Myosin heavy chain expression in zebrafish and slow muscle composition.

In the zebrafish embryo, two distinct classes of muscle fibers have been described in the forming myotome that arise from topographically separable precursor populations. Based entirely on cross-reactivity with antibodies raised against mammalian and chick myosin heavy chain isoforms slow twitch muscle has been shown to arise exclusively from "adaxial" myoblasts, which migrate from their origin flanking the notochord to form a single layer of subcutaneous differentiated muscle cells. The remainder of the myotome differentiates behind this migration as muscle fibers recognized by anti-fast myosin heavy chain (MyHC) antibodies. To identify unambiguous molecular markers of cell fate in the myotome, we have characterized genes encoding zebrafish fast and slow MyHC. Using phylogenetic and expression analysis, we demonstrate that these genes are definitive molecular markers of slow and fast twitch fates. We also demonstrate that zebrafish embryonic slow twitch muscle co-expresses both slow and fast twitch MyHC isoforms, a property that they share with primary fibers of the amniote myotome.

Calcium-dependent adhesion is necessary for the maintenance of prosomeres.

Cell adhesion has been suggested to function in the establishment and maintenance of the segmental organization of the central nervous system. Here we tested the role of different classes of adhesion molecules in prosencephalic segmentation. Specifically, we examined the ability of progenitors from different prosomeres to reintegrate and differentiate within various brain regions after selective maintenance or removal of different classes of calcium-dependent versus -independent surface molecules. This analysis implicates calcium-dependent adhesion molecules as central to the maintenance of prosomeres. Only conditions that spared calcium-dependent adhesion systems but ablated more general (calcium-independent) adhesion systems resulted in prosomere-specific integration after transplantation. Among the members of this class of adhesion molecules, R-cadherin shows a striking pattern of prosomeric expression during development. To test whether expression of this molecule was sufficient to direct progenitor integration to prosomeres expressing R-cadherin, we used a retroviral-mediated gain-of-function approach. We found that progenitors originally isolated from prosomere P2 (a region which does not express R-cadherin), when forced to express this molecule, can now integrate more readily into R-cadherin-expressing regions, such as the cortex, the ventral thalamus, and the hypothalamus. Nonetheless, our analysis suggests that while calcium-dependent molecules are able to direct prosomere-specific integration, they are not sufficient to induce progenitors to change their regional identity. While diencephalic progenitors from R-cadherin-expressing regions of prosomere 5 could integrate into R-cadherin-expressing regions of the cortex, they did not express the cortex-specific gene Emx1 or the telencephalic-specific gene Bf-1. Furthermore, diencephalic progenitors that integrate heterotopically into the cortex do not persist postnatally, whereas the same progenitors survive and differentiate when they integrate homotopically into the diencephalon. Together our results implicate calcium-dependent adhesion molecules as key mediators of prosomeric organization but suggest that they are not sufficient to bestow regional identities.

HrpZ(Psph) from the plant pathogen Pseudomonas syringae pv. phaseolicola binds to lipid bilayers and forms an ion-conducting pore in vitro.

The hrp gene clusters of plant pathogenic bacteria control pathogenicity on their host plants and ability to elicit the hypersensitive reaction in resistant plants. Some hrp gene products constitute elements of the type III secretion system, by which effector proteins are exported and delivered into plant cells. Here, we show that the hrpZ gene product from the bean halo-blight pathogen, Pseudomonas syringae pv. phaseolicola (HrpZ(Psph)), is secreted in an hrp-dependent manner in P. syringae pv. phaseolicola and exported by the type III secretion system in the mammalian pathogen Yersinia enterocolitica. HrpZ(Psph) was found to associate stably with liposomes and synthetic bilayer membranes. Under symmetric ionic conditions, addition of 2 nM of purified recombinant HrpZ(Psph) to the cis compartment of planar lipid bilayers provoked an ion current with a large unitary conductivity of 207 pS. HrpZ(Psph)-related proteins from P. syringae pv. tomato or syringae triggered ion currents similar to those stimulated by HrpZ(Psph). The HrpZ(Psph)-mediated ion-conducting pore was permeable for cations but did not mediate fluxes of Cl-. Such pore-forming activity may allow nutrient release and/or delivery of virulence factors during bacterial colonization of host plants.

