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.     

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).     

Analyses of segment-specific expression of alkaline phosphatase activity in the mesoderm of the oligochaete annelid Tubifex: implications for specification of segmental identity

In the embryos of the oligochaete annelid Tubifex, segments VII and VIII specifically express mesodermal alkaline phophatase (ALP) activity in the ventrolateral region. In this study, we examined whether this segment-specific expression of ALP activity depends on external cues. Cell lineage analyses show that the ALP-expressing cells originate from M teloblasts. Furthermore, a set of teloblast-ablation experiments demonstrated that the seventh and eighth primary m blast cells (m7 and m8) produced from M teloblasts give rise to ALP-expressing cells in segments VII and VIII, respectively, and that primary m blast cells other than m7 and m8 lack the ability to generate ALP-expressing progeny cells. The results of another set of blastomere-ablation experiments suggest that ALP-expressing cells emerge independently of interactions with surrounding tissues. Teloblast-transplantation experiments demonstrated that m8 can generate ALP-expressing cells in an ectopical position, suggesting that it is unlikely that ALP activity emerges in response to the positional cues residing in the embryo. These results suggest that m7 and m8 are exclusively specified as precursors of ALP-expressing cells at the time of their birth from M teloblasts. We propose that segmental identities in primary m blast cells of the Tubifex embryo 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.     

Selective feeding by the aquatic oligochaete Tubifex tubifex (Tubificidae,Clitellata)

The particle size distribution of faecal pellets produced by the tubificid worm Tubifex tubifex in laboratory culture, was measured with a Coulter® Multisizer. The faecal material from worms cultured in a range of sediments was composed of particles with a mean diameter of less than 63 m, and only a few isolated larger particles were found by microscopic analysis. This suggests that this species actively selects the silt-clay fraction, avoiding larger sand particles. A more detailed analysis of faeces revealed that about 75%, by volume, was composed of particles with a mean diameter < 25m, and the mode was < 10m. T. tubifex fed selectively on the organic rich particles of the sediment, and this feeding was independent of particle size. Measurement of the organic content of faeces (measured as % loss on ignition) showed that they had a consistently higher organic content than the sediment, considered as whole sediments or the <63 m sieved fraction. On the basis of these results, we hypothesise that this species exhibits two levels of selectivity in its feeding behaviour. Thus selection is primarily based on particle size, avoiding the ingestion of sand particles and also, on the preferential selection of particles associated with organic material, within the fine (silt-clay) fraction of the sediment.     

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.     

Head segmentation in the embryo of the Colorado beetleLeptinotarsa decemlineata as seen with anti-en immunostaining

Segmentation in the head of the embryo of the Colorado beetleLeptinotarsa decemlineata is described on the basis of anti-engrailed (en) immunostaining of germ band stages. Six segmental units can be identified with this technique. Three segmentalen stripes can be distinguished in the gnathal region, a weak stripe interrupted medially shows the intercalary segment rudiment, a pair of oblique stripes indicate the antennal segment, and one pair of preantennalen spots are taken to indicate a sixth segment. In the broad head lobes of the beetle the spacing of the six segmental units as demarcated byen regions is similar to that in other parts of the germ band. The results are discussed with respect to old and new data concerning the number of head segments and origin of the compound eye in insects.     

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.     

Behaviour of centrosomes in early Tubifex embryos: asymmetric segregation and mitotic cycle-dependent duplication

