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D A Weisblat

Publications and source records attributed to D A Weisblat.

At least 19 recordsLinked to original sources
PubMed1992

An investigation of the specification of unequal cleavages in leech embryos.

Leech embryos develop via stereotyped cell divisions, many of which are unequal. The first division generates identifiable cells, blastomeres AB and CD, which normally follow distinct developmental pathways. When these two cells are dissociated and cultured in isolation, their fates remain distinct and are reminiscent of normal development, but their typical cleavage patterns are disrupted; cell AB undergoes relatively few cell divisions, giving rise to a variable number of macromeres and micromeres, while cell CD cleaves many times, usually forming a poorly organized set of macromeres, embryonic stem cells (teloblasts), and micromeres. We have investigated the hypothesis that the abnormal cleavage pattern of isolated CD blastomeres is due to removal of mechanical constraints normally imposed by cell AB. We find that when cell CD is constrained in vitro to mimic its in vivo shape, it cleaves more normally.

Animals↗
PubMed1992

Expression of a Wnt gene in embryonic epithelium of the leech.

A new member of the Wnt class of cell-cell communication molecules was identified in the leech Helobdella triserialis, on the basis of a conserved 86 amino acid coding sequence and exon structure. This gene, htr-wnt-A, is not an obvious homolog of any one of the previously described wnt class proteins. The embryonic expression of htr-wnt-A has been characterized at the cellular level, using nonradioactive in situ hybridization and polyclonal antibodies generated via a novel method of antigen presentation. Subcellular localization of the htr-wnt-A protein was examined by the use of immunofluorescence and confocal microscopy. htr-wnt-A is among the first zygotically expressed genes in Helobdella, appearing first in a single cell of the eight-cell embryo. In early development it is expressed within a stereotyped subset of micromeres and later, in a seemingly dynamic and stochastic pattern, by cells in a micromere-derived provisional embryonic epithelium. Its spatial and temporal expression pattern make it a candidate for participation in the regulation of cell fate in the O/P equivalence group.

Amino Acid Sequence↗
PubMed1992

Cytoplasmic and cortical determinants interact to specify ectoderm and mesoderm in the leech embryo.

In leech embryos, segmental ectoderm and mesoderm are produced by a pair of sister cells located near the animal and vegetal poles, respectively. We have investigated the mechanism that localizes ectodermal and mesodermal fates along the animal-vegetal axis. The results of cleavage arrest and cell ablation experiments suggest that the full range of normal cell interactions are not required for this process. However, when the animal and vegetal hemispheres are separated by re-orientation of the first cleavage plane from meridional to equatorial, the ectodermal fate co-segregates with the animal hemisphere and the mesodermal fate with the vegetal hemisphere. Two pools of yolk-deficient cytoplasm, called teloplasm, are located at the animal and vegetal poles of the zygote, but separation of the animal and vegetal teloplasms is not sufficient for the segregation of ectodermal and mesodermal fates. Rather, complete segregation of fates requires an equatorial cleavage orientation that separates not only the two teloplasms, but also the animal and vegetal cortical regions. This, in conjunction with previous findings, indicates that ectodermal determinants are localized to the cell cortex in the animal hemisphere of the zygote. We propose that these determinants segregate to the ectodermal precursor and interact with factors in teloplasm to transform the fate of this cell from a mesodermal ground state to ectoderm.

Animals↗
PubMed1991

Cloning and sequencing of a leech homolog to the Drosophila engrailed gene.

We have cloned and sequenced a homolog (ht-en) to the Drosophila engrailed (en) gene from the glossiphoniid leech, Helobdella triserialis. Amino acid comparisons of the ht-en homeodomain and C-terminal residues with the corresponding residues encoded by en-class genes of other species reveal 75-79% sequence identity. In addition, the ht-en sequence appears to have a serine-rich region 16 residues C-terminal from the homeodomain, which by analogy to Drosophila may be a target site for phosphorylation. The leech gene encodes some amino acid substitutions for residues that are highly conserved in other species. These are found within the second and third of the three putative helices of the homeodomain, and in both of the intervening turn regions.

Amino Acid Sequence↗
PubMed1991

Transcription in leech: mRNA synthesis is required for early cleavages in Helobdella embryos.

