The insertion of mesenchyme cells into the ectoderm during differentiation in Sea urchin embryos

Summary During the course of sea urchin development, from early blastula to pluteus larva, there are two major visible processes toward which all activities seem to be focused. They are the differentiation of the larval skeleton by the primary mesenchyme cells and the differentiation of the primitive gut by the secondary mesenchyme cells. These activities take place within the shell-like layer of epithelial cells, or ectodermal wall. The interactive role of the ectodermal wall with the mesenchyme cells is not yet clearly understood. A number of earlier studies have proposed that the ectoderm may have an inductive influence on the mesenchyme cells and that its inner surface forms a molecular template for guiding the mesenchyme cells. In this report, we suggest an additional role for the ectodermal wall. We show that some primary mesenchyme cells and secondary mesenchyme cells insert between the cells of the ectodermal wall in order to firmly anchor the anlage of the larval skeleton and primitive gut during differentiation. This mechanism may provide a physical basis for maintaining the stable positional relationship of the anlage during development.     

A stochastic model for chemotaxis based on the ordered extension of pseudopods

Many amoeboid cells move by extending pseudopods. Here I present a new stochastic model for chemotaxis that is based on pseudopod extensions by Dictyostelium cells. In the absence of external cues, pseudopod extension is highly ordered with two types of pseudopods: de novo formation of a pseudopod at the cell body in random directions, and alternating right/left splitting of an existing pseudopod that leads to a persistent zig-zag trajectory. We measured the directional probabilities of the extension of splitting and de novo pseudopods in chemoattractant gradients with different steepness. Very shallow cAMP gradients can bias the direction of splitting pseudopods, but the bias is not perfect. Orientation of de novo pseudopods require much steeper cAMP gradients and can be more precise. These measured probabilities of pseudopod directions were used to obtain an analytical model for chemotaxis of cell populations. Measured chemotaxis of wild-type cells and mutants with specific defects in these stochastic pseudopod properties are similar to predictions of the model. These results show that combining splitting and de novo pseudopods is a very effective way for cells to obtain very high sensitivity to stable gradient and still be responsive to changes in the direction of the gradient.     

An excitable cortex and memory model successfully predicts new pseudopod dynamics

Motile eukaryotic cells migrate with directional persistence by alternating left and right turns, even in the absence of external cues. For example, Dictyostelium discoideum cells crawl by extending distinct pseudopods in an alternating right-left pattern. The mechanisms underlying this zig-zag behavior, however, remain unknown. Here we propose a new Excitable Cortex and Memory (EC&M) model for understanding the alternating, zig-zag extension of pseudopods. Incorporating elements of previous models, we consider the cell cortex as an excitable system and include global inhibition of new pseudopods while a pseudopod is active. With the novel hypothesis that pseudopod activity makes the local cortex temporarily more excitable--thus creating a memory of previous pseudopod locations--the model reproduces experimentally observed zig-zag behavior. Furthermore, the EC&M model makes four new predictions concerning pseudopod dynamics. To test these predictions we develop an algorithm that detects pseudopods via hierarchical clustering of individual membrane extensions. Data from cell-tracking experiments agrees with all four predictions of the model, revealing that pseudopod placement is a non-Markovian process affected by the dynamics of previous pseudopods. The model is also compatible with known limits of chemotactic sensitivity. In addition to providing a predictive approach to studying eukaryotic cell motion, the EC&M model provides a general framework for future models, and suggests directions for new research regarding the molecular mechanisms underlying directional persistence.     

