Widespread RNA segregation in a spiralian embryo

Asymmetric cell divisions are a crucial mode of cell fate specification in multicellular organisms, but their relative contribution to early embryonic patterning varies among taxa. In the embryo of the mollusc Ilyanassa, most of the early cell divisions are overtly asymmetric. During Ilyanassa early cleavage, mRNAs for several conserved developmental patterning genes localize to interphase centrosomes, and then during division they move to a portion of the cortex that will be inherited by one daughter cell. Here we report an unbiased survey of RNA localization in the Ilyanassa embryo, and examine the overall patterns of centrosomal localization during early development. We find that 3-4% of RNAs are specifically localized to centrosomes during early development, and the remainder are either ubiquitously distributed throughout the cytoplasm or weakly enriched on centrosomes compared with levels in the cytoplasm. We observe centrosomal localization of RNAs in all cells from zygote through the fifth cleavage cycle, and asymmetric RNA segregation in all divisions after the four-cell stage. Remarkably, each specifically localized message is found on centrosomes in a unique subset of cells during early cleavages, and most are found in unique sets of cells at the 24-cell stage. Several specifically localized RNAs are homologous to developmental regulatory proteins in other embryos. These results demonstrate that the mechanisms of localization and segregation are extraordinarily intricate in this system, and suggest that these events are involved in cell fate specification across all lineages in the early Ilyanassa embryo. We propose that greater reliance on segregation of determinants in early cleavage increases constraint on cleavage patterns in molluscs and other spiralian groups.     

Development of blastomere clones in the Ilyanassa embryo: transformation of the spiralian blastula into the larval body plan

Spiralian embryogenesis is deeply conserved and seems to have been in place in the last common ancestor of the large assemblage of protostome phyla known as the Lophotrochozoa. While the blastula fate maps of several spiralian embryos have been determined, little is known about the events that link the early embryo and the larva. For all cells in the Ilyanassa blastula, we determined the clonal morphology at four time points between the blastula and veliger stages. We found that ectomesoderm comes mostly from 3a and 3b, but also from 2c and 2b. We also observed the ingression and early proliferation of 3a- and 3b-derived ectomesoderm. We found cells in the 2b clone that marked the anterior edge of the blastopore and later the mouth and cells in the 3c/3d clones that marked the posterior edges of these structures. This demonstrates directly that the mouth forms in the same location as the blastopore. In the development of the shell field, we observed dramatic cell migration events that invert the positions of the 2b and 2d clones that contribute to the shell. Using time-lapse imaging, we followed and described the cleavage pattern of the conserved endomesodermal blast cell, 4d, up to 4d?+?45 h, when there were 52 cells in the clone. Our results show the growth and movement of clones derived from cells of the spiralian blastula as they transform into the trochophore-like and veliger stages. They have implications for the evolution of the shell in gastropods, the origins of mesoderm in spiralians, and the evolution of mouth formation in metazoans.     

Cell Determination and Organogenesis in Molluscan Development: A Reappraisal Based on Deletion Experiments in Ilyanassa

The vegetal region of the Ilyanassa egg, incorporated into thepolar lobe and segregated to the D blastomere, appears to havean organizing influence in development. The results of deletingindividual micromeres of the first three quartets, and the 4dcell, indicate that most of these cells play rather distinctivedevelopmental roles. Both cytoplasmic segregation and embryonicinduction are believed to be involved in determining the fateof cells during development in Ilyanassa. Comparisons are madewith other forms.     

Spiralian quartet developmental potential is regulated by specific localization elements that mediate asymmetric RNA segregation

Spiralian embryos are found in a large group of invertebrate phyla but are largely uncharacterized at a molecular level. These embryos are thought to be particularly reliant on autonomous cues for patterning, and thus represent potentially useful models for understanding asymmetric cell division. The series of asymmetric divisions that produce the micromere quartets are particularly important for patterning because they subdivide the animal-vegetal axis into tiers of cells with different developmental potentials. In the embryo of the snail Ilyanassa, the IoLR5 RNA is specifically segregated to the first quartet cells during the third cleavage. Here, we show that this RNA, and later the protein, are maintained in the 1q(121) cells and their descendents throughout development. Some IoLR5-expressing cells become internalized and join the developing cerebral ganglia. Knockdown of IoLR5 protein results in loss of the larval eyes, which normally develop in association with these ganglia. Segregation of this RNA to the first quartet cells does not occur if centrosomal localization is bypassed. We show that the specific inheritance of the RNA by the first quartet cells is driven by a discrete RNA sequence in the 3' UTR that is necessary and sufficient for localization and segregation, and that localization of another RNA to the first quartet is mediated by a similar element. These results demonstrate that micromere quartet identity, a hallmark of the ancient spiralian developmental program, is controlled in part by specific RNA localization motifs.     

