Blastomere Transplantation Research Articles

Cleavage Blastomere Deletion and Transplantation to Test Cell Fate Commitment in Xenopus.

Fate maps identify the precursors of an organ, and tracing the members of a blastomere lineage over time shows how its descendants come to populate that organ. The fates of the individual blastomeres of the two- to 32-cell Xenopus embryo have been fully mapped to reveal which cells are the major contributors to various cell types, tissues, and organs. However, because these fate maps were produced in the normal embryo, they do not reveal whether a precursor blastomere is competent to give rise to additional tissues or is already committed to its fate-mapped repertoire of descendants. To identify the mechanisms by which a cell's fate is committed, one needs to expose the cell to different experimental environments. If the cell's fate is determined, it will express its normal fate or gene expression profile in novel environments, whereas if it is not yet determined it will express different fates or gene expression profiles when exposed to novel external factors or neighboring cells. This protocol describes two techniques for testing cell fate commitment: single cell deletion and single cell transplantation. Deleting a blastomere allows one to test whether the deleted cell is required for the remaining cells to produce their normal, specific cell fates. Transplanting a blastomere to a novel location in a host embryo allows one to test whether the transplanted cell is committed to produce its normal fate-mapped repertoire, or whether it is still competent to respond to novel cell-cell interactions.

Migration of primordial germ cells during late embryogenesis of pikeperch Sander lucioperca relative to blastomere transplantation

Pikeperch Sander lucioperca is a valuable fish in Europe, and basic information about its embryonic development, especially primordial germ cell (PGC) migration, is important for use in biotechnology. We categorized pikeperch embryonic development into six stages as in other fish species: zygote, cleavage, blastula, gastrula, segmentation, and hatching and described PGC migration. PGCs were visualized by injection of synthesized green fluorescent protein (GFP) within the 3’untranslated region (UTR) mRNA of nanos3. GFP-positive PGCs appeared in all embryos at approximately 100% epiboly. Time-lapse imaging revealed the PGC migration pattern from their initial appearance to location at the gonadal ridge. We conducted blastomere transplantation (BT) at the blastula stage. Donor embryos were labelled with GFP-nos3 3’UTR mRNA and tetramethylrhodamine dextran to label PGCs and somatic cells, respectively. Twelve BT chimeras were produced, with eight surviving to hatching. All exhibited donor-derived somatic cells in the developing body. The PGCs from donor embryos were observed to migrate towards the gonad region of the host embryos. Our results indicated that BT can be successfully applied in pikeperch, and these findings may be useful to produce germline chimeras in percids.

Early asymmetric cues triggering the dorsal/ventral gene regulatory network of the sea urchin embryo.

Dorsal/ventral (DV) patterning of the sea urchin embryo relies on a ventrally-localized organizer expressing Nodal, a pivotal regulator of the DV gene regulatory network. However, the inceptive mechanisms imposing the symmetry-breaking are incompletely understood. In Paracentrotus lividus, the Hbox12 homeodomain-containing repressor is expressed by prospective dorsal cells, spatially facing and preceding the onset of nodal transcription. We report that Hbox12 misexpression provokes DV abnormalities, attenuating nodal and nodal-dependent transcription. Reciprocally, impairing hbox12 function disrupts DV polarity by allowing ectopic expression of nodal. Clonal loss-of-function, inflicted by blastomere transplantation or gene-transfer assays, highlights that DV polarization requires Hbox12 action in dorsal cells. Remarkably, the localized knock-down of nodal restores DV polarity of embryos lacking hbox12 function. Finally, we show that hbox12 is a dorsal-specific negative modulator of the p38-MAPK activity, which is required for nodal expression. Altogether, our results suggest that Hbox12 function is essential for proper positioning of the DV organizer.

