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.     

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.     

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.     

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.     

The durations and compositions of cell cycles in embryos of the leech, Helobdella triserialis

When tritiated thymidine triphosphate ([(3)H]TTP) or its immunohistochemically detectable analogue, bromodeoxyuridine triphosphate (BrdUTP), is injected into blastomeres of leech embryos it passes throughout the entire embryo and is rapidly incorporated (within 2 min after injection) into nuclei of cells synthesizing DNA (S phase). In the same embryos a DNA-specific stain can be used to identify cells in mitosis (M phase) or nonreplicative interphase (G(1) or G(2) phase) on the basis of nuclear or chromosomal morphology. Using this procedure, we have determined the lengths and compositions of the mitotic cell cycles of identifiable cells in early embryos of the leech, Helobdella triserialis, and have analysed how the cell cycles change during the first seven stages of development. The relatively short cell cycles of the early blastomeres comprise not only phases of M and S, but also postreplicative gap (G(2)) phases. The lengthening of the cell cycles that occurs as development progresses is primarily accomplished by an increase in the length of G(2) and secondarily by an increase in the length of S and,in some instances, the addition of a prereplicative gap(G(1)) phase; M phase remains relatively constant. These data suggest that the durations of the cell cycles of embryonic cells are regulated by a variety of mechanisms.     

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.     

Host specificity in Metamera sillasenorum, n. sp., a gregarine parasite of the leech Helobdella triserialis with notes on transmission dynamics

Eugregarines of the suborder Septatorina are apicomplexan parasites that are found mainly in arthropods. Some exceptions are species in the Metameridae that contains the only 5 septate gregarines recorded from annelids. The type genus is Metamera Duke, 1910 with 2 species, Metamera schubergi Duke, 1910, in European Glossiphonia complanata, and Metamera reynoldsi Jones, 1943, from North American G. complanata. Over the summers of 1995-1998, in Keith County, Nebraska, septate gregarines were found in the glossiphoniid leech Helobdella triserialis. The gregarines were determined to be a new species of Metamera, herein named Metamera sillasenorum. Measurements of size and body proportions of over 600 gregarines and 50 oocysts showed differences from measurements of M. schubergi and M. reynoldsi, and secondary septa in the deutomerite were rarely observed. Field observations indicated that M. sillasenorum is probably host specific. In the laboratory, leeches also exhibited strong feeding preferences; e.g., H. triserialis and G. complanata consumed only snails, whereas Helobdella stagnalis consumed only oligochaetes. Infection experiments demonstrated that freshwater snails ingest the oocysts and are required as mechanical vectors. Oocysts were passed unaltered through the snails' intestines. Glossiphonia complanata did not become infected regardless of heavy exposure to oocysts, although only 5 G. complanata were used in the experiments. The results show that host specificity of M. sillasenorum is most likely due to a combination of host-feeding habits and host-parasite compatibility.     

Micromere lineages in the glossiphoniid leech Helobdella

In leech embryos, segmental mesoderm and ectoderm arise from teloblasts by lineages that are already relatively well characterized. Here, we present data concerning the early divisions and the definitive fate maps of the micromeres, a group of 25 small cells that arise during the modified spiral cleavage in leech (Helobdella robusta) and contribute to most of the nonsegmental tissues of the adult. Three noteworthy results of this work are as follows. (1) The c"' and dm' clones (3d and 3c in traditional nomenclature) give rise to a hitherto undescribed network of fibers that run from one end of the embryo to the other. (2) The clones of micromeres b" and b"' (2b and 3b in traditional nomenclature) die in normal development; the b" clone can be rescued to assume the normal c" fate if micromere c" or its clone are ablated in early development. (3) Two qualitative differences in micromere fates are seen between H. robusta (Sacramento) and another Helobdella sp. (Galt). First, in Helobdella sp. (Galt), the clone of micromere b" does not normally die, and contributes a subset of the cells arising exclusively from c" in H. robusta (Sacramento). Second, in Helobdella sp. (Galt), micromere c"' makes no definitive contribution, whereas micromere dm' gives rise to cells equivalent to those arising from c"' and dm' in H. robusta (Sacramento).     

