Temporal mechanisms of myogenic specification in human induced pluripotent stem cells.

Understanding the mechanisms of myogenesis in human induced pluripotent stem cells (hiPSCs) is a prerequisite to achieving patient-specific therapy for diseases of skeletal muscle. hiPSCs of different origin show distinctive kinetics and ability to differentiate into myocytes. To address the unique cellular and temporal context of hiPSC differentiation, we perform a longitudinal comparison of the transcriptomic profiles of three hiPSC lines that display differential myogenic specification, one robust and two blunted. We detail temporal differences in mechanisms that lead to robust myogenic specification. We show gene expression signatures of putative cell subpopulations and extracellular matrix components that may support myogenesis. Furthermore, we show that targeted knockdown of ZIC3 at the outset of differentiation leads to improved myogenic specification in blunted hiPSC lines. Our study suggests that ß-catenin transcriptional cofactors mediate cross-talk between multiple cellular processes and exogenous cues to facilitate specification of hiPSCs to mesoderm lineage, leading to robust myogenesis.

Immunoglobulin heavy-chain enhancer requires one or more tissue-specific factors.

Enhancer sequences are regulatory regions that greatly increase transcription of certain eukaryotic genes. An immunoglobulin heavy-chain variable gene segment is moved from a region lacking enhancer activity to a position adjacent to the known heavy-chain enhancer early in B-cell maturation. In lymphoid cells, the heavy-chain and SV40 enhancers bind a common factor essential for enhancer function. In contrast, fibroblast cells contain a functionally distinct factor that is used by the SV40 but not by the heavy-chain enhancer. The existence of different factors in these cells may explain the previously described lymphoid cell specificity of the heavy-chain enhancer.

Platelet-derived growth factor A chain is maternally encoded in Xenopus embryos.

Transcription of zygotic genes does not occur in early Xenopus embryos until the mid-blastula transition, 6 to 7 hours after fertilization. Before this time, development is directed by maternal proteins and messenger RNAs stored within the egg. Two different forms of the A chain of platelet-derived growth factor (PDGF) are shown here to be encoded by maternal messenger RNAs. The two forms closely resemble human PDGF; however, the long form contains a hydrophobic region near the carboxyl terminus. The presence of PDGF messenger RNA in the embryo supports the idea that endogenous growth factors act at the earliest stages of embryogenesis.

Transcriptional enhancer elements in the mouse immunoglobulin heavy chain locus.

Two regions in the immunoglobulin heavy chain locus were tested for their ability to enhance transcription of the SV40 early promoter. A portion of the intervening sequence between the heavy chain joining region (Jh) and the constant region of the mu chain (Cmu) can enhance transcription when it is cloned either 5' or 3' to the SV40 early promoter. The region between C alpha and the alpha switch site, which occurs 5' to the translocated c-myc oncogene in many murine plasmacytomas, does not show transcriptional enhancer activity in this assay.

Spatially distinct head and heart inducers within the Xenopus organizer region.

BACKGROUND: The mouse anterior visceral endoderm, an extraembryonic tissue, expresses several genes essential for normal development of structures rostral to the anterior limit of the notochord and has been termed the head organizer. This tissue also has heart-inducing activity and expresses mCer1 which, like its Xenopus homolog cerberus, can induce markers of cardiac specification and anterior neural tissue when ectopically expressed. We investigated the relationship between head and heart induction in Xenopus embryos, which lack extraembryonic tissues. RESULTS: We found three regions of gene expression in the Xenopus organizer: deep endoderm, which expressed cerberus; prechordal mesoderm, which showed overlapping but non-identical expression of genes characteristic of the murine head organizer, such as XHex and XANF-1; and leading-edge dorsoanterior endoderm, which expressed both cerberus and a subset of the genes expressed by the prechordal mesoderm. Microsurgical ablation of the cerberus-expressing endoderm decreased the incidence of heart, but not head, formation. Removal of prechordal mesoderm, in contrast, caused deficits of anterior head structures. Finally, although misexpression of cerberus induced ectopic heads, it was unable to induce genes thought to participate in head induction. CONCLUSIONS: In Xenopus, the cerberus-expressing endoderm is required for heart, but not head, inducing activity. Therefore, this tissue is not the topological equivalent of the murine anterior visceral endoderm. We propose that, in Xenopus, cerberus is redundant to other bone morphogenetic protein (BMP) and Wnt antagonists located in prechordal mesoderm for head induction, but may be necessary for heart induction.

