Specification of ectodermal teloblast lineages in embryos of the oligochaete annelid Tubifex: involvement of novel cell-cell interactions

In embryos of clitellate annelids (i.e. oligochaetes and leeches), four ectodermal teloblasts (ectoteloblasts N, O, P and Q) are generated on either side through a stereotyped sequence of cell divisions of a proteloblast, NOPQ. The four ectoteloblasts assume distinct fates and produce bandlets of smaller progeny cells, which join together to form an ectodermal germ band. The pattern of the germ band, with respect to the ventrodorsal order of the bandlets, has been highly preserved in clitellate annelids. We show that specification of ectoteloblast lineages in the oligochaete annelid Tubifex involves cell interaction networks distinct from those in leeches. Cell ablation experiments have shown that fates of teloblasts N, P and Q in Tubifex embryos are determined rigidly as early as their birth. In contrast, the O teloblast and its progeny are initially pluripotent and their fate becomes restricted to the O fate through an inductive signal emanating from the P lineage. In the absence of this signal, the O lineage assumes the P fate. These results differ significantly from those obtained in embryos of the leech Helobdella, suggesting the diversity of patterning mechanisms that give rise to germ bands with similar morphological pattern.     

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.     

Generation of Bilateral Symmetry in the Ectoderm of the Tubifex Embryo: Involvement of Cell–cell Interactions

In embryos of the oligochaete annelid Tubifex, most ectodermal tissues are derived from four bilateral pairs of embryonic stem cells called teloblasts (ectoteloblasts N, O, P and Q). Ectoteloblasts are generated on both left and right sides of the embryo through an invariable sequence of cell divisions of a proteloblast, NOPQ, and they are positioned in a mirror symmetric pattern relative to the embryonic midline. This mirror symmetry of ectoteloblast arrangement gives rise to the generation of bilateral symmetry in the ectoderm. Here we review results of our recent experiments on Tubifex tubifex that were designed to gain an insight into the mechanisms underlying the generation of the bilaterally symmetric organization of ectoteloblasts. Cell transplantation experiments have shown that nascent NOPQ cells can be polarized according to positional information residing in the embryo. If a left NOPQ cell is transplanted to the right side of a host embryo, it exhibits polarity comparable to that of right NOPQ cells. It has also been shown that contact between NOPQ cells serves as an external cue for their polarization. Another series of cell transplantation experiments have suggested that the competence of NOPQ cells to respond to external cues becomes undetectable shortly before the production of the first teloblast (N) from the NOPQ cell. Another series of experiments utilizing cell ablation techniques have shown that teloblasts N, P and Q are specified to express the N, P and Q fates, respectively, as early as their birth. In contrast, the O teloblast and its progeny are initially pluripotent and their fate becomes restricted through inductive signals emanating from its sister P lineage. On the basis of these findings, we have proposed a model for polarization of ectodermal teloblastogenesis in the Tubifex embryo.     

Microdissection Studies on the Polarity of Unequal Division in Grasshopper Neuroblasts: II. Cell Division in Binucleate Neuroblasts

The grasshopper neuroblast divides unequally to produce two types of cells: a large daughter neuroblast that contains a doughnut-shaped nucleus and repeats unequal division with definite polarity, and a small daughter ganglion cell that has a spherical nucleus with low mitotic activity. Binucleate neuroblasts were induced by preventing cytokinesis in the course of microdissection experiments, and subsequent divisions were traced to analyze the factors that determine the polarity of unequal division.
In binucleate neuroblasts, both daughter chromosome groups developed into neuroblast-type nuclei. Mitosis of the two nuclei proceeded synchronously. Although the axes of the two mitotic apparatuses formed at late prophase were random in direction, they became parallel with the original division axis at metaphase. The two mitotic apparatuses shifted simultaneously toward the ganglion cell side during anaphase, just as in normal neuroblasts, and the binucleate cell divided unequally. These findings showed that the poearity of unequal division is strictly maintained in grasshpper neuroblasts, even when they contain two nuclei.     

Cell lineage studies in the crayfish Cherax destructor (Crustacea,Decapoda) : germ band formation,segmentation, and early neurogenesis

