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ABSTRACT: Cell therapy in animal models of Parkinson's disease (PD) is effective after intrastriatal grafting of dopamine (DA) neurons, whereas intranigral transplantation of dopaminergic cells does not cause consistent behavioral recovery. One strategy to promote axonal growth of dopaminergic neurons from the substantia nigra (SN) to the striatum is degradation of inhibitory components such as chondroitin sulphate proteoglycans (CSPG). An alternative is the guidance of DA axons by chemotropic agents. Semaphorins 3A and 3C enhance axonal growth of embryonic stem (ES) cell-derived dopaminergic neurons in vitro, while Semaphorin 3C also attracts them. We asked whether intranigral transplantation of DA neurons, combined with either degradation of CSPG or with grafts of Semaphorin 3-expressing cells, towards the striatum, is effective in establishing a new nigrostriatal dopaminergic pathway in rats with unilateral depletion of DA neurons. We found depolarization-induced DA release in dorsal striatum, DA axonal projections from SN to striatum, and concomitant behavioral improvement in Semaphorin 3-treated animals. These effects were absent in animals that received intranigral transplants combined with Chondroitinase ABC treatment, although partial degradation of CSPG was observed. These results are evidence that Semaphorin 3-directed long-distance axonal growth of dopaminergic neurons, resulting in behavioral improvement, is possible in adult diseased brains.Molecular Therapy (2013); doi:10.1038/mt.2013.78.
Molecular Therapy 06/2013; · 6.87 Impact Factor
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ABSTRACT: Chemotropic proteins guide neuronal projections to their final target during embryo development and are useful to guide axons of neurons used in transplantation therapies. Site-specific delivery of the proteins however is needed for their application in the brain to avoid degradation and pleiotropic affects. In the present study we report the use of Poly (ethylene glycol)-Silica (PEG-Si) nanocomposite gel with thixotropic properties that make it injectable and suitable for delivery of the chemotropic protein semaphorin 3A. PEG-Si gel forms a functional gradient of semaphorin that enhances axon outgrowth of dopaminergic neurons from rat embryos or differentiated from stem cells in culture. It is not cytotoxic and its properties allowed its injection into the striatum without inflammatory response in the short term. Long term implantation however led to an increase in macrophages and glial cells. The inflammatory response could have resulted from non-degraded silica particles, as observed in biodegradation assays.
Journal of Materials Science Materials in Medicine 07/2011; 22(9):2097-109. · 2.32 Impact Factor
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ABSTRACT: During early vertebrate forebrain development, pioneer axons establish a symmetrical scaffold descending longitudinally through the rostral forebrain, thus forming the tract of the postoptic commissure (TPOC). In mouse embryos, this tract begins to appear at embryonic day 9.5 (E9.5) as a bundle of axons tightly constrained at a specific dorsoventral level. We have characterized the participation of the Slit chemorepellants and their Robo receptors in the control of TPOC axon projection. In E9.5-E11.5 mouse embryos, Robo1 and Robo2 are expressed in the nucleus origin of the TPOC (nTPOC), and Slit expression domains flank the TPOC trajectory. These findings suggested that these proteins are important factors in the dorsoventral positioning of the TPOC axons. Consistently with this role, Slit2 inhibited TPOC axon growth in collagen gel cultures, and interfering with Robo function in cultured embryos induced projection errors in TPOC axons. Moreover, absence of both Slit1 and Slit2 or Robo1 and Robo2 in mutant mouse embryos revealed aberrant TPOC trajectories, resulting in abnormal spreading of the tract and misprojections into both ventral and dorsal tissues. These results reveal that Slit-Robo signaling regulates the dorsoventral position of this pioneer tract in the developing forebrain.