Phylogeny and revision of the leech genus Helobdella (Glossiphoniidae) based on mitochondrial gene sequences and morphological data and a special consideration of the triserialis complex

The relationships, as well as identification of species, within Helobdella (Glossiphoniidae) were explored through phylogenetic analysis and through an overview of the historical systematics of the genus. The phylogeny was determined using morphological data and the mitochondrial gene sequences of cytochrome c oxidase subunit I and nicotinamide adenine dinucleotide dehydrogenase subunit I. A broad representation of 15 ingroup species was sampled, including 10 individuals from South America. Outgroup taxa included five species of Haementeria . Cladistic analysis of all available data resulted in one most parsimonious tree. Results shed light on genetic divergence of members classified as the same species, including those that are not monophyletic. Historically, external morphological characters have played a significant role in contributing to the confusion in the classification of H. triserialis, H. papillata, H. lineata and H. fusca in North America. Re-evaluation of Verril's Clepsine papillifera var. b and var. d in a phylogenetic context provides a solution. Additionally, the genera Adaetobdella, Acritobdella, Dacnobdella and Gloiobdella created by Ringuelet are returned to Helobdella based on overlapping morphological characters.     

Cell death in late embryogenesis of the leech Helobdella

Cell death was characterized during stages 8 and 9 in the leech Helobdella with a modified terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method. Using confocal analysis, the positions of dying cells were compared to rows of cells expressing the leech engrailed protein ht-en and to fluorescently marked cell lineages. Dying cells were present in diverse tissues. Some dying cells were in no obvious pattern, and others were in segmentally iterated patterns. Particular attention was paid to the ectoderm and mesoderm, where most of the cells examined died over a period equivalent to 1–4 h at 25°C. Segmentally iterated rows of dying cells were observed in the mesoderm just beneath the nf-derived ht-en expressing cell rows at a time when ht-en expressing cells were beginning to disappear. The position of these dying cell rows was consistent with a role in the partial deterioration of the septum. Received: 12 October 1998 / Accepted: 8 February 1999     

Regional differences in BMP-dependence of dorsoventral patterning in the leech Helobdella

In the leech Helobdella, the ectoderm exhibits a high degree of morphological homonomy between body segments, but pattern elements in lateral ectoderm arise via distinct cell lineages in the segments of the rostral and midbody regions. In each of the four rostral segments, a complete set of ventrolateral (O fate) and dorsolateral (P fate) ectodermal pattern elements arises from a single founder cell, op. In the 28 midbody and caudal segments, however, there are two initially indeterminate o/p founder cells; the more dorsal of these is induced to adopt the P fate by BMP5-8 emanating from the dorsalmost ectoderm, while the more ventral cell assumes the O fate. Previous work has suggested that the dorsoventral patterning of O and P fates differs in the rostral region, but the role of BMP signaling in those segments has not been investigated. We show here that suppression of dorsal BMP5-8 signaling (which effects a P-to-O fate change in the midbody) has no effect on the patterning of O and P fates in the rostral region. Furthermore, ectopic expression of BMP5-8 in the ventral ectoderm (which induces an O-to-P fate change in the midbody) has no effect in the rostral region. Finally, expression of a dominant-negative BMP receptor (which induces a P-to-O fate change in the midbody) fails to affect O/P patterning in the rostral region. Thus, the rostral segments appear to use some mechanism other than BMP signaling to pattern O and P cell fates along the dorsoventral axis. From a mechanistic standpoint, the OP lineage of the rostral segments and the O-P equivalence group of the midbody and caudal segments constitute distinct developmental modules that rely to differing degrees on positional cues from surrounding ectoderm in order to specify homonomous cell fates.     

