Regionalization and segmentation of the leech

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

Applications of mRNA injections for analyzing cell lineage and asymmetric cell divisions during segmentation in the leech Helobdella robusta

Synthetic mRNAs can be injected to achieve transient gene expression even for 'non-model' organisms in which genetic approaches are not feasible. Here, we have used this technique to express proteins that can serve as lineage tracers or reporters of cellular events in embryos of the glossiphoniid leech Helobdella robusta (phylum Annelida). As representatives of the proposed super-phylum Lophotrochozoa, glossiphoniid leeches are of interest for developmental and evolutionary comparisons. Their embryos are suitable for microinjection, but no genetic approaches are currently available. We have injected segmentation stem cells (teloblasts) with mRNAs encoding nuclear localized green fluorescent protein (nGFP) and its spectral variants, and have used tandem injections of nGFP mRNA followed by antisense morpholino oligomer (AS MO), to label single blast cell clones. These techniques permit high resolution cell lineage tracing in living embryos. We have applied them to the primary neurogenic (N) lineage, in which alternate segmental founder cells (nf and ns blast cells) contribute distinct sets of progeny to the segmental ganglia. The nf and ns blast cell clones exhibit strikingly different cell division patterns: the increase in cell number within the nf clone is roughly linear, while that in the ns clone is almost exponential. To analyze spindle dynamics in the asymmetric divisions of individual blast cells, we have injected teloblasts with mRNA encoding a tau::GFP fusion protein. Our results show that the asymmetric divisions of n blast cells result from a posterior shift of both the spindle within the cell and the midbody within the mitotic spindle, with differential regulation of these processes between nf and ns.     

Cell interactions in the developing leech embryo

The stereotyped pattern of cell commitments during leech embryogenesis is described. The nature of cell commitments during segmentation differs significantly between leech and fruit fly. Despite the constancy of cell fate assignments in normal development, ablation experiments show that cell interactions are essential in setting some of these commitments. Interacting cells follow a positionally determined hierarchy of fate choices. For other cells, which appear to have fates fixed from birth, the possibility of determinative interactions between mother and daughter cells is discussed.     

Cell differentiation lineage in the prostate

Prostatic epithelium consists mainly of luminal and basal cells, which are presumed to differentiate from common progenitor/stem cells. We hypothesize that progenitor/stem cells are highly concentrated in the embryonic urogenital sinus epithelium from which prostatic epithelial buds develop. We further hypothesize that these epithelial progenitor/stem cells are also present within the basal compartment of adult prostatic epithelium and that the spectrum of differentiation markers of embryonic and adult progenitor/stem cells will be similar. The present study demonstrates that the majority of cells in embryonic urogenital sinus epithelium and developing prostatic epithelium (rat, mouse, and human) co-expressed luminal cytokeratins 8 and 18 (CK8, CK18), the basal cell cytokeratins (CK14, CK5), p63, and the so-called transitional or intermediate cell markers, cytokeratin 19 (CK19) and glutathione-S-transferase-pi (GSTpi). The majority of luminal cells in adult rodent and human prostates only expressed luminal markers (CK8, CK18), while the basal epithelial cell compartment contained several distinct subpopulations. In the adult prostate, the predominant basal epithelial subpopulation expressed the classical basal cell markers (CK5, CK14, p63) as well as CK19 and GSTpi. However, a small fraction of adult prostatic basal epithelial cells co-expressed the full spectrum of basal and luminal epithelial cell markers (CK5, CK14, CK8, CK18, CK19, p63, GSTpi). This adult prostatic basal epithelial cell subpopulation, thus, exhibited a cell differentiation marker profile similar to that expressed in embryonic urogenital sinus epithelium. These rare adult prostatic basal epithelial cells are proposed to be the progenitor/stem cell population. Thus, we propose that at all stages (embryonic to adult) prostatic epithelial progenitor/stem cells maintain a differentiation marker profile similar to that of the original embryonic progenitor of the prostate, namely urogenital sinus epithelium. Adult progenitor/stem cells co-express both luminal cell, basal cell, and intermediate cell markers. These progenitor/stem cells differentiate into mature luminal cells by maintaining CK8 and CK18, and losing all other makers. Progenitor/stem cells also give rise to mature basal cells by maintaining CK5, CK14, p63, CK19, and GSTpi and losing K8 and K18. Thus, adult prostate basal and luminal cells are proposed to be derived from a common pleuripotent progenitor/stem cell in the basal compartment that maintains its embryonic profile of differentiation markers from embryonic to adult stages.     