An antibody raised against a highly conserved peptide of -tubulin (Joshi et al. 1992) recognized a 50 kDa polypeptide in centrosomes in Tubifex embryos. Centrosomes labelled with this antibody are found at both poles of the first meiotic spindle and at the inner pole of the second meiotic spindle. At the transition to the second meiosis, there is no change in morphology of the centrosomes which are retained in the egg proper. In contrast, as the second meiosis proceeds from anaphase to telophase, centrosomes labelled with the antibody gradually become smaller, but are still recognized as tiny dots; each egg exhibits no more than one tiny dot. The first cleavage spindles exhibit a centrosome at one pole but not at the other. The spindle pole with a centrosome forms an aster which is inherited by the larger cell, CD, of the two-cell embryo; the centrosome-free spindle pole then becomes anastral and is segregated to a smaller cell AB. Centrosomes are present in the C and D cell lineages but not in the A and B lineages, at least up to the eighth cleavage cycle. During cleavage stages, centrosomes duplicate early in telophase of each mitosis, and their size changes in a cell cycle-specific fashion. Centrosomes which otherwise duplicate asynchronously in separate cells do so synchronously in a common cytoplasm. Centrosome duplication is inhibited by nocodazole but not by cytochalasin D. An examination of embryos treated with cycloheximide or aphidicolin also suggests that centrosome duplication during cleavages requires protein synthesis but no DNA replication per se. These results suggest that the centrosome cycle in Tubifex blastomeres is linked to the mitotic cycle more closely than is that in other animals.     

UFO in the Arabidopsis inflorescence apex is required for floral-meristem identity and bract suppression

The UNUSUAL FLORAL ORGANS (UFO) gene of Arabidopsis encodes an F-box protein required for the determination of floral-organ and floral-meristem identity. Mutation of UFO leads to dramatic changes in floral-organ type which are well-characterized whereas inflorescence defects are more subtle and less understood. These defects include an increase in the number of secondary inflorescences, nodes that alternate between forming flowers and secondary inflorescences, and nodes in which a single flower is subtended by a bract. Here, we show how inflorescence defects correlate with the abnormal development of floral primordia and establish a temporal requirement for UFO in this process. At the inflorescence apex of ufo mutants, newly formed primordia are initially bract-like. Expression of the floral-meristem identity genes LFY and AP1 are confined to a relatively small adaxial region of these primordia with expression of the bract-identity marker FIL observed in cells that comprise the balance of the primordia. Proliferation of cells in the adaxial region of these early primordia is delayed by several nodes such that primordia appear “chimeric” at several nodes, having visible floral and bract components. However, by late stage 2 of floral development, growth of the bract generally ceases and is overtaken by development of the floral primordium. This abnormal pattern of floral meristem development is not rescued by expression of UFO from the AP1 promoter, indicating that UFO is required prior to AP1 activation for normal development of floral primordia. We propose that UFO and LFY are jointly required in the inflorescence meristem to both promote floral meristem development and inhibit, in a non-cell autonomous manner, growth of the bract.Shelley R. Hepworth and Jennifer E. Klenz contributed equally to this work.     

Synchronization of somatic embryogenesis from carrot cells at high frequency as a basis for the mass production of embryos

Synchronization of somatic embryogenesis at high frequency is a useful system for the mass production of embryos. Many attempts have been carried out, however, it was difficult to obtain the system in which most of the initial embryogenic cells or cell clusters synchronously differentiate to embryos. In carrot suspension cultures, high frequency, synchronous embryogenesis systems (following three systems) have been established.(1) Small spherical single cells from suspension cultures obtained by sieving and density gradient centrifugation in Percoll solutions differentiated to embryogenic cell clusters at high frequency when they were cultured in a medium containing 2,4-dichlorophenoxyacetic acid (0.05 micromolar), zeatin (1 micromolar) and mannitol (0.2 molar). (2) Embryogenic cell clusters from suspension cultures obtained by sieving, density gradient centrifugation in Ficoll solutions, and subsequent centrifugation at a low speed for a short time synchronously differentiated to embryos, especially globular embryos at high frequency, when they were cultured in a medium containing zeatin (0.1 micromolar) but no auxin. (3) Embryogenic cell clusters obtained by above method are cultured at cell densities of 2×10³ cell clusters ml^-1. Globular embryos which were sieved from embryos induced synchronously differentiated to torpedo-shaped embryos at high frequency when they were cultured at densities below 150 globular embryos ml^-1.Using these systems, the whole process of embryogenesis from single cells to whole plants could be synchronously induced at high frequency.Abbreviations ABA abscissic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellin A₃ - IAA indoleacetic acid - NAA naphthylacetic acid     

