High potential for formation and persistence of chimeras following aggregated larval settlement in the broadcast spawning coral, Acropora millepora

In sessile modular marine invertebrates, chimeras can originate from fusions of closely settling larvae or of colonies that come into contact through growth or movement. While it has been shown that juveniles of brooding corals fuse under experimental conditions, chimera formation in broadcast spawning corals, the most abundant group of reef corals, has not been examined. This study explores the capacity of the broadcast spawning coral Acropora millepora to form chimeras under experimental conditions and to persist as chimeras in the field. Under experimental conditions, 1.5-fold more larvae settled in aggregations than solitarily, and analyses of nine microsatellite loci revealed that 50 per cent of juveniles tested harboured different genotypes within the same colony. Significantly, some chimeric colonies persisted for 23 months post-settlement, when the study ended. Genotypes within persisting chimeric colonies all showed a high level of relatedness, whereas rejecting colonies displayed variable levels of relatedness. The nearly threefold greater sizes of chimeras compared with solitary juveniles, from settlement through to at least three months, suggest that chimerism is likely to be an important strategy for maximizing survival of vulnerable early life-history stages of corals, although longer-term studies are required to more fully explore the potential benefits of chimerism.     

Natural chimerism in colonial urochordates

Natural chimeras are commonly distributed in the wild, challenging the traditional paradigm for the advantages of genetically homogenous entities, where uniclonality prevents within-organism conflicts. This essay focuses on the last two-decade studies on chimerism in the cosmopolitan urochordate Botryllus schlosseri, enlightening and focusing the idea of multichimeras as a primary tool for fending off the pervasiveness of super parasitic germ lines. Interacting Botryllus colonies may fuse or reject each other based on allelic compatibility on a single highly polymorphic gene locus. After fusion and establishment of a chimera, a second tier of allorecognition is developed, expressed as genetically controlled morphological resorption of one of the chimeric partners. This is followed by the third tier of allorecognition where somatic and germ cell lineages parasitism are developed. Studies revealed a complex network of costs and few suggested benefits for the state of chimerism in botryllid ascidians. Two life history traits (diversification of allorecognition allele repertoire, colonial programmed lifespan) were considered as selected to combat the major cost of chimeric associated germ cell parasitism. Three other ecological traits (heterosis, settlement of kin larvae in aggregates, multichimerism) have been suggested as selected to enhance the existence of chimerism in botryllid ascidians. Recent results revealing a fine-tuning of the chimerical somatic genetic components in response to changes in environmental conditions are discussed. Results further elucidate the possible existence of multichimeras, each made of several genotypes. It is proposed that natural multichimeras form more stable and vigorous entities, depicting a unique way for domesticating consortia of selfish cells that may otherwise seriously threaten survivorship of the entity.     

Chimerism in Wild Adult Populations of the Broadcast Spawning Coral Acropora millepora on the Great Barrier Reef

Background

Chimeras are organisms containing tissues or cells of two or more genetically distinct individuals, and are known to exist in at least nine phyla of protists, plants, and animals. Although widespread and common in marine invertebrates, the extent of chimerism in wild populations of reef corals is unknown.

Methodology/Principal Findings

The extent of chimerism was explored within two populations of a common coral, Acropora millepora, on the Great Barrier Reef, Australia, by using up to 12 polymorphic DNA microsatellite loci. At least 2% and 5% of Magnetic Island and Pelorus Island populations of A. millepora, respectively, were found to be chimeras (3% overall), based on conservative estimates. A slightly less conservative estimate indicated that 5% of colonies in each population were chimeras. These values are likely to be vast underestimates of the true extent of chimerism, as our sampling protocol was restricted to a maximum of eight branches per colony, while most colonies consist of hundreds of branches. Genotypes within chimeric corals showed high relatedness, indicating that genetic similarity is a prerequisite for long-term acceptance of non-self genotypes within coral colonies.

Conclusions/Significance

While some brooding corals have been shown to form genetic chimeras in their early life history stages under experimental conditions, this study provides the first genetic evidence of the occurrence of coral chimeras in the wild and of chimerism in a broadcast spawning species. We hypothesize that chimerism is more widespread in corals than previously thought, and suggest that this has important implications for their resilience, potentially enhancing their capacity to compete for space and respond to stressors such as pathogen infection.     