Nucleic acid synthesis in the normal and lobeless embryo of Ilyanassa obsoleta.

The RNA and DNA content of the normal and lobeless embryo of the Ilyanassa embryo was measured at several stages of embryogenesis. In the lobeless embryo a delay in the net accumulation of RNA and a decrease in the rate of DNA synthesis were observed. It is suggested that the failure of some of the lobe dependent organs to differentiate may be caused by a reduced rate of proliferation of determined embryonic cells.     

Identification of morphogenetic capability limitations via a single starfish embryo/larva reconstruction method

Reconstruction of a starfish embryo provides unique morphogenesis during the developmental process that is not observed in normal development. Here, we established a novel method for reconstruction from single embryos/larvae. By using this method, we investigated the morphogenetic capabilities in critical steps during the reconstruction process as showed by the reconstructed embryos generated from embryos/larvae at the six developmental stages, or from segregated ectodermal and/or endomesodermal cells. Additionally, the novel method addressed several problems found in prior methods related to reproducibly generating reconstructed embryos. In the reconstructions from the various stage embryos/larvae, the morphogenetic capabilities were substantively reduced in the reconstructed embryos generated from 3‐day bipinnaria (3dBp). The combination experiments using ectodermal or endomesodermal cells segregated from 2dBp or 3dBp showed a reduction of the morphogenetic capabilities in both cells types in 3dBp. The reconstructed embryos generated from ectodermal or endomesodermal cells segregated from 2dBp possessed partial morphological features, such as formation of the epithelium or blastopore, but all failed to develop into bipinnariae. These results indicate two limitations of the morphogenetic capabilities during the reconstruction process. Firstly, the morphogenetic capabilities to reconstruct an embryo are considerably reduced between 2dBp and 3dBp. Secondly, cells specified as ectoderm or endomesoderm possess limited morphogenetic capabilities to reconstruct bipinnaria. Furthermore, our results demonstrate that the interaction between these specified cell types is required for reconstruction.     

Nucleic Acid Chemistry of the Ilyanassa Embryo

A morphological study of Ilyanassa embryogenesis was made fromsections of several stages and the nucleic acid content wasmeasured throughout the course of development. The kineticsof uptake and incorporation of radioactive precursors into RNAwas determined. The RNAs synthesized at different stages ofembryogenesis were fractionated by methylated albumin kieselguhrchromatography, and the polyadenylation of RNA during developmentwas measured. From these studies it was concluded that the Ilyanassa embryo,beginning with the 4-cell stage, synthesizes dRNA, rRNA, 4SRNA, and 5S RNA throughout all stages of development. The extensivesynthesis of dRNA and the higher proportion of RNAs polyadenylatedby the pregastrula embryo was considered to be of particularsignificance. The localization of nucleic acids and nucleic acid precursorsin the polar lobe and the effect of removing the polar lobeon nucleic synthesis was reviewed. Recent work on the ultrastructure of the Ilyanassa egg was reviewedin relation to the problem of ooplasmic segregation. The discoveryof a new cytoplasmic organelle in the Ilyanassa oocyte was reviewed.     

Kinetics of carrot somatic embryo development in suspension culture

Somatic embryos in liquid culture can serve as a mass cloning system in a plant propagation program. A quantitative formulation of embryo development obtained from cell suspension cultures is used to develop a segregated kinetic model. The model is based on standard classification schemes as previously developed by plant physiologists. Dependent variables include carbohydrate concentrations (sucrose, fructose, and glucose) and biomass apportioned among the inoculum (free single cells, cell clusters), normal developmental stages, and aberrant cell and embryo types. Good agreement between the model and experimental results is indicated and allows for a rigorous approach to media optimization and reactor scaleup for embryo formation.     

Transplantation of a polyembryonic wasp embryo: a technique for transferring endoparasitic embryo into the host egg

Concealed development of many animal embryos prevents examination of development and limits the application of embryo manipulation techniques aimed at understanding developmental processes. In embryos developing in utero, such as in mammals, it is necessary to dissect embryos from the mother and, upon manipulative intervention, to implant them back into the recipient. Parasitic wasps present a promising system for understanding the evolution of early developmental processes. In basal ectoparasitic species that lay eggs on the surface of the host, it is possible to adapt embryo manipulation techniques developed in Drosophila. However, their derived endoparasitic relatives, which exhibit various modifications of developmental programs, undergo concealed development within the host body. For example, the parasitic polyembryonic wasp Copidosoma floridanum oviposits an egg into the egg of the host moth Trichoplusia ni. The host larva emerges and the parasite undergoes development within the host body, preventing embryo manipulation as a means of examining developmental regulation. Here we present a protocol for embryo transfer that allows the transplantation of C. floridanum egg into the host egg. This approach opens a new avenue in the application of various embryo manipulation techniques aimed at understanding the evolution of embryogenesis in endoparasitic Hymenoptera. In addition, this approach has potential for the development of other tools in C. floridanum, such as transgenesis and reverse genetics, which can also be extended to other endoparasitic species.     

