitary expression of pituitary somatotrophs or the appearance of GH in the circulation. The extrapituitary expression of GH in embryos has also been shown to be of functional relevance in a number of species, since the immunoneutralization of endogenous GH or the blockade of GH production is accompanied by growth impairment or cellular apoptosis. The extrapituitary expression of the GH gene also persists in some central and peripheral tissues postnatally, which may reflect its continued functional importance and physiological or pathophysiological significance. The expression and functional relevance of extrapituitary GH, particularly during embryonic growth, is the focus of this brief review.


Assuntos
Hormônio do Crescimento/metabolismo , Hipófise/crescimento & desenvolvimento , Hipófise/metabolismo , Animais , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Hormônio do Crescimento/genética , Hipófise/embriologia , Somatotrofos/metabolismo
7.
Methods Mol Biol ; 2765: 107-126, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38381336

RESUMO

CircRNAs are covalently closed RNA molecules gaining increasing attention over the years. Initially considered mere splicing errors, circRNAs are now recognized as a novel class of endogenous, conserved RNAs, expressed in many different species. The unique structure, the low levels of expression, and the almost complete sequence overlap with the cognate linear RNA make their detection and quantification challenging. Moreover, it has become crucial to prove the circular nature of the targeted transcript and unequivocally distinguish the circRNA from its linear counterpart. Nowadays, the most widely used technique to quantify circRNA expression is real-time quantitative PCR (qPCR). However, in the particular case of quantification of circles, it shows several technical shortcomings which affect the accuracy of the quantification. To precisely assess circRNA expression level, droplet digital PCR (ddPCR) is rapidly taking over for the more popular qPCR. In this chapter, we describe the detailed procedure based on droplets partitioning to quantify both linear and circRNA abundancy and demonstrate the circularity of the transcript under study with high precision, in a single experiment.

8.
J Neurosci ; 32(1): 223-8, 2012 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-22219284

RESUMO

When neurons exit the cell cycle after their terminal mitosis, they detach from the apical surface of the neuroepithelium. Despite the fact that this detachment is crucial for further neurogenesis and neuronal migration, the underlying mechanisms are still not understood. Here, taking advantage of the genetics and imaging possibilities of the zebrafish retina as a model system, we show by knockdown experiments that the guidance molecule Slit1b and its receptor Robo3 are required for apical retraction of retinal ganglion cells (RGCs). In contrast, N-cadherin seems to be responsible for maintenance of apical attachment, as expression of dominant-negative N-cadherin causes RGCs to lose apical attachments prematurely and rescues retraction in slit1b morphants. These results suggest that Slit-Robo signaling downregulates N-cadherin activity to allow apical retraction in newly generated RGCs.


Assuntos
Caderinas/fisiologia , Diferenciação Celular/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Receptores Imunológicos/fisiologia , Retina/embriologia , Células Ganglionares da Retina/metabolismo , Proteínas de Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Retina/citologia , Células Ganglionares da Retina/citologia , Peixe-Zebra
9.
Sci Adv ; 6(38)2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32948587

RESUMO

We report the construction of artificial cells that chemically communicate with mammalian cells under physiological conditions. The artificial cells respond to the presence of a small molecule in the environment by synthesizing and releasing a potent protein signal, brain-derived neurotrophic factor. Genetically controlled artificial cells communicate with engineered human embryonic kidney cells and murine neural stem cells. The data suggest that artificial cells are a versatile chassis for the in situ synthesis and on-demand release of chemical signals that elicit desired phenotypic changes of eukaryotic cells, including neuronal differentiation. In the future, artificial cells could be engineered to go beyond the capabilities of typical smart drug delivery vehicles by synthesizing and delivering specific therapeutic molecules tailored to distinct physiological conditions.

