Multiple inductive signals are involved in the development of the ctenophore Mnemiopsis leidyi.

Ctenophores possess eight longitudinally arrayed rows of comb plate cilia. Previous intracellular cell lineage analysis has shown that these comb rows are derived from two embryonic lineages, both daughters of the four e(1) micromeres (e(11) and e(12)) and a single daughter of the four m(1) micromeres (the m(12) micromeres). Although isolated e(1) micromeres will spontaneously generate comb plates, cell deletion experiments have shown that no comb plates appear during embryogenesis following the removal of e(1) descendents. Thus, the m(1) lineage requires the inductive interaction of the e(1) lineage to contribute to comb plate formation. Here we show that, although m(12) cells are normally the only m(1) derivatives to contribute to comb plate formation, m(11) cells are capable of generating comb plates in the absence m(12) cells. The reason that m(11) cells do not normally make comb rows may be attributable either to their more remote location relative to critical signaling centers (e.g., e(1) descendants) or to inhibitory signals that may be provided by other nearby cells such as sister cells m(12). In addition, we show that the signals provided by the e(1) lineage are not sufficient for m(1)-derived comb plate formation. Signals provided by endomesodermal progeny of either the E or the M lineages (the 3E or 2M macromeres) are also required.     

A provisional epithelium in leech embryo: Cellular origins and influence on a developmental equivalence group

Segmental tissues of glossiphoniid leeches arise from rostrocaudally arrayed columns (bandlets) of segmental founder cells (primary m, n, o, p, and q blast cells) which undergo stereotyped sublineages to generate identifiable subsets of definitive progeny. The bandlets lie at the surface of the embryo beneath the squamous epithelium of a transient embryonic covering called the provisional integument. This "provisional epithelium" derives from microsomes produced during the early cleavage divisions. Previous experiments have shown that the primary o and p blast cells constitute an equivalence group, i.e., are initially developmentally equipotent and undergo hierarchical interactions which cause them to assume distinct O and P fates. Here, we examine the role of the provisional epithelium in determining the fates of the underlying o and p blast cells. Experiments entailing the microinjection of individual micromeres with cell lineage tracers show that, at stages 7-8 of normal development, the epithelium comprises coherent and relatively stereotyped domains derived from particular micromeres. Upon photoablating domains of epithelium labeled with photosensitizing lineage tracer, the normal assignment of O fates is disturbed; o blast cells divide symmetrically (as p blast cells do) and some supernumerary definitive progeny expressing P fates arise within the O lineage. We therefore conclude that the epithelium is essential for generation and/or reception of signal(s) by which the o and p blast cells' normally determine their fates. Finally, a new tracer substance, biotinylated fixable dextran (BFD), is described which was essential for this study by virtue of its superior resistance to photobleaching and which offers several other advantages as well.     

Regulation and regeneration in the ctenophore Mnemiopsis leidyi

Lobate ctenophores (tentaculates) generally exhibit a remarkable ability to regenerate missing structures as adults. On the other hand, their embryos exhibit a highly mosaic behavior when cut into halves or when specific cells are ablated. These deficient embryos do not exhibit embryonic regulation, and generate incomplete adult body plans. Under certain conditions, however, these deficient animals are subsequently able to replace the missing structures during the adult phase in a process referred to as "post-regeneration." We have determined that successful post-regeneration can be predicted on the basis of a modified polar coordinate model, and the rules of intercalary regeneration, as defined by French et al. (V. French, P. J. Bryant, and S. V. Bryant, 1976, Science 193, 969-981.) The model makes certain assumptions about the organization of the ctenophore body plan that fit well with what we have determined on the basis of cell lineage fates maps, and their twofold rotational ("biradial") symmetry. The results suggest that cells composing the ctenophore adult body plan possess positional information, which is utilized to reconstruct the adult body plan. More specifically, we have found that the progeny of three specific cell lineages are required to support post-regeneration of the comb rows (the e(1), e(2), and m(1) micromeres). Furthermore, post-regeneration of the comb rows involves a suite of cell-cell inductive interactions, which are similar to those that take place during their embryonic formation. The significance of these findings is discussed in terms of the organization of the ctenophore body plan, and the mechanisms involved in cell fate specification. This situation is also contrasted with that of the atentaculate ctenophores, which are unable to undergo post-regeneration.     

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.     

