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PubMed1987

Extension and retraction of axonal projections by some developing neurons in the leech depends upon the existence of neighboring homologues. I. The HA cells.

The role of homologues in the establishment of the pattern of axonal projections of identified segmentally homologous neurons was investigated by means of selective cell ablation and dye injection. The cells studied were the bilateral pairs of heart accessory (HA) neurons found in the fifth and sixth segmental ganglia of the leech ventral nerve cord. Homologues start their morphological differentiation with identical axonal projections, and segmental differences are manifested later, when specific branches stop growing and disappear. The deletion of single HA cells at early stages, however, permits these branches to survive in their ipsilateral homologues and to grow and take over the projections of the deleted neurons. In addition, if both HA homologues on the same side of the nerve cord, or three of the four HA cells, are deleted in an animal, the remaining HA cells often extend novel projections. These observations suggest that either competition for targets, inputs or growth factors, or direct interactions among homologous cells may play a role in the differentiation of segment specific patterns of axonal projections.

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↗
PubMed1989

Parallel processing and selection of the responses to serotonin during reinnervation of an identified leech neuron.

In an attempt to define the mechanism of synaptic specificity, we have been studying pairs of identified leech neurons isolated in tissue culture. The cultured neurons reform specific synapses when paired with appropriate partners in the absence of other cell types. In recent studies, we have examined in detail the reformation of a serotoninergic synapse between the Retzius cell and one of its targets, the pressure sensitive (P) cell. The P cell in vivo and its soma in vitro have two types of responses to serotonin (5-HT). From voltage clamp analysis of cultured P cells, we demonstrated the parallel activation of chloride (gCls) and monovalent cation (gCations) channels coupled to distinct receptor subtypes and gated by separate second messengers. Only gCls was activated by 5-HT released from the presynaptic Retzius cell both in vivo and in vitro. This demonstrates the remarkable specificity of the reformation of this synapse in culture since only the correct 5-HT receptor subtype is activated. An 80% reduction of gCations was observed in P cells that had failed to be innervated by Retzius cells in culture, suggesting that gCations may be lost prior to synapse formation. Retzius cells depleted of 5-HT also reduced gCations in the paired P cells and incubating single P cells in 5-HT did not reduce gCations. In addition, aldehyde-fixed Retzius cells were able to selectively reduce gCations when paired with P cells. We conclude that the loss of gCations was due to contact between the neurons. The early clearing of counter-effective receptor subtypes may be a prelude to synapse formation.

Animals↗
PubMed1990

Competition or inhibition? Developmental strategies in the establishment of peripheral projections by leech neurons.

Leech neurons, like those of other invertebrates and those of vertebrates, undergo specific interactions during development which serve to define their adult morphologies and synaptic connections. We review here several observations and experiments that illustrate these interactions. In particular, we consider how they shape and constrain peripheral arborizations and whether the evidence favors competition or inhibition as their mode of action.

Animals↗
PubMed1991

Using fluorescence photoablation to study the regeneration of singly cut leech axons.

The regeneration of the axons of leech Retzius cells was compared following two different methods of axonal severing: (1) a crush of the whole connective that includes the Retzius axon; and (2) photoablation of a small segment of only the Retzius axon. The photoablation was carried out after filling the Retzius cell with Lucifer Yellow (LY). Several tests were carried out to determine whether the photoablation actually severed the axon. These included (1) using the lipophilic membrane probe DiI as an indicator of membrane severance (2) electron microscopic examination of the photoablated axon after filling it with horseradish peroxidase (HRP); and (3) filling the Retzius cell first with HRP, then photoablating, and looking for the disappearance of the HRP in the photoablated region. These and other observations indicated that the photoablated axon was actually severed. Two differences were seen in the regeneration of the Retzius axon after crush versus after photoablation. First, the sprouting following crush was far more disorganized, and included significantly more lateral spread. Second, after photoablation, over 70% of the axons, upon refilling with LY after 3 days or more, showed the newly introduced LY suddenly extending far down the distal segment, indicating that the proximal and distal segments had become reconnected. This was never seen following a crush. The photoablated axons did not pass HRP into the distal segment, suggesting that the reconnection was not by fusion, but perhaps by a gap junction. The results show that axonal regeneration can take a dramatically different form than it does following a standard crush procedure if, instead, the axon is severed in a way that preserves the structural integrity of the surrounding tissue.