Tal1 regulates the formation of intercellular junctions and the maintenance of identity in the endocardium

The endocardium forms the inner lining of the heart tube, where it enables blood flow and also interacts with the myocardium during the formation of valves and trabeculae. Although a number of studies have identified regulators in the morphogenesis of the myocardium, relatively little is known about the molecules that control endocardial morphogenesis. Prior work has implicated the bHLH transcription factor Tal1 in endocardial tube formation: in zebrafish embryos lacking Tal1, endocardial cells form a disorganized mass within the ventricle and do not populate the atrium. Through blastomere transplantation, we find that tal1 plays a cell-autonomous role in regulating endocardial extension, suggesting that Tal1 activity influences the behavior of individual endocardial cells. The defects in endocardial behavior in tal1-deficient embryos originate during the earliest steps of endocardial morphogenesis: tal1-deficient endocardial cells fail to generate a cohesive monolayer at the midline and instead pack tightly together into a multi-layered aggregate. Moreover, the tight junction protein ZO-1 is mislocalized in the tal1-deficient endocardium, indicating a defect in intercellular junction formation. In addition, we find that the tal1-deficient endocardium fails to maintain its identity; over time, a progressively increasing number of tal1-deficient endocardial cells initiate myocardial gene expression. However, the onset of defects in intercellular junction formation precedes the onset of ectopic myocardial gene expression in the tal1-deficient endocardium. We therefore propose a model in which Tal1 has distinct roles in regulating the formation of endocardial intercellular junctions and maintaining endocardial identity.

Specific expression of a TRIM-containing factor in ectoderm cells affects the skeletal morphogenetic program of the sea urchin embryo

In the indirect developing sea urchin embryo, the primary mesenchyme cells (PMCs) acquire most of the positional and temporal information from the overlying ectoderm for skeletal initiation and growth. In this study, we characterize the function of the novel gene strim1, which encodes a tripartite motif-containing (TRIM) protein, that adds to the list of genes constituting the epithelial-mesenchymal signaling network. We report that strim1 is expressed in ectoderm regions adjacent to the bilateral clusters of PMCs and that its misexpression leads to severe skeletal abnormalities. Reciprocally, knock down of strim1 function abrogates PMC positioning and blocks skeletogenesis. Blastomere transplantation experiments establish that the defects in PMC patterning, number and skeletal growth depend upon strim1 misexpression in ectoderm cells. Furthermore, clonal expression of strim1 into knocked down embryos locally restores skeletogenesis. We also provide evidence that the Otp and Pax2/5/8 regulators, as well as FGFA, but not VEGF, ligand act downstream to strim1 in ectoderm cells, and that strim1 triggers the expression of the PMC marker sm30, an ectoderm-signaling dependent gene. We conclude that the strim1 function elicits specific gene expression both in ectoderm cells and PMCs to guide the skeletal biomineralization during morphogenesis.

Production of loach (Misgurnus anguillicaudatus) germ-line chimera using transplantation of primordial germ cells isolated from cryopreserved blastomeres1

An efficient procedure for the cryopreservation of fish blastomeres followed by restoration through germ-line chimera formation was established. Blastomeres of the loach (Misgurnus anguillicaudatus) were cryopreserved in 250-µL straws in Eagle's minimum essential medium with various concentrations of dimethyl-sulfoxide (0, 5, 10, 15, and 20%), and the best concentration was combined with glycerol (1, 2, and 4%) and external cryoprotectants (1 or 2% sucrose; 2, 5, or 10% fetal bovine serum; 1 or 2% BSA). Postthaw viability of the blastomeres was used to optimize cryopreservation conditions. Donor blastomeres were injected with zebrafish green fluorescence protein-nos1 3' untranslated region mRNA and biotin dextran before cryopreservation in the optimal freeze medium. Host embryos were injected with zebrafish DsRed-nos1 3' untranslated region mRNA and reared to the blastula stage. Donor blastomeres were thawed at 25 °C for 10 s and transplanted to the host embryos either immediately or after incubation for 16 h at 20 °C. Donor and host primordial germ cell migration was visualized with fluorescent imaging during the early stages of embryogenesis, and also by histology in 4-d-old embryos. Transplantation of blastomeres immediately after thawing gave decreased hatching rates (approximately 3%) and generated a smaller percentage of germ-line chimeras (approximately 1.1%). In contrast, incubation of a cryopreserved sample for 16 h followed by transplantation of the green fluorescence protein-positive blastomeres improved the hatching rate to 90%, and successfully produced presumable germ-line chimeras at a rate of 16.5%. The improved survival rates and germ-line chimerism may be an effective method for gene banking and subsequent reconstitution of endangered fish genotypes.

