Population genomics of sexual and asexual lineages in fissiparous ribbon worms (Lineus, Nemertea): hybridization, polyploidy and the Meselson effect.

Comparative population genetics in asexual vs. sexual species offers the opportunity to investigate the impact of asexuality on genome evolution. Here, we analyse coding sequence polymorphism and divergence patterns in the fascinating Lineus ribbon worms, a group of marine, carnivorous nemerteans with unusual regeneration abilities, and in which asexual reproduction by fissiparity is documented. The population genomics of the fissiparous L. pseudolacteus is characterized by an extremely high level of heterozygosity and unexpectedly elevated πN /πS ratio, in apparent agreement with theoretical expectations under clonal evolution. Analysis of among-species allele sharing and read-count distribution, however, reveals that L. pseudolacteus is a triploid hybrid between Atlantic populations of L. sanguineus and L. lacteus. We model and quantify the relative impact of hybridity, polyploidy and asexuality on molecular variation patterns in L. pseudolacteus and conclude that (i) the peculiarities of L. pseudolacteus population genomics result in the first place from hybridization and (ii) the accumulation of new mutations through the Meselson effect is more than compensated by processes of heterozygosity erosion, such as gene conversion or gene copy loss. This study illustrates the complexity of the evolutionary processes associated with asexuality and identifies L. pseudolacteus as a promising model to study the first steps of polyploid genome evolution in an asexual context.

Lineus as a model for studying developmental processes in animals reconstructed from adult pieces.

The difficulties that prevent reconstruction of animals by piecing together body fragments from several adults are overcome by using nemerteans of the genus Lineus. For 25 years I have managed to make viable composite worms by grafting body parts cut out of Lineus specimens either from the same clonal strain (syngeneically reconstructed worms) or from the same species (allogeneically reconstructed worms) or else from different species (xenogeneically reconstructed worms). Body reconstruction has usually been carried out orthotopically, i.e., the components of composite animals have been selected so as to be anatomically complementary. However, reconstruction has been made heterotopically when it was essential to obtain morphogenetic events in the adults. Here, I shall review some of the developmental processes that took place in such reconstructed animals. First, I shall report immune responses in composite worms derived from various combinations of body pieces grafted together. Second, I shall study sex differentiation during gonad development in growing or regenerating chimeric worms made by the grafting of male and female components. I shall refer also to gonadogenesis in the asexual progeny of bipartite chimeras derived from lateral body halves of both sexes fused together (clones of bilaterally allophenic worms). Third, I shall analyze regulative processes (regeneration, transgeneration) during localized morphogenesis occurring in heterotopically reconstructed worms. The data show how reconstructed Lineus may be exploited to increase our knowledge of developmental mechanisms, especially in the misunderstood field of organismal pattern homeostasis.

Expression of sex-specific molecular markers in clones of bipartite allophenic nemertines produced by somatic embryogenesis from Lineus sanguineus male/female chimera fragments.

SDS-PAGE electrophoresis showed major sex-specific proteins in sexually maturing and mature Lineus sanguineus. These "egg-specific" (145, 78 and 40 kDa) and "sperm-specific" (55,52 and 28 kDa) proteins are useful for studying sex differentiation in bilaterally allophenic worms produced by asexual reproduction of bipartite male/female chimeric worms. This study was carried out on 2 symmetrical clones of bilaterally allophenic worms, derived by somatic embryogenesis from fragments transected from chimeras obtained by exchange-grafting lateral body halves of male and female specimens, and from their asexually-derived progeny. The electrophoretic patterns of proteins extracted from sexually immature, maturing and mature allophenic animals from the 5th to the 19th year of cloning, showed the presence of all female-specific markers and the absence of male-specific markers. There was also complete biochemical feminization of the male halves. The synthesis of the only egg-specific molecules in initially male lateral body halves means that the long-term cloning results in the total repression of genes encoding sperm-specific proteins, since genetically male determinant-bearing cells can randomly re-express the testis characteristic as fertile but rudimentary male gonads.

Immunocompetent cells requisite for graft rejection in Lineus (Invertebrata, Nemertea).

