Establishment of the dorsoventral axis in nemertean embryos: Evolutionary considerations of spiralian development

The Nemertea represent one of a number of invertebrate phyla that display a highly conserved pattern of cell division known as spiral cleavage. The fates of the early blastomeres are known for representatives of some spiralian phyla (i.e., molluscs and annelids) and in these species there appears to be a high degree of conservation in the ultimate fates of particular embryonic cells. The first two cleavage planes bear an invariant relationship to the symmetry properties of the future larval and adult body plan. To investigate whether these properties of spiralian embryo-genesis are shared (conserved) amongst members of other spiralian phyla, individual blastomeres in two- and four-cell embryos of the nemertean, Nemertopsis bivittata, were microinjected with bi-otinylated dextran lineage tracers. N. bivittata is a direct-developing hoplonemertean that forms a nonfeeding larva. When individual blastomeres are injected at the two-cell stage, two sets of complementary labeling patterns (a total of four different patterns) were observed in the ectoderm of the larvae. When cells were injected at the four-cell stage, four different patterns were observed that represented subsets of the four patterns observed in the previous experiment. Unlike the case in the annelids and molluscs, in which the first cleavage plane bears a strict 45° angular relationship to the future dorsoventral axis, the first cleavage plane in N. bivittata can bear one of two different relationships relative to the larval/adult dorsoventral axis. In half the cases examined, the first cleavage plane corresponded roughly to the plane of bilateral symmetry, and in the rest, it lay along a frontal plane. A similar result was observed for the embryos of the indirect-developing heteronemertean, Cerebratutus lacteus. These results indicate that the fates of the four cell quadrants in nemerteans are not directly homologous to those in other spira-lians, such as the annelids and molluscs. For instance, no single cell quadrant appears to contribute a greater share to the formation of ectoderm, as is the case in the formation of the post-trochal region by the D-cell quadrant in annelids and mol-luscs. Rather, two adjacent cell quadrants contribute nearly equally to the formation of dorsal or ventral ectoderm in the larvae. Possible explanations for the determination of dorsoventrality in nemerte-ans, as well as implications of these findings regarding the evolution of spiralian development, are discussed. © 1994 Wiley-Liss, Inc.     

Fertilization potential and polyspermy prevention in the egg of the nemertean, Cerebratulus lacteus

We investigated the electrical properties of the egg of the nemertean worm Cerebratulus, and found evidence that an electrically-mediated polyspermy block operates for a period of about 1 hr after fertilization. At fertilization, in natural or artificial sea water, the membrane potential shifts from its resting level of about -66 mV to a peak of about +43 mV, and in most cases remains greater than 0 mV for more than 1 hr. The average potential during the first 30 min is +22 +/- 8 mV (SD, n = 12). When the external Na+ concentration is reduced from 486 to 51 mM (choline substituted) the fertilization potential amplitude is reduced; the average potential during the first 30 min is -27 +/- 21 mV (SD, n = 5). Eggs inseminated in 51 mM Na+ sea water become polyspermic, indicating that polyspermy prevention depends on an electrically-mediated mechanism. The electrical block is required for about 60 min, since transfer to 51 mM Na+ sea water during this period results in polyspermy. During the first hour following fertilization, the egg is also developing a permanent, nonelectrical block; the degree of polyspermy which results upon transfer to low Na+ sea water decreases progressively with time. The permanent block appears to be at the level of the egg plasma membrane or glycocalyx, since the egg envelope is not a barrier to sperm penetration, nor does its removal induce polyspermy. Electron micrographs show no obvious changes in the morphology of the extracellular layers, plasma membrane or cortex of the egg after fertilization.     

Localized axis determinant in the early cleavage embryo of the goldfish, Carassius auratus

