Archenteron elongation in the sea urchin embryo is a microtubule-independent process

Earlier studies using colchicine (L. G. Tilney and J. R. Gibbins, 1969, J. Cell Sci. 5, 195-210) had suggested that intact microtubules (MTs) are necessary for archenteron elongation during the second phase of sea urchin gastrulation (secondary invagination), presumably by allowing secondary mesenchyme cells (SMCs) to extend their long filopodial processes. In light of subsequently discovered effects of colchicine on other cellular processes, the role of MTs in archenteron elongation in the sea urchin, Lytechinus pictus, has been reexamined. Immunofluorescent staining of ectodermal fragments and isolated archenterons reveals a characteristic pattern of MTs in the ectoderm and endoderm during gastrulation. Ectodermal cells exhibit arrays of MTs radiating away from the region of the basal body/ciliary rootlet and extending along the periphery of the cell, whereas endodermal cells exhibit a similar array of peripheral MTs emanating from the region of the apical ciliary rootlet facing the lumen of the archenteron. MTs are found primarily at the bases of the filopodia of normal SMCs. beta-Lumicolchicine (0.1 mM), an analog of colchicine which does not bind tubulin, inhibits secondary invagination, indicating that the effects previously ascribed to the disruption of MTs are probably due to the effects of colchicine on other cellular processes. The MT inhibitor nocodazole (5-10 micrograms/ml) added prior to secondary invagination does not prevent gastrulation or spontaneous exogastrulation, even though indirect immunofluorescence indicates that cytoplasmic MTs are completely disrupted in drug-treated embryos. Transverse tissue sections indicate that a comparable amount of cell rearrangement occurs in nocodazole-treated and control embryos. Significantly, SMCs in nocodazole-treated embryos often detach prematurely from the tip of the gut rudiment and extend abnormally large broad lamellipodial protrusions but are also capable of extending long slender filopodia comparable in length to those of control embryos. These results indicate that cytoplasmic MTs are not essential for either filopodial extension by SMCs or for the active epithelial cell rearrangement which accompanies elongation during sea urchin gastrulation.     

Transitions between epithelial and mesenchymal states in development and disease

The ancestors of modern Metazoa were constructed in large part by the foldings and distortions of two-dimensional sheets of epithelial cells. This changed approximately 600 million years ago with the evolution of mesenchymal cells. These cells arise as the result of epithelial cell delamination through a reprogramming process called an epithelial to mesenchymal transition (EMT) [Shook D, Keller R. Mechanisms, mechanics and function of epithelial-mesenchymal transitions in early development. Mech Dev 2003;120:1351-83; Thiery JP, Sleeman JP. Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol 2006;7:131-42]. Because mesenchymal cells are free to migrate through the body cavity, the evolution of the mesenchyme opened up new avenues for morphological plasticity, as cells evolved the ability to take up new positions within the embryo and to participate in novel cell-cell interactions; forming new types of internal tissues and organs such as muscle and bone [Thiery JP, Sleeman, JP. Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol 2006;7:131-42; Hay ED, Zuk A. Transformations between epithelium and mesenchyme: normal, pathological, and experimentally induced. Am J Kidney Dis 1995;26:678-90]. After migrating to a suitable site, mesenchymal cells coalesce and re-polarize to form secondary epithelia, in a so-called mesenchymal-epithelial transition (MET). Such switches between mesenchymal and epithelial states are a frequent feature of Metazoan gastrulation [Hay ED, Zuk A. Transformations between epithelium and mesenchyme: normal, pathological, and experimentally induced. Am J Kidney Dis 1995;26:678-90] and the neural crest lineage [Duband JL, Monier F, Delannet M, Newgreen D. Epitheliu-mmesenchyme transition during neural crest development. Acta Anat 1995;154:63-78]. Significantly, however, when hijacked during the development of cancer, the ability of cells to undergo EMT, to leave the primary tumor and to undergo MET at secondary sites can have devastating consequences on the organism, allowing tumor cells derived from epithelia to invade surrounding tissues and spread through the host [Thiery JP, Sleeman JP. Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol 2006;7:131-42; Hay ED, Zuk A. Transformations between epithelium and mesenchyme: normal, pathological, and experimentally induced. Am J Kidney Dis 1995;26:678-90]. Thus, the molecular and cellular mechanisms underpinning EMT are both an essential feature of Metazoan development and an important area of biomedical research. In this review, we discuss the common molecular and cellular mechanisms involved in EMT in both cases.     