Evolutionary origins of vertebrate appendicular muscle.

The evolution of terrestrial tetrapod species heralded a transition in locomotor strategies. While most fish species use the undulating contractions of the axial musculature to generate propulsive force, tetrapods also rely on the appendicular muscles of the limbs to generate movement. Despite the fossil record generating an understanding of the way in which the appendicular skeleton has evolved to provide the scaffold for tetrapod limb musculature, there is, by contrast, almost no information as to how this musculature arose. Here we examine fin muscle formation within two extant classes of fish. We find that in the teleost, zebrafish, fin muscles arise from migratory mesenchymal precursor cells that possess molecular and morphogenetic identity with the limb muscle precursors of tetrapod species. Chondrichthyan dogfish embryos, however, use the primitive mechanism of direct epithelial somitic extensions to derive the muscles of the fin. We conclude that the genetic mechanism controlling formation of tetrapod limb muscles evolved before the Sarcopterygian radiation.

Slow muscle induction by Hedgehog signalling in vitro.

Muscles are composed of several fibre types, the precise combination of which determines muscle function. Whereas neonatal and adult fibre type is influenced by a number of extrinsic factors, such as neural input and muscle load, there is little knowledge of how muscle cells are initially determined in the early embryo. In the zebrafish, fibres of the slow twitch class arise from precociously specified myoblasts that lie close to the midline whereas the remainder of the myotome differentiates as fast myosin expressing muscle. In vivo evidence has suggested the Sonic Hedgehog glycoprotein, secreted from the notochord, controls the formation of slow twitch and fast twitch muscle fates. Here we describe an in vitro culture system that we have developed to test directly the ability of zebrafish myoblasts to respond to exogenous Sonic Hedgehog peptide. We find that Sonic Hedgehog peptide can control the binary cell fate choice of embryonic zebrafish myoblasts in vitro. We have also used this culture system to assay the relative activities of different Hedgehog-family proteins and to investigate the possible involvement of heterotrimeric G-proteins in Hedgehog signal transduction.

Yersinia enterocolitica type III secretion-translocation system: channel formation by secreted Yops.

'Type III secretion' allows extracellular adherent bacteria to inject bacterial effector proteins into the cytosol of their animal or plant host cells. In the archetypal Yersinia system the secreted proteins are called Yops. Some of them are intracellular effectors, while YopB and YopD have been shown by genetic analyses to be dedicated to the translocation of these effectors. Here, the secretion of Yops by Y.enterocolitica was induced in the presence of liposomes, and some Yops, including YopB and YopD, were found to be inserted into liposomes. The proteoliposomes were fused to a planar lipid membrane to characterize the putative pore-forming properties of the lipid-bound Yops. Electrophysiological experiments revealed the presence of channels with a 105 pS conductance and no ionic selectivity. Channels with those properties were generated by mutants devoid of the effectors and by lcrG mutants, as well as by wild-type bacteria. In contrast, mutants devoid of YopB did not generate channels and mutants devoid of YopD led to current fluctuations that were different from those observed with wild-type bacteria. The observed channel could be responsible for the translocation of Yop effectors.

Insertion of a Yop translocation pore into the macrophage plasma membrane by Yersinia enterocolitica: requirement for translocators YopB and YopD, but not LcrG.

