Positive and negative regulation of Shh signalling in vertebrate retinal development.

Cell-to-cell communication is fundamental for embryo development and subsequent tissue homeostasis. This communication is often mediated by a small number of signaling pathways in which a secreted ligand binds to the surface of a target cell, thereby activating signal transduction. In vertebrate neural development, these signaling mechanisms are repeatedly used to obtain different and context-dependent outcomes. Part of the versatility of these communication mechanisms depends on their finely tuned regulation that controls timing, spatial localization, and duration of the signaling. The existence of secreted antagonists, which prevent ligand-receptor interaction, is an efficient mechanism to regulate some of these pathways. The Hedgehog family of signaling proteins, however, activates a pathway that is controlled largely by the positive or negative activity of membrane-bound proteins such as Cdon, Boc, Gas1, or Megalin/LRP2. In this review, we will use the development of the vertebrate retina, from its early specification to neurogenesis, to discuss whether there is an advantage to the use of such regulators, pointing to unresolved or controversial issues.

Conductas promotoras de salud en alimentación saludable y actividad física en conductores de la locomoción colectiva de una empresa privada de la ciudad de Puerto Montt, Chile / Health-promoting behaviors regarding healthy diet and physical activity in drivers in a private collective transporting company in Puerto Montt, Chile

Resumen

Objetivo: se analizó las conductas promotoras de salud en cuanto a alimentación saludable y actividad física, en conductores de la locomoción colectiva de una empresa privada de la ciudad de Puerto Montt, Chile.

Metodología: se desarrolló un estudio cuantitativo, descriptivo, no experimental y transversal. Se utilizó un cuestionario de elaboración propia que incluía los aspectos conceptuales del Modelo de Promoción de la Salud, complementado con el test AUDIT y Test de Fagerstörm, el cual se aplicó a 38 individuos, quienes cumplieron los criterios de inclusión. Se analizó la información separando a los participantes con y sin patologías crónicas diagnosticadas con el fin de evitar el sesgo en los resultados.

Resultados: el total de los sujetos fueron hombres, arrojando una alta prevalencia de sobrepeso, obesidad abdominal, cifras tensionales elevadas e hiperglicemia. El 74% presentó una alimentación inadecuada, alcanzando el 90,9% en los sujetos que presentan enfermedades crónicas. En cuanto a la actividad física, el 61% refirió no realizarla, llegando al 81,8% del total de los individuos que presentan patologías crónicas; para las dos conductas evaluadas, la familia sería la principal red de apoyo y aspectos laborales como los horarios de trabajo y el entorno laboral, serían las principales barreras para la acción.

Conclusiones: los conductores presentan factores de riesgo condicionantes potencialmente modificables, que conllevan una alta probabilidad de desencadenar enfermedades cardiovasculares y que requieren intervención sanitaria que permita el fomento de la actividad física y alimentación saludable, para lograr establecerlas como conductas promotoras de la salud.

Abstract

Purpose: health-promoting behaviors regarding healthy diet and physical activity were analyzed in drivers in a private collective transporting company in Puerto Montt, Chile.

Methods: a quantitative, descriptive, non-experimental, cross-sectional study was carried out. We developed a questionnaire including conceptual items from Health promotion Model, supplemented with AUDIT test and Fagerstörm test. The questionnaire was completed by 38 participants meeting inclusion criteria. Data analyses were performed separately for participants with and without previously diagnosed chronic diseases to avoid bias risk in results.

Results: all participants were men and showed a high prevalence of overweight, abdominal obesity, high blood pressure and hyperglycemia. An inappropriate diet was observed in 74% of all participants, and in 90.9% of participants with chronic diseases. No physical activity was performed in 61% of all participants and in 81.8% of participants with chronic diseases. For both evaluated behaviors, family was the main support resource, and working aspects, such as working times and environment, appeared to be the main barriers to action.

Conclusions: drivers show several potentially modifiable risk factors, leading to a high probability of cardiovascular diseases, which requires health care interventions aiming at physical activity and health diet promotion, so that such behaviors become established as health-promoting activities.

Objetivo: analisar condutas promotoras de saúde, quanto à alimentação saudável e atividade física, em motoristas de locomoção coletiva de uma empresa privada da cidade de Puerto Montt, Chile.

