Evaluation of reference genes for reverse transcription-quantitative PCR assays in organs of zebrafish exposed to glyphosate-based herbicide, Roundup.

Roundup is a glyphosate-based herbicide (GBH) widely used in agriculture and may cause toxic effects in non-target organisms. Model organisms, as zebrafish, and analysis of gene expression by reverse transcription-quantitative PCR (RT-qPCR) could be used to better understand the Roundup toxicity. A prerequisite for RT-qPCR is the availability of appropriate reference genes; however, they have not been described for Roundup-exposed fish. The aim of this study was to evaluate the expression stability of six reference genes (rpl8, ß-act, gapdh, b2m, ef1α, hprt1) and one expressed repetitive element (hatn10) in organs of males (brain, gill, testis) and females (ovary) of zebrafish exposed to Roundup WG at three concentrations (0.065, 0.65 and 6.5 mg N-(phosphonomethyl) glycine/l) for 7 days. Genes were ranked by geNorm, NormFinder, BestKeeper, Delta C t and RefFinder, and their best combinations were determined by geNorm and NormFinder programs. The two most stable ranked genes were specific to each organ: gill (ß-act; rpl8); brain (rpl8; ß-act); testis (ef1α; gapdh); and ovary (rpl8; hprt1). The cat transcript level was used to evaluate the effect of normalization with these reference genes. These are the first suitable reference genes described for the analysis of gene expression in organs of Roundup-exposed zebrafish, and will allow investigations of the molecular mechanisms of Roundup toxicity.

Distribution of microglial cells in the cerebral hemispheres of embryonic and neonatal chicks.

The distribution, morphology and morphometry of microglial cells in the chick cerebral hemispheres from embryonic day 4 (E4) to the first neonatal day (P1) were studied by histochemical labeling with a tomato (Lycopersicon esculentum) lectin. The histochemical analysis revealed lectin-reactive cells in the nervous parenchyma on day E4. Between E4 (5.7 +/- 1.35 mm length) and E17 (8.25 +/- 1.2 mm length), the lectin-reactive cells were identified as ameboid microglia and observed starting from the subventricular layer, distributed throughout the mantle layer and in the proximity of the blood vessels. After day E13, the lectin-reactive cells exhibited elongated forms with small branched processes, and were considered primitive ramified microglia. Later, between E18 (5.85 +/- 1.5 mm cell body length) and P1 (3.25 +/- 0.6 mm cell body length), cells with more elongated branched processes were observed, constituting the ramified microglia. Our findings provide additional information on the migration and differentiation of microglial cells, whose ramified form is observed at the end of embryonic development. The present paper focused on the arrangement of microglial cells in developing cerebral hemispheres of embryonic and neonatal chicks, which are little studied in the literature. Details of morphology, morphometry and spatial distribution of microglial cells contributed to the understanding of bird and mammal central nervous system ontogeny. Furthermore, the identification and localization of microglial cells during the normal development could be used as a morphological guide for embryonic brain injury researches.

Distribution of microglial cells in the cerebral hemispheres of embryonic and neonatal chicks

The distribution, morphology and morphometry of microglial cells in the chick cerebral hemispheres from embryonic day 4 (E4) to the first neonatal day (P1) were studied by histochemical labeling with a tomato (Lycopersicon esculentum) lectin. The histochemical analysis revealed lectin-reactive cells in the nervous parenchyma on day E4. Between E4 (5.7 ± 1.35 mm length) and E17 (8.25 ± 1.2 mm length), the lectin-reactive cells were identified as ameboid microglia and observed starting from the subventricular layer, distributed throughout the mantle layer and in the proximity of the blood vessels. After day E13, the lectin-reactive cells exhibited elongated forms with small branched processes, and were considered primitive ramified microglia. Later, between E18 (5.85 ± 1.5 mm cell body length) and P1 (3.25 ± 0.6 mm cell body length), cells with more elongated branched processes were observed, constituting the ramified microglia. Our findings provide additional information on the migration and differentiation of microglial cells, whose ramified form is observed at the end of embryonic development. The present paper focused on the arrangement of microglial cells in developing cerebral hemispheres of embryonic and neonatal chicks, which are little studied in the literature. Details of morphology, morphometry and spatial distribution of microglial cells contributed to the understanding of bird and mammal central nervous system ontogeny. Furthermore, the identification and localization of microglial cells during the normal development could be used as a morphological guide for embryonic brain injury researches.

Ação da insulina na morfogênese de embrioes de Gallus gallus domesticus / The action of insulin in the morphogenesis of Gallus gallus domesticus embryos

Aspectos concernentes à morfogênese de Gallus gallus domesticus viabilizam estudos relacionados à ação da insulina sobre estruturas e topografia embrionárias. Na temperatura de 37,5ºC, ovos foram incubados por 24 h, injetados com 5 ml de insulina de suínos em 3 concentrações e reincubados por mais 72 h. As características morfológicas de 80 embriões foram avaliadas e, de acordo com a organização apresentada, classificados em 5 níveis de morfogênese. Em 21 embriões submetidos aos testes com a insulina registrou-se dismorfismo generalizado (4o nível), enquanto nos de experimento de controle foi verificado morfogênese-padrão (1o nível) e dismorfismo localizado (3o nível). Aqueles espécimes mostraram corpo com dimensões reduzidas, caracterizado por limites ântero-dorsal organizados em uma projeção cefálica e regiões posterior-ventral alteradas, evidenciando um padrão de anormalidades na determinação do eixo ântero-posterior, que indica a ação específica da insulina na morfogênese embrionária no período de 96 horas de incubação.

Características do desenvolvimento embrionário de Gallus gallus domesticus, em temperaturas e períodos diferentes de incubaçäo / Features of the embryonic development of Gallus gallus domesticus in different temperatures and times of incubation

Objetivando avaliar o desenvolvimento embrionário de Gallus gallus domesticus, foram incubados ovos nas temperaturas de 34,0ºC, 37,5ºC e 40,0ºC, nos períodos de 48, 72 e 96 horas. Avaliaram-se em 81 embriöes o comprimento cefalocaudal, o peso total e 6 características morfológicas, utilizando-se um protocolo referindo o desenvolvimento como padräo, acelerado ou defasado. Os dados relativos ao nível de diferenciaçäo das estruturas embrionárias demonstram que os fatores temperatura e tempo de incubaçäo interagem nos processos de morfogênese