Cell death in the oligodendrocyte lineage

We have recently found that about 50% of newly formed oligodendrocytes normally die in the developing rat optic nerve. When purified oligodendrocytes or their precursors are cultured in the absence of serum or added signalling molecules, they die rapidly with the characteristics of programmed cell death. This death is prevented either by the addition of medium conditioned by cultures of their normal neighboring cells in the developing optic nerve, or by the addition of platelet-derived growth factor (PDGF) or insulin-like growth factors (IGFs). Increasing PDGF in the developing optic nerve decreases normal oligodendrocyte death by up to 90% and doubles the number of oligodendrocytes, suggesting that this normally occurring glial cell death might result from a competition for limiting amounts of survival signals. These results suggest that competition for limiting amounts of survival factors is not confined to developing neurons, and raise the possibility that a similar mechanism may be responsible for some naturally occurring cell deaths in nonneural tissues. © 1992 John Wiley & Sons, Inc.     

The segmentation of the gnathobdellid leeches with special reference to the Indian leech Hirudinaria and medicinal leech Hirudo

Large number of annuli in Hirudinea are not true segments, and in the absence of spacious bodycavity and septa in adult no decision was taken regarding limit of a somite, until Gratiolet 1862 recognised a segment by colour marking, repetition of nephridial openings, and especially by the presence of segmental receptors, distinguishing first annulus of a segment. Whitman 1884 gave precision to these determinations and analyzed morphology of leeches to logical completeness. He recognised that though Hirudinaria and Hirudo have 102 body annuli and posterior sucker, true segments are only 26 plus 7. Castle ('00) and Moore ('00) proposed a new scheme of segmentation, with segmental receptor bearing annulus, as central annulus of a complete somite, with nerve ganglion, like that of other annelids, in center of a segment. They orientated everything roundabout the ganglion without noticing distorted fate of organ system. In this paper both the views are compared. Morphological and embryological studies reveal that the annulus bearing the segmental receptors in uniformly first annulus of all segments, including incomplete segments at the two extremities, with nerve ganglion in first annulus of the segment. Clitellum occupies three natural segments, IX, X, XI; crop caeca, nephridia, testis sacs, haemocoelomic channels and “rhomboidal figures” formed by ventrolaterals, all make a complete unit, well integrated in such segment. Conclusive evidence comes from the presence of septa at the level of each nerve ganglion in embryos of Hirudinaria. These observations corroborate Gratiolet and Whitman's view.     

Cell death in the oligodendrocyte lineage.

We have recently found that about 50% of newly formed oligodendrocytes normally die in the developing rat optic nerve. When purified oligodendrocytes or their precursors are cultured in the absence of serum or added signalling molecules, they die rapidly with the characteristics of programmed cell death. This death is prevented either by the addition of medium conditioned by cultures of their normal neighboring cells in the developing optic nerve, or by the addition of platelet-derived growth factor (PDGF) or insulin-like growth factors (IGFs). Increasing PDGF in the developing optic nerve decreases normal oligodendrocyte death by up to 90% and doubles the number of oligodendrocytes, suggesting that this normally occurring glial cell death might result from a competition for limiting amounts of survival signals. These results suggest that competition for limiting amounts of survival factors is not confined to developing neurons, and raise the possibility that a similar mechanism may be responsible for some naturally occurring cell deaths in nonneural tissues.     

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     

Cell lineage: Compartments and Capricious

Until recently, little was known about the mechanisms that prevent cell migration across compartment boundaries in Drosophila. A new report suggests that the lineage restriction between the dorsal and ventral compartments of the developing wing relies in part on the transmembrane proteins, Capricious and Tartan.     

Cell lineage in mammalian craniofacial mesenchyme

We have analysed the contributions of neural crest and mesoderm to mammalian craniofacial mesenchyme and its derivatives by cell lineage tracing experiments in mouse embryos, using the permanent genetic markers Wnt1-cre for neural crest and Mesp1-cre for mesoderm, combined with the Rosa26 reporter. At the end of neural crest cell migration (E9.5) the two patterns are reciprocal, with a mutual boundary just posterior to the eye. Mesodermal cells expressing endothelial markers (angioblasts) are found not to respect this boundary; they are associated with the migrating neural crest from the 5-somite stage, and by E9.5 they form a pre-endothelial meshwork throughout the cranial mesenchyme. Mesodermal cells of the myogenic lineage also migrate with neural crest cells, as the branchial arches form. By E17.5 the neural crest-mesoderm boundary in the subectodermal mesenchyme becomes out of register with that of the underlying skeletogenic layer, which is between the frontal and parietal bones. At E13.5 the primordia of these bones lie basolateral to the brain, extending towards the vertex of the skull during the following 4-5 days. We used DiI labelling of the bone primordia in ex-utero E13.5 embryos to distinguish between two possibilities for the origin of the frontal and parietal bones: (1) recruitment from adjacent connective tissue or (2) proliferation of the original primordia. The results clearly demonstrated that the bone primordia extend vertically by intrinsic growth, without detectable recruitment of adjacent mesenchymal cells.     