Metodologia: realizou-se um estudo quantitativo, descritivo, não experimental e transversal. Utilizou-se um questionário de elaboração própria que incluía aspetos conceituais do Modelo de Promoção da Saúde, complementando o teste AUDIT e o Teste de Fagerstörm, o qual se aplicou em 38 indivíduos que cumpriam com os critérios de inclusão. Analisou-se a informação separando os participantes com e sem patologias crônicas diagnosticadas, com o fim de evitar viés nos resultados.

Resultados: todos os sujeitos foram homens, evidenciando alta prevalência de sobrepeso, obesidade abdominal, cifras tensionais elevadas e hiperglicemia. O 74% apresento alimentação inadequada, chegando a 90,9% nos sujeitos com enfermidades crônicas. Quanto à atividade física, 61% referi não realizada, chegando a 81,8% do total dos indivíduos que apresentam patologias crônicas; para as condutas avaliadas, a família seria a principal rede de apoio e aspectos laborais, como horários de trabalho e clima laboral, seriam as principais barreiras para a ação.

Conclusões: os motoristas apresentam fatores de risco condicionantes potencialmente as modificáveis, que acarretam uma alta probabilidade de desencadear doenças cardiovasculares, e que requerem intervenção sanitária que permita o fomento da atividade física e alimentação saudável para lograr estabelecê-las como condutas promotoras de saúde.

High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9.

The use of CRISPR/Cas9 as a genome-editing tool in various model organisms has radically changed targeted mutagenesis. Here, we present a high-throughput targeted mutagenesis pipeline using CRISPR/Cas9 technology in zebrafish that will make possible both saturation mutagenesis of the genome and large-scale phenotyping efforts. We describe a cloning-free single-guide RNA (sgRNA) synthesis, coupled with streamlined mutant identification methods utilizing fluorescent PCR and multiplexed, high-throughput sequencing. We report germline transmission data from 162 loci targeting 83 genes in the zebrafish genome, in which we obtained a 99% success rate for generating mutations and an average germline transmission rate of 28%. We verified 678 unique alleles from 58 genes by high-throughput sequencing. We demonstrate that our method can be used for efficient multiplexed gene targeting. We also demonstrate that phenotyping can be done in the F1 generation by inbreeding two injected founder fish, significantly reducing animal husbandry and time. This study compares germline transmission data from CRISPR/Cas9 with those of TALENs and ZFNs and shows that efficiency of CRISPR/Cas9 is sixfold more efficient than other techniques. We show that the majority of published "rules" for efficient sgRNA design do not effectively predict germline transmission rates in zebrafish, with the exception of a GG or GA dinucleotide genomic match at the 5' end of the sgRNA. Finally, we show that predicted off-target mutagenesis is of low concern for in vivo genetic studies.

Phenotype-driven chemical screening in zebrafish for compounds that inhibit collective cell migration identifies multiple pathways potentially involved in metastatic invasion.

In the last decade, high-throughput chemical screening has become the dominant approach for discovering novel compounds with therapeutic properties. Automated screening using in vitro or cultured cell assays have yielded thousands of candidate drugs for a variety of biological targets, but these approaches have not resulted in an increase in drug discovery despite major increases in expenditures. In contrast, phenotype-driven screens have shown a much stronger success rate, which is why we developed an in vivo assay using transgenic zebrafish with a GFP-marked migrating posterior lateral line primordium (PLLp) to identify compounds that influence collective cell migration. We then conducted a high-throughput screen using a compound library of 2160 annotated bioactive synthetic compounds and 800 natural products to identify molecules that block normal PLLp migration. We identified 165 compounds that interfere with primordium migration without overt toxicity in vivo. Selected compounds were confirmed in their migration-blocking activity by using additional assays for cell migration. We then proved the screen to be successful in identifying anti-metastatic compounds active in vivo by performing orthotopic tumor implantation assays in mice. We demonstrated that the Src inhibitor SU6656, identified in our screen, can be used to suppress the metastatic capacity of a highly aggressive mammary tumor cell line. Finally, we used CRISPR/Cas9-targeted mutagenesis in zebrafish to genetically validate predicted targets of compounds. This approach demonstrates that the migrating PLLp in zebrafish can be used for large-scale, high-throughput screening for compounds that inhibit collective cell migration and, potentially, anti-metastatic compounds.