The Yersinia survival strategy is based on its ability to inject effector Yops into the cytosol of host cells. Translocation of these effectors across the eukaryotic cell membrane requires YopB, YopD and LcrG, but the mechanism is unclear. An effector polymutant of Y. pseudotuberculosis has a YopB-dependent contact haemolytic activity, indicating that YopB participates in the formation of a pore in the cell membrane. Here, we have investigated the formation of such a pore in the plasma membrane of macrophages. Infection of PU5-1.8 macrophages with an effector polymutant Y. enterocolitica led to complete flattening of the cells, similar to treatment with the pore-forming streptolysin O from Streptococcus pyogenes. Upon infection, cells released the low-molecular-weight marker BCECF (623 Da) but not the high-molecular-weight lactate dehydrogenase, indicating that there was no membrane lysis but, rather, insertion of a pore of small size into the macrophage plasma membrane. Permeation to lucifer yellow CH (443 Da) but not to Texas red-X phalloidin (1490 Da) supported this hypothesis. All these events were found to be dependent not only on translocator YopB as expected but also on YopD, which was required equally. In contrast, LcrG was not necessary. Consistently, lysis of sheep erythrocytes was also dependent on YopB and YopD, but not on LcrG.

Role of SycD, the chaperone of the Yersinia Yop translocators YopB and YopD.

Extracellular Yersinia adhering at the surface of a eukaryotic cell translocate effector Yops across the plasma membrane of the cell by a mechanism requiring YopD and YopB, the latter probably mediating pore formation. We studied the role of SycD, the intrabacterial chaperone of YopD. By producing GST-YopB hybrid proteins and SycD in Escherichia coli, we observed that SycD also binds specifically to YopB and that this binding reduces the toxicity of GST-YopB in E. coli. By analysis of a series of truncated GST-YopB proteins, we observed that SycD does not bind to a discrete segment of YopB. Using the same approach, we observed that YopD can also bind to YopB. Binding between YopB and YopD occurred even in the presence of SycD, and a complex composed of these three proteins could be immunoprecipitated from the cytoplasm of Yersinia. In a sycD mutant, the intracellular pool of YopB and YopD was greatly reduced unless the lcrV gene was also deleted. As LcrV is known to interact with YopB and YopD and to promote their secretion, we speculate that SycD prevents a premature association between YopB-YopD and LcrV.

The virulence plasmid of Yersinia, an antihost genome.

The 70-kb virulence plasmid enables Yersinia spp. (Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica) to survive and multiply in the lymphoid tissues of their host. It encodes the Yop virulon, an integrated system allowing extracellular bacteria to disarm the cells involved in the immune response, to disrupt their communications, or even to induce their apoptosis by the injection of bacterial effector proteins. This system consists of the Yop proteins and their dedicated type III secretion apparatus, called Ysc. The Ysc apparatus is composed of some 25 proteins including a secretin. Most of the Yops fall into two groups. Some of them are the intracellular effectors (YopE, YopH, YpkA/YopO, YopP/YopJ, YopM, and YopT), while the others (YopB, YopD, and LcrV) form the translocation apparatus that is deployed at the bacterial surface to deliver the effectors into the eukaryotic cells, across their plasma membrane. Yop secretion is triggered by contact with eukaryotic cells and controlled by proteins of the virulon including YopN, TyeA, and LcrG, which are thought to form a plug complex closing the bacterial secretion channel. The proper operation of the system also requires small individual chaperones, called the Syc proteins, in the bacterial cytosol. Transcription of the genes is controlled both by temperature and by the activity of the secretion apparatus. The virulence plasmid of Y. enterocolitica and Y. pseudotuberculosis also encodes the adhesin YadA. The virulence plasmid contains some evolutionary remnants including, in Y. enterocolitica, an operon encoding resistance to arsenic compounds.

Telencephalic progenitors maintain anteroposterior identities cell autonomously.

Grafting experiments have demonstrated that determination of anteroposterior (AP) identity is an early step in neural patterning that precedes dorsoventral (DV) specification [1,2]. These studies used pieces of tissue, however, rather than individual cells to address this question. It thus remains unclear whether the maintenance of AP identity is a cell-autonomous property or a result of signaling between cells within the grafted tissue. Previously, we and others [3-5] have used transplants of dissociated brain cells to show that individual telencephalic precursor cells can adopt host-specific DV identities when they integrate within novel regions of the telencephalon. We have now undertaken a set of transplantations during the same mid-neurogenic period used in the previous studies to assess the ability of telencephalic progenitors to integrate and differentiate into more posterior regions of the neuraxis. We observed that telencephalic progenitors were capable of integrating and migrating within different AP levels of the central nervous system (CNS). Despite this, we found that telencephalic progenitors that integrated within the diencephalon and the mesencephalon continued to express a telencephalic marker until adulthood. We speculate that during neurogenesis individual progenitors are determined in terms of their AP but not their DV identity. Hence, AP identity is maintained cell autonomously within individual progenitors.

