eXtraembryonic ENdoderm (XEN) stem cells produce factors that activate heart formation

Background

Initial specification of cardiomyocytes in the mouse results from interactions between the extraembryonic anterior visceral endoderm (AVE) and the nascent mesoderm. However the mechanism by which AVE activates cardiogenesis is not well understood, and the identity of specific cardiogenic factors in the endoderm remains elusive. Most mammalian studies of the cardiogenic potential of the endoderm have relied on the use of cell lines that are similar to the heart-inducing AVE. These include the embryonal-carcinoma-derived cell lines, END2 and PYS2. The recent development of protocols to isolate eXtraembryonic ENdoderm (XEN) stem cells, representing the extraembryonic endoderm lineage, from blastocyst stage mouse embryos offers new tools for the genetic dissection of cardiogenesis.

Methodology/Principal Findings

Here, we demonstrate that XEN cell-conditioned media (CM) enhances cardiogenesis during Embryoid Body (EB) differentiation of mouse embryonic stem (ES) cells in a manner comparable to PYS2-CM and END2-CM. Addition of CM from each of these three cell lines enhanced the percentage of EBs that formed beating areas, but ultimately, only XEN-CM and PYS2-CM increased the total number of cardiomyocytes that formed. Furthermore, our observations revealed that both contact-independent and contact-dependent factors are required to mediate the full cardiogenic potential of the endoderm. Finally, we used gene array comparison to identify factors in these cell lines that could mediate their cardiogenic potential.

Conclusions/Significance

These studies represent the first step in the use of XEN cells as a molecular genetic tool to study cardiomyocyte differentiation. Not only are XEN cells functionally similar to the heart-inducing AVE, but also can be used for the genetic dissection of the cardiogenic potential of AVE, since they can be isolated from both wild type and mutant blastocysts. These studies further demonstrate the importance of both contact-dependent and contact-independent factors in cardiogenesis and identify potential heart-inducing proteins in the endoderm.     

Lithium inhibits glycogen synthase kinase-3 activity and mimics Wingless signalling in intact cells

Background Exposing eukaryotic cells to lithium ions (Li⁺) during development has marked effects on cell fate and organization. The phenotypic consequences of Li⁺ treatment on Xenopus embryos and sporulating Dictyostelium are similar to the effects of inhibition or disruption, respectively, of a highly conserved protein serine/threonine kinase, glycogen synthase kinase-3 (GSK-3). In Drosophila, the GSK-3 homologue is encoded by zw3^sgg, a segment-polarity gene involved in embryogenesis that acts downstream of wg. In higher eukaryotes, GSK-3 has been implicated in signal transduction pathways downstream of phosphoinositide 3-kinase and mitogen-activated protein kinases.Results We investigated the effect of Li⁺ on the activity of the GSK-3 family. At physiological doses, Li⁺ inhibits the activity of human GSK-3β and Drosophila Zw3^Sgg, but has no effect on other protein kinases. The effect of Li⁺ on GSK-3 is reversible in vitro. Treatment of cells with Li⁺ inhibits GSK-3-dependent phosphorylation of the microtubule-associated protein Tau. Li⁺ treatment of Drosophila S2 cells and rat PC12 cells induces accumulation of cytoplasmic Armadillo/β-catenin, demonstrating that Li⁺ can mimic Wingless signalling in intact cells, consistent with its inhibition of GSK-3.Conclusions Li⁺ acts as a specific inhibitor of the GSK-3 family of protein kinases in vitro and in intact cells, and mimics Wingless signalling. This reveals a possible molecular mechanism of Li⁺ action on development and differentiation.     