Journal of Neuroscience Research 06/2011; 89(10):1531-41. · 2.74 Impact Factor
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ABSTRACT: Embryonic neurogenesis is controlled by the activation of specific genetic programs. In the hypothalamus, neuronal thyrotropin-releasing hormone (TRH) populations control important physiological process, including energy homeostasis and autonomic function; however, the genetic program leading to the TRH expression is poorly understood. Here, we show that the Klf4 gene, encoding the transcription factor Krüppel-like factor 4 (Klf4), was expressed in the rat hypothalamus during development and regulated Trh expression. In rat fetal hypothalamic cells Klf4 regulated Trh promoter activity through CACCC and GC motifs present on the Trh gene promoter. Accordingly, hypothalamic Trh expression was down-regulated at embryonic day 15 in the Klf4(-/-) mice resulting in diminished bioactive peptide levels. Although at the neonatal stage the Trh transcript levels of the Klf4(-/-) mice were normal, the reduction in peptide levels persisted. Thus, our data indicate that Klf4 plays a key role in the maturation of TRH expression in hypothalamic neurons.
Molecular and Cellular Endocrinology 02/2011; 333(2):127-33. · 4.19 Impact Factor
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Amaya Miquelajáuregui, Alfredo Varela-Echavarría,
M Laura Ceci,
Fernando García-Moreno,
Itzel Ricaño,
Kimmi Hoang,
Daniela Frade-Pérez,
Carlos Portera-Cailliau,
Elisa Tamariz,
Juan A De Carlos,
Heiner Westphal,
Yangu Zhao
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ABSTRACT: Cajal-Retzius (C-R) cells play important roles in the lamination of the mammalian cortex via reelin secretion. The genetic mechanisms underlying the development of these neurons have just begun to be unraveled. Here, we show that two closely related LIM-homeobox genes Lhx1 and Lhx5 are expressed in reelin+ cells in various regions in the mouse telencephalon at or adjacent to sites where the C-R cells are generated, including the cortical hem, the mantle region of the septal/retrobulbar area, and the ventral pallium. Whereas Lhx5 is expressed in all of these reelin-expressing domains, Lhx1 is preferentially expressed in the septal area and in a continuous domain spanning from lateral olfactory region to caudomedial territories. Genetic ablation of Lhx5 results in decreased reelin+ and p73+ cells in the neocortical anlage, in the cortical hem, and in the septal, olfactory, and caudomedial telencephalic regions. The overall reduction in number of C-R cells in Lhx5 mutants is accompanied by formation of ectopic reelin+ cell clusters at the caudal telencephalon. Based on differential expression of molecular markers and by fluorescent cell tracing in cultured embryos, we located the origin of reelin+ ectopic cell clusters at the caudomedial telencephalic region. We also confirmed the existence of a normal migration stream of reelin+ cells from the caudomedial area to telencephalic olfactory territories in wild-type embryos. These results reveal a complex role for Lhx5 in regulating the development and normal distribution of C-R cells in the developing forebrain.
Journal of Neuroscience 08/2010; 30(31):10551-62. · 7.11 Impact Factor
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ABSTRACT: Class 3 Semaphorins are a subfamily of chemotropic molecules implicated in the projection of dopaminergic neurons from the ventral mesencephalon and in the formation of the nigrostriatal pathway (NSP) during embryonic development. In humans, loss of mesencephalic dopaminergic neurons leads to Parkinson's disease (PD). Cell replacement therapy with dopaminergic neurons generated from embryonic stem cells (ES-TH(+)) is being actively explored in models of PD. Among several requisites for this approach to work are adequate reconstruction of the NSP and correct innervation of normal striatal targets by dopaminergic axons. In this work, we characterized the response of ES-TH(+) neurons to semaphorins 3A, 3C, and 3F and compared it with that of tyrosine hidroxylase-positive neurons (TH(+)) obtained from embryonic ventral mesencephalon (VM-TH(+)). We observed that similar proportions of ES-TH(+) and VM-TH(+) neurons express semaphorin receptors neuropilins 1 and 2. Furthermore, the axons of both populations responded very similarly to semaphorin exposure: semaphorin 3A increased axon length, and semaphorin 3C attracted axons and increased their length. These effects were mediated by neuropilins, insofar as addition of blocking antibodies against these proteins reduced the effects on axonal growth and attraction, and only TH(+) axons expressing neuropilins responded to the semaphorins analyzed. The observations reported here show phenotypic similarities between VM-TH(+) and ES-TH(+) neurons and suggest that semaphorins 3A and 3C could be employed to guide axons of grafted ES-TH(+) in therapeutic protocols for PD.