The Yersinia Yop virulon: LcrV is required for extrusion of the translocators YopB and YopD.

LcrV, an essential piece of the Yop virulon, is encoded by the large lcrGVsycDyopBD operon. In spite of repeated efforts, the role of LcrV in the Yop virulon remains elusive. In an attempt to clarify this, we engineered a complete deletion of lcrV in the pYV plasmid of Yersinia enterocolitica E40 and characterized the phenotype of the mutant. Complementation experiments showed that the mutation was not polar with regard to yopB and yopD. Nevertheless the mutation abolished secretion of YopB and YopD, while secretion of the other Yops was unaffected or even increased. Northern blot analysis showed that transcription of yopD was not affected. YopD could be detected inside the bacteria, showing that the lack of its secretion was not due to a lack of translation or to proteolysis. This indicated that LcrV is specifically involved in the process of release of YopB and YopD. We then investigated the possible interactions between LcrV and YopB or YopD. We constructed a glutathione S-transferase-LcrV hybrid protein, and we observed that either YopB or YopD could be copurified with it. The same approach showed that LcrV also interacts with LcrG but not with the chaperone SycD. Using deletants of lcrV, we then identified a definite LcrG-binding domain in the C terminus of LcrV.

A short-range signal restricts cell movement between telencephalic proliferative zones.

During telencephalic development, a boundary develops that restricts cell movement between the dorsal cortical and basal striatal proliferative zones. In this study, the appearance of this boundary and the mechanism by which cell movement is restricted were examined through a number of approaches. The general pattern of neuronal dispersion was examined both with an early neuronal marker and through the focal application of DiI to telencephalic explants. Both methods revealed that, although tangential neuronal dispersion is present throughout much of the telencephalon, it is restricted within the boundary region separating dorsal and ventral telencephalic proliferative zones. To examine the cellular mechanism underlying this boundary restriction, dissociated cells from the striatum were placed within both areas of the boundary, where dispersion is limited, and areas within the cortex, where significant cellular dispersion occurs. Cells placed within the boundary region remain round and extend only thin processes, whereas progenitors placed onto the cortical ventricular zone away from this boundary are able to migrate extensively. This suggests that the boundary inhibits directly the migration of cells. To examine whether the signal inhibiting dispersion within the boundary region acts as a long- or short-range cue, we apposed explants of boundary and nonboundary regions in vitro. Within these explants we found that migration was neither inhibited in nonboundary regions nor induced in boundary regions. This suggests that the boundary between dorsal and ventral telencephalon isolates these respective environments through either a contact-dependent or a short-range diffusible mechanism.

Virulence and arsenic resistance in Yersiniae.

The genus Yersinia contains three pathogenic species: Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica. Only a few biotypes and serotypes of Y. enterocolitica are pathogenic, and these form two distinct groups: some are of low virulence, and they are encountered worldwide; others, mainly encountered in North America, are markedly more virulent. All pathogenic yersiniae possess a 70-kb virulence plasmid called pYV which encodes secreted antihost proteins called Yops as well as a type III secretion machinery that is required for Yop secretion. Genes encoding Yop synthesis and secretion are tightly clustered in three quadrants of the pYV plasmid. We show here that in the low-virulence strains of Y. enterocolitica, the fourth quadrant of the plasmid contains a new class II transposon, Tn2502. This transposon encodes a defective transposase, but transposition can be complemented in trans by Tn2501, another class II transposon. Tn2502 was not detected in the pYV plasmids of the more virulent American strains of Y. enterocolitica, of Y. pseudotuberculosis, and of Y. pestis. Tn2502 confers arsenite and arsenate resistance. This resistance involves four genes; three are homologous to the arsRBC genes present on the Escherichia coli chromosome, but no homolog of the fourth one, arsH, has been found. The systematic presence of such a resistance operon on a virulence plasmid is unusual and could be related to the recent spread of low-virulence Y. enterocolitica strains. The presence of this ars operon also constitutes the first significant difference between the pYV plasmids from different Yersinia species.

