Pattern formation in embryos of the oligochaete annelid Tubifex: cellular basis for segmentation and specification of segmental identity

The embryonic origin of metameric segmentation was examined in the oligochaete Tubifex using lineage tracers. Segments in Tubifex embryos arise from five bilateral pairs of longitudinal coherent columns (bandlets) of primary blast cells which are generated by five bilateral pairs of embryonic stem cells called teloblasts (M, N, O, P and Q). As development proceeds, an initially linear array of blast cells in each ectodermal bandlet gradually changes its shape in a lineage-specific manner. These morphogenetic changes result in the formation of distinct cell clumps, which are separated from the bandlet to serve as segmental elements (SEs). SEs in the N and Q lineages are each comprised of clones of two consecutive primary blast cells. In contrast, in the O and P lineages, individual blast cell clones are distributed across SE boundaries; each SE is a mixture of a part of the preceding anterior clone and a part of the next posterior clone. Morphogenetic events, including segmentation, in an ectodermal bandlet proceed normally in the absence of neighboring ectodermal bandlets. Without the underlying mesoderm, separated SEs fail to space themselves at regular intervals along the anteroposterior axis. It is suggested that ectodermal segmentation in Tubifex consists of two stages; autonomous morphogenesis of each bandlet leading to generation of SEs, and the ensuing mesoderm-dependent alignment of separated SEs. In contrast, metameric segmentation in the mesoderm (M lineage) is a one-step process in that it arises from an initially simple organization (i.e. a linear series) of primary m-blast cells, which individually serve as a founder cell of each segment. The boundary between mesodermal segments is determined autonomously. The results of a set of cell ablation and transplantation experiments, using alkaline phosphatase activity as a biochemical marker for segments VII and VIII suggest that segmental identities in primary m-blast cells are determined according to the genealogical position in the M lineage and that the M teloblast possesses a developmental program through which the sequence of blast cell identities is determined.     

Embryonic expression of a decapentaplegic gene in the oligochaete annelid Tubifex tubifex

We have cloned and characterized the expression of a decapentaplegic homologue (designated Ttu-dpp) from the oligochaete annelid Tubifex tubifex. RT-PCR analysis and in situ hybridization revealed that Ttu-dpp begins to be expressed around the time of the onset of ectodermal germ band (GB) elongation (i.e., the onset of gastrulation). At this time, Ttu-dpp expression is detected in the anteriormost part of the GBs. As development proceeds and the GBs elongate, the domain of Ttu-dpp-expressing cells extends posteriorly. Then Ttu-dpp-expressing cells within the GB are divided into two groups: one group occurs along the ventral midline and coincides with the domain of ventral ganglia; the other is located more dorsally. The latter group of Ttu-dpp-expressing cells subsequently undergoes dorsalward expansion, which results in the formation of a lateral stripe of cells in every segment except the first (i.e., segment I). In embryos that undergo body elongation (that is one of the last morphogenetic movements occurring prior to hatchout), Ttu-dpp expression in the lateral region is confined to setal sacs, which are arranged in the same transverse plane around the periphery of each segment (except segment I).     

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.     

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.     

Generation of bilateral symmetry in Anthozoa: a model

Polyps of Anthozoa usually display bilateral symmetry with respect to their mouth opening, to their pharynx, and in particular to the arrangement of their mesenteries. Mesenteries, which are endodermal folds running from the apical to the basal end of the body, subdivide the gastric cavity into pouches. They form in a bilateral symmetric sequence. In this article I propose that early in polyp development the endoderm subdivides successively into three different types of compartments. A mesentery forms at the border between compartments. Two of the compartments are homologous to those of Scyphozoa. They form by mutual activation of cell states that locally exclude each other. The third compartment leads to siphonoglyph formation and is an evolutionary innovation of the Anthozoa. The mechanism that controls the number and spatial arrangement of the third type of compartment changes the radial symmetry into a bilateral one and occasionally into a different one. The dynamics of its formation indicate an activator-inhibitor mechanism. Computer models are provided that reproduce decision steps in the generation of the mesenteries.     

The existence of all three ParaHox genes in the clitellate annelid, Perionyx excavatus

A ParaHox gene cluster is composed of three genes (Gsx, Xlox, and Cdx). It has been proposed that all three ParaHox genes were present in the last common ancestor to the lophotrochozoan protostomes and the deuterostomes and that gene loss event has occurred in the ecdysozoan lineage. In this paper, we report the existence of all three ParaHox genes in Perionyx excavatus, a clitellate annelid. Although orthologs of each of the three ParaHox genes were previously discovered from other lopotrochozoan taxa, this study constitutes the first reported isolation of all three ParaHox genes in the same clitellate species.Bum Joon Park and Sung-Jin Cho contributed equally to this work.     