Syndecan and tenascin expression is induced by epithelial-mesenchymal interactions in embryonic tooth mesenchyme

Morphogenesis of embryonic organs is regulated by epithelial-mesenchymal interactions associating with changes in the extracellular matrix (ECM). The response of the cells to the changes in the ECM must involve integral cell surface molecules that recognize their matrix ligand and initiate transmission of signal intracellularly. We have studied the expression of the cell surface proteoglycan, syndecan, which is a matrix receptor for epithelial cells (Saunders, S., M. Jalkanen, S. O'Farrell, and M. Bernfield. J. Cell Biol. In press.), and the matrix glycoprotein, tenascin, which has been proposed to be involved in epithelial-mesenchymal interactions (Chiquet-Ehrismann, R., E. J. Mackie, C. A. Pearson, and T. Sakakura. 1986. Cell. 47:131-139) in experimental tissue recombinations of dental epithelium and mesenchyme. Our earlier studies have shown that in mouse embryos both syndecan and tenascin are intensely expressed in the condensing dental mesenchyme surrounding the epithelial bud (Thesleff, I., M. Jalkanen, S. Vainio, and M. Bernfield. 1988. Dev. Biol. 129:565-572; Thesleff, I., E. Mackie, S. Vainio, and R. Chiquet-Ehrismann. 1987. Development. 101:289-296). Analysis of rat-mouse tissue recombinants by a monoclonal antibody against the murine syndecan showed that the presumptive dental epithelium induces the expression of syndecan in the underlying mesenchyme. The expression of tenascin was induced in the dental mesenchyme in the same area as syndecan. The syndecan and tenascin positive areas increased with time of epithelial-mesenchymal contact. Other ECM molecules, laminin, type III collagen, and fibronectin, did not show a staining pattern similar to that of syndecan and tenascin. Oral epithelium from older embryos had lost its ability to induce syndecan expression but the presumptive dental epithelium induced syndecan expression even in oral mesenchyme of older embryos. Our results indicate that the expression of syndecan and tenascin in the tooth mesenchyme is regulated by epithelial-mesenchymal interactions. Because of their early appearance, syndecan and tenascin may be used to study the molecular regulation of this interaction. The similar distribution patterns of syndecan and tenascin in vivo and in vitro and their early appearance as a result of epithelial-mesenchymal interaction suggest that these molecules may be involved in the condensation and differentiation of dental mesenchymal cells.     

Profilin-actin complexes directly elongate actin filaments at the barbed end.

We demonstrate that the profilin-G-actin complex can elongate actin filaments directly at the barbed end but cannot bind to the pointed end. During elongation, the profilin-actin complex binds to the barbed filament end, whereupon profilin is released, leaving the actin molecule behind. This was first proposed by Tilney [Tilney, L. G., et al. (1983) J. Cell Biol. 97, 112-124] and demonstrated by Pollard and Cooper [(1984) Biochemistry 23, 6631-6641] by electron microscopy. We show that a model without any outside energy supply, in contrast to the mechanism proposed by Pollard and Cooper, can be fitted to our and their [Kaiser et al. (1986) J. Cell Biol. 102, 221-226] findings. Input of outside energy is necessary only if profilin-mediated elongation continues after free G-actin has been lowered to or below the critical concentration observed at the barbed end in the absence of profilin.     