Summary The cell division pattern of the germ band of Cherax destructor is described from gastrulation to segmentation, limb bud formation, and early neurogenesis. The naupliar segments are formed almost simultaneously from scattered ectoderm cells arranged in a V-shaped germ disc, anterior to the blastopore. No specific cell division pattern is recognisable. The post-naupliar segments are formed successively from front to rear. Most post-naupliar material is budded by a ring of about 39 to 46 ectoteloblasts, which are differentiated successively and in situ in front of the telson ectoderm. The ectoteloblasts give rise to 15 descendant cell rows by unequal divisions in an anterior direction, following a mediolateral mitotic wave. Scattered blastoderm cells of non-ectoteloblastic origin in front of the ectoteloblast descendants and behind the mandibular region are also arranged in rows. Despite their different origins, teloblastic and non-teloblastic rows cleave twice by mediolateral mitotic waves to form 4 regular descendant rows each. Thereafter, the resulting grid-like pattern is dissolved by stereotyped differential cleavages. Neuroblasts are formed during these differential cleavages and segmentation becomes visible. Each ectoderm row represents a parasegmental unit. Therefore, the segmental boundary lies within the area covered by the descendants of 1 row. Segmental structures (limbs, ganglia) are composed of derivatives of 2 ectoderm rows. The results are compared with the early development of other crustaceans and insects in relation to mechanisms of germ band formation, segmentation, neurogenesis, and evolution.     

Role of intercellular contacts in generating an asymmetric mitotic apparatus in the Tubifex embryo

The 2-cell stage embryo of Tubifex is composed of a smaller cell, AB, and a larger cell, CD. At the second cleavage, the CD-cell divides unequally. The mitotic apparatus (MA) involved in this division is organized asymmetrically: the MA pole to be segregated to a smaller cell is flattened and truncated, and associated with the anterior cortex facing the AB-cell, while the other pole is symmetric and located more centrally. The present study was undertaken to elucidate the mechanism that generates asymmetry in the MA organization in CD-cells. When CD-cell nuclei, which are normally located near the anterior cortex, were displaced toward the posterior end of the cell (i.e. opposite AB-cells) by centrifugation, MA assembled ectopically there, and were bilaterally symmetric in organization. Similar symmetric MA were formed in isolated CD-cells, which divided more equally than intact cells. This equality of cell division was dramatically reduced if the anterior surface of isolated CD-cells formed contact with other cells, such as AB-, C- and 4D-cells. The MA that formed in these reconstituted embryos were asymmetric in organization; one MA pole was always found to be truncated and apposed to the cortical site at the cell contact. Symmetric MA were also observed in cytochalasin-treated embryos. Together with the finding that one of the MA poles is physically attached to the anterior cortex of the intact CD-cell, these results suggest that factors generating asymmetry in the spatial organization of MA poles reside at the anterior cortex of the CD-cell and that this cortical mechanism is dependent upon cell contacts.     

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.     

Unequal cell division and chromatin differentiation in pollen grain cells

Pinus pollen grains, normally developing, were subjected to centrifugal force, low temperature and caffeine solution. In the former two treatments, daughter cells with some abnormal directions of division, abnormal volume and chromatin dispersion were induced in pollen grains treated. Regardless of the direction of division, of the two daughter cells produced by the unequal division, the larger one contained strongly dispersed chromatin and the smaller one weakly dispersed chromatin. In the two daughter cells produced by approximately equal division, the chromatin was dispersed strongly to a similar degree, and by halfway unequal division, chromatin in the larger cell was dispersed strongly and in the smaller one intermediately. Chromatin in bi-nucleate cells induced by caffeine treatment was dispersed strongly to an identical degree. It is suggested that for the occurrence of heteronomous chromatin configuration in natural pollen grains the unequal cell division was indispensable, although the axis of division didn't directly contribute. After both the treatments of centrifugation and low temperature during microspore and embryonal cell divisions, the affected daughter cells divided in terms of the certain fixed axis of division and chromatin dispersion, instead of exhibiting abnormal development.     

D quadrant specification in the leech Helobdella: Actomyosin contractility controls the unequal cleavage of the CD blastomere

The unequal division of the CD blastomere at second cleavage is critical in establishing the second embryonic axis in the leech Helobdella, as in other unequally cleaving spiralians. When CD divides, the larger D and smaller C blastomeres arise invariantly on the left and right sides of the embryo, respectively. Here we show that stereotyped cellular dynamics, including the formation of an intercellular blastocoel, culminate in a morphological left-right asymmetry in the 2-cell embryo, which precedes cytokinesis and predicts the chirality of the second cleavage. In contrast to the unequal first cleavage, the unequal second cleavage does not result from down-regulation of one centrosome, nor from an asymmetry within the spindle itself. Instead, the unequal cleavage of the CD cell entails a symmetric mitotic apparatus moving and anisotropically growing rightward in an actomyosin-dependent process. Our data reveal that mechanisms controlling the establishment of the D quadrant differ fundamentally even among the monophyletic clitellate annelids. Thus, while the homologous spiral cleavage pattern is highly conserved in this clade, it has diverged significantly at the level of cell biological mechanisms. This combination of operational conservation and mechanistic divergence begins to explain how the spiral cleavage program has remained so refractory to change while, paradoxically, accommodating numerous modifications throughout evolution.     

