Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “functional experiments”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 721 records · Page 40Linked to original sources
PubMed2006

Role of cyclophilin D in the resistance of brain mitochondria to the permeability transition.

The mitochondrial permeability transition (MPT) is involved in both necrosis and apoptosis. Cyclophilin D (CypD) is an important component of the MPT. Brain mitochondria are more resistant to the MPT when compared to heart or liver mitochondria. We found that this increased resistance correlates with low expression of CypD in brain when compared to heart or liver. In newborn rats, sensitivity of brain mitochondria to the MPT and CypD expression are significantly higher than in mature animals. In an in vitro model of neuronal development, mitochondria in differentiated neuronal-like cells exert a higher calcium threshold toward MPT induction and express significantly less CypD when compared to undifferentiated precursor cells. Gain and loss of function experiments confirm the role of CypD in sensitivity to the MPT. Together our data indicate that the increased calcium threshold of brain mitochondria to the MPT correlates with low expression of CypD in brain; and that neuronal cells lose CypD during differentiation and become less sensitive to the MPT induction. This may be a protection mechanism that raises the threshold of brain tissue against injuries.

Aging↗
PubMed2005

Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo.

Within the vertebrate nervous system, the presence of many different lineages of neurons and glia complicates the molecular characterization of single neuronal populations. In order to elucidate molecular mechanisms underlying the specification and development of corticospinal motor neurons (CSMN), we purified CSMN at distinct stages of development in vivo and compared their gene expression to two other pure populations of cortical projection neurons: callosal projection neurons and corticotectal projection neurons. We found genes that are potentially instructive for CSMN development, as well as genes that are excluded from CSMN and are restricted to other populations of neurons, even within the same cortical layer. Loss-of-function experiments in null mutant mice for Ctip2 (also known as Bcl11b), one of the newly characterized genes, demonstrate that it plays a critical role in the development of CSMN axonal projections to the spinal cord in vivo, confirming that we identified central genetic determinants of the CSMN population.

Animals↗
PubMedDate not supplied

Functional genomics as an emerging strategy for the investigation of central mechanisms in experimental hypertension.

Centrally mediated increases in sympathetic nerve activity and attenuated arterial baroreflexes contribute to the pathogenesis of hypertension. Despite the characterization of cellular and physiological mechanisms that regulate blood pressure and alterations that contribute to hypertension, the genetic and molecular basis of this pathophysiology remains poorly understood. Strategies to identify genes that contribute to central pathophysiologic mechanisms in hypertension include integrative biochemistry and physiology as well as functional genomics. This article summarizes recent progress in applying functional genomics to elucidate the genetic basis of altered central blood pressure regulatory mechanisms in hypertension. We describe approaches others and we have undertaken to investigate gene expression profiles in hypertensive models in order to identify genes that contribute to the pathogenesis of hypertension. Finally, we provide the readers a roadmap for negotiating the route from experimental findings of gene expression profiling to translating their therapeutic potential. The combination of gene expression profiling and the phenotypic characterization of in vitro and in vivo loss or gain of function experiments for candidate genes have the potential to identify genes involved in the pathogenesis of hypertension and may present novel targets for therapy.

Animals↗
PubMed2006

Effects of different human chorionic gonadotrophin preparations on trophoblast differentiation.

Recent evidence from the literature suggested that hCG preparations purified from urine of pregnant women, which are widely used in in vitro studies and IVF programs, may contain contaminants such as EGF. To determine the putative biological effects of the contaminating growth factor, we here investigated distinct trophoblast differentiation processes in the presence of various hCG compounds. Western blot analyses indicated that treatment of trophoblastic SGHPL-5 cells and purified term trophoblasts with potentially EGF-contaminated hCG (hCG-A) resulted in auto-phosphorylation of the EGF receptor at tyrosine 1173 whereas supplementation of another urine-purified hCG preparation (hCG-B), recombinant holo-hCG or recombinant alphahCG had no effects. Phosphorylation was specifically blocked by the EGF receptor inhibitor PD153035. Urinary hCG-A was most effective in promoting invasion of SGHPL-5 cells through Matrigel-coated transwells, but increased invasiveness was also observed in the presence of hCG-B or recombinant holo-hCG. Similarly, the extent of syncytialisation of term trophoblasts, quantitated by nuclei in desmoplakin-negative areas, was highest upon addition of hCG-A or recombinant EGF as a control. PD153035 reduced invasion and fusion of trophoblasts supplemented with hCG-A, but did not diminish the effects provoked by hCG-B. In conclusion, the data suggest that the EGF contamination of hCG considerably affects trophoblast function. Experiments using EGF-free hCG preparations demonstrate that the hormone increases trophoblast invasion and syncytialisation.