Zygotic transcription was analyzed in embryos of the glossiphoniid leech Helobdella triserialis by autoradiographic detection of tritiated uridine incorporated in the presence or absence of low concentrations of alpha-amanitin. RNA synthesis was first detected after the second cleavage and alpha-amanitin-sensitive RNA synthesis was first detected during the divisions yielding the embryonic stem cells, or teloblasts. RNA synthesis increased as development progressed, and the bulk of alpha-amanitin-sensitive RNA synthesis was found in two classes of cells, the blast cells, which are the progeny of the teloblasts, and the micromere-derived cells. The time during which zygotic gene products are required was determined by observing the developmental consequences of alpha-amanitin exposure. Zygotes microinjected with alpha-amanitin underwent the first several cleavages with normal timing and symmetry, but underwent aberrant cleavages and produced supernumerary large blastomeres during the time that the control embryos generated teloblasts. Once the teloblasts were formed, the microinjection of alpha-amanitin did not affect the production of blast cells by the teloblasts, but it did block the divisions and movements of the blast cells and the micromere-derived cells. These data suggest that zygotic transcription is activated during the early cleavages of Helobdella embryos and that newly synthesized transcripts are required for the generation of teloblasts. Thus, there is an early, critical period of messenger RNA synthesis essential for teloblast production that is distinct from the later phase of messenger RNA synthesis required for cell divisions and cell movements during gastrulation.

Amanitins↗
PubMed1991

Segmental expression of an engrailed-class gene during early development and neurogenesis in an annelid.

ht-en protein, an annelid homolog of the Drosophila engrailed protein, is expressed during both early development and neurogenesis in embryos of the leech, Helobdella triserialis. In Helobdella as in Drosophila, early expression is in segmentally iterated stripes of cells within the posterior portion of the segment and later expression is in cells of the segmental ganglia. These findings suggest that dual expression of an en-class gene was present in a common ancestor of annelids and arthropods.

Animals↗
PubMed1989

Factors specifying cell lineages in the leech.

As in arthropods, several major organ systems in leeches, including body musculature, nervous system and nephridia, are organized into a fixed number of longitudinally iterated units called segments. Many cells, especially neurons, can be uniquely identified from segment to segment. Leech embryos comprise identified cells, which facilitates developmental analysis. So far as it is known, cell lineages in leech are largely determinate. Prior to first cleavage, cytoplasmic reorganization generates domains of yolk-deficient cytoplasm called teloplasm. In situ hybridization experiments suggest that teloplasm is enriched for polyadenylated RNAs. During the first three, unequal cell divisions, teloplasm is segregated to macromere D'; normally, this cell alone cleaves further to generate five bilateral pairs of embryonic stem cells, M, N, O/P and Q teloblasts. Centrifugation experiments have shown a causal link between inheritance of teloplasm and the cleavage pattern that generates teloblasts. Teloblasts undergo highly unequal divisions, generating a longitudinal array of segmental founder cells called m, n, o, p and q blast cells, from which the definitive segmental tissues arise via further stereotyped cell divisions. Microinjecting new-born teloblasts or their precursors with polyadenylic acid induces the formation of supernumerary teloblasts. This discovery permits further analyses of factors specifying the five cell lines generating segmental tissues of the leech.

Animals↗
PubMed1989

Teloplasm formation in a leech, Helobdella triserialis, is a microtubule-dependent process.

Fertilized eggs of the leech Helobdella triserialis undergo a cytoplasmic reorganization which generates domains of nonyolky cytoplasm, called teloplasm, at the animal and vegetal poles. The segregation of teloplasm to one cell of the eight-cell embryo is responsible for a unique developmental fate of that cell, i.e., to give rise to segmental ectoderm and mesoderm. We have studied the cytoplasmic movements that generate teloplasm using time-lapse video microscopy; the formation and migration of rings of nonyolky cytoplasm were visualized using transmitted light, while the movements of mitochondria into these rings were monitored with epifluorescence after labeling embryos with rhodamine 123, a fluorescent mitochondrial dye. To examine the likelihood that cytoskeletal elements play a role in the mechanism of teloplasm formation in Helobdella, we examined the distribution of microtubules and microfilaments during the first cell cycle by indirect immunofluorescence and rhodamine-phalloidin labeling, respectively. The cortex of the early embryo contained a network of microtubules many of which were oriented parallel to the cell surface. As teloplasm formation ensued, microtubule networks became concentrated in the animal and the vegetal cortex relative to the equatorial cortex. More extensive microtubule arrays were found within the rings of teloplasm. Actin filaments appeared in the form of narrow rings in the cortex, but these varied apparently randomly from embryo to embryo in terms of number, size, and position. The role of microtubules and microfilaments in teloplasm formation was tested using depolymerizing agents. Teloplasm formation was blocked by microtubule inhibitors, but not by microfilament inhibitors. These results differ significantly from those obtained in embryos of the oligochaete Tubifex hattai, suggesting that the presumably homologous cytoplasmic reorganizations seen in these two annelids have different cytoskeletal dependencies.

Actin Cytoskeleton↗
PubMed1989

Animal and vegetal teloplasms mix in the early embryo of the leech, Helobdella triserialis.