Cell lineage and fate map of the primary somatoblast of the polychaete annelid Capitella teleta

Like most polychaete annelids, Capitella teleta (formerly Capitella sp. I) exhibits a highly stereotypic program of early development known as spiral cleavage. Animals with spiral cleavage have diverse body plans, and homologous embryonic cells can be readily identified among distantly related animals. Spiralian embryos are particularly amenable to studies of fate-mapping, and larval fates of identified cells are conserved among diverse taxa. One cell of particular importance in spiralian development is 2d, or the primary somatoblast, which generates ectoderm of the body posterior to the prototroch. We are interested in the evolution of the primary somatoblast, and thus far, the 2d sublineage has only been analyzed in a few species. In Capitella teleta, 2d generates ectoderm of the segmented trunk and post-segmental pygidium. In this study, development of the 2d lineage was characterized in detail through intracellular injections of DiI, and time-lapse as well as confocal microscopy to analyze cleavage patterns and the fates of larval cells. Analysis of cleavage patterns reveals that the first bilateral division in the 2d sublineage occurs with the division of 2d112, the same 2d daughter cell that first divides bilaterally in the polychaete Platynereis dumerilii. Larval fates of blastomeres 2d1, 2d2, 2d11, 2d12, 2d112, 2d1121, and 2d1122 were determined. All cells show stereotypic descendant clones that are consistent with segregation within sublineages. In the first few divisions of the 2d sublineage, larval-specific structures (neurotroch and telotroch) and pygidial ectoderm are segregated from segmental ectoderm and ventral nerve cord. The daughters of the first bilateral division, 2d1121 and 2d1122, generate the right and left halves of the segmental ectoderm and ventral nerve cord respectively, although the clones are consistently asymmetric across the dorsal midline. The pattern of cleavage divisions and the fates of the 2d daughters in Capitella teleta are compared to those in other spiralians with special attention to annelids.     

Fate maps of the first quartet micromeres in the gastropod Ilyanassa obsoleta.

Cell fate specification in the gastropod mollusc Ilyanassa obsoleta involves both cell autonomous and inductive mechanisms, which depend on determinants localized first in the polar lobe and then in the D quadrant of the embryo. A complete cell lineage is lacking for this embryo and is essential for a critical interpretation of previous experimental results and an analysis of the mechanisms at the molecular level. Lineages of the first quartet micromeres were followed using Lucifer Yellow dextran as a tracer. The tracer was injected into individual first quartet micromeres using iontophoresis and patterns of fluorescence were analyzed in the larva after 8 days of development. Fluorescence was limited to head structures, including eyes, tentacles and velum. Structures on the left side were derived from 1a and 1d micromeres; 1a gave rise to the left eye, including the lens. Right side structures were derived from the 1c micromere and 1b contributed to the apical plate between the eyes and symmetrically to both sides of the velum. First quartet lineage data are compared with results from previous cell ablation experiments and with lineage data from other species.     

The development of bioluminescence in the ctenophore Mnemiopsis leidyi

The photocytes of the ctenophore Mnemiopsis have a discontinuous distribution along the radial canal between the sites where the comb plate cilia cells are located on the side of the canal which contains the testes. They are separated from the lumen of the canal by a population of gastric cells. Cytologically these cells are characterized by a condensed nucleus and cytoplasm which stains lightly with basophilic dyes.The ability of the ctenophore embryo to produce light appears at the developmental stage when the comb plate cilia first begin to grow out. At this stage four light-producing areas are present; each area corresponds to one quadrant of the adult animal. At the sites of light production, a population of cells can be identified that have some of the cytological properties of the photocytes of the adult animal. Within 8–10 hr after light production begins there is a 10-fold increase in the amount of light produced by an embryo and a cytological maturation of its photocytes; during this time period there is no increase in photocyte number. At about the time the embryo begins to feed, each light-producing region splits into two regions, each of which corresponds to a radial canal.During the process of embryogenesis the photocyte cell lineage is first segregated from non-photocytes at the differential division which gives the 8-cell stage embryo. The M macromere lineage goes on to form photocytes, but the E macromere lineage does not. The M macromeres form a micromere at the aboral pole of the embryo at each of the next two cleavages; during these cleavages the potential for photocyte differentiation continues to segregate with the M macromeres. During the division which gives the 64-cell stage the M macromeres divide equally; the potential for photocyte differentiation segregates with the M macromeres nearest the oral-aboral axis. M macromeres which are isolated from the embryo at the 8-, 16-, or 32-cell stage of development will continue to cleave as though they were part of a normal embryo and differentiate to form photocytes.The events that are responsible for the differential division during the formation of the 8-cell stage embryo have been studied by centrifuging eggs to produce fragments of different cytoplasmic composition. Egg fragments which contain only cortical cytoplasm differentiate comb plate cilia cells, but do not produce photocytes. Cortical fragments with a small amount of yolk differentiate comb plate cilia cells and photocytes. Both the M and E macromeres from cortical fragments with no yolk produce comb plate cilia. Only M macromeres containing yolk form photocytes; if an M macromere forms photocytes it does not form comb plate cilia.     

