Nerve-dependent changes in clonable myoblast populations

Between the 3rd and 12th days of development at least four distinct classes of clonable myoblast are present in chick embryo leg skeletal muscle (N. K. White, P. H. Bonner, D. R. Nelson, and S. D. Hauschka, 1975, Develop. Biol.44, 346–361). In the present study, the behavior of each class has been examined quantitatively by clonal growth and differentiation of cells derived from denervated and normal embryos. Embryos functionally denervated by cauterization of the posterior spinal cord or by injection of the neuromuscular blocking agent d-tubocurarine exhibit changes in the two broad categories of clonable myoblast—fresh medium-sufficient (FMS) and conditioned medium-requiring (CMR) muscle cells. Clonal analysis of cells derived from leg muscle tissue of 10- to 12-day-old embryos denervated early in development (Days 3 to 6) has shown that the proportion of FMS clonable myoblasts is reduced to about 60% of the level found in normally innervated leg muscle. The CMR class is composed of three subclasses: CMR-I, CMR-II, and CMR-III (White et al., 1975). Clonal analysis of cells from denervated muscle has shown that, while the total CMR clone proportion is unchanged, the three subclasses are rearranged. The proportions of CMR-I and CMR-II muscle clones are greatly increased and the CMR-III subclass is severely reduced or absent when compared to clones derived from normally innervated leg muscle tissue.     

Pattern formation and determination in the antenna of the homoeotic mutant Antennapedia of Drosophila melanogaster

The development of the antenna in the antennal-leg homoeotic mutant Antennapedia (Antp^R) was investigated using somatic crossing-over to mark clones of cells in Antp^R antennal appendages. Antp^R antennae ranged from a nearly normal antenna to a nearly normal leg. The arrangement of clones of marked bristles and cuticle in the more antennalike antennae was similar to the wild type antenna, and that of the leglike antennae was similar to the wild-type leg. The contiguity of clones argued against extensive individual cell migration. The regions occupied by homoeotic leg varied considerably between different Antp^R antennae. Observation of Antp^R antennae in these phenotypic mosaics showed that specific leg parts replaced specific antennal parts. Even small groups of leg sensilla appeared only at precise locations in the antenna. These results suggest that homoeotic leg cells and antennal cells can both respond to the same positional information or prepattern. An analysis of clone size provided estimates for cell number in the Antp^R antenna. It was found that cell numbers in the wild-type and Antp^R antennae are about the same until the third instar, when the Antp^R cells start dividing more rapidly than wild type. Previous work had shown that clonal inheritance of a commitment for homoeotic leg also did not occur prior to the early third instar. It is suggested that determination for homoeotic leg occurs in the early third instar, and that thereafter this commitment is inherited by the progeny of the determined cells. The increase in growth rate is probably due to a faster growth rate in cells with a leg commitment than in cells with an antennal commitment. The results suggest that, once initiated, determination may be of two types—a clonally inherited determination (for example, to be homoeotic leg) and an environmental determination (for example, to be a specific part of a homoeotic leg). Clonal inheritance of determination in normal embryonic development and in sex determination in intersexes is discussed.     

Comparison of phenotypic and genetic clone delineation in quaking aspen, <Emphasis Type="Italic">Populus tremuloides</Emphasis>

Key message

Clonal delineation at nuclear microsatellites and phenotypic traits showed high correspondence and revealed an important role of both sexual and clonal reproduction for stand genetic structure.