Diversification of Cardiomyogenic Cell Lineages in Vitro

The ability of undifferentiated cardiogenic mesoderm to generate diversified myogenic phenotypes was assayed in a minimal culture system. During cardiogenesis in vivo, the anterior and posterior segments of the avian heart have distinct patterns of contractile protein gene expression when they first differentiate. To assess the potential of undifferentiated cardiogenic tissue to diversify into distinct anterior and posterior lineages prior to heart formation, cardiogenic mesoderm and endoderm were removed together from the embryo at Hamburger and Hamilton stages 4-8. Explants from each of these stages differentiated in defined medium as indicated by the expression of muscle-specific genes. However, the ability to express the atrial-specific myosin heavy chain (AMHC1) mRNA was confined to posterior cardiac progenitors. Diversification was not dependent on anterior endoderm, suggesting that inductive interactions between the mesoderm and endoderm are not necessary to maintain diversified cardiac lineages after stage 4. The diversified potential of explanted cardiogenic tissue was altered with retinoic acid treatment, resulting in the activation of AMHC1 gene expression in the anterior progenitors. Anterior cardiogenic cells removed from the embryo at stage 8, when the heart begins to differentiate in vivo, are not susceptible to the alteration of diversified phenotype by retinoic acid treatment. Therefore, the potential to form distinct cardiomyogenic cell lineages is present in the anterior lateral plate mesoderm soon after gastrulation and the maturation of these lineages in a positionally dependent manner is maintained in a simple defined culture system in vitro.     

Cyclic GMP is not involved in neural induction inXenopus laevis

Summary Recent evidence indicates an important role for cell-surface mediated signal transduction in embryonic induction. We, therefore, started a systematic search to identify signal transduction pathways which are activated during embryonic induction and specifically during neural induction. We showed previously that the protein kinase C and cAMP pathways mediate neural induction inXenopus laevis. Here, we investigated whether cGMP is also involved in the early development of the nervous system. We measured the cGMP content of whole embryos at embryonic stages which mark important events in the early development of the nervous system, as well as in the developing neural tissue itself, after this was induced from ectoderm by dorsal mesoderm. No changes in cGMP content were found, either in whole embryos at different developmental stages, or in developing neural tissue from these stages. We also found no evidence for the presence of nitroprusside stimulatable guanylate cyclase in these developmental stages. A cGMP analogue, 8-Br-cGMP, was not able to induce neural tissue, either alone or in combination with known neural inducers, the phorbol ester TPA and 8-Br-cAMP. 8-Br-cGMP also had no negative influence on the neural inducing ability of dorsal mesoderm or TPA, alone or in combination with 8-Br-cAMP. We conclude that cGMP has no role in the early development of the central nervous system inXenopus laevis. This conclusion underlines the specificity of the signal transduction pathways (PKC and cAMP pathways) that do mediate neural induction.     

Evolutionary origin of the insect wing via integration of two developmental modules

SUMMARY Insect wing is a key evolutionary innovation for insect radiation, but its origins and intermediate forms are absent from the fossil record. To understand the ancestral state of the wing, expression of three key regulatory genes in insect wing development, wingless (wg), vestigial (vg), and apterous (ap) was studied in two basal insects, mayfly and bristletail. These basal insects develop dorsal limb branches, tracheal gill and stylus, respectively, that have been considered candidates for wing origin. Here we show that wg and vg are expressed in primordia for tracheal gill and stylus. Those primordia are all located in the lateral body region marked by down‐regulation of early segmental wg stripes, but differ in their dorsal–ventral position, indicating their positions drifted within the lateral body region. On the other hand, ap expression was detected in terga of mayfly and bristletail. Notably, the extensive outgrowth of the paranotal lobe of apterygote bristletail developed from the border of ap‐expressing tergal margin, and also expressed wg and vg. The data suggest that two regulatory modules involving wg–vg are present in apterygote insects: one associated with lateral body region and induces stick‐like dorsal limb branches, the other associated with the boundary of dorsal and lateral body regions and the flat outgrowth of their interface. A combinatorial model is proposed in which dorsal limb branch was incorporated into dorsal–lateral boundary and acquired flat limb morphology through integration of the two wg–vg modules, allowing rapid evolution of the wing.     

The convergence of Notch and MAPK signaling specifies the blood progenitor fate in the Drosophila mesoderm

Blood progenitors arise from a pool of pluripotential cells (“hemangioblasts”) within the Drosophila embryonic mesoderm. The fact that the cardiogenic mesoderm consists of only a small number of highly stereotypically patterned cells that can be queried individually regarding their gene expression in normal and mutant embryos is one of the significant advantages that Drosophila offers to dissect the mechanism specifying the fate of these cells. We show in this paper that the expression of the Notch ligand Delta (Dl) reveals segmentally reiterated mesodermal clusters (“cardiogenic clusters”) that constitute the cardiogenic mesoderm. These clusters give rise to cardioblasts, blood progenitors and nephrocytes. Cardioblasts emerging from the cardiogenic clusters accumulate high levels of Dl, which is required to prevent more cells from adopting the cardioblast fate. In embryos lacking Dl function, all cells of the cardiogenic clusters become cardioblasts, and blood progenitors are lacking. Concomitant activation of the Mitogen Activated Protein Kinase (MAPK) pathway by Epidermal Growth Factor Receptor (EGFR) and Fibroblast Growth Factor Receptor (FGFR) is required for the specification and maintenance of the cardiogenic mesoderm; in addition, the spatially restricted localization of some of the FGFR ligands may be instrumental in controlling the spatial restriction of the Dl ligand to presumptive cardioblasts.     