Precocious cleavage furrows simultaneously move and ingress when kinetochore microtubules are depolymerized in <Emphasis Type="Italic">Mesostoma ehrenbergii</Emphasis> spermatocytes

A “precocious” cleavage furrow develops and ingresses during early prometaphase in Mesostoma ehrenbergii spermatocytes (Forer and Pickett-Heaps Eur J Cell Biol 89:607-618, 2010). In response to chromosome movements which regularly occur during prometaphase and that alter the balance of chromosomes in the two half-spindles, the precocious furrow shifts its position along the cell, moving 2–3 μm towards the half cell with fewer chromosomes (Ferraro-Gideon et al. Cell Biol Int 37:892-898, 2013). This process continues until proper segregation is achieved and the cell enters anaphase with the cleavage furrow again in the middle of the cell. At anaphase, the furrow recommences ingression. Spindle microtubules (MTs) are implicated in various furrow positioning models, and our experiments studied the responses of the precocious furrows to the absence of spindle MTs. We depolymerized spindle MTs during prometaphase using various concentrations of nocodazole (NOC) and colcemid. The expected result is that the furrow should regress and chromosomes remain in the midzone of the cell (Cassimeris et al. J Cell Sci 96:9-15, 1990). Instead, the furrows commenced ingression and all three bivalent chromosomes moved to one pole while the univalent chromosomes, that usually reside at the two poles, either remained at their poles or moved to the opposite pole along with the bivalents, as described elsewhere (Fegaras and Forer 2018). The microtubules were completely depolymerized by the drugs, as indicated by immunofluorescence staining of treated cells (Fegaras and Forer 2018), and in the absence of microtubules, the furrows often ingressed (in 33/61 cells) at a rate similar to normal anaphase ingression (~?1 μm/min), while often simultaneously moving toward one pole. Thus, these results indicate that in the absence of anaphase and of spindle microtubules, cleavage furrows resume ingression.     

HSP90 function is required for morphogenesis in ascidian and echinoid embryos

Treatment of embryos of the ascidians Boltenia villosa and Cnemidocarpa finmarkiensis and the sea urchin Strongylocentrotus purpuratus with the anti-HSP90 drugs geldanamycin and radicicol caused morphogenetic arrest. All embryonic stages during which obvious morphogenesis was observed were sensitive to treatment, including formation of the sea urchin blastular epithelium. Arrested embryos were viable for many hours to days post-treatment, indicating a low general toxicity of these drugs. Morphogenetic movements including gastrulation and migration (but not ingression) of sea urchin primary and secondary mesenchyme cells were arrested 8-10 h after treatment began. Cell division and developmentally regulated expression of some genes continued after morphogenesis was arrested. Anti-HSP90 drugs cause selective inactivation or degradation of proteins with which the protein chaperone HSP90 interacts. Therefore, morphogenetic arrest subsequent to the disruption of HSP90 function may result from the reduction in concentration, or activity, of client proteins required for morphogenetic movements of cells. The use of these drugs may provide a means to identify novel activities or proteins involved in morphogenesis.     

Elongated Microvilli on Vegetal Pole Cells in Sea Urchin Embryos

The ultrastructure of cells in the vegetal pole region of sea urchin embryos during early development to the mesenchyme blastula stage was examined by scanning electron microscopy. Vegetal pole cells in the ectoderm with longer microvilli than those of neighboring cells were first detectable at the early blastula stage just before hatching. These cells with elongated microvilli remained in the central region of the vegetal plate when most vegetal plate cells ingressed into the blastocoel to form primary mesenchyme. When first detectable in the sea urchin, Anthocidaris crassispina , four vegetal pole cells had elongated microvilli, but at the time of primary mesenchyme cell ingression, the number of cells with elongated microvilli had increased to eight, apparently by cell division. These vegetal pole cells were wedge-shaped with a broad surface adhering to the hyaline layer at the time of primary mesenchyme cell ingression. SEM observation of the outer surface of embryos showed that the microvilli extended into the hyaline layer. The reinforced attachment of vegetal pole cells to the hyaline layer through their elongated microvilli may explain why these cells could remain at the vegetal pole when the surrounding cells ingressed into the blastocoel as primary mesenchyme cells.     

