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M Mercola

Publications and source records attributed to M Mercola.

At least 19 recordsLinked to original sources
PubMed2002

Isolation and characterization of Xenopus Hey-1: a downstream mediator of Notch signaling.

Regulation of Notch signaling likely occurs, at least in part, at the level of basic helix-loop-helix (bHLH) transcription factors that function downstream of Suppressor of Hairless (Su(H)) in the Notch pathway. To begin to characterize modulation of Notch signaling during organogenesis, we examined the bHLH transcription factor, XHey-1 (hairy related-1) in early Xenopus laevis embryos. XHey-1 is expressed in numerous tissues during early development including the somites, head, embryonic kidneys, and heart. Importantly, the expression of XHey-1 was significantly altered in response to perturbation of Notch signaling by means of inducible constructs that served to either activate or suppress Notch signaling through Su(H) in a temporally controlled manner.

Animals↗
PubMed2001

Wnt antagonism initiates cardiogenesis in Xenopus laevis.

Heart induction in Xenopus occurs in paired regions of the dorsoanterior mesoderm in response to signals from the Spemann organizer and underlying dorsoanterior endoderm. These tissues together are sufficient to induce heart formation in noncardiogenic ventral marginal zone mesoderm. Similarly, in avians the underlying definitive endoderm induces cardiogenesis in precardiac mesoderm. Heart-inducing factors in amphibians are not known, and although certain BMPs and FGFs can mimic aspects of cardiogenesis in avians, neither can induce the full range of activities elicited by the inducing tissues. Here we report that the Wnt antagonists Dkk-1 and Crescent can induce heart formation in explants of ventral marginal zone mesoderm. Other Wnt antagonists, including the frizzled domain-containing proteins Frzb and Szl, lacked this activity. Unlike Wnt antagonism, inhibition of BMP signaling did not promote cardiogenesis. Ectopic expression of GSK3beta, which inhibits beta-catenin-mediated Wnt signaling, also induced cardiogenesis in ventral mesoderm. Analysis of Wnt proteins expressed during gastrulation revealed that Wnt3A and Wnt8, but not Wnt5A or Wnt11, inhibited endogenous heart induction. These results indicate that diffusion of Dkk-1 and Crescent from the organizer initiate cardiogenesis in adjacent mesoderm by establishing a zone of low Wnt3A and Wnt8 activity.

Animals↗
PubMed2001

TGF-beta superfamily signaling and left-right asymmetry.

Despite an outwardly bilaterally symmetrical appearance, most internal organs of vertebrates display considerable left-right (LR) asymmetry in their anatomy and physiology. The orientation of LR asymmetry with respect to the dorsoventral and anteroposterior body axes is invariant such that fewer than 1 in 10,000 individuals exhibit organ reversals. The stereotypic orientation of LR asymmetry is ensured by distinct left- and right-side signal transduction pathways that are initiated by divergent members of the transforming growth factor-beta (TGF-beta) superfamily of secreted proteins. During early embryogenesis, the TGF-beta-like protein Nodal (or a Nodal-related ortholog) is expressed by the left lateral plate mesoderm and provides essential LR cues to the developing organs. In chick embryos at least, bone morphogenetic protein (BMP) signaling is active on the right side of the embryo and must be inhibited on the left in order for Nodal to be expressed. Thus, at a key point in the determination of LR asymmetry, left-sided signaling is mediated by the transcription factors Smad2 and Smad3 (regulated by Nodal), whereas signaling on the right depends on Smad1 and Smad5 (which are regulated by BMP). This review summarizes the considerable progress that has been made in recent years in understanding the complex network of feedback and feedforward circuitry that regulates both the left- and right-sided pathways. Also discussed is the problem of how signal transduction mediated by the Smad proteins can pattern LR asymmetry without interfering with coincident dorsoventral patterning, which relies on the same Smad proteins.

Animals↗
PubMed2001

Left-right asymmetry determination in vertebrates.