Abstract

Quaking aspen (Populus tremuloides Michx.) grows throughout the northern and central portions of North America. Reproduction occurs both sexually via seeds and clonally from root suckers. Clonal delineation using morphological/phenological traits, and more recently, highly variable nuclear microsatellites have shown considerable variation in the size of clonal assemblies, and the relative importance of sexual versus clonal reproduction across the species range. In order to provide reliable estimates of genet size (N/G; ramets per sampled genet) and genotypic diversity (G/N; genets/ramets), and to compare genetic and phenotypic clone delineation, we characterized 181 sampled stems (ramets) at seven nuclear microsatellites, and morphological and phenological traits from six clones (genet size ≥11). Genotypic diversity was moderate (G/N = 0.18) and within the range reported in other studies across North America. Multivariate statistics revealed a high correspondence between genetic and phenotypic clone delineation, both with and without predefined genetic groups (94.2 %, 81.7 %). Moderate average genet size (5.6 ramets per genet) and the occurrence of genetically distinct single-ramet genets surrounded by larger genets suggested intermediate levels of sexual reproduction contributing to the genetic structure of this stand. Significant differences among genets were found for phenological and morphological traits such as bark thickness and leaf shape. However, most clones showed no significant differences in diameter growth which was likely caused by poor drainage in this high clay soil that inhibited the expression of genetic differences in growth.

     

Parameters of cell competition in the compartments of the wing disc of Drosophila.

When compartments in the wing disc of Drosophila are mosaic for two populations of cells, one of which is dividing more slowly, then the slower-growing population tends to be eliminated. The phenomenon leading to the disappearance of nonlethal, slowly dividing Minute cells was termed cell competition by Morata and Ripoll (1975) [Morata, G., and Ripoll, P. (1975). Develop. Biol.427, 211–221]. In this paper the different parameters of cell competition are explored. Starvation of the larvae rescued the Minute clones and permitted the following observations: The Minute clones grow to some extent before being out-competed. Prior to their disappearance, they become fragmented into small patches. Cell competition is greater in the centre of compartments than in the boundary regions. Possible causes of cell competition are discussed, as well as the hypothesis that the phenomenon may be related to the control of growth.     

Differentiation of chick embryo myoblasts is transiently sensitive to functional denervation

Clonal analysis of myoblast differentiation has been used to assess effects of denervation on developing skeletal muscle: chick embryo legs denervated by spinal cord cautery yield reduced proportions of clonable myoblasts (P. H. Bonner, 1978, Develop. Biol., 66, 207–219). The present work examines the effects on clonable myoblasts of functional denervation by d-tubocurarine. Curare treatment during the third or fourth days of embryonic development had no effect on clonable myoblasts later in development, treatment during the fifth or sixth days resulted in reduced proportions of clonable myoblasts, and treatment during the eighth or ninth days again had no effect. Clonal analysis of treated and control embryo leg muscle cells was performed between Days 10 and 18. Embryos were also permanently denervated by spinal cord cautery late in the sixth day. These embryos showed no effect of denervation on clonable myoblast proportion. It is concluded that the differentiation of skeletal muscle myoblasts is affected by interference with normal nerve-muscle relationships only during a “window” of sensitivity and that this “window” extends approximately from Hamburger and Hamilton stage 27 to stage 30.     

Drosophila twin spot clones reveal cell division dynamics in regenerating imaginal discs

Cell proliferation is required for tissue regeneration, yet the dynamics of proliferation during regeneration are not well understood. Here we investigated the proliferation of eye and leg regeneration in fragments of Drosophila imaginal discs. Using twin spot clones, we followed the proliferation and fates of sister cells arising from the same mother cell in the regeneration blastema. We show that the mother cell gives rise to two sisters that participate equally in regeneration. However, when cells switch disc identity and transdetermine to another fate, they fail to turn off the cell cycle and continue dividing long after regeneration is complete. We further demonstrate that the regeneration blastema moves as a sweep of proliferation, in which cells are displaced. Our results suggest that regenerating cells stop dividing once the missing parts are formed, but if they undergo a switch in cell fate, the proliferation clock is reset.     

Clonal analysis of vertebrate myogenesis: VII. Heritability of muscle colony type through sequential subclonal passages in vitro

Muscle colony-forming (MCF) cells from vertebrate limb buds show stage-specific differences in media requirements and clonal morphology (N. K. White, P. H. Bonner, D. R. Nelson, and S. D. Hauschka, 1975, Develop. Biol.44, 346–361). The cell lineage relationship, if any, between the different myogenic cell types is unknown. To investigate whether early MCF cells convert or mature into the late type, MCF cells from stage 23–33 chick embryo leg buds were grown in vitro and sequentially subcloned. Results indicate that colony type, no matter what the stage of origin, is heritable in vitro through at least five passages (45 in vitro doublings) or until the subclones reach proliferative senescence. These results are discussed with respect to various alternatives for the generation of MCF cell diversity during limb development.     