The adenomatous polyposis coli protein unambiguously localizes to microtubule plus ends and is involved in establishing parallel arrays of microtubule bundles in highly polarized epithelial cells

Loss of full-length adenomatous polyposis coli (APC) protein correlates with the development of colon cancers in familial and sporadic cases. In addition to its role in regulating β-catenin levels in the Wnt signaling pathway, the APC protein is implicated in regulating cytoskeletal organization. APC stabilizes microtubules in vivo and in vitro, and this may play a role in cell migration (Näthke, I.S., C.L. Adams, P. Polakis, J.H. Sellin, and W.J. Nelson. 1996. J. Cell Biol. 134:165–179; Mimori-Kiyosue, Y., N. Shiina, and S. Tsukita. 2000. J. Cell Biol. 148:505–517; Zumbrunn, J., K. Inoshita, A.A. Hyman, and I.S. Näthke. 2001. Curr. Biol. 11:44–49) and in the attachment of microtubules to kinetochores during mitosis (Fodde, R., J. Kuipers, C. Rosenberg, R. Smits, M. Kielman, C. Gaspar, J.H. van Es, C. Breukel, J. Wiegant, R.H. Giles, and H. Clevers. 2001. Nat. Cell Biol. 3:433–438; Kaplan, K.B., A. Burds, J.R. Swedlow, S.S. Bekir, P.K. Sorger, and I.S. Näthke. 2001. Nat. Cell Biol. 3:429–432). The localization of endogenous APC protein is complex: actin- and microtubule-dependent pools of APC have been identified in cultured cells (Näthke et al., 1996; Mimori-Kiyosue et al., 2000; Reinacher-Schick, A., and B.M. Gumbiner. 2001. J. Cell Biol. 152:491–502; Rosin-Arbesfeld, R., G. Ihrke, and M. Bienz. 2001. EMBO J. 20:5929–5939). However, the localization of APC in tissues has not been identified at high resolution. Here, we show that in fully polarized epithelial cells from the inner ear, endogenous APC protein associates with the plus ends of microtubules located at the basal plasma membrane. Consistent with a role for APC in supporting the cytoskeletal organization of epithelial cells in vivo, the number of microtubules is significantly reduced in apico-basal arrays of microtubule bundles isolated from mice heterozygous for APC.     

Posttranslational modification of distinct microtubule subpopulations during cell polarization and differentiation in the mouse preimplantation embryo

During the course of preimplantation development, the cells of the mouse embryo undergo both a major subcellular reorganization (at the time of compaction) and, subsequently, a process of differentiation as the phenotypes of trophectoderm and inner cell mass cell types diverge. We have used antibodies specific for tyrosinated (Kilmartin, J. V., B. Wright, and C. Milstein. 1982. J. Cell Biol. 93:576-582) and acetylated (Piperno, G., and M. T. Fuller. 1985. J. Cell Biol. 101:2085-2094) alpha-tubulin in immunofluorescence studies and found that subsets of microtubules can be distinguished within and between cells during the course of these events. Whereas all microtubules contained tyrosinated alpha-tubulin, acetylated alpha-tubulin was detected only in a subpopulation, located predominantly in the cell cortices. Striking differences developed between the distribution of the two populations during the course of development. Firstly, whereas the microtubule population as a whole tends to redistribute towards the apical domain of cells as they polarize during compaction (Houliston, E., S. J. Pickering, and B. Maro. 1987. J. Cell Biol. 104:1299-1308), the microtubules recognized by the antiacetylated alpha-tubulin antibody became enriched in the basal part of the cell cortex. After asymmetric division of polarized cells to generate two distinct cell types (termed inside and outside cells) we found that, despite the relative abundance of microtubules in outside cells, acetylated microtubules accumulated preferentially in inside cells. Treatment with nocodazole demonstrated that within each cell type acetylated microtubules were the more stable ones; however, the difference in composition of the microtubule network between cell types was not accompanied by a greater stability of the microtubule network in inside cells.     

Microfilaments, cell shape changes, and the formation of primary mesenchyme in sea urchin embryos.

Primary mesenchyme formation in sea urchin embryos occurs when a subset of epithelial cells of the blastula move from the epithelial layer into the blastocoel. The role of microfilaments in producing the cell shape changes that characterize this process, referred to as ingression, was investigated in this study. f-Actin was localized by confocal microscopy using labeled phalloidin. The distribution of f-actin was observed before, during, and after ingression and was correlated with cellular movements. Prior to the onset of ingression, staining became intense in the apical region of putative primary mesenchyme and disappeared following the completion of mesenchyme formation. The apical end of these cells constricted coincidentally with the appearance of the intensified staining, indicating that f-actin may be involved in this constriction. In addition, papaverine, a smooth muscle cell relaxant that interferes with microfilament-based contraction, and that was shown in this study to inhibit cytokinesis, diminished apical constriction and delayed ingression. Despite this interference with apical constriction, the basal surface of ingressing cells protruded into the blastocoel. It is suggested that apical constriction, while not necessary for ingression, does contribute to the efficient production of mesenchyme and that protrusion of the basal surface results from changes that occur independent of apical constriction.     