A distinctive and essential feature of the vertebrate body is a pronounced left-right asymmetry of internal organs and the central nervous system. Remarkably, the direction of left-right asymmetry is consistent among all normal individuals in a species and, for many organs, is also conserved across species, despite the normal health of individuals with mirror-image anatomy. The mechanisms that determine stereotypic left-right asymmetry have fascinated biologists for over a century. Only recently, however, has our understanding of the left-right patterning been pushed forward by links to specific genes and proteins. Here we examine the molecular biology of the three principal steps in left-right determination: breaking bilateral symmetry, propagation and reinforcement of pattern, and the translation of pattern into asymmetric organ morphogenesis.

Animals↗
PubMed2000

Notch regulates cell fate in the developing pronephros.

The mechanisms that regulate cell fate within the pronephros are poorly understood but are important for the subsequent development of the urogenital system and show many similarities to nephrogenesis in the definitive kidney. Dynamic expression of Notch-1, Serrate-1, and Delta-1 in the developing Xenopus pronephros suggests a role for this pathway in cell fate segregation. Misactivation of Notch signaling using conditionally active forms of either Notch-1 or RBP-J/Su(H) proteins prevented normal duct formation and the proper expression of genetic markers of duct cell differentiation. Inhibition of endogenous Notch signaling elicited the opposite effect. Taken together with the mRNA expression patterns, these data suggest that endogenous Notch signaling functions to inhibit duct differentiation in the dorsoanterior region of the anlage where cells are normally fated to form tubules. In addition, elevated Notch signaling in the pronephric anlage both perturbed the characteristic pattern of the differentiated tubule network and increased the expression of early markers of pronephric precursor cells, Pax-2 and Wilms' tumor suppressor gene (Wt-1). We propose that Notch signaling plays a previously unrecognized role in the early selection of duct and tubule cell fates as well as functioning subsequently to control tubule cell patterning and development.

Animals↗
PubMed2000

Subdivision of the cardiac Nkx2.5 expression domain into myogenic and nonmyogenic compartments.

Nkx2.5 is expressed in the cardiogenic mesoderm of avian, mouse, and amphibian embryos. To understand how various cardiac fates within this domain are apportioned, we fate mapped the mesodermal XNkx2.5 domain of neural tube stage Xenopus embryos. The lateral portions of the XNkx2.5 expression domain in the neural tube stage embryo (stage 22) form the dorsal mesocardium and roof of the pericardial cavity while the intervening ventral region closes to form the myocardial tube. XNkx2.5 expression is maintained throughout the period of heart tube morphogenesis and differentiation of myocardial, mesocardial, and pericardial tissues. A series of microsurgical experiments showed that myocardial differentiation in the lateral portion of the field is suppressed during normal development by signals from the prospective myocardium and by tissues located more dorsally in the embryo, in particular the neural tube. These signals combine to block myogenesis downstream of XNkx2.5 and at or above the level of contractile protein gene expression. We propose that the entire XNkx2.5/heart field is transiently specified as cardiomyogenic. Suppression of this program redirects lateral cells to adopt dorsal mesocardial and dorsal pericardial fates and subdivides the field into distinct myogenic and nonmyogenic compartments.

Animals↗
PubMed2000

Expression of connexin 30 in Xenopus embryos and its involvement in hatching gland function.

Connexins are a family of proteins that assemble to form gap junction channels. Cell-cell communication through gap junctions mediates many important events in embryogenesis, including limb patterning, lens physiology, neuronal function, left-right asymmetry, and secretion from gland tissue. We studied the expression of connexin 30 (Cx30) in the Xenopus embryo and find that it is expressed in the developing hatching gland and pronephros. To determine whether its expression plays a functional role in the activity of the hatching gland, we exposed pre-hatching embryos to drugs that block gap junctional communication. This resulted in a continuation of normal growth and development but specifically abolished hatching. The treatment did not affect Cx30 or Xenopus hatching enzyme transcription, suggesting a post-transcriptional effect on Cx30 gap junctions. We conclude that junctional communication, possibly mediated by Cx30, is involved in secretion of hatching enzyme in Xenopus.

