Patterning of the vertebrate ventral spinal cord

We review investigations that have lead to a model of how the ventral spinal cord of higher vertebrate embryos is patterned during development. Central to this model is the secreted morphogen protein, Sonic hedgehog. There is now considerable evidence that this molecule acts in a concentration-dependent manner to direct the development of the spinal cord. Recent studies have suggested that two classes of homeodomain proteins are induced by threshold concentrations of Sonic hedgehog. Reciprocal inhibition between the two classes acts to convert the continuous gradient of Sonic hedgehog into defined domains of transcription factor expression. However, a number of aspects of ventral spinal cord patterning remain to be elucidated. Some issues currently under investigation involve temporal aspects of Shh-signalling, the role of other signals in ventral patterning and the characterisation of ventral interneurons. In this review, we discuss the current state of knowledge of these issues and present some preliminary studies aimed at furthering understanding of these processes in spinal cord patterning.

Genes induced by growth hormone in a model of adipogenic differentiation

A substantial number of GH regulated genes have been reported in mature hepatocytes. but genes involved in GH-initiated cell differentiation have not yet been identified. Here we have studied a, ell-characterised model of GH-dependent differentiation, adipogenesis of 3T3-F442A preadipocytes, to identify genes rapidly induced by GH. Using the suppression subtractive hybridisation technique, we have identified eight genes induced within 60 min of GH treatment, and verified these by northern analysis. Six were identifiable as Stat 2. Stat 3, thrombospondin-1. oncostatin M receptor beta chain. a DEAD box RNA helicase. and muscleblind. a developmental transcription factor. Bioinformatic approaches assigned one of the two remaining unknown genes as a novel 436 residue serine,threonine kinase. As each of the identified genes hake important developmental roles. they may be important in initiating GH-induced adipogenesis. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

The VCAM-1 gene that encodes the vascular cell adhesion molecule is a target of the Sry-related high mobility group box gene, Sox18

VCAM-1 (vascular cell adhesion molecule-1) and Sox18 are involved in vascular development. VCAM-1 is an important adhesion molecule that is expressed on endothelial cells and has a critical role in endothelial activation, inflammation, lymphatic pathophysiology, and atherogenesis. The Sry-related high mobility group box factor Sox18 has previously been implicated in endothelial pathologies. Mutations in human and mouse Sox18 leads to hypotrichosis and lymphedema. Furthermore, both Sox18 and VCAM-1 have very similar spatio-temporal patterns of expression, which is suggestive of crosstalk. We use biochemical techniques, cell culture systems, and the ragged opossum (RaOP) mouse model with a naturally occurring mutation in Sox18 to demonstrate that VCAM-1 is an important target of Sox18. Transfection, site-specific mutagenesis, and gel shift analyses demonstrated that Sox18 directly targeted and trans-activated VCAM-1 expression. Importantly, the naturally occurring Sox18 mutant attenuates the expression and activation of VCAM-1 in vitro. Furthermore, in vivo quantitation of VCAM-1 mRNA levels in wild type and RaOP mice demonstrates that RaOP animals show a dramatic and significant reduction in VCAM-1 mRNA expression in lung, skin, and skeletal muscle. Our observation that the VCAM-1 gene is an important target of SOX18 provides the first molecular insights into the vascular abnormalities in the mouse mutant ragged and the human hypotrichosis-lymphedematelangiectasia disorder.

Sox18 mutations in the ragged mouse alleles ragged-like and opossum

The ragged (Ra) spontaneous mouse mutant is characterised by abnormalities in its coat and cardiovascular system. Four alleles are known and we have previously described mutations in the transcription factor gene Sox18 in the Ra and Ra-J alleles. We report here Sox18 mutations in the remaining two ragged alleles, opossum (Ra-op) and ragged-like (Ragl). The single-base deletions cause a C-terminal frameshift, abolishing transcriptional trans-activation and impairing interaction with the partner protein MEF2C. The nature of these mutations, together with the near-normal phenotype of Sox18-null mice, suggests that the ragged mutant SOX18 proteins act in a dominant-negative fashion. The four ragged mutants represent an allelic series that reveal SOX18 structure-function relationships and implicate related SOX proteins in cardiovascular and hair follicle development. (C) 2003 Wiley-Liss, Inc.