Asymmetric Numb distribution is critical for asymmetric cell division of mouse cerebral cortical stem cells and neuroblasts

Stem cells and neuroblasts derived from mouse embryos undergo repeated asymmetric cell divisions, generating neural lineage trees similar to those of invertebrates. In Drosophila, unequal distribution of Numb protein during mitosis produces asymmetric cell divisions and consequently diverse neural cell fates. We investigated whether a mouse homologue m-numb had a similar role during mouse cortical development. Progenitor cells isolated from the embryonic mouse cortex were followed as they underwent their next cell division in vitro. Numb distribution was predominantly asymmetric during asymmetric cell divisions yielding a beta-tubulin III(-) progenitor and a beta-tubulin III(+) neuronal cell (P/N divisions) and predominantly symmetric during divisions producing two neurons (N/N divisions). Cells from the numb knockout mouse underwent significantly fewer asymmetric P/N divisions compared to wild type, indicating a causal role for Numb. When progenitor cells derived from early (E10) cortex undergo P/N divisions, both daughters express the progenitor marker Nestin, indicating their immature state, and Numb segregates into the P or N daughter with similar frequency. In contrast, when progenitor cells derived from later E13 cortex (during active neurogenesis in vivo) undergo P/N divisions they produce a Nestin(+) progenitor and a Nestin(-) neuronal daughter, and Numb segregates preferentially into the neuronal daughter. Thus during mouse cortical neurogenesis, as in Drosophila neurogenesis, asymmetric segregation of Numb could inhibit Notch activity in one daughter to induce neuronal differentiation. At terminal divisions generating two neurons, Numb was symmetrically distributed in approximately 80% of pairs and asymmetrically in 20%. We found a significant association between Numb distribution and morphology: most sisters of neuron pairs with symmetric Numb were similar and most with asymmetric Numb were different. Developing cortical neurons with Numb had longer processes than those without. Numb is expressed by neuroblasts and stem cells and can be asymmetrically segregated by both. These data indicate Numb has an important role in generating asymmetric cell divisions and diverse cell fates during mouse cortical development.     

Cellular Symmetry Breaking during Caenorhabditis elegans Development

The nematode worm Caenorhabditis elegans has produced a wellspring of insights into mechanisms that govern cellular symmetry breaking during animal development. Here we focus on two highly conserved systems that underlie many of the key symmetry-breaking events that occur during embryonic and larval development in the worm. One involves the interplay between Par proteins, Rho GTPases, and the actomyosin cytoskeleton and mediates asymmetric cell divisions that establish the germline. The other uses elements of the Wnt signaling pathway and a highly reiterative mechanism that distinguishes anterior from posterior daughter cell fates. Much of what we know about these systems comes from intensive study of a few key events—Par/Rho/actomyosin-mediated polarization of the zygote in response to a sperm-derived cue and the Wnt-mediated induction of endoderm at the four-cell stage. However, a growing body of work is revealing how C. elegans exploits elements/variants of these systems to accomplish a diversity of symmetry-breaking tasks throughout embryonic and larval development.Over the past few decades, the C. elegans embryo has become a premiere system for studying cellular symmetry breaking in a developmental context. During C. elegans development, nearly every division produces daughter cells with different developmental trajectories. In some cases, these differences are imposed on daughters before or after division through inductive signals, but many of these divisions are intrinsically asymmetric—an initial symmetry-breaking step creates polarized distributions or activities of factors that control developmental potential. Registration of the cleavage plane with the axis of polarity then ensures differential inheritance of these potentials. With respect to cell fates, the output of these asymmetric divisions is amazingly diverse, yet the embryo seems to accomplish this diversity through variants of a few conserved symmetry-breaking systems. Thus the C. elegans embryo provides an exceptional opportunity to explore not only the core mechanisms underlying cellular symmetry breaking, but also how evolution can reconfigure these mechanisms to do different but related jobs in multiple contexts.In this review, we focus most of our attention on two conserved systems that together account for much of the cellular asymmetry observed during C. elegans embryogenesis. The first, which is best known for its role in the early asymmetric cell divisions that segregate germline from the soma, involves a complex interplay between Par proteins, Rho-family GTPases, and the actomyosin cytoskeleton. Interestingly, the embryo exploits elements of this same system to break symmetry during cleavage furrow specification and to establish apicobasal polarity in early embryonic cells and in the first true embryonic epithelia. The second system we focus on involves an unusual application of WNT signaling pathway components and is used reiteratively throughout embryonic and larval development to distinguish anterior and posterior daughter cell fates. Rather than comprehensively review these systems, we highlight topics not extensively covered in other reviews.     