Administration, Topical↗
PubMed2000

Serrate and Notch specify cell fates in the heart field by suppressing cardiomyogenesis.

Notch signaling mediates numerous developmental cell fate decisions in organisms ranging from flies to humans, resulting in the generation of multiple cell types from equipotential precursors. In this paper, we present evidence that activation of Notch by its ligand Serrate apportions myogenic and non-myogenic cell fates within the early Xenopus heart field. The crescent-shaped field of heart mesoderm is specified initially as cardiomyogenic. While the ventral region of the field forms the myocardial tube, the dorsolateral portions lose myogenic potency and form the dorsal mesocardium and pericardial roof (Raffin, M., Leong, L. M., Rones, M. S., Sparrow, D., Mohun, T. and Mercola, M. (2000) Dev. Biol., 218, 326-340). The local interactions that establish or maintain the distinct myocardial and non-myocardial domains have never been described. Here we show that Xenopus Notch1 (Xotch) and Serrate1 are expressed in overlapping patterns in the early heart field. Conditional activation or inhibition of the Notch pathway with inducible dominant negative or active forms of the RBP-J/Suppressor of Hairless [Su(H)] transcription factor indicated that activation of Notch feeds back on Serrate1 gene expression to localize transcripts more dorsolaterally than those of Notch1, with overlap in the region of the developing mesocardium. Moreover, Notch pathway activation decreased myocardial gene expression and increased expression of a marker of the mesocardium and pericardial roof, whereas inhibition of Notch signaling had the opposite effect. Activation or inhibition of Notch also regulated contribution of individual cells to the myocardium. Importantly, expression of Nkx2. 5 and Gata4 remained largely unaffected, indicating that Notch signaling functions downstream of heart field specification. We conclude that Notch signaling through Su(H) suppresses cardiomyogenesis and that this activity is essential for the correct specification of myocardial and non-myocardial cell fates.

Animals↗
PubMed1999

Cerberus regulates left-right asymmetry of the embryonic head and heart.

BACKGROUND: Most of the molecules known to regulate left-right asymmetry in vertebrate embryos are expressed on the left side of the future trunk region of the embryo. Members of the protein family comprising Cerberus and the putative tumour suppressor Dan have not before been implicated in left-right asymmetry. In Xenopus, these proteins have been shown to antagonise members of the transforming growth factor beta (TGF-beta) and Wnt families of signalling proteins. RESULTS: Chick Cerberus (cCer) was found to be expressed in the left head mesenchyme and in the left flank of the embryo. Expression on the left side of the head was controlled by Sonic hedgehog (Shh) acting through the TGF-beta family member Nodal; in the flank, cCer was also regulated by Shh, but independently of Nodal. Surprisingly, although no known targets of Cerberus are expressed asymmetrically on the right side of the embryo at these stages, misexpression of cCer on this side of the embryo led to upregulation of the transcription factor Pitx2 and reversal of the direction of heart and head turning, apparently as independent events. Consistent with the possibility that cCer may be acting on bilaterally expressed TGF-beta family members such as the bone morphogenetic proteins (BMPs), this result was mimicked by right-sided misexpression of the BMP antagonist, Noggin. CONCLUSIONS: Our findings suggest that cCer maintains a delicate balance of different TGF-beta family members involved in laterality decisions, and reveal the existence of partially overlapping molecular pathways regulating left-right asymmetry in the head and trunk of the embryo.

Amino Acid Sequence↗
PubMed1999

Embryological basis for cardiac left-right asymmetry.

Asymmetric heart tube looping and chamber morphogenesis is a complex process that is just beginning to be understood at the genetic level. Rightward looping is the first embryological manifestation of consistently oriented, left-right asymmetric development of nearly all visceral organs. Intuitively, invariant anatomical asymmetry must derive from a novel mechanism capable of integrating dorsoventral and anteroposterior information. The details of this process are emerging for several vertebrates and reveal that overall left-right asymmetry, once polarized with respect to dorsoventral and anteroposterior axes, unfolds through distinct left- and right-sided programs of gene expression. These, in turn, regulate expression of cardiac and chamber-specific genes which guide heart morphogenesis and differentiation.

