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.     

Developmental interdeterminacy in embryos of the leech Helobdella triserialis

In embryonic development of the leech Helobdella triserialis, each of the four paired positionally identifiable, ectodermal teloblasts (N, O, P, and Q) generates a bandlet of blast cell progeny that merges with ipsilateral bandlets into a germinal band. Left and right germinal bands coalesce into the germinal plate which gives rise to the segmental tissues of the leech and wherein the progeny of each teloblast generate a characteristic pattern of epidermal and neuronal cells. Experiments reported here show that the positionally identified O teloblast sometimes generates the P pattern and vice versa. The reversal of these teloblasts' generative identities was shown to correspond to the formation of chiasmata by their blast cell bandlets, so that the positions of their bandlets in the germinal band are reversed as well. Thus it is the position of the bandlet in the germinal band, rather than the position of the parent teloblast, which correlates with the fate of o and p blast cells. Moreover, two types of ablation experiments have shown that, in the absence of generative P teloblast progeny, those cells which would normally generate the O pattern take on a new fate and give rise to the P pattern in the nervous system, both at the gross pattern level in the segmental ganglia, and at the level of identified neurons in the peripheral nervous system. If related, these phenomena suggest that the O and P teloblasts, which derive from the symmetric cleavage of the OP proteloblasts, have a common developmental pluripotency. And in that case, the fates of their progeny are determined hierarchically on the basis of relative position in the nascent germinal band, with P-type fate being preferred.     

Early differences between alternate n blast cells in leech embryo

Segmentally iterated tissues of the mature leech comprise five distinct sets of definitive progeny that arise from chains of blast cells (m, n, o, p, and q bandlets) produced by five bilateral pairs of stem cells (M, N, O/P, O/P, and Q teloblasts). In each n and q bandlet, two blast cells are needed to generate one set of hemisegmental progeny, and two alternating classes of blast cells (nf and ns, qf and qs) can be distinguished after their first divisions. Furthermore, two distinct subsets of definitive N and Q progeny exist within each hemisegment. Here we first show that there is fixed correspondence between the class of blast cell and the subset of final progeny: ns cells contribute mainly anterior ganglionic neurons and epidermal cells; nf cells contribute mainly posterior ganglionic neurons, peripheral neurons and neuropil glia; qs cells contribute both ventral and dorsal progeny; and qf cells contribute only dorsal progeny. Second, ablation studies indicate that the two classes of n blast cells do not behave as an equivalence group in the germinal band. Finally, we show that the cycles giving rise to nf and ns blast cells differ. These data suggest that cellular interactions within the germinal band may not be critical in establishing the distinct nf and ns cell fates and that, conversely, differences between the two classes of n blast cells may be established at birth.     

Cell lineage analysis of pattern formation in the Tubifex embryo. II. Segmentation in the ectoderm

Ectodermal segmentation in the oligochaete annelid Tubifex is a process of separation of 50-microm-wide blocks of cells from the initially continuous ectodermal germ band (GB), a cell sheet consisting of four bandlets of blast cells derived from ectoteloblasts (N, O, P and Q). In this study, using intracellular lineage tracers, we characterized the morphogenetic processes that give rise to formation of these ectodermal segments. The formation of ectodermal segments began with formation of fissures, first on the ventral side and then on the dorsal side of the GB; the unification of these fissures gave rise to separation of a 50-microm-wide block of approximately 30 cells from the ectodermal GB. A set of experiments in which individual ectoteloblasts were labeled showed that as development proceeded, an initially linear array of blast cells in each ectodermal bandlet gradually changed its shape and that its contour became indented in a lineage-specific manner. These morphogenetic changes resulted in the formation of distinct cell clumps, which were separated from the bandlet to serve as segmental elements (SEs). SEs in the N and Q lineages were each comprised of clones of two consecutive primary blast cells. In contrast, in the O and P lineages, individual blast cell clones were distributed across SE boundaries; each SE was a mixture of a part of a more anterior clone and a part of the next more posterior clone. Morphogenetic events, including segmentation, in an ectodermal bandlet proceeded normally in the absence of neighboring ectodermal bandlets. Without the underlying mesoderm, separated SEs failed to space themselves at regular intervals along the anteroposterior axis. We suggest 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.     

Segmentation of the central nervous system in leech

Central nervous system (CNS) in leech comprises segmentally iterated progeny derived from five embryonic lineages (M, N, O, P and Q). Segmentation of the leech CNS is characterized by the formation of a series of transverse fissures that subdivide initially continuous columns of segmental founder cells in the N lineage into distinct ganglionic primordia. We have examined the relationship between the N lineage cells that separate to form the fissures and lateral ectodermal and mesodermal derivatives by differentially labeling cells with intracellular lineage tracers and antibodies. Although subsets of both lateral ectoderm and muscle fibers contact N lineage cells at or near the time of fissure formation, ablation experiments suggest that these contacts are not required for initiating fissure formation. It appears, therefore, that this aspect of segmentation occurs autonomously within the N lineage. To support this idea, we present evidence that fundamental differences exist between alternating ganglionic precursor cells (nf and ns primary blast cells) within the N lineage. Specifically, ablation of an nf primary blast cell sometimes resulted in the fusion of ipsilateral hemi-ganglia, while ablation of an ns primary blast cell often caused a 'slippage' of blast cells posterior to the lesion. Also, differences in cell behavior were observed in biochemically arrested nf and ns primary blast cells. Collectively, these results lead to a model of segmentation in the leech CNS that is based upon differences in cell adhesion and/or cell motility between the alternating nf and ns primary blast cells. We note that the segmentation processes described here occur well prior to the expression of the leech engrailed-class gene in the N lineage.     

