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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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. 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 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.     

Epidermal cell lineage.

The epidermis is a stratified squamous epithelium, which is under a constant state of proliferation, commitment, differentiation, and elimination so that the functional integrity of the tissue is maintained. The intact epidermis has the ability to respond to diverse environmental stimuli by continuous turnover to maintain its normal homeostasis throughout an organism's life. This is achieved by a tightly regulated balance between stem cell self-renewal and the generation of a population of cells that undergo a limited number of more rapid (amplifying) transit divisions before giving rise to nonproliferative, terminally differentiating cells. This process makes it an excellent model system to study lineage, commitment, and differentiation, although neither the identity of epidermal stem cells nor the precise steps and regulators that lead to mature epidermal cells have yet been determined. Furthermore, the identities of genes that initiate epidermal progenitor commitment to the epidermal lineage, from putative epidermal stem cells, are unknown. This is mainly due to the lack of an in vitro model system, as well as the lack of specific reagents, to study the early events in epidermal lineage. Our recent development of a differentiating embryonic stem cell model for epidermal lineage now offers the opportunity to analyze the factors that regulate epidermal lineage. These studies will provide new insight into epidermal lineage and lead to a better understanding of various hyperproliferative skin diseases such as psoriasis and cancer.     

Postembryonic nongonadal cell lineages of the nematode Panagrellus redivivus: Description and comparison with those of Caenorhabditis elegans

The postembryonic nongonadal cell lineages of the nematode Panagrellus redivivus are described and compared with those of Caenorhabditis elegans. The newly hatched larvae of P. redivivus females and males and C. elegans hermaphrodites and males are very similar. An almost identical set of blast cells divides postembryonically in P. redivivus and C. elegans to produce similar changes in the neuronal, muscular, hypodermal, and digestive systems. Most of these cell lineages are invariant; however, there is substantial variability in the number of cell divisions in the relatively extensive lineages of the lateral hypodermis of P. redivivus. Typically, in P. redivivus females, 55 blast cells generate 635 surviving progeny and 29 cell deaths; in P. redivivus males, 59 blast cells generate 758 surviving progeny and 35 cell deaths. The lineages generating the cells of the male tails of P. redivivus and C. elegans are almost identical; thus, the grossly different characteristics of these structures must reflect differences in the morphogenesis of cells equivalent in lineage history. Laser ablation experiments demonstrate that the gonad induces vulva development and that cell-cell interactions are important in specifying the fates of hypodermal precursor cells. The lateral hypodermal lineages provide striking examples of the apparent construction of complex lineages from modular sublineages; one simple pattern of cell divisions and cell fates occurs 70 times in the P. redivivus female. The differences in cell lineage between P. redivivus and C. elegans are relatively minor, and many appear to have involved two types of evolutionary change: the replacement of sublineages, and the modification of sublineages by the four classes of lineage transformations previously proposed based on a comparison of P. redivivus and C. elegans gonadal cell lineages (Sternberg and Horvitz, 1981). These types of differences suggest that the genetic programming of cell lineage includes instructions specifying where and when a particular sublineage is utilized, and other instructions specifying the nature of that sublineage.     

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.     

Regionalization and segmentation of the leech

Regionalization and segmentation of the leech body plan have been examined by numerous approaches over the years. A wealth of knowledge has accumulated regarding the normally invariant cell lineages of the leech and the degree of developmental plasticity that is possible in each cell line in early development and in neurogenesis. Homologues of genes that control regionalization and segmentation in Drosophila have been cloned from the leech and the expression patterns reveal conserved features with those in Drosophila and other organisms. Possible developmental functions of the en-class proteins in spatial and temporal modes of segment formation are discussed in light of leech and Drosophila development. Annelida and Arthropoda cell lineages of engrailed-class gene expression are compared in leech blast cell clones and crustacean parasegments. In addition, future directions for molecular analysis of segmentation of the leech are summarized. © 1995 John Wiley & Sons, Inc.     

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

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