Animals↗
PubMed1994

Cell surface contact mediates neuronal recognition and synapse formation between two identified leech neurons.

An early event in the formation of the serotonergic synapse by the Retzius (R) onto the pressure-sensitive (P) neurons of the leech is the elimination of an extrasynaptic response to transmitter from sites of contact on the postsynaptic cell. This event during synapse formation is cell-specific in that it is elicited in vitro by contact with the presynaptic R cell but not with other neurons. In the study reported here, we investigated the nature of this interaction between R and P neurons. The loss of the extrasynaptic response of the P cell was elicited by contact with R cells fixed in a mild paraformaldehyde solution, but not by R cells treated with the proteolytic enzyme trypsin prior to fixation. As well, a variety of lectins were assayed for their ability to interfere with synapse formation. The transmitter responses of P cells plated on lectin-coated substrates were unaffected. However, exposure of the R cell to the lectin wheat germ agglutinin (WGA), but not to other lectins, prior to pairing prevented the loss of the extrasynaptic response in contacted P cells and blocked the formation of the R-P synapse in culture. We conclude that recognition by the P cell of the R cell during synapse formation may be mediated by an R cell-specific surface protein which binds wheat germ agglutinin.

Animals↗
PubMed1994

Specific pathway selection by the early projections of individual peripheral sensory neurons in the embryonic medicinal leech.

In leech, the central annulus of each midbody segment possesses seven pairs of sensilla, which are mixed clusters of primary peripheral sensory neurons that extend their axons into the CNS where they segregate into distinct fascicles. Pathway selection by individual afferent growth cones of sensillar neurons was examined by double labeling using intracellular dye-filling with antibody labeling in early Hirudo medicinalis embryos. The monoclonal antibody Lan3-2 was used because sensillar neuronal tracts are specifically labeled by this antibody. Examining 68 individually filled neurons we found that sensillar neuron growth cones bifurcate within the CNS, that they project long filopodia capable of sampling the local environment, and that all of them appeared to choose a single particular CNS fascicle without apparent retraction or realignment of growth cones. Furthermore, each side of the bifurcating afferent growth cones always chose the same fascicle, implying a specific choice of a distinct labeled pathway. By dye-filling individual central neurons (P-cells), we show that there are centrally projecting axons present at the time sensillar afferents enter the ganglionic primordia and select a particular fascicle, and we confirm that at least the dorsal peripheral nerve is likely to be pioneered by central neurons, not by the peripheral afferents. In the sensillum studied here, we found examples of sensory neurons extending axons into one of all the available fascicles. Thus, an individual embryonic sensillum possesses a heterogeneous population of afferents with respect to the central fascicle chosen. This is consistent with the idea that segregation into distinct axon fascicles may be based upon functional differences between individual afferent neurons. Our findings argue strongly in favor of specific pathway selection by afferents in this system and are consistent with previous suggestions that there exists a hierarchy of cues, including surface glycoconjugates that mediate navigation of the sensillar growth cones and the fasciculation of their axons.

Animals↗
PubMed1995

Filarin, a novel invertebrate intermediate filament protein present in axons and perikarya of developing and mature leech neurons.

