A freely diffusible form of Sonic hedgehog mediates long-range signalling

The secreted protein Sonic hedgehog (Shh) exerts many of its patterning effects through a combination of short- and long-range signalling. Three distinct mechanisms, which are not necessarily mutually exclusive, have been proposed to account for the long-range effects of Shh: simple diffusion of Shh, a relay mechanism in which Shh activates secondary signals, and direct delivery of Shh through cytoplasmic extensions, termed cytonemes. Although there is much data (using soluble recombinant Shh (ShhN)) to support the simple diffusion model of long-range Shh signalling, there has been little evidence to date for a native form of Shh that is freely diffusible and not membrane-associated. Here we provide evidence for a freely diffusible form of Shh (s-ShhNp) that is cholesterol modified, multimeric and biologically potent. We further demonstrate that the availability of s-ShhNp is regulated by two functional antagonists of the Shh pathway, Patched (Ptc) and Hedgehog-interacting protein (Hip). Finally, we show a gradient of s-ShhNp across the anterior-posterior axis of the chick limb, demonstrating the physiological relevance of s-ShhNp.     

Sonic hedgehog function in chondrichthyan fins and the evolution of appendage patterning

The genetic mechanisms regulating tetrapod limb development are well characterized, but how they were assembled during evolution and their function in basal vertebrates is poorly understood. Initial studies report that chondrichthyans, the most primitive extant vertebrates with paired appendages, differ from ray-finned fish and tetrapods in having Sonic hedgehog (Shh)-independent patterning of the appendage skeleton. Here we demonstrate that chondrichthyans share patterns of appendage Shh expression, Shh appendage-specific regulatory DNA, and Shh function with ray-finned fish and tetrapods. These studies demonstrate that some aspects of Shh function are deeply conserved in vertebrate phylogeny, but also highlight how the evolution of Shh regulation may underlie major morphological changes during appendage evolution.     

Suppression of Notch signalling by the COUP-TFII transcription factor regulates vein identity

Arteries and veins are anatomically, functionally and molecularly distinct. The current model of arterial-venous identity proposes that binding of vascular endothelial growth factor to its heterodimeric receptor--Flk1 and neuropilin 1 (NP-1; also called Nrp1)--activates the Notch signalling pathway in the endothelium, causing induction of ephrin B2 expression and suppression of ephrin receptor B4 expression to establish arterial identity. Little is known about vein identity except that it involves ephrin receptor B4 expression, because Notch signalling is not activated in veins; an unresolved question is how vein identity is regulated. Here, we show that COUP-TFII (also known as Nr2f2), a member of the orphan nuclear receptor superfamily, is specifically expressed in venous but not arterial endothelium. Ablation of COUP-TFII in endothelial cells enables veins to acquire arterial characteristics, including the expression of arterial markers NP-1 and Notch signalling molecules, and the generation of haematopoietic cell clusters. Furthermore, ectopic expression of COUP-TFII in endothelial cells results in the fusion of veins and arteries in transgenic mouse embryos. Thus, COUP-TFII has a critical role in repressing Notch signalling to maintain vein identity, which suggests that vein identity is under genetic control and is not derived by a default pathway.     

FGF-induced vesicular release of Sonic hedgehog and retinoic acid in leftward nodal flow is critical for left-right determination

The precise specification of left-right asymmetry is an essential process for patterning internal organs in vertebrates. In mouse embryonic development, the symmetry-breaking process in left-right determination is initiated by a leftward extraembryonic fluid flow on the surface of the ventral node. However, it is not known whether the signal transduction mechanism of this flow is chemical or mechanical. Here we show that fibroblast growth factor (FGF) signalling triggers secretion of membrane-sheathed objects 0.3-5 microm in diameter termed 'nodal vesicular parcels' (NVPs) that carry Sonic hedgehog and retinoic acid. These NVPs are transported leftward by the fluid flow and eventually fragment close to the left wall of the ventral node. The silencing effects of the FGF-receptor inhibitor SU5402 on NVP secretion and on a downstream rise in Ca2+ were sufficiently reversed by exogenous Sonic hedgehog peptide or retinoic acid, suggesting that FGF-triggered surface accumulation of cargo morphogens may be essential for launching NVPs. Thus, we propose that NVP flow is a new mode of extracellular transport that forms a left-right gradient of morphogens.     

Control of neuronal fate by the Drosophila segmentation gene even-skipped

The central nervous system (CNS) contains a remarkable diversity of cell types. The molecular basis for generating this neuronal diversity is poorly understood. Much is known, however, about the regulatory genes which control segmentation and segment identity during early Drosophila embryogenesis. Interestingly, most of the segmentation and homoeotic genes in Drosophila, as well as many of their vertebrate homologues, are expressed during the development of the nervous system (for example, ref. 3). Are these genes involved in specifying the identity of individual neurons during neurogenesis, just as they specify the identity of cells during segmentation? We previously described the CNS expression of the segmentation gene fushi tarazu (ftz) and showed that ftz CNS expression is involved in the determination of an identified neuron. Here we show that another segmentation gene, even-skipped (eve), is expressed in a different but overlapping subset of neurons. Temperature-sensitive inactivation of the eve protein during neurogenesis alters the fate of two of these neurons. Our results indicate that the nuclear protein products of the eve and ftz segmentation genes are components of the mechanism controlling cell fate during neuronal development.     