The protein kinase kin1 is required for cellular symmetry in fission yeast

The fission yeast Schizosaccharomyces pombe is a highly polarized unicellular eukaryote with two opposite growing poles in which F-actin cytoskeleton is focused. The KIN1/PAR-1/MARK protein family is composed of conserved eukaryotic serine/threonine kinases which are involved in cell polarity, microtubule stability or cell cycle regulation. Here, we investigate the function of the fission yeast KIN1/PAR-1/MARK member, kin1p. Using a deletion allele (kin1Delta), we show that kin1 mutation promotes a delay in septation. Kin1p regulates the structure of the new cell end after cytokinesis by modulating cell wall remodeling. Abnormal shaped interphase kin1Delta cells misplace F-actin patches and the premitotic nucleus. Thus, mitotic kin1Delta cells misposition the F-actin ring assembly site that is dependent on the position of the interphase nucleus. The resulting asymmetric cell division produces daughter cells with distinct shapes. Overexpressed kin1p accumulates asymmetrically at the cell cortex and affects cell shape, F-actin organization and microtubules. Our results suggest that correct dosage of kin1p at the cortex is required for spatial organization of the fission yeast cell.     

Asymmetrization of first cleavage by transient disassembly of one spindle pole aster in the leech Helobdella robusta

Unequal first cleavage is characteristic of a diverse group of protostome animals. In the nematode Caenorhabditis elegans, unequal first cleavage is achieved through the interaction of an apparently symmetric mitotic spindle apparatus with a clearly polarized cell cortex. In the clitellate annelid Tubifex tubifex, by contrast, the spindle is monastral and contains only one gamma-tubulin-reactive centrosome; this monastral spindle is inherently asymmetric throughout mitosis. Here, we have used immunostaining for beta- and gamma-tubulin to follow spindle dynamics during the unequal first cleavage in another clitellate annelid, the leech Helobdella robusta. We find that the mitotic spindle is diastral and symmetric through early metaphase, then becomes asymmetric following the transient down-regulation of one centrosome, as judged by gamma-tubulin immunofluorescence. Low levels of drugs that affect microtubule dynamics can symmetrize the first cleavage without affecting the gamma-tubulin dynamics. Our results provide a striking example of the evolvability of cellular mechanisms underlying an unambiguously homologous developmental process.     

Grandparental stem cells in leech segmentation: Differences in CDC42 expression are correlated with an alternating pattern of blast cell fates

Embryonic segmentation in clitellate annelids (oligochaetes and leeches) is a cell lineage-driven process. Embryos of these worms generate a posterior growth zone consisting of 5 bilateral pairs of identified segmentation stem cells (teloblasts), each of which produces a column of segmental founder cells (blast cells). Each blast cell generates a lineage-specific clone via a stereotyped sequence of cell divisions, which are typically unequal both in terms of the relative size of the sister cells and in the progeny to which they give rise. In two of the five teloblast lineages, including the ventralmost, primary neurogenic (N) lineage, the blast cells adopt two different fates, designated nf and ns, in exact alternation within the blast cell column; this is termed a grandparental stem cell lineage. To lay groundwork for investigating unequal divisions in the leech Helobdella, we have surveyed the Helobdella robusta genome for genes encoding orthologs of the Rho family GTPases, including the rho, rac and cdc42 sub-families, which are known to be involved in multiple processes involving cell polarization in other systems. We find that, in contrast to most other known systems the Helobdella genome contains two cdc42 orthologs, one of which is expressed at higher levels in the ns blast cells than in nf blast cells. We also demonstrate that the asymmetric divisions of the primary nf and ns blast cells are regulated by the polarized distribution of the activated form of the Cdc42 protein, rather than by the overall level of expression. Our results provide the first molecular insights into the mechanisms of the grandparental stem cell lineages, a novel, yet evolutionarily ancient stem cell division pattern. Our results also provide an example in which asymmetries in the distribution of Cdc42 activity, rather than in the overall levels of Cdc42 protein, are important regulating unequal divisions in animal cells.     