Ontogeny of the holothurian larval nervous system: evolution of larval forms

Echinoderm larvae share numerous features of neuroanatomy. However, there are substantial differences in specific aspects of neural structure and ontogeny between the dipleurula-like larvae of asteroids and the pluteus larvae of echinoids. To help identify apomorphic features, we have examined the ontogeny of the dipleurula-like auricularia larva of the sea cucumber, Holothuria atra. Neural precursors arise in the apical ectoderm of gastrulae and appear to originate in bilateral clusters of cells. The cells differentiate without extensive migration, and they align with the developing ciliary bands and begin neurogenesis. Neurites project along the ciliary bands and do not appear to extend beneath either the oral or aboral epidermis. Apical serotonergic cells are associated with the preoral loops of the ciliary bands and do not form a substantial commissure. Paired, tripartite connectives form on either side of the larval mouth that connect the pre-oral, post-oral, and lateral ciliary bands. Holothurian larvae share with hemichordates and bipinnariae a similar organization of the apical organ, suggesting that the more highly structured apical organ of the pluteus is a derived feature. However, the auricularia larva shares with the pluteus larva of echinoids several features of neural ontogeny. Both have a bilateral origin of neural precursors in ectoderm adjacent to presumptive ciliary bands, and the presumptive neurons move only a few cell diameters before undergoing neurogenesis. The development of the holothurian nervous systems suggests that the extensive migration of neural precursors in asteroids is a derived feature. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Monoclonal Antibodies against Differentiating Mesenchyme Cells in Larvae of the Ascidian Halocynthia roretzi

Mechanisms of cell specification of mesenchyme during ascidian embryogenesis are poorly understood. This is because no good molecular markers have been available to evaluate differentiation of the mesenchyme cells. To obtain molecular markers of mesenchyme differentiation, we established monoclonal antibodies, Mch-1 and Mch-3, that recognize antigens present in the mesenchyme cells of the larva of Halocynthia roretzi. The antigens recognized by both antibodies start to be detectable in the mesenchyme cells at the late tailbud stage. The Mch-3 antibody specifically recognized all mesenchyme cells of the larva, whereas the Mch-1 antibody stained the cells only in the anterior portions of mesenchyme clusters in the trunk region of the larva. The Mch-1 antibody also stained trunk lateral cells. In addition, both antibodies recognized the mesenchyme cells in the ventro-lateral boundary between endoderm and epidermis that are migrating to the anterior head region of the larva. The partial embryos that originated from the mesenchymelineage cells at the 8-cell stage expressed the Mch-1 and Mch-3 antigens. The Mch-1 and Mch-3 antibodies will be useful as immunological probes for studying the specification mechanisms of mesenchyme cells.     

Ponticulin plays a role in the positional stabilization of pseudopods

Ponticulin is a 17-kD glycoprotein that represents a major high affinity link between the plasma membrane and the cortical actin network of Dictyostelium. To assess the role of ponticulin in pseudopod extension and retraction, the motile behavior of two independently generated mutants lacking ponticulin was analyzed using computer- assisted two- and three-dimensional motion analysis systems. More than half of the lateral pseudopods formed off the substratum by ponticulin- minus cells slipped relative to the substratum during extension and retraction. In contrast, all pseudopods formed off the substratum by wild-type cells were positionally fixed in relation to the substratum. Ponticulin-minus cells also formed a greater proportion of both anterior and lateral pseudopods off the substratum and absorbed a greater proportion of lateral pseudopods into the uropod than wild-type cells. In a spatial gradient of cAMP, ponticulin-minus cells were less efficient in tracking the source of chemoattractant. Since ponticulin- minus cells extend and retract pseudopods with the same time course as wild-type cells, these behavioral defects in ponticulin-minus cells appear to be the consequence of pseudopod slippage. These results demonstrate that pseudopods formed off the substratum by wild-type cells are positionally fixed in relation to the substratum, that ponticulin is required for positional stabilization, and that the loss of ponticulin and the concomitant loss of positional stability of pseudopods correlate with a decrease in the efficiency of chemotaxis.     

Expression of an Otx gene in the adult rudiment and the developing central nervous system in the vestibula larva of the sea urchin Holopneustes purpurescens

Expression of the Otx gene, HprOtx, from the sea urchin Holopneustes purpurescens, is described during the development of the adult echinoid rudiment in the vestibula larva of this species. The adult rudiment forms directly after gastrulation in the vestibula larva since, unlike the pluteus larva of most other sea urchin species, it is not a feeding larva. The expression is described during the period from hatching to a late vestibula larva. At hatching, HprOtx is expressed throughout the ectoderm of the gastrula. A short time later, expression is absent from the ectoderm on the oral side of the gastrula where the vestibule will form. In an early vestibula larva, HprOtx is not expressed in the ectodermal floor of the vestibule but is expressed in an asymmetric pattern in the aboral ectoderm. As the vestibule invaginates, HprOtx is newly expressed in the ectodermal floor of the vestibule as it develops into the neuroectoderm that is the anlage of the circum-oral central nervous system. The expression is at first in the central part of the floor, then it extends outwards to the ectoderm around the five primary podia and to the epineural folds between the podia. The epineural folds later close to form the radial nerves and the circum-oral nerve ring. In a late vestibula larva, HprOtx is expressed in the radial nerves and the nerve ring. The expression of an Otx gene in the developing echinoid central nervous system is interpreted as an instance of conserved gene expression in echinoderm development.     