Antecerebral ends from donors of one Lineus species (L. sanguineus) were grafted onto bispecific recipients previously constructed from two other Lineus species (denoted L. ruber----L. lacteus because the anterior component of chimeras was from L. ruber and the posterior component was from L. lacteus) and onto monospecific controls. Histological examination of areas where the tissues from L. sanguineus and L. ruber had been brought into contact by grafting always showed, at early stages, (6 to 20 days postgrafting), a great deal of difference depending upon whether the recipients were monospecific L. ruber or bispecific L. ruber----L. lacteus: only in grafts onto the former was there lysis of gland cells, connective tissue, muscular fibers, and finally epidermis. We attribute this lytic process to a strongly and rapidly cytotoxic action of lymphocyte-like cells from the L. ruber intestinal segment and the absence of lysis during the same stage in grafts onto composite recipients and monospecific L. lacteus to weak, delayed actions of immunocytes from the L. lacteus intestinal segment. Subsequent phagocytosis of material from lysed cell of grafts in the process of being rejected was effected by wandering amebocytes usually involved in destruction of degenerating "self" components, as in oosorption and resorptive processes after fasting. This work supports the existence of immunocytes at an early phylogenetic level.

Wrist arthrodesis using a dynamic compression plate.

We performed 45 wrist arthrodeses in 43 patients by a modification of the AO technique using the dynamic compression plate. Radiological follow-up was obtained in 41 wrists; all had united at a mean of ten weeks. Clinical follow-up was obtained in 32 wrists. Subjectively, the surgical outcome was satisfactory in 26, marginally satisfactory in two and unsatisfactory in four. This method is safe and reliable. The plate can be contoured to allow a variety of positions of fusion, and gives rigid immobilisation. The rate of union is higher than that for other techniques.

[Effects of gonad-inhibiting factor from the brain upon morphology and metabolism in germ cells and sexula somatic cells of nemertines (author's transl)]. / Effets du facteur cérébralgonado-inhibiteur (FGI) sur la morphologie et le métabolisme des cellules sexualisées germinales et somatiques des nemertiens.

After the deprivation of gonad-inhibiting factor (GIF) from the brain in adult Nemertines of both sexes, the sexual characters appear and/or grow rapidly. Very early, RNA synthesis is promoted in young oocytes, DNA synthesis is increased in spermatogonia and primary spermatocytes and protein synthesis is significantly increased in germ cells and sexual somatic cells. The brain origin of GIF suggests a polypeptide nature whereas its effects are those of sex steroid hormones in Vertebrates. GIF could be a regulating hormone which would inhibit the secretion of tropic hormone(s) produced by unknown cells in proximity to the sexual targets.

Structural and functional relationships between nuclear bodies and the nucleolus-DNA body complex in the oocyte of Amphiporus lactifloreus.

The localization, structure and function of two types of nuclear bodies have been investigated by cytological and cytochemical electron microscopy methods in oocytes from the Hoplonemertean, Amphiporus lactifloreus. Type I nuclear bodies differentiate in contact with the nucleolus-DNA body complex, whereas type II nuclear bodies develop close to the diplotenic chromosomal axes. The structure of type I and type II spherical nuclear bodies, 4-5 micron in width, results from the association of a fibrillar reticulum with some dense included regions. The cytochemical findings following the use of osmium-ammine reaction for DNA and silver reaction for NOR proteins support the hypothesis that type II nuclear bodies, derived from the extranucleolar area, as well as type I nuclear bodies, derived from the nucleolar complex, may be involved in ribosomal biogenesis.

An ultrastructural and cytochemical study of the nucleolus-DNA body complex of Amphiporus lactifloreus oocytes.