 The teleost dorsoventral axis cannot be distinguished morphologically before gastrulation. In order to examine whether the yolk cell affects axis determination, we bisect early cleavage embryos of the goldfish, Carassius auratus. When the vegetal yolk hemisphere is removed by bisection along the equatorial plane at the 2-cell stage, the embryos develop abnormally and exhibit a symmetrical morphology. No dorsal structures, such as notochord, somites and neural tube, differentiate and no embryonic shield is formed during gastrulation. In addition, no goosecoid mRNA is expressed before gastrulation. The frequency of abnormality decreases as the age at which the vegetal yolk hemisphere is removed increases. Most embryos removed at the 32-cell stage develop normally. Their morphological phenotype is similar to that of a Xenopus ventralized embryo generated by ultraviolet irradiation on the vegetal hemisphere soon after fertilization. We also observed that, when the embryos were bisected along the first cleavage plane at the 2-cell stage, the proportion of pairs of embryos of which one embryo developed normally was 44.8%. These results indicate that the vegetal yolk hemisphere of the early cleavage embryo of the goldfish contains axis determination factor(s), which are necessary for generation of dorsal structures. Furthermore, it is suggested that these determinant(s) are distributed asymmetrically within the vegetal yolk hemisphere. Received: 25 May 1996 / Accepted: 19 September 1996     

Beta-catenin is required for the establishment of vegetal embryonic fates in the nemertean, Cerebratulus lacteus

Downstream components of the canonical Wnt signaling pathway that result in the nuclear localization of beta-catenin are involved in diverse developmental processes including the formation of the mesendoderm, the regulation of axial properties and asymmetric cell divisions in a wide array of metazoans. The nemertean worm, Cerebratulus lacteus, represents a member of the understudied lophotrochozoan clade that exhibits a highly stereotyped spiral cleavage program in which ectodermal, endodermal, and mesodermal origins are known from intracellular fate mapping studies. Here, the embryonic distribution of beta-catenin protein was studied using injection of synthetic mRNA, encoding GFP-tagged beta-catenin, into fertilized eggs. During the early cleavage stages beta-catenin was destabilized/degraded in animal hemisphere blastomeres and became localized to the nuclei of the four vegetal-most cells at the 64-cell stage, which give rise to definitive larval and adult endoderm. Functional assays indicate that beta-catenin plays a key role in the development of the endoderm. Morpholino knockdown of endogenous beta-catenin, as confirmed by Western analysis, resulted in the failure to gastrulate, absence of the gut and an animalized phenotype in the resulting larvae, including the formation of ectopic (anterior) apical organ tissue with elongated apical tuft cilia and no indications of dorsoventral polarity. Similarly, over-expression of the cytoplasmic domain of cadherin or a beta-catenin-engrailed repressor fusion construct prevented endoderm formation and generated the same animalized phenotype. Injections of mRNA encoding either a stabilized, constitutively activated form of beta-catenin or a dominant negative form of GSK3-beta converted all or nearly all cells into endodermal fates expressing gut-specific esterase. Thus, beta-catenin appears to be both necessary and sufficient to promote endoderm formation in C. lacteus, consistent with its role in endoderm and endomesoderm formation in anthozoan cnidarians, ascidians, and echinoderms. Consistent with the results of other studies, beta-catenin may be viewed as playing a role in the development of posterior/vegetal larval fates (i.e., endoderm) in C. lacteus. However, unlike the case found in polychaete annelid and soil nematode embryos, there is no evidence for a role of beta-catenin in regulating cell fates and asymmetric cell divisions along the entire anterior-posterior axis.     

Restricting oxygen supply to the prospective dorsal side does not reverse axis polarity in embryos of Xenopus laevis

During the first cell cycle, the prospective dorsal side of the embryo of Xenopus laevis becomes enriched in mitochondria relative to the ventral side. This differential distribution of mitochondria persists throughout early development, but it is not known if it is of functional significance, since there do not appear to be dorsal-ventral differences in metabolic rate. However, the unilateral anaerobiosis experiments of Landström and Løvtrup do suggest a role for energy metabolism in determining axis polarity. These experiments apparently show that restricting oxygen supply to the prospective dorsal side causes a reversal of dorsal-ventral axis polarity. We have reinvestigated this point using cell-marking techniques. We find that although gastrulation is initiated at the open end of the tube, the polarity of neural plate development is unaffected. Thus, definitive dorsal-ventral polarity is not affected by the experimental treatment, and it is unlikely that gradients of energy metabolism have a role in specifying axis polarity in X. laevis.     