Etiology of ovarian failure in blepharophimosis ptosis epicanthus inversus syndrome: FOXL2 is a conserved, early-acting gene in vertebrate ovarian development

Blepharophimosis ptosis epicanthus inversus syndrome (BPES) is a human disorder caused by mutations in the forkhead transcription factor gene FOXL2 and is characterized by facial dysmorphology combined in some cases with ovarian failure. To better understand the role of FOXL2 in the etiology of ovarian failure in BPES, we examined its expression in embryonic ovaries of mice, chickens, and red-eared slider turtles, representatives of three phylogenetically distant vertebrate groups that have different mechanisms of sex determination. Expression of Foxl2 was detected in early ovaries of all three species around the time of sex determination and was associated with both somatic and germ cell populations in mice. Expression was sexually dimorphic in all cases. Sequence analysis of turtle and chicken FoxL2 orthologues indicated an unusually high degree of structural conservation during evolution. FoxL2 was found to be autosomal in chickens, and therefore unlikely to represent the dominant ovarian-determining gene that has been postulated to exist as a possible explanation for female heterogamety in birds. Our observations suggest that BPES may result from early abnormalities in regulating the development of the fetal ovary, rather than premature degeneration of the postnatal or adult ovary. Further, our results suggest that FOXL2 is a highly conserved early regulator of vertebrate ovarian development.

SOX8 is expressed during testis differentiation in mice and synergizes with SF1 to activate the Amh promoter in vitro

Sox8 is a member of the Sox family of developmental transcription factor genes and is closely related to Sox9, a key gene in the testis determination pathway in mammals. Like Sox9, Sox8 is expressed in the developing mouse testis around the time of sex determination, suggesting that it might play a role in regulating the expression of testis-specific genes. An early step in male sex differentiation is the expression of anti-Mullerian hormone (AMH) in Sertoli cells. Expression of the Amh gene during sex differentiation requires the interaction of several transcription factors, including SF1, SOX9, GATA4, WT1, and DAX1. Here we show that SOX8 may also be involved in regulating the expression of Amh. Expression of Sox8 begins just prior to that of Amh at 12 days post coitum (dpc) in mouse testes and continues beyond 16 dpc in Sertoli cells. In vitro assays showed that SOX8 binds specifically to SOX binding sites within the Amh minimal promoter and, like SOX9, acts synergistically with SF1 through direct protein-protein interaction to enhance Amh expression, albeit at lower levels compared with SOX9. SOX8 and SOX9 appear to have arisen from a common ancestral gene and may have retained some common functions during sexual development. Our data provide the first evidence that SOX8 may partially compensate for the reduced SOX9 activity in campomelic dysplasia and substitute for Sox9 where Sox9 is either not expressed or expressed too late to be involved in sex determination or regulation of Amh expression.

The receptor tyrosine kinase regulator sprouty1 is a target of the tumor suppressor WT1 and important for kidney development

WT1 encodes a transcription factor involved in kidney development and tumorigenesis. Using representational difference analysis, we identified a new set of WT1 targets, including a homologue of the Drosophila receptor tyrosine kinase regulator, sprouty. Sprouty1 was up-regulated in cell lines expressing wild-type but not mutant WT1. WT1 bound to the endogenous sprouty1 promoter in vivo and directly regulated sprouty1 through an early growth response gene-1 binding site. Expression of Sprouty1 and WT1 overlapped in the developing metanephric mesenchyme, and Sprouty1, like WT1, plays a key role in the early steps of glomerulus formation. Disruption of Sprouty1 expression in embryonic kidney explants by antisense oligonucleotides reduced condensation of the metanephric mesenchyme, leading to a decreased number of glomeruli. In addition, sprouty1 was expressed in the ureteric tree and antisense-treated ureteric trees had cystic lumens. Therefore, sprouty1 represents a physiologically relevant target gene of WT1 during kidney development.