Cell Differentiation↗
PubMed2004

A southpaw joins the roster: the role of the zebrafish nodal-related gene southpaw in cardiac LR asymmetry.

The establishment of the left-right (LR) asymmetry of the zebrafish heart involves at least two discrete events: cardiac jogging and cardiac looping. The nodal-related gene southpaw is required for both aspects of heart LR asymmetry as well as for LR patterning of other visceral organs. Reductions in southpaw activity abolish the normal LR biases of jogging and looping and uncouple the normal correlation between jogging and looping polarities. These loss-of-function experiments also reveal that southpaw is required for organizing at least the initial phase of diencephalic LR asymmetry and suggest that heart and brain LR patterning may be coordinated by southpaw signaling originating within the anterior, left lateral plate mesoderm.

Animals↗
PubMed2005

A lentiviral expression system demonstrates that L* protein of Theiler's murine encephalomyelitis virus (TMEV) is essential for virus growth in a murine macrophage-like cell line.

The DA subgroup strains of Theiler's murine encephalomyelitis virus (TMEV) synthesize L* protein, which is translated out of frame with the polyprotein from an alternative AUG, 13 nucleotides downstream from the authentic polyprotein AUG. By a 'loss of function' experiment using a mutant virus, DAL*-1, in which the L* AUG is mutated to an ACG, L* protein is shown to play an important role in virus persistence, TMEV-induced demyelination, and virus growth in macrophages. In the present study, we established an L* protein-expressed macrophage-like cell line and confirmed the importance of L* protein in virus growth in this cell line.

Animals↗
PubMed2004

Role of the ERK-mediated signaling pathway in mesenchyme formation and differentiation in the sea urchin embryo.

Mesoderm and mesodermal structures in the sea urchin embryo are entirely generated by two embryologically distinct populations of mesenchyme cells: the primary (PMC) and the secondary (SMC) mesenchyme cells. We have identified the extracellular signal-regulated kinase (ERK) as a key component of the regulatory machinery that controls the formation of both these cell types. ERK is activated in a spatial-temporal manner, which coincides with the epithelial-mesenchyme transition (EMT) of the prospective PMCs and SMCs. Here, we show that ERK controls EMT of both primary and secondary mesenchyme cells. Loss and gain of function experiments demonstrate that ERK signaling is not required for the early specification of either PMCs or SMCs, but controls the maintenance and/or the enhancement of expression levels of regulatory genes which participate in the process of specification of these cell types. In addition, ERK-mediated signaling is essential for the transcription of terminal differentiation genes encoding proteins that define the final structures generated by PMCs and SMCs. Our findings suggest that ERK has a central pan-mesodermal role in coupling EMT and terminal differentiation of all mesenchymal cell types in the sea urchin embryo.

Animals↗
PubMed2004

Expression of an NK2 homeodomain gene in the apical ectoderm defines a new territory in the early sea urchin embryo.

We have identified an NK2 family homeodomain transcription factor, SpNK2.1, in the sea urchin Strongylocentrotus purpuratus whose transcripts are initially detected within the apical plate ectoderm of the hatching blastula and are confined to the apical organ at least through 2 weeks of development. Protein localization studies demonstrate that SpNK2.1 is restricted to the apical plate epithelium, but is excluded from the nucleus of serotonergic neurons. The expression profile of SpNK2.1 is dictated via two separate regulatory systems. Initially, SpNK2.1 is restricted to the apical pole domain by beta-catenin-dependent processes operating along the animal-vegetal axis, as evidenced by an expansion of SpNK2.1 expression upon cadherin overexpression. Starting at gastrulation, expression in the apical plate is maintained by SpDri, the sea urchin orthologue of dead ringer. Abrogation of SpDri results in the downregulation of SpNK2.1 after gastrulation, but SpDri is not necessary for the initial activation of SpNK2.1. Loss of function experiments using SpNK2.1-specific morpholino antisense oligonucleotides and SpNK2.1 overexpression experiments do not disrupt embryonic development and have no effect upon the development of neuronal components of the apical organ. Nonetheless, SpNK2.1 defines a new early territory of the sea urchin embryo.

Animals↗
PubMed2004

An artificial promoter construct for heat-inducible misexpression during fish embryogenesis.

