Expression of 'segmentation' genes during larval and juvenile development in the polychaetes Capitella sp. I and H. elegans

Polychaete annelids and arthropods are both segmented protostome invertebrates. To investigate whether the segmented body plan of these two phyla share a common molecular ground pattern, we report the developmental expression of orthologues of the arthropod segment polarity genes engrailed (en), hedgehog (hh), and wingless (wg/Wnt1) in larval and juvenile stages of the polychaete annelid Capitella sp. I and en in a second polychaete, Hydroides elegans. Temporally, neither Wnt1 nor hh are detected in the segmented region of the larval body until after morphological segmentation is apparent. Expression of CapI-Wnt1 is limited to a ring of ectoderm marking the future anus during larval segmentation. CapI-hh is expressed in a ring of the hindgut internal to that of CapI-Wnt1, as well as in a subset of ventral nerve cord neurons, anterior gut tissue, and mesoderm. In both H. elegans and Capitella sp. I, en is expressed in a spatially and temporally dynamic manner in segmentally iterated structures as well as a population of cells that migrate internally from ectoderm to mesoderm, possibly representing a population of ecto-mesodermal precursors. Significantly, the expression patterns we report for wg, en, and hh orthologues in Capitella sp. I and for en in larval development of H. elegans are not comparable to the highly conserved ectodermal segment polarity pattern observed in arthropods at any life history stage, consistent with distinct origins of segmentation between annelids and arthropods.     

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.     

Cell Lineage of the Ilyanassa Embryo: Evolutionary Acceleration of Regional Differentiation during Early Development

Cell lineage studies in mollusk embryos have documented numerous variations on the lophotrochozoan theme of spiral cleavage. In the experimentally tractable embryo of the mud snail Ilyanassa, cell lineage has previously been described only up to the 29-cell stage. Here I provide a chronology of cell divisions in Ilyanassa to the stage of 84 cells (about 16 hours after first cleavage at 23°C), and show spatial arrangements of identified nuclei at stages ranging from 27 to 84 cells. During this period the spiral cleavage pattern gives way to a bilaterally symmetric, dorsoventrally polarized pattern of mitotic timing and geometry. At the same time, the mesentoblast cell 4d rapidly proliferates to form twelve cells lying deep to the dorsal ectoderm. The onset of epiboly coincides with a period of mitotic quiescence throughout the ectoderm. As in other gastropod embryos, cell cycle lengths vary widely and predictably according to cell identity, and many of the longest cell cycles occur in small daughters of highly asymmetric divisions. While Ilyanassa shares many features of embryonic cell lineage with two other caenogastropod genera, Crepidula and Bithynia, it is distinguished by a general tendency toward earlier and more pronounced diversification of cell division pattern along axes of later differential growth.     

Ephrin-Eph signalling drives the asymmetric division of notochord/neural precursors in Ciona embryos

Asymmetric cell divisions produce two sibling cells with distinct fates, providing an important means of generating cell diversity in developing embryos. Many examples of such cell divisions have been described, but so far only a limited number of the underlying mechanisms have been elucidated. Here, we have uncovered a novel mechanism controlling an asymmetric cell division in the ascidian embryo. This division produces one notochord and one neural precursor. Differential activation of extracellular-signal-regulated kinase (ERK) between the sibling cells determines their distinct fates, with ERK activation promoting notochord fate. We first demonstrate that the segregation of notochord and neural fates is an autonomous property of the mother cell and that the mother cell acquires this functional polarity via interactions with neighbouring ectoderm precursors. We show that these cellular interactions are mediated by the ephrin-Eph signalling system, previously implicated in controlling cell movement and adhesion. Disruption of contacts with the signalling cells or inhibition of the ephrin-Eph signal results in the symmetric division of the mother cell, generating two notochord precursors. Finally, we demonstrate that the ephrin-Eph signal acts via attenuation of ERK activation in the neural-fated daughter cell. We propose a model whereby directional ephrin-Eph signals functionally polarise the notochord/neural mother cell, leading to asymmetric modulation of the FGF-Ras-ERK pathway between the daughter cells and, thus, to their differential fate specification.     