Animals↗
PubMed1999

Zebrafish narrowminded suggests a genetic link between formation of neural crest and primary sensory neurons.

In the developing vertebrate nervous system, both neural crest and sensory neurons form at the boundary between non-neural ectoderm and the neural plate. From an in situ hybridization based expression analysis screen, we have identified a novel zebrafish mutation, narrowminded (nrd), which reduces the number of early neural crest cells and eliminates Rohon-Beard (RB) sensory neurons. Mosaic analysis has shown that the mutation acts cell autonomously suggesting that nrd is involved in either the reception or interpretation of signals at the lateral neural plate boundary. Characterization of the mutant phenotype indicates that nrd is required for a primary wave of neural crest cell formation during which progenitors generate both RB sensory neurons and neural crest cells. Moreover, the early deficit in neural crest cells in nrd homozygotes is compensated later in development. Thus, we propose that a later wave can compensate for the loss of early neural crest cells but, interestingly, not the RB sensory neurons. We discuss the implications of these findings for the possibility that RB sensory neurons and neural crest cells share a common evolutionary origin.

Animals↗
PubMed1999

Gap junction-mediated transfer of left-right patterning signals in the early chick blastoderm is upstream of Shh asymmetry in the node.

Invariant patterning of left-right asymmetry during embryogenesis depends upon a cascade of inductive and repressive interactions between asymmetrically expressed genes. Different cascades of asymmetric genes distinguish the left and right sides of the embryo and are maintained by a midline barrier. As such, the left and right sides of an embryo can be viewed as distinct and autonomous fields. Here we describe a series of experiments that indicate that the initiation of these programs requires communication between the two sides of the blastoderm. When deprived of either the left or the right lateral halves of the blastoderm, embryos are incapable of patterning normal left-right gene expression at Hensen's node. Not only are both flanks required, suggesting that there is no single signaling source for LR pattern, but the blastoderm must be intact. These results are consistent with our previously proposed model in which the orientation of LR asymmetry in the frog, Xenopus laevis, depends on large-scale partitioning of LR determinants through intercellular gap junction channels (M. Levin and M. Mercola (1998) Developmental Biology 203, 90-105). Here we evaluate whether gap junctional communication is required for the LR asymmetry in the chick, where it is possible to order early events relative to the well-characterized left and right hierarchies of gene expression. Treatment of cultured chick embryos with lindane, which diminishes gap junctional communication, frequently unbiased normal LR asymmetry of Shh and Nodal gene expression, causing the normally left-sided program to be recapitulated symmetrically on the right side of the embryo. A survey of early expression of connexin mRNAs revealed that Cx43 is present throughout the blastoderm at Hamburger-Hamilton stage 2-3, prior to known asymmetric gene expression. Application of antisense oligodeoxynucleotides or blocking antibody to cultured embryos also resulted in bilateral expression of Shh and Nodal transcripts. Importantly, the node and primitive streak at these stages lack Cx43 mRNA. This result, together with the requirement for an intact blastoderm, suggests that the path of communication through gap junction channels circumvents the node and streak. We propose that left-right information is transferred unidirectionally throughout the epiblast by gap junction channels in order to pattern left-sided Shh expression at Hensen's node.

Animals↗
PubMed1998

Gap junctions are involved in the early generation of left-right asymmetry.