Asymmetric cell divisions in flowering plants - one mother, "two-many" daughters

Plant development shows a fascinating range of asymmetric cell divisions. Over the years, however, cellular differentiation has been interpreted mostly in terms of a mother cell dividing mitotically to produce two daughter cells of different fates. This popular view has masked the significance of an entirely different cell fate specification pathway, where the mother cell first becomes a coenocyte and then cellularizes to simultaneously produce more than two specialized daughter cells. The "one mother - two different daughters" pathways rely on spindle-assisted mechanisms, such as translocation of the nucleus/spindle to a specific cellular site and orientation of the spindle, which are coordinated with cell-specific allocation of cell fate determinants and cytokinesis. By contrast, during "coenocyte-cellularization" pathways, the spindle-assisted mechanisms are irrelevant since cell fate specification emerges only after the nuclear divisions are complete, and the number of specialized daughter cells produced depends on the developmental context. The key events, such as the formation of a coenocyte and migration of the nuclei to specific cellular locations, are coordinated with cellularization by unique types of cell wall formation. Both one mother - two different daughters and the coenocyte-cellularization pathways are used by higher plants in precise spatial and time windows during development. In both the pathways, epigenetic regulation of gene expression is crucial not only for cell fate specification but also for its maintenance through cell lineage. In this review, the focus is on the coenocyte-cellularization pathways in the context of our current understanding of the asymmetric cell divisions. Instances where cell differentiation does not involve an asymmetric division are also discussed to provide a comprehensive account of cell differentiation.     

tcl-2 encodes a novel protein that acts synergistically with Wnt signaling pathways in C. elegans

Mutations in tcl-2 cause defects in the specification of the fates of the descendants of the TL and TR blast cells, whose polarity is regulated by lin-44/Wnt and lin-17/frizzled, during Caenorhabditis elegans development. In wild-type animals, POP-1/TCF/LEF, is asymmetrically distributed to the T cell daughters, resulting in a higher level of POP-1 in the nucleus of the anterior daughter. The POP-1 asymmetric distribution is controlled by lin-44 and lin-17. However, in tcl-2 mutants, POP-1 is equally distributed to T cell daughters as is observed in lin-17 mutants, indicating that, like lin-17, tcl-2 functions upstream of pop-1. In addition, tcl-2 mutations cause defects in the development of the gonad and the specification of fate of the posterior daughter of the P12 cell, both of which are controlled by the Wnt pathway. Double mutant analyses indicate that tcl-2 can act synergistically with the Wnt pathway to control gonad development as well as P12 descendant cell fate specification. tcl-2 encodes a novel protein. A functional tcl-2::gfp construct was weakly expressed in the nuclei of the T cell and its descendants. Our results suggest that tcl-2 functions with Wnt pathways to control T cell fate specification, gonad development, and P12 cell fate specification.     

The effects of altering the position of cleavage planes on the process of localization of developmental potential in ctenophores.

During the transition from the four- to the eight-cell stage in ctenophore embryos, each blastomere produces one daughter cell with the potential to form comb plate cilia and one daughter cell that does not have this potential. If the second cleavage in a two-cell embryo is blocked, at the next cleavage these embryos frequently form four blastomeres which have the configuration of the blastomeres in a normal eight-cell embryo. At this division there is also a segregation of comb plate-forming potential. By compressing a two-cell embryo in a plane perpendicular to the first plane of cleavage it is possible to produce a four-cell blastomere configuration that is identical to that produced following the inhibition of the second cleavage. However, under these circumstances the segregation of comb plate potential does not occur. These results suggest that the appropriate plane of cleavage must take place for a given cleavage cycle, in order for localizations of developmental potential to be properly positioned within blastomeres.     

Pattern formation during vulval development in C. elegans

Previous studies have shown that the development of the vulva of the C. elegans hermaphrodite involves six multipotential hypodermal cells as well as the gonadal anchor cell, which induces vulval formation. Our further examination of the interactions among these seven cells has led to the following model. Each hypodermal precursor cell becomes determined to adopt one of its three potential fates; each of these fates is to generate a particular cell lineage. In the absence of cellular interactions each precursor cell will generate the nonvulval cell lineage; an inductive signal from the anchor cell is required for a precursor cell to generate either of the two types of vulval cell lineages. The inductive signal is spatially graded, and the potency of the signal specifies which lineage is expressed by each of the tripotential precursor cells.     