Cell fate analysis of teloblasts in the Tubifex embryo by intracellular injection of HRP

As in other clitellate annelids, embryonic development in the oligochaete Tubifex is characterized by the generation of five bilateral pairs of teloblasts (designated M, N, O, P and Q), which serve as embryonic stem cells to produce germ bands on either side of the embryo. A large part of the tissues comprising body segments has been assigned to the progenies of the teloblasts; however, the developmental fate of each teloblast has been inferred only from its initial position in the embryo. In the present study, the fate of the progenies of each teloblast was followed by means of intracellular injection of a tracer enzyme, horseradish peroxidase. Cell fate maps for teloblasts in the Tubifex embryo were constructed. M teloblasts gave rise to nearly all of the mesodermal tissues, which included circular and longitudinal muscles, coelomic walls, nephridia (in segments VII and VIII) and primordial germ cells (in segments X and XI). Although few in number, M teloblasts also contributed cells to the ventral ganglion. Similarly, each of the ectoteloblasts, N, O, P and Q, made a topographically characteristic contribution to the ectodermal tissues such as the nervous system (i.e. ganglionic cells and peripheral neurones) and epidermis, all of which exhibited a segmentally repeated distribution pattern. The P and Q teloblasts uniquely gave rise to additional ectodermal tissues, namely ventral and dorsal setal sacs, respectively. Furthermore, O teloblasts made a contribution to the nephridiopores in segments VII and VIII as well. These results confirm the previously held view that ectoteloblasts and mesoteloblasts are the main source of ectodermal and mesodermal segmental tissues, respectively, but also suggest that all of the teloblasts produce more types of tissue than has previously been thought.     

Establishment of segment polarity in the ectoderm of the leech Helobdella

The segmented ectoderm and mesoderm of the leech arise via a stereotyped cell lineage from embryonic stem cells called teloblasts. Each teloblast gives rise to a column of primary blast cell daughters, and the blast cells generate descendant clones that serve as the segmental repeats of their particular teloblast lineage. We have examined the mechanism by which the leech primary blast cell clones acquire segment polarity - i.e. a fixed sequence of positional values ordered along the anteroposterior axis of the segmental repeat. In the O and P teloblast lineages, the earliest divisions of the primary blast cell segregate anterior and posterior cell fates along the anteroposterior axis. Using a laser microbeam, we ablated single cells from both o and p blast cell clones at stages when the clone was two to four cells in length. The developmental fate of the remaining cells was characterized with rhodamine-dextran lineage tracer. Twelve different progeny cells were ablated, and in every case the ablation eliminated the normal descendants of the ablated cell while having little or no detectable effect on the developmental fate of the remaining cells. This included experiments in which we specifically ablated those blast cell progeny that are known to express the engrailed gene, or their lineal precursors. These findings confirm and extend a previous study by showing that the establishment of segment polarity in the leech ectoderm is largely independent of cell interactions conveyed along the anteroposterior axis. Both intercellular signaling and engrailed expression play an important role in the segment polarity specification of the Drosophila embryo, and our findings suggest that there may be little or no conservation of this developmental mechanism between those two organisms.     

Cell lineage,cell-cell interaction,and segment formation in the ectoderm of a glossiphoniid leech embryo

Cell division patterns and cell-cell interactions in the germinal bands of the glossiphoniid leech Helobdella triserialis were studied with the aid of a cell lineage tracer dye. Each germinal band of the Helobdella embryo consists of five columns, or bandlets, of primary blast cells, designated as the mesodermal m bandlet and ectodermal n, o, p, and q bandlets. Primary blast cells of each ectodermal bandlet appear to undergo stereotyped, lineage-specific cell divisions. The metameric segmentation pattern of the leech thus appears to arise through a series of segmentally iterated, stereotyped cell divisions of serially homologous primary blast cell clones. Cell-cell interactions were studied by means of cell ablations. With one exception, blast cells underwent their stereotyped divisions without regard to the presence or absence of their normal neighbors. In the one exceptional case, o blast cells underwent divisions normally characteristic of p blast cells when their normal neighboring p bandlet was deleted. However, both o and p blast cells underwent their normal stereotyped divisions when their neighboring m, n, and q bandlets were deleted. It is proposed that the differential choice of pathway by the o and p blast cells depends upon their relative position with respect to each other and to a polarity cue external to the germinal band.     

