Zebrafish slit2 and slit3 act together to regulate retinal axon crossing at the midline

Slit-Robo signaling regulates midline crossing of commissural axons in different systems. In zebrafish, all retinofugal axons cross at the optic chiasm to innervate the contralateral tectum. Here, the mutant for the Robo2 receptor presents severe axon guidance defects, which were not completely reproduced in a Slit2 ligand null mutant. Since slit3 is also expressed around this area at the stage of axon crossing, we decided to analyze the possibility that it collaborates with Slit2 in this process. We found that the disruption of slit3 expression by sgRNA-Cas9 injection caused similar, albeit slightly milder, defects than those of the slit2 mutant, while the same treatment in the slit2−/−mz background caused much more severe defects, comparable to those observed in robo2 mutants. Tracking analysis of in vivo time-lapse experiments indicated differential but complementary functions of these secreted factors in the correction of axon turn errors around the optic chiasm. Interestingly, RT-qPCR analysis showed a mild increase in slit2 expression in slit3-deficient embryos, but not the opposite. Our observations support the previously proposed “repulsive channel” model for Slit-Robo action at the optic chiasm, with both Slits acting in different manners, most probably relating to their different spatial expression patterns.ANII:FCE_1_2014_1_498

Regulación multihormonal de la expresión del gen de hormona de crecimiento (GH) de rata

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de BioquímicaPeer reviewe

Early thyroid hormone-induced gene expression changes in N2a-β neuroblastoma cells

Thyroid hormone has long been known to regulate neural development. Hypothyroidism during pregnancy and early postnatal period has severe neurological consequences including even mental retardation. The purpose of this study was to characterize gene expression pattern during thyroid hormone-induced differentiation of neuro-2a β cells in order to select >direct response genes> for further analysis. In this neuroblastoma cell line, thyroid hormone blocks proliferation and induces differentiation. Changes in gene expression level were examined after a T3 treatment of 3 and 24 h using cDNA arrays. Sixteen genes were significantly up-regulated and 79 down-regulated by T3 treatment. Five up-regulated genes not previously described as regulated by thyroid hormone and selected for their putative significance to understand T3 action on cell differentiation, were verified by RT-PCR analysis. The transcription factors Phox2a and basic helix-loop-helix domain containing, class B2 mRNAs exhibited a clear increase after 3- and 24-h treatment. The guanine-nucleotide exchange factor RalGDS was greatly up-regulated after 3-h treatment but not 24 h after. The results suggest an early involvement of these genes in T3 action during neuroblastoma cell differentiation probably mediating later changes in gene expression pattern. © 2010 Springer Science+Business Media, LLC.This work was supported by grants from TWAS and Comisión Sectorial de Investigación Científica (Universidad de la República, Uruguay) and Comisión Interministerial de Ciencia y Tecnología, grant number SAF2009-11150.Peer Reviewe

Retinoic acid regulates growth hormone gene expression

VITAMIN A is required for normal growth and development, and retinoic acid (RA) may be the active metabolite in this process. Recent evidence indicates that RA acts through binding to a nuclear receptorwhich belongs to the steroid/thyroid hormone receptor superfamily. The receptors seem to associate with hormone-response elements in the target genes resulting in the activation (or inhibition) of transcription. Although no interaction of RA-receptor complex with specific DNA sequences has yet been reported, the homology of the different receptors suggests their mechanisms of action are similar. We therefore examined whether the effects of RA on growth could be related to changes in the expression of the growth hormone gene which is known to be transcriptionally regulated by both thyroid and glucocorticoid hormones. Our results show that RA controls growth hormone production in pituitary GHl cells and that its effect is synergistic with that caused by these hormones. © 1989 Nature Publishing Group

Expression of the growth hormone gene and the pituitary-specific transcription factor GHF-1 in diabetic rats

