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A recent study reveals that the propagation of intercellular calcium signals is closely associated with the generation of convergent extension movements during Xenopus gastrulation. Such signals provide a mechanism whereby large populations of cells can communicate to generate orchestrated cell movements.
We have investigated the importance of two small nested genes (p19 and p22) located near the 3' end of the genome of tomato bushy stunt virus (TBSV) for infectivity in several hosts. Our results show that both genes are dispensable for replication and transcription and that the p19 gene encodes a soluble protein, whereas the p22 gene specifies a membrane-associated protein. Assays using TBSV derivatives that have the beta-glucuronidase gene substituted for the capsid protein gene demonstrate that p22 is required for cell-to-cell movement in all plants tested. Mutations inactivating p19 ameliorate the severe necrotic systemic symptoms elicited by wild-type TBSV in Nicotiana benthamiana and Nicotiana clevelandii, but p19 does not obviously affect movement in these hosts. However, in some local lesion hosts p19 influences the lesion diameter, which suggests that it has an auxiliary host-dependent role in movement. This notion is supported by the observation that p19 is required for long-distance spread of TBSV in spinach and for systemic infection of pepper plants. Thus, movement of TBSV is regulated by two nested genes; p22 governs cell-to-cell movement and p19 has a host-specific role in systemic invasion.
The JAK/STAT pathway was originally identified in mammals. Studies of this pathway in the mouse have revealed that JAK/STAT signaling plays a central role during hematopoeisis and other developmental processes. The role of JAK/STAT signaling in blood appears to be conserved throughout evolution, as it is also required during fly hematopoeisis. Studies in Dictyostelium, Drosophila, and zebrafish have shown that the JAK/STAT pathway is also required in an unusually broad set of developmental decisions, including cell proliferation, cell fate determination, cell migration, planar polarity, convergent extension, and immunity. There is increasing evidence that the versatility of this pathway relies on its cooperation with other signal transduction pathways. In this review, we discuss the components of the JAK/STAT pathway in model organisms and what is known about its requirement in cellular and developmental processes. In particular, we emphasize recent insights into the role that this pathway plays in the control of cell movement.
The role of bean common mosaic necrosis potyvirus (BCMNV) and lettuce mosaic potyvirus (LMV) proteins was investigated in terms of their capacity to function as viral movement proteins (MPs). Using Escherichia coli-expressed proteins and microinjection techniques, direct evidence was obtained that both the potyviral capsid protein (CP) and helper component- proteinase (HC-Pro) function in this capacity, in that both proteins (a) trafficked from cell to cell, (b) induced an increase in plasmodesmal size exclusion limit, and (c) facilitated cell-to-cell movement of viral RNA. CP and HC-Pro mutants were also produced and used in microinjection experiments. Mutations in the core region of the CP either impaired (single and double amino acid substitution mutants) or abolished (triple amino acid substitution mutant) cell-to-cell movement, as did C-terminal deletion mutants in HC-Pro. The BCMNV P1, CI, NIa, and NIb proteins did not exhibit viral MP properties, but NIa and NIb proteins were found to accumulate within the nuclei of injected cells. These results further establish the multifunctional nature of the potyvirus CP and HC-Pro.
This contribution represents a continuation of our studies of a gradient in convergent cell movement in the germ ring (GR) during Fundulus gastrulation (Trinkaus et al. [1992] J. Exp. Zool., 261:40-61). In our previous study, we discovered that cells in the dorsal GR nearest the embryonic shield (ES) move toward the ES at a net faster rate than those farther away and that this is due to less meandering. Those farther away meander more. These data suggest the hypothesis that there is a gradient of cues that direct cells to the ES. If so, cells in the ventral GR, farthest from the ES, should meander even more and hence show little or no convergence toward the ES. To test this hypothesis, we have traced the trajectories of individual cells in the midventral GR during midepiboly and have found that, although the general motile behavior of ventral GR cells is the same as that of dorsal cells, they do indeed meander much more and, as a result, show little or no directional movement toward the ES. Taken together, these results indicate that cells of the germ ring move up a gradient in directionality as they converge toward their target, the embryonic shield. One possible explanation for this is that the embryonic shield attracts cells to itself.