Beside spatial distribution, timing of gene expression is a key parameter controlling gene function during embryonic development. Gain-of-function experiments can therefore have quite opposing results, depending on the time of gene activation. Induction techniques are necessary to control timing in these experiments from outside of the organism. Natural heat shock promoters constitute a simple inducible misexpression system, the main disadvantage is a high background level of expression. We present here a new heat stress-inducible bidirectional promoter consisting of multimerized heat shock elements (HSE). The simplified architecture of this promoter largely improves the properties needed for an efficient induction system: dramatically reduced background activity, improved inducibility, and loss of all tissue specific components. Based on this new artificial promoter, we present a transient induction system for fish embryos. Application of this new induction system for Fgf8 misexpression during embryonic development reveals different windows of competence during eye development. A dramatic early phenotype resulting in loss of the eyes is observed for conventional mRNA injection. Later activation, by using our inducible promoter, uncovers different eye phenotypes like cyclopic eyes. Even after 14 days, an efficient heat stress response could be evoked in the injected embryos. The HSE promoter therefore represents a new artificial heat shock promoter with superior properties, making possible transient experiments with inducible misexpression at various stages of development.

Animals↗
PubMed2004

A new role for BMP5 during limb development acting through the synergic activation of Smad and MAPK pathways.

In an attempt to identify new genes implicated in the control of programmed cell death during limb development, we have generated a cDNA library from the regressing interdigital tissue of chicken embryos. We have analyzed 804 sequences from this library and identified 23 genes involved in apoptosis in different models. One of the genes that came up in the screening was the Bone Morphogenetic Protein family member, Bmp5, that has not been previously involved in the control of apoptosis during limb development. In agreement with a possible role in the control of cell death, Bmp5 exhibited a regulated pattern of expression in the interdigital tissue. Transcripts of Bmp5 and BMP5 protein were abundant within the cytoplasm of the fragmenting apoptotic interdigital cells in a way suggesting that delivery of BMPs into the tissue is potentiated during apoptosis. Gain-of-function experiments demonstrated that BMP5 has the same effect as other interdigital BMPs inducing apoptosis in the undifferentiated mesoderm and growth in the prechondrogenic mesenchyme. We have characterized both Smad proteins and MAPK p38 as intracellular effectors for the action of BMPs in the developing limb autopod. Activation of Smad signaling involves the receptor-regulated genes Smad1 and -8, and the inhibitory Smad6, and results in both the upregulation of gene transcription and protein phosphorylation with subsequent nuclear translocation. MAPK p38 is also quickly phosphorylated after BMP stimulation in the limb mesoderm. Treatment with the inhibitor of p38, SB203580, revealed that there are interdigital genes induced by BMPs in a p38-dependent manner (DKK, Snail and FGFr3), and genes induced in a p38-independent manner (BAMBI, Msx2 and Smads). Together, our results suggest that Smad and MAPK pathways act synergistically in the BMP pathway controlling limb development.

Animals↗
PubMed2004

Role of BMP signaling and the homeoprotein Iroquois in the specification of the cranial placodal field.

Different types of placodes originate at the anterior border of the neural plate but it is still an unresolved question whether individual placodes arise as distinct ectodermal specializations in situ or whether all or a subset of the placodes originate from a common preplacodal field. We have analyzed the expression and function of the homeoprotein Iro1 in Xenopus and zebrafish embryos, and we have compared its expression with several preplacodal and placodal markers. Our results indicate that the iro1 genes are expressed in the preplacodal region, being one of the earliest markers for this area. We show that an interaction between the neural plate and the epidermis is able to induce the expression of several preplacodal markers, including Xiro1, by a similar mechanism to that previously shown for neural crest induction. In addition, we analyzed the role of BMP in the specification of the preplacodal field by studying the expression of the preplacodal markers Six1, Xiro1, and several specific placodal markers. We experimentally modified the level of BMP activity by three different methods. First, we implanted beads soaked with noggin in early neurula stage Xenopus embryos; second, we injected the mRNA that encodes a dominant negative of the BMP receptor into Xenopus and zebrafish embryos; and third, we grafted cells expressing chordin into zebrafish embryos. The results obtained using all three methods show that a reduction in the level of BMP activity leads to an expansion of the preplacodal and placodal region similar to what has been described for neural crest regions. By using conditional constructs of Xiro1, we performed gain and loss of function experiments. We show that Xiro1 play an important role in the specification of both the preplacodal field as well as individual placodes. We have also used inducible dominant negative and activator constructs of Notch signaling components to analyze the role of these factors on placodal development. Our results indicate that the a precise level of BMP activity is required to induce the neural plate border, including placodes and neural crest cells, that in this border the iro1 gene is activated, and that this activation is required for the specification of the placodes.

Animals↗
PubMed2004

Specific regulation of cyclins D1 and D2 by FGF and Shh signaling coordinates cell cycle progression, patterning, and differentiation during early steps of spinal cord development.

