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.     

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.     

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 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.     

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.     

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.     

Immunoglobulin gene organisation and expression in haemopoietic stem cell leukaemia

We have analysed the organisation and expression of mu genes in the granulocytic phase and in the lymphoid and myeloid blast crises of Philadelphia chromosome (Ph1) chronic granulocytic leukaemia (CGL), a leukaemia which is known to arise in multipotential stem cells. We find that mu chain gene rearrangement occurs exclusively in lymphoid blast crisis leading in some, but not all, cases to the synthesis of small amounts of cytoplasmic mu chains characteristic of early pre-B lymphocytes. In Southern blots, only one or two rearranged mu chain genes are seen, suggesting that a clonal event leading to blast crisis can occur in a committed B cell precursor rather than in the multipotential stem cell precursor, in which the Ph1 chromosome originated. The pattern of mu gene rearrangement observed in Ph1 CGL blast crisis is compared with that in normal B cells, other B lineage malignancies, myeloid leukaemias and T cell leukaemias.     

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.     

Leech neurogenesis. I. Positional commitment of neural precursor cells

This paper reports analyses of the differentiation and distribution of identified peripheral neurons and central 5-HT-containing neurons in embryos of the glossiphoniid leech Theromyzon rude that have been deprived of one of the bilaterally paired major ectodermal cell lines called the n bandlets. Cells descended from a lone surviving n bandlet were abnormally distributed across both sides of the ventral midline. Nevertheless, they produced the complement of identified neurons that they would have produced in a normal embryo. Neurons produced by cells that crossed the midline occupied the normal positions of their absent homologs, as demonstrated by morphometric analysis of normal and n-bandlet-deprived ganglia. Ablations of ectodermal cell lines other than the n bandlets (o and p, or q) allowed the formation of normal distributions of neurons descended from the n bandlets. These results are interpreted as showing that neural precursor cells are committed to occupy particular positions before reaching those positions and probably use positional cues of predominantly nonectodermal origin to recognize those positions. Together, the results reported here and in the accompanying paper (S. Torrence, M. Law, and D. Stuart, 1989, Dev. Biol. 136, 40-60) suggest that ectodermal cells that are committed to give rise to specific neurons use cues provided by the mesoderm to find positions appropriate to their fates.     

Nanos is required in somatic blast cell lineages in the posterior of a mollusk embryo

During animal development, blast cell lineages are generated by repeated divisions of a mother cell into a series of daughter cells, often with a specific series of distinct fates. Nanos is a translational regulator that is involved in germline development in diverse animals and also involved in somatic patterning in insects. Recently, Nanos was found to be required for maintenance of stem cell divisions in the Drosophila germline. We have found that in the mollusk Ilyanassa, Nanos messenger RNA and protein are specifically localized in the mesendodermal blast cell lineage derived from the strongly conserved 4d cell. Nanos activity is required for differentiation of multiple tissues that are derived from the 4d cell, showing that IoNanos is required for somatic development in this embryo. At the cellular level, we show that IoNanos activity is required for the highly stereotyped cleavage pattern of the 4d lineage, the proliferative capacity of the blast cells, and the marked asymmetry of the blast cell divisions. These results suggest that IoNanos is involved in regulating blast cell behaviors in the 4d lineage.     

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.     

A provisional epithelium in leech embryo: Cellular origins and influence on a developmental equivalence group

Segmental tissues of glossiphoniid leeches arise from rostrocaudally arrayed columns (bandlets) of segmental founder cells (primary m, n, o, p, and q blast cells) which undergo stereotyped sublineages to generate identifiable subsets of definitive progeny. The bandlets lie at the surface of the embryo beneath the squamous epithelium of a transient embryonic covering called the provisional integument. This "provisional epithelium" derives from microsomes produced during the early cleavage divisions. Previous experiments have shown that the primary o and p blast cells constitute an equivalence group, i.e., are initially developmentally equipotent and undergo hierarchical interactions which cause them to assume distinct O and P fates. Here, we examine the role of the provisional epithelium in determining the fates of the underlying o and p blast cells. Experiments entailing the microinjection of individual micromeres with cell lineage tracers show that, at stages 7-8 of normal development, the epithelium comprises coherent and relatively stereotyped domains derived from particular micromeres. Upon photoablating domains of epithelium labeled with photosensitizing lineage tracer, the normal assignment of O fates is disturbed; o blast cells divide symmetrically (as p blast cells do) and some supernumerary definitive progeny expressing P fates arise within the O lineage. We therefore conclude that the epithelium is essential for generation and/or reception of signal(s) by which the o and p blast cells' normally determine their fates. Finally, a new tracer substance, biotinylated fixable dextran (BFD), is described which was essential for this study by virtue of its superior resistance to photobleaching and which offers several other advantages as well.     