Fluorescent activated cell sorting (FACS) combined with gene expression microarrays for transcription enrichment profiling of zebrafish lateral line cells.

Transgenic lines carrying fluorescent reporter genes like GFP have been of great value in the elucidation of developmental features and physiological processes in various animal models, including zebrafish. The lateral line (LL), which is a fish specific superficial sensory organ, is an emerging organ model for studying complex cellular processes in the context of the whole living animal. Cell migration, mechanosensory cell development/differentiation and regeneration are some examples. This sensory system is made of superficial and sparse small sensory patches called neuromasts, with less than 50 cells in any given patch. The paucity of cells is a real problem in any effort to characterize those cells at the transcriptional level. We describe here a method which we applied to efficiently separate subpopulation of cells of the LL, using two distinct stable transgenic zebrafish lines, Tg(cldnb:gfp) and Tg(tnks1bp1:EGFP). In both cases, the GFP positive (GFP+) cells were separated from the remainder of the animal by using a Fluorescent Activated Cell Sorter (FACS). The transcripts of the GFP+ cells were subsequently analyzed on gene expression microarrays. The combination of FACS and microarrays is an efficient method to establish a transcriptional signature for discrete cell populations which would otherwise be masked in whole animal preparation.

Transcriptional signature of accessory cells in the lateral line, using the Tnk1bp1:EGFP transgenic zebrafish line.

BACKGROUND: Because of the structural and molecular similarities between the two systems, the lateral line, a fish and amphibian specific sensory organ, has been widely used in zebrafish as a model to study the development/biology of neuroepithelia of the inner ear. Both organs have hair cells, which are the mechanoreceptor cells, and supporting cells providing other functions to the epithelium. In most vertebrates (excluding mammals), supporting cells comprise a pool of progenitors that replace damaged or dead hair cells. However, the lack of regenerative capacity in mammals is the single leading cause for acquired hearing disorders in humans. RESULTS: In an effort to understand the regenerative process of hair cells in fish, we characterized and cloned an egfp transgenic stable fish line that trapped tnks1bp1, a highly conserved gene that has been implicated in the maintenance of telomeres' length. We then used this Tg(tnks1bp1:EGFP) line in a FACsorting strategy combined with microarrays to identify new molecular markers for supporting cells. CONCLUSIONS: We present a Tg(tnks1bp1:EGFP) stable transgenic line, which we used to establish a transcriptional profile of supporting cells in the zebrafish lateral line. Therefore we are providing a new set of markers specific for supporting cells as well as candidates for functional analysis of this important cell type. This will prove to be a valuable tool for the study of regeneration in the lateral line of zebrafish in particular and for regeneration of neuroepithelia in general.

Sublethal concentrations of waterborne copper induce cellular stress and cell death in zebrafish embryos and larvae.

Copper is an essential ion that forms part of the active sites of many proteins. At the same time, an excess of this metal produces free radicals that are toxic for cells and organisms. Fish have been used extensively to study the effects of metals, including copper, present in food or the environment. It has been shown that different metals induce different adaptive responses in adult fish. However, until now, scant information has been available about the responses that are induced by waterborne copper during early life stages of fish. Here, acute toxicity tests and LC50 curves have been generated for zebrafish larvae exposed to dissolved copper sulphate at different concentrations and for different treatment times. We determined that the larvae incorporate and accumulate copper present in the medium in a concentration-dependent manner, resulting in changes in gene expression. Using a transgenic fish line that expresses enhanced green fluorescent protein (EGFP) under the hsp70 promoter, we monitored tissue-specific stress responses to waterborne copper by following expression of the reporter. Furthermore, TUNEL assays revealed which tissues are more susceptible to cell death after exposure to copper. Our results establish a framework for the analysis of whole-organism management of excess external copper in developing aquatic animals.

