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M Shankland

Publications and source records attributed to M Shankland.

At least 19 recordsLinked to original sources
PubMed1998

Expression of the head gene Lox22-Otx in the leech Helobdella and the origin of the bilaterian body plan.

The orthodenticle orthologue Lox22-Otx was isolated from an annelid worm, the leech Helobdella triserialis. In situ hybridization reveals that embryonic expression of Lox22-Otx RNA is primarily restricted to an unsegmented head domain, including tissues in the foregut, surface ectoderm, and the head ganglion of the central nervous system. The patterns of head expression form concentric rings about the stomadeum and mark tissue domains that exhibit discrete behaviors during later morphogenesis and differentiation. Expression was also observed in one to two bilateral pairs of neurons in each segmental ganglion or neuromere of the body trunk. The largely head-specific expression of Lox22-Otx in this annelid species supports data from two other bilaterian phyla in suggesting the existence of a genetically defined head/trunk distinction. We suggest here that this head/trunk distinction is a synapomorphy of the Bilateria as a whole, and that it reflects the body plan of an early bilaterian ancestor. In addition, we discuss the possibility that the radial organization of gene expression and cell lineages in the leech's head domain may reflect the symmetry properties of a prebilaterian ancestor that had a radially symmetric body plan.

Amino Acid Sequence↗
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↗
PubMed1997

Conserved anterior boundaries of Hox gene expression in the central nervous system of the leech Helobdella.

Molecular developmental studies of fly and mouse embryos have shown that the identity of individual body segments is controlled by a suite of homeobox-containing genes called the Hox cluster. To examine the conservation of this patterning mechanism in other segmented phyla, we here describe four Hox gene homologs isolated from glossiphoniid leeches of the genus Helobdella. Based on sequence similarity and phylogenetic analysis, the leech genes Lox7, Lox6, Lox20, and Lox5 are deemed to be orthologs of the Drosophila genes lab, Dfd, Scr, and Antp, respectively. Sequence similarities between Lox5 and Antp outside the homeodomain and phylogenetic reconstructions suggest that the Antennapedia family of Hox genes (as defined by Bürglin, 1994) had already expanded to include at least two discrete Antp and Ubx/abdA precursors prior to the annelid/arthropod divergence. In situ hybridization reveals that the four Lox genes described in this study are all expressed at high levels within the segmented portion of the central nervous system (CNS), with variable levels of expression in the segmental mesoderm. Little or no expression was seen in peripheral ectoderm or endoderm, or in the unsegmented head region (prostomium). Each Lox gene has a distinct anterior expression boundary within one of the four rostral segments, and the anterior-posterior (AP) order of these expression boundaries is identical to that reported for the orthologous Hox gene products in fly and mouse. This finding supports the idea that the process of AP axis differentiation is conserved among the higher metazoan phyla with respect to the regional expression of individual Hox genes along that axis. One unusual feature of leech Hox genes is the observation that some genes are only expressed during later development -- beginning at the time of terminal cell differentiation -- whereas others begin expression at a much earlier stage, and their RNA ceases to be detectable shortly after the onset of expression of the 'late' Hox genes. The functional significance of this temporal disparity is unknown, but it is noteworthy that only the two 'early' Hox genes display high levels of mesodermal expression.

Amino Acid Sequence↗
PubMed1996

Identification and characterization of a hunchback orthologue, Lzf2, and its expression during leech embryogenesis.