Journal of Neuroscience Research 10/2009; 88(5):971-80. · 2.74 Impact Factor
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ABSTRACT: In the adult brain, neuroblasts originating in the subventricular zone migrate through the rostral migratory stream to the olfactory bulb. While migrating, neuroblasts undergo progressive differentiation until reaching their final locations and fates. Because molecules involved in migration may also exert differentiating effects on young neurons, the identification of factors that support migration could also shed light on the processes of adult neuroblast differentiation. This is the case for members of the family of semaphorins and of its cognate receptors, the neuropilins. Here, we have evaluated the presence of semaphorin-3A and of its receptor neuropilin-1 along the rostral migratory stream in young and adult mice by using immunocytochemical, histochemical, and in situ hybridization techniques. Our morphological studies show that semaphorin-3A and neuropilin-1 are both mainly expressed on endothelial cells along the rostral migratory stream during postnatal development. Our results suggest that endothelial cells constitute the primary source and target of semaphorin-3A along the rostral migratory stream. Moreover, the present work outlines the potential role of blood vessels on neuroblast migration in the postnatal rostral migratory stream.
Cell and Tissue Research 07/2008; 333(2):175-84. · 3.11 Impact Factor
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ABSTRACT: Information from both sides of the brain is integrated by axons that project across the midline of the central nervous system via numerous commissures present at all axial levels. Despite the accumulated experimental evidence, questions remain regarding the formation of commissures in the presence of strong repulsive signals in the ventral midline. Studies from invertebrates suggest that interaction at the midline between homologous axons of specific decussating neurons contributes to efficient midline crossing, but such evidence is lacking in vertebrate systems. We performed experiments to determine whether commissural axons of the caudal region of the hindbrain interact with their contralateral counterparts at the ventral midline and to evaluate the relevance of this reciprocal interaction. Double anterograde axon labeling with lipophilic tracers revealed close apposition between growth cones of contralateral pioneer decussating axons at the midline. Later, we detected fasciculation between contralateral axons that is maintained even after they have crossed the midline. Blocking axon projections unilaterally with a solid mechanical barrier decreased dramatically the midline crossing of the equivalent population from the contralateral side. Decussation was also blocked by a unilateral barrier permeable to diffusible molecules but not by an axon-permeable barrier. These results suggest that in the caudal region of the hindbrain, midline crossing is facilitated by interactions between decussating contralateral axon partners.
Developmental Neurobiology 03/2008; 68(3):349-64. · 3.55 Impact Factor
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ABSTRACT: By analyzing the mechanisms that govern dopaminergic axon pathfinding from the midbrain to the striatum in embryonic rat brains, we identified neuroepithelial regions that exert chemotropic effects on mesencephalic dopaminergic axons. Explants from the pretectum and the striatum showed an attractive effect, whereas those from the midhindbrain boundary, the dorsal thalamus, and the ventral thalamus had no effect. Expression of semaphorin (Sema) 3C and Sema3F in the pretectum and of Sema3A in the striatum suggested a role for these axon guidance molecules in dopaminergic axon pathfinding. When expressed in HEK293 cell aggregates, Sema3C had an attractive effect and enhanced axon growth, Sema3A enhanced axon growth, and Sema3F had a repulsive effect on dopaminergic axons. Antineuropilin-1 and antineuropilin-2 antibodies reduced attraction by the pretectum, whereas attraction by the striatum was not affected by the presence of antineuropilin-1 antibodies. Moreover, neuropilin-1- and neuropilin-2-soluble Fc chimeras reduced the attraction by the pretectum. These results suggest that semaphorins may help to establish the dopaminergic projection from the midbrain to the striatum during embryonic development.
The Journal of Comparative Neurology 02/2008; 506(3):387-97. · 3.81 Impact Factor
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ABSTRACT: Con objeto de obtener subcultivos seriales de preadipocitos del tejido adiposo bovino y luego tratar de diferenciarlos a adipocitos, se compararon dos medios de cultivo. Se obtuvieron muestras de tejido adiposo subcutáneo y de omento de animales adultos de 3 a 4 años de edad, y de grasa perirrenal y de omento de fetos en el último tercio de la gestación. En el caso de células adultas, se establecieron subcultivos; sin embargo no pudieron ser diferenciadas. Para el tejido adiposo fetal, se establecieron subcultivos y los fibroblastos perirrenales fueron capaces de diferenciarse con el medio apropiado (DMEM/ F-12 1:1; 10 µg/ml insulina; 0.25 µM dexametasona; 10 µg/ml lipoproteínas de muy baja densidad; 100 µg/ml estreptomicina; 100 UI/ml penicilina). Estos resultados podrían servir de base para obtener un modelo que permita estudiar los mecanismos moleculares de diferenciación de preadipocitos en tejido adiposo bovino.