Diabetes in the rat is associated with poor growth and decreased GH in the pituitary. In this study we have examined whether this reduction reflects an impairment of GH gene expression. Diabetes was induced by the administration of streptozotocin (7 mg/100 g BW), and 18 days later, GH content, GH mRNA, and GH transcription rate were determined. GH mRNA levels were reduced by more than 80% in the pituitaries of diabetic rats, which had a similarly reduced GH content. The differences observed in transcription fully account for the changes in mRNA concentration, since the transcription rate of the gene was also reduced by a factor of 10 in the diabetic pituitaries. Insulin therapy (3 U/15 days) partially restored these parameters. The expression of the specific transcription factor GHF-1/Pit-1 in diabetic rats was also analyzed. Both GHF-1 mRNA levels and the binding of nuclear proteins to an oligodeoxynucleotide conforming to the GHF-1 proximal binding site in the promoter of the GH gene were normal in the diabetic pituitaries, thus excluding the possibility that decreased availability of this factor could be responsible for the decreased GH transcription. Since diabetes produced an approximately 3-fold reduction of circulating T3, the potential role of thyroid hormones on GH gene expression was also evaluated in thyroidectomized and thyroidectomized diabetic rats. Thyroidectomy decreased GH and GH mRNA to less than 5% of the values found in intact animals, and a single saturating injection of T3 (250 μg/100 g BW) resulted in a 8- to 10-fold induction of GH mRNA after 6 h. This response was markedly depressed in thyroidectomized diabetic rats, in which T3 produced only a minor increase in GH mRNA. Administration of insulin alone to these animals did not alter GH mRNA, but partially restored the response to T3, and GH mRNA levels increased 3- to 4-fold in this group. The changes in GH mRNA were not accompanied by concomitant changes in the abundance of GHF-1. These results show that transcription of the rat GH gene is altered in diabetes, and that insulin is required for a normal regulation of the GH gene by thyroid hormone

Regulation of thyroid hormone receptor and c-erbA mRNA levels by butyrate in neuroblastoma (N2A) and glioma (C6) cells

Butyrate produced a biphasic modulation of the thyroid hormone receptor in neuroblastoma N2A cells increasing receptor number by 20-35% at concentrations 0.25-0.75 mM and decreasing receptor levels by 30-55% at 2-4 mM. The half-life of the receptor, as assessed by its disappearance after incubation with 18 μM cycloheximide was 8.4 hr in control cells and 10.3 hr and 5.0 hr in cells incubated with 0.25 and 4 mM butyrate, respectively. This compound increased the abundance of multyacetylated forms of histone H4 from 30% in control cells to almost 70% with butyrate 4 mM. In glioma C6 cells, the fatty acid produced a dose-dependent increase of receptor levels (up to 3-4-fold with 2-5 mM butyrate) and had little effect in increasing multiacetylation (from 30% in controls to 42-46% with 2-5 mM butyrate). Recent studies have shown that the c-erbA proto-oncogen codes for the thyroid hormone receptor. In N2A and C6 cells, 2 c-erbA-related mRNAs, one measuring 2.6 kb and the other 6 kb, were detected. Both forms were differently regulated by butyrate. This compound decreased the abundance of the 2.6 kb forms in both cell types, even at the concentrations at which there was an elevation of receptor levels. Only the largest mRNA correlated with receptor concentration increasing by 2-3-fold after treatment of C6 cells with butyrate, and undergoing a smaller but biphasic change in N2A cells. Our data suggest that modification of chromatin structure probably secondary to acetylation induces changes in thyroid hormone receptor levels in neuroblastoma and glioma cells by affecting both receptor stability and receptor mRNA levels

Informe final del proyecto: Aspectos motivacionales del comportamiento sexual de ratas hembras durante la adolescencia y sus bases neurales

Durante la adolescencia el cerebro transita por grandes cambios que se reflejan en un perfil comportamental único. Las ratas, al igual que otros mamíferos, comienzan a expresar interés sexual durante la adolescencia, sin embargo los circuitos neurales que lo controlan están aún en desarrollo. Este proyecto caracterizó el desarrollo de la motivación sexual (tiempo y energía invertidos en interactuar con una pareja) y la ejecución de este comportamiento de ratas hembras durante la adolescencia, y los cambios en los circuitos neurales subyacentes. Determinamos que la motivación sexual (búsqueda del macho) de las hembras aumenta a lo largo de la adolescencia, si bien su comportamiento sexual durante la cópula no difiere del de las adultas. De forma interesante, las adolescentes exhiben juego y alta exploración social, conductas típicas de este período, durante la cópula. El menor interés sexual de las adolescentes se acompañó de una activación diferente de áreas cerebrales que regulan este comportamiento en relación a las adultas. Observamos a su vez, variaciones en la expresión de receptores del neurotransmisor dopamina en el sistema mesocorticolímbico (sistema general de recompensa del cerebro) entre adolescentes y adultas, y una menor sensibilidad de las adolescentes al efecto de la cocaína (droga que aumenta la dopamina en este sistema) sobre la motivación sexual. En conjunto, estos resultados muestran que el comportamiento sexual se expresa durante la adolescencia, pero la motivación sexual, a diferencia de la social, es baja en este período. Este perfil comportamental probablemente se asocie a la inmadurez del sistema mesocorticolímbico, que difiere entre ambas edades. Este proyecto contribuyó a la compresión de los cambios que ocurren durante un período único de la vida de los mamíferos en las hembras, y abre perspectivas centradas en entender cómo madura el cerebro y el comportamiento social a lo largo del desarrollo.Agencia Nacional de Investigación e Innovació