10.
Gen Comp Endocrinol ; 163(1-2): 63-9, 2009 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-19344664

RESUMO

Growth hormone (GH) is found in the retina and vitreous of the chick embryo, where it appears to act as a growth and differentiation factor, having neuroprotective effects on retinal ganglion cells (RGCs). Here, we review the molecular mechanisms of the anti-apoptotic effect of GH in chick RGCs. GH treatment of RGCs reduces Akt levels, while raising Akt-phos levels, consistent with a role for Akt signaling pathways in the GH neuroprotective action. The induction of apoptosis by immunoneutralization with GH antiserum is accompanied by an increase in caspase-3 and caspase-9 activation, and also PARP-1 cleavage. Calpain activation also appears to be a major caspase-independent pathway to PARP-1 cleavage and apoptosis in these cells, supporting the view that caspase and calpain inhibitors are major neuroprotective agents for RGCs, and that pathways that activate both caspases and calpains are important for the anti-apoptotic actions of GH in these cells. These pathways involve the activation of cytosolic tyrosine kinases (Trks) and extracellular-signal-related kinases (ERKs). Occupation of the GH receptor by GH involves downstream intracellular Trk pathways. The Akt and Trk pathways appear to converge on the activation of cAMP response element binding protein (CREB), which is able to initiate transcription of pro- or anti-apoptotic genes. These results indicate that the action of GH in the neuroprotection of embryonic RGCs involves pathways common to with other neurotrophins, and that GH can be considered to be a growth and differentiation factor in the development of the embryonic retina. We have also investigated the relationship between the overlapping anti-apoptotic effects of GH and insulin-like growth factor-1 (IGF-1), two functionally closely related factors. We find that simultaneous immunoneutralization of GH and IGF-1 does not increase the level of apoptosis in the cultures above that achieved by immunoneutralization of GH alone. We therefore conclude that the neuroprotective actions of GH in the developing retina are likely mediated in large part through the action of IGF-1.


Assuntos
Galinhas/fisiologia , Hormônio do Crescimento/metabolismo , Retina/embriologia , Retina/metabolismo , Transdução de Sinais , Animais , Embrião de Galinha , Galinhas/crescimento & desenvolvimento , Galinhas/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Células Ganglionares da Retina/metabolismo , Transdução de Sinais/fisiologia
11.
J Mol Neurosci ; 31(3): 261-71, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17726230

RESUMO

A novel variant of chicken growth hormone (cGH) that is severely truncated has recently been discovered in the neural retina. It is, however, unknown whether this protein binds to GH receptors (GHRs) and has biological activity. This possibility has therefore been addressed by homology modeling, using human (h)GH as a template because it is the only GH molecule with a crystal structure and because hGH binds to cGH receptors (cGHRs). Most of the residues of the small (s)cGH model fitted the hGH template, apart from two restricted regions from Ser 12 to Gln 20 and from Ser 55 to Val 58. The scGH model differs, however, from hGH in structure: hGH is composed of a four-helix bundle, whereas scGH has three main helices. Helices 2, 3, and 4 of hGH correspond to helices 1, 2, and 3 of scGH, but they are longer by one, four, and one residues, respectively. The secondary structure of the C-terminus of scGH is therefore similar to C-terminal hGH. The N-terminus of scGH is, however, severely truncated, lacking the residues of the full-length molecule derived from exons 1, 2, and 3. The N-terminus of scGH also includes 20 residues derived from intron C of full-length cGH. The predicted structure of its N-terminus has no classical secondary structure (alpha-helix or beta-sheet), whereas the N-terminus of hGH is composed of helix 1 and two mini-helices located between helix 1 and 2. This difference in ribbon structure results in a difference in the overall shape of the scGH model and hGH. The possibility that scGH could bind to a GHR dimer was assessed by examining the primary and hypothetical tertiary structure of scGH. hGH binds the extracellular domain (ECD) of two GHRs sequentially at its binding site 1 (or high affinity site) then at its binding site 2 (or low affinity site). Sequence alignment of scGH with hGH demonstrates that scGH lacks three key residues (of 14) at site 1 and nine residues (of 15) at site 2. It is therefore unlikely that tight binding of ECD1 to the site 1 of scGH could occur. scGH also lacks most of the site 2 residues, suggesting that it is unlikely that ECD2 would bind to the scGH model. In summary, we have developed a novel, structural model of scGH, with implications for its putative actions through classical GHRs.