Neurulation, formation of the neural tube, a crucial event of early embryogenesis, is believed to be driven by the coordination of a number of diverse morphogenetic cell behaviors. Such behaviors include changes in cell number (division, death), cell shape and size (wedging, palisading and spreading), cell position (rearrangement or intercalation) and cell-cell and cell-matrix associations (including inductive interactions). The focus of this essay is on epiblast cell movements and their role in shaping and bending of the neural plate. Neurulation is a multifactorial process requiring both intrinsic (within the neural plate) and extrinsic (outside the neural plate) forces. The origin and movements of three populations of epiblast cells have been studied in avian embryos by constructing quail/chick transplantation chimeras and by labeling cells in situ with identifiable, heritable markers. MHP (median hinge-point neurepithelial) cells originate principally from a midline epiblast area rostral to and overlapping Hensen's node. In addition, a few caudal MHP cells originate from paranodal epiblast areas. MHP cells stream down the length of the midline neuraxis in the wake of the regressing Hensen's node. This streaming occurs as a result of cell division (presumably oriented so that daughter cells are placed into the longitudinal plane rather than into the transverse plane) and rearrangement (intercalation), resulting in a narrowing of the width of the MHP region with a concomitant increase in its length. L (lateral neurepithelial) cells originate from paired epiblast areas flanking the rostral portion of the primitive streak, and they stream down the length of the lateral neuraxis concomitant with regression of Hensen's node.(ABSTRACT TRUNCATED AT 250 WORDS)
To study the effect of cell-to-cell movement of potato virus X (PVX) on the size-exclusion limit of plasmodesmata we have microinjected virus particles and fluorescently labelled dextrans (F-dextrans) with average molecular mass of 4.4, 10, and 20 kDa into Nicotiana clevelandii trichome cells. A vector construct of PVX expressing GUS (PVX.GUS) moved from the injected cell and modified the plasmodesmata to allow passage of the 4.4- and 10-kDa F-dextrans but not the 20-kDa F-dextran. A mutant PVX with a deletion in the gene for the 25-kDa protein accumulated in inoculated protoplasts to the same level as the wild-type PVX. This mutant virus did not move from the injected cell and did not modify the plasmodesmata to allow passage of the 10-kDa F-dextran. However, the 4.4-kDa F-dextran moved from the cells infected with the mutant virus. These data therefore illustrate that movement of PVX is associated with modification of plasmodesmata. The full plasmodesmatal modification requires a functional 25-kDa protein but partial modification occurs when this protein is present in a mutant form.
Potato virus X (PVX) requires three proteins, p25, p12, and p8, encoded by the triple gene block plus the coat protein (CP) for cell-to-cell movement. When each of these proteins was co-expressed with a cytosolic green fluorescent protein (GFP) in the epidermal cells of Nicotiana benthamiana by the microprojectile bombardment-mediated gene delivery method, only p12 enhanced diffusion of co-expressed GFP, indicating an ability to alter plasmodesmal permeability. p25, p12, and CP, expressed transiently in the initially infected cells, transcomplemented the corresponding movement-defective mutants to spread through two or more cell boundaries. Thus, these proteins probably move from cell to cell with the genomic RNA. In contrast, p8 only functioned intracellularly and was not absolutely required for cell-to-cell movement. Since overexpression of p12 overcame the p8 deficiency, p8 appears to facilitate the functioning of p12, presumably by mediating its intracellular trafficking. Considering the likelihood that p12 and p8 are membrane proteins, it is suggested that intercellular as well as intracellular movement of PVX involves a membrane-mediated process.
We used cDNA microarray analysis to screen for FGF target genes in Xenopus embryos treated with the FGFR1 inhibitor SU5402, and identified neurotrophin receptor homolog (NRH) as an FGF target. Causing gain of NRH function by NRH mRNA or loss of NRH function using a Morpholino antisense-oligonucleotide (Mo) led to gastrulation defects without affecting mesoderm differentiation. Depletion of NRH by the Mo perturbed the polarization of cells in the dorsal marginal zone (DMZ), thereby inhibiting the intercalation of the cells during convergent extension as well as the filopodia formation on DMZ cells. Deletion analysis showed that the carboxyl-terminal region of NRH, which includes the "death domain," was necessary and sufficient to rescue gastrulation defects and to induce the protrusive cell morphology. Furthermore, we found that the FGF signal was both capable of inducing filopodia in animal cap cells, where they do not normally form, and necessary for filopodia formation in DMZ cells. Finally, we demonstrated that FGF required NRH function to induce normal DMZ cell morphology. This study is the first to identify an in vivo role for FGF in the regulation of cell morphology, and we have linked this function to the control of gastrulation cell movements via NRH.