In the vertebrate embryo, spinal cord elongation requires FGF signaling that promotes the continuous development of the posterior nervous system by maintaining a stem zone of proliferating neural progenitors. Those escaping the caudal neural stem zone, which is expressed to Shh signal, initiate ventral patterning in the neural groove before starting neuronal differentiation in the neural tube. Here we investigated the integration of D-type cyclins, known to govern cell cycle progression under the control of extracellular signals, in the program of spinal cord maturation. In chicken embryo, we find that cyclin D2 is preferentially expressed in the posterior neural plate, whereas cyclin D1 appears in the neural groove. We demonstrated by loss- and gain-of-function experiments that FGF signaling maintains cyclin D2 in the immature caudal neural epithelium, while Shh activates cyclin D1 in the neural groove. Moreover, forced maintenance of cyclin D1 or D2 in the neural tube favors proliferation at the expense of neuronal differentiation. These results contribute to our understanding of how the cell cycle control can be linked to the patterning programs to influence the balance between proliferation and neuronal differentiation in discrete progenitors domains.

Animals↗
PubMed2004

A balance between the anti-apoptotic activity of Slug and the apoptotic activity of msx1 is required for the proper development of the neural crest.

We have studied the pattern of programmed cell death in the neural crest and analyzed how it is controlled by the activity of the transcription factors Slug and msx1. Our results indicate that apoptosis is more prevalent in the neural folds than in the rest of the neural ectoderm. Through gain- and loss-of-function experiments with inducible forms of both Slug and msx1 genes, we showed that Slug acts as an anti-apoptotic factor whereas msx1 promotes cell death, either in the neural folds of the whole embryos, in isolated or induced neural crest and in animal cap assays. The protective effect of expressing Slug can be reversed by expressing the apoptotic factor Bax, while the apoptosis promoted by msx1 can be abolished by expressing the Xenopus homologue of Bcl2 (XR11). Furthermore, we show that Slug and msx1 control the transcription of XR11 and several caspases required for programmed cell death. In addition, expression of Bax or Bcl2, produced similar effects on the survival of the neural crest and on the development of its derivatives to those produced by altering the activity of Slug or msx1. Finally, we show that in the neural crest, the region of the neural folds where Slug is expressed, cells undergo less apoptosis, than in the region where the msx1 gene is expressed, which correspond to cells adjacent to the neural crest. We show that the expression of Slug and msx1 controls cell death in certain areas of the neural folds, and we discuss how this equilibrium is necessary to generate sharp boundaries in the neural crest territory, and to precisely control cell number among neural crest derivatives.

Animals↗
PubMed2005

Evidence that nervy, the Drosophila homolog of ETO/MTG8, promotes mechanosensory organ development by enhancing Notch signaling.

In the imaginal tissue of developing fruit flies, achaete (ac) and scute (sc) expression defines a group of neurally-competent cells called the proneural cluster (PNC). From the PNC, a single cell, the sensory organ precursor (SOP), is selected as the adult mechanosensory organ precursor. The SOP expresses high levels of ac and sc and sends a strong Delta (Dl) signal, which activates the Notch (N) receptor in neighboring cells, preventing them from also adopting a neural fate. Previous work has determined how ac and sc expression in the PNC and SOP is regulated, but less is known about SOP-specific factors that promote SOP fate. Here, we describe the role of nervy (nvy), the Drosophila homolog of the mammalian proto-oncogene ETO, in mechanosensory organ formation. Nvy is specifically expressed in the SOP, where it interacts with the Ac and Sc DNA binding partner Daughterless (Da) and affects the expression of Ac and Sc targets. nvy loss- and gain-of-function experiments suggest that nvy reinforces, but is not absolutely required for, the SOP fate. We propose a model in which nvy acts downstream of ac and sc to promote the SOP fate by transiently strengthening the Dl signal emanating from the SOP.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Identification of an unexpected link between the Shh pathway and a G2/M regulator, the phosphatase CDC25B.