The Egg Cortex Problem as Seen Through the Squid Eye

In many spiralian embryos it has been possible to demonstratethat embryonic development is partially controlled by cytoplasmicfactors located at or in the surface of the fertilized egg andcleaving embryo. In the embryo of the squid Loligo pealei, apattern of developmental information can be demonstrated toexist at the surface, or the egg cortex, of the fertilized butuncleaved embryo. The informational pattern apparently is releasedor activated during the time of the cytoplasmic streaming whichforms the blastodisc. Eventually this developmental informationalpattern is imposed upon the blastoderm cells that come to coverorgan-specific regions of the yolk syncytium which was derivedfrom the egg cortex. Ultrastructural studies demonstrate manyintercellular connections between the yolk syncytium and theblastoderm and between the cells of the blastoderm itself. Duringoogenesis there are regional differences in the follicular syncytiumwhich suggests that the pattern of developmental informationmay arise in the ovary and be retained in a latent state untiltriggered by fertilization.     

Arabidopsis DDB1a and DDB1b are critical for embryo development

DNA DAMAGED BINDING PROTEIN 1 (DDB1) is a highly conserved protein of around 125 kDa. It serves as a substrate adaptor subunit to a CUL4-based E3 ubiquitin ligase within the ubiquitin proteasome pathway. However, based on a set of three beta-propellers, the protein is able to mediate various protein–protein interactions, suggesting that it participates in many developmental and physiological processes in the plant. Arabidopsis encodes for two closely related DDB1 proteins, named DDB1a and DDB1b. While loss-of DDB1a does not severely affect development, loss-of DDB1b has been reported to result in an embryo lethal phenotype. Here we describe two novel ddb1b T-DNA insertion mutants that are not embryo lethal, which we utilized as genetic tools to dissect DDB1b from DDB1a function. Information generated by these studies showed that the C-terminal part of the DDB1 proteins is critical for specific protein–protein interactions. In addition, we demonstrated that DDB1a, like DDB1b, is critical for embryo development, and that both proteins have distinct functions in whole plant development.     

What studies of turbellarian embryos can tell us about the evolution of development mechanisms

In spiralian embryos determination of the axes of bilateral symmetry is associated with D quadrant specification. This can occur late through equal cleavage and cell interactions (conditional specification) or by the four-cell stage through unequal cleavage and cytoplasmic localization (autonomous specification). Freeman & Lundelius (1992) suggest that in spiralian coelomates the former method is ancestral and the latter derived, with evolutionary pressure to shorten metamorphosis resulting in early D quadrant determination through unequal cleavage and appearance of adult features in the larvae. Because of the key phylogenetic position of the turbellarian platyhelminthes, understanding the method of axis specification in this group is important in evaluating the hypothesis. Polyclad development, with equal quartet spiral cleavage, is believed to represent the most primitive condition among living turbellarians and has been examined experimentally in Hoploplana inquilina. Blastomere deletions at the two and four-cell stage produce larvae that are abnormal in morphology and symmetry, indicating that early development is not regulative, and also establish that the embryo does not have an invariant cell lineage. Deletions of micromeres and macromeres at the eight-cell stage indicate that cell interactions are involved in dorso-ventral axis determination, with cross-furrow macromeres playing a more significant role than non-cross-furrow cells. The results support the idea that conditional specification is the primitive developmental mode that characterized the common ancestor of the turbellarians and spiralian coelomates. Evolutionary trends in development in polyclads and other turbellarian orders are discussed.     