Sublethal concentrations of waterborne copper induce cellular stress and cell death in zebrafish embryos and larvae

Copper is an essential ion that forms part of the active sites of many proteins. At the same time, an excess of this metal produces free radicals that are toxic for cells and organisms. Fish have been used extensively to study the effects of metals, including copper, present in food or the environment. It has been shown that different metals induce different adaptive responses in adult fish. However, until now, scant information has been available about the responses that are induced by waterborne copper during early life stages of fish. Here, acute toxicity tests and LC50 curves have been generated for zebrafish larvae exposed to dissolved copper sulphate at different concentrations and for different treatment times. We determined that the larvae incorporate and accumulate copper present in the medium in a concentration-dependent manner, resulting in changes in gene expression. Using a transgenic fish line that expresses enhanced green fluorescent protein (EGFP) under the hsp70 promoter, we monitored tissue-specific stress responses to waterborne copper by following expression of the reporter. Furthermore, TUNEL assays revealed which tissues are more susceptible to cell death after exposure to copper. Our results establish a framework for the analysis of whole-organism management of excess external copper in developing aquatic animals.

A high-throughput chemically induced inflammation assay in zebrafish.

BACKGROUND: Studies on innate immunity have benefited from the introduction of zebrafish as a model system. Transgenic fish expressing fluorescent proteins in leukocyte populations allow direct, quantitative visualization of an inflammatory response in vivo. It has been proposed that this animal model can be used for high-throughput screens aimed at the identification of novel immunomodulatory lead compounds. However, current assays require invasive manipulation of fish individually, thus preventing high-content screening. RESULTS: Here we show that specific, noninvasive damage to lateral line neuromast cells can induce a robust acute inflammatory response. Exposure of fish larvae to sublethal concentrations of copper sulfate selectively damages the sensory hair cell population inducing infiltration of leukocytes to neuromasts within 20 minutes. Inflammation can be assayed in real time using transgenic fish expressing fluorescent proteins in leukocytes or by histochemical assays in fixed larvae. We demonstrate the usefulness of this method for chemical and genetic screens to detect the effect of immunomodulatory compounds and mutations affecting the leukocyte response. Moreover, we transformed the assay into a high-throughput screening method by using a customized automated imaging and processing system that quantifies the magnitude of the inflammatory reaction. CONCLUSIONS: This approach allows rapid screening of thousands of compounds or mutagenized zebrafish for effects on inflammation and enables the identification of novel players in the regulation of innate immunity and potential lead compounds toward new immunomodulatory therapies. We have called this method the chemically induced inflammation assay, or ChIn assay. See Commentary article: http://www.biomedcentral.com/1741-7007/8/148.

Molecular dissection of the migrating posterior lateral line primordium during early development in zebrafish.

BACKGROUND: Development of the posterior lateral line (PLL) system in zebrafish involves cell migration, proliferation and differentiation of mechanosensory cells. The PLL forms when cranial placodal cells delaminate and become a coherent, migratory primordium that traverses the length of the fish to form this sensory system. As it migrates, the primordium deposits groups of cells called neuromasts, the specialized organs that contain the mechanosensory hair cells. Therefore the primordium provides both a model for studying collective directional cell migration and the differentiation of sensory cells from multipotent progenitor cells. RESULTS: Through the combined use of transgenic fish, Fluorescence Activated Cell Sorting and microarray analysis we identified a repertoire of key genes expressed in the migrating primordium and in differentiated neuromasts. We validated the specific expression in the primordium of a subset of the identified sequences by quantitative RT-PCR, and by in situ hybridization. We also show that interfering with the function of two genes, f11r and cd9b, defects in primordium migration are induced. Finally, pathway construction revealed functional relationships among the genes enriched in the migrating cell population. CONCLUSIONS: Our results demonstrate that this is a robust approach to globally analyze tissue-specific expression and we predict that many of the genes identified in this study will show critical functions in developmental events involving collective cell migration and possibly in pathological situations such as tumor metastasis.

Phoenix is required for mechanosensory hair cell regeneration in the zebrafish lateral line.