Characterization of human serum N-acetylmuramyl-L-alanine amidase purified by affinity chromatography.

Human serum N-acetylmuramyl-L-alanine amidase was purified to homogeneity by a relatively short procedure including affinity chromatography. For this purpose, a specific adsorbent was prepared by coupling the main substrate of the enzyme, N-acetyl-muramyl-L-alanine-gamma-D-glutamyl-L-meso-2,6[3,4,5-3H] diaminopimelic acid, to a divinylsulfone agarose gel. The enzyme is unable to hydrolyze this muramylpeptide when it is attached as a ligand to the gel, whereas a high affinity is conserved. In addition to affinity chromatography, the presented purification scheme includes ion exchange chromatography on DEAE-Sepharose and molecular sieving on Superdex 200. The enzyme was purified 739-fold with a yield of 22.5%. One single band at 135 kDa was obtained on native gradient PAGE. Gradient PAGE in denaturing conditions gave one single band at 74 kDa, which was lowered to 64 kDa when the enzyme was denatured in nonreducing conditions. This suggests that the native enzyme is a dimer consisting of two subunits of identical molecular weight with only intramolecular disulfide bonds. Isoelectric focusing gave one single band at pI 5.0. Glycan detection before and after treatment with N-glycosidase F showed that the enzyme is a glycoprotein. Further analysis by lectin immuno detection on dot blots confirmed that the enzyme is an N-glycosylated protein of complex type with sialic acid, terminally linked alpha (2-->6) to galactose or N-acetylgalactosamine. The 15 amino acid N-terminal sequence was determined by microsequence analysis.

Mutations located on both F1 and F2 subunits of the Newcastle disease virus fusion protein confer resistance to neutralization with monoclonal antibodies.

The fusion gene sequence of six Newcastle disease virus escape mutants revealed that residues important for the integrity of antigenic site 1 and antigenic site 2 were located, respectively, on the F2 subunit and within the cysteine-rich domain of the F1 subunit. We further report the antibody-binding capacity of these mutants.

The hemagglutinin-neuraminidase (HN) gene of Newcastle disease virus strain Italien (ndv Italien): comparison with HNs of other strains and expression by a vaccinia recombinant.

A cDNA library was constructed with poly(A+) mRNA from cells infected with the virulent Italien NDV strain. A clone that hybridized to the HN gene mRNA was sequenced. A long open reading-frame encodes for a protein of 571 amino acids, with a calculated molecular weight of 61,900, including 13 cysteine residues and six potential glycosylation sites. To define the sequence changes that occurred in the avian paramyxovirus hemagglutinin-neuraminidase (HN) during the evolution of virulence, we have studied the HNs of the virulent Italien NDV strain, the mesovirulent Beaudette strain and the nonvirulent Hitchner strain. The majority of amino acid variations are conservative changes but they cluster at 4 preferential sites in the putative head of HN. The clusters of amino acid substitutions are intimately associated or overlap with regions of HN rich in charged amino acid residues and in cysteines. The latter are conserved not only between HNs from all 3 NDV strains but also between HNs of 4 different paramyxoviruses, NDV, SV 5, Sendai and PI 3. The HN coding sequence was inserted into the genome of vaccinia virus under the control of vaccinia P 7.5 K transcriptional regulatory sequences. Expression of native HN proteins at the surface of recombinant HN vaccinia-infected cells was demonstrated by indirect immunofluorescence with 2 anti-HN monoclonals.