Spontaneous frequency of a developmental mutant in Volvox

Two types of mutants, those resistant to the base analog 5-bromo-2′-deoxyuridine (BrdU) and somatic regenerator (SR) mutants, have been analyzed in Volvox carteri. In somatic regenerator mutants, the somatic cells which are normally terminally differentiated dedifferentiate and regenerate gonidia [Sessoms, A., and Huskey, R. J. (1973). Proc. Nat. Acad. Sci. USA70, 1335–1338; Starr, R. C. (1970). Develop. Biol. Suppl.4, 59–100]. The SR phenotype allows recovery of SR mutations arising in somatic cells, since such somatic cells would regenerate gonidia and give rise to mutant clones. Mutants of any phenotype other than SR can only be recovered if the mutation first appears in a gonidium. Since the somatic cells are 100-fold more numerous than reproductive cells (gonidia), we have determined the spontaneous frequency of both somatic regenerator mutants and mutations to BrdU resistance in order to determine if the SR mutation exerts its effect in the gonidium or in the somatic cell. The two frequencies were found to be nearly identical, suggesting that the SR mutation must first appear in a gonidium in order to be expressed.     

Leg regeneration in insects. An experimental analysis in Drosophila and a new interpretation.

Fragments from prospective distal regions of Drosophila male foreleg imaginal discs failed to undergo proximal intercalary regeneration across leg segment borders when mechanically intermixed and cultured for 8 days with various fragments from prospective proximal disc regions. The failure of the distal cells to regenerate proximal leg segments was not due to a general restriction in their developmental potentials: Distal fragments, when deprived of their distal-most tips, regenerated in the distal direction at a high frequency. It is concluded that there exist in Drosophila leg discs the same restrictions with respect to regeneration along the proximodistal leg axis as had been previously observed in legs of several hemimetabolous insect species: Intersegmental discontinuities between grafted tissue pieces are not eliminated by intercalation. Based on the available evidence in hemimetabolous insects and in Drosophila, a new interpretation of the different aspects of regeneration in insect legs is offered. It is proposed that the two categories of regulative fields observed in insect legs, the leg segment fields and the whole leg field, represent the units of regulation for two fundamentally different regulative pathways that a cell at a wound edge can follow, the intercalative pathway and the terminal pathway, respectively. It is suggested that the criterion used by cells at healing wounds to choose between the two pathways is the difference in circumferential positional information between juxtaposed cells. The intercalative regulative pathway is switched on when cells with disparities in their axial positional information, or cells with less than maximal disparities in their circumferential information, contact one another. The terminal regulative pathway is triggered whenever cells with maximal circumferential disparities come into contact.     

Zebrafish as a model to assess cancer heterogeneity,progression and relapse

Clonal evolution is the process by which genetic and epigenetic diversity is created within malignant tumor cells. This process culminates in a heterogeneous tumor, consisting of multiple subpopulations of cancer cells that often do not contain the same underlying mutations. Continuous selective pressure permits outgrowth of clones that harbor lesions that are capable of enhancing disease progression, including those that contribute to therapy resistance, metastasis and relapse. Clonal evolution and the resulting intratumoral heterogeneity pose a substantial challenge to biomarker identification, personalized cancer therapies and the discovery of underlying driver mutations in cancer. The purpose of this Review is to highlight the unique strengths of zebrafish cancer models in assessing the roles that intratumoral heterogeneity and clonal evolution play in cancer, including transgenesis, imaging technologies, high-throughput cell transplantation approaches and in vivo single-cell functional assays.KEY WORDS: Cancer stem cell, Fluorescence, Intratumoral, Single cell, Targeted therapy, Tumor     