Regulation of PDGF signalling and vascular remodelling by peroxiredoxin II

Platelet-derived growth factor (PDGF) is a potent mitogenic and migratory factor that regulates the tyrosine phosphorylation of a variety of signalling proteins via intracellular production of H2O2 (refs 1, 2-3). Mammalian 2-Cys peroxiredoxin type II (Prx II; gene symbol Prdx2) is a cellular peroxidase that eliminates endogenous H2O2 produced in response to growth factors such as PDGF and epidermal growth factor; however, its involvement in growth factor signalling is largely unknown. Here we show that Prx II is a negative regulator of PDGF signalling. Prx II deficiency results in increased production of H2O2, enhanced activation of PDGF receptor (PDGFR) and phospholipase Cgamma1, and subsequently increased cell proliferation and migration in response to PDGF. These responses are suppressed by expression of wild-type Prx II, but not an inactive mutant. Notably, Prx II is recruited to PDGFR upon PDGF stimulation, and suppresses protein tyrosine phosphatase inactivation. Prx II also leads to the suppression of PDGFR activation in primary culture and a murine restenosis model, including PDGF-dependent neointimal thickening of vascular smooth muscle cells. These results demonstrate a localized role for endogenous H2O2 in PDGF signalling, and indicate a biological function of Prx II in cardiovascular disease.     

低掺量水泥改善级配碎石力学和收缩规律分析

为了研究低掺量水泥改善级配碎石力学和收缩性能规律,提高对低水泥掺量基层结构的认识,通过不同龄期与水泥掺量下的无侧限抗压强度、劈裂强度、加州承载比、抗压回弹模量和干燥收缩等试验,揭示了力学和收缩变化的规律,建立了强度和模量等四个力学参数随龄期和水泥掺量变化的对数增长预测模型,以及上述参数对应龄期之间的相互转化关系模型。研究结果表明龄期和水泥掺量对混合料力学和收缩性能影响显著,推荐水泥掺量在2.0%~2.5%范围内较为合适。     

Proliferation and differentiation of neuronal stem cells regulated by nerve growth factor

Nerve growth factor plays an important part in neuron-target interactions in the late embryonic and adult brain. We now report that this growth factor controls the proliferation of neuronal precursors in a defined culture system of cells derived from the early embryonic brain. Neuronal precursor cells were identified by expression of the intermediate filament protein nestin. These cells proliferate in response to nerve growth factor but only after they have been exposed to basic fibroblast growth factor. On withdrawal of nerve growth factor, the proliferative cells differentiate into neurons. Thus, in combination with other growth factors, nerve growth factor regulates the proliferation and terminal differentiation of neuroepithelial stem cells.     

Hedgehog signalling activity of Smoothened requires phosphorylation by protein kinase A and casein kinase I

The Hedgehog (Hh) family of secreted proteins governs cell growth and patterning in animal development. The Hh signal is transduced by the seven-transmembrane protein Smoothened (Smo); however, the mechanism by which Smo is regulated remains largely unknown. Here we show that protein kinase A (PKA) and casein kinase I (CKI) regulate Smo cell-surface accumulation and activity in response to Hh. Blocking PKA or CKI activity in the Drosophila wing disc prevents Hh-induced Smo accumulation and attenuates pathway activity, whereas increasing PKA activity promotes Smo accumulation and pathway activation. We show that PKA and CKI phosphorylate Smo at several sites, and that phosphorylation-deficient forms of Smo fail to accumulate on the cell surface and are unable to transduce the Hh signal. Conversely, phosphorylation-mimicking Smo variants show constitutive cell-surface expression and signalling activity. Furthermore, we find that the levels of Smo cell-surface expression and activity correlate with its levels of phosphorylation. Our data indicate that Hh induces progressive Smo phosphorylation by PKA and CKI, leading to elevation of Smo cell-surface levels and signalling activity.     

The behaviour of Drosophila adult hindgut stem cells is controlled by Wnt and Hh signalling

The intestinal tract maintains proper function by replacing aged cells with freshly produced cells that arise from a population of self-renewing intestinal stem cells (ISCs). In the mammalian intestine, ISC self renewal, amplification and differentiation take place along the crypt-villus axis, and are controlled by the Wnt and hedgehog (Hh) signalling pathways. However, little is known about the mechanisms that specify ISCs within the developing intestinal epithelium, or about the signalling centres that help maintain them in their self-renewing stem cell state. Here we show that in adult Drosophila melanogaster, ISCs of the posterior intestine (hindgut) are confined to an anterior narrow segment, which we name the hindgut proliferation zone (HPZ). Within the HPZ, self renewal of ISCs, as well as subsequent proliferation and differentiation of ISC descendants, are controlled by locally emanating Wingless (Wg, a Drosophila Wnt homologue) and Hh signals. The anteriorly restricted expression of Wg in the HPZ acts as a niche signal that maintains cells in a slow-cycling, self-renewing mode. As cells divide and move posteriorly away from the Wg source, they enter a phase of rapid proliferation. During this phase, Hh signal is required for exiting the cell cycle and the onset of differentiation. The HPZ, with its characteristic proliferation dynamics and signalling properties, is set up during the embryonic phase and becomes active in the larva, where it generates all adult hindgut cells including ISCs. The mechanism and genetic control of cell renewal in the Drosophila HPZ exhibits a large degree of similarity with what is seen in the mammalian intestine. Our analysis of the Drosophila HPZ provides an insight into the specification and control of stem cells, highlighting the way in which the spatial pattern of signals that promote self renewal, growth and differentiation is set up within a genetically tractable model system.     

