Cell lineage and segmentation in the leech

Segments in the leech arise by the proliferation of longitudinally arrayed bandlets of blast cells derived from ten identifiable embryonic stem cells, two M, two N, four O/P and two Q teloblasts. In each bandlet, older blast cells lie ahead of those born later. By using microinjected cell lineage tracers it was shown previously that the teloblasts give rise to characteristic cell patterns made up of segmentally iterated complements of progeny designated as M, N, O, P and Q kinship groups. When a teloblast is injected after it has begun generating blast cells, a boundary is observed later in development between anterior, unlabelled progeny of blast cells produced before injection and posterior, labelled progeny of blast cells produced after injection. We have examined such boundaries in detail to establish the precise relationship between blast cell clones and segments, with the following conclusions: (i) in the M, O and P cell lines, one blast cell generates one segmental complement of progeny, but serially homologous blast clones intermix so that no segment boundaries can be defined based on primary blast cell clones; (ii) in the N and Q cell lines, two blast cells are required to generate a complete segmental complement of progeny; (iii) in the process of forming the germinal plate, cells derived from the N and Q teloblasts move past those derived from the M and O/P teloblasts, so that consegmental blast cell clones do not come into register until well after the establishment of segmentally iterated units within each bandlet.     

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.     

Cell interactions in the developing epidermis of the leech Helobdella triserialis

In embryonic development of the leech Helobdella triserialis, each of the four paired ectodermal teloblasts contributes some progeny to a characteristic dorsal or ventral territory of the epidermis. To ascertain the relative roles of cell lineage and cell interactions in generating the highly regular epidermal distribution pattern of the various ectodermal cell lines, a series of experiments was carried out in which the ablation of particular teloblasts was combined with the intracellular injection of cell lineage tracers. The results showed that, after the ablation of an OP proteloblast, or of an O, P, or Q teloblast, the epidermal progeny of the remaining ipsilateral and contralateral teloblasts spread into the territory normally occupied by the epidermal progeny of the ablated teloblast. In this spreading process, cells may cross the ventral midline but not the dorsal midline. The spread of epidermal progeny of one teloblast in response to ablation of another teloblast is contrasted with the failure of the neuronal progeny of one teloblast to replace any missing neural tissue. It appears, therefore, that all epidermal cell lines are of equal developmental potential, regardless of their teloblast of origin, with the eventual location of any epidermal cell in the body wall being governed by interactions between cells within the developing epidermis.     

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.     

A distinct patterning mechanism of O and P cell fates in the development of the rostral segments of the leech Helobdella robusta: implications for the evolutionary dissociation of developmental pathway and morphological outcome

Despite a high degree of homonomy in the segmental organization of the ectoderm, the body plan of the leech is divided into two zones based on the distinct cell lineage patterns that give rise to the O/P portion of the segmental ectoderm. In the midbody and caudal segments, each segmental repeat of ectoderm arises in part from one 'o' blast cell and one 'p' blast cell. These two blast cells are positionally specified to distinct O and P fates, and give rise to differentiated descendant cells called O and P pattern elements, respectively. In the rostral segments, each segmental repeat of O and P pattern elements arises from a single 'op' blast cell. Based on their developmental fates and their responses to the ablation of neighboring cells, the granddaughters of the primary op blast cell are categorized into two O-type cells and two P-type cells. The O-type cells do not require the presence of the rest of the op blast cell clone for their normal development. By contrast, normal development of the P-type cells depends upon interactions with the other OP sublineages. Additional experiments showed that the O-type cells are the source of a repressive signal involved in the normal fate specification of the P-type cells. Our data suggest that the cell interactions involved in fate specification differ substantially in the rostral and midbody segments, even though the set of differentiated descendants produced by the rostral OP pathway and the midbody O and P pathways are very similar.     

Ectodermal interactions during neurogenesis in the glossiphoniid leech Helobdella triserialis

Morphogenetic cell interactions during development were studied by combining cell ablation and cell lineage tracing techniques in embryos of the leech Helobdella triserialis. Ablation of an identified ectodermal teloblast, or teloblast precursor blastomere, on one side of an early embryo was often found to result in the later abnormal migration of the progeny cells of the corresponding contralateral, nonablated teloblast to the ablated side of the embryo; such abnormal migration was termed “midline violation.” Two different kinds of midline violation were observed. Crossover: after ablation of an N teloblast individual stem cell progeny of the contralateral N teloblast sometimes cross the ventral midline of the germinal plate of the embryo. Switching: after ablation of an OPQ teloblast precursor bandlets of stem cells produced by the contralateral O, P, or Q teloblasts sometimes switch to the germinal band of the ablated side at the site of origin of the germinal bands. The occurrence of crossover and switching shows that the eventual site occupied by a progeny cell of a particular teloblast is not automatically determined by its lineage, but also depends on interactions with other cells. Midline violation in the leech embryo CNS does not constitute true regulation, however, since the restoration of neurons to the ablated side is accompanied by a neuron deficit on the nonablated side. The occurrence of the two distinct kinds of midline violation, crossover and switching, may be explained by the relative position of the stem cell bandlets within the germinal bands, and by the geometrical features of the formation of the germinal plate from the germinal bands.     