Chemotaxis in shallow gradients is mediated independently of PtdIns 3-kinase by biased choices between random protrusions

Current models of eukaryotic chemotaxis propose that directional sensing causes localized generation of new pseudopods. However, quantitative analysis of pseudopod generation suggests a fundamentally different mechanism for chemotaxis in shallow gradients: first, pseudopods in multiple cell types are usually generated when existing ones bifurcate and are rarely made de novo; second, in Dictyostelium cells in shallow chemoattractant gradients, pseudopods are made at the same rate whether cells are moving up or down gradients. The location and direction of new pseudopods are random within the range allowed by bifurcation and are not oriented by chemoattractants. Thus, pseudopod generation is controlled independently of chemotactic signalling. Third, directional sensing is mediated by maintaining the most accurate existing pseudopod, rather than through the generation of new ones. Finally, the phosphatidylinositol 3-kinase (PI(3)K) inhibitor LY294002 affects the frequency of pseudopod generation, but not the accuracy of selection, suggesting that PI(3)K regulates the underlying mechanism of cell movement, rather than control of direction.     

Does ADP-Ribosylation of Proteins in Nuclei Contribute to Ectoderm Cell Differentiation in Sea Urchin Embryos?

In nuclei of sea urchin embryos, marked increase in ADP-ribosyltransferase activity followed by its decrease occurrs in the pre-hatching and post-hatching periods with peaks of activity at the morula and gastrula stages. Increase in its activity was blocked by cycloheximide in the pre- and post-hatching periods and by actinomycin D only in the post-hatching period. Embryo wall cells (ectoderm cells) isolated from gastrulae exhibited markedly higher activity of this enzyme than archenteron cells and mesenchyme cells. Probably, the increase in the activity of this enzyme in the post-hatching period results from expression of the gene for this enzyme mainly in ectoderm cells. In the post-hatching period, the activity increased more in animalized embryos than in normal ones, and increased little in vegetalized embryos. 3-Aminobenzamide (3-ABA), as well as luminol and nicotinamide, inhibited formation of ectoderm structures more than that of endoderm structures, such as the archenteron, in normal and animalized embryos, but had no appreciable effect on morphogenesis in vegetalized embryos. The reaction catalyzed by ADP-ribosyltransferase probably contributes to ectoderm cell differentiation. Treatment of embryos with 3-ABA in the pre-hatching period had little inhibitory effect on the morphogenesis in the post-hatching period, though it caused death of many embryos.     

Chemotaxis: a feedback-based computational model robustly predicts multiple aspects of real cell behaviour

The mechanism of eukaryotic chemotaxis remains unclear despite intensive study. The most frequently described mechanism acts through attractants causing actin polymerization, in turn leading to pseudopod formation and cell movement. We recently proposed an alternative mechanism, supported by several lines of data, in which pseudopods are made by a self-generated cycle. If chemoattractants are present, they modulate the cycle rather than directly causing actin polymerization. The aim of this work is to test the explanatory and predictive powers of such pseudopod-based models to predict the complex behaviour of cells in chemotaxis. We have now tested the effectiveness of this mechanism using a computational model of cell movement and chemotaxis based on pseudopod autocatalysis. The model reproduces a surprisingly wide range of existing data about cell movement and chemotaxis. It simulates cell polarization and persistence without stimuli and selection of accurate pseudopods when chemoattractant gradients are present. It predicts both bias of pseudopod position in low chemoattractant gradients and--unexpectedly--lateral pseudopod initiation in high gradients. To test the predictive ability of the model, we looked for untested and novel predictions. One prediction from the model is that the angle between successive pseudopods at the front of the cell will increase in proportion to the difference between the cell's direction and the direction of the gradient. We measured the angles between pseudopods in chemotaxing Dictyostelium cells under different conditions and found the results agreed with the model extremely well. Our model and data together suggest that in rapidly moving cells like Dictyostelium and neutrophils an intrinsic pseudopod cycle lies at the heart of cell motility. This implies that the mechanism behind chemotaxis relies on modification of intrinsic pseudopod behaviour, more than generation of new pseudopods or actin polymerization by chemoattractants.     