Técnica Pecuaria en México. 01/2008;
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ABSTRACT: Semaphorins constitute a family of signaling molecules with functions in axon pathfinding and neuronal migration. Neuropilins 1 and 2 have been identified as the ligand-binding component of semaphorin receptors. Both ligands and receptors are expressed in embryonic and adult organs in complementary and sometimes redundant patterns. In the present work, we compared the brain expression patterns of the class III semaphorins 3A, 3C, and 3F and neuropilins 1 and 2 between mouse and chick embryos at early developmental stages. Our studies revealed that expression of semaphorins is restricted in some cases to neuromeric transverse domains, to specific neuromeric boundaries, and to specific neuronal populations. Moreover, our studies also revealed coexpression of neuropilins and one or more semaphorins in some of the different expression sites. Comparison of the expression patterns between mouse and chick embryos showed large similarities, but important differences were also detected.
Brain Research 02/2006; 1067(1):126-37. · 2.73 Impact Factor
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ABSTRACT: The previous findings that both a long and a short type 1 deiodinase (D1) mRNA are present in different tissues and that the D1 gene contains two potential polyA signals suggest that the two mRNAs result from differential polyA signal usage. In this study, we examined the properties of the two D1 mRNAs generated in HEK 293 cells by the alternative use of each of the poly A signals in order to ascertain the potential regulatory role of the 3'UTR of this gene. Our results showed that the long mRNA is less stable, but that it is translated more efficiently than the short mRNA. The net result of these differences is a higher D1 activity with the long message. These data suggest that the D1 3'UTR may play an important role in regulating the stability and translational efficiency of the D1 mRNA, both of which could be physiologically relevant when the demand for D1 activity is high.
Endocrine 09/2005; 27(3):219-25. · 1.42 Impact Factor
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ABSTRACT: Hindbrain reticulospinal neurons are involved in complex neural functions that are mediated by spinal elements, including posture control and modulation of respiration and cardiovascular function. Recent descriptive studies with chick, mouse, and rat embryos have provided anatomical insight into the development of the different reticulospinal nuclei and the establishment of their axonal projection pathways into the spinal cord. In this study, we have addressed the molecular control of this process. Retrograde labeling of reticulospinal neurons in chick and mouse embryos combined with immunostaining for the homeodomain factors Lhx1/Lhx5, Lhx3/Lhx4, and Chx10 have defined transcriptional codes that label subsets of neurons with different axon projection patterns. Gain of function and loss of function experiments using in ovo electroporation implicate these transcription factors in the determination of reticulospinal neuron identity. Furthermore, our studies reveal novel gene interactions between the transcription factors analyzed that may determine the final patterns of reticulospinal axon projection.
Molecular and Cellular Neuroscience 02/2005; 28(1):30-41. · 3.66 Impact Factor
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ABSTRACT: Vitamin A is essential for vertebrate embryonic development; dietary carotenoids are the primary source of vitamin A since animals cannot synthesize it de novo. To study the role of beta-carotene during embryonic development, we analyzed in chick embryos the expression of beta,beta-carotene 15,15'-oxygenase (beta-oxy) which cleaves beta-carotene to produce two molecules of retinal. Beta-oxy transcripts were detected in one-and-a-half- to five-day-old embryo homogenates and in situ hybridization in five-day-old embryos, revealing their presence in tissues including the central nervous system, lungs, limbs, and cardiovascular system. Moreover, we detected beta-oxy enzymatic activity in extracts from five-day-old embryos as well as small amounts of beta-carotene in the egg yolk. These results indicate that beta-oxy is present during early developmental stages, raising the possibility that yolk-stored beta-carotene is utilized as a source of vitamin A. Thus, our results suggest that beta-carotene could play an important role in early avian embryonic development as a local source of vitamin A in specific tissues.