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.     

Evolutionary diversification of specification mechanisms within the O/P equivalence group of the leech genus Helobdella

Developmental fates and cell lineage patterns are highly conserved in the teloblast lineages that give rise to the segmental ectoderm of clitellate annelids. But previous studies have shown that the pathways involved in specification of the ventrolateral O lineage and the dorsolateral P lineage differ to some degree in distantly related clitellate species such as the leeches Helobdella and Theromyzon, and the sludgeworm Tubifex. To examine this developmental variation at a lower taxonomic level, we have explored the specification pathways of the O and P lineages in the leech genus Helobdella. In leech, the O and P lineages arise from a developmental equivalence group of O/P teloblasts. In this study, we demonstrate that the cell-cell interactions involved in cell fate specification of the O/P equivalence group differ among three laboratory colonies of closely related species. In two populations, the Q lineage is necessary to specify the P fate in the dorsalmost O/P lineage, but in the third population the P fate can be specified by a redundant pathway involving the M lineage. We also observe interspecific variation in the role played by cell interactions within the O/P equivalence group, and in the apparent significance of extrinsic signals from the micromere cell lineages. Our data suggest that cell fate specification in the O/P equivalence group is a complex process that involves multiple cell-cell interactions, and that the developmental architecture of the O/P equivalence group has undergone evolutionary diversification in closely related species, despite maintaining a conserved morphology.     

The state of engrailed expression is not clonally transmitted during early Drosophila development.

In Drosophila embryos, boundaries of lineage restriction separate groups of cells, or compartments. Engrailed is essential for specification of the posterior compartment of each segment, and its expression is thought to mark this compartment. Using a new photo-activatable lineage tracer, we followed the progeny of single embryonic cells marked at the blastoderm stage. No clones straddled the anterior edges of engrailed stripes (the parasegment border). However, posterior cells of each stripe lose engrailed expression, producing mixed clones. We suggest that stable expression of engrailed by cells at the anterior edge of the stripe reflects, not cell-intrinsic mechanisms, but proximity to cells that produce Wingless, an extracellular signal needed for maintenance of engrailed expression. If control of posterior cell fate parallels control of engrailed expression, cell fate is initially responsive to cell environment and cell fate determination is a later event.     

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.     

Gangliogenesis in leech: morphogenetic processes leading to segmentation in the central nervous system

 Using intracellular lineage tracers to study the main neurogenic lineage (N lineage) of the glossiphoniid leech embryo, we have characterized events leading from continuous columns of segmental founder cells (nf and ns primary blast cells) to discrete, segmentally iterated ganglia. The separation between prospective ganglia was first evident as a fissure between the posterior boundary of nf- and the anterior boundary of ns-derived progeny. We also identified the sublineages of nf-derived cells that contribute parallel stripes of cells to each segment. These stripes of cells project ventrolaterally from the dorsolateral margin of each nascent ganglion to the ventral body wall. The position and orientation of the stripes suggests that they play a role in forming the posterior segmental nerve; they are not coincident with the ganglionic boundary, and they form well after the separation of ganglionic primordia. Previous work has shown that cells in the anterior stripe express the leech engrailed-class gene. Thus, in contrast to the role of cells expressing engrailed in Drosophila, the stripes of N-derived cells expressing an engrailed-class gene in leech do not seem to play a direct role in segmentation or segment polarity. Received: 10 October 1997 / Accepted: 12 December 1997     

Cell lineage analysis of pattern formation in the Tubifex embryo. I. Segmentation in the mesoderm.

Annelids are strongly segmented animals that display a high degree of metamerism in their body plan. The embryonic origin of metameric segmentation was examined in an oligochaete annelid Tubifex using lineage tracers. Segmental organization arises sequentially in the anterior-to-posterior direction along the longitudinal axis of the mesodermal germ band, a coherent column of primary blast cells that are produced from the mesodermal teloblast. Shortly after its birth, each primary blast cell undergoes a spatiotemporally stereotyped sequence of cell divisions to generate three classes of cells (in terms of cell size), which together give rise to a distinct cell cluster. Each cluster is composed of descendants of a single primary blast cell; there is no intermingling of cells between adjacent clusters. Relatively small-sized cells in each cluster become localized at its periphery, and they form coelomic walls including an intersegmental septum to establish individuality of segments. A set of cell ablation experiments showed that these features of mesodermal segmentation are not affected by the absence of the overlying ectodermal germ band. These results suggest that each primary blast cell serves as a founder cell of each mesodermal segment and that the boundary between segments is determined autonomously. It is concluded that the metameric body plan of Tubifex arises from an initially simple organization (i.e., a linear series) of segmental founder cells.     

