MAP kinase activation in avian cardiovascular development.

Signaling pathways mediated by receptor tyrosine kinases (RTK) and mitogen-activated protein kinase (MAPK) activation have multiple functions in the developing cardiovascular system. The localization of diphosphorylated extracellular signal regulated kinase (dp-ERK) was monitored as an indicator of MAPK activation in the forming heart and vasculature of avian embryos. Sustained dp-ERK expression was observed in vascular endothelial cells of embryonic and extraembryonic origins. Although dp-ERK was not detected during early cardiac lineage induction, MAPK activation was observed in the epicardial, endocardial, and myocardial compartments during heart chamber formation. Endocardial expression of dp-ERK in the valve primordia and heart chambers may reflect differential cell growth associated with RTK signaling in the heart. dp-ERK localization in the epicardium, subepicardial fibroblasts, myocardial fibroblasts, and coronary vessels is consistent with MAPK activation in epicardial-derived cell lineages. The complex temporal-spatial regulation of dp-ERK in the heart supports diverse regulatory functions for RTK signaling in different cell populations, including the endocardium, myocardium, and epicardial-derived cells during cardiac organogenesis.     

Sprouty1 regulates neural and endothelial differentiation of mouse embryonic stem cells

Fibroblast growth factor (FGF) signaling is implicated in the control of pluripotency and lineage differentiation of both human and mouse embryonic stem cells (mESCs). FGF4 dependent stimulation of ERK1/2 signaling triggers transition of pluripotent ESCs from self-renewal and lineage commitment. In this study, Sprouty 1 (Spry1) expression was observed in undifferentiated mESCs, where it modulated ERK1/2 activity. Spry1 was confirmed as dispensable for the maintenance of self-renewal. However, suppression of Spry1 expression and subsequent activation of ERK1/2 signaling promoted neural differentiation and inhibited endothelial differentiation of mESCs. Moreover, evidence is presented which indicates that SHP2, a major determinant of balance between mESC self-renewal and differentiation, directly regulates Spry1 activity to modulate ERK1/2 signaling and lineage-specific differentiation in mESCs. Our results show that Spry1 has an essential role in the lineage specific differentiation of mESCs.     

Heart development before beating

During heart development at the pregastrula stage, prospective heart cells reside in the posterior lateral region of the epiblast layer. Interaction of tissues between the posterior epiblast and hypoblast is necessary to generate the future heart mesoderm. Signaling regulating the interaction involves fibroblast growth factor (FGF)-8, Nodal, bone morphogenetic protein (BMP)-antagonist, and canonical Wnt and acts on the posterior epiblast to induce the expression of genes specific for the anterior lateral mesoderm. At the early gastrula stage, prospective heart cells accumulate at the posterior midline and migrate to the anterior region of the primitive streak. During gastrulation, future heart cells leave the primitive streak and migrate anterolaterally to form the left and right anterior lateral plate mesoderm including the precardiac mesoderm. At this stage, prospective heart cells receive endoderm-derived signals, including BMP, FGF, and Wnt-antagonist, and thereby become committed to the heart lineage. At the neurula stage, the left and right precardiac mesoderm move to the ventral midline and fuse, resulting in the formation of a single primitive heart tube. Therefore, a two-step signaling cascade, which includes tissue interaction between epiblast and hypoblast at the blastula stage and endoderm-derived signals during gastrulation, is required to generate a beating heart.

Progressive induction of caudal neural character by graded Wnt signaling

Early in differentiation, all neural cells have a rostral character. Only later do posteriorly positioned neural cells acquire characteristics of caudal forebrain, midbrain and hindbrain cells. Caudalization of neural tissue in the chick embryo apparently involves the convergent actions of (i) fibroblast growth factor (FGF) signaling and (ii) signaling from the caudal paraxial mesoderm, or 'PMC activity', which has not yet been defined molecularly. Here we report evidence that Wnt signaling underlies PMC activity, and show that Wnt signals act directly and in a graded manner on anterior neural cells to induce their progressive differentiation into caudal forebrain, midbrain and hindbrain cells.     

The rostro-caudal position of cardiac myocytes affect their fate.