Invariant left-right asymmetry of the visceral organs is a fundamental feature of vertebrate embryogenesis. While a cascade of asymmetrically expressed genes has been described, the embryonic mechanism that orients the left-right axis relative to the dorsoventral and anteroposterior axes (a prerequisite for asymmetric gene expression) is unknown. We propose that this process involves dorsoventral differences in cell-cell communication through gap junctions composed of connexin proteins. Global modulation of gap junctional states in Xenopus embryos by pharmacological agents specifically induced heterotaxia involving mirror-image reversals of heart, gut, and gall bladder. Greatest sensitivity was observed between st. 5 and st. 12, well before the onset of organogenesis. Moreover, heterotaxia was also induced following microinjection of dominant negative and wild-type connexin mRNAs to modify the endogenous dorsoventral difference in junctional communication. Heterotaxia was induced by either blocking gap junction communication (GJC) dorsally or by introducing communication ventrally (but not the reverse). Both connexin misexpression and exposure to GJC-modifying drugs altered expression of the normally left-sided gene XNR-1, demonstrating that GJC functions upstream of XNR-1 in the pathway that patterns left-right asymmetry. Finally, lineage analysis to follow the progeny of microinjected cells indicated that they generally do not contribute the asymmetric organs. Together with the early sensitivity window, this suggests that GJC functions as part of a fundamental, early aspect of left-right patterning. In addition, we show that a potential regulatory mutation in Connexin43 is sufficient to cause heterotaxia. Despite uncertainty about the prevalence of the serine364 to proline substitution reported in human patients with laterality defects, the mutant protein is both a mild hypomorph and a potent antimorph as determined by the effect of its expression on left-right patterning. Taken together, our data suggest that endogenous dorsoventral differences in GJC within the early embryo are needed to consistently orient left-right asymmetry.

Animals↗
PubMed1998

PDGF mediates cardiac microvascular communication.

The diversity of cellular and tissue functions within organs requires that local communication circuits control distinct populations of cells. Recently, we reported that cardiac myocytes regulate the expression of both von Willebrand factor (vWF) and a transgene with elements of the vWF promoter in a subpopulation of cardiac microvascular endothelial cells (J. Cell Biol. 138:1117). The present study explores this communication. Histological examination of the cardiac microvasculature revealed colocalization of the vWF transgene with the PDGF alpha-receptor. Transcript analysis demonstrated that in vitro cardiac microvascular endothelial cells constitutively express PDGF-A, but not B. Cardiac myocytes induced endothelial expression of PDGF-B, resulting in PDGF-AB. Protein measurement and transcript analysis revealed that PDGF-AB, but not PDGF-AA, induced endothelial expression of vWF and its transgene. Antibody neutralization of PDGF-AB blocked the myocyte-mediated induction. Immunostaining demonstrated that vWF induction is confined to PDGF alpha-receptor-positive endothelial cells. Similar experiments revealed that the PDGF-AB/alpha-receptor communication also induces expression of vascular endothelial growth factor and Flk-1, critical components of angiogenesis. The existence of this communication pathway was confirmed in vivo. Injection of PDGF-AB neutralizing antibody into the amniotic fluid surrounding murine embryos extinguished expression of the transgene. In summary, these studies suggest that environmental induction of PDGF-AB/alpha-receptor interaction is central to the regulation of cardiac microvascular endothelial cell hemostatic and angiogenic activity.

Animals↗
PubMed1998

Small-molecule control of insulin and PDGF receptor signaling and the role of membrane attachment.

BACKGROUND: Receptor tyrosine kinases (RTKs) regulate the proliferation, differentiation and metabolism of cells, and play key roles in tissue repair, tumorigenesis and development. To facilitate the study of RTKs, we have made conditional alleles that encode monomeric forms of the normally heterotetrameric insulin receptor and monomeric platelet-derived growth factor (PDGF) beta receptors fused to the FK506-binding protein 12 (FKBP12). The chimeric receptors can be induced to undergo dimerization or oligomerization by a small synthetic molecule called FK1012, and the consequences were studied in cells and embryonic tissues. RESULTS: When equipped with an amino-terminal plasma membrane localization sequence and expressed in HEK293 cells, these chimeric receptors could signal to downstream targets as indicated by the FK1012-dependent activation of p70 S6 kinase (p70(S6k)) and mitogen-activated protein (MAP) kinase. In Xenopus embryos, the engineered PDGF receptor protein induced the formation of mesoderm from animal-pole explants in an FK1012-dependent manner. A cytosolic variant of the protein underwent efficient transphosphorylation, yet failed to activate appreciably either p70(S6k) or MAP kinase following treatment with FK1012. These results provide evidence of a requirement for membrane localization of RTKs, consistent with current models of RTK signaling. CONCLUSION: We have developed an approach using the small molecule FK1012 to conditionally activate chimeric proteins containing FKBP fused to the insulin receptor or to the PDGF beta receptor. Using this system, we were able to induce mesoderm formation in Xenopus animal-cap tissue and to demonstrate that membrane localization is required for RTK signaling in transfected cells. This system should allow the further dissection of RTK-mediated pathways.