Developmental origin of segmental differences in the leech ectoderm: survival and differentiation of the distal tubule cell is determined by the host segment

The body plan of the adult leech is metameric, with each hemisegmental complement of ectodermal and mesodermal tissues being produced from a set of seven serially repeated embryonic blast cells. Previous studies have shown that homologous o blast cells give rise to an almost identical complement of descendant cells in each of the 21 abdominal segments, but that one o blast cell derivative--the distalmost cell of the nephridial tubule--is only present in 15 abdominal segments in the mature leech. Here we show that all o blast cells generate a presumptive distal tubule cell and that this cell migrates to its normal position in all abdominal segments. However, in segments which normally do not contain the mesodermal portion of the nephridium, the distal tubule cell dies before undergoing its terminal morphological differentiation. To ascertain whether the fate of the distal tubule cell is determined by its lineage history or by the segmental environment into which it is born, we utilized a previously described procedure for altering the segmental register between different embryonic cell lines. This procedure allowed us to effectively transplant o blast cells into more posterior segments prior to the cell divisions which generate their descendant clones. The results indicate that the survival or death of the distal tubule cell is determined by the identity of the host segment and that a given distal tubule cell could be effectively murdered or rescued by slipping its blast cell precursor into an appropriate segment. These findings suggest that the segment-specific pattern of distal tubule cell survival is not inherent to the O cell line, but arises from interactions with surrounding tissues.     

Primary culture of single ectodermal precursors of Drosophila reveals a dorsoventral prepattern of intrinsic neurogenic and epidermogenic capabilities at the early gastrula stage.

We have analyzed the development in vitro of individual precursor cells from the presumptive truncal segmental ectoderm of the Drosophila embryo to study the intrinsic component in the determination of cell fate. For each cultured cell, the original position within as well as the developmental stage of the donor embryo were known. Cells removed from the ventral neurogenic region develop neural clones. Cells from the dorsal ectoderm and from the dorsalmost part of the ventral neurogenic ectoderm develop epidermal clones. These two classes of clones differ with respect to their division pattern, adhesiveness, cell morphologies and the expression of cell-specific markers. Mixed neural/epidermal clones were obtained from a fraction of precursors at almost all dorsoventral sites. We conclude that, at the onset of gastrulation, precursor cells of the truncal segmental ectoderm already have the capability to develop as either neuroblasts or epidermoblasts in the absence of further cell interactions. At the same time, positional cues distributed along the dorsoventral axis equip precursors with intrinsic preferences towards the neural or epidermal fate, thus defining a prepattern of high neurogenic preferences ventrally, and high epidermogenic preferences dorsally. It is likely that this prepattern is involved in defining the extent of the ventral neurogenic and dorsal epidermogenic regions of the ectoderm. The roles of intrinsic capabilities versus extrinsic influences in the regulation of the characteristic pattern of segregation of the two lineages are discussed.     

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.     

A single cell analysis of engrailed expression in the early embryonic brain of the grasshopper Schistocerca gregaria: ontogeny and identity of the secondary headspot cells

The expression pattern of the segment polarity gene engrailed was studied at the single cell level in the protocerebrum of the early embryonic brain of the grasshopper Schistocerca gregaria, the neuromere containing the secondary headspot cells. The engrailed protein is first expressed in the protocerebrum at about 22% of embryogenesis by a group of identified neuroblasts bordering the antennal lobe. The number of immunoreactive neuroblasts increases up to 26% of embryogenesis and then rapidly declines so that by 30% only the three most posterior remain immunoreactive. These three neuroblasts become incorporated into the developing antennal lobe of the deutocerebrum. Subsequently, there is a progressive re-expression of the engrailed protein in the protocerebrum by the so-called six secondary headspot cells. These are the first born sibling progeny of three identified protocerebral neuroblasts which themselves expressed the engrailed protein prior to generating their lineages, and so represents a reacquisition of engrailed expression within identified clones. The secondary headspot cells are neurons which direct axonal processes into the developing optic tract and so contribute to the primary axon scaffold of the brain. From our analysis of their ontogeny, we conclude that the secondary headspot cells do not represent a segmental border in the brain.     

Differentiation of the O and P cell lines in the embryo of the leech. II. Genealogical relationship of descendant pattern elements in alternative developmental pathways

The p blast cells are a group of embryonic precursors found in the ectodermal cell layer of the leech germinal band. Each p blast cell normally undergoes the same invariant sequence of cell divisions and gives rise to a precisely defined set of uniquely identifiable neuronal and epidermal descendants in the mature leech. In the present paper, various of the p blast cell progeny were injected with a fluorescent lineage tracer in order to characterize the cellular composition of their descendant clones, and the results show that there is a stereotyped segregation of descendant cell fates through the first three p blast cell divisions. Previous work has shown that neurons and epidermal specializations which normally descend from the p blast cell will arise from a different precursor--the o blast cell--in response to ablation of the neighboring P cell line and that if the o blast cell is at a certain stage of differentiation when the ablation is performed it will produce only a subset of the normal P descendants. Comparison with the present findings indicates that under those conditions the o blast cell clone is not simply recapitulating a branch of the normal p blast cell lineage, but rather manifests an alternative lineage in which P descendants exhibit an abnormal genealogical relationship. Thus, even though normal leech development comprises a nearly invariant cell lineage, lineage relationships are open to considerable reorganization under experimental conditions.