Lzf1 and Lzf2 are leech zinc finger (Lzf) genes that are shown to be orthologues to the Drosophila gap gene hunchback (hb). Neither in situ hybridization nor RT-PCR detected Lzf1 transcripts in leech embryos or adults. Lzf2 expression was examined in leech embryos at various stages by in situ hybridization. Lzf2 is expressed continually throughout the early embryonic cleavage divisions, including the period during which the embryo forms stem cells that will give rise to the segmented tissues of the adult. At the time of segmental pattern formation, Lzf2 RNA is expressed uniformly along the length of the segmented trunk in both the ectodermal and mesodermal tissues. This is in contrast to the anteriorly restricted gradient of hb RNA shown to be critical to the normal anteroposterior (AP) patterning of the insect embryo. Thus, this leech orthologue of hb does not appear to play a comparable role in the patterning of the AP axis. In addition, Lzf2 is expressed during organogenesis in segmentally restricted patterns in the central nervous system, the gut, and epidermally derived structures. Lzf2 is the first hb orthologue to be characterized in detail outside of insects and its expression pattern suggests that hb may have acquired a gap gene function in arthropods or insects after their phyletic separation from the annelids.

Amino Acid Sequence↗
PubMed1996

Segmental diversification of an identified leech neuron correlates with the segmental domain in which it expresses Lox2, a member of the Hox gene family.

The cellular colocalization of LOX2 protein and small cardioactive peptide (SCP)-like immunoreactivity was studied in the nerve cord of the glossiphoniid leech Helobdella triserialis. Of the six neurons that express SCP in the midbody segments 7 to 17, only one, the MPS neuron, expresses LOX2 protein. The medial paired SCP (MPS) neurons are segmentally repeated and can be divided into three contiguous segmental domains according to cell body size and the timing and level of SCP expression. MPS neurons located in the anterior and middle segmental domains express LOX2 protein. In the middle domain, large MPS neurons begin to accumulate SCP shortly after the end of embryonic development, whereas in the anterior domain the MPS neurons are smaller and begin to express SCP at a later stage. In the posterior domain the MPS neurons exhibit a third phenotype -- they have large cell bodies, express low levels of SCP starting from the midjuvenile stage, and do not show detectable LOX2 expression. Lineage tracer injections showed that the MPS neurons arise from a stereotyped cell lineage and are descended from the O teloblast stem cell. In midbody ganglia 2 to 6 and 18 to 21, there are lineally homologous neurons that do not express either LOX2 protein or SCP. Thus, the boundaries of LOX2 expression coincide precisely with two of the segmental boundaries of MPS differentiation, suggesting that expression of LOX2 at the level of this single identified neuron governs some, but not all, aspects of the neuron's segmental diversification.

Animals↗
PubMed1995

Formation and specification of neurons during the development of the leech central nervous system.

In the leech embryo, neurogenesis takes place within the context of a stereotyped cell lineage. The prospective germ layers are formed during the early cleavage divisions by the reorganization and segregation of circumscribed domains within the cytoplasm of the fertilized egg. The majority of central neurons arise from the ectoderm, and central neuroblasts are distributed throughout both the length and width of each ectodermal hemisegment. Much of the segmental ganglion arises from medial neuroblasts, but there are also lateral ectodermal neuroblasts and mesodermal neuroblasts that migrate into the nascent ganglion from peripheral sites of origin. Some of these migratory cells are committed to neurogenesis prior to reaching their central destination. In addition, the leech embryo exhibits a secondary phase of neurogenesis that is restricted to the two sex segment ganglia. Secondary neurogenesis requires that a mitogenic or trophic signal be conveyed from the peripherally located male sex organ to a particular set of centrally located neuroblasts, apparently via already differentiated central neurons that innervate the sex organ. The differential specification of neuronal phenotypes within the leech central nervous system occurs in multiple steps. Some aspects of a neuron's identity are already specified at the time of its terminal cell division and would seem to involve the lineal inheritance of developmental commitments made by one of the neuron's progenitors. This lineage-based identity can then be modified by interactions between the postmitotic neuron and other neurons or non-neuronal target cells encountered during its terminal differentiation.

Animals↗
PubMed1994

Leech segmentation: a molecular perspective.