Local cell interactions and the control of gastrulation in the sea urchin embryo

The sea urchin embryo is a good model system for studying the role of mechanical and cell-cell interactions during epithelial invagination, cell rearrangement and mesenchymal patterning in the gastrula. The mechanisms underlying the initial invagination of the archenteron have been surprisingly elusive; several possible mechanisms are discussed. In contrast to its initial invagination, the cellular basis for the elongation of the archenteron is better understood: both autonomous epithelial cell rearrangement and further rearrangement driven by secondary mesenchyme cells appear to be involved. Experiments indicate that patterning of freely migrating primary mesenchyme cells and secondary mesenchyme cells residing in the tip of the archenteron relies to a large extent on information resident in the ectoderm. Interactions between cells in the early embryo and later cell-cell interactions are both required for the establishment of ectodermal pattern information. Surprisingly, in the case of the oral ectoderm the fixation of pattern information does not occur until immediately prior to gastrulation.     

The origin of pigment cells in embryos of the sea urchin Strongylocentrotus purpuratus

A monoclonal antibody (SP1/20.3.1) that recognizes a cell surface epitope expressed by pigment cells in the pluteus larva of Strongylocentrotus purpuratus has been produced. Using indirect immunofluorescence, the epitope is first detected in nonpigmented cells of the vegetal plate after primary mesenchyme ingression. Between the beginning of gastrulation, and when the archenteron is one-third the distance across the blastocoel, SP1/20.3.1-positive cells are free within the blastocoel, at the tip of the archenteron, and dispersed within the blastoderm. Cells at the tip of the archenteron, and mesenchyme near the tip in later stages of gastrulation (secondary mesenchyme), do not express the SP1/20.3.1 antigen. By the completion of gastrulation all SP1/20.3.1-positive cells are dispersed throughout the epidermis. It has been concluded that in S. purpuratus pigment cell precursors are released from the vegetal plate during the initial phase of gastrulation. The cells migrate first to the vegetal ectoderm, and subsequently disperse throughout the ectoderm and develop pigment granules.     

Long-range interactions in mammalian platelet aggregation. II. The role of platelet pseudopod number and length.

In part 1, we reported that human (H) platelets, activated with high concentrations (10 microM) of adenosine diphosphate, aggregate under Brownian diffusion (nonstirred, platelet-rich plasma) with an apparent efficiency of collision (alpha B) approximately 4 times and 8 times larger than observed, respectively, for canine (C) and rabbit (R) platelets. Further evaluations of parallel inhibition of alpha B and shape change suggested a central role for platelet pseudopods in mediating the long-range interactions associated with the elevated alpha B values. We found that greater than 90% of all platelet contacts in the doublets and triplets formed were via at least one pseudopod. We therefore compared pseudopod number and length per platelet generated by approximately 30 s post ADP activation in nonstirred PRP from human, canine, and rabbit donors, using phase-contrast, video-enhanced microscopy of fixed platelets. Theoretical calculations assessing the effects of pseudopod length and number on the collision frequency enhanced by an increased radius of a collision sphere supported the experimental observations that approximately 3 or 4 pseudopods per human or canine platelet, and approximately 5 or 6 pseudopods per rabbit platelet yield optimal alpha B values, with the average pseudopod length: approximately 3:2:1 for H/C/R, paralleling the alpha B differences. After correcting for effects of pseudopods and platelet size on platelet diffusion and sedimentation, it still appeared that the small number of long pseudopods formed on human platelets could largely explain the unusually large alpha B values. The quantitative discrepancies between theory and experiment do not appear related to time-dependent refractoriness within the less than 60 s of observation, but may be related to biochemical differences in dynamics and surface density of adhesive (sticky sites) present on the pseudopod surface.     