During chick embryogenesis, cells destined to form cardiac myocytes are located within the primitive streak at stage 3 in the same relative anterior-posterior distribution as in the prelooped heart. The most rostral cells contribute to the extreme anterior pole of the heart, the bulbus cordis, and the most caudal to the extreme posterior end, the sinoatrial region. After gastrulation, these cells commit to the myocyte lineage and, retaining their relative positions, migrate to the anterior lateral plate. From stages 5 to 10 they diversify into atrial and ventricular myocytes, with the former located posteriorly and the latter, anteriorly. To determine the effect of a change in the rostro-caudal position of these cells on their diversification, anterior lateral plate mesoderm and the underlying endoderm were cut and rotated 180 degrees along the longitudinal axis, at stages 4-8. The subsequent diversification of these precursor cells into atrial and ventricular myocytes was examined using lineage-specific markers. Our results showed that altering location along the longitudinal axis through stage 6 changed the normal fate of a precursor cell. The orientation of the overlying ectoderm did not alter normal morphogenesis or determination of fate.     

vHnf1 regulates specification of caudal rhombomere identity in the chick hindbrain.

The homeobox-containing gene variant hepatocyte nuclear factor-1 (vHnf1) has recently been shown to be involved in zebrafish caudal hindbrain specification, notably in the activation of MafB and Kro x 20 expression. We have explored this regulatory network in the chick by in ovo electroporation in the neural tube. We show that mis-expression of vHnf1 confers caudal identity to more anterior regions of the hindbrain. Ectopic expression of mvHnf1 leads to ectopic activation of MafB and Kro x 20, and downregulation of Hoxb1 in rhombomere 4. Unexpectedly, mvhnf1 strongly upregulates Fgf3 expression throughout the hindbrain, in both a cell-autonomous and a non-cell-autonomous manner. Blockade of FGF signaling correlates with a selective loss of MafB and Kro x 20 expression, without affecting the expression of vHnf1, Fgf3, or Hoxb1. Based on these observations, we propose that in chick, as in zebrafish, vHnf1 acts with FGF to promote caudal hindbrain identity by activating MafB and Kro x 20 expression. However, our data suggest differences in the vHnf1 downstream cascade in different vertebrates.     

Specific glycosaminoglycans modulate neural specification of mouse embryonic stem cells

Mouse embryonic stem (mES) cells express a low sulfated form of heparan sulfate (HS). HS chains displayed by ES cells and their progeny become more complex and more sulfated during progression from pluripotency to neuroectodermal precursors. Sulfated epitopes are important for recognition and binding of a variety of ligands including members of the fibroblast growth factor (FGF) family. We demonstrated previously that mES cells lacking HS cannot undergo neural specification but this activity can be recovered by adding soluble heparin, a highly sulfated glycosaminoglycan (GAG). Therefore, we hypothesized that soluble GAGs might be used to support neural differentiation of HS competent cells and that the mechanisms underlying this activity might provide useful information about the signaling pathways critical for loss of pluripotency and early lineage commitment. In this study, we demonstrate that specific HS/heparin polysaccharides support formation of Sox1(+) neural progenitor cells from wild-type ES cells. This effect is dependent on sulfation pattern, concentration, and length of saccharide. Using a selective inhibitor of FGF signal transduction, we show that heparin modulates signaling events regulating exit from pluripotency and commitment to primitive ectoderm and subsequently neuroectoderm. Interestingly, we were also able to demonstrate that multiple receptor tyrosine kinases were influenced by HS in this system. This suggests roles for additional factors, possibly in cell proliferation or protection from apoptosis, during the process of neural specification. Therefore, we conclude that soluble GAGs or synthetic mimics could be considered as suitable low-cost factors for addition to ES cell differentiation regimes.     

Coordinate expression of Fgf8, Otx2, Bmp4, and Shh in the rostral prosencephalon during development of the telencephalic and optic vesicles.