Regeneration and transdetermination: The role of wingless and its regulation

Imaginal discs of Drosophila have the remarkable ability to regenerate. After fragmentation wound healing occurs, ectopic wg is induced and a blastema is formed. In some, but not all fragments, the blastema will replace missing structures and a few cells can become more plastic and transdetermine to structures of other discs. A series of systematic cuts through the first leg disc revealed that a cut must transect the dorsal-proximal disc area and that the fragment must also include wg-competent cells. Fragments that fail to both transdetermine and regenerate missing structures will do both when provided with exogenous Wg, demonstrating the necessity of Wg in regenerative processes. In intact leg discs ubiquitously expressed low levels of Wg also leads to blastema formation, regeneration and transdetermination. Two days after exogenous wg induction the endogenous gene is activated, leading to elevated levels of Wg in the dorsal aspect of the leg disc. We identified a wg enhancer that regulates ectopic wg expression. Deletion of this enhancer increases transdetermination, but lowers the amount of ectopic Wg. We speculate that this lessens repression of dpp dorsally, and thus creates a permissive condition under which the balance of ectopic Wg and Dpp is favorable for transdetermination.     

Human Mesenchymal Stem Cells Self-Renew and Differentiate According to a Deterministic Hierarchy

Background

Mesenchymal progenitor cells (MPCs) have been isolated from a variety of connective tissues, and are commonly called “mesenchymal stem cells” (MSCs). A stem cell is defined as having robust clonal self-renewal and multilineage differentiation potential. Accordingly, the term “MSC” has been criticised, as there is little data demonstrating self-renewal of definitive single-cell-derived (SCD) clonal populations from a mesenchymal cell source.

Methodology/Principal Findings

Here we show that a tractable MPC population, human umbilical cord perivascular cells (HUCPVCs), was capable of multilineage differentiation in vitro and, more importantly, contributed to rapid connective tissue healing in vivo by producing bone, cartilage and fibrous stroma. Furthermore, HUCPVCs exhibit a high clonogenic frequency, allowing us to isolate definitive SCD parent and daughter clones from mixed gender suspensions as determined by Y-chromosome fluorescent in situ hybridization.

Conclusions/Significance

Analysis of the multilineage differentiation capacity of SCD parent clones and daughter clones enabled us to formulate a new hierarchical schema for MSC self-renewal and differentiation in which a self-renewing multipotent MSC gives rise to more restricted self-renewing progenitors that gradually lose differentiation potential until a state of complete restriction to the fibroblast is reached.     

Fingerprinting Microbial Assemblages from the Oxic/Anoxic Chemocline of the Black Sea

Biomass samples from the Black Sea collected in 1988 were analyzed for SSU genes from Bacteria and Archaea after 10 years of storage at −80°C. Both clonal libraries and direct fingerprinting by terminal restriction fragment length polymorphism (T-RFLP) analyses were used to assess the microbial community. Uniform and discrete depth distributions of different SSU phylotypes were observed. However, most recombinant clones were not restricted to a specific depth in the water column, and many of the major T-RFLP peaks remain uncharacterized. Of the clones obtained, an -Proteobacteria and a Pseudoalteromonas-like clone accounted for major peaks in the fingerprint, while deeply branching lineages of α- and γ-Proteobacteria were associated with smaller peaks. Additionally, members were found among both the δ-Proteobacteria related to sulfate reducers and the Archaea related to phylotypes from the ANME groups that anaerobically oxidize methane.     

Types of troponin components during development of chicken skeletal muscle

Changes in troponin components during development of chicken skeletal muscles have been investigated by using electrophoretic, immunoelectrophoretic, and immunoelectron microscopic methods. Previous reports (S. V. Perry and H. A. Cole, 1974, Biochem. J.141, 733–743; J. M. Wilkinson, 1978, Biochem. J.169, 229–238) pointed out that breast and leg muscles of adult chicken contain different types of troponin-T (TN-T), i.e., breast- and leg-type TN-T, respectively. However, the present paper indicates that the embryonic breast muscle contains leg-type TN-T. As development progresses two types of TN-T, i.e., breast- and leg-type TN-T, are found, and finally breast-type TN-T becomes the only species of TN-T present in the breast muscle. This change is well coordinated with the change of tropomyosin in the breast muscle. In contrast, the leg muscle contains leg-type TN-T through all the developmental stages. Leg-type TN-T is present in myogenic cells in vitro, irrespective of their origin, whether from the breast or leg muscle. The types of troponin-I and troponin-C in both breast and leg muscles do not change during development. The significance of the changes in the types of TN-T is discussed in terms of differential gene expression during development of chicken breast and leg muscles.     