Intracolonial genetic variation in the scleractinian coral Seriatopora hystrix

In recent years, increasing numbers of studies revealed intraorganismal genetic variation, primarily in modular organisms like plants or colonial marine invertebrates. Two underlying mechanisms are distinguished: Mosaicism is caused by somatic mutation, whereas chimerism originates from allogeneic fusion. We investigated the occurrence of intracolonial genetic variation at microsatellite loci in five natural populations of the scleractinian coral Seriatopora hystrix on the Great Barrier Reef. This coral is a widely distributed, brooding species that is at present a target of intensive population genetic research on reproduction and dispersal patterns. From each of 155 S. hystrix colonies, either two or three samples were genotyped at five or six loci. Twenty-seven (~17%) genetically heterogeneous colonies were found. Statistical analyses indicated the occurrence of both mosaicism and chimerism. In most cases, intracolonial variation was found only at a single allele. Our analyses suggest that somatic mutations present a major source of genetic heterogeneity within a single colony. Moreover, we observed large, apparently stable chimeric colonies that harbored clearly distinct genotypes and contrast these findings with the patterns typically observed in laboratory-based experiments. We discuss the error that mosaicism and chimerism introduce into population genetic analyses.     

Development of either split tolerance or robust tolerance along with humoral tolerance to donor and third-party alloantigens in nonmyeloablative mixed chimeras

Hematopoietic chimerism is considered to generate robust allogeneic tolerance; however, tissue rejection by chimeras can occur. This "split tolerance" can result from immunity toward tissue-specific Ags not expressed by hematopoietic cells. Known to occur in chimeric recipients of skin grafts, it has not often been reported for other donor tissues. Because chimerism is viewed as a potential approach to induce islet transplantation tolerance, we generated mixed bone marrow chimerism in the tolerance-resistant NOD mouse and tested for split tolerance. An unusual multilevel split tolerance developed in NOD chimeras, but not chimeric B6 controls. NOD chimeras demonstrated persistent T cell chimerism but rejected other donor hematopoietic cells, including B cells. NOD chimeras also showed partial donor alloreactivity. Furthermore, NOD chimeras were split tolerant to donor skin transplants and even donor islet transplants, unlike control B6 chimeras. Surprisingly, islet rejection was not a result of autoimmunity, since NOD chimeras did not reject syngeneic islets. Split tolerance was linked to non-MHC genes of the NOD genetic background and was manifested recessively in F(1) studies. Also, NOD chimeras but not B6 chimeras could generate serum alloantibodies, although at greatly reduced levels compared with nonchimeric controls. Surprisingly, the alloantibody response was sufficiently cross-reactive that chimerism-induced humoral tolerance extended to third-party cells. These data identify split tolerance, generated by a tolerance-resistant genetic background, as an important new limitation to the chimerism approach. In contrast, the possibility of humoral tolerance to multiple donors is potentially beneficial.     

Coalescing red algae exhibit noninvasive,reversible chimerism

Chimerism is produced by the somatic fusion of two or more genetically distinct conspecific individuals. In animals, the main cost of fusion is competition between genetically different cell lineages and the probability of original cell line replacement by more competitive invasive lines, which limits its natural frequency (3%–5%). In red and brown seaweeds, chimerism is widespread (27%–53%), seemingly without the negative outcomes described for animals. The rigidity of cell walls in macroalgae prevents cell motility and invasions. In addition, in moving waters, most somatic fusions involve the holdfast. Histological observations in laboratory‐built bicolor macroalgal chimeras indicated that upright axes emerge from the base of plants by proliferation and vertical growth of discrete cell groups that include one or just a few of the cell lineages occurring in the holdfasts. Laboratory experiments showed growth competition between cell lineages, thus explaining lineage segregation during growth along originally chimeric erect axes. Genotyping of the axes showed more heterogeneous tissues basally, but apically more homogeneous ones, generating a vertical gradient of allele abundance and diversity. The few chimeric primary branches produced, eventually became homogenous after repeated branching. Therefore, coalescing macroagae exhibit a unique pattern of post‐fusion growth, with the capacity to reverse chimerism. This pattern is significantly different from those in animals and land plants, suggesting chimerism is a biologically heterogeneous concept.     