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed.

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed.

Developmental potential of embryonic cells in a nucleocytoplasmic hybrid formed using a goldfish haploid nucleus and loach egg cytoplasm

In teleosts, viable nucleocytoplasmic hybrids, formed by combining a nucleus from one species with the egg cytoplasm of another, have been used as one of the methods for breed improvement in aquaculture, but have been little exploited for developmental biology studies. Here, we used an artificial androgenesis technique to form nucleocytoplasmic hybrids comprising a goldfish haploid nucleus and loach egg cytoplasm. These hybrids were used to investigate interactions between the nucleus and cytoplasm during embryonic development. Additionally, the developmental characteristics of embryonic cells of nucleocytoplasmic hybrids were examined in chimeras produced by transplantation of blastomeres into recipient loach or goldfish embryos. We found that the nucleocytoplasmic hybrids arrested at the dome stage of embryonic development and did not form any gastrula structures. The goosecoid (gsc) and no tail (ntl) genes were expressed normally before gastrulation in nucleocytoplasmic hybrids, similar to diploid loach. However, expression of the gsc and ntl genes was not maintained in nucleocytoplasmic hybrids. In chimeric embryos, blastomeres derived from nucleocytoplasmic hybrids were found to mix with the cells of recipient loach embryos at the gastrula stage. The transplanted blastomeres formed small clusters at the somitogenesis stage and, finally, small spots at the hatching stage. In contrast, when the blastomeres were transplanted into goldfish embryos, the transplanted blastomeres aggregated in the chimeric embryos. Thus, embryonic cells from nucleocytoplasmic hybrids that arrest before gastrulation could survive beyond the somitogenesis stage depending on the cytoplasmic environment in the recipient embryos.

Inter-species transplantation and migration of primordial germ cells in cyprinid fish

Primordial germ cells (PGCs) are the only cells in developing embryos that can transmit genetic information to the next generation. PGCs therefore have considerable potential value for gene banking and cryopreservation, particularly via production of donor gametes using germ-line chimeras. In some animal species, including teleost fish, the feasibility of using PGC transplantation to obtain donor-derived offspring, within and between species, has been demonstrated. Successful use of PGC transplantation to produce germ-line chimeras is absolutely dependent on the migration of the transplanted cells from the site of transplantation to the host gonadal region. Here, we induced germ-line chimeras between teleost species using two different protocols: blastomere transplantation and single PGC transplantation. We evaluated the methods using the rate of successful migration of transplanted PGCs to the gonadal region of the host embryo. First, we transplanted blastomeres from zebrafish, pearl danio, goldfish, or loach into blastula-stage zebrafish embryos. Some somatic cells, derived from donor blastomeres, were co-transplanted with the PGCs and formed aggregates in the host embryos; a low efficiency of PGC transfer was achieved. Second, a single PGC from the donor species was transplanted into a zebrafish embryo. In all inter-species combinations, the donor PGC migrated toward the gonadal region of the host embryo at a comparatively high rate, regardless of the phylogenetic relationship of the donor and host species. These transplantation experiments showed that the mechanism of PGC migration is highly conserved beyond the family barrier in fish and that transplantation of a single PGC is an efficient method for producing inter-species germ-line chimeras.