Informe final del proyecto: Aspectos motivacionales del comportamiento sexual de ratas hembras durante la adolescencia y sus bases neurales

Durante la adolescencia el cerebro transita por grandes cambios que se reflejan en un perfil comportamental único. Las ratas, al igual que otros mamíferos, comienzan a expresar interés sexual durante la adolescencia, sin embargo los circuitos neurales que lo controlan están aún en desarrollo. Este proyecto caracterizó el desarrollo de la motivación sexual (tiempo y energía invertidos en interactuar con una pareja) y la ejecución de este comportamiento de ratas hembras durante la adolescencia, y los cambios en los circuitos neurales subyacentes. Determinamos que la motivación sexual (búsqueda del macho) de las hembras aumenta a lo largo de la adolescencia, si bien su comportamiento sexual durante la cópula no difiere del de las adultas. De forma interesante, las adolescentes exhiben juego y alta exploración social, conductas típicas de este período, durante la cópula. El menor interés sexual de las adolescentes se acompañó de una activación diferente de áreas cerebrales que regulan este comportamiento en relación a las adultas. Observamos a su vez, variaciones en la expresión de receptores del neurotransmisor dopamina en el sistema mesocorticolímbico (sistema general de recompensa del cerebro) entre adolescentes y adultas, y una menor sensibilidad de las adolescentes al efecto de la cocaína (droga que aumenta la dopamina en este sistema) sobre la motivación sexual. En conjunto, estos resultados muestran que el comportamiento sexual se expresa durante la adolescencia, pero la motivación sexual, a diferencia de la social, es baja en este período. Este perfil comportamental probablemente se asocie a la inmadurez del sistema mesocorticolímbico, que difiere entre ambas edades. Este proyecto contribuyó a la compresión de los cambios que ocurren durante un período único de la vida de los mamíferos en las hembras, y abre perspectivas centradas en entender cómo madura el cerebro y el comportamiento social a lo largo del desarrollo.Agencia Nacional de Investigación e Innovació

Evidence of two co-circulating genetic lineages of canine distemper virus in South America

Canine distemper virus (CDV) is the etiological agent of a multisystemic infection that affects different species of carnivores and is responsible for one of the main diseases suffered by dogs. Recent data have shown a worldwide increase in the incidence of the disease, including in vaccinated dog populations, which necessitates the analysis of circulating strains. The hemagglutinin (H) gene, which encodes the major antigenic viral protein, has been widely used to determine the degree of genetic variability and to associate CDVs in different worldwide circulating lineages. Here, we obtained the sequence of the first full-length H gene of field South American CDV strains and compared it with sequences of worldwide circulating field strains and vaccine viruses. In South America, we detect two co-circulating lineages with different prevalences: the Europe 1 lineage and a new South America 2 lineage. The Europe 1 lineage was the most prevalent in South America, and we suggest renaming it the Europe 1/South America 1 lineage. The South America 2 lineage was found only in Argentina and appears related to wild CDV strains. All South American CDV strains showed high amino-acid divergence from vaccine strains. This genetic variability may be a possible factor leading to the resurgence of distemper cases in vaccinated dog populations.Fil: Panzera, Yanina. Universidad de la República; UruguayFil: Gallo Calderon, Marina Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. Cesar Milstein". Fundación Pablo Cassara. Instituto de Ciencia y Tecnología ; ArgentinaFil: Sarute, Nicolás. Universidad de la República; UruguayFil: Guasco, Soledad. Universidad de la República; UruguayFil: Cardeillac, Arianne. Universidad de la República; UruguayFil: Bonilla, Braulio. Universidad de la República; UruguayFil: Hernández, Martín. Universidad de la República; UruguayFil: Francia, Lourdes. Universidad de la República; UruguayFil: Bedó, Gabriela. Universidad de la República; UruguayFil: la Torre, Jose Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. Cesar Milstein". Fundación Pablo Cassara. Instituto de Ciencia y Tecnología ; ArgentinaFil: Pérez, Ruben. Universidad de la República; Urugua