C. elegans HAM-1 positions the cleavage plane and regulates apoptosis in asymmetric neuroblast divisions

Asymmetric cell division occurs when a mother cell divides to generate two distinct daughter cells, a process that promotes the generation of cellular diversity in metazoans. During Caenorhabditis elegans development, the asymmetric divisions of neural progenitors generate neurons, neural support cells and apoptotic cells. C. elegans HAM-1 is an asymmetrically distributed cortical protein that regulates several of these asymmetric neuroblast divisions. Here, we show that HAM-1 is a novel protein and define residues important for HAM-1 function and distribution to the cell cortex. Our phenotypic analysis of ham-1 mutant embryos suggests that HAM-1 controls only neuroblast divisions that produce apoptotic cells. Moreover, ham-1 mutant embryos contain many unusually large cell-death corpses. An investigation of this corpse phenotype revealed that it results from a reversal of neuroblast polarity. A misplacement of the neuroblast cleavage plane generates daughter cells of abnormal size, with the apoptotic daughters larger than normal. Thus, HAM-1 regulates the position of the cleavage plane, apoptosis and mitotic potential in C. elegans asymmetric cell divisions.     

Origins of radial symmetry identified in an echinoderm during adult development and the inferred axes of ancestral bilateral symmetry

How the radial body plan of echinoderms is related to the bilateral body plan of their deuterostome relatives, the hemichordates and the chordates, has been a long-standing problem. Now, using direct development in a sea urchin, I show that the first radially arranged structures, the five primary podia, form from a dorsal and a ventral hydrocoele at the oral end of the archenteron. There is a bilateral plane of symmetry through the podia, the mouth, the archenteron and the blastopore. This adult bilateral plane is thus homologous with the bilateral plane of bilateral metazoans and a relationship between the radial and bilateral body plans is identified. I conclude that echinoderms retain and use the bilateral patterning genes of the common deuterostome ancestor. Homologies with the early echinoderms of the Cambrian era and between the dorsal hydrocoele, the chordate notochord and the proboscis coelom of hemichordates become evident.     

On the roles of Notch, Delta, kuzbanian, and inscuteable during the development of Drosophila embryonic neuroblast lineages

The generation of cellular diversity in the nervous system involves the mechanism of asymmetric cell division. Besides an array of molecules, including the Par protein cassette, a heterotrimeric G protein signalling complex, Inscuteable plays a major role in controlling asymmetric cell division, which ultimately leads to differential activation of the Notch signalling pathway and correct specification of the two daughter cells. In this context, Notch is required to be active in one sibling and inactive in the other. Here, we investigated the requirement of genes previously known to play key roles in sibling cell fate specification such as members of the Notch signalling pathway, e.g., Notch (N), Delta (Dl), and kuzbanian (kuz) and a crucial regulator of asymmetric cell division, inscuteable (insc) throughout lineage progression of 4 neuroblasts (NB1-1, MP2, NB4-2, and NB7-1). Notch-mediated cell fate specification defects were cell-autonomous and were observed in all neuroblast lineages even in cells born from late ganglion mother cells (GMC) within the lineages. We also show that Dl functions non-autonomously during NB lineage progression and clonal cells do not require Dl from within the clone. This suggests that within a NB lineage Dl is dispensable for sibling cell fate specification. Furthermore, we provide evidence that kuz is involved in sibling cell fate specification in the central nervous system. It is cell-autonomously required in the same postmitotic cells which also depend on Notch function. This indicates that KUZ is required to facilitate a functional Notch signal in the Notch-dependent cell for correct cell fate specification. Finally, we show that three neuroblast lineages (NB1-1, NB4-2, and NB7-1) require insc function for sibling cell fate specification in cells born from early GMCs whereas insc is not required in cells born from later GMCs of the same lineages. Thus, there is differential requirement for insc for cell fate specification depending on the stage of lineage progression of NBs.     

Growth and cellular patterns in the petal epidermis of Antirrhinum majus: empirical studies

Background and Aims

Analysis of cellular patterns in plant organs provides information about the orientation of cell divisions and predominant growth directions. Such an approach was employed in the present study in order to characterize growth of the asymmetrical wild-type dorsal petal and the symmetrical dorsalized petal of the backpetals mutant in Antirrhinum majus. The aims were to determine how growth in an initially symmetrical petal primordium leads to the development of mature petals differing in their symmetry, and to determine how specific cellular patterns in the petal epidermis are formed.

Methods

Cellular patterns in the epidermis in both petal types over consecutive developmental stages were visualized and characterized quantitatively in terms of cell wall orientation and predominant types of four-cell packets. The data obtained were interpreted in terms of principal directions of growth (PDGs).