Stepwise commitment of blast cell fates during the positional specification of the O and P cell lines in the leech embryo

The o and p bandlets of the leech embryo are parallel columns of ectodermal blast cells which are identified by their relative positions, and which during normal embryogenesis follow distinct developmental pathways. A previous study showed that o blast cells are initially capable of following either the O or P pathway, and suggested that commitment to the O pathway depends upon interaction with the adjacent p bandlet. To better understand the nature and timing of this interaction we examined the fate of o blast cells whose p blast cell neighbors had been selectively ablated by photoexcitation of a fluorescent lineage tracer. If an o blast cell has not yet begun its secondary divisions, its normal commitment to the O pathway can be effectively prevented by ablation of the adjacent p bandlet. Comparing the outcome of progressively later lesions reveals that the progeny of the o blast cell become committed to the O pathway in a series of three discrete steps, and that these steps occur around the time of the first three blast cell divisions. Each of the three events affects a different subset of elements within the blast cell clone, and apparently commits those elements to either the O or P pathway depending upon the presence or absence of the other bandlet. These changes in blast cell fate are coextensive with the lesion along the bandlet's length, suggesting that the interaction of the two bandlets is localized to neighboring cells.     

Applications of mRNA injections for analyzing cell lineage and asymmetric cell divisions during segmentation in the leech Helobdella robusta

Synthetic mRNAs can be injected to achieve transient gene expression even for 'non-model' organisms in which genetic approaches are not feasible. Here, we have used this technique to express proteins that can serve as lineage tracers or reporters of cellular events in embryos of the glossiphoniid leech Helobdella robusta (phylum Annelida). As representatives of the proposed super-phylum Lophotrochozoa, glossiphoniid leeches are of interest for developmental and evolutionary comparisons. Their embryos are suitable for microinjection, but no genetic approaches are currently available. We have injected segmentation stem cells (teloblasts) with mRNAs encoding nuclear localized green fluorescent protein (nGFP) and its spectral variants, and have used tandem injections of nGFP mRNA followed by antisense morpholino oligomer (AS MO), to label single blast cell clones. These techniques permit high resolution cell lineage tracing in living embryos. We have applied them to the primary neurogenic (N) lineage, in which alternate segmental founder cells (nf and ns blast cells) contribute distinct sets of progeny to the segmental ganglia. The nf and ns blast cell clones exhibit strikingly different cell division patterns: the increase in cell number within the nf clone is roughly linear, while that in the ns clone is almost exponential. To analyze spindle dynamics in the asymmetric divisions of individual blast cells, we have injected teloblasts with mRNA encoding a tau::GFP fusion protein. Our results show that the asymmetric divisions of n blast cells result from a posterior shift of both the spindle within the cell and the midbody within the mitotic spindle, with differential regulation of these processes between nf and ns.     

Embryonic origins of cells in the leech Helobdella triserialis

To ascertain the embryonic origins of the cells in various tissues of the leech Helobdella triserialis, horseradish peroxidase (HRP) was injected as a cell lineage tracer into all identified blastomeres of the early embryo in turn, except for a few of the micromeres, and the resulting distribution of HRP-labeled cells was then examined in the late embryo. In this way it was found that in every body segment a topographically characteristic set of neurons in the ganglion and body wall and a characteristic territory of the epidermis is derived from each of the four paired ectodermal teloblasts N, O/P, O/P, and Q, whereas the muscles, nephridia, and connective tissue, as well as a few presumptive neurons in each segmental ganglion, are derived from the paired mesodermal teloblast, M. Each topographically characteristic, segmentally iterated set of neurons descended from a given teloblast is designated as a kinship group. However, the prostomial (nonsegmental) epidermis and the neurons of the supraesophageal ganglion were found to be derived from the a, b, c, and d micromere quartet to which the A, B, C, and D blastomeres give rise at the dorsal pole of the embryo. The superficial epithelium of the provisional integument, which covers the surface of the embryo midway through development and is sloughed off at the time of body closure, was found to be derived from the a, b, c, and d micromere quartet, as well as from other micromeres produced in the course of teloblast formation. The contractile fibers of the provisional integument were found to be derived from the paired M teloblast. These results demonstrate that development of the leech embryo proceeds according to a highly stereotyped pattern, in the sense that a particular identifiable blastomere of the early embryo regularly gives rise to a particular set of cells of the adult (or provisional embryonic) tissues.     

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 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.     

Determination of cleavage pattern in embryonic blast cells of the leech

The o blast cells of the leech embryo become committed to one of two alternative cleavage geometries shortly before they divide. Cleavage geometry depends upon the presence or absence of the adjoining p bandlet, and if that bandlet is ablated, the pattern of o blast cell cleavages will undergo an abrupt transition several hours later. Previous work has shown that the oblast cell becomes committed to the formation of a particular complement of postmitotic descendants early in its differentiation, but the present findings suggest that cleavage pattern and descendant fate are determined at separate commitment events.     

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.     

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.