Binary cell death decision regulated by unequal partitioning of Numb at mitosis

An important issue in Metazoan development is to understand the mechanisms that lead to stereotyped patterns of programmed cell death. In particular, cells programmed to die may arise from asymmetric cell divisions. The mechanisms underlying such binary cell death decisions are unknown. We describe here a Drosophila sensory organ lineage that generates a single multidentritic neuron in the embryo. This lineage involves two asymmetric divisions. Following each division, one of the two daughter cells expresses the pro-apoptotic genes reaper and grim and subsequently dies. The protein Numb appears to be specifically inherited by the daughter cell that does not die. Numb is necessary and sufficient to prevent apoptosis in this lineage. Conversely, activated Notch is sufficient to trigger death in this lineage. These results show that binary cell death decision can be regulated by the unequal segregation of Numb at mitosis. Our study also indicates that regulation of programmed cell death modulates the final pattern of sensory organs in a segment-specific manner.     

Long-Term Live Cell Imaging and Automated 4D Analysis of Drosophila Neuroblast Lineages

The developing Drosophila brain is a well-studied model system for neurogenesis and stem cell biology. In the Drosophila central brain, around 200 neural stem cells called neuroblasts undergo repeated rounds of asymmetric cell division. These divisions typically generate a larger self-renewing neuroblast and a smaller ganglion mother cell that undergoes one terminal division to create two differentiating neurons. Although single mitotic divisions of neuroblasts can easily be imaged in real time, the lack of long term imaging procedures has limited the use of neuroblast live imaging for lineage analysis. Here we describe a method that allows live imaging of cultured Drosophila neuroblasts over multiple cell cycles for up to 24 hours. We describe a 4D image analysis protocol that can be used to extract cell cycle times and growth rates from the resulting movies in an automated manner. We use it to perform lineage analysis in type II neuroblasts where clonal analysis has indicated the presence of a transit-amplifying population that potentiates the number of neurons. Indeed, our experiments verify type II lineages and provide quantitative parameters for all cell types in those lineages. As defects in type II neuroblast lineages can result in brain tumor formation, our lineage analysis method will allow more detailed and quantitative analysis of tumorigenesis and asymmetric cell division in the Drosophila brain.     

Embryonic cell lineages in the nervous system of the Glossiphoniid leech Helobdella triserialis

The lines of descent of cells of the nervous system of the leech Helobdella triserialis have been ascertained by injection of horseradish peroxidase (HRP) as a tracer into identified cells of early embryos. Such experiments show that the nervous system of the leech has several discrete embryological origins. Some of the neurons on one side of each of the segmental ganglia derive from a single cell, the ipsilateral N ectoteloblast. Other neurons derive from a different precursor cell, the ipsilateral OPQ cell that gives rise to the O, P, and Q ectoteloblasts. The positions within the ganglion of neuronal populations derived from each of these sources are relatively invariant from segment to segment and from specimen to specimen. Other nerve cord cells derive from the mesoteloblast M; of these four per segment appear to be the precursors of the muscle cells of the connective. The A, B, or C macromeres contribute cells to the supraesophageal ganglion. In preparations in which an N ectoteloblast was injected with HRP after production of its bandlet of n stem cells had begun, the boundary between unstained (rostral) and stained (caudal) tissues can fall within a ganglion or between ganglia. This suggests that each hemiganglion contains the descendants of more than one, and probably two, n stem cells.     

Drosophila E-cadherin regulates the orientation of asymmetric cell division in the sensory organ lineage

BACKGROUND: Generation of cell-fate diversity in Metazoan depends in part on asymmetric cell divisions in which cell-fate determinants are asymmetrically distributed in the mother cell and unequally partitioned between daughter cells. The polarization of the mother cell is a prerequisite to the unequal segregation of cell-fate determinants. In the Drosophila bristle lineage, two distinct mechanisms are known to define the axis of polarity of the pI and pIIb cells. Frizzled (Fz) signaling regulates the planar orientation of the pI division, while Inscuteable (Insc) directs the apical-basal polarity of the pIIb cell. The orientation of the asymmetric division of the pIIa cell is identical to the one of its mother cell, the pI cell, but, in contrast, is regulated by an unknown Insc- and Fz-independent mechanism. RESULTS: DE-Cadherin-Catenin complexes are shown to localize at the cell contact between the two cells born from the asymmetric division of the pI cell. The mitotic spindle of the dividing pIIa cell rotates to line up with asymmetrically localized DE-Cadherin-Catenin complexes. While a complete loss of DE-Cadherin function disrupts the apical-basal polarity of the epithelium, both a partial loss of DE-Cadherin function and expression of a dominant-negative form of DE-Cadherin affect the orientation of the pIIa division. Furthermore, expression of dominant-negative DE-Cadherin also affects the position of Partner of Inscuteable (Pins) and Bazooka, two asymmetrically localized proteins known to regulate cell polarity. These results show that asymmetrically distributed Cad regulates the orientation of asymmetric cell division. CONCLUSIONS: We describe a novel mechanism involving a specialized Cad-containing cortical region by which a daughter cell divides with the same orientation as its mother cell.     