Key Results

Both petal types grew predominantly along the proximo-distal axis. Anticlinal cell walls in the epidermis exhibited a characteristic fountain-like pattern that was only slightly modified in time. New cell walls were mostly perpendicular to PDG trajectories, but this alignment could change with wall age.

Conclusions

The results indicate that the predominant orientation of cell division planes and the fountain-like cellular pattern observed in both petal types may be related to PDGs. The difference in symmetry between the two petal types arises because PDG trajectories in the field of growth rates (growth field) controlling petal growth undergo gradual redefinition. This redefinition probably takes place in both petal types but only in the wild-type does it eventually lead to asymmetry in the growth field. Two scenarios of how redefinition of PDGs may contribute to this asymmetry are considered.     

Cytoskeletal dynamics ofApedinella radians (Pedinellophyceae) II. Cell division and maintenance of cell polarity and symmetry

Summary The dynamics of the cytoskeletal proteins centrin, actin, and tubulin were followed during cell division in the unicellular phytoflagellateApedinella radians (Pedinellophyceae). Three centrin, or centrin-like, components appear to coordinate independent developmental events during cell division. The first component, basal body centrin, maintains a physical link between basal bodies and the anterior nuclear membrane. Basal body centrin divides in two at metaphase, and each portion segregates with two basal bodies at anaphase. As the positioning of basal bodies defines the anterior region of the cell, basal body centrin appears to play a role in maintaining cell polarity throughout the cell cycle. The second centrin component consists of an array of filamentous bundles arranged as a six-pointed star. During cell division, the star undergoes a conformational change resulting in two distinct centrin triangles, one distributed to each daughter cell, suggesting that centrin filamentous bundles are involved in maintaining cell (radial) symmetry. The third centrin component is transient and associates with the spindle poles, emerging prior to mitosis and remaining until late anaphase/early telophase. Spindle pole centrin establishes temporary horizontal bipolarity, thereby establishing the spindle axis. Unlike centrin filamentous bundles, actin filamentous bundles depolymerize prior to mitosis, indicating they do not influence cell symmetry during cell division. Mitosis is described for the first time in a pedinellid and features a closed spindle, the absence of rhizoplasts and a persistent spindle.     

A novel noncanonical Wnt pathway is involved in the regulation of the asymmetric B cell division in C. elegans

The polarities of several cells that divide asymmetrically during Caenorhabditis elegans development are controlled by Wnt signaling. LIN-44/Wnt and LIN-17/Fz control the polarities of cells in the tail of developing C. elegans larvae, including the male-specific blast cell, B, that divides asymmetrically to generate a larger anterior daughter and a smaller posterior daughter. We determined that WRM-1 and the major canonical Wnt pathway components: BAR-1, SGG-1/GSK-3 and PRY-1/Axin were not involved in the control of B cell polarity. However, POP-1/Tcf is involved and is asymmetrically distributed to the B daughter nuclei, as it is in many cell divisions during C. elegans development. Aspects of the B cell division are reminiscent of the divisions controlled by the planar cell polarity (PCP) pathway that has been described in both Drosophila and vertebrate systems. We identified C. elegans homologs of Wnt/PCP signaling components and have determined that many of them appear to be involved in the regulation of B cell polarity. Specifically, MIG-5/Dsh, RHO-1/RhoA and LET-502/ROCK appear to play major roles, while other PCP components appear to play minor roles. We conclude that a noncanonical Wnt pathway, which is different from other Wnt pathways in C. elegans, regulates B cell polarity.     

Expression pattern of annelid <Emphasis Type="Italic">Zic</Emphasis> in embryonic development of the oligochaete <Emphasis Type="Italic">Tubifex tubifex</Emphasis>

Embryonic expression of a Zic homologue (Ttu-Zic) was examined in the oligochaete annelid Tubifex tubifex. The body plan of T. tubifex is characterized by obvious segmentation in the ectoderm and mesoderm. Ttu-Zic expression is detected in the mesodermal germ band and a subset of micromere descendants. Ttu-Zic is transiently expressed in primary m-blast cells (i.e., founder cells of mesodermal segments) as early as the time of their birth from M teloblasts. During its development, each mesodermal segment experiences two additional phases of Ttu-Zic expression. Ttu-Zic expression in micromere descendants is seen on the anterior surfaces of embryos undergoing teloblastogenesis; subsequently, these cells proliferate to form bilateral clusters, which then become internalized. Finally, clusters of Ttu-Zic-expressing cells are found in the center of the prostomium, corresponding to the cerebral ganglion. The Ttu-Zic expression profile in the early embryogenesis of T. tubifex may be homologous to those of evolutionarily distant animals.