Previous studies suggest that Fgf8 has a key role in regulating vertebrate development. In the rostral head of the embryonic chicken, there are increasing numbers of separate Fgf8 domains; these are present in tissues that appear to have previously expressed Otx2. As Fgf8 expression becomes established, Otx2 expression weakens, but remains in cells abutting the Fgf8 expression domain. These Fgf8 expression domains are closely associated with tissues expressing Bmp4 and Shh. Based on analogy with the embryonic limb, we suggest that Fgf8, Bmp4 and Shh function together in patterning regions of the embryonic head. Gene expression changes are particularly prominent in 14-21 somite stage embryos in the rostral forebrain, during early morphogenesis of the telencephalic and optic vesicles, when several new interfaces of Fgf8, Bmp4 and Shh are generated. To gain insights into the functions of fibroblast growth factor 8 (FGF8) in the embryonic forebrain, we studied the effects of implanting beads containing this protein in the dorsal prosencephalon of embryonic day 2 chicken embryos. Ectopic FGF8 had profound effects on morphogenesis of the telencephalic and optic vesicles. It disrupted formation of the optic stalk and caused a transformation of the pigment epithelium into neural retina. Within the telencephalon, FGF8 beads frequently induced a sulcus that had features of an ectopic rostral midline. The sulcus separated the telencephalon into rostral and caudal vesicles. Furthermore, we present evidence that FGF8 can regulate regionalization of the prosencephalon through inhibition of Otx2 and Emx2 expression. Thus, these experiments provide evidence that FGF8 can regulate both morphogenesis and patterning of the rostral prosencephalon (telencephalic and optic vesicles). FGF8 beads can induce midline properties (e.g. a sulcus) and can modulate the specification and differentiation of adjacent tissues. We suggest that some of these effects are through regulating the expression of homeobox genes (Otx2 and Emx2) that are known to participate in forebrain patterning.     

Expression and localization of fibroblast growth factor (FGF)23 and Klotho in the spleen: its physiological and functional implications

The FGF23–Klotho signaling axis is known to exert anti-aging effects via calcium–phosphorus metabolism. In mice deficient in FGF23–Klotho signaling, however, the number of splenocytes is reduced. FGF23 is expressed in both bone and spleen, with regulation of its production differing in these organs. As FGF23–Klotho signaling may play an immunological role in the spleen, splenocytes in male C57BL/6J mice were assayed for expression of Klotho or FGF23 by flow cytometry and immunohistochemistry. Cells that expressed Klotho included CD45R/B220⁺ CD21/CD35⁺ CD1d⁺ CD43^? marginal zone B cells. These cells also expressed FGF receptor 1, indicating that Klotho-positive B cells could respond to FGF23. Plasmacytoid dendritic cells (pDCs) with CD11c⁺ CD45R/B220⁺ CD11b^? CD8α^? were found to produce FGF23. Klotho-positive cells and FGF23-producing cells were present in close proximity to each other, suggesting that FGF23 produced by pDCs may act within a limited area. These findings indicate that FGF23–Klotho signaling could play a biological or immunological role in the spleen.     

Functions and mechanisms of receptor tyrosine kinase Torso signaling: lessons from Drosophila embryonic terminal development.

The Torso receptor tyrosine kinase (RTK) is required for cell fate specification in the terminal regions (head and tail) of the early Drosophila embryo. Torso contains a split tyrosine kinase domain and belongs to the type III subgroup of the RTK superfamily that also includes the platelet-derived growth factor receptors, stem cell or steel factor receptor c-Kit proto-oncoprotein, colony-stimulating factor-1 receptor, and vascular endothelial growth factor receptor. The Torso pathway has been a model system for studying RTK signal transduction. Genetic and biochemical studies of Torso signaling have provided valuable insights into the biological functions and mechanisms of RTK signaling during early Drosophila embryogenesis.     