Cell fate polarization in ascidian mesenchyme/muscle precursors by directed FGF signaling and role for an additional ectodermal FGF antagonizing signal in notochord/nerve cord precursors

Asymmetric cell division plays a fundamental role in generating various types of embryonic cell. In ascidian embryos, asymmetric cell divisions occur in the vegetal hemisphere in a manner similar to those found in Caenorhabditis elegans. Early divisions in embryos of both species involve inductive events on a single mother cell that result in production of daughters with different cell fates. Here we show in the ascidian Halocynthia roretzi that polarity of muscle/mesenchyme mother precursors is determined solely by the direction from which the FGF9/16/20 signal is presented, a role similar to that of Wnt signaling in the EMS and T cell divisions in C. elegans. However, polarity of nerve cord/notochord mother precursors is determined by possible antagonistic action between the FGF signal and a signal from anterior ectoderm, providing a new mechanism underlying asymmetric cell division. The ectoderm signal suppresses MAPK activation and expression of Hr-FoxA, which encodes an intrinsic competence factor for notochord induction, in the nerve cord lineage.     

The cleavage program in the 2d cell lineage of Tubifex embryos

Early development in clitellate annelids is characterized by a highly stereotyped sequence of unequal, spiral cleavages. Cell 2d (i.e., the second micromere of the D quadrant) in the oligochaete Tubifex tubifex also undergoes an evolutionarily conserved sequence of cell division to produce four bilateral pairs of ectodermal teloblasts that act as embryonic stem cells. This study was conducted to characterize each of the 15 rounds of cell division that occur in the 2d cell lineage in this clitellate. After its occurrence, cell 2d undergoes three rounds of highly unequal divisions, giving off the first smaller daughter cell toward the posterior right of the larger daughter cell, the second cell toward the posterior left, and the third cell toward the anterior side of the cell; the larger daughter cell that results from the third division (i.e., the great-granddaughter cell of 2d) then divides equally into a bilateral pair of NOPQ proteloblasts. Cell NOPQ on either side of the embryo undergoes 11 rounds of cell division, during which ectoteloblasts N, Q, and O/P are produced in this order. After its appearance, NOPQ undergoes highly unequal divisions twice cutting off the smaller cells toward the anterior end of the embryo and then divides almost equally into ectoteloblast N and proteloblast OPQ. After its appearance, OPQ undergoes highly unequal divisions twice giving off the first smaller cell toward the anterior and the second smaller cell toward the posterior of the embryo and then divides almost equally into ectoteloblast Q and proteloblast OP. Finally, OP undergoes highly unequal division four times after its birth budding off the smaller cells toward the anterior and then cleaves equally into ectoteloblasts O and P. In the unequally dividing cells of the 2d cell lineage, the mitotic apparatus (MA), which forms at the cell's center, moves eccentrically toward the cortical site where the smaller cell will be given off. The moving MA is oriented perpendicular to the surface it approaches, and its peripheral pole becomes closely associated with the cell cortex. In contrast, the MA involved in the equal divisions remains in the cell center throughout mitosis. The key features of the cleavage program in the 2d cell lineage are discussed in light of the present observations. The mechanical aspects of unequal cleavage in the 2d cell lineage and the modes of specification of MA orientation are discussed. A comparison of the cleavage mode in the 2d cell lineage is also performed among six selected clitellate annelid species.     

Control of Cleavage Spindle Orientation in Caenorhabditis Elegans: The Role of the Genes Par-2 and Par-3

Polarized asymmetric divisions play important roles in the development of plants and animals. The first two embryonic cleavages of Caenorhabditis elegans provide an opportunity to study the mechanisms controlling polarized asymmetric divisions. The first cleavage is unequal, producing daughters with different sizes and fates. The daughter blastomeres divide with different orientations at the second cleavage; the anterior blastomere divides equally across the long axis of the egg, whereas the posterior blastomere divides unequally along the long axis. We report here the results of our analysis of the genes par-2 and par-3 with respect to their contribution to the polarity of these division. Strong loss-of-function mutations in both genes lead to an equal first cleavage and an altered second cleavage. Interestingly, the mutations exhibit striking gene-specific differences at the second cleavage. The par-2 mutations lead to transverse spindle orientations in both blastomeres, whereas par-3 mutations lead to longitudinal spindle orientations in both blastomeres. The spindle orientation defects correlate with defects in centrosome movements during both the first and the second cell cycle. Temperature shift experiments with par-2(it5ts) indicate that the par-2(+) activity is not required after the two-cell stage. Analysis of double mutants shows that par-3 is epistatic to par-2. We propose a model wherein par-2(+) and par-3(+) act in concert during the first cell cycle to affect asymmetric modification of the cytoskeleton. This polar modification leads to different behaviors of centrosomes in the anterior and posterior and leads ultimately to blastomere-specific spindle orientations at the second cleavage.     