In humans, the absence or irreversible loss of hair cells, the sensory mechanoreceptors in the cochlea, accounts for a large majority of acquired and congenital hearing disorders. In the auditory and vestibular neuroepithelia of the inner ear, hair cells are accompanied by another cell type called supporting cells. This second cell population has been described as having stem cell-like properties, allowing efficient hair cell replacement during embryonic and larval/fetal development of all vertebrates. However, mammals lose their regenerative capacity in most inner ear neuroepithelia in postnatal life. Remarkably, reptiles, birds, amphibians, and fish are different in that they can regenerate hair cells throughout their lifespan. The lateral line in amphibians and in fish is an additional sensory organ, which is used to detect water movements and is comprised of neuroepithelial patches, called neuromasts. These are similar in ultra-structure to the inner ear's neuroepithelia and they share the expression of various molecular markers. We examined the regeneration process in hair cells of the lateral line of zebrafish larvae carrying a retroviral integration in a previously uncharacterized gene, phoenix (pho). Phoenix mutant larvae develop normally and display a morphologically intact lateral line. However, after ablation of hair cells with copper or neomycin, their regeneration in pho mutants is severely impaired. We show that proliferation in the supporting cells is strongly decreased after damage to hair cells and correlates with the reduction of newly formed hair cells in the regenerating phoenix mutant neuromasts. The retroviral integration linked to the phenotype is in a novel gene with no known homologs showing high expression in neuromast supporting cells. Whereas its role during early development of the lateral line remains to be addressed, in later larval stages phoenix defines a new class of proteins implicated in hair cell regeneration.

Progressive iron accumulation induces a biphasic change in the glutathione content of neuroblastoma cells.

Glutathione (GSH) constitutes the single most important antioxidant in neurons, whereas iron causes oxidative stress that leads to cell damage and death. Although GSH and iron produce opposite effects on redox cell status, no mechanistic relationships between iron and GSH metabolism are known. In this work, we evaluated in SH-SY5Y neuroblastoma cells the effects of iron accumulation on intracellular GSH metabolism. After 2 d exposure to increasing concentrations of iron, cells underwent concentration-dependent iron accumulation and a biphasic change in intracellular GSH levels. Increasing iron from 1 to 5 microM resulted in a marked increase in intracellular oxidative stress and increased GSH levels. Increased GSH levels were due to increased synthesis. Further increases in iron concentration led to significant reduction in both reduced (GSH) and total (GSH + (2 x GSSG)) glutathione. Cell exposure to high iron concentrations (20-80 microM) was associated with a marked decrease in the GSH/GSSG molar ratio and the GSH half-cell reduction potential. Moreover, increasing iron from 40 to 80 microM resulted in loss of cell viability. Iron loading did not change GSH reductase activity but induced significant increases in GSH peroxidase and GSH transferase activities. The changes in GSH homeostasis reported here recapitulate several of those observed in Parkinson's disease substantia nigra. These results support a model by which progressive iron accumulation leads to a progressive decrease in GSH content and cell reduction potential, which finally results in impaired cell integrity.

Concentración de consultas por morbilidad en menores de 6 años: Consultorio Dr. J. Symon O., 1997-1999 / Morbidity consultation in children under 6 years old: José Symon Ojeda, MD. clinic, 1997-1999

Se describe una muestra aleatoria de 468 niños de 0 a 5 años bajo control en el consultorio J. Symon Ojeda, del área norte de Santiago, que acudieron por enfermedad a solicitar atención médica y fueron seguidos durante un año. Se determina frecuencia de consultas y diagnósticos por año de edad y sexo. Los "diagnósticos diferentes" en cada niño comprende las distintas enfermedades diagnosticadas en cada uno en 12 meses, no equivalentes a episodios y/o consultas. No se hace distinción entre diagnósticos de afecciones respiratorias altas ni entre dermatológicos. Se calculan percentiles y se analiza con dócimas no paramétricas. La frecuencia de consultas varió entre 1 y 50, la mitad de los niños consultó 5 veces o menos y el 75 por ciento 9 o menos. Un 3, 2 por ciento sobrepasó las 20 atenciones. Promedio 6,7 y DS 5,68. Se identificaron entre 1 y 15 enfermedades en un mismo niño, la mitad presentó 4 o menos y 75 por ciento acumuló a lo más 6. Sólo en 3,8 por ciento se identificó más de 10 diagnósticos. El promedio fue de 4,8 y la DS 3,02. No hay diferencias por sexo. La correlación es positiva entre número de consultas y número de diagnósticos. Los menores de 3 años consultaron más que los de 3 a 5 años. Los índices calculados son más altos que otros publicados, pero estos corresponden a una metodología distinta y más depurada. Se trata del seguimiento de consultantes y no de un cuociente entre consultas y beneficiarios