Chimeric honeybees (Apis mellifera) produced by transplantation of embryonic cells into pre-gastrula stage embryos and detection of chimerism by use of microsatellite markers

The production of chimeras, by use of cell transplantation, has proved to be highly valuable in studies of development by providing insights into cell fate, differentiation, and developmental potential. So far, chimeric honeybees have been created by nuclear transfer technologies. We have developed protocols to produce chimeric honeybees by use of cell transplantation. Embryonic cells were transplanted between pre-gastrula stage embryos (32-34 hr after oviposition) and hatched larvae were reared in vitro for 4 days. Chimeric individuals were detected by use of microsatellite analysis and a conservative estimation approach. 4.8% of embryos, posteriorly injected with embryonic cells, developed into chimeric honeybee larvae. By injection of cells pre-stained with fluorescent cell tracer dye, we studied the integration of transplanted cells in the developing embryos. Number of injected cells varied from 0 to 50 and cells remained and multiplied mainly in the area of injection.     

Viability of blastocysts produced by aggregation of two half-embryos in the mouse

Although methods for production of chimeras from early cleavage stages have been well established, little research has been directed toward production of genetically identical chimeric offspring. This study was designed to examine survival of blastocysts produced by aggregation of two halved eight-cell stage embryos from two different mouse strains. Four blastomeres of an eight-cell embryo from a pigmented strain were aggregated with four blastomeres of an eight-cell embryo from a nonpigmented strain. Aggregates were cultured for 48 h and transferred as blastocysts to synchronized recipients of three treatment groups. Viability was determined by examining the number of offspring produced relative to the number of blastocysts transferred. Thirty-nine pups were born from 375 transferred blastocysts (10%), with 16 pups displaying coat-color chimerism. Both nonmanipulated eight-cell embryos cultured for 48 h (P < 0.05) and chimeric blastocysts (P < 0.001) displayed lower embryo survival after transfer to recipients than noncultured, nonmanipulated blastocysts used as controls. Viability of chimeric blastocysts was also lower than that of nonmanipulated embryos cultured for the same period and transferred to the same recipients (P < 0.001). Although posttransfer survival of chimeric blastocysts was low, the birth of morphologically normal offspring demonstrated that production of chimeras from half embryos was compatible with survival. Improvements in this procedure may be useful for production of tenetically identical chimeras from outbred populations, such as those commonly found in domestic livestock species.     

An alternative simple method for mass production of chimeric embryos by coculturing denuded embryos and embryonic stem cells in Eppendorf vials

The generation of germline competent chimeric mice via embryonic stem (ES) cells is a crucial step in developing gene-manipulated mouse models. To date, techniques for generating chimeric mice include direct microinjection of ES cells into the cavity of 3.5-d post-coitum (dpc) blastocysts and aggregating or coculturing 2.5 dpc zona pellucida-free (denuded) embryos with ES cells. We present here a procedure that is simple and reproducible for mass producing (10-150 embryos/vial/time) chimeric embryos by coculturing denuded 8-cell embryos and morula in 0.8 mL KSOM-AA medium containing 5 x 10(5)mL-1 purified green fluorescence protein-expressing ES cells (either fresh or thawed) in an 1.7 mL Eppendorf vial for 3h. The resulting chimeras had substantial levels of chimerism and high germline transmission rates. Therefore, the method developed in this study can provide a simple and mass reproducible alternative method (to germline transmitter chimeric mice), without technological and instrumental difficulties, for generating chimeric embryos.     

"Living together apart": the hidden genetic diversity of sponge populations

Intraorganism genetic stability is assumed in most organisms. However, here we show for the first time intraorganism genetic heterogeneity in natural populations of marine sponges. A total of 36 different multilocus genotypes (MLGs) were detected in 13 individuals of Scopalina lophyropoda sampled at 4 distant points within each sponge. All genotypes (showing a mosaic distribution), were transmitted to the progeny, thus contributing to the high genetic diversity and low clonality reported for this species, although its populations are small and structured and show high fission rates. There did not seem to be intraindividual genotype conflicts; on the contrary, chimeric individuals are expected to show low mortality thanks to the differential mortality of their different MLGs. This novel mechanism may also counterbalance genetic constraints in other benthic invertebrate species. The presence of sponge chimerism also suggests that results from previous population genetics studies could have been misinterpreted.     