Primitive neural stem cells from the mammalian epiblast differentiate to definitive neural stem cells under the control of Notch signaling

Basic fibroblast growth factor (FGF2)-responsive definitive neural stem cells first appear in embryonic day 8.5 (E8.5) mouse embryos, but not in earlier embryos, although neural tissue exists at E7.5. Here, we demonstrate that leukemia inhibitory factor-dependent (but not FGF2-dependent) sphere-forming cells are present in the earlier (E5.5-E7.5) mouse embryo. The resultant clonal sphere cells possess self-renewal capacity and neural multipotentiality, cardinal features of the neural stem cell. However, they also retain some nonneural properties, suggesting that they are the in vivo cells' equivalent of the primitive neural stem cells that form in vitro from embryonic stem cells. The generation of the in vivo primitive neural stem cell was independent of Notch signaling, but the activation of the Notch pathway was important for the transition from the primitive to full definitive neural stem cell properties and for the maintenance of the definitive neural stem cell state.     

Gastrulation in the spider Zygiella x-notata involves three distinct phases of cell internalization.

The cell movements of gastrulation were analyzed in embryos of the spider Zygiella x-notata, using time-lapse video, cell tracing, and improved histology. Cells are internalized near the center of the germ disc in three distinct phases. First, cumulus mesenchyme cells ingress and migrate as a group beneath the superficial layer. Second, mass internalization through a blastopore yields a diffusely organized deep layer. Third, superficial cells accumulate at the center of the germ disc to form the caudal bud. The floor is internalized, and the caudal bud moves over the nascent dorsal field to form the caudal lobe. This pattern of gastrulation differs from the canonical pattern described in the historical literature: (1) the cumulus of Z. x-notata is completely formed before any other cells internalize; and (2) the caudal lobe is formed by means of the caudal bud, which is a locus of cell internalization.     

Reduced notch activity is associated with an impaired marginal zone B cell development and function in Sly1 mutant mice

MZ B cells represent a distinct lineage of naive B lymphocytes, apart from FO B cells and peritoneal B1 cells, and mediate humoral immune responses against blood-borne type 2 T-independent antigens. Regulation of MZ B cell development involves the Notch receptor signaling, the intensity of B cell receptor signals, and cell compartmentalization by adhesion and chemokine receptors. Our previous work showed that gene-targeted mice expressing a truncated form of the putative signaling adapter protein SLy1 exhibit reduced numbers of a splenic B cell population enriched in MZ B cells. Here, we demonstrate that Sly1(d/d) mice exhibit a partial, but selective, block in the transition from pre-MZ to mature MZ B cells. Development of both T1 and T2 precursor subsets and FO B cells was normal in Sly1(d/d) mice. Consistent with the loss of MZ B cells, the production of antigen-specific IgM antibodies following immunization with pneumococcal polysaccharides was severely impaired in Sly1(d/d) mice. Importantly, expression of the Notch signaling mediator RBP-J and the Notch target genes Hes-1 and Hes-5 was markedly reduced in MZ but not FO B cells of Sly1(d/d) mice. In contrast, B cell receptor signaling, expression and function of LFA-1 and alpha4-integrins, and expression of chemokine receptors appeared intact in Sly1(d/d) cells. Collectively, these results provide strong evidence that SLy1 is important for the generation and function of MZ B cells and suggest a novel link between SLy1 and the activity of the Notch pathway in the development of MZ B cells.     

Cyclooxygenase-1-derived PGE2 promotes cell motility via the G-protein-coupled EP4 receptor during vertebrate gastrulation

Gastrulation is a fundamental process during embryogenesis that shapes proper body architecture and establishes three germ layers through coordinated cellular actions of proliferation, fate specification, and movement. Although many molecular pathways involved in the specification of cell fate and polarity during vertebrate gastrulation have been identified, little is known of the signaling that imparts cell motility. Here we show that prostaglandin E(2) (PGE(2)) production by microsomal PGE(2) synthase (Ptges) is essential for gastrulation movements in zebrafish. Furthermore, PGE(2) signaling regulates morphogenetic movements of convergence and extension as well as epiboly through the G-protein-coupled PGE(2) receptor (EP4) via phosphatidylinositol 3-kinase (PI3K)/Akt. EP4 signaling is not required for proper cell shape or persistence of migration, but rather it promotes optimal cell migration speed during gastrulation. This work demonstrates a critical requirement of PGE(2) signaling in promoting cell motility through the COX-1-Ptges-EP4 pathway, a previously unrecognized role for this biologically active lipid in early animal development.