Cleavage and gastrulation of the dendrobranchiate shrimp Penaeus monodon (Crustacea,Malacostraca, Decapoda)

The cleavage pattern of the black tiger shrimp Penaeus monodon was analyzed from the first division until gastrulation. Observations were based on microscopy combined with the use of fluorescent dyes, histological techniques, and computer based three-dimensional reconstructions. Early cleavage is holoblastic and follows a stereotypic pattern, which largely corresponds to what is known from other dendrobranchiate decapods. However, for the first time in this group, we report the presence of an intracellular structure throughout early development. This intracellular body (icb) marks the lineage of one of the two enlarged and division-delayed mesendoderm cells that initiate gastrulation. The identity of the icb as a natural marker and putative determinant of the germ line and its implications on the establishment of the body axes are discussed. The icb as a landmark reveals that the same stereotypic cell division pattern can lead to different fates of individual cells. Hence, the results of this study permit an additional approach to study the relation between cell lineage pattern and the identity of cell lineages.     

An investigation of the specification of unequal cleavages in leech embryos.

Leech embryos develop via stereotyped cell divisions, many of which are unequal. The first division generates identifiable cells, blastomeres AB and CD, which normally follow distinct developmental pathways. When these two cells are dissociated and cultured in isolation, their fates remain distinct and are reminiscent of normal development, but their typical cleavage patterns are disrupted; cell AB undergoes relatively few cell divisions, giving rise to a variable number of macromeres and micromeres, while cell CD cleaves many times, usually forming a poorly organized set of macromeres, embryonic stem cells (teloblasts), and micromeres. We have investigated the hypothesis that the abnormal cleavage pattern of isolated CD blastomeres is due to removal of mechanical constraints normally imposed by cell AB. We find that when cell CD is constrained in vitro to mimic its in vivo shape, it cleaves more normally.     

Specification of polarity of teloblastogenesis in the oligochaete annelid Tubifex: cellular basis for bilateral symmetry in the ectoderm

Ectodermal teloblastogenesis in the oligochaete annelid Tubifex is a spatiotemporally regulated process that gives rise to four bilateral pairs of ectoteloblasts (N, O, P, and Q) that assume distinct fates. Ectoteloblasts on either side of the embryo arise from an invariable sequence of asymmetric cell divisions of a proteloblast, NOPQ, which occur with a defined orientation with respect to the embryonic axes: the N teloblast is generated first and located ventralmost, and the Q teloblast, which is generated next, is located dorsalmost; finally, the O and P teloblasts are generated by almost equal division of their precursor cell, OP. Polarity of teloblastogenesis on one side of the embryo is a mirror image of the other; this mirror symmetry of ectoteloblasts about the embryo's midline gives rise to the bilaterally symmetric organization of the ectoderm. In this study, we examined whether cellular interactions are involved in specification of polarity of asymmetric cell divisions in NOPQ cells. A set of cell transplantation experiments demonstrated that NOPQ cells are initially uncommitted in terms of division pattern and cell fates: If a left NOPQ cell is transplanted to the right side of a host embryo, it exhibits a polarity comparable to that of right NOPQ cells. The results of another set of cell transplantation experiments suggest that contact between NOPQ cells serves as an external cue for their polarization, irrespective of their position in the embryo, and that in the absence of host NOPQ cells, transplanted NOPQ cells can be polarized according to positional information residing in the host embryo. The competence of NOPQ cells to respond to external cues tapers down before their division into N and OPQ. A set of cell ablation experiments demonstrated that neighboring cells such as posteriorly located M teloblasts and anterolaterally located micromeres play a role in controlling spatial aspects of NOPQ's behavior that gives rise to their division along the dorsoventral axis. These results suggest that NOPQ cells, which do not initially have a rigidly fixed polarity, become polarized through external cues. Possible sources of signals for this polarizing induction are discussed in the light of the present results.     