Germ-line chimera by lower-part blastoderm transplantation between diploid goldfish and triploid crucian carp

Germ-line chimerism was successfully induced by blastoderm transplantation from donor triploid crucian carp, which reproduces gynogenetically, to recipient diploid goldfish, which reproduces bisexually. Lower part of donor blastoderm including primordial germ cells (PGCs) was sandwiched between recipient blastoderm at the mid- to late-blastula stage. When donor grafts were prepared from intact embryos or ventralized ones by removing vegetal yolk hemisphere at the 1- to 2-cell stage, malformations including double axes were observed in the resultant chimeras transplanted with grafts from intact embryos at the hatching stage, while a few malformations in those from ventralized embryos. PGCs originated from donor grafts were observed around the gonadal anlage at 10 days post-fertilization in chimeras. When ploidy of erythrocytes and epidermal cells in chimeric fish was examined by flow-cytometry, no triploid cells were detected at 1- and 5-year-old chimeras. Three-year-old chimeric fish (n=5) laid eggs originated from the donor together with those from the recipient. The frequency of eggs from the donor crucian carp blastoderm varied from 3.1 to 89.3% between chimeras.     

Viable Chimeras between Embryonal Carcinoma Cells and Mouse Embryos: Comparison of Aggregation and Injection Methods

An embryonal carcinoma (EC) cell line having the ability to form chimeric mice was isolated from embryo-derived teratocarcinomas experimentally induced in BALB/cCrSlc mice. This EC cell line, B242 g, was one of the 5 EC cell lines pre-selected based on the ability to incorporate into blastocysts by means of aggregating with 8-cell mouse embryos.
Using the B242g EC cells, the effectiveness of producing chimeras was compared between two currently available techniques, aggregation and injection, by examining chimerism of the midgestationally recovered conceptuses and live-born mice. The present result revealed that EC cells studied here were able to form chimeras more efficiently by injection as compared to aggregation method.     

Efficiency of cell culture derivation from blastula embryos and of chimera formation in the medaka (Oryzias latipes) depends on donor genotype and passage number

 Embryonic stem (ES) cells from early vertebrate embryos only rarely retain their full developmental potential under in vitro culture conditions, but undergo differentiation and lose their ability for chimeric embryogenesis. This is reflected by the fact that the ES cell technology to date could only be fully developed in mice. In the fish Oryzias latipes, the medaka, one ES-like cell line, MES1, has been established which gives rise to a high frequency of somatic chimeras but a low degree of chimerism. Here we have tested the effect of donor genotype and cultivation time on the efficiency of cell culture derivation and on chimera formation. The HB12A, HB32C and HNI strains of medaka most efficiently and reproducibly give rise to blastula-derived cell cultures that produce pigmented chimeras in albino hosts. Seven chimeras grew to male or female adults with normal fertility, although none of them showed obvious donor germline contribution. During prolonged in vitro propagation the frequency of chimeras and the degree of chimerism dropped to a value retained in the long-term cultured MES1 cells. Obviously, genetic factors in host/donor compatibility and physiological changes during prolonged in vitro culture may compromise, but do not abolish, the developmental potential of medaka ES-like cells. Thus, elucidation of conditions that will expand the developmental potential of medaka blastula cell cultures should lead to a further improvement towards establishment of the ES cell technology in medaka. Received: 5 June 1998 / Accepted: 6 July 1998     

Growth and long-term somatic and germline chimerism following fusion of juvenile Botryllus schlosseri

The colonial ascidian Botryllus schlosseri undergoes a histocompatibility reaction that can result in vascular fusion of distinct genotypes, creating a chimera. Chimerism has both potential benefits, such as an immediate increase in size that may enhance growth rates, and costs. For the latter, the presence of multiple genotypes in a chimera can lead to competition between genetically distinct stem cell lineages, resulting in complete replacement of somatic and germline tissues by a single genotype. Although fusion can occur at any point after metamorphosis, previous studies have focused on chimeras created from sexually mature adults, where no benefit to chimerism has been documented. Here we focus on the costs and benefits of fusion between juveniles, characterizing growth rates and patterns of somatic and germline chimerism after natural and controlled fusion events. We also compared outcomes between low- and high-density growth conditions, the latter more likely representative of what occurs in natural populations. We found that growth rates were density-dependent, and that only chimeras grew under high-density conditions. We also observed a positional component to a post-fusion event called resorption, indicating that extrinsic factors were important in this process. Patterns of germline and somatic chimerism and dominance in chimeras made from fused juveniles were equivalent to those after fusion of sexually mature adults, and there were no age-related differences in these processes. Finally, by using genetic markers that could retrospectively assign genotypes, we also found that the majority of individual testes in a chimera were clonally derived.     