A cellular basis for polarized-light vision in rainbow trout

Polarized light sensitivity was examined in single units of the rainbow trout (Oncorhynchus mykiss) torus semicircularis, a sub-tectal visual area with a high degree of ultraviolet sensitivity. First, chromatically isolated torus units with inputs from each of the four cone mechanisms found in the trout visual system were separately examined for e-vector sensitivity. UV ON-response units showed polarization sensitivity for vertical ly (0° and 180°) polarized stimuli, while ON-response units of the short, middle and long cone mechanisms were not polarization sensitive. No OFF-response units of the UV or short cone mechanism were observed, but OFF-response units of the middle and long cone mechanisms show polarization sensitivity for horizontally (90°) polarized stimuli. Second, e-vector sensitivity was observed in color-coded units which received inputs from more than one cone mechanism and showed different sign responses (ON or OFF) at different points of the spectral sensitivity curve. Biphasic units which had ON input from the UV cone mechanism and OFF inputs from the middle and long cone mechanisms showed polarization opponency. This opponency was observed with a 380 nm stimulus when the threshold sensitivities of the alpha-band absorption peak of the UV mechanism and the beta-band absorption peak of the middle and long cone mechanisms were equal. We believe that biphasic torus units provide a possible cellular basis for polarized light vision in rainbow trout.Abbreviations UV ultraviolet - S short - M middle - L long - PS polarization sensitivity - TS torus semicircularis - ONR optic nerve response     

Changes in endogenous polyamines during the formation of somatic embryos from isogenic lines of Dactylis glomerata L. with different regenerative capacities

The endogenous levels of polyamines (PAs) in leaf-base explants isolated from plants of two isogenic lines of Dactylis glomerata L., differing in their competence for somatic embryogenesis, were compared. Leaf-bases isolated from plants with a high level of competence for somatic embryogenesis (HEC) contained four times the level of polyamines compared to those isolated from plants with a low level of competence for somatic embryogenesis (LEC). When the levels of individual polyamines in the HEC and LEC lines were compared, leaf-bases from plants of the HEC line had much lower PUT/SPD ratios than those from the LEC line. When changes in the levels of PAs were monitored during the first 28 d of culture, on a medium which promotes initiation of somatic embryogenesis, leaf-base cultures from plants of the HEC line showed a 50% increase in the levels of PAs during the first 7 d of culture, after which time levels began to decline. By day 21, levels had dropped below those found in freshly isolated leaf bases. While PUT and SPM levels increased by about 30%, the greatest increase was shown by SPD, which increased by more than 100% during the first 7 d of culture, before declining. In contrast much smaller changes in PA levels were found when leaf-bases from plants of the LEC line were cultured.     

Calcium increases the yield of somatic embryos in carrot embryogenic suspension cultures

An upward shift in the concentration of calcium present in the medium during somatic embryogenesis increased the number of embryos produced approximately two-fold. This was observed when embryogenic suspension cells grown in 2,4-D medium with the normal calcium concentration of 10^–3 M were transferred to hormone-free medium containing 10^–2 M calcium and when embryogenic suspension cells grown in 2,4-D medium containing 10^–4 M calcium were transferred to hormone-free medium with 10^–3 M calcium. At calcium concentrations between 6·10^–3 and 10^–2 M globular stage somatic embryos were found in cultures supplemented with 2·10^–6 M of 2,4-D indicating that elevated calcium counteracts the inhibitory effect of 2,4-D on somatic embryogenesis. No qualitative changes were found in the pattern of extracellular polypeptides as a result of growth and embryogenesis in media with different calcium concentrations.     