Growth patterns during segmentation in the two polychaete annelids, Capitella sp. I and Hydroides elegans: comparisons at distinct life history stages

Many animals generate new body segments sequentially from a posterior growth zone, and this is generally thought to be the case for the annelids. Most annelids, including polychaetes, have an indirect life cycle and generate their earliest segments during larval life. We have characterized the nature of the growth zone in two polychaetes, Hydroides elegans and Capitella sp. I, during both larval and juvenile stages of segment formation by examining cell division patterns with 5-bromo-2'-deoxyuridine incorporation. Cell division patterns show commonalities between the two species, even though they have distinct body plans and life history characteristics. In both polychaetes, larval segments arise from a field of dividing cells located in lateral regions of the body, rather than from a localized posterior growth zone. Circumferential expansion of the forming segmental tissue is particularly pronounced in Capitella sp. I. Post-metamorphic segments, in contrast, originate from a classical posterior growth zone, with the exception of four posterior thoracic segments of H. elegans, which appear to arise from an area in the middle of the body, indicating plasticity of segment-generating mechanisms present in different annelid life histories. The distinct nature of larval versus juvenile growth zones in H. elegans and Capitella sp. I raises the question of the mechanistic relationship between these two growth zones. The results of this study increase our understanding of the cellular origins of segments in annelids, and serve as a basis for interpretation of molecular expression patterns associated with segment formation in polychaetes.     

β-catenin and early development in the gastropod, Crepidula fornicata

This study describes the early expression and function of β-catenin in the gastropod, Crepidula fornicata. In other bilaterians β-catenin functions in cell adhesion, gastrulation, and cell signaling, which is related to the establishment of the dorso-ventral axis and mesendoderm. Here, we studied the distribution of β-catenin mRNA and protein in C. fornicata via whole mount in situ hybridization and by expressing GFP-tagged β-catenin in vivo. During early cleavage, β-catenin mRNA and protein appear to be broadly localized to all cells in the early embryo. The mRNA tends to be concentrated at inter-phase centrosomes in these cells. At later stages, the mRNA is predominantly in the vegetal macromeres, and subsequently in the rudiment of the hindgut, stomodeum, and velar lobes. Expression of full-length GFP-tagged protein suggests that there is no active mechanism to degrade β-catenin within cells of the early embryos prior to the 25-cell stage. However, by the second day of development, when the fourth quartet micromeres have formed, β-catenin becomes selectively stabilized in the progeny of the 4d mesentoblast (e.g., ML and MR and their daughters) and is missing from most other blastomeres, including vegetal macromeres. Over the next 2 days of development, during subsequent divisions of 4d, β-catenin protein becomes progressively degraded, along the proximo-distal axes, within the progeny of the paired mesendodermal bands. The cells located at the tips of the mesodermal bands (2?mL2 and 2?mR2) are the last to contain this protein, which is no longer detected after 4 days of development. In animals like C. fornicata, which undergo a spiral cleavage program (e.g., molluscs, annelids, nemerteans, and polyclad flatworms), the mesentoblast or 4d cell represents the progenitor of endomesoderm (forming hindgut, internal and external kidneys, and various muscles). Therefore, the selective stabilization of β-catenin in the progeny of 4d in C. fornicata is consistent with arguments that a basic, ancestral role of β-catenin lies in the formation of endomesodermal fates. Experiments using a truncated β-catenin clone show that the regions located in the C-terminus, distal to the 11th armadillo repeat, are required for normal stabilization/degradation of β-catenin protein within the embryo. Microinjection of translation blocking β-catenin morpholinos into zygotes led to the down-regulation of β-catenin expression. This resulted in the subsequent failure of gastrulation, but did not interfere with the formation and early cleavage of 4d, although there were no discernable differentiated cell fates in these defective embryos. These results are compared with those obtained in other metazoans.     

Cell lineage,axis formation,and the origin of germ layers in the amphipod crustacean Orchestia cavimana