The role of unequal cleavage and the polar lobe in the segregation of developmental potential during first cleavage in the embryo of Chætopterus variopedatus

Summary The inequality of the first cleavage division of the Chætopterus embryo is caused by the production of a small polar lobe and the internal shifting of the first cleavage spindle. This division produces a two-celled embryo containing a small AB and a large CD blastomere. These blastomeres have different morphogenetic potentials. Only the larvae resulting from isolated CD blastomeres are able to form bioluminescent photocytes, eyes and lateral hooked bristles. The removal of the polar lobe during first cleavage does not have a great effect on development. These lobeless embryos display a normal pattern of cleavages through the time of mesentoblast formation. The resulting larvae are essentially normal, however they do not form functional photocytes. If the CD cell is isolated after the removal of the first polar lobe, the resulting larva is virtually identical to those formed by the intact CD cell except it lacks the photocyte cells. These results indicate that two separate pathways are involved in the segregation of developmental or morphogenetic potential which takes place during first cleavage. One set of factors, which are necessary for photocyte formation, are associated with the first polar lobe. Other factors that are necessary for the formation of the eyes and lateral hooked bristles are segregated by the unequal cleavage which results from an internal shifting of the cleavage spindle. The removal of a large portion of the vegetal region of the embryo during first cleavage leads to the production of larvae which display a decreased ability to form eyes and lateral hooked bristles. These embryos frequently display an abnormal pattern of cleavages. They do not form the primary somatoblast or the mesentoblast. These results indicate that the vegetal region of the CD cell of Chætopterus is analogous to polar lobes which have been studied in other species, and is therefore important in the specification of the D quadrant. These features of the first cleavage of Chætopterus are a combination of those displayed by forms with direct unequal cleavage and other forms which cleave unequally through the production of large polar lobes. The significance of these findings is discussed relative to the origins of these different types of unequal cleavage.     

The production and elimination of supernumerary blast cells in the leech embryo

 Different species of leech vary greatly in body size but all have 32 body segments. It is unclear how the development of this precise number of segments is regulated, although it is known that the teloblasts of the early leech embryo initially produce more than the required numbers of segment founder cells (blast cells). We used fluorescent dextrans to show that the M teloblast of the Helobdella robusta embryo produces a variable number of additional (supernumerary) cells. These cells fail to enter the germinal band (which contains cells of all lineages and gives rise to the adult leech), but detach from its posterior end and disappear. Our observations suggest that some suffer an increase in membrane permeability while others fuse with the M teloblasts, but that they do not undergo apoptosis. The supernumerary cells of different lineages detach from the germinal band at different times, suggesting that detachment is not triggered by a global signal acting simultaneously on all lineages. We tested the hypothesis that the elimination of the supernumerary m blast cells results from a requirement of m blast cells for close interactions with cells of the other lineages for their survival, a condition that would not be achieved by the last-born m blast cells that fail to enter the germinal band. We cultured isolated M teloblasts and found that they do produce blast cells that themselves divide, indicating that cells of the M lineage can survive in the absence of any interactions with cells of the other lineages. Received: 17 August 1998 / Accepted: 20 November 1998     

Expression of alphaVbeta3 integrin in the chick embryo aortic endothelium

The integrin chain alphaV, expressed in association with beta3, by cells of the megakaryocytic/thrombocytic and endothelial lineages is thought to play an important role in angiogenesis. alphaVbeta3 expression by endothelial cells is not constitutive but induced by various stimuli in avian and human models. Here the developmental pattern of alphaVbeta3 expression was analysed in the chick embryo by immunocytochemistry, using a specific monoclonal antibody. On day 2 of development alphaVbeta3 expression was restricted to rare cells in the blood stream, in the embryo proper and in the yolk sac blood islands. AlphaVbeta3 expression by endothelial cells became detectable on day 3 and was restricted to the dorsal aorta. Interestingly it was absent from the intra-aortic hemopoietic clusters (E3.5) which, as we have showed previously, express the alphaIIbbeta3 integrin and display progenitor potentialities. However the endothelium underlying intra-embryonic hemopoietic clusters expressed this integrin. In contrast E6-7 para-aortic hemopoietic foci contained numerous alphaVbeta3 positive cells. Both alphaVbeta3 and alphaIIbbeta3 were expressed in these latter hemopoietic sites, while alphaVbeta3 was still selectively expressed by the aortic endothelium until E6. Thereafter, at E7 the pulmonary artery also expressed it. Since alphaIIbbeta3 is expressed by avian and murine multilineage hemopoietic progenitors, we then studied the hemopoietic potentialities of alphaVbeta3/alphaIIbeta3 double positive cells from embryonic bone marrow differentiating in vitro in erythro-myeloid conditions. Thrombocytic, erythroid and myeloid progenitor potentialities were found within the cell population expressing both beta3 integrins.     