Cell lineage in plant development.

Lineage analyses in several plant species demonstrate that meristematic cells proliferate in a predictable manner to form the differentiated tissues of the mature shoot system. These studies also demonstrate, however, that the fates of meristematic cells are not absolutely dependent on their lineage. This variability indicates that interactions between cells must play a role in morphogenesis.     

Cell lineage in the developing neural tube.

Acquisition of cell type specific properties in the spinal cord is a process of sequential restriction in developmental potential. A multipotent stem cell of the nervous system, the neuroepithelial cell, generates central nervous system and peripheral nervous system derivatives via the generation of intermediate lineage restricted precursors that differ from each other and from neuroepithelial cells. Intermediate lineage restricted neuronal and glial precursors termed neuronal restricted precursors and glial restricted precursors, respectively, have been identified. Differentiation is influenced by extrinsic environmental signals that are stage and cell type specific. Analysis in multiple species illustrates similarities between chick, rat, mouse, and human cell differentiation. The utility of obtaining these precursor cell types for gene discovery, drug screening, and therapeutic applications is discussed.     

Cell lineage conversion in the sea urchin embryo

The mesoderm of the sea urchin embryo conventionally is divided into two populations of cells; the primary mesenchyme cells (PMCs), which produce the larval skeleton, and the secondary mesenchyme cells (SMCs), which differentiate into a variety of cell types but do not participate in skeletogenesis. In this study we examine the morphogenesis of embryos from which the PMCs have been removed microsurgically. We confirm the observation of Fukushi (1962) that embryos lacking PMCs form a complete skeleton, although in a delayed fashion. We demonstrate by microsurgical and cell marking experiments that the appearance of skeletogenic cells in such PMC-deficient embryos is due exclusively to the conversion of other cells to the PMC phenotype. Time-lapse video recordings of PMC-deficient embryos indicate that the converting cells are a subpopulation of late-ingressing SMCs. The conversion of these cells to the skeletogenic phenotype is accompanied by their de novo expression of cell surface determinants normally unique to PMCs, as shown by binding of wheat germ agglutinin and a PMC-specific monoclonal antibody. Cell transplantation and cell marking experiments have been carried out to determine the number of SMCs that convert when intermediate numbers of PMCs are present in the embryo. These experiments indicate that the number of converting SMCs is inversely proportional to the number of PMCs in the blastocoel. In addition, they show that PMCs and converted SMCs cooperate to produce a skeleton that is correct in both size and configuration. This regulatory system should shed light on the nature of cell-cell interactions that control cell differentiation and on the way in which evolutionary processes modify developmental programs.     

Gangliogenesis in leech: morphogenetic processes leading to segmentation in the central nervous system

 Using intracellular lineage tracers to study the main neurogenic lineage (N lineage) of the glossiphoniid leech embryo, we have characterized events leading from continuous columns of segmental founder cells (nf and ns primary blast cells) to discrete, segmentally iterated ganglia. The separation between prospective ganglia was first evident as a fissure between the posterior boundary of nf- and the anterior boundary of ns-derived progeny. We also identified the sublineages of nf-derived cells that contribute parallel stripes of cells to each segment. These stripes of cells project ventrolaterally from the dorsolateral margin of each nascent ganglion to the ventral body wall. The position and orientation of the stripes suggests that they play a role in forming the posterior segmental nerve; they are not coincident with the ganglionic boundary, and they form well after the separation of ganglionic primordia. Previous work has shown that cells in the anterior stripe express the leech engrailed-class gene. Thus, in contrast to the role of cells expressing engrailed in Drosophila, the stripes of N-derived cells expressing an engrailed-class gene in leech do not seem to play a direct role in segmentation or segment polarity. Received: 10 October 1997 / Accepted: 12 December 1997     

Cell lineage in plant development.

Lineage analyses in several plant species demonstrate that meristematic cells proliferate in a predictable manner to form the differentiated tissues of the mature shoot system. These studies also demonstrate, however, that the fates of meristematic cells are not absolutely dependent on their lineage. This variability indicates that interactions between cells must play a role in morphogenesis.