Alleles↗
PubMed1998

Evolutionary conservation of mechanisms upstream of asymmetric Nodal expression: reconciling chick and Xenopus.

Recent experiments have suggested a pathway of genes that regulate left-right asymmetry in vertebrate embryogenesis. The most downstream member of this cascade is nodal (XNR-1 in frogs), which is expressed in the left-side lateral mesoderm. Previous work in the chick [Levin, 1998] suggests that an inductive interaction by Shh (Sonic hedgehog) present at the midline was needed for the left-sided expression of nodal, which by default would not be expressed. Interestingly, it has been reported [Lohr et al., 1997] that in Xenopus, right-side mesoderm that is explanted at st. 15 and allowed to develop in culture, goes on to express nodal, suggesting that lateral mesoderm expresses this gene by default and that a repression of nodal by the midline is needed to achieve asymmetry. Such a contradiction raises interesting questions about the degree of conservation of the mechanisms upstream of nodal asymmetry and, in general, about the differences in the LR pathway among species. Thus we examined this issue directly. We show that in the chick, as in the frog, explanted mesoderm from both sides does, indeed, go on to express nodal, including both the medial and lateral expression domains. Ectopic nodal expression in the medial domain on the right side is not sufficient to induce an ectopic lateral domain. We also show that explanted lateral tissue regenerates node/notochord structures exhibiting Shh expression. Furthermore, we show that Xenopus explants done at st. 15 also regenerate notochord by the stage at which XNR-1 would be expressed. Thus explants are not isolated from the influence of the midline. In contrast to the midline repressor model previously suggested [Lohr et al., 1997] to explain the presence of nodal expression in explants, we propose that the expression is due to induction by signals secreted by regenerating node and notochord tissue (Shh in the chick). Thus our results are consistent with Shh being necessary for nodal induction in both species, and we provide an explanation for both sets of data in terms of a single conserved mechanism upstream of nodal expression.

Animals↗
PubMed1997

Organizer induction determines left-right asymmetry in Xenopus.

Vertebrates appear bilaterally symmetrical but have considerable left-right (LR) asymmetry in the anatomy and placement of internal organs such as the heart. Although a number of asymmetrically expressed genes are known to affect LR patterning, both the initial source of asymmetry and the mechanism that correctly orients the LR axis remain controversial. In this study, we show that the induction of dorsal organizing centers in the embryo can orient LR asymmetry. Ectopic organizing centers were induced by microinjection of mRNA encoding a variety of body axis duplicating proteins, including members of the Wnt signal transduction pathway. The ectopic and primary body axes form side-by-side conjoined twins, with the secondary axis developing as either the left or right sibling. In all cases, correct LR asymmetry was observed in the left twin, regardless of whether it was derived from the primary axis or induced de novo by injection of Xwnt-8, beta-catenin, or Siamois mRNA. In contrast, the right twin was generally unbiased, regardless of the origin of the left body axis, as seen in many instances of experimentally induced and spontaneous conjoined twins. An unanticipated exception was that right twins induced by beta-catenin and Siamois, two downstream effectors of Wnt signaling, exhibited predominately normal heart looping, even when they formed the right twin. Taken together, these results indicate that LR asymmetry is locally oriented as a consequence of Wnt signaling through beta-catenin and Siamois. We discuss the possibility that signals upstream of beta-catenin and Siamois might be required in order for a right sibling to be randomized.

Animals↗