Embryos of the amphipod crustacean Orchestia cavimana are examined during cleavage, gastrulation, and segmentation by using in vivo labelling. Single blastomeres of the 8- and 16-cell stages were labelled with DiI to trace cell lineages. Early cleavage follows a distinct pattern and the a/p and d/v body axes are already determined at the 4- and 8-cell stages, respectively. In these stages, the germinal rudiment and the naupliar mesoderm can be traced back to a single blastomere each. In addition, the ectoderm and the postnaupliar mesoderm are separated into right and left components. At the16-cell stage, naupliar ectoderm is divided from the postnaupliar ectoderm, and extraembryonic lineages are separated from postnaupliar mesoderm and endoderm. From our investigation, it is evident that the cleavage pattern and cell lineage of Orchestia cavimana are not of the spiral type. Furthermore, the results of the labelling show many differences to cleavage patterns and cell lineages in other crustaceans, in particular, other Malacostraca. The cleavage and cell lineage patterns of the amphipod Orchestia are certainly derived within Malacostraca, whose ancestral cleavage mode was most likely of the superficial type. On the other hand, Orchestia exhibits a stereotyped cell division pattern during formation and differentiation of the germ band that is typical for malacostracans. Hence, a derived (apomorphic) early cleavage pattern is the ontogenetic basis for an evolutionarily older cell division pattern of advanced developmental stages. O. cavimana offers the possibility to trace the lineages and the fates of cells from early developmental stages up to the formation of segmental structures, including neurogenesis at a level of resolution that is not matched by any other arthropod system.     

First cell fate decisions and spatial patterning in the early mouse embryo

A growing body of evidence indicates that although the early mouse embryo retains flexibility in responding to perturbations, its patterning is initiated at the earliest developmental stages. There are a few spatial cues that are able to influence the pattern of cleavage divisions: one of these lies in the vicinity of the previous meiotic division, the second is associated with the sperm entry and, related to this, the third is the cell shape. Furthermore, the first cleavage separates the zygote into two cells that tend to follow distinguishable fates: one contributes mainly to the embryonic part of the blastocyst, and the other to the abembryonic. The cumulative effect of the early asymmetries generated through cleavage might lead to asymmetric interactions between the first lineages of cells. This could influence development of patterning after implantation. These early polarity cues serve to bias patterning and not as definitive determinants.     

Mitotic spindle orientation distinguishes stem cell and terminal modes of neuron production in the early spinal cord

Despite great insight into the molecular mechanisms that specify neuronal cell type in the spinal cord, cell behaviour underlying neuron production in this tissue is largely unknown. In other neuroepithelia, divisions with a perpendicular cleavage plane at the apical surface generate symmetrical cell fates, whereas a parallel cleavage plane generates asymmetric daughters, a neuron and a progenitor in a stem cell mode, and has been linked to the acquisition of neuron-generating ability. Using a novel long-term imaging assay, we have monitored single cells in chick spinal cord as they transit mitosis and daughter cells become neurons or divide again. We reveal new morphologies accompanying neuron birth and show that neurons are generated concurrently by asymmetric and terminal symmetric divisions. Strikingly, divisions that generate two progenitors or a progenitor and a neuron both exhibit a wide range of cleavage plane orientations and only divisions that produce two neurons have an exclusively perpendicular orientation. Neuron-generating progenitors are also distinguished by lengthening cell cycle times, a finding supported by cell cycle acceleration on exposure to fibroblast growth factor (FGF), an inhibitor of neuronal differentiation. This study provides a novel, dynamic view of spinal cord neurogenesis and supports a model in which cleavage plane orientation/mitotic spindle position does not assign neuron-generating ability, but functions subsequent to this step to distinguish stem cell and terminal modes of neuron production.     

Competence and commitment of Caenorhabditis elegans vulval precursor cells.

Multipotent Caenorhabditis elegans vulval precursor cells (VPCs) choose among three fates (1 degrees, 2 degrees, and 3 degrees ) in response to two intercellular signals: the EGF family growth factor LIN-3 induces 1 degrees fates at high levels and 2 degrees fates at low levels; and a signal via the receptor LIN-12 induces 2 degrees fates. If the level of LIN-3 signal is reduced by a lin-3 hypomorphic mutation, the daughters of the VPC closest to the anchor cell (AC), P6.p, are induced by the AC. By expressing LIN-3 as a function of time in LIN-3-deficient animals, we find that both VPCs and the daughters of VPCs are competent to respond to LIN-3, and VPC daughters lose competence after fusing with the hypodermis. We also demonstrate that the daughters of VPCs specified to be 2 degrees can respond to LIN-3, indicating that 2 degrees VPCs are not irreversibly committed. We propose that maintenance of VPC competence after the first cell cycle and the prioritization of the 1 degrees fate help ensure that P6.p will become 1 degrees. This mechanism of competence regulation might have been maintained from ancestral nematode species that used induction both before and after VPC division and serves to maximize the probability that a functional vulva is formed.