The lan 3-8 monoclonal antibody recognizes a 63 kD antigen that is associated with the cytoskeleton in leech neurons. We have used this antibody to clone a novel invertebrate neuronal intermediate filament protein, filarin, by screening an expression vector library. A full-length clone of 2.2 kb identified by the antibody was isolated and sequenced. The protein contains a coiled-coil rod domain typical of the superfamily of intermediate filament proteins flanked by unique N- and C-terminal domains. The highest homology of filarin is to the alternatively spliced squid brain intermediate filament protein (Szaro et al., 1991, J. Biol. Chem. 266:15035-15041), the only other invertebrate neuronal intermediate filament in the data bank. However, apart from extensive homology in the two end regions of the rod domain, the similarity of the two proteins is limited to the general coiled-coil structure of intermediate filaments. Thus, filarin may represent a novel type of invertebrate neuronal intermediate filament protein. Filarin contains the extra 6 heptads characteristic of lamins and of all cytoplasmic invertebrate intermediate filaments analyzed so far. By Northern analysis, it appears that filarin is not alternatively spliced, since only a single transcript of 2.2 kb is recognized by the clone. Using the lan 3-8 antibody to follow its developmental expression, we found that filarin is present in all known neurons in the central and peripheral nervous system.

Amino Acid Sequence↗
PubMed1993

Selective pathway choice of a single central axonal fascicle by a subset of peripheral neurons during leech development.

In the CNS of leech, the central projections of peripheral sensory neurons segregate into three distinct axonal tracts during early development. We have previously shown that a subset of these neurons, recognized by the monoclonal antibody lan 4-2, projects axons into only one of these fascicles (Johansen et al., 1992, Neuron 8, 599). Here we report on a developmental and biochemical characterization of another fascicle-specific antigen labeled by the monoclonal antibody lan 3-6. By immunocytochemistry and double labelings we demonstrate that the lan 3-6 epitope is expressed only by a small subgroup of the peripheral neurons in Macrobdella embryos. The axons of these neurons selectively fasciculate in the CNS, but to only one of the three lan 3-2-positive tracts, which is different from the previously described lan 4-2-positive tract. These observations support the existence of a hierarchy of guidance cues mediating specific tract formation in this system. A biochemical analysis of the antigen suggests that it is likely to be a glycosylated protein with a molecular weight of approximately 200 kDa. Thus, the restricted expression of the lan 3-6 antigen and its biochemical properties are consistent with the hypothesis that this antigen may be playing a role in axonal guidance.

Animals↗
PubMed1994

Lox1, an Antennapedia-class homeobox gene, is expressed during leech gangliogenesis in both transient and stable central neurons.

The leech homeobox gene Lox1 contains a homeodomain homologous to those of Sex combs reduced and Antennapedia of Drosophila, and to homeodomains of the vertebrate Hox groups 5 and 6. Lox1 expression was detected during late gangliogenesis in 15-20 pairs of central neurons repeated in most segments, suggesting that Lox1 may play a role in the differentiation of these cells. The monoclonal antibody Laz1-1 identified two pairs of Lox1-expressing neurons as the Bipolar cells and the L1 neurons. The L1 neurons were detected late in gangliogenesis and became stable neurons. The Bipolar cells appeared very early in gangliogenesis, extended processes in the longitudinal connective nerves, and then degenerated, suggesting that they play a role in establishing the longitudinal tracts of the central nerve cord.

Amino Acid Sequence↗
PubMed1994

Formation of the male pronucleus, organization of the first interphase monaster, and establishment of a perinuclear plasm domain in the egg of the glossiphoniid leech Theromyzon rude.

Whole-mounted or sectioned eggs of the glossiphoniid leech Theromyzon rude were studied under the dissecting, fluorescence, light, and electron microscope. The egg is often penetrated by a single sperm that enters the animal hemisphere and becomes subjected to migration block. The latter is released shortly before or after discharge of the first pole cell, when the sperm centrosome initiates aster formation, the nucleus begins to be untwisted, and its chromatin decondensed. Sperm centration occurs along one side of the egg and appears to follow an arc-like trajectory as a result of vegetal and inward movements affected by colchicine and cytochalasin B but not by taxol. Results indicate that growing microtubules are needed for both movements, whereas actin filaments are essential for the vegetalward movement only. The sperm centrosome becomes the main microtubule organizing center (MTOC) of the egg and concomitantly originates the elaborate first interphase monaster. Additional peripherally situated MTOC form cytaster-like bodies whose visualization is improved by taxol treatment. A voluminous centrosphere, formed around the sperm centrosome, becomes a center of organelle accumulation, giving rise to a perinuclear plasm domain. This process seems to involve both import and replication of organelles.