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.     

Embryonic development of the Glossiphoniid leech Theromyzon rude: Characterization of developmental stages

The embryonic development of the leech Theromyzon rude was studied under the dissecting microscope. Embryos were examined both live and after acid treatment that solubilizes the yolk, or vitelloplasm, and renders the embryos transparent. Most of the remaining cytoplasm, or teloplasm, of the uncleaved egg is passed on to five pairs of germinal cells, or teloblasts. Teloblasts arise sequentially from a set of precursor cells, or proteloblasts, that divide according to a modified spiral cleavage pattern. Each teloblast buds off a succession of smaller stem cells, which form a single row, or germinal bandlet, and to which the teloblast passes on its teloplasm. The five bilateral pairs of germinal bandlets thus produced give rise to most of the embryonic structures. A new notational system for the designation of proteloblasts, teloblasts, and their stem cells has been introduced. Development of the embryo, from the uncleaved egg to the completion of the gut, has been divided into 10 stages. At 14°C, completion of these 10 stages takes approximately 850 hr from the time the egg is laid.     

How far does cell lineage influence cell fate specification in crustacean embryos?

In crustaceans, invariant cell lineages have been shown to occur (i) in early cleavages of several taxa and (ii) in the course of formation and differentiation of the post-naupliar germ bands in malacostracans. Work on early cleavages is still in its infancy. In contrast, the generation and proliferation of mesoteloblasts and ectoteloblasts and the subsequent proliferation and differentiation of bandlet cells have been studied in members of several subgroups of Malacostraca. Similarities and differences have been determined in order to interpret the interdependencies of the steps in the differentiation process. Some of these steps are highly conserved, as in the case of the generation of four pairs of mesoteloblasts, others are prone to phylogenetic change, as in the case of the primary ring of 19 ectoteloblasts which has been altered at least twice in evolution. A stereotyped cleavage pattern in the germ band has been shown to be independent of the origin of the precursor cells. The question whether neuroblasts in crustaceans and insects are homologous or are the result of convergent evolution is still open. However, the homology of early differentiating neurons in crustaceans and insects seems to be well established. In addition, similarities in the expression patterns of the engrailed gene are likely to be homologous and point to a close relationship between these two groups.     

CpG-Specific Common Commitment in Caspase-Dependent and -Independent Cell Deaths

Cell death in mammals seems to have caspase-dependent and -independent pathways unlike that in Caenorhabditis elegans where CED-3 protease activation is the central command. A recent suggestion to define apoptosis as the caspase-dependent or caspase-committed cell death form and leave cell death committed by other pathways as just cell death was meant to categorize the apparent divergence in mammalian cell death pathways. However, we show CpG oligonucleotides (ODN) blocking caspase-dependent fas(CD95) ligand-mediated apoptosis as well as caspase-independent etoposide-mediated apoptosis and etoposide–zVAD-mediated necrosis. CpG specificity was demonstrated by reversing the CpG motif or replacing it with a methylated motif (mCpG) which failed to inhibit. CpG ODN blocked CpG-specific DNA cleavage by rare-cutting NotI restriction, which produced a megabase cleavage pattern similar to that in the fasL and etoposide cell death inductions. CpG ODN inhibition was similar to that by CpG-specific SssI methylase. A common CpG-specific commitment point preceding caspase-dependent and -independent cell death pathways was suggested. CpG-specific modulation is a key epigenetic mechanism in genomic imprinting, resisting nuclease restriction, and patterning of chromatin conformations. It is now shown to have a powerful effect modulating cell death.