Dual functions of the heartless fibroblast growth factor receptor in development of the Drosophila embryonic mesoderm

The Drosophila embryonic mesoderm forms by invagination of the ventral-most blastoderm cells. Subsequent development of this germ layer involves the dorsolateral migration of the internalized cells and expansion by cell division, followed by the specification of particular cell fates through the coordinate actions of both intrinsic and extrinsic regulatory mechanisms. The latter include several intercellular signals that function across germ layers. These processes combine to generate a diversity of mesodermal subtypes, including the cardial and pericardial cells of the heart or dorsal vessel, a complete set of somatic muscle founders each with its unique identity, a population of cells that form the visceral musculature, and other cells that develop into hemocytes and the fat body. Here, we review recent evidence for the involvement of a fibroblast growth factor receptor (FGFR) encoded by the heartless (htl) gene in early directional migration of the Drosophila mesoderm. In addition, we provide new data that 1) demonstrate a second role for Htl in promoting the specification of the precursors to certain cardiac and somatic muscle cells in the Drosophila embryo, independent of its cell migration function, 2) suggest that Ras and at least one other signal transduction pathway act downstream of Htl, and 3) establish a functional relationship between the Ras pathway and Tinman (Tin), a homeodomain factor that is essential for specifying some of the same dorsal mesodermal cells that are dependent on Htl. Finally, parallels between requirements for FGFR signaling in Drosophila and vertebrate mesoderm development are considered. Dev. Genet. 22:212–229, 1998. © 1998 Wiley-Liss, Inc.     

Thec-kit receptor transduces the stem cell factor-triggered growth signal in murine interleukin-3-dependent cell line

The stem cell factor is a glycoprotein hormone which regulates the proliferation and differentiation of primitive hematopoietic cells through its interaction with a tyrosine kinase transmembrane receptor which is encoded by thec-kit proto-oncogene. To examine whether a murinec-kit receptor can be functional in murine interleukin-3 (mlL-3)-dependent hematopoietic cell line, we introduced the murinec-kit cDNA into mlL-3-dependent pro-B cell line Ba/F3. One of the resulting clones, Ba/F3 clone BF-K96, expressed the 140 kDa protein recognized by anti-c-kit monoclonal antibody and the expressedc-kit receptor protein on the cell surface bound to a radiolabeled soluble form of murine stem cell factor (mSCF) with high affinity. BF-K96 clone expressing thec-kit receptor could proliferate in response to mSCF in the absence of mlL-3. The cell clone could also grow in co-culture with mouse 3T3 cells which are endogeneously expressing a membrane-associated type of mSCF on their cell surfaces. These findings demonstrate that thec-kit receptor expressed on mlL-3-dependent hematopoietic cell line Ba/F3 transduce the mSCF-dependent growth signal, indicating that established cell clone will provide a unique cellular system for the study of SCF/c-kit signal transduction mechanism.Abbreviations SCF stem cell factor - IL interleukin - CSF colony stimulating factor - IMDM Iscove's Modified Dulbecco's Medium - DME Dulbecco's Modified Eagle's Medium - FCS fetal calf serum - PCR polymerase chain reaction - EDTA ethylenediaminetetra-acetic acid; sodium dodecyl sulfate     