Electron microscopic determination of the actin filament end at which cytochalasin B blocks monomer addition using the acrosomal actin bundle from horseshoe crab sperm

G-actin freed from exogenous ATP was added to the pieces of isolated acrosomal actin bundles from horseshoe crab sperm to form filaments as reported earlier (Tilney, L.G., Bonder, E.M., & DeRosier, D.J. (1981) J. Cell Biol. 90, 485-494). The growth of a filament was far more rapid at one end (the preferred end) than the other end. These ends were shown to correspond to the barbed and pointed ends, respectively, by decoration of the filaments with myosin subfragment 1. Cytochalasin B inhibited the monomer addition at the preferred end. This technique is useful in determining the ends to which actin filament end-binding proteins from nonmuscle cells bind, which are considered to regulate the actin polymerization in the cells.     

Elevation of Cyclic AMP-Dependent Protein Kinase Activity during Migration of Primary Mesenchyme Cell in Sand Dollar Blastulae

Concetration of intracellular cyclic AMP (cAMP), and activities of adenylate cyclase and cAMP-dependent protein kinase were examined in swimming and mesenchyme blastulae and primary mesenchyme cells (PMCs) of the sand dollar, Clypeaster japonicus , respectively. In mesenchyme blastulae, the concentration of cAMP increased 45% from that in swimming blastulae. PMCs contained a concentration of cAMP 40% higher than that in whole embryos at the mesenchyme blastula stage. The activity of adenylate cyclase in mesenchyme blastulae was 100% higher than that in swimming blastulae. The activites of cAMP-dependent protein kinase in whole embryos at the above two developmental stages, on the other hand, were quite similar to each other. However, in PMCs the activity of the enzyme was conspicuously higher than that in these embryos, and it reached 190% higher than that in these embryos. Inhibition of cAMP-dependent protein kinase activity by a synthetic inhibitor, H8, caused severe inhibition of PMC migration but it did not exert any effect on PMC ingression. These results suggest that the cAMP-dependent protein kinase activity is involved in PMC migration, but not in PMC ingression.     

Localization of Mad2 to Kinetochores Depends on Microtubule Attachment, Not Tension

A single unattached kinetochore can delay anaphase onset in mitotic tissue culture cells (Rieder, C.L., A. Schultz, R. Cole, G. Sluder. 1994. J. Cell Biol. 127:1301–1310). Kinetochores in vertebrate cells contain multiple binding sites, and tension is generated at kinetochores after attachment to the plus ends of spindle microtubules. Checkpoint component Mad2 localizes selectively to unattached kinetochores (Chen, R.-H., J.C. Waters, E.D. Salmon, and A.W. Murray. 1996. Science. 274:242–246; Li, Y., and R. Benezra. Science. 274: 246–248) and disappears from kinetochores by late metaphase, when chromosomes are properly attached to the spindle. Here we show that Mad2 is lost from PtK₁ cell kinetochores as they accumulate microtubules and re-binds previously attached kinetochores after microtubules are depolymerized with nocodazole. We also show that when kinetochore microtubules in metaphase cells are stabilized with taxol, tension at kinetochores is lost. The phosphoepitope 3f3/2, which has been shown to become dephosphorylated in response to tension at the kinetochore (Nicklas, R.B., S.C. Ward, and G.J. Gorbsky. 1995. J. Cell Biol. 130:929–939), is phosphorylated on all 22 kinetochores after tension is reduced with taxol. In contrast, Mad2 only localized to an average of 2.6 out of the 22 kinetochores in taxol-treated PtK₁ cells. Therefore, loss of tension at kinetochores occupied by microtubules is insufficient to induce Mad2 to accumulate on kinetochores, whereas unattached kinetochores consistently bind Mad2. We also found that microinjecting antibodies against Mad2 caused cells arrested with taxol to exit mitosis after ~12 min, while uninjected cells remained in mitosis for at least 6 h, demonstrating that Mad2 is necessary for maintenance of the taxol-induced mitotic arrest. We conclude that kinetochore microtubule attachment stops the Mad2 interactions at kinetochores which are important for inhibiting anaphase onset.     