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.

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.

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.

Effect of geraniol on rat cardiomyocytes and its potential use as a cardioprotective natural compound.

AIMS: Reactive oxygen species (ROS) are generated in the ischaemic myocardium especially during early reperfusion and affect myocardial function and viability. Monoterpenes have been proposed to play beneficial roles in diverse physiological systems; however, the mechanisms of action remain largely unknown. This study aims to assess the effect of monoterpene geraniol (GOH) on neonatal rat ventricular cardiomyocytes (NRVCs) subjected to oxidative stress. MAIN METHODS: We used an in vitro model of hypoxia-reoxygenation. Cardioprotective (AMPK) and cardiotoxic (ERK1/2, ROS) signaling indicators were measured. Assays were performed by fluorogenic probes, MTT assays and Western-blots. KEY FINDINGS: We determined that the addition of GOH (5-200µM) to cultured normoxic and hypoxic-NRVCs diminished the endogenous production of ROS in stressed cardiomyocytes. We observed that GOH treatment increased pAMPK levels and decreased pERK1/2 levels in cultured NRVCs. SIGNIFICANCE: This report suggests that GOH is a candidate cardioprotective natural compound that operates by blunting the oxidative stress signaling that is normally induced by hypoxia-reoxygenation.

Rapid, complete and reversible transformation by v-sis precedes irreversible transformation.

v-sis is the oncogene of simian sarcoma virus, but whether tumor growth is maintained by v-sis expression alone or requires additional changes is unknown. To distinguish these possibilities we studied a model of reversible transformation including tumorigenicity using NIH3T3 cells bearing a metallothionein promoter-v-sis construction. Cells subcultured from 10 out of 18 tumors from athymic mice, all less than 0.1 g and less than or equal to 21 days in age, reverted to a normal phenotype but exhibited transformation upon addition of zinc as judged by morphology, growth rate, saturation density and anchorage independence of growth. Thus, activation of v-sis alone is sufficient for initiation and early autocrine-based growth of tumors. However, the cells from the remaining and predominantly larger, 0.5 +/- 0.7 g, tumors did not revert and exhibited zinc-independent transformation as judged by the same criteria. Southern analysis and examination of the regulation of v-sis product expression in cells derived from these tumors showed no change in zinc-dependent and reversible regulation of v-sis sequences. These results suggest that subsequent tumor growth strongly favors acquisition of additional irreversible change(s) in the tumor cell genome at high frequency (44%). Thus an early event of a multistep process stimulated by v-sis-dependent transformation best accounts for the sum of results.

The interaction of Cibacron Blue F3GA with troponin and its subunits.

Binding of troponin to Cibacron Blue F3GA-agarose column and its selective release from the gel in the presence of 0.5 M KCl provides the basis for a new purification method. The two-step procedure consists of isoelectric precipitation of tropomyosin and chromatography of the resultant crude troponin supernatant on Affi-Gel Blue column. Adsorption of troponin to the immobilized dye appears to occur through the troponin-T subunit. Troponin-I and troponin-C do not bind to the blue agarose column, whereas troponin-T binds to it very tightly. Binding of the dye to troponin-T prevents formation of troponin-T-troponin-C complex, but does not interfere with direct interaction of troponin-T with troponin-I. The activity of troponin in conferring calcium sensitivity on actomyosin ATPase is not affected by Cibacron Blue. Circular dichroism and difference absorption measurements of complexes of the blue dye with troponin and its subunits reveal the presence of a tight binding site on whole troponin and on troponin-T (KA greater than or equal to 10(6) M). The existence of weak binding sites for the dye on troponin and all of its subunits is deduced from difference absorption studies. Cibacron Blue appears to be a sensitive probe for subunit interactions in troponin.

Distribution and functions of platelet-derived growth factors and their receptors during embryogenesis.