Progress in nemertean biology: development and phylogeny

This paper reviews progress in developmental biology and phylogenyof the Nemertea, a common but poorly studied spiralian taxonof considerable ecological and evolutionary significance. Analysesof reproductive biology (including calcium dynamics during fertilizationand oocyte maturation), larval morphology and development anddevelopmental genetics have significantly extended our knowledgeof spiralian developmental biology. Developmental genetics studieshave in addition provided characters useful for reconstructingmetazoan phylogeny. Reinvestigation of the cell lineage of Cerebratuluslacteus using fluorescent tracers revealed that endomesodermforms from the 4d cell as in other spiralians and that ectomesodermis derived from the 3a and 3b cells as in annelids, echiuransand molluscs. Studies examining blastomere specification showthat cell fates are established precociously in direct developersand later in indirect developers. Morphological characters usedto estimate the phylogenetic position of nemerteans are criticallyre-evaluated, and cladistic analyses of morphology reveal thatconflicting hypotheses of nemertean relationships result becauseof different provisional homology statements. Analyses thatinclude disputed homology statements (1, gliointerstitial cellsystem 2, coelomic circulatory system) suggest that nemerteansform the sister taxon to the coelomate spiralian taxa ratherthan the sister taxon to Platyhelminthes. Analyses of smallsubunit rRNA (18S rDNA) sequences alone or in combination withmorphological characters support the inclusion of the nemerteansin a spiralian coelomate clade nested within a more inclusivelophotrochozoan clade. Ongoing evaluation of nemertean relationshipswith mitochondrial gene rearrangements and other molecular charactersis discussed.     

Differential regulation of imprinting in the murine embryo and placenta by the Dlk1-Dio3 imprinting control region

Genomic imprinting is an epigenetic mechanism controlling parental-origin-specific gene expression. Perturbing the parental origin of the distal portion of mouse chromosome 12 causes alterations in the dosage of imprinted genes resulting in embryonic lethality and developmental abnormalities of both embryo and placenta. A 1 Mb imprinted domain identified on distal chromosome 12 contains three paternally expressed protein-coding genes and multiple non-coding RNA genes, including snoRNAs and microRNAs, expressed from the maternally inherited chromosome. An intergenic, parental-origin-specific differentially methylated region, the IG-DMR, which is unmethylated on the maternally inherited chromosome, is necessary for the repression of the paternally expressed protein-coding genes and for activation of the maternally expressed non-coding RNAs: its absence causes the maternal chromosome to behave like the paternally inherited one. Here, we characterise the developmental consequences of this epigenotype switch and compare these with phenotypes associated with paternal uniparental disomy of mouse chromosome 12. The results show that the embryonic defects described for uniparental disomy embryos can be attributed to this one cluster of imprinted genes on distal chromosome 12 and that these defects alone, and not the mutant placenta, can cause prenatal lethality. In the placenta, the absence of the IG-DMR has no phenotypic consequence. Loss of repression of the protein-coding genes occurs but the non-coding RNAs are not repressed on the maternally inherited chromosome. This indicates that the mechanism of action of the IG-DMR is different in the embryo and the placenta and suggests that the epigenetic control of imprinting differs in these two lineages.     

Competence of blastomeres for the expression of molecular tissue markers is acquired by diverse mechanisms in the embryo of Platynereis (Annelida)

This paper is devoted to the role of cell divisions for the establishment of histospecificity in the embryo of the spiralian, Platynereis dumerilii (Annelida). We have incubated successive cleavage stages in cytochalasin B (CCB) and observed whether the cells thereafter were able to acquire the competence for expressing histospecific antigens of larval gland cells (labelled by the monoclonal antibody OI64) and of neural components of the ventral nerve cord (labelled by mAb OI7 or by testing acety1cholinesterase activity), respectively. Incubation in CCB results in permanent cleavage arrest, but does not necessarily interfere with biochemical differentiation of such markers. Synthesis of the differentiation marker specific for larval gland cells does not require any cleavages but this capacity becomes restricted to the 1a and 1b cell lines if cleavages are allowed to occur. In contrast, the progenitors of neural cells need at least 6.5 h of normal development before they acquire the competence to synthesize two neural differentiation markers, which can be demonstrated after at least two more days of development. Thus, prespecifying and diversifying cleavages are a prerequisite for neurogenesis and production of the investigated neural markers. Competence for the expression of histospecific antigens may also depend on cell-cell interactions. If 20–24 h old embryos are treated with puromycin, pioneering fibres fail to grow out from a pair of posterior nerve cell progenitors as they would have done normally 24–48 h after fertilization. Concomitantly, a number of potential nerve cells which now do not come into contact with pioneering fibres do not express the neural antigen. This suggests that a local inductive stimulus from the pioneering fibres normally imprints cell fate onto ventral plate cells and turns them into neuroblasts. Correspondence to: A.W.C. Dorresteijn