Animals↗
PubMed1995

Kinetics of the inhibition of axonal defasciculation and arborization mediated by carbohydrate markers in the embryonic leech.

We studied carbohydrate interactions that mediate the targeting of sensory afferents in the synaptic neuropil of segmental central nervous system (CNS) ganglia in the embryonic leech. First, we determined the rate of sensory afferent development in vivo, and then we devised a culture system that permits the normal patterning of their projections in the CNS and PNS to proceed at 92% the normal rate. Using this in vitro system, we analyzed the mannose-specific recognition that mediates the defasciculation and arborization of sensory afferents in the CNS neuropil after they have tracked through peripheral nerves as a tight axon bundle. Sensory afferent defasciculation and arborization in the neuropil were inhibited by culturing embryos in Fab fragments directed against the mannose-containing surface epitope of sensory afferents. We demonstrate that the rate at which separately extending axons or their branches are lost from the neuropil can be modeled by a first-order decay process. These kinetic studies indicate that the loss of each separately extending axon or branch is an independent event. This suggests that sensory afferent projections extend autonomously across the target region in the search of their appropriate postsynaptic partners.

Animals↗
PubMed1995

Peripheral neurons depend on CNS-derived guidance cues for proper navigation during leech development.

In leech, major nerve pathways are pioneered by CNS neurons and evidence from dye-injection and antibody experiments suggest that they serve as guides for later differentiating neurons. In this study we have directly tested this hypothesis by examining the consequences of CNS ablation on the navigation in the periphery of a well-defined population of afferent sensory neurons. We show that in the absence of CNS-derived axons the axonal growth cones of this population of peripheral neurons extend with little directionality and instead of forming orderly projections, default into forming circular fasciculated pathways with each other. This suggests that CNS-derived guidance cues are absolutely required for the correct navigation of these peripheral sensory neurons.

Animals↗
PubMed1995

Formation of the female pronucleus and reorganization and disassembly of the first interphase cytoskeleton in the egg of the glossiphoniid leech Theromyzon rude.

Eggs of the leech Theromyzon rude were studied under dissecting, fluorescence, light, and electron microscopes. The sperm-derived centrosome divides and separates at early first interphase. The female pronucleus forms beside the male pronucleus at the centrally located perinuclear plasm domain, by fusion and remodelling of karyomeres that have descended from the animal pole at the tip of an ooplasmic process (centripetal ooplasmic flow). Retraction of this process (centrifugal ooplasmic flow) appears to return ooplasm to the animal pole. The reversible flow of ooplasm occurs along a subset of monaster microtubules that exhibit delayed depolymerization, and organelles thus move in both directions. Depolymerization of the first interphase monaster fibers seems to occur as a regular wave, moving from the internally located perinuclear plasm to the peripherally situated egg poles. However, disassembly of the ectoplasmic monaster fibers is preceded by furrowing and reorganization of both microtubules and microfilaments along polar rings and meridional bands. Bipolar contraction of microfilaments and shortening of microtubules lead to organelle concentration at the egg poles.

Actins↗
PubMed1997

Mesoderm is required for the formation of a segmented endodermal cell layer in the leech Helobdella.