Direct seeding of grapevine somatic embryos and regeneration of plants

Summary Somatic embryos of grapevine (Vitis vinifera L.) ‘Chardonnay’ were produced from liquid suspension cultures. Mature somatic embryos were blot dried briefly in the laminar flow hood and germinated directly in Magenta GA-7 Vessels^™ containing one of the following potting media: (1) sand, (2) commercial potting mixture (CPM), or (3) CPM overlaid with sand. Each vessel containing 20 ml of distilled water and the potting medium was sterilized by autoclaving for 30 min and cooled overnight before inoculating the somatic embryos. Five somatic embryos were placed in each vessel under aseptic conditions. The vessels were closed and incubated at 26±2°C, 16 h photoperiod at 75 μmol s⁻¹ m⁻² light intensity. Results revealed that CPM overlaid with sand was best for plant development. There was more contamination of somatic embryos on pure CPM. Since direct seeding bypasses at least two subcultures in agar medium, it has implications for use of somatic embryos as ‘synthetic seeds’ for clonal plant production. This study shows that somatic embryos of grapevine can be handled with some of the convenience of seeds, emphasizing the feasibility for further automating in vitro plant production, which might be especially useful for new varieties where propagation material is limited.     

Artificial seeds in barley: encapsulation of microspore-derived embryos

Summary An in vitro culture system has been developed for barley (Hordeum vulgare), which yields high frequencies of high quality microspore-derived embryos without an intervening callus phase. The embryos are very similar to zygotic embryos with regard to their morphology and germination capacity. These embryos were encapsulated in sodium alginate to produce individual beads containing one embryo each. In accordance with the literature, these beads are denoted artificial seeds. The artificial seeds germinated well and with a root system superior to that of non-encapsulated embryos. The artificial seeds also maintained their germination capacity for at least 6 months, whereas non-encapsulated embryos did not survive more than 2 weeks in storage. Artificial seeds, thus, probably provide a simple and universal delivery system of in vitro plantlets to greenhouse or field.     

Activator proteins for sulphatide hydrolysis and GM1-ganglioside hydrolysis. Probable identity on the basis of their co-purification,properties, ligand binding and immunochemical interactions

The concurrent purification of the activator protein for sulphatide hydrolysis and for G_M1-ganglioside hydrolysis including chromat ofocusing and hydrophobic chromatography stages is described. The purified preparation has a pl of 4.2 and the sub-unit M_r is 10 000. The stoichiometry of binding of sulphatide and ganglioside to the protein is very similar. Both activities are removed in similar proportions on binding to IgG purified from antisera raised against the activator protein. The probable identity of the activator protein for sulphatide hydrolysis with that for G_M1-ganglioside hydrolysis and a molecular explanation for this identity are discussed.     

Developmental pattern formation of somatic embryos induced in cell suspension cultures of cowpea [Vigna unguiculata (L.) Walp]

The sequence of events in the functional body pattern formation during the somatic embryo development in cowpea suspensions is described under three heads. Early stages of somatic embryogenesis were characterized by both periclinal and anticlinal cell divisions. Differentiation of the protoderm cell layer by periclinal divisions marked the commencement of somatic embryogenesis. The most critical events appear to be the formation of apical meristems, establishment of apical-basal patterns of symmetry, and cellular organization in oblong-stage somatic embryo for the transition to torpedo and cotyledonary-stage somatic embryos. Two different stages of mature embryos showing distinct morphology, classified based on the number of cotyledons and their ability to convert into plantlets, were visualized. Repeated mitotic divisions of the sub-epidermal cell layers marked the induction of proembryogenic mass (PEM) in the embryogenic calli. The first division plane was periclinally-oriented, the second anticlinally-oriented, and the subsequent division planes appeared in any direction, leading to clusters of proembryogenic clumps. Differentiation of the protoderm layer marks the beginning of the structural differentiation in globular stage. Incipient procambium formation is the first sign of somatic embryo transition. Axial elongation of inner isodiametric cells of the globular somatic embryo followed by the change in the growth axis of the procambium is an important event in oblong-stage somatic embryo. Vacuolation in the ground meristem of torpedo-stage embryo begins the process of histodifferentiation. Three major embryonic tissue systems; shoot apical meristem, root apical meristem, and the differentiation of procambial strands, are visible in torpedo-stage somatic embryo. Monocotyledonary-stage somatic embryo induced both the shoot apical meristem and two leaf primordia compared to the ansiocotyledonary somatic embryo.