International Journal for Vitamin and Nutrition Research 04/2004; 74(2):116-22. · 0.88 Impact Factor
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ABSTRACT: We have addressed the control of longitudinal axon pathfinding in the developing hindbrain, including the caudal projections of reticular and raphe neurons. To test potential sources of guidance signals, we assessed axon outgrowth from embryonic rat hindbrain explants cultured in collagen gels at a distance from explants of midbrain-hindbrain boundary (isthmus), caudal hindbrain, or cervical spinal cord. Our results showed that the isthmus inhibited caudally directed axon outgrowth by 80% relative to controls, whereas rostrally directed axon outgrowth was unaffected. Moreover, caudal hindbrain or cervical spinal cord explants did not inhibit caudal axons. Immunohistochemistry for reticular and raphe neuronal markers indicated that the caudal, but not the rostral projections of these neuronal subpopulations were inhibited by isthmic explants. Companion studies in chick embryos showed that, when the hindbrain was surgically separated from the isthmus, caudal reticulospinal axon projections failed to form and that descending pioneer axons of the medial longitudinal fasciculus (MLF) play an important role in the caudal reticulospinal projection. Taken together, these results suggest that diffusible chemorepellent or nonpermissive signals from the isthmus and substrate-anchored signals on the pioneer MLF axons are involved in the caudal direction of reticulospinal projections and might influence other longitudinal axon projections in the brainstem.
Developmental Biology 04/2003; 255(1):99-112. · 4.07 Impact Factor
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ABSTRACT: Endothelial cells perform a large array of physiological functions that are influenced by their cellular heterogeneity in the different vascular beds. Vein endothelial cells isolated from the umbilical cords are commonly used to study vascular endothelium. Primary cultures of these cells, however, have low proliferative capacity and a limited life span. We have immortalized bovine umbilical vein endothelial cells (BUVEC) by transfection with an expression vector containing the human papillomavirus type 16 E6E7 oncogenes. Expression of E6E7 extended the life span of BUVEC from 40 to more than 1-20 cell replication cycles with no signs of senescence. Four immortalized clones were isolated and found to maintain endothelial cell properties, such as the uptake of acetylated low density lipoprotein, the expression of the von Willebrand protein, the binding of endothelial cell-specific lectins and proliferative responses to the specific endothelial cell mitogen, vascular endothelial growth factor. Moreover, clone BVE-E6E7-1, like its wild-type counterparts, expressed prolactin mRNA and decreased its proliferation in response to the anti-angiogenic 16-kDa fragment of prolactin. This clone showed little signs of genetic instability as revealed by centrosome and chromosome number analysis. Thus, immortalized E6E7 BUVEC cell lines retain endothelial cell characteristics and could facilitate studies to investigate the action of regulatory factors of vascular endothelium. Moreover, being the first non-human umbilical vein endothelial cell lines, their use should provide insights into the mechanisms governing species-related heterogeneity of endothelial cells.
European Journal of Cell Biology 02/2002; 81(1):1-8. · 2.81 Impact Factor
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ABSTRACT: Con objeto de obtener subcultivos seriales de preadipocitos del tejido adiposo bovino y luego tratar de diferenciarlos a adipocitos, se compararon dos medios de cultivo. Se obtuvieron muestras de tejido adiposo subcutaneo y de omento de animales adultos de 3 a 4 anos de edad, y de grasa perirrenal y de omento de fetos en el ultimo tercio de la gestacion. En el caso de celulas adultas, se establecieron subcultivos; sin embargo no pudieron ser diferenciadas. Para el tejido adiposo fetal, se establecieron subcultivos y los fibroblastos perirrenales fueron capaces de diferenciarse con el medio apropiado (DMEM/ F-12 1:1; 10 �Êg/ml insulina; 0.25 �ÊM dexametasona; 10 �Êg/ml lipoproteinas de muy baja densidad; 100 �Êg/ml estreptomicina; 100 UI/ml penicilina). Estos resultados podrian servir de base para obtener un modelo que permita estudiar los mecanismos moleculares de diferenciacion de preadipocitos en tejido adiposo bovino.
Técnica pecuaria en México, Vol. 46, Nº. 2, 2008, pags. 195-204.