Assuntos
Galinhas , Hormônio do Crescimento/química , Hormônio do Crescimento/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Retina/química , Sequência de Aminoácidos , Animais , Hormônio do Crescimento/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia Estrutural de Proteína
12.
Pediatr Endocrinol Rev ; 5(1): 510-5, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17925792

RESUMO

While growth hormone (GH) is obligatory for postnatal growth, its possible involvement in ocular development and vision is poorly understood. The eye is, however, a target site for GH action and GH production and GH may have endocrine, autocrine and/or paracrine roles in ocular development. The importance of GH in ocular development is demonstrated by the ocular abnormalities that can occur in patients with pituitary GH excess or GH deficiency. Clinical and basic studies supporting roles for GH in ocular development are the focus of this brief review.


Assuntos
Hormônio do Crescimento , Hormônio do Crescimento Humano , Humanos , Hipófise
13.
Cell Rep ; 18(5): 1171-1186, 2017 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-28147273

RESUMO

During brain wiring, cue-induced axon behaviors such as directional steering and branching are aided by localized mRNA translation. Different guidance cues elicit translation of subsets of mRNAs that differentially regulate the cytoskeleton, yet little is understood about how specific mRNAs are selected for translation. MicroRNAs (miRNAs) are critical translational regulators that act through a sequence-specific mechanism. Here, we investigate the local role of miRNAs in mRNA-specific translation during pathfinding of Xenopus laevis retinal ganglion cell (RGC) axons. Among a rich repertoire of axonal miRNAs, miR-182 is identified as the most abundant. Loss of miR-182 causes RGC axon targeting defects in vivo and impairs Slit2-induced growth cone (GC) repulsion. We find that miR-182 targets cofilin-1 mRNA, silencing its translation, and Slit2 rapidly relieves the repression without causing miR-182 degradation. Our data support a model whereby miR-182 reversibly gates the selection of transcripts for fast translation depending on the extrinsic cue.


Assuntos
Orientação de Axônios/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , MicroRNAs/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Biossíntese de Proteínas/fisiologia , RNA Mensageiro/metabolismo , Animais , Axônios/metabolismo , Regulação da Expressão Gênica/fisiologia , Cones de Crescimento/metabolismo , Células Ganglionares da Retina/metabolismo , Xenopus laevis/metabolismo
14.
J Mol Neurosci ; 28(3): 257-64, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16691013

RESUMO

Growth hormone (GH) mRNA and protein have recently been localized in the neural retina of embryonic chicks, in which exogenous GH promotes cell survival. GH is also expressed in the rat CNS, in which it has neuroprotective roles, although its presence in the rat neural retina is unknown and is the focus of the present study. GH immunoreactivity, to a 22-kDa protein, was present in extracts of fetal (embryonic day [ED]17) eyes and in extracts from the neural retinas of newborn pups, comparable to GH immunoreactivity in pituitary extracts. The GH immunoreactivity in the neural retina was widespread but was most intense in large rounded cells in the retinal ganglion cell (RGC) layer and in the optic fiber layer derived from the axons of the RGCs. A 693-bp cDNA was also generated by the RT-PCR of RNA extracted from the eyes of ED17 rats and from the neural retinas and eyes of newborn rats, when amplified in the presence of oligonucleotide primers for the rat GH cDNA. Expression of the GH gene in the neural retina was also shown by specific in situ hybridization of an antisense GH riboprobe to cells in the neural retina, particularly those in the RGC layers of fetal and adult rat eyes. These results demonstrate GH expression in the neural retinas of fetal, newborn, and adult rats, in which retinal GH might have neuroprotective roles.


Assuntos
Hormônio do Crescimento/metabolismo , Retina/química , Animais , Animais Recém-Nascidos , Hormônio do Crescimento/genética , Hibridização In Situ , Ratos , Ratos Sprague-Dawley , Retina/citologia , Retina/embriologia
15.
Neuroreport ; 17(16): 1715-8, 2006 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-17047459

RESUMO

Growth hormone has recently been shown to be expressed in the retinal ganglion cells of embryonic chicks, in which it induces cell survival during neurogenesis. The mechanism of this action has been examined in neural retina explants from 6-day-old and 8-day-old embryos that were incubated for 48 h in 10 M growth hormone, to reduce the number of spontaneous apoptotic cells. This anti-apoptotic action was accompanied by a reduction in caspase-3 expression and, at embryonic day 8, by reduced expression of apoptosis inducing factor-1, which is caspase independent. These actions were specific, as other genes involved in apoptotic signaling (bcl-2, bcl-x, bid and inhibitor of apoptosis protein-1) were unaffected. These results therefore demonstrate caspase-dependent and caspase-independent pathways in growth hormone-induced retinal cell survival.