The homeobox gene Lox3 is expressed in a segmentally iterated pattern within the endoderm of the leech Helobdella. We use that expression here to study endoderm differentiation following experimental ablations of mesoderm. Lox3 RNA was first detected by in situ hybridization at the stage when a definitive cellular endoderm is formed from its syncytial precursor and was never observed in derivatives of other germ layers. Expression is initially distributed throughout the endoderm, but rapidly disappears from specific regions of the nascent gut wall so as to produce a pattern of segmental stripes. The stripe pattern differs markedly between midgut organs, with thin stripes of Lox3 expression in the intercaecal constrictions of the crop and wide stripes of Lox3 expression marking the caecal bulges of the intestine. Lox3 expression in the rectum is not obviously segmental. Ablation of segmental mesoderm in the early Helobdella embryo prevents the formation of definitive endoderm and the expression of Lox3 RNA and leads to abnormalities in the morphogenesis of the gut tube. These endodermal deficits are precisely coextensive with the zone of mesodermal deficiency, suggesting that the mesoderm normally acts to promote the formation of the endodermal cell layer via local cell interactions. The segmental pattern of Lox3 expression is largely unaffected in portions of the endoderm surrounding such deficits, suggesting that endodermal segmentation is not established by lateral interactions within that tissue layer. Rather, we propose that the segmental organization of the endoderm is imprinted by vertical interactions with the segmental mesoderm.

Amino Acid Sequence↗
PubMed2000

Ectopic CNS projections guide peripheral neuron axons along novel pathways in leech embryos.

Previous studies have indicated that the formation of stereotyped segmental nerves in leech embryos depends on the interactions between CNS projections and ingrowing afferents from peripheral neurons. Especially, CNS-ablation experiments have suggested that CNS-derived guidance cues are required for the correct navigation of several groups of peripheral sensory neurons. In order to directly test this hypothesis we have performed transplantations of CNS ganglia into ectopic sites in segments from which the resident ganglia have been removed. We find that the transplanted ganglia extend numerous axons distributed roughly equally in all directions. When these CNS projections reach and make contact with peripheral sensory axons they are used as guides for peripheral neurons to grow toward and into the ectopic ganglia even when this means following novel pathways that cross the midline and/or segmental boundaries. The peripheral sensory axons turn and grow toward the ectopic ganglia only when in physical contact with CNS axons, suggesting that diffusible chemoattractants are not a factor. These results demonstrate that the guidance cues provided by ectopic CNS projections are both necessary and sufficient to steer peripheral sensory neuron axons into the CNS.

Animals↗
PubMed2002

Maternal and zygotic expression of a nanos-class gene in the leech Helobdella robusta: primordial germ cells arise from segmental mesoderm.

The nanos-class gene of the leech Helobdella robusta (Hro-nos) is present as a maternal transcript whose levels decay during cleavage; HRO-NOS protein is more abundant in the D quadrant cells relative to the A, B, and C quadrants; and HRO-NOS is more abundant in the ectodermal precursor cell (DNOPQ) than in its sister mesodermal precursor (DM) (Pilon and Weisblat, 1997). Here, using in situ hybridization, we show that Hro-nos mRNA is broadly distributed throughout the zygote, is concentrated in both animal and vegetal teloplasm during stage 1 and is at higher levels in DNOPQ than in DM at stage 4b. Hro-nos expression increases after stage 7, as judged by in situ hybridization, developmental RT-PCR, and western blots; this increase must therefore represent later zygotic expression. Of particular interest, during stages 9 and 10, each of 11 mid-body segments (M8-M18) has a pair of Hro-nos positive "spots" comprising of one or two large cells each. These spots later disappear in an anteroposterior progression. We find that these Hro-nos-expressing cells are of mesodermal origin, arising in a segmentally iterated manner from the M lineage, and correspond to cells previously proposed as primordial germ cells (PGCs; Bürger, 1891; Weisblat and Shankland, 1985). These results support the proposal that nanos-class genes functioned in the specification of germline cells in the ancestral bilaterian and possibly in a separate process related to embryonic polarity in the ancestral protostome.

Animals↗