The components of the nucleolus-DNA body complex of oocytes from the hoplonemertean, Amphiporus lactifloreus, were studied by electron microscopy and cytochemistry. In the early stages of meiotic prophase I, the nucleus of female germ cells was characterized by a large heterochromatic body. From this body a polymorphic nucleolar apparatus, the nucleolus-DNA body complex, subsequently arose. This nucleolar complex was made up of spherical components termed nucleolar spherulae, which contained fibrillar electron-opaque material with cortical 20 nm dense granules, and characteristic ribbon-shaped components termed nucleolar lamellae also with dense granules. The latter components originated from the former by a process of self-peeling of the spherula cortex. A high level of transcriptional activity was located in the nucleolar spherulae which also contributed with the nucleolar lamellae to the formation of many secondary nucleoli and granular bodies. By ultrastructural cytochemistry a number of DNA-containing structures were visualized in nucleolar areas: first, cytochemically well-characterized DNA of partly decondensed chromatin was demonstrated in the thick cortex of spherulae and the inner part of lamellae arising from spherula cortex; second, more dispersed chromatin fibers, related with the cortical component, were revealed in the core of nucleolar spherulae, where ribonucleoproteins were also seen. A thin layer of ribonucleoproteins was also detected in the edge of both spherulae and lamellae of the nucleolar complex. These findings, which show that the nucleolar complex of previtellogenic oocytes of hoplonemerteans is basically dual, are discussed in terms of two synchronous functions: a) the start of the transcription in the nucleolus, b) dispersal of the intranuclear rDNA as a prerequisite to subsequent nucleolar transcription activity.

[Occurrence of circulating immunocytes in the process of graft rejection in nemertines of the genus Lineus (acelomate invertebrates)]. / Intervention d'immunocytes circulants dans le rejet des greffes chez les némertiens du genre Lineus (invertébrés, Acoelomates).

Performed upon bispecific Lineus chimaeras of a suitable constitution, the grafting of tissues excised from nemerteans of a third Lineus species resulted in a confrontation between graft cells, fixed cells from one chimaera component and mobile cells which originate from the second chimaera component. Survival of such grafts was dependent only upon compatibility in the "graft cells/mobile cells of recipient" interspecific combination. This result shows evidence for the existence of circulating immunocompetent cells which are responsible for the specific immune response to tissue transplantation in nemerteans of the genus Lineus.

Immune characteristics of graft rejection in nemerteans of the genus Lineus.

The rejection of xenogeneic grafts in marine worms of the genus Lineus (Nemertea) gives evidence for the occurrence of immune mechanisms in these invertebrates. First, second-set response is anamnestic with a three-month memory component. Second, the accelerated rejection of second-set grafts occurs anywhere in the body of the recipient, that is to say it is systemic. Third, the anamnestic response is species-specific since it takes place only when second grafts are from donors of the same species as that of the first set. It is therefore plausible that the reaction to xenogeneic grafts is a cell-mediated immune mechanism and that the self-nonself discrimination may be a function of nemertean cells specialized for recognition at the species level and for memory.

[Synthesis of RNAs and proteins in hoplonemertinian oocytes with and without the effect of gonadostatin (GIH)]. / Synthèse de RNAs et de protéines dans les oocytes d'hoplonémertes soumis et soustraits à l'influence de la gonadostatine (GIH).

Deprivation of gonadostatin (gonad-inhibiting hormone: GIH) performed during the quiescent period of the annual reproductive cycle in the hoplonemertean, Amphiporus lactifloreus, resulted in precocious sexual maturation. In female worms, the oometrical analysis showed that the hormone target, the ovary, responds to GIH deprivation by accelerated growth of a part of the oocyte population. The qualitative and quantitative histoautoradiographic examination of [3H]uridine and [3H]leucine incorporation enabled us to study RNAs and protein synthesis prerequisite and requisite for the oocyte growth. Early and continuous stimulation of protein synthesis in precociously growing oocytes was testified to by an increase in [3H]leucine incorporation into their ooplasm from day 6. On the other hand, later sequential stimulation of RNAs synthesis was observed, since a strong activation (induction?) of nucleoplasmic RNAs synthesis (tRNA and/or mRNA) from day 10 only took over from the slightly higher rDNA transcriptional activity seen in nucleolus from day 6 to day 9. By forcing the oocyte population of the ovary to retain a juvenile status, the inhibition of traduction into oocytes constitutes the earlier effect of gonadostatin. For bioassays by labelled precursor incorporation labelled aminoacids are the best candidates.

Sex differentiation in bilaterally allophenic animals produced by cloning of two bipartite male/female chimaeras of Lineus sanguineus.