Sea urchin primary mesenchyme cells: relation of cell polarity to the epithelial-mesenchymal transformation

In euechinoid sea urchin embryos, a subset of epithelial cells in the wall of the blastula become pulsatile, elongate, lose connections with their neighboring cells, and move into the blastocoel to form the primary mesenchyme cells. The Golgi apparatus and microtubule organizing center (MTOC) are located at the apical end of these epithelial cells. We show that as primary mesenchyme cells begin to move into the blastocoel, the Golgi apparatus and MTOC move to a new position adjacent to the apical side of the nucleus. They do not move to a position between the nucleus and the leading (i.e., basal) end of the cell as they do in cultured fibroblasts undergoing directed migration. In addition, we have inhibited the movement of membranous vesicles to the cell surface by incubating embryos in the ionophore monensin. We have used antibodies to msp130, a primary mesenchyme cell surface-specific glycoprotein, to demonstrate that monensin inhibits the movement of msp130-containing vesicles to the cell surface. Despite the inhibition of membrane shuttling by monensin, primary mesenchyme cells ingress on schedule and display normal cell-shape changes. We draw two conclusions from our data. First, the cellular elongation that characterizes ingression is not due to the local insertion of membrane at the leading (basal) end of the cell. Second, ingression does not depend upon establishment of the same cell polarity required for fibroblasts to carry out directed cell migration.     

Microtubules are involved in anterior-posterior axis formation in C. elegans embryos

Microtubules deliver positional signals and are required for establishing polarity in many different organisms and cell types. In Caenorhabditis elegans embryos, posterior polarity is induced by an unknown centrosome-dependent signal. Whether microtubules are involved in this signaling process has been the subject of controversy. Although early studies supported such an involvement (O'Connell, K.F., K.N. Maxwell, and J.G. White. 2000. Dev. Biol. 222:55-70; Wallenfang, M.R., and G. Seydoux. 2000. Nature. 408:89-92; Hamill, D.R., A.F. Severson, J.C. Carter, and B. Bowerman. 2002. Dev. Cell. 3:673-684), recent work involving RNA interference knockdown of tubulin led to the conclusion that centrosomes induce polarity independently of microtubules (Cowan, C.R., and A.A. Hyman. 2004. Nature. 431:92-96; Sonneville, R., and P. Gonczy. 2004. Development. 131: 3527-3543). In this study, we investigate the consequences of tubulin knockdown on polarity signaling. We find that tubulin depletion delays polarity induction relative to wild type and that polarity only occurs when a small, late-growing microtubule aster is visible at the centrosome. We also show that the process of a normal meiosis produces a microtubule-dependent polarity signal and that the relative levels of anterior and posterior PAR (partitioning defective) polarity proteins influence the response to polarity signaling. Our results support a role for microtubules in the induction of embryonic polarity in C. elegans.     

The origin of spicule-forming cells in a 'primitive' sea urchin (Eucidaris tribuloides) which appears to lack primary mesenchyme cells

The calcareous larval skeleton of euechinoid sea urchins is synthesized by primary mesenchyme cells which ingress prior to gastrulation. In embryos of the cidaroid sea urchin Eucidaris tribuloides, no mesenchyme cells ingress before gastrulation, yet larvae later contain skeletons. This apparent paradox is resolved by immunochemical, cell lineage and morphological evidence showing that spicule-forming cells of Eucidaris are homologous to primary mesenchyme cells of euechinoids. In particular, these two cell types share expression of two cell lineage-specific gene products, are derived from the same cellular precursors, the micromeres, and undergo a similar migratory phase prior to skeletogenesis. Despite these similarities, there are far fewer spicule-forming cells in Eucidaris than in typical euechinoids and they assume a different pattern during spiculogenesis. The homology between Eucidaris spicule-forming cells and euechinoid primary mesenchyme cells indicates that a heterochrony in the time of spicule-forming cell ingression has occurred since the divergence of their respective lineages.