Daughter cells of Saccharomyces cerevisiae from old mothers display a reduced life span

The yeast Saccharomyces cerevisiae typically divides asymmetrically to give a large mother cell and a smaller daughter cell. As mother cells become old, they enlarge and produce daughter cells that are larger than daughters derived from young mother cells. We found that occasional daughter cells were indistinguishable in size from their mothers, giving rise to a symmetric division. The frequency of symmetric divisions became greater as mother cells aged and reached a maximum occurrence of 30% in mothers undergoing their last cell division. Symmetric divisions occurred similarly in rad9 and ste12 mutants. Strikingly, daughters from old mothers, whether they arose from symmetric divisions or not, displayed reduced life spans relative to daughters from young mothers. Because daughters from old mothers were larger than daughters from young mothers, we investigated whether an increased size per se shortened life span and found that it did not. These findings are consistent with a model for aging that invokes a senescence substance which accumulates in old mother cells and is inherited by their daughters.     

Mutations in the Caenorhabditis elegans Gene vab-3 Reveal Distinct Roles in Fate Specification and Unequal Cytokinesis in an Asymmetric Cell Division

Asymmetric cell divisions in which a precursor cell distributes fate potential unequally between the two daughter cells represent one of the major mechanisms for fate specification during development. Such mechanisms suggest at least two distinct cellular activities: factors that act to establish asymmetry in the precursor cell and factors that are distributed or activated unequally and function to make the daughter cells different from each other. In Caenorhabditis elegans , cytokinesis of the first division of the male-specific postembryonic blast cell B is unequal, and the two daughters adopt different fates. Others have observed that the genes lin-17 and lin-44 are required, respectively, to establish and to orient this asymmetric division. Mutations in lin-17 and lin-44 coordinately disrupt cytokinesis and fate specification. We describe the function of the gene vab-3 in the B cell lineage. Mutations in vab-3 disrupt the fate of the anterior daughter of B, B.a. However, unlike lin-17 and lin-44 , mutations in vab-3 can disrupt fate without the corresponding disruption of unequal cytokinesis. Analysis of lin-17; vab-3 double mutants suggests that vab-3 acts after lin-17 for B.a fate specification. Double mutant analysis has also identified additional functions of lin-17 in the B lineage subsequent to this first division.     

Ephrin-mediated restriction of ERK1/2 activity delimits the number of pigment cells in the Ciona CNS

Recent evidence suggests that ascidian pigment cells are related to neural crest-derived melanocytes of vertebrates. Using live-imaging, we determine a revised cell lineage of the pigment cells in Ciona intestinalis embryos. The neural precursors undergo successive rounds of anterior–posterior (A–P) oriented cell divisions, starting at the blastula 64-cell stage. A previously unrecognized fourth A–P oriented cell division in the pigment cell lineage leads to the generation of the post-mitotic pigment cell precursors. We provide evidence that MEK/ERK signals are required for pigment cell specification until approximately 30 min after the final cell division has taken place. Following each of the four A–P oriented cell divisions, ERK1/2 is differentially activated in the posterior sister cells, into which the pigment cell lineage segregates. Eph/ephrin signals are critical during the third A–P oriented cell division to spatially restrict ERK1/2 activation to the posterior daughter cell. Targeted inhibition of Eph/ephrin signals results in, at neurula stages, anterior expansion of both ERK1/2 activation and a pigment cell lineage marker and subsequently, at larval stages, supernumerary pigment cells. We discuss the implications of these findings with respect to the evolution of the vertebrate neural crest.     

Roles for polarity and nuclear determinants in specifying daughter cell fates after an asymmetric cell division in the maize leaf

Asymmetric cell divisions occur repeatedly during plant development, but the mechanisms by which daughter cells are directed to adopt different fates are not well understood [1,2]. Previous studies have demonstrated roles for positional information in specification of daughter cell fates following asymmetric divisions in the embryo [3] and root [4]. Unequally inherited cytoplasmic determinants have also been proposed to specify daughter cell fates after some asymmetric cell divisions in plants [1,2,5], but direct evidence is lacking. Here we investigate the requirements for specification of stomatal subsidiary cell fate in the maize leaf by analyzing four mutants disrupting the asymmetric divisions of subsidiary mother cells (SMCs). We show that subsidiary cell fate does not depend on proper localization of the new cell wall during the SMC division, and is not specified by positional information acting on daughter cells after completion of the division. Instead, our data suggest that specification of subsidiary cell fate depends on polarization of SMCs and on inheritance of the appropriate daughter nucleus. We thus provide evidence of a role for unequal inheritance of an intracellular determinant in specification of cell fate after an asymmetric plant cell division.