Assuntos
Caspase 3/fisiologia , Hormônio do Crescimento/fisiologia , Células Ganglionares da Retina/enzimologia , Animais , Apoptose , Fator de Indução de Apoptose/biossíntese , Caspase 3/biossíntese , Sobrevivência Celular , Embrião de Galinha , Desenvolvimento Embrionário , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , RNA Mensageiro/biossíntese , Células Ganglionares da Retina/citologia , Técnicas de Cultura de Tecidos
16.
Int J Dev Biol ; 60(4-6): 151-7, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27389985

RESUMO

Waves of Bone Morphogenetic Proteins (BMPs) and their antagonists are present during initial eye development, but their possible roles in retinogenesis are still unknown. We have recently shown that noggin 1, a BMP antagonist, renders pluripotent cells able to differentiate into retinal precursors, and might be involved in the maintenance of retinal structures in the adult vertebrate eye. Here, we report that noggin 1, differently from noggin 2 and noggin 4, is expressed during all phases of Xenopus laevis retinal development. Gain-of-function experiments by electroporation in the optic vesicle show that overexpression of noggin 1 significantly decreases the number of bipolar cells in the inner nuclear layer of the retina, without significantly affecting the generation of the other retinal cell types. Our data suggest that BMP signaling could be involved in the differentiation of retinal progenitors into specific retinal subtypes during late phases of vertebrate retinal development.


Assuntos
Proteínas de Transporte/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Retina/metabolismo , Proteínas de Xenopus/genética , Animais , Proteínas de Transporte/metabolismo , Proteínas de Homeodomínio/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Retina/embriologia , Neurônios Retinianos/citologia , Neurônios Retinianos/metabolismo , Transdução de Sinais/fisiologia , Proteínas de Xenopus/metabolismo , Xenopus laevis
17.
Endocrinology ; 144(12): 5459-68, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12960021

RESUMO

The presence of GH and GH mRNA in the eyes of embryonic chicks is controversial and has, therefore, been further examined. In this study, GH cDNAs identical in size and sequence to the full-length pituitary transcript were generated by RT-PCR from mRNA extracted from the neural retinas of embryonic day (ED) 7 chick embryo eyes. GH immunoreactivity in the neural retina of embryos was primarily associated with proteins of 15 and 16 kDa, whereas only trace amounts of monomer (22-25 kDa) GH, the most abundant form in the pituitary gland, were present. GH immunoreactivity was also present in the vitreous humor, although this was associated only with the 15-kDa protein. After hatch, retinal proteins with GH immunoreactivity of 15 and 16 kDa were present neonatally but not after 42 d of age. The GH immunoreactivity in the neural retina of ED8 embryos was widespread, although GH staining was particularly abundant in retinal ganglion cells (RGCs). Full-length GH mRNA was similarly located, by in situ hybridization, throughout the neural retina and concentrated in cells in the RGC layer. The neural retina is also a site of GH action because 10-6 m chicken GH greatly increased (4- to 5-fold) the content of IGF-1 mRNA in 48-h cultured ED8 neural retinas. These results demonstrate the presence of full-length GH mRNA in the neural retina of chick embryos, in which GH immunoreactivity is primarily associated with RGCs and submonomer GH proteins of 15-16 kDa. These results also demonstrate GH action in the neural retina of embryos and suggest hitherto unsuspected roles for GH in retinal development and/or ocular function.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Hormônio do Crescimento/genética , Retina/embriologia , Retina/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Embrião de Galinha , Galinhas , Hormônio do Crescimento/metabolismo , Imuno-Histoquímica , Fator de Crescimento Insulin-Like I/genética , Dados de Sequência Molecular , RNA Mensageiro/análise
18.
Front Cell Neurosci ; 8: 78, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24672429

RESUMO

Brain wiring is a highly intricate process in which trillions of neuronal connections are established. Its initial phase is particularly crucial in establishing the general framework of neuronal circuits. During this early step, differentiating neurons extend axons, which reach their target by navigating through a complex environment with extreme precision. Research in the past 20 years has unraveled a vast and complex array of chemotropic cues that guide the leading tip of axons, the growth cone, throughout its journey. Tight regulation of these cues, and of their receptors and signaling pathways, is necessary for the high degree of accuracy required during circuit formation. However, little is known about the nature of regulatory molecules or mechanisms fine-tuning axonal cue response. Here we review recent, and somewhat fragmented, research on the possibility that microRNAs (miRNAs) could be key fine-tuning regulatory molecules in axon guidance. miRNAs appear to shape long-range axon guidance, fasciculation and targeting. We also present several lines of evidence suggesting that miRNAs could have a compartmentalized and differential action at the cell soma, and within axons and growth cones.