WNT-mediated relocalization of dishevelled proteins

Summary TheWnt family of proto-oncogenes encodes secreted signaling proteins that are required for mouse development. TheDrosophila Wnt homolog, thewingless (Wg) segment polarity gene, mediates a signal transduction pathway in which the downstream elements appear to be conserved through evolution. One such element, thedishevelled gene product, becomes hyperphosphorylated and translocates to the plasma membrane in response to Wg (Yanagawa et al., 1995). We report here that the mouseDishevelle-1 (Dvl-1) andDishevelled-2 genes encode proteins that are differentially localized inWnt-overexpressing PC12 cell lines (PC12/Wnt). WhereasDvl-1 andDvl-2 proteins are limited to the soluble fraction of parental PC12 cells, PC12/Wnt cells display a subset ofDvl-1 protein associated with the membrane andDvl-2 protein with the cytoskeletal fraction. These results suggest a conserved role forDvl inWnt/wg signal transduction.     

misshapen encodes a protein kinase involved in cell shape control in Drosophila

We have identified a novel protein kinase encoded by the misshapen gene, which is required for the normal shape and orientation of Drosophila photoreceptor cells. misshapen is also expressed in the embryonic mesoderm, pole plasm and other sites of cell shape change or movement. We propose that msn may act in a signal transduction pathway leading to cytoskeletal re-arrangements.     

Requirement ofwingless signaling andengrailed action in the development and differentiation of reproductive system inDrosophila

The segment polarity geneswingless (wg) andengrailed (en) have been shown to play important roles in pattern formation at different stages ofDrosophila development in the thoracic imaginai discs. We have studied the patterns of expression of these genes in genital discs from wild type larvae, pupae and pharate adults and also from hetero-allelic mutant combinations of these genes. Our results suggest that these genes play vital roles in the normal development and differentiation of genital discs and gonads. In the absence of normalwg oren functions, the flies showed a complete lack of internal accessory reproductive organs and specific defects in the external genitalia. In addition, the testes in such males were small, rounded and with an abnormal cellular organization, although the ovaries in females appeared normal. Temperature shift experiments using the conditional mutant allele ofwg, (wg ^IL-114 ) indicated a requirement ofwg signaling from second instar onwards for normal development and differentiation of the accessory reproductive organs. Using a heat-shock allele (Hs-wg) we also show that the spatially regulated expression ofwg as a pre-requisite for normal development and differentiation. Based on the expression patterns ofen andhedgehog (hh) we suggest that even in the genital disc development and differentiation the action ofen is mediated throughhh.     

Autonomy of the wingless mutation in Drosophila melanogaster

Summary T(Y;2) translocations were used to cytologically localise the wingless locus of Drosophila melanogaster. We found that an existing T(Y;2), which is an insertion of a segment of 2L into the Y chromosome, has wg ⁺ within this insert. This Y chromosome was used to generate an attached XY chromosome containing wg ⁺. The mutation claret-nondisjunctional (ca ^nd) was used to induce the loss of this XY chromosome and thus generate gynandromorphs with wg ¹/wg ¹ male tissue and wg ⁺/wg ¹/wg ¹ female tissue. Analysis of these gynanders demonstrated that a genotypically wingless mutant hemithorax is usually also phenotypically mutant in these half body mosaics; thus wg ¹ is discautonomous. This observation is of interest as it is known that wg is not cell autonomous.     

Lack of regulation in the heart forming region of avian embryos

The ability to regenerate a heart after ablation of cardiogenic mesoderm has been demonstrated in early stage fish and amphibian embryos but this type of regulation of the heart field has not been seen in avians or mammals. The regulative potential of the cardiogenic mesoderm was examined in avian embryos and related to the spatial expression of genes implicated in early cardiogenesis. With the identification of early cardiac regulators such as bmp-2 and nkx-2.5, it is now possible to reconcile classical embryological studies with molecular mechanisms of cardiac lineage determination in vivo. The most anterior lateral embryonic cells were identified as the region that becomes the heart and removal of all or any subset of these cells resulted in the loss of corresponding cardiac structures. In addition, removal of the lateral heart forming mesoderm while leaving the lateral endoderm intact also results in loss of cardiac structures. Thus the medial anterior mesoderm cannot be recruited into the heart lineage in vivo even in the presence of potentially cardiac inducing endoderm. In situ analysis demonstrated that genes involved in early events of cardiogenesis such as bone morphogenetic protein 2 (bmp-2) and nkx-2.5 are expressed coincidentally with the mapped far lateral heart forming region. The activin type IIa receptor (actR-IIa) is a potential mediator of BMP signaling since it is expressed throughout the anterior mesoderm with the highest level of expression occurring in the lateral prospective heart cells. The posterior boundary of actR-IIa is consistent with the posterior boundary of nkx-2.5 expression, supporting a model whereby ActR-IIa is involved in restricting the heart forming region to an anterior subset of lateral cells exposed to BMP-2. Analysis of the cardiogenic potential of the lateral plate mesoderm posterior to nkx-2.5 and actR-IIa expression demonstrated that these cells are not cardiogenic in vitro and that removal of these cells from the embryo does not result in loss of heart tissue in vivo. Thus, the region of the avian embryo that will become the heart is defined medially, laterally, and posteriorly by nkx-2.5 gene expression. Removal of all or part of the nkx-2.5 expressing region results in the loss of corresponding heart structures, demonstrating the inability of the chick embryo to regenerate cardiac tissue in vivo at stages after nkx-2.5 expression is initiated.     