Complex three-dimensional waves of excitation can explain the observed cell movement pattern in Dictyostelium slugs. Here we show that these three-dimensional waves can be produced by a realistic model for the cAMP relay system [Martiel, J. L. & Goldbeter, A. (1987) Biophys J. 52, 807-828]. The conversion of scroll waves in the prestalk zone of the slug into planar wave fronts in the prespore zone can result from a smaller fraction of relaying cells in the prespore zone. Further, we show that the cAMP concentrations to which cells in a slug are exposed over time display a simple pattern, despite the complex spatial geometry of the waves. This cAMP distribution agrees well with observed patterns of cAMP-regulated cell type-specific gene expression. The core of the spiral, which is a region of low cAMP concentration, might direct expression of stalk-specific genes during culmination.
Co-inoculation of Nicotiana benthamiana plants with in vitro transcripts of both genomic and short defective interfering (DI) RNAs of Cymbidium ringspot virus results in an accumulation of de novo generated DI RNA dimers. Time-course analysis of DI RNA accumulation in the inoculated leaves showed early accumulation of DI RNA dimers followed by increased levels of DI RNA monomers. In contrast, DI RNA dimers were barely detectable in systems where cell-to-cell movement does not take place (protoplasts) or is less important (monomeric DI RNA-expressing transgenic plants). Our results also demonstrated that the size of DI RNAs is important in the colonization of inoculated leaves, suggesting that DI RNA dimers are quickly selected for cell-to-cell movement if short DI RNA monomers are used for infection.
Although our understanding of the regulation of cellular actin and its control during the development of invertebrates is increasing, the question as to how such actin dynamics are regulated differentially across the vertebrate embryo to effect its relatively complex morphogenetic cell movements remains poorly understood. Intercellular signaling that provides spatial and temporal cues to modulate the subcellular localization and activity of actin regulatory molecules represents one important mechanism. Here we explore whether the localized gene expression of specific actin regulatory molecules represents another developmental mechanism. We have identified a cap1 homolog and a novel guanine nucleotide exchange factor (GEF), quattro (quo), that share a restricted gene expression domain in the anterior mesendoderm of the zebrafish gastrula. Each gene is required for specific cellular behaviors during the anterior migration of this tissue; furthermore, cap1 regulates cortical actin distribution specifically in these cells. Finally, although cap1 and quo are autonomously required for the normal behaviors of these cells, they are also nonautonomously required for convergence and extension movements of posterior tissues. Our results provide direct evidence for the deployment of developmentally restricted actin-regulatory molecules in the control of morphogenetic cell movements during vertebrate development.
Mound formation in the cellular slime mould Dictyostelium results from the chemotactic aggregation of competent cells. Periodic cAMP signals propagate as multiarmed spiral waves and coordinate the movement of the cells. In the late aggregate stage the cells differentiate into prespore and several prestalk cell types. Prestalk cells sort out chemotactically to form the tip, which then controls all further development. The tip organises cell movement via a scroll wave that converts to planar waves in the prespore zone leading to rotational cell movement in the tip and periodic forward movement in the prespore zone. Expression of an activated G alpha1 protein under its own promoter leads to a severely altered morphogenesis from the mound stage onwards. Instead of forming a tipped mound, the cells form a ring-shaped structure without tip. Wave propagation pattern and dynamics during aggregation and mound formation in the mutant are indistinguishable from the parental strain AX3. However, at the time of tip formation the spiral waves that organise the late aggregate do not evolve in a scroll-organising centre in the tip but transform into a circularly closed (twisted) scroll ring wave. This leads to the formation of a doughnut-shaped aggregate. During further development, the doughnut increases in diameter and the twisted scroll wave converts into a train of planar waves, resulting in periodic rotational cell movement. Although biochemical consequences resulting from this mutation are still unclear, it must affect prestalk cell differentiation. The mutant produces the normal proportion of prespore cells but is unable to form functional prestalk cells, i.e., prestalk cells with an ability to sort out from the prespore cells and form a prestalk zone. Failure of sorting leads to an altered signal geometry, ring-shaped scroll waves, that then directs ring formation. This mutant demonstrates the importance of prestalk cell sorting for the stabilisation of the scroll wave that organises the tip.