The Wilms' Tumour Suppressor Protein, WT1, Undergoes CRM1-independent Nucleocytoplasmic Shuttling

The Wilms' tumour suppressor gene (WT1) encodes a zinc finger-containing nuclear protein essential for kidney and urogenital development. Initially considered a transcription factor, there is mounting evidence that WT1 has a role in post-transcriptional processing. Using the interspecies heterokaryon assay, we have demonstrated that WT1 can undergo nucleocytoplasmic shuttling. We have also mapped the region responsible for nuclear export to residues 182-324. Our data add further complexity to the role of WT1 in trancriptional and post-transcriptional regulation. (C) 2003 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

E2F modulates keratinocyte squamous differentiation. Implications for E2F inhibition in squamous cell carcinoma

E2F regulation is essential for normal cell cycle progression. Therefore, it is not surprising that squamous cell carcinoma cell lines (SCC) overexpress E2F1 and exhibit deregulated E2F activity when compared with normal keratinocytes. Indeed, deliberate E2F1 deregulation has been shown to induce hyperplasia and skin tumor formation. In this study, we report on a dual role for E2F as a mediator of keratinocyte proliferation and modulator of squamous differentiation. Overexpression of E2F isoforms in confluent primary keratinocyte cultures resulted in suppression of differentiation-associated markers. Moreover, we found that the DNA binding domain and the trans-activation domain of E2F1 are important in mediating suppression of differentiation. Use of a dominant/negative form of E2F1 ( E2F d/n) found that E2F inhibition alone is sufficient to suppress the activity of proliferation-associated markers but is not capable of inducing differentiation markers. However, if the E2F d/n is expressed in differentiated keratinocytes, differentiation marker activity is further induced, suggesting that E2F may act as a modulator of squamous differentiation. We therefore examined the effects of E2F d/n in a differentiation- insensitive SCC cell line. We found that treatment with the differentiating agent, 12-O-tetradecanoyl- phorbol-13-acetate (TPA), or expression of E2F d/n alone had no effect on differentiation markers. However, a combination of E2F d/n + TPA induced the expression of differentiation markers. Combined, these data indicate that E2F may play a key role in keratinocyte differentiation. These data also illustrate the unique potential of anti-E2F therapies in arresting proliferation and inducing differentiation of SCCs.

Site-directed mutagenesis of the ATM promoter: Consequences for response to proliferation and ionizing radiation

Although ATM, the protein defective in ataxia-telangiectasia (A-T), is activated primarily by radiation, there is also evidence that expression of the protein can be regulated by both radiation and growth factors. Computer analysis of the ATM promoter proximal 700-bp sequence reveals a number of potentially important cis-regulatory sequences. Using nucleotide substitutions to delete putative functional elements in the promoter of ATM, we examined the importance of some of these sites for both the basal and the radiation-induced activity of the promoter. In lymphoblastoid cells, most of the mutations in transcription factor consensus sequences [Sp1(1), Sp1(2), Cre, Ets, Xre, gammaIre(2), a modified AP1 site (Fse), and GCF] reduced basal activity to various extents, whereas others [gammaIre(1), NF1, Myb] left basal activity unaffected. In human skin fibroblasts, results were generally the same, but the basal activity varied up to 8-fold in these and other cell lines. Radiation activated the promoter approximately 2.5-fold in serum-starved lymphoblastoid cells, reaching a maximum by 3 hr, and all mutated elements equally blocked this activation. Reduction in Sp1 and AP1 DNA binding activity by serum starvation was rapidly reversed by exposure of cells to radiation. This reduction was not evident in A-T cells, and the response to radiation was less marked. Data provided for interaction between ATM and Sp1 by protein binding and co-immunoprecipitation could explain the altered regulation of Sp1 in A-T cells. The data described here provide additional evidence that basal and radiation-induced regulation of the ATM promoter is under multifactorial control. (C) 2003 Wiley-Liss, Inc.