Sonic hedgehog (Shh) signaling controls numerous aspects of vertebrate development, including proliferation of precursors in different organs. Identification of molecules that link the Shh pathway to cell cycle machinery is therefore of major importance for an understanding of the mechanisms underlying Shh-dependent proliferation. Here, we show that an actor in the control of entry into mitosis, the phosphatase CDC25B, is transcriptionally upregulated by the Shh/Gli pathway. Unlike other G2/M regulators, CDC25B is highly expressed in domains of Shh activity, including the ventral neural tube and the posterior limb bud. Loss- and gain-of-function experiments reveal that Shh contributes to CDC25B transcriptional activation in the neural tube both of chick and mouse embryos. Moreover, CDC25B transcripts are absent from the posterior limb bud of Shh-/- mice, while anterior grafts of Shh-expressing cells in the chicken limb bud induce ectopic CDC25B expression. Arresting the cell cycle does not reduce the level of CDC25B expression in the neural tube strongly suggesting that the upregulation of CDC25B is not an indirect consequence of the Shh-dependent proliferation. These data reveal an unexpected developmental link between the Shh pathway and a participant in G2/M control.

Animals↗
PubMed2006

Apelin, the ligand for the endothelial G-protein-coupled receptor, APJ, is a potent angiogenic factor required for normal vascular development of the frog embryo.

The peptide growth factor apelin is the high affinity ligand for the G-protein-coupled receptor APJ. During embryonic development of mouse and frog, APJ receptor is expressed at high levels in endothelial precursor cells and in nascent vascular structures. Characterization of Xenopus apelin shows that the sequence of the bioactive region of the peptide is perfectly conserved between frogs and mammals. Embryonic expression studies indicate that apelin is expressed in, or immediately adjacent to, a subset of the developing vascular structures, particularly the intersegmental vessels. Experimental inhibition of either apelin or APJ expression, using antisense morpholino oligos, results in elimination or disruption of intersegmental vessels in a majority of embryos. In gain of function experiments, apelin peptide is a potent angiogenic factor when tested using two in vivo angiogenesis assays, the frog embryo and the chicken chorioallantoic membrane. Furthermore, studies using the mouse brain microvascular cell line bEnd.3 show that apelin acts as a mitogenic, chemotactic and anti-apoptotic agent for endothelial cells in culture. Finally, we show that, similar to a number of other angiogenic factors, expression of the apelin gene is increased under conditions of hypoxia. Taken together, these studies indicate that apelin is required for normal vascular development in the frog embryo and has properties consistent with a role during normal and pathological angiogenesis.

Amino Acid Sequence↗
PubMed2005

Regulation of lymphangiogenesis--from cell fate determination to vessel remodeling.

Lymphatic vessels are important for the maintenance of normal tissue fluid balance, immune surveillance and adsorption of digested fats. During the past decade, the identification of lymphatic-specific markers and growth factors has enabled detailed studies of the lymphatic system, and gain- and loss-of-function experiments have greatly increased our understanding of the mechanisms of normal lymphatic development. Understanding the basic biology has provided novel insights into the pathologic conditions of the lymphatic system that contribute to lymphedema, inflammation or lymphatic metastasis, and opened possibilities for the development of better therapeutic strategies. Here we review the current knowledge about the molecular mechanisms regulating the development of the lymphatic vasculature; of the differentiation of lymphatic endothelial cells, of the regulation of the growth of lymphatic vessels, and of remodeling of the vasculature into a network consisting of lymphatic capillaries and collecting lymphatic vessels. Furthermore, we will discuss the molecular mechanisms involved in the pathological conditions of the lymphatic vessels.

Angiopoietins↗
PubMed2001

Dimerization co-factor of hepatocyte nuclear factor 1/pterin-4alpha-carbinolamine dehydratase is necessary for pigmentation in Xenopus and overexpressed in primary human melanoma lesions.

Dimerization co-factor of hepatocyte nuclear factor 1 (HNF1)/pterin-4alpha-carbinolamine dehydratase (DCoH/PCD) is both a positive co-factor of the HNF1 homeobox transcription factors and thus involved in gene regulation as well as an enzyme catalyzing the regeneration of tetrahydrobiopterin. Dysfunction of DCoH/PCD is associated with the human disorders hyperphenylalaninemia and vitiligo. In Xenopus, overexpression of the protein during development induces ectopic pigmentation. In this study loss of function experiments using DCoH/PCD-specific antibodies demonstrated that the protein is also absolutely necessary for pigment cell formation in Xenopus. In normal human skin DCoH/PCD protein is weakly expressed in the basal layer of the epidermis that consists of keratinocytes and melanocytes. Whereas only 4 of 25 benign nevi reacted with DCoH/PCD-specific antibodies, high protein levels were detectable in melanoma cell lines and 13 of 15 primary malignant melanoma lesions. The comparison with the commonly used melanoma markers S100 and HMB45 demonstrated that DCoH/PCD has an overlapping but distinct expression pattern in melanoma lesions. In addition to human colon cancer, this is the second report about the overexpression of DCoH/PCD in human tumor cells indicating that the protein might be involved in cancerogenesis.

Animals↗