A new triple staining method for double in situ hybridization in combination with cell lineage tracing in whole‐mount Xenopus embryos

To analyze cell to cell interaction effects on cell differentiation, we developed a new triple staining method for double in situ hybridization with cell lineage tracing in whole-mount Xenopus embryos. The method provides high color contrast views, and also enabled us to examine inside the embryos. Wild-type embryos whose blastomere(s) had been injected with a cell lineage tracer were cultured, fixed, hemisectioned when necessary, and first served for the double in situ hybridization, with two sequential chromogenic reactions. They were postfixed, and the labeled cells were retraced immunohistochemically. Finally, the pigment of the embryos was bleached to obtain a clear view. We applied this method to a blastomere transplantation experiment to examine whether the spatial gene expression patterns along the anteroposterior axis can be induced by cell to cell interactions. The presumptive organizer of a 32-cell embryo was replaced by the labeled presumptive epidermis of another synchronous embryo. The resultant triple-stained late gastrula showed quite similar anteroposterior expression patterns of gsc and Xbra to those of a normal embryo in the axial mesoderm derived from the transplanted presumptive epidermis, indicating that cell to cell interactions had induced these patterns.

Developmental biotechnology for aquaculture, with special reference to surrogate production in teleost fishes

This review introduces surrogate production as a new technique for fish-seed production in aquaculture. Surrogate production in fish is a technique used to obtain the gametes of a certain genotype through the gonad of another genotype. It is achieved by inducing germ-line chimerism between different species during early development. Primordial germ cells (PGCs) are the key material of this technique to induce germ-line chimera. In several species, it has been reported that PGCs differentiated from the blastomeres inherited some maternally supplied mRNA located in the terminal regions of the early cleavage furrows. PGCs from donor species (or strains) are isolated and transplanted into host species to induce the germ-line chimera. Four methods for inducing germ-line chimera are described: blastomere transplantation, blastoderm-graft transplantation, transplantation of PGC from the genital ridge, and transplantation visualised PGC with GFP fluorescence. Several problems preventing the successful induction of germ-line chimera in various fish species are discussed. Surrogate production, however, opens the possibility of efficient fish-seed production and effective breeding and transfer of biodiversity to an aquaculture strain. Conservation and efficient utilisation of genetic resources will be achieved through surrogate production combined with the cryopreservation of PGCs.

Repression of mesodermal fate byfoxa, a key endoderm regulator of the sea urchin embryo

The foxa gene is an integral component of the endoderm specification subcircuit of the endomesoderm gene regulatory network in the Strongylocentrotus purpuratus embryo. Its transcripts become confined to veg2, then veg1 endodermal territories, and, following gastrulation, throughout the gut. It is also expressed in the stomodeal ectoderm. gatae and otx genes provide input into the pregastrular regulatory system of foxa, and Foxa represses its own transcription, resulting in an oscillatory temporal expression profile. Here, we report three separate essential functions of the foxa gene: it represses mesodermal fate in the veg2 endomesoderm; it is required in postgastrular development for the expression of gut-specific genes; and it is necessary for stomodaeum formation. If its expression is reduced by a morpholino, more endomesoderm cells become pigment and other mesenchymal cell types, less gut is specified, and the larva has no mouth. Experiments in which blastomere transplantation is combined with foxa MASO treatment demonstrate that, in the normal endoderm, a crucial role of Foxa is to repress gcm expression in response to a Notch signal, and hence to repress mesodermal fate. Chimeric recombination experiments in which veg2, veg1 or ectoderm cells contained foxa MASO show which region of foxa expression controls each of the three functions. These experiments show that the foxa gene is a component of three distinct embryonic gene regulatory networks.

Blastomere transplantation in human embryos may be a treatment for single gene diseases