Two bipartite chimaeras were constructed in Lineus sanguineus by grafting the lateral halves from a phenotypically dark-brown male onto the anatomically complementary halves from a phenotypically light-brown female. Regeneration of a large number of pieces transected from these two bilaterally allogeneic chimaeras produced two clones of bilaterally allophenic nemertines (male/female and female/male). Sex differentiation in the cloned worms started with a transitory stage of gonad developmental autonomy, termed the primary gynandromorphous state; at this stage there were young testes in the originally male lateral halves and juvenile ovaries in the originally female ones, the only abnormality then was that the ovarian development was more advanced than the testicular development relative to those in male and female controls. Then, unilateral sex reversal occurred, with feminization of the testes, i.e. oogenesis took the place of spermatogenesis in the many male gonads located in either the right or the left side of allophenic worms according to the symmetry patterns of the two clones. Finally, when the gonads reached maturity, both sides allophenic L. sanguineus contained only ovaries with ripe oocytes. The complete feminization of these allophenic worms and the previously observed masculinization of 'heterosexual' chimaeras in L. ruber suggest that a diffusible factor controls gonadal differentiation in worms of the prevailing sex, which is the female sex in L. sanguineus and the male sex in L. ruber.

Reverse homeosis in homeotically reconstructed ribbonworms.

Homeosis is the replacement of one body part by another, which may be caused by either developmental or genetic variations. It is particularly obvious in segmented animals, like insects, in which one body segment may be transformed into another. However, homeosis also occurs in animals without overt segmentation that also have detailed positional information specifying their body plan. By grafting, we have artificially generated homeotic ribbonworms of the species Lineus ruber with a duplicated ocellar region replacing the postocellar region anterior to the brain. Such chimeric animals are capable of complete morphogenetic regulation of the anterior-posterior (A-P) pattern. The missing postocellar region is restored by intercalary regeneration, and the anterior duplicated ocellar region is eliminated by a process called transgeneration. Thus, homeosis is reversed, and a completely normal pattern along the A-P axis is restored. This reverse homeosis involves the elimination of the syngeneic eyes and the survival of the grafted allogeneic eye region. LsPax-6, the Lineus sanguineus ortholog of the mammalian Pax-6 gene, which is considered to be a master control gene for eye morphogenesis, is expressed specifically in regenerating, regenerated, and intact eye regions. Our data show that ribbonworm eyes are either maintained or they regress according to their position along the A-P axis, even though there are no obvious segmental boundaries. This system allows us to test the function of LsPax-6 protein not only during eye regeneration but also during maintenance and regression of the eyes.

Homeobox genes in the ribbonworm Lineus sanguineus: evolutionary implications.

From our current understanding of the genetic basis of development and pattern formation in Drosophila and vertebrates it is commonly thought that clusters of Hox genes sculpt the morphology of animals in specific body regions. Based on Hox gene conservation throughout the animal kingdom it is proposed that these genes and their role in pattern formation evolved early during the evolution of metazoans. Knowledge of the history of Hox genes will lead to a better understanding of the role of Hox genes in the evolution of animal body plans. To infer Hox gene evolution, reliable data on lower chordates and invertebrates are crucial. Among the lower triploblasts, the body plan of the ribbonworm Lineus (nemertini) appears to be close to the common ancestral condition of protostomes and deuterostomes. In this paper we present the isolation and identification of Hox genes in Lineus sanguineus. We find that the Lineus genome contains a single cluster of at least six Hox genes: two anterior-class genes, three middle-class genes, and one posterior-class gene. Each of the genes can be definitely assigned to an ortholog group on the basis of its homeobox and its flanking sequences. The most closely related homeodomain sequences are invariably found among the mouse or Amphioxus orthologs, rather than Drosophila and other invertebrates. This suggests that the ribbonworms have diverged relatively little from the last common ancestors of protostomes and deuterostomes, the urbilateria.

External fixation of distal radial fractures.

From February 1995 until December 1997 we treated 48 patients aged between 26 and 79 years for distal radial fractures with external fixation. They were treated in an Arbeitsgemeinschaft fur Osteosynthestragen (A.O.) construct for periods of 5-10 weeks, the mean being 6 weeks. Forty patients were available for follow up. Our indications for this procedure were: severe comminuted displaced intra-articular fracture, severe shortening and inability to achieve fixation with other methods. The majority of patients had A.O. Classification type C fractures. Patients at follow up had objective clinical and radiological assessment as well as a subjective functional evaluation. Results were calculated using Stewart's modification of Gartland and Werley's assessment criteria. Functionally 90% achieved an excellent or good result. Radiographically 95% had an excellent or good result.