High variation in clonal vs. sexual reproduction in populations of the wild strawberry, Fragaria virginiana (Rosaceae)

Background and Aims

Many plants reproduce both clonally and sexually, and the balance between the two modes of reproduction will vary among populations. Clonal reproduction was characterized in three populations of the wild strawberry, Fragaria virginiana, to determine the extent that reproductive mode varied locally between sites. The study sites were fragmented woodlands in Cook County, Illinois, USA.

Methods

A total of 95 strawberry ramets were sampled from the three sites via transects. Ramets were mapped and genotyped at five variable microsatellite loci. The variability at these five loci was sufficient to assign plants to clones with high confidence, and the spatial pattern of genets was mapped at each site.

Key Results

A total of 27 distinct multilocus genotypes were identified. Of these, 18 genotypes were detected only once, with the remaining nine detected in multiple ramets. The largest clone was identified in 16 ramets. No genets were shared between sites, and each site exhibited markedly different clonal and sexual recruitment patterns, ranging from two non-overlapping and widespread genets to 19 distinct genets. Only one flowering genet was female; the remainder were hermaphrodites.

Conclusions

Local population history or fine-scale ecological differences can result in dramatically different reproductive patterns at small spatial scales. This finding may be fairly widespread among clonal plant species, and studies that aim to characterize reproductive modes in species capable of asexual reproduction need to evaluate reproductive modes in multiple populations and sites.Key words: Clonal structure, gynodioecy, Fragaria virginiana, microsatellites, population genetic structure     

A clonal analysis reveals early developmental restrictions in the Drosophila head.

Head development of Drosophila melanogaster was studied by forming, in a background of Minute (M) cells, clones whose cell division rate was higher (M⁺). By studying such clones true developmental restrictions on clonal growth may be revealed, and not restrictions on clones which are just the result of interactions between neighboring cells. Pigment, bristle, and trichome markers allowed clone detection in both the compound eye and in much of the head cuticle. Clones were formed by X-ray-induced mitotic recombination at three stages in the first and second instar. Initial experiments determined some parameters of cell division in the compound eye and verified the differential division rate of M⁺ cells growing in an M background, and vice versa. The earliest and most striking developmental restriction on clonal growth divides the head into a dorsal and a ventral compartment. No evidence for anterior-posterior compartmentalization was found. By 75 hr of development in Minute flies, several lines of developmental restriction are formed which subdivide these two compartments. Evidence is presented which supports these conclusions: One subdivision may contain cells of different clonal origin, cells derived from the same progenitor blastoderm cell may be in different subdivisions, and each subdivision is formed from a group of contiguous cells.     

Influences of the genetic neighborhood on ramet reproductive success in a clonal desert cactus

Clonal structure in clonal plants can affect sexual reproduction. Individual ramets can decrease reproduction if their neighbors are ramets of the same genet due to inbreeding depression or self-incompatibility. We assessed ramet reproductive success in the partial self-incompatible Ferocactus robustus (Cactaceae) as a function of floral display size in focal ramets and floral display size and clonal structure of their reproductive neighborhoods. Ramets were labeled, sized in number of stems, mapped and genetically identified through RAPD markers in one population. A pollen dispersal area of 15-m radius was established for each ramet to determine the clonal diversity in the neighborhoods. Flower production and fruit set were counted on a monthly basis during one reproductive season as a surrogate of ramet fitness. We expected a decrease in individual ramet reproductive success as a function of the number of reproductive ramets of the same genet in the neighborhood. A total of 272 sampled ramets revealed 116 multilocus genotypes, showing high clonal diversity in the population (G/N = 0.43, D = 0.98). Clonal diversity of neighborhoods ranged from 0.06 to 1 and fruit set varied from 0 to 76.9%. Individual ramet reproductive success was influenced by (1) mate availability, (2) floral display size of a genet within the reproductive neighborhood, and (3) the proportion of distinguishable genotypes. Floral display size of genets and ramets coupled with the genetic diversity within the reproductive neighborhood determines the low sexual reproduction in F. robustus.     