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 role of cell lineage in development

Studies of the role of cell lineage in development began in the latter part of the 19th century, fell into decline in the early part of the 20th, and were revived about 20 years ago. This recent revival was accompanied by the introduction of new and powerful analytical techniques. Concepts of importance for cell lineage studies include the principal division modes by which a cell may give rise to its descendant clone (proliferative, stem cell and diversifying); developmental determinacy, or indeterminacy, which refer to the degree to which the normal cleavage pattern of the early embryo and the developmental fate of its individual cells is, or is not, the same in specimen after specimen; commitment, which refers to the restriction of the developmental potential of a pluripotent embryonic cell; and equivalence group, which refers to two or more equivalently pluripotent cell clones that normally take on different fates but of which under abnormal conditions one clone can take on the fate of another. Cell lineage can be inferred to have a causative role in developmental cell fate in embryos in which induced changes in cell division patterns lead to changes in cell fate. Moreover, such a causative role of cell lineage is suggested by cases where homologous cell types characteristic of a symmetrical and longitudinally metameric body plan arise via homologous cell lineages. The developmental pathways of commitment to particular cell fates proceed according to a mixed typologic and topographic hierarchy, which appears to reflect an evolutionary compromise between maximizing the ease of ordering the spatial distribution of the determinants of commitment and minimizing the need for migration of differentially committed embryonic cells. Comparison of the developmental cell lineages in leeches and insects indicates that the early course of embryogenesis is radically different in these phyletically related taxa. This evolutionary divergence of the course of early embryogenesis appears to be attributable to an increasing prevalence of polyclonal rather than monoclonal commitment in the phylogenetic line leading from an annelid-like ancestor to insects.     

Cellular interactions in early C. elegans embryos

In normal development both the anterior and posterior blastomeres in a 2-cell C. elegans embryo produce some descendants that become muscles. We show that cellular interactions appear to be necessary in order for the anterior blastomere to produce these muscles. The anterior blastomere does not produce any muscle descendants after either the posterior blastomere or one of the daughters of the posterior blastomere is removed from the egg. Moreover, we demonstrate that a daughter of the anterior blastomere that normally does not produce muscles appears capable of generating muscles when interchanged with its sister, a cell that normally does produce muscles. Embryos develop normally after these blastomeres are interchanged, suggesting that cellular interactions play a major role in determining the fates of some cells in early embryogenesis.     

Visualization of calcium transients controlling orientation of ciliary beat

To image changes in intraciliary Ca controlling ciliary motility, we microinjected Ca Green dextran, a visible wavelength fluorescent Ca indicator, into eggs or two cell stages of the ctenophore Mnemiopsis leidyi. The embryos developed normally into free-swimming, approximately 0.5 mm cydippid larvae with cells and ciliary comb plates (approximately 100 microns long) loaded with the dye. Comb plates of larvae, like those of adult ctenophores, undergo spontaneous or electrically stimulated reversal of beat direction, triggered by Ca influx through voltage-sensitive Ca channels. Comb plates of larvae loaded with Ca Green dextran emit spontaneous or electrically stimulated fluorescent flashes along the entire length of their cilia, correlated with ciliary reversal. Fluorescence intensity peaks rapidly (34-50 ms), then slowly falls to resting level in approximately 1 s. Electrically stimulated Ca Green emissions often increase in steps to a maximum value near the end of the stimulus pulse train, and slowly decline in 1-2 s. In both spontaneous and electrically stimulated flashes, measurements at multiple sites along a single comb plate show that Ca Green fluorescence rises within 17 ms (1 video field) and to a similar relative extent above resting level from base to tip of the cilia. The decline of fluorescence intensity also begins simultaneously and proceeds at similar rates along the ciliary length. Ca-free sea water reversibly abolishes spontaneous and electrically stimulated Ca Green ciliary emissions as well as reversed beating. Calculations of Ca diffusion from the ciliary base show that Ca must enter the comb plate along the entire length of the ciliary membranes. The voltage-dependent Ca channels mediating changes in beat direction are therefore distributed over the length of the comb plate cilia. The observed rapid and virtually instantaneous Ca signal throughout the intraciliary space may be necessary for reprogramming the pattern of dynein activity responsible for reorientation of the ciliary beat cycle.