A variety of leech homeobox genes have been identified by homology with genes that are known to bring about the regionalization and segmentation of the anteroposterior body axis in other organisms. Embryonic expression patterns suggest a number of interphyletic similarities in the way that these genes are utilized. However, several interesting differences have also been observed. In particular, transplantation experiments in the leech embryo have shown that axially aligned patterns of homeobox gene expression are not specified by a global pattern of positional cues. Rather, the leech independently establishes anteroposterior patterns of gene expression in each of five discrete stem cell lineages, and these patterns are brought into their final alignment through a process of morphogenetic assembly.

Animals↗
PubMed1994

An axial domain of HOM/Hox gene expression is formed by morphogenetic alignment of independently specified cell lineages in the leech Helobdella.

The homeobox gene Lox2, a member of the HOM/Hox gene class, is expressed in a restricted domain along the anteroposterior (A-P) body axis of the leech Helobdella. The segmental tissues of the leech embryo arise from the parallel merger of five distinct and bilaterally paired cell lineages generated by embryonic stem cells or teloblasts. Injection of cell lineage tracers coupled with anti-LOX2 immunochemistry reveals that all five teloblast lineages generate central nervous system neurons that express the LOX2 protein, and that each lineage expresses LOX2 within a similar domain of body segments. Some lineally identified neurons display anti-LOX2 immunoreactivity over the entire expression domain, but the OM7 neuron has a distinctively high level of LOX2 expression, which is restricted to the seventh midbody ganglion. To ascertain the role of positional information in the axial patterning of LOX2 expression, we performed focal cell ablations that displaced one or another of the teloblast lineages out of segmental register with the other axial tissues. Such displacements brought about a corresponding shift in the LOX2 expression of the perturbed lineage, and had little or no effect on the LOX2 expression of the other, unperturbed lineages. This result indicates that the axial domain of LOX2 expression is not specified by positional cues acting coordinately across the various teloblast lineages, nor would it seem that the expression domain is imprinted from one lineage to the others. Rather, the different teloblast lineages acquire their axial patterns independently, and secondarily bring these patterns into alignment along the A-P axis through a process of morphogenetic assembly.

Animals↗
PubMed1993

Lox10, a member of the NK-2 homeobox gene class, is expressed in a segmental pattern in the endoderm and in the cephalic nervous system of the leech Helobdella.

A novel leech homeobox gene, Lox10, is shown to encode a homeodomain sequence characteristic of a phyletically widespread NK-2 homeobox gene class. Lox10 expression was examined in leech embryos of various ages by in situ hybridization. In the unsegmented cephalic region, Lox10 RNA is expressed in a subset of the cells descended from the a' and b' micromeres, including a small cluster of cells, believed to be postmitotic neurons, within the supraesophageal ganglion of the central nervous system. Hybridization signal was not detected in either the mesoderm or ectoderm of the trunk segments, and the apparent restriction of Lox10 ectodermal expression to the nonsegmented cephalic domain resembles the restricted forebrain expression pattern of its mammalian homologues. Lox10 is also expressed within the endodermal tissues of the leech midgut, which arises by cellularization from a polynucleate syncytium. Endodermal expression is organized into a pattern of transverse stripes and spots which are aligned with the intersegmental septa, and which prefigure the pattern of gut wall constrictions observed at later stages of development. Lox10 is the first molecular marker of segmentally periodic endoderm differentiation reported for any animal species.

Amino Acid Sequence↗
PubMed1992

Lox2, a putative leech segment identity gene, is expressed in the same segmental domain in different stem cell lineages.