Two populations of pluripotent stem cells in planarians <Emphasis Type="Italic">Girardia tigrina</Emphasis>

The positional differences in the regenerative capability of individual body parts of the planarian Girardia (Dugesia) tigrina were analyzed. The paper shows the significance of the size and positional differences of individual fragments of planarians for their regenerative capabilities, as well as the fundamental difference in the mechanisms of the head and tail blastema formation. A scheme of regeneration that includes two populations of pluripotent stem cells called neoblasts is suggested. The two populations of neoblasts differ in their role and distribution along the planarian body. Specifically, the population of neoblasts involved in the formation of any blastema migrates to the nearest blastema, and the population participating only in the creation of the head blastema migrates along the planarian body, following the gradient of biomass of the damaged axons arising after the amputation of the head end. The maximal size of the head blastema was found in the fragment obtained after cutting off the head fragment at the eye level, and the maximal portion of all pluripotent stem cells migrating into two blastemas was found in the fragment obtained by cutting the planarian above the mouth, followed by cutting off the head fragment at the eye level.     

Large-Scale Clonal Analysis Reveals Unexpected Complexity in Surface Ectoderm Morphogenesis

Background

Understanding the series of morphogenetic processes that underlie the making of embryo structures is a highly topical issue in developmental biology, essential for interpreting the massive molecular data currently available. In mouse embryo, long-term in vivo analysis of cell behaviours and movements is difficult because of the development in utero and the impossibility of long-term culture.

Methodology/Principal Findings

We improved and combined two genetic methods of clonal analysis that together make practicable large-scale production of labelled clones. Using these methods we performed a clonal analysis of surface ectoderm (SE), a poorly understood structure, for a period that includes gastrulation and the establishment of the body plan. We show that SE formation starts with the definition at early gastrulation of a pool of founder cells that is already dorso-ventrally organized. This pool is then regionalized antero-posteriorly into three pools giving rise to head, trunk and tail. Each pool uses its own combination of cell rearrangements and mode of proliferation for elongation, despite a common clonal strategy that consists in disposing along the antero-posterior axis precursors of dorso-ventrally-oriented stripes of cells.

Conclusions/Significance

We propose that these series of morphogenetic processes are organized temporally and spatially in a posterior zone of the embryo crucial for elongation. The variety of cell behaviours used by SE precursor cells indicates that these precursors are not equivalent, regardless of a common clonal origin and a common clonal strategy. Another major result is the finding that there are founder cells that contribute only to the head and tail. This surprising observation together with others can be integrated with ideas about the origin of axial tissues in bilaterians.     

Development of SCAR marker for sex identification in dioecious Garcinia gummi-gutta

Key message

We have developed sex-specific SCAR marker for the identification of dioecious Garcinia gummi - gutta (L.), which is useful for the selection of G. gummi - gutta at seedling stage and for plantation programmes.

Abstract

Garcinia gummi-gutta (L.) Robs. is a dioecious fruit yielding tree, which is naturally distributed as well as cultivated in the orchards in Western Ghat regions of India. A sex-linked DNA fragment was identified in Garcinia gummi-gutta (L.) Robs. by screening 150 randomly amplified polymorphic DNA primers and only one of them (OPBD20) showed different amplification band pattern associated with sex type. This sex-linked fragment was converted into male-specific sequence-characterized amplified region (SCAR) marker, CAM-566. The primers deigned in this study (OPBD20F and OPBD20R) correctly differentiated 12 male and 12 female plants at high annealing temperatures. Thus, a 556-bp band was amplified in male samples but not in female ones. Nevertheless, it should be noted that the fragments from both sexes were amplified at relatively low annealing temperatures. Additionally, the developed SCAR marker successfully identified the sexes of ten sex-unknown samples. Therefore, it can be used as an effective, convenient and reliable tool for sex determination in such dioecious species.