Platelet-derived growth factors (PDGFs) are soluble proteins that mediate intercellular signaling via receptor tyrosine kinases. The patterns of PDGF and PDGF receptor expression during embryogenesis are complex and dynamic and suggest that signaling can be autocrine or paracrine, depending on the particular tissue and the stage of development. Mesenchymal cells throughout the embryo and within some developing organs produce PDGF receptors, whereas their ligands are often produced by adjacent epithelial or endothelial cells. Disruption of PDGF signaling in the embryo leads to morphogenetic defects and embryonic or perinatal lethality. Tissues that are particularly susceptible to the absence of PDGF signaling are migrating mesoderm cells during gastrulation, nonneuronal neural crest cell derivatives, and kidney mesangial cells. These tissues share the common feature of undergoing epithelial-mesenchymal transitions. We review current knowledge of the distribution of PDGF ligands and receptors and discuss how this distribution may relate to several roles for PDGF during embryogenesis, particularly the regulation of mesenchymal cell behavior.

Xenopus laevis cellular retinoic acid-binding protein: temporal and spatial expression pattern during early embryogenesis.

There is increasing evidence that retinoic acid (RA) has a role in establishing normal axial patterns during Xenopus laevis embryo-genesis. Several types of retinoid binding proteins are thought to mediate the effects of RA. We report the isolation of a cDNA, named xCRABP-b, which encodes a X. laevis cellular retinoic acid-binding protein (xCRABP). This cDNA hybridises to a transcript in gastrular stage embryos of approximately 3 kb, much larger than those CRABP transcripts expressed in mice. The expression of the xCRABP mRNA is generally restricted to tissues which are sensitive to the teratogenic effects of excess RA. It is likely, that during normal X. laevis embryogenesis, concentrations of RA in RA-responsive cells are modulated by the xCRABP gene product.

Localization of PDGF A and PDGFR alpha mRNA in Xenopus embryos suggests signalling from neural ectoderm and pharyngeal endoderm to neural crest cells.

In situ hybridization analysis of Xenopus laevis embryos reveals that mRNA encoding the platelet-derived growth factor alpha receptor (PDGFR alpha) is expressed in cephalic neural crest masses prior to migration from the future neural tube and during their migration into the visceral arches. The analysis of fluorescently labeled neural crest tissue transplanted to unlabeled host embryos demonstrates that neural crest cells are the only detectable source of PDGFR alpha mRNA within visceral arches. Transcripts encoding PDGF A are present in neural ectoderm, otic vesicle and pharyngeal endoderm. Their location suggests that PDGF A provides a signal, first from the neural epithelium and later from the otic vesicle and pharyngeal endoderm, to cephalic neural crest cells during their migration in the arch region.

Expression of mouse PDGF-A and PDGF alpha-receptor genes during pre- and post-implantation development: evidence for a developmental shift from an autocrine to a paracrine mode of action.

We examined the expression of platelet-derived growth factor (PDGF)-A and the PDGF alpha-receptor in pre-implantation and early post-implantation mouse embryos. At two-cell and blastocyst stages, all cells express mRNA and protein for both ligand and receptor. In contrast, early post-implantation embryos express PDGF-A chain mRNA in both embryonic ectoderm and in the ectoderm lining the ectoplacental cavity, while mRNA for PDGF alpha-receptor is localized to the mesoderm layers of both embryonic and extra-embryonic membranes. At days 3.5 and 7.5, receptors are demonstrably functional in response to exogenous PDGF-AA. We propose that chronic autostimulation of PDGF alpha-receptors occurs in pre-implantation embryos, whereas, following implantation, early mesoderm development is dependent on stimulation by ectodermally produced PDGF-A.

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.

Endoderm and cardiogenesis new insights.

Classic studies of vertebrate heart development have implicated the endoderm in an inductive role, based on its ability to induce rhythmic beating in explants of presumptive heart mesoderm. Recent experiments, aided by the use of heart-specific molecular markers, have defined discrete phases of cardiogenesis that depend on endodermal signals for functional contractility. In addition, the ability of the endoderm to generate a beating heart from tissues fated to form other cell types suggests that endoderm may also be involved in the initial specification of the early heart field.

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.

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.