19.
Nat Neurosci ; 16(2): 166-73, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23292679

RESUMO

Cell adhesion molecules and diffusible cues both regulate axon pathfinding, yet how these two modes of signaling interact is poorly understood. The homophilic cell adhesion molecule NF-protocadherin (NFPC) is expressed in the mid-dorsal optic tract neuroepithelium and in the axons of developing retinal ganglion cells (RGC) in Xenopus laevis. Here we report that targeted disruption of NFPC function in RGC axons or the optic tract neuroepithelium results in unexpectedly localized pathfinding defects at the caudal turn in the mid-optic tract. Semaphorin 3A (Sema3A), which lies adjacent to this turn, stimulates rapid, protein synthesis-dependent increases in growth cone NFPC and its cofactor, TAF1, in vitro. In vivo, growth cones exhibit marked increases in NFPC translation reporter activity in this mid-optic tract region that are attenuated by blocking neuropilin-1 function. Our results suggest that translation-linked coupling between regionally localized diffusible cues and cell adhesion can help axons navigate discrete segments of the pathway.


Assuntos
Axônios/fisiologia , Caderinas/fisiologia , Sinais (Psicologia) , Biossíntese de Proteínas , Células Ganglionares da Retina/citologia , Transdução de Sinais/fisiologia , Vias Visuais/fisiologia , Proteínas de Xenopus/fisiologia , Análise de Variância , Animais , Anisomicina/farmacologia , Células COS , Caderinas/genética , Chlorocebus aethiops , Eletroporação , Embrião não Mamífero , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia Confocal , Células Neuroepiteliais/fisiologia , Técnicas de Cultura de Órgãos , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/fisiologia , Inibidores da Síntese de Proteínas/farmacologia , Protocaderinas , Retina/citologia , Semaforina-3A/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Fatores de Transcrição/metabolismo , Transfecção , Proteínas de Xenopus/genética , Xenopus laevis
20.
Mol Brain ; 4: 40, 2011 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-22051374

RESUMO

BACKGROUND: The small non-coding microRNAs play an important role in development by regulating protein translation, but their involvement in axon guidance is unknown. Here, we investigated the role of microRNA-134 (miR-134) in chemotropic guidance of nerve growth cones. RESULTS: We found that miR-134 is highly expressed in the neural tube of Xenopus embryos. Fluorescent in situ hybridization also showed that miR-134 is enriched in the growth cones of Xenopus spinal neurons in culture. Importantly, overexpression of miR-134 mimics or antisense inhibitors blocked protein synthesis (PS)-dependent attractive responses of Xenopus growth cones to a gradient of brain-derived neurotrophic factor (BDNF). However, miR-134 mimics or inhibitors had no effect on PS-independent bidirectional responses of Xenopus growth cones to bone morphogenic protein 7 (BMP7). Our data further showed that Xenopus LIM kinase 1 (Xlimk1) mRNA is a potential target of miR-134 regulation. CONCLUSIONS: These findings demonstrate a role for miR-134 in translation-dependent guidance of nerve growth cones. Different guidance cues may act through distinct signaling pathways to elicit PS-dependent and -independent mechanisms to steer growth cones in response to a wide array of spatiotemporal cues during development.


Assuntos
Quimiotaxia , Cones de Crescimento/metabolismo , MicroRNAs/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Quimiotaxia/efeitos dos fármacos , Imunofluorescência , Regulação da Expressão Gênica/efeitos dos fármacos , Cones de Crescimento/efeitos dos fármacos , Cones de Crescimento/enzimologia , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Modelos Biológicos , Oligonucleotídeos Antissenso/farmacologia , Fosforilação/efeitos dos fármacos , Xenopus , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo
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