Cyclin A, cyclin B and stringlike are regulated separately in cell cycle arrested trochoblasts of Patella vulgata embryos

 Trochoblasts are the first cells to differentiate during the development of spiralian embryos. Differentiation is accompanied by a cell division arrest. In embryos of the limpet Patella vulgata, the participation of cell cycle-regulating factors in trochoblast arrest was analysed as a first step to unravel its cause. We determined the cell cycle phase in which the trochoblasts are arrested by analysing the subcellular locations of mitotic cyclins. The results show that the trochoblasts are most likely arrested in the G2 phase. This was supported by measurement of the DNA content in trochoblast nuclei after the last division. Trochoblasts complete their final division at the sixth mitotic cycle. This mitotic cycle resembles the first postblastoderm cell cycle of Drosophila, in which mitotic activity is controlled by expression of the string gene. As failure of string expression results in cell cycle arrest in the G2 phase, negative regulation of a Patella string homolog could be responsible for trochoblast arrest. Although Stl messengers disappeared from trochoblasts during their final division, expression was observed again 20 min later. Messengers remained present in all trochoblasts at low levels during further development. Thus, expression of the stringlike gene allows the cell cycle arrest of these cells, whereas in Drosophila cells arrested in division lack string messengers. Received: 10 February 1997 / Accepted: 23 November 1997     

Tension-dependent collective cell movements in the early gastrula ectoderm of Xenopus laevis embryos

Ventral ectodermal explants taken from early gastrula embryos of Xenopus laevis were artificially stretched either by two opposite concentrated forces or by a distributed force applied to the internal explant’s layer. These modes of stretching reflect different mechanical situations taking place in the normal development. Two main types of kinematic response to the applied tensions were detected. First, by 15 min after the onset of concentrated stretching a substantial proportion of the explant’s cells exhibited a concerted movement towards the closest point of the applied stretching force. We define this movement as tensotaxis. Later, under both concentrated and distributed stretching, most of the cell’s trajectories became reoriented perpendicular to the stretching force, and the cells started to intercalate between each other, both horizontally and vertically. This was accompanied by extensive elongation of the outer ectodermal cells and reconstruction of cell-cell contacts. The intercalation movements led first to a considerable reduction in the stretch-induced tensions and then to the formation of peculiar bipolar ”embryoid” shapes. The type and intensity of the morphomechanical responses did not depend upon the orientation of a stretching force in relation to the embryonic axes. We discuss the interactions of the passive and active components in tension-dependent cell movements and their relations to normal morphogenetic events. Received: 26 April 1999 / Accepted: 30 August 1999     

Flow cytometric analysis of the chromosomes and stability of a wheat cell-culture line

A rapidly growing, long-term suspension culture derived from Triticum aestivum L. (wheat) was synchronized using hydroxyurea and colchicine, and a chromosome suspension with chromosomes was made. After staining with the DNA-specific fluorochromes Hoechst 33258 and Chromomycin univariate and bivariate flow-cytometry histograms showed 15 clearly resolved peaks corresponding to individual chromosome types or groups of chromosomes with similar DNA contents. The flow karyotype was closely similar to a histogram of DNA content measurements of Feulgen-stained chromosomes made by microdensitometry. We were able to show the stability of the flow karyotype of the cell line over a year, while a parallel subculture had a slightly different, stable, karyotype following different growth conditions. The data indicate that flow cytometric analysis of plant karyotypes enables accurate, statistically precise chromosome classification and karyotyping of cereals. There was little overlap between individual flow-histogram peaks, so the method is useful for flow sorting and the construction of chromosome specific-recombinant DNA libraries. Using bivariate analysis, the AT:GC ratio of all the chromosomes was remarkably similar, in striking contrast to mammalian flow karyotypes. We speculate about a fundamental difference in organization and homogenization of DNA sequences between chromosomes within mammalian and plant genomes. Received: 24 April 1996 / Accepted: 24 May 1996     

Buccal capsule development as a consideration for phylogenetic analysis of Rhabditida (Nemata)

 Bacterial feeding nematodes in the order Rhabditida including Zeldia punctata (Cephalobidae) and Caenorhabditis elegans (Rhabditidae) differ profoundly in the buccal capsule parts and associated cells. We carried out a range of tests to determine which buccal capsule parts and cells are evolutionarily homologous between the representative species of the two families. Tests included reconstruction of the buccal capsule and procorpus with transmission electron microscopy (TEM), nuclei position and morphology using 4,6-diamidino-2-phenylindole (DAPI) staining, and cell lineage using four dimensional (4D) microscopy. The lining of the buccal capsule of Z. punctata and additional Cephalobidae includes four sets of muscular radial cells, ma, mb, mc and md, in contrast to C. elegans and additional Rhabditidae, which has two sets of epithelial cells (e1, e3) and two sets of muscle cells (m1, m2). Cell lineage of a nematode closely related to Z. punctata, Cephalobus cubaensis, supports the hypothesis that in cephalobids the e1 and e3 cells become hypodermal cells or are programmed to die. Our findings contradict all previous hypotheses of buccal capsule homology, and suggest instead that ma and mb in Z. punctata are homologous to m1 and m2 in C. elegans respectively. We also hypothesize that ma and mb could be homologous to primary and secondary sets of stylet-protractor muscle cells in the plant parasitic Tylenchida. Received: 24 March 1998 / Accepted: 24 July 1998     