The segmented tissues of the adult leech arise from a set of five, bilaterally paired embryonic stem cells via a stereotyped sequence of cell lineage. Individual segments exhibit unique patterns of cell differentiation, and previous studies have suggested that each stem cell lineage establishes at least some aspects of its own segmental specificity autonomously. In this paper, we describe a putative leech segment identity gene, Lox2, and examine its expression in the various stem cell lineages. Both sequence analysis and the segmental pattern of Lox2 expression suggest a specific homology to the fruitfly segment identity genes Ubx and abdA. In situ hybridization reveals a cellular accumulation of Lox2 RNA over a contiguous domain of 16 midbody segments (M6-M21), including postmitotic neurons, muscles and the differentiating genitalia. Lox2 transcripts were not detected at the stage when segment identities are first established, suggesting that Lox2 gene products may not be part of the initial specification process. Individual stem cell lineages were labeled by intracellular injection of fluorescent tracers, and single cell colocalization of lineage tracer and hybridization reaction product revealed expression of Lox2 RNA in the progeny of four different stem cells. The segmental domain of Lox2 RNA was very similar in the various stem cell lineages, despite the fact that some stem cells generate one founder cell/segment, whereas other stem cells generate two founder cells/segment.

Amino Acid Sequence↗
PubMed1991

Origin of segmental identity in the development of the leech nervous system.

The leech embryo develops its segmental body plan by means of a stereotyped cell lineage. Each hemilateral segment arises from a small set of embryonic blast cells via a comparable sequence of formative cell divisions, and for the most part, lineally homologous cells manifest similar patterns of differentiation in the various hemisegments. Nonetheless, some identified central neurons undergo segment-specific or laterally asymmetric patterns of neuropeptide expression and/or cell death. Certain aspects of this regional diversification result from competitive cell interactions which occur at the level of the postmitotic neuron. However, the neuron's segmental identity is lineally determined, being inherited from its blast cell progenitor over several intervening rounds of mitosis. To learn more about the molecular basis of this phenomenon, we have isolated and begun to characterize leech homeobox genes which are related to the genes that govern segmental identity in other organisms.

Amino Acid Sequence↗
PubMed1990

Intrinsic segmental identity of segmental founder cells of the leech embryo.

Segmentation occurs in several animal phyla, and the cellular mechanisms generating this structural periodicity vary considerably. In the leech, an annelid worm, segmental founder cells arise through a fixed cell lineage (Fig. 1), and come together in a longitudinally repeating array through a stereotyped pattern of morphogenesis. In this paper we demonstrate that founder cells forced to differentiate in a foreign segmental environment give rise to their normal, segment-specific clones of neuronal descendants, even in segments in which those neuronal phenotypes would not normally be observed. These findings indicate that the individual founder cells possess segmental identity at or shortly after the time of their birth, and further suggest that such identities are established by a mechanism in which the parent stem cell 'counts' mitotic cycles.

Animals↗
PubMed1990

Neuronal competition determines the spatial pattern of neuropeptide expression by identified neurons of the leech.

Staining adult and embryonic leech ventral nerve cords with antibodies raised against the molluscan neuropeptides small cardioactive peptide B (SCP) and FMRFamide results in segment-specific and bilaterally asymmetric patterns of cell staining. One immunoreactive neuron, the RAS interneuron, is present in only four rostral segmental ganglia, while another, the CAS interneuron, is restricted to the four most caudal abdominal ganglia and tail. In addition to their segment-specific distributions, only one RAS or CAS cell is found in each segmental ganglion, and they alternate sides between adjacent ganglia (either L-R-L-R or R-L-R-L) with a fidelity of about 95%. This paper utilizes cell deletion techniques to investigate the determination of the asymmetric and alternating pattern of RAS and CAS neurons. We show that developmentally equivalent RAS and CAS homologs are present on both sides of the appropriate ganglia, and that within each ganglion one of the initially paired homologs loses the ability to assume the immunoreactive RAS or CAS fate 2-3 days after axonogenesis has begun. These experiments suggest that there is a competitive interaction between bilateral homologs which ensures that only one mature RAS/CAS neuron is formed per ganglion, and that contralateral RAS/CAS neurons are not required in the same or adjacent ganglia for the determination of the RAS or CAS developmental pathways. Nerve cord transections between ganglia in the CAS domain can alter the spatial pattern of CAS neuron determination, confirming that both bilateral homologs retain the ability to express neuropeptide until late embryonic stages, and suggesting that the alternating pattern of RAS/CAS cells requires communication between adjacent ganglia through the longitudinal connectives.