In host plants, cell-to-cell spread of tobacco mosaic virus (TMV) presumably occurs through intercellular connections, the plasmodesmata. TMV movement is mediated by a specific virus-encoded single-strand nucleic acid-binding protein, P30. The mechanism by which P30 operates is largely unknown. Here, we demonstrate that P30 expressed in transgenic plants is a phosphoprotein. We have developed an assay for in vitro phosphorylation of purified P30 by plant cell wall fractions and have localized the phosphorylation sites to amino acid residues Ser-258, Thr-261, and Ser-265. Interestingly, the P30 phosphorylation sites do not correspond to any known consensus phosphorylation sites for protein kinases. While P30 binding to single-stranded DNA (ssDNA) was shown to involve Thr-261, phosphorylation of this residue does not appear to play a role in binding activity. The protein kinase activity contained in the cell wall fractions was developmentally regulated, expressed predominantly in leaves. Within a leaf, this protein kinase activity increased with leaf maturation and correlated with the reported development of secondary plasmodesmata, sites of P30 accumulation. We suggest that phosphorylation may represent a mechanism for the host plant to sequester P30 following its localization to cell walls.
Anti-neutrophil monoclonal antibody PMN7C3 (IgG3) recognizes glycoproteins bearing the oligosaccharide lacto-N-fucopentaose III, including the C3bi receptor, LFA-1, and p150,95 on the plasma membrane and a group of granule-associated glycoproteins. We have previously shown that binding of this antibody to polymorphonuclear leukocytes (PMNs) stimulates a transient rise in cytosolic free calcium concentration but does not trigger the neutrophil respiratory burst. We now demonstrate that binding of PMN7C3 (and five other monoclonal antibodies recognizing the same antigen) to human neutrophils activates several other cellular responses. Addition of PMN7C3 to monolayers of neutrophils induces a rapid change in cell shape followed by pseudopod formation and increased migration. With incubation at 37 degrees C, the neutrophils aggregate in clusters (leukoagglutination). Quantitation of cell movement in a multiwell chemotaxis assembly or by migration of PMNs under agarose revealed that PMN7C3 is both chemotactic and chemokinetic. Pretreatment with the antibody inhibits subsequent chemotactic response to other stimuli. Monoclonal antibodies binding to other neutrophil antigens do not mimic these effects. These data suggest that cell movement and adhesion can be triggered independently from the respiratory burst. PMN7C3 may be a useful probe with which to study the events that link receptor-ligand binding to cellular response.
Mutant Dictyostelium cells lacking any of the component polypeptides of myosin II exhibit developmental defects. To define myosin's role in establishing Dictyostelium's developmental pattern, we have rescued myosin function in a myosin regulatory light chain null mutant (mlcR-) using cell-type-specific promoters. While mlcR- cells fail to progress beyond the mound stage, expression of RLC from the prestalk promoter, ecmA, produces culminants with normal stalks but with defects in spore cell localization. When GFP-marked prestalk and prespore cells expressing ecmA-RLC are mixed with wild-type cells, the mislocalization of prestalk cells, but not prespore cells, is rescued. Time-lapse video recording of ecmA-RLC cells showed that the posterior prespore zone failed to undergo a contraction important for the upward movement of prespore cells. Prespore cells marked with green fluorescent protein (GFP) failed to move toward the tip with the spiral motion typical of wild type. In contrast, expression of RLC in prespore cells using the psA promoter produced balloon-like structures reminiscent of sorocarps but lacking stalks. GFP-labeled prespore cells showed a spiral movement toward the top of the structures. Expression of RLC from the psA promoter restores the normal localization of psA-GFP cells, but not ecmA-GFP cells. These results define two distinct, myosin-dependent movements that are required for establishing a Dictyostelium fruiting body: stalk extension and active movement of the prespore zone that ensures proper placement of the spores atop the stalk. The approach used in these studies provides a direct means of testing the role of cell motility in distinct cell types during a morphogenetic program.
The conversion of an epithelial monolayer into a multilayered structure consisting of the three germ layers, ectoderm, mesoderm and endoderm, constitutes a conserved theme in the early development of animals. This is accomplished by morphogenetic movements that occur during gastrulation and serve not only to generate shape but also to ensure that cells receive the right signals at the right time. Recent evidence of the role of molecular interactions facilitated by cell movements in continuously defining the chick 'organizer' during gastrulation challenges the notion that it is a fixed cell population derived from an exclusive cell lineage.
Pollination involves an interaction between the female tissues (stigma, style and ovary) and the male gametophyte or the pollen tube cell, which contains the sperm cells. Freezing methods now allow us to visualize the extracellular matrices that guide pollen tubes to the ovary. Adhesion of the pollen tube to these specialized extracellular matrices might be a mechanism of guidance and tube cell movement in the style. In lily, the stylar adhesion molecules are a pectin and a small, basic cysteine-rich protein, both of which are necessary to induce tube cell adhesion to an artificial, in vitro style matrix.