Ectopic expression of Xenopus noggin RNA induces complete secondary body axes in embryos of the direct developing frog Eleutherodactylus coqui

We tested the effects of noggin RNA from Xenopus laevis on axis induction in embryos of a direct developing frog, Eleutherodactylus coqui. We microinjected noggin RNA into one blastomere of 4-cell embryos at the site close to the animal pole, and found that overexpression of noggin RNA is not only sufficient to induce additional axes but also induces heads with eyes. We also injected noggin RNA into 8-cell or 16-cell embryos in various sites, including the marginal zone, above the marginal zone, and the vegetal pole, and found the formation of a complete secondary axis in all three types of injection. These effects of X. laevis noggin RNA on the E. coqui embryo are remarkably different from those found in the X. laevis embryo itself. It has been shown previously that overexpression of noggin RNA on the ventral side of the normal X. laevis embryo induces only a partial axis, with no head structures. We show here that the failure of noggin induction of a complete axis when overexpressed on the ventral side of the X. laevis embryos is not due to an insufficient amount of RNA injected. Also, the failure is unlikely due to inhibition from the primary axis since noggin RNA can induce duplicated head structures on opposite sides of UV-irradiated X. laevis embryos. There appear to be fundamental differences in the responses of E. coqui and X. laevis embryos to exogenous noggin RNA. We propose that these differences stem from an alteration in cytoplasmic arrangements that occurred during evolution of this large egg. Received: 26 July 1999 / Accepted: 1 September 1999     

Genetic variance, coefficient of parentage, and genetic distance of six soybean populations

Plant breeders would like to predict which biparental populations will have the largest genetic variance. If the population genetic variance could be predicted using coefficient of parentage or genetic distance estimates based on molecular marker data, breeders could choose parents that produced segregating populations with a large genetic variance. Three biparental soybean {Glycine max (L.) Merr.} populations were developed by crossing parents that were closely related, based on pedigree relationships. Three additional biparental populations were developed by crossing parents that were assumed to be unrelated. The genetic variance of each population was estimated for yield, lodging, physiological maturity, and plant height. Coefficient of parentage was calculated for each pair of parents used to develop the segregating populations. Genetic distance was determined, based on the number of random amplified polymorphic markers (RAPD) that were polymorphic for each pair of parents. Genetic distance was not associated with the coefficient of parentage or the magnitude of the genetic variance. The genetic variance pooled across the three closely related populations was smaller than the genetic variance pooled across the three populations derived from crossing unrelated parents for all four traits that were evaluated. Received: 24 April 1996 / Accepted: 17 May 1996     

Skeletogenic potential of induced secondary mesenchyme cells derived from the presumptive ectoderm in echinoid embryos

 During the normal development of echinoids, an animal cap consisting of 8 mesomeres in a 16-cell stage embryo differentiates exclusively into ectoderm. Micromeres in an embryo at the same stage differentiate into primary mesenchyme cells (PMC) and coelomic pouch constituents. An animal cap and a quartet of micromeres were isolated from a 16-cell stage embryo and recombined to make a chimeric embryo devoid of presumptive endoderm and secondary mesenchyme cells (SMC). The PMC in the chimeric embryo were completely removed at the mesenchyme blastula stage. The PMC-depleted chimeric embryos formed an archenteron derived from the mesomeres. Some secondary mesenchyme-like cells (induced SMC) were released from the archenteron tip. A considerable fraction of the induced SMC formed the typical mesenchyme pattern after migrating into the vegetal region, synthesized skeletogenic mesenchyme cell-surface protein (msp130) and produced the larval skeleton. These findings indicate that induced SMC derived from the presumptive ectoderm have the same nature as natural SMC in both the timing of their release and their skeletogenic potential expressed in the absence of PMC. Received: 14 November 1996 / Accepted: 30 December 1996     