Animals↗
PubMed1990

Interactions between adjacent ganglia bring about the bilaterally alternating differentiation of RAS and CAS neurons in the leech nerve cord.

Antibodies to small cardioactive peptide (SCP) label a segmentally iterated subset of cells in the leech nerve cord, including the previously identified alternating SCP (AS) neurons. Unlike the majority of leech neurons, these cells are asymmetrically distributed in the adult nerve cord. Moreover, each AS neuron shows a strong tendency to lie on alternate right and left sides in successive ganglia. Previous work has shown that these unpaired neurons arise from bilaterally paired embryonic homologues, only 1 of which takes on the mature immunoreactive phenotype. The 2 AS homologues within a ganglion compete for this fate, in that either the right or the left homologue will become a mature AS neuron with a high degree of reliability if its contralateral homologue is ablated during embryogenesis. In this paper, we demonstrate the existence of interactions between neurons in adjacent ganglia that could account for the alternation of sides observed during normal development. The unilateral ablation of a single AS homologue neuron forced its contralateral homologue to take on the mature AS fate, and this consistently biased the side of AS development in adjacent, unlesioned ganglia both anterior and posterior to the lesion. One of the AS neurons, the caudal alternating SCP (CAS) cell, was injected with Lucifer yellow in adult nerve cords and was shown to have a large primary axon that extends into more anterior ganglia, as well as other, finer axons that are variable in number and arrangement. If the interganglionic interaction of AS neuron homologues is mediated by their primary axons, signals of developmental import must be transmitted both anterogradely and retrogradely along the axon's length. The present results indicate that the development of individual AS neurons is influenced by homologous cells located in the same and neighboring ganglia and suggest that the final, multisegmental patterning of the AS neuron distribution is not predetermined, but rather, arises as an emergent property of the cell interactions that occur during nervous system differentiation.

Animals↗
PubMed1989

Segmental specificity and lateral asymmetry in the differentiation of developmentally homologous neurons during leech embryogenesis.

This paper describes the embryonic development of three leech neurons which undergo spatially regulated patterns of differentiation. In leeches, the nervous system arises from an iterated array of embryonic cell lineages, and each neuron is represented by a set of bilaterally symmetric and segmentally repeated homologs. Two of the cells discussed here, the neurons nz4 and mz3, stain with antibodies to the neuropeptides SCP and FMRFamide during the course of their embryonic differentiation, but only a subset of the initially immunoreactive homologs continue to express this immunoreactivity into postembryonic life. Those nz4 cells which retain immunoreactivity are referred to as RAS neurons, and the persistently immunoreactive mz3 cells referred to as CAS neurons. The subset of homologs which show persistent expression is segment specific, such that the mature RAS and CAS neurons occupy different segmental domains. In addition, both neurons display a final pattern of expression which is laterally asymmetric, with only one of the two homologs in each segment maintaining the RAS or CAS phenotype. Asymmetric differentiation can occur in either orientation for any given segment, although there is a very strong tendency for the persistently immunoreactive cells to lie on opposite sides of successive segments. The fate of the transiently immunoreactive homologs is unclear, but labeling with intracellular lineage tracers suggests that there are some mz3 neurons which survive late into postemobryonic life and never express detectable levels of immunoreactivity. Intracellular lineage tracers also allowed us to follow the development of a third neuron, mz4, which does not stain for either peptide. The mz4 neuron is initially paired, but undergoes an asymmetric pattern of cell death which also shows a strong tendency to alternate sides in successive segments. These spatially coordinated patterns of neuronal survival and/or differentiation suggest that cell interactions play a role in determining the developmental choices made by individual neurons, and a subsequent paper will characterize those interactions through experimental manipulation.

Animals↗
PubMed1988

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.

Abdomen↗