Effect of blastomere sex and fluorescent labelling on the development of bovine chimeric embryos reconstituted at the four-cell stage

The development rate of bovine chimeric embryos reconstituted at the 4-cell stage is relatively low. If chimerism is to be used as an approach in producing transgenic livestock, it is important to investigate whether this rate is affected by the sex of the blastomeres being combined and if all blastomeres survive equally well. In Experiment 1, blastomeres from 4-cell stage embryos were inserted into surrogate zonae pellucidae either in pairs to reconstitute 4-cell chimeras, or as the original sets of four to make handled controls. The development of chimeras with one pair of blastomeres labelled with PKH26-GL was also investigated. The rate of development into blastocysts was similar in chimeras with unlabelled blastomeres (23%) and in those in which one pair of blastomeres was labelled (26%) and was lower (P < 0.001) than in the handled and IVF control groups (43 and 58%, respectively). Labelled cells were distributed approximately evenly between ICM and trophoblast. In Experiment 2, the effect of sex differences between pairs of blastomeres in chimeras was investigated; chimeras were reconstituted from pairs of blastomeres taken from 4-cell embryos in which the remaining pair was sexed by PCR. No significant differences according to the sex of constituent blastomeres were detectable (mixed sex, 27%; males, 24%; females, 21%; P > 0.05). These results suggest that, in addition to the negative effects of micromanipulation, factors other than the sex of the blastomeres are involved in the reduced rate of development of chimeric bovine embryos. They also confirm the usefulness of PKH26-GL labelling for tracking the progeny of cleaving bovine blastomeres at least to the blastocyst stage.     

Cryopreserved Morulae can be used to Efficiently Generate Germline-transmitting Chimeras by Blastocyst Injection

The production of chimeric mice is a complex process, requiring the careful coordination of tissue culture cell growth, production of a large number (30–75) of competent blastocysts and the availability of appropriately timed pseudo pregnant female mice. Failure at any of these steps can impinge upon the rapid production of chimeras. One potential improvement for the efficient generation of chimeric mice would be the utilization of cryopreserved embryos suitable for injection. C57Bl/6 morulae were frozen using a standard 2-step protocol with ethylene glycol as the cryopreservation agent. We determined that cryopreserved morulae could thaw, culture to blastocyst stage in KSOM media and survive injection at rates equivalent to control embryos. Cryopreserved morulae were also equivalent to controls at all later stages in the process of production of chimeric mice, including birth rate, percentage chimerism of resulting animals and ability to produce germline progeny. Hence, cryopreservation of morulae for blastocyst injection is a suitable option to enhance the efficiency of chimeric mouse generation.     

Attempts to enhance production of porcine chimeras from embryonic germ cells and preimplantation embryos

Porcine embryonic germ (EG) cells share common features with porcine embryonic stem (ES) cells, including morphology, alkaline phosphatase activity and capacity for in vitro differentiation. Porcine EG cells are also capable of in vivo development by producing chimeras after blastocyst injection; however, the proportion of injected embryos that yield a chimera and the proportion of cells contributed by the cultured cells in each chimera are too low for practical use in genetic manipulation. Moreover, somatic, but not germ-line chimerism, has been reported from blastocyst injection using porcine ES or EG cells. To test whether efficiency of chimera production from blastocyst injection can be improved upon by changing the host embryo, we used as host embryos four groups according to developmental stage or length in culture: fresh 4-cell and 8-cell stage embryos subsequently cultured into blastocysts, fresh morulae, fresh blastocysts, and cultured blastocysts. Injection and embryo transfer of fresh and cultured blastocysts produced similar percentages of live piglets (17% versus 19%). Four piglets were judged to have a small degree of pigmentation chimerism, but microsatellite analysis failed to confirm chimerism in these or other piglets. Polymerase chain reaction analysis for detection of the porcine SRY gene in female piglets born from embryos injected with male EG cells identified six chimeras, at least one, but not more than two, from each treatment. Chimerism was confirmed in two putative pigmentation chimeras and in four piglets without overt signs of chimerism. The low percentage of injected embryos that yielded a chimera and the small contribution by EG cells to development of each confirmed chimera indicated that procedural changes in how EG cells were combined with host embryos were unsuccessful in increasing the likelihood that porcine EG cells will participate in embryonic development. Alternatively, our results suggested that improvements are needed in EG cell isolation and culture procedures to ensure in vitro maintenance of EG cell developmental capacity.