Objective To determine whether human embryos accept blastomere transplants and integrate them normally into the architecture of the developing embryo. Design A human blastomere transplantation model, involving 44 cryopreserved embryos that were specifically donated to research. Setting Academically affiliated private infertility center. Patient(s) Forty-four human embryos. Intervention(s) In 21 experiments, one, two, or three blastomeres were transplanted, using standard microsurgical techniques that are widely used in preimplantation genetic diagnosis (PGD). Embryos were thawed and gender was determined, using established PGD techniques. Male (xy) blastomeres were then transplanted into female (xx) day 3 embryos, and the xy cells were tracked through blastocyst stage (days 5–6) and into the hatching period (day 6), using fluorescent in situ hybridization (FISH). Main outcome measure(s) Degree and location of xy cell integration into xx embryos. Result(s) High-quality recipient embryos (with 4 to 10 cells) developed uniformly into normal blastocyst stage embryos in 12 of 12 experiments (100%) and integrated donor blastomeres into their architecture, with apparently even distribution of daughter cells; this integration was documented in inner cell mass as well as in trophoectoderm. The intensity of this distribution appeared to correlate with the number of blastomeres transferred. Among nine abnormally developing embryos, only three (33%) demonstrated a normal distribution of offspring donor cells. Conclusion(s) High-quality embryos appear to have the ability to integrate donor blastomeres. Because the treatment of single gene diseases does not require successful treatment of all cells, blastomere transplantation could be explored as a treatment option, which also would greatly enhance efficiency and utilization of preimplantation genetic diagnosis.

Blastomere transplantation in early human embryos

Objective: We previously reported the successful transplantation and integration of donor blastomeres into recipient embryos (ESHRE, Madrid, Spain, July 1, 2003). The objective of this study was to determine whether the degree of integration of the donor transplant into the recipient embryo changed, based on the number of blastomeres transplanted.

Production of germ-line chimeras in rainbow trout by blastomere transplantation.

We describe a technique for producing germ-line chimeric rainbow trout, Oncorhynchus mykiss, by microinjection of the isolated blastomeres. FITC-labeled donor cells and non-labeled recipient embryos at various developmental stages between the early blastula and early gastrula stages were used for cell transplantation. The chimera formation rate and the degree of donor cell distribution in recipient embryos were evaluated at both the late gastrula stage (5 days post fertilization (dpf)) and the 40-somite stage (10 dpf). Among the six combinations of developmental stages of donor and recipient embryos, the combination of midblastula (2.5 dpf) donor cells and early blastula (1.5 dpf) recipient embryos gave the highest chimera formation rate and the best distribution pattern of donor cells. Using this combination, chimeric rainbow trout were produced with donor blastomeres from dominant orange-colored mutant embryos and wild-type recipient embryos. Of the 238 chimeric embryos produced, 28 (12%) hatched normally and 14 of the 28 fry (50%) had donor-derived orange body color. To test for germ-line transmission of donor cells, gametes obtained from the matured chimeras were fertilized with gametes from wild-type fish. Of the 19 matured chimeras, 6 (32%) yielded donor-derived orange-colored progeny, in addition to wild-type siblings. The contribution rates of donor cells in the germ-line ranged from 0.3 to 14%. This technique for producing germ-line chimeras should be a powerful tool for cell-mediated gene transfer in rainbow trout. Especially, if body color mutants are used for either donor cells or the host embryos, it will be possible to easily concentrate F1 transgenic embryos derived from transplanted donor cells by body color screening. Mol. Reprod. Dev. 59: 380-389, 2001.

Cloned Piglets Born after Nuclear Transplantation of Embryonic Blastomeres into Porcine Oocytes Matured In Vivo

Nuclear transplantation in the pig is more difficult than in other domestic animals and only one embryonic nuclear transplantation (NT) pig has been born to date. In this study, reconstituted porcine embryos were produced by electrofusion of blastomeres from in vivo four-cell embryos to enucleated in vivo or in vitro matured (IVM) oocytes. Nuclear transfer using cumulus cells as nuclear donors was also conducted. When blastomeres were used as donors, the electrofusion rate was significantly higher in oocytes matured in vivo (91.5%) than in those matured in vitro (66.1%) (p < 0.01). After fusion, the NT embryos reconstituted from in vivo matured oocytes developed to blastocysts at a rate of 10.3% after culture in rabbit oviducts for up to 5 days, while only 5.9% of the NT embryos reconstructed from in vitro matured oocytes developed to blastocyst stage. Electrofusion rate of cumulus cell nuclei with enucleated IVM oocytes was lower (47.6%) and only 1.5% (2/136) of the reconstituted eggs developed in vitro to morula stage, and 1.9% developed to blastocysts when cultured in the ligated rabbit oviducts. Transfer of 94 embryos reconstructed by blastomere NT with in vivo matured oocytes to five synchronous recipients resulted in the birth of two cloned piglets. No piglet was born following transfer to two recipients of embryos (n = 39) derived from NT with in vitro matured oocytes. The results demonstrate that in vivo matured oocytes are better recipients than those matured in vitro for pig cloning.