Essential role for TIRAP in activation of the signalling cascade shared by TLR2 and TLR4

Signal transduction through Toll-like receptors (TLRs) originates from their intracellular Toll/interleukin-1 receptor (TIR) domain, which binds to MyD88, a common adaptor protein containing a TIR domain. Although cytokine production is completely abolished in MyD88-deficient mice, some responses to lipopolysaccharide (LPS), including the induction of interferon-inducible genes and the maturation of dendritic cells, are still observed. Another adaptor, TIRAP (also known as Mal), has been cloned as a molecule that specifically associates with TLR4 and thus may be responsible for the MyD88-independent response. Here we report that LPS-induced splenocyte proliferation and cytokine production are abolished in mice lacking TIRAP. As in MyD88-deficient mice, LPS activation of the nuclear factor NF-kappaB and mitogen-activated protein kinases is induced with delayed kinetics in TIRAP-deficient mice. Expression of interferon-inducible genes and the maturation of dendritic cells is observed in these mice; they also show defective response to TLR2 ligands, but not to stimuli that activate TLR3, TLR7 or TLR9. In contrast to previous suggestions, our results show that TIRAP is not specific to TLR4 signalling and does not participate in the MyD88-independent pathway. Instead, TIRAP has a crucial role in the MyD88-dependent signalling pathway shared by TLR2 and TLR4.     

Regulation of intracellular calcium by a signalling complex of IRAG, IP3 receptor and cGMP kinase Ibeta

Calcium release from the endoplasmic reticulum controls a number of cellular processes, including proliferation and contraction of smooth muscle and other cells. Calcium release from inositol 1,4,5-trisphosphate (IP3)-sensitive stores is negatively regulated by binding of calmodulin to the IP3 receptor (IP3R) and the NO/cGMP/cGMP kinase I (cGKI) signalling pathway. Activation of cGKI decreases IP3-stimulated elevations in intracellular calcium, induces smooth muscle relaxation and contributes to the antiproliferative and pro-apoptotic effects of NO/cGMP. Here we show that, in microsomal smooth muscle membranes, cGKIbeta phosphorylated the IP3R and cGKIbeta, and a protein of relative molecular mass 125,000 which we now identify as the IP3R-associated cGMP kinase substrate (IRAG). These proteins were co-immunoprecipitated by antibodies directed against cGKI, IP3R or IRAG. IRAG was found in many tissues including aorta, trachea and uterus, and was localized perinuclearly after heterologous expression in COS-7 cells. Bradykinin-stimulated calcium release was not affected by the expression of either IRAG or cGKIbeta, which we tested in the absence and presence of cGMP. However, calcium release was inhibited after co-expression of IRAG and cGKIbeta in the presence of cGMP. These results identify IRAG as an essential NO/cGKI-dependent regulator of IP3-induced calcium release.     

Down-regulation of αGal epitopes by co-transfection of α1,3-galactosidase gene and α1,2–fucosyltransferase gene

The polycarbohydrate structure of Galα1- 3Ga1β1-4GluNAc-R （known as αGal epitopes of xenoantigen）, produced by α1-3-galactosyltransferase （α1,3-GT） in the course of animal development, is the major xenoantigen on the cell surface of porcine which causes hyperacute rejection in pig-to-human xenotransplantation. Alpha-1,3-galactosidase （AGL）, a hydrolytic enzyme, can remove the terminal α1,3-galactosyi from the Galα1-3Galβ1-4GluNAc-R structure resulting in cleaning αGai epitopes from the porcine cells. Aipha-1,2-fucosyitransferase （HT） can modify the surface carbohydrate phenotype of porcine cells, bringing about reduction of αGai epitopes expression. In this study, human AGL and HT gene were co-transfected to porcine fetal fibroblast （PFFb） in equimolar concentration to reduce the xenoantigen. Gene and protein of hAGL and HT were both detected to express at high level by RT-PCR and Western blot, respectively. There was an 84% reduction in αGai xenoantigen and an 82% increase in H antigen as assayed by flow cytometry in the AGL and HT gene co-transfected PFFb. The number and morphology of transgenic PFFb chromosome were normal. Findings indicate that Galα1-3Gal epitopes of PFFb could be down regulated by AGL and HT co-transfection without deleterious effects on the chromosomal profile of the transgenic ceil.