Effects of rosiglitazone and linoleic acid on human preadipocyte differentiation

Background Peroxisome proliferator activated receptor gamma (PPARgamma) is a ligand-activated transcription factor known to be central to both adipose tissue development and insulin action. Growth of adipose tissue requires differentiation of preadipocytes with acquisition of specific cellular functions including insulin sensitivity, leptin secretion and the capacity to store triglyceride. Dietary fatty acids and members of the thiazolidinedione class of compounds have been reported to influence adipogenesis at the transcriptional level. Here, we compare the effects of a dietary fatty acid, linoleic acid, and a thiazolidinedione, rosiglitazone, on biochemical and functional aspects of human preadipocyte differentiation in vitro . Materials and methods Human omental and subcutaneous preadipocytes were subcultured 2-3 times and subsequently differentiated for 21 days in the presence of either linoleic acid or rosiglitazone. Differentiation was assessed using a number of biochemical and functional parameters. Results Omental and subcutaneous preadipocytes differentiated in the presence of linoleic acid showed marked cytoplasmic triacylglycerol accumulation however, no biochemical markers of differentiation (LPL expression, G3PDH gene expression and enzyme activity and leptin expression or secretion) were detected. In contrast, treatment of these cells with rosiglitazone induced full biochemical differentiation as judged by all markers assessed, despite comparatively little lipid accumulation. The rosiglitazone effects were subcutaneous depot-specific. Cells treated with linoleic acid showed decreased glucose uptake cf rosiglitazone-treated cells. A luciferase reporter assay demonstrated that rosiglitazone potently activates h-peroxisome proliferator activated receptor gamma while linoleic acid had no effect. Conclusions These studies demonstrate that (a) human preadipocytes have the potential to accumulate triacylglycerol irrespective of their stage of biochemical differentiation; (b) while omental preadipocytes are refractory to biochemical differentiation in vitro , they are able to accumulate triacylglycerol; and (c) rosiglitazone and linoleic acid may exert their effects via different biochemical pathways.

Análise Imuno-histoquímica de hipóxia renal em modelo de isquemia/reperfusão tratado com Fator Estimulador de Colônia de Granulócitos

O rim demonstra uma capacidade singular em reparar-se após danos locais, no entanto, depois de acometido, as chances de desenvolvimento de lesões renais elevam-se. A patofisiologia da isquemia/reperfusão (IR) é complexa porque há ocorrência simultânea de danos celulares e inflamação. O decréscimo na quantidade de oxigênio requer um sistema capaz de evitar seus efeitos prejudiciais e uma maquinaria molecular HIF (Hypoxia Inducible Factor), um complexo, atua como fator de transcrição de diversos genes desde os da regulação da proliferação celular e apoptose até a sinalização para angiogênese. O Fator Estimulador de Colônia de Granulócitos (G-CSF) é uma glicoproteína conhecida pela sua capacidade de promover a sobrevivência, proliferação e diferenciação de células estimulando a recuperação aos efeitos advindos da IR. Com o intuito de observar as influências dessas proteínas foi realizada uma análise semi-quantitativa de amostras renais submetidas ou não à IR, usando-se descrições microscópicas morfológicas e imunohistoquímicas, com os cálculos e gráficos estatísticos foram feitos no software GraphPad Prism®. Das análises morfológicas, constatou-se que as lesões características de IR foram observadas em espécimes não tratados: bolhas em epitélio tubular; vacuolização citoplasmática, distalização tubular e congestão luminal. De forma análoga, foi encontrada nos tratados, contudo em estágios menos avançados e em animais controle, não foi houve esta diferença tissular. As análises de microscopia eletrônica demonstraram alteração na barreira filtrante com concomitante perda de outras características glomerulares. Aos animais controle foi observada a arquitetura típica, ao passo que para os animais tratados notou-se conservação da barreira. A presença de HIF-1α nos rins contralaterais demonstrouse significante quando comparadas às amostras isquêmicas e tratadas (p<0,05). Já a ocorrência da mesma proteína em rins isquêmicos não apresentou qualquer diferença. Analisando-se a proteína VEGF foi comprovado que em rins contralaterais não há diferença estatística, contudo nos rins esquerdos há significância entre os três grupos (p<0,05). Já a correlação entre estas duas proteínas não se mostrou estatisticamente significante. Em relação às atividades de proliferação e morte celulares, todos os três grupos foram significantes entre si (p<0,05). Ao que concerne o tratamento, foi demonstrada a atividade protetora do medicamento e uma possível interação molecular com a HIF, enquanto que a ativação desta proteína corrobora sua rota metabólica já previamente descrita.

Avaliação da proteção conferida pela via de sinalização do Nrf2 num modelo experimental de sobrecarga de ferro in vivo