Endogenous Wnt/β-Catenin Signaling Is Required for Cardiac Differentiation in Human Embryonic Stem Cells

Background

Wnt/β-catenin signaling is an important regulator of differentiation and morphogenesis that can also control stem cell fates. Our group has developed an efficient protocol to generate cardiomyocytes from human embryonic stem (ES) cells via induction with activin A and BMP4.

Methodology/Principal Findings

We tested the hypothesis that Wnt/β-catenin signals control both early mesoderm induction and later cardiac differentiation in this system. Addition of exogenous Wnt3a at the time of induction enhanced cardiac differentiation, while early inhibition of endogenous Wnt/β-catenin signaling with Dkk1 inhibited cardiac differentiation, as indicated by quantitative RT-PCR analysis for β-myosin heavy chain (β-MHC), cardiac troponin T (cTnT), Nkx2.5, and flow cytometry analysis for sarcomeric myosin heavy chain (sMHC). Conversely, late antagonism of endogenously produced Wnts enhanced cardiogenesis, indicating a biphasic role for the pathway in human cardiac differentiation. Using quantitative RT-PCR, we show that canonical Wnt ligand expression is induced by activin A/BMP4 treatment, and the extent of early Wnt ligand expression can predict the subsequent efficiency of cardiogenesis. Measurement of Brachyury expression showed that addition of Wnt3a enhances mesoderm induction, whereas blockade of endogenously produced Wnts markedly inhibits mesoderm formation. Finally, we show that Wnt/β-catenin signaling is required for Smad1 activation by BMP4.

Conclusions/Significance

Our data indicate that induction of mesoderm and subsequent cardiac differentiation from human ES cells requires fine-tuned cross talk between activin A/BMP4 and Wnt/β-catenin pathways. Controlling these pathways permits efficient generation of cardiomyocytes for basic studies or cardiac repair applications.     

Calcium and secondary CPK signaling in plants in response to herbivore attack

Plant Ca²⁺ signals are involved in a sizable array of intracellular signaling pathways after pest invasion. Upon herbivore feeding there is a dramatic Ca²⁺ influx, followed by the activation of Ca²⁺-dependent signal transduction pathways that include interacting downstream networks of kinases for defense responses. Notably, Ca²⁺-binding sensory proteins such as Ca²⁺-dependent protein kinases (CPKs) have recently been documented to mediate the signaling following Ca²⁺ influx after herbivory, in phytohormone-independent manners. Here, we review the sequence of signal transductions triggered by herbivory-evoked Ca²⁺ signaling leading to CPK actions for defense responses, and discuss in a comparative way the involvement of CPKs in the signal transduction of a variety of other biotic and abiotic stresses.     

hpc2研究进展

生物个体的胚胎发育以及细胞的增殖、分化,都同时受到多种基因的严格调控,PcG基因家族就是一类重要的发育相关基因.而hPc2基因是人PcG基因家族中的一个重要成员,其编码的HPC2蛋白,不仅可以和HPH、BMI-1以及RING1等其他人类PcG蛋白结合形成HPC/HPH PcG复合体,以蛋白复合体的形式参与对homeotic基因的表达抑制,以维持机体的正常发育以及细胞的增殖和定向分化,还发现它能与其他多种蛋白质相结合,提示HPC2可能具有多种功能.因此,对hPc2的深入研究不仅有助于进一步阐明PcG基因家族的作用机理,扩展人们对基因表达调控的认识,还有助于发现PcG基因家族与其他信号转导通路的联系,更好地理解细胞信号网络系统.