Hox gene duplication and deployment in the annelid leech Helobdella

SUMMARY The segmented leeches are members of the phylum Annelida within the Lophotrochozoa. Here, we describe the isolation of a new Hox gene, Lox18 , in the leech Helobdella triserialis. Phylogenetic analysis indicates that Lox18 is a Deformed ( Dfd   ) ortholog. H. triserialis has at least two Dfd orthologs, Lox18 and the previously described Lox6 ( Kourakis et al. 1997 ; Wong and Macagno 1998 ), indicating that these genes duplicated after the last common ancestor of annelids and arthropods. Although the temporal appearance of Lox18 message is similar to that of Lox6 , the spatial pattern is different. Lox18 does not have a sharply defined anterior border of expression in the second neuromere of the subesophageal ganglion of the central nervous system (CNS) as does Lox6 , but is expressed uniformly in a small subset of cells in the longitudinal connectives and lateral roots in every segment of the CNS along the entire anterior-posterior (AP) axis. Even though Lox18 shares greater sequence similarity within the homeodomain and flanking regions to Drosophila Dfd than to the previously isolated Lox6 , its expression pattern suggests that its function has diverged from the ancestral Hox function. Previous sampling has indicated that the last common ancestor of protostomes and deuterostomes had as many as 10 clustered Hox genes representing distinct paralogy groups ( Irvine et al. 1997 ; de Rosa et al. 1999 ); leech Hox genes may have undergone subsequent and independent cluster or genome-wide duplication. These results point to the need for total genome level understanding for key members of the Lophotrochozoa.     

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.     

A new molecular logic for BMP-mediated dorsoventral patterning in the leech Helobdella

Bone morphogenetic protein (BMP) signaling is broadly implicated in dorsoventral (DV) patterning of bilaterally symmetric animals [1-3], and its role in axial patterning apparently predates the birth of Bilateria [4-7]. In fly and vertebrate embryos, BMPs and their antagonists (primarily Sog/chordin) diffuse and interact to generate signaling gradients that pattern fields of cells [8-10]. Work in other species reveals diversity in essential facets of this ancient patterning process, however. Here, we report that BMP signaling patterns the DV axis of segmental ectoderm in the leech Helobdella, a clitellate annelid (superphylum Lophotrochozoa) featuring stereotyped developmental cell lineages, but the detailed mechanisms of DV patterning in Helobdella differ markedly from fly and vertebrates. In Helobdella, BMP2/4s are expressed broadly, rather than in dorsal territory, whereas a dorsally expressed BMP5-8 specifies dorsal fate by short-range signaling. A BMP antagonist, gremlin, is upregulated by BMP5-8 in dorsolateral, rather than ventral territory, and yet the BMP-antagonizing activity of gremlin is required for normal ventral cell fates. Gremlin promotes ventral fates without disrupting dorsal fates by selectively inhibiting BMP2/4s, not BMP5-8. Thus, DV patterning in the development of the leech revealed unexpected evolutionary plasticity of the conserved BMP patterning system, presumably reflecting its adaptation to different modes of embryogenesis.     

The reproductive biology of the leech Helobdella stagnalis (L.) in Utah Lake, Utah

The reproductive biology of the glossiphoniid leech Helobdella stagnalis in Utah Lake, Utah was studied for an 18-month period from June 1970 to December 1971. In this lake it was found that the overwintering adult leeches produce two broods of young in May and June. Evidence is presented to support the idea that the same individual can produce two broods. Two complete cycles of gametogenesis also take place. Continuous water temperatures were recorded at the study site to correlate with reproductive activity. Gametogenesis appears to be temperature dependent with the first generation of sperm taking 4 months to be produced in the fall and winter months while the second generation of sperm is produced in 6 weeks during the spring. Egg production follows a similar pattem.     

Social group size, potential sperm competition and reproductive investment in a hermaphroditic leech, Helobdella papillornata (Euhirudinea: Glossiphoniidae)

Social group size may affect the potential for sperm competition, and this in turn may favour ontogenetic adjustments in testicular mass according to the likely requirements for sperm and spermatophore production. In a number of comparative analyses of testis mass among vertebrate species that differ in mating system or social organization, increasing potential for sperm competition is associated with larger testis size. Intraspecific phenotypic plasticity should be able to produce the same pattern if social group size is heterogenous and reflects differing degrees of average sperm competition, but this intraspecific effect is less well studied. We tested the effect of social groups on both male and female investment in the simultaneously hermaphroditic leech, Helobdella papillornata. Leeches were placed in groups of one, two, four or eight. Sexual investment at the onset of reproductive maturity was quantified as the total testisac volume for male function and total egg volume for female function. We found that testisac volume (statistically adjusted for body size) showed a significant increase with increasing group size. Total egg volume (also adjusted for body size) was unaffected by group size. Our findings indicate adaptive developmental plasticity in male gonad investment in response to the potential for sperm competition.