O ferro é encontrado em praticamente todos os seres vivos, sendo um cofator para proteínas que desempenham funções essenciais à vida. Nos mamíferos, a maioria do ferro está incorporada na hemoglobina ou armazenado no fígado, ligado à ferritina. É absorvido pelos enterócitos, sendo a principal forma de controlo dos seus níveis. A sobrecarga de ferro pode levar a hemocromatose, podendo ser tóxica para vários órgãos. O fator de transcrição Nrf2 é importante na ativação de genes citoprotetores em situações de stress oxidativo/eletrofílico, colocando-se a hipótese de que poderá estar envolvido na resposta à progressão de doença devido à sobrecarga de ferro. Com o objetivo de determinar se a via do Nrf2 representa uma proteção contra a toxicidade do ferro a nível hepático, foram realizadas duas experiências nas quais murganhos C57BL/6 (B6) e Nrf2-/- machos foram alimentados com dieta standard ou com dieta enriquecida em ferro carbonilo (FeC) (0,5% ou 2,0%). Os resultados demonstram sobrecarga de ferro nos animais que receberam dieta enriquecida, sendo que os que receberam FeC 2,0% apresentaram níveis mais elevados de ferro hepático e sérico, bem como da saturação da transferrina. Os murganhos Nrf2-/- são mais suscetíveis a esta acumulação, mostrando evidências patológicas mais graves, nomeadamente necrose hepatocítica e infiltração de células inflamatórias. A deleção do Nrf2 associado a uma dieta suplementada com FeC 2,0% parece não ser suficiente para o desenvolvimento de fibrose hepática. O estudo da expressão de genes e proteínas do metabolismo do ferro mostrou que os animais B6 e Nrf2-/- são igualmente capazes de responder à sobrecarga de ferro, sugerindo que a sua diferente suscetibilidade à toxicidade do ferro não se deverá a uma regulação ineficiente da homeostasia do Fe. A dieta com FeC 2,0% aumentou a expressão de dois genes alvo do Nrf2, Nqo1 e Gsta1, o que não se verificou com os genes e proteínas GCLC e GCLM. A expressão de genes pró-inflamatórios não mostrou evidências de inflamação nestes animais. Foi demonstrado que os animais Nrf2-/- são mais suscetíveis à toxicidade do ferro, concluindo-se que a via do Nrf2 é ativada em resposta a uma dieta contendo quantidades excessivas de FeC e que confere proteção contra a acumulação de ferro em murganhos B6.

Protection against oxidative stress through SUA7/TFIIB regulation in Saccharomyces cerevisiae

The general transcription factor TFIIB, encoded by SUA7 in Saccharomyces cerevisiae, is required for transcription activation but apparently of a specific subset of genes, for example, linked with mitochondrial activity and hence with oxidative environments. Therefore, studying SUA7/TFIIB as a potential target of oxidative stress is fundamental. We found that controlled SUA7 expression under oxidative conditions occurs at transcriptional and mRNA stability levels. Both regulatory events are associated with the transcription activator Yap1 in distinct ways: Yap1 affects SUA7 transcription up regulation in exponentially growing cells facing oxidative signals; the absence of this activator per se contributes to increase SUA7 mRNA stability. However, unlike SUA7 mRNA, TFIIB abundance is not altered on oxidative signals. The biological impact of this preferential regulation of SUA7 mRNA pool is revealed by the partial suppression of cellular oxidative sensitivity by SUA7 overexpression, and supported by the insights on the existence of a novel RNA-binding factor, acting as an oxidative sensor, which regulates mRNA stability. Taken together the results point out a primarily cellular commitment to guarantee SUA7 mRNA levels under oxidative environments.

Cellular polarity in aging: role of redox regulation and nutrition

Cellular polarity concerns the spatial asymmetric organization of cellular components and structures. Such organization is important not only for biological behavior at the individual cell level, but also for the 3D organization of tissues and organs in living organisms. Processes like cell migration and motility, asymmetric inheritance, and spatial organization of daughter cells in tissues are all dependent of cell polarity. Many of these processes are compromised during aging and cellular senescence. For example, permeability epithelium barriers are leakier during aging; elderly people have impaired vascular function and increased frequency of cancer, and asymmetrical inheritance is compromised in senescent cells, including stem cells. Here, we review the cellular regulation of polarity, as well as the signaling mechanisms and respective redox regulation of the pathways involved in defining cellular polarity. Emphasis will be put on the role of cytoskeleton and the AMP-activated protein kinase pathway. We also discuss how nutrients can affect polarity-dependent processes, both by direct exposure of the gastrointestinal epithelium to nutrients and by indirect effects elicited by the metabolism of nutrients, such as activation of antioxidant response and phase-II detoxification enzymes through the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2). In summary, cellular polarity emerges as a key process whose redox deregulation is hypothesized to have a central role in aging and cellular senescence.