Regulation and activation of phytoene synthase, a key enzyme in carotenoid biosynthesis, during photomorphogenesis

 During photomorphogenesis in higher plants, a coordinated increase occurs in the chlorophyll and carotenoid contents. The carotenoid level is under phytochrome control, as reflected by the light regulation of the mRNA level of phytoene synthase (PSY), the first enzyme in the carotenoid biosynthetic pathway. We investigated PSY protein levels, enzymatic activity and topological localization during photomorphogenesis. The results revealed that PSY protein levels and enzymatic activity increase during de-etiolation and that the enzyme is localized at thylakoid membranes in mature chloroplasts. However, under certain light conditions (e.g., far-red light) the increases in PSY mRNA and protein levels are not accompanied by an increase in enzymatic activity. Under those conditions, PSY is localized in the prolamellar body fraction in a mostly enzymatically inactive form. Subsequent illumination of dark-grown and/or in far-red light grown seedlings with white light causes the decay of these structures and a topological relocalization of PSY to developing thylakoids which results in its enzymatic activation. This light-dependent mechanism of enzymatic activation of PSY in carotenoid biosynthesis shares common features with the regulation of the NADPH:protochlorophyllide oxidoreductase, the first light-regulated enzyme in chlorophyll biosynthesis. The mechanism of regulation described here may contribute to ensuring a spatially and temporally coordinated increase in both carotenoid and chlorophyll contents. Received: 14 February 2000 / Accepted: 15 March 2000     

Generalized monotonicity, integral conditions and partial survival

 An interesting paper of Vance and Coddington concerning the scalar equation x˙=xf (t, x) is improved by a drastic weakening of the assumptions. Surprising as it may seem, this weakening simplifies the analysis and leads to stronger conclusions. An extension to scalar inequalities of similar structure is applied to a large class of vector equations describing the evolution of n interacting species. Thus we obtain new criteria for extinction and for partial survival, in the sense that each of the separate species may come arbitrarily close to extinction but the population as a whole does not. Received: 5 March 1999 / Revised version: 24 August 1999     

The role of MAPK signaling in patterning and establishing axial symmetry in the gastropod Haliotis asinina

Gastropods are members of the Spiralia, a diverse group of invertebrates that share a common early developmental program, which includes spiral cleavage and a larval trochophore stage. The spiral cleavage program results in the division of the embryo into four quadrants. Specification of the dorsal (D) quadrant is intimately linked with body plan organization and in equally cleaving gastropods occurs when one of the vegetal macromeres makes contact with overlying micromeres and receives an inductive signal that activates a MAPK signaling cascade. Following the induction of the 3D macromere, the embryo begins to gastrulate and assumes a bilateral cleavage pattern. Here we inhibit MAPK activation in 3D with U0126 and examine its effect on the formation and patterning of the trochophore, using a suite of territory-specific markers. The head (pretrochal) region appears to maintain quadri-radial symmetry in U0126-treated embryos, supporting a role for MAPK signaling in 3D in establishing dorsoventral polarity in this region. Posterior (posttrochal) structures - larval musculature, shell and foot - fail to develop in MAPK inhibited trochophores. Inhibition of 3D specification by an alternative method - monensin treatment - yields similar abnormal trochophores. However, genes that are normally expressed in the ectodermal structures (shell and foot) are detected in U0126- and monensin-perturbed larvae in patterns that suggest that this region has latent dorsoventral polarity that is manifested even in the absence of D quadrant specification.     

ribbon, raw, and zipper have distinct functions in reshaping the Drosophila cytoskeleton

rib and raw mutations prevent cells in a number of tissues from assuming specialized shapes, resulting in abnormal tubular epithelia and failure of morphogenetic movements such as dorsal closure. Mutations of zip, which encodes the nonmuscle myosin heavy chain, suppress the phenotypes of rib and raw, suggesting that rib and raw are not directly required for myosin function. Abnormal formation of the actin cytoskeletal structures underlying embryonic cuticular hairs suggests possible roles for rib and raw in organizing the actin cytoskeleton. The actin prehair structures are absent in rib mutants and abnormally shaped in raw mutants, indicating that the two genes have different functions required for organizing the actin cytoskeleton. Received: 4 December 1998 / Accepted: 26 January 1999