Genetic deletion of ABP-120 alters the three-dimensional organization of actin filaments in Dictyostelium pseudopods

This study extends the observations on the defects in pseudopod formation of ABP-120+ and ABP-120- cells by a detailed morphological and biochemical analysis of the actin based cytoskeleton. Both ABP-120+ and ABP-120- cells polymerize the same amount of F-actin in response to stimulation with cAMP. However, unlike ABP-120+ cells, ABP-120- cells do not incorporate actin into the Triton X-100-insoluble cytoskeleton at 30-50 s, the time when ABP-120 is incorporated into the cytoskeleton and when pseudopods are extended after cAMP stimulation in wild-type cells. By confocal and electron microscopy, pseudopods extended by ABP- 120- cells are not as large or thick as those produced by ABP-120+ cells and in the electron microscope, an altered filament network is found in pseudopods of ABP-120- cells when compared to pseudopods of ABP-120+ cells. The actin filaments found in areas of pseudopods in ABP- 120+ cells either before or after stimulation were long, straight, and arranged into space filling orthogonal networks. Protrusions of ABP-120- cells are less three-dimensional, denser, and filled with multiple foci of aggregated filaments consistent with collapse of the filament network due to the absence of ABP-120-mediated cross-linking activity. The different organization of actin filaments may account for the diminished size of protrusions observed in living and fixed ABP-120- cells compared to ABP-120+ cells and is consistent with the role of ABP- 120 in regulating pseudopod extension through its cross-linking of actin filaments.     

Cell dissociation experiments reveal that positional information operates in the chicken frontonasal mass

In this study we examined the role of cell-cell affinity in patterning the avian frontonasal mass-the facial prominence that forms the prenasal cartilage and premaxillary bone. Reconstituted cell pellets derived from undifferentiated, frontonasal mass mesenchyme were recombined with facial epithelium and grafted to host embryos to continue development. We determined that the cells reestablished a recognizable frontonasal mass pattern and were able to induce egg teeth in overlying ectoderm. Further analysis revealed there were region-specific differences in the cartilage patterns such that central recombinations were more likely to form a straight cartilage rod, whereas lateral mesenchyme pellets were more likely to form complex, branched cartilage patterns. The basis for the pattern differences was that central mesenchyme cells showed preferential clustering in the cartilage condensations in the center of the graft, whereas lateral cells were spread throughout as determined by dye labeling and quail chicken chimeras. The disruption of cell contacts temporarily delayed onset of gene expression but by 48 h both Msx2 and Dlx5 were expressed. Msx2, in particular, had very clear edges to the expression domains and often the pattern of expression correlated with type of cartilage morphology. Together, these data suggest that an important patterning mechanism in the face is the ability of mesenchymal cells to sort out according to position and that Msx2 may help repress chondrogenic potential in the lateral frontonasal mass.     

Localized VEGF signaling from ectoderm to mesenchyme cells controls morphogenesis of the sea urchin embryo skeleton

During development, cell migration plays an important role in morphogenetic processes. The construction of the skeleton of the sea urchin embryo by a small number of cells, the primary mesenchyme cells (PMCs), offers a remarkable model to study cell migration and its involvement in morphogenesis. During gastrulation, PMCs migrate and become positioned along the ectodermal wall following a stereotypical pattern that determines skeleton morphology. Previous studies have shown that interactions between ectoderm and PMCs regulate several aspects of skeletal morphogenesis, but little is known at the molecular level. Here we show that VEGF signaling between ectoderm and PMCs is crucial in this process. The VEGF receptor (VEGFR) is expressed exclusively in PMCs, whereas VEGF expression is restricted to two small areas of the ectoderm, in front of the positions where the ventrolateral PMC clusters that initiate skeletogenesis will form. Overexpression of VEGF leads to skeletal abnormalities, whereas inhibition of VEGF/VEGFR signaling results in incorrect positioning of the PMCs, downregulation of PMC-specific genes and loss of skeleton. We present evidence that localized VEGF acts as both a guidance cue and a differentiation signal, providing a crucial link between the positioning and differentiation of the migrating PMCs and leading to morphogenesis of the embryonic skeleton.