In embryos of the glossiphoniid leech, Helobdella triserialis, as in many annelids, cytoplasmic reorganization prior to first cleavage generates distinct animal and vegetal domains of yolk-deficient cytoplasm, called teloplasm. Both domains are sequestered to the D' macromere, progenitor of the definitive segmental tissues, during the first three rounds of cell division. And it has been believed that during the fourth round of cell division, the obliquely equatorial cleavage of macromere D' cleanly segregates animal teloplasm into an ectodermal precursor, cell DNOPQ, and vegetal teloplasm into a mesodermal precursor, cell DM. But here we report a hitherto unobserved cytoplasmic rearrangement between the second and the fourth divisions that seems to mix the animal and vegetal domains of teloplasm. The newly observed rearrangement consists of the movement of vegetal teloplasm toward the animal pole of cell D' between the second and the fourth cell divisions. Animal and vegetal teloplasms form a single pool of teloplasm in cell D' which is then divided between DM and DNOPQ at the fourth division. The movement of teloplasm was inferred by examination of embryos fixed and sectioned between the second and the fourth rounds of cleavage and was confirmed in living embryos microinjected with rhodamine 123, a fluorescent mitochondrial stain.

Animals↗
PubMed1988

Segmentation in leech development.

Segments in glossiphoniid leeches, such as Helobdella triserialis, are the products of sterotyped cell lineages that yield identifiable cells from first cleavage. Cell lines generating segmental tissues are separated from those generating prostomial tissues early in development. Segments arise from five bilateral pairs of longitudinal columns of primary blast cells that are generated by five bilateral pairs of embryonic stem cells called teloblasts. There are four ectodermal cell lines (N, O, P and Q) and one mesodermal cell line (M) on each side of the embryo. In normal development, each cell line generates a segmentally iterated set of identified definitive progeny comprising a mixture of cell types. In the M, O and P cell lines, each blast cell generates one segment's worth of definitive progeny (segmental complement). But the clones of blast cells in each of these three cell lines interdigitate longitudinally with cells of the adjacent clones from the same line, so that the clone of an individual m, o and p blast cell is distributed across more than one segment. Thus, there is no simple clonal basis for morphologically defined segments. In the N and Q cell lines, two blast cells are required to produce one segmental complement of definitive progeny; in each of these two cell lines, two classes of blast cells (nf and ns, qf and qs) are produced in exact alternation. Primary n and q blast cells are about the same size and are produced at the same rate as blast cells for the o and p bandlets, but the longitudinal extent of their clones is roughly half that of the o and p blast cells' clones.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

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.

Animals↗
PubMed1987

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.

Animals↗
PubMed1987

Cell lineage, cell death, and the developmental origin of identified serotonin- and dopamine-containing neurons in the leech.

The nervous system of the glossiphoniid leech includes segmentally iterated neurons that contain serotonin (5-HT) and dopamine. These have been investigated in Helobdella triserialis, Theromyzon rude, and Haementeria ghilianii. Five types of 5-HT neurons are identified by immunocytochemistry in the abdominal ganglia of the ventral nerve cord: the bilaterally paired Retzius, anteromedial, ventrolateral and dorsolateral neurons, and the unpaired posteromedial (pm) neuron. Three types of bilaterally paired dopamine neurons are identified by glyoxylic acid-induced fluorescence in the segmental body wall: MD, LD1, and LD2. Each left or right half of the segmental complement of the leech nervous system is known to develop from 6 distinct ectodermal primary blast cells (ns, nf, o, p, qs, and qf). To identify the blast cells of origin of the 5-HT and dopamine neurons, fluorescent cell lineage tracers were injected into the various precursors of the blast cells in early (stage 6) embryos. The embryos were then raised until their 5-HT and dopamine neurons could be scored (stage 11) for the presence or absence of lineage tracer. We find that the Retzius, anteromedial, and posteromedial 5-HT neurons are derived from the ns blast cell, while the ventrolateral and dorsolateral 5-HT neurons are derived from the nf blast cell. The unpaired pm 5-HT neuron arises as one of a bilateral pair of neurons, of which one later dies. Whether the left or right pm neuron survives in any given ganglion is the consequence of some form of competitive interaction between cells derived from the left and right n primary blast cells, possibly between the left and right pm neurons themselves. We find that, of the dopamine neurons, the LD1 neuron is derived from the o blast cell, the LD2 neurons from the p blast cell, and the MD neuron from one of the 2 kinds of q blast cells. These results show that the 5-HT and dopamine neurons arise from 5 different primary blast cells in a highly determinate manner, and they support the view that cells of a similar phenotype need not be closely related in the developmental cell lineage tree.

Animals↗
PubMed1985

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.

Animals↗