Irradiation of fish embryos prior to blastomere transfer boosts the colonisation of their gonads by donor-derived gametes.

Blastomere transplantation into fish blastula embryos results in somatic chimeras, which generally provide null or a small proportion of gametes derived from the donor. This may partly explain why none of the ES-like cell lines established from fish embryos has contributed to the germline of chimeras when transplanted at the blastula stage. Here, we report that a moderate gamma-irradiation of recipient embryos, followed by transplantation of dispersed blastomeres, considerably enhances the proportion of donor-derived gametes (53% versus 5% in average). In fish, the resulting protocol should maximise the pluripotency level measured in vivo for embryonic cell lines and for cultured germ cells.

Development of porcine embryos following microsurgical blastomere transplantation

The objective of these studies was to characterize the in vitro and in vivo development of porcine embryos following microsurgical blastomere transplantation. In Experiment 1, 60 four-cell embryos recovered on Day 3 of gestation (Day 0 = onset of estrus), were subjected to either blastomere transplantation (micromanipulated; n = 30) or no microsurgical procedure (control; n = 30). Four-cell embryos were cultured in modified Kreb's Ringer bicarbonate medium supplemented with 0.4% bovine serum albumin and 10% bovine calf serum. After 72 h in culture, embryos were stained and the number of nuclei per embryo was determined. Blastomere transplantation had no effect on embryonic development in vitro as the numbers of micromanipulated and control embryos that reached the 12–24 and ≥25 nuclei stages were similar ( P<0.40). In Experiment 2, 64 four- to eight-cell embryos recovered on Day 3 of gestation were distributed across two treatments: control ( n=33) or micromanipulated ( n=31). Micromanipulated embryos each received a single blastomere from a donor embryo at the same stage of development. After culture for 4–5 h, seven recipients which exhibited estrus within 24 h of the donors, received either control or host embryos. Seventy-two hours later, the transferred embryos were recovered from the recipient females, stained and the number of nuclei per embryo was determined. The proportion of micromanipulated and control embryos recovered was not different. In comparison with control embryos, a greater ( P<0.02) proportion of micromanipulated embryos contained <50 nuclei. The mean number of nuclei present in embryos which reached the >100 nuclei stage was greater ( P<0.0004) for micromanipulated than for control embryos (173±20 and 125±4, respectively). While blastomere transplantation appears to have no detrimental effect on development of porcine embryos in vitro, it may reduce the proportion of embryos developing beyond the 50 cell stage in vivo.

Sex chromosome chimaerism and the transmission of blood group genes by tetraparental rams.

A previously published study of 4 rams, 3 of them tetraparental chimaeras, has been extended. All were born subsequent to transplantation of separated blastomeres into intact 4--8-cell embryos which were transferred to recipient ewes. All were phenotypically normal males. Study of blood lymphocytes and red cell antigens showed that two were XX/XY chimaeras, one an XY/XY chimaera. Chimaerism was not identified in the 4th animal. The new data show that the previously reported decline in proportions of red cells derived from the one cell line has continued, although the proportions of lymphocytes entering mitosis in culture have remained nearly constant. A decline of potassium concentration in the red cells of one animal and of transferrin type in another also continued. Analysis of blood types in the offspring of the 4 rams revealed that both cell lines of the XY/XY chimaera must have produced functional spermatozoa whereas the genes transmitted by the remaining 3 rams gave evidence of spermatozoa from one cell line only.