Tetraploid Embryos Research Articles

Keratinocyte-specific Expression of Fatty Acid Transport Protein 4 Rescues the Wrinkle-free Phenotype in Slc27a4/Fatp4 Mutant Mice

FATP4 (fatty acid transport protein 4; also known as SLC27A4) is the most widely expressed member of a family of six long chain fatty acid transporters. FATP4 is highly expressed in enterocytes and has therefore been proposed to be a major importer of dietary fatty acids. Two independent mutations in Fatp4 cause mice to be born with thick, tight, shiny, "wrinkle-free" skin and a defective skin barrier; they die within hours of birth from dehydration and restricted movements. In contrast, induced keratinocyte-specific deficiency of FATP4 in adult mice causes only mild skin abnormalities. Therefore, whether the loss of FATP4 from skin or a systemic gestational metabolic defect causes the severe skin defects and neonatal lethality remain important unanswered questions. To investigate the basis for the phenotype, we first generated wild-type tetraploid/mutant diploid aggregates that should lead to rescue of any abnormalities caused by loss of FATP4 from the placenta. However, the skin phenotype was not ameliorated. We then generated transgenic mice expressing exogenous FATP4 either widely or specifically in suprabasal keratinocytes, and we bred the transgenes onto the Fatp4(-/-) background. Both modes of FATP4 expression led to rescue of the neonatally lethal skin defects, and the resulting mice were viable and fertile. Keratinocyte expression of an FATP4 variant with mutations in the acyl-CoA synthetase domain did not provide any degree of rescue. We conclude that expression of FATP4 with an intact acyl-CoA synthetase domain in suprabasal keratinocytes is necessary for normal skin development and that FATP4 functions in establishing the cornified envelope.

Developmental and adult phenotyping directly from mutant embryonic stem cells

Tetraploid embryo complementation assay has shown that mouse ES cells alone are capable of supporting embryonic development and adult life of mice. Newly established F(1) hybrid ES cells allow the production of ES cell-derived animals at a high enough efficiency to directly make ES cell-based genetics feasible. Here we report the establishment and characterization of 12 new F(1) hybrid ES cell lines and the use of one of the best (G4) in a gain- and loss-of-function genetic study, where the in vivo phenotypes were assessed directly from ES cell-derived embryos. We found the generation of G4 ES cell-derived animals to be very efficient. Furthermore, even after two consecutive rounds of genetic modifications, the majority of transgenic lines retained the original potential of the parental lines; with 10-40% of chimeras producing ES cell-derived animals/embryos. Using these genetically altered ES cells, this success rate, in most cases, permitted the derivation of a sufficient number of mutants for initial phenotypic analyses only a few weeks after the establishment of the cell lines. Although the experimental design has to take into account a moderate level of uncontrolled damage on ES cell lines, our proof-of-principle experiment provides useful data to assist future designs harnessing the power of this technology to accelerate our understanding of gene function.

Notch2 is required for formation of the placental circulatory system, but not for cell-type specification in the developing mouse placenta

We have previously reported that a mutation in the ankyrin repeats of mouse Notch2 results in embryonic lethality by embryonic day 11.5 (E11.5), showing developmental retardation at E10.5. This indicated that Notch2 plays an essential role in postimplantation development in mice. Here, we demonstrate that whole embryo culture can circumvent developmental retardation of Notch2 mutant embryos for up to 1 day, suggesting that the lethality was primarily caused by extraembryonic defects. Histological examinations revealed delayed entry of maternal blood into the mutant placenta and poor blood sinus formation at later stages. Notch2-expressing cells appeared around maternal blood sinuses. Specification of trophoblast subtypes appeared not to be drastically disturbed and expression of presumptive downstream genes of Notch2 signaling was not altered by the Notch2 mutation. Thus, in the developing mouse placenta, Notch2 is unlikely to be involved in cell fate decisions, but rather participates in formation of maternal blood sinuses. In aggregation chimeras with wild-type tetraploid embryos, the mutant embryos developed normally until E12.5, but died before E13.5. The chimeric placentas showed a restored maternal blood sinus formation when compared with the mutant placentas, but not at the level of wild-type diploid placentas. Therefore, it was concluded that the mutant suffers from defects in maternal blood sinus formation. Thus, Notch2 is not cell autonomously required for the early cell fate determination of subtypes of trophoblast cells, but plays an indispensable role in the formation of maternal blood sinuses in the developing mouse placenta.

Passage number affects the pluripotency of mouse embryonic stem cells as judged by tetraploid embryo aggregation

The aim of this study was to determine whether the number of passages affected the developmental pluripotency of embryonic stem (ES) cells as measured by the attainment of adult fertile mice derived from embryonic stem (ES) cell/tetraploid embryo complementation. Thirty-six newborns were produced by the aggregation of tetraploid embryos and hybrid ES cells after various numbers of passages. These newborns were entirely derived from ES cells as judged by microsatellite DNA, coat-color phenotype, and germline transmission. Although 15 survived to adulthood, 17 died of respiratory failure, and four were eaten by their foster mother. From the 15 mice that reached adulthood and that could reproduce, none arose from ES cells at passage level 15 or more. All 15 arose from cells at passages 3-11. Our results demonstrate that the number of passages affects the developmental pluripotency of ES cells.

Development of rat tetraploid and chimeric embryos aggregated with diploid cells

In the present study, we examined the preimplantation and postimplantation development of rat tetraploid embryos produced by electrofusion of 2-cell-stage embryos. Developmental rate of tetraploid embryos to morula or blastocyst stage was 93% (56/60) and similar to that found in diploid embryos (95%, 55/58). After embryo transfer, rat tetraploid embryos showed implantation and survived until day 8 of pregnancy, however the conceptuses were aberrant on day 9. In mouse, tetraploid embryos have the ability to support the development of blastomeres that cannot develop independently. As shown in the present study, a pair of diploid blastomeres from the rat 8-cell-stage embryo degenerated immediately after implantation. Therefore, we examined whether rat tetraploid embryos have the ability to support the development of 2/8 blastomeres. We produced chimeric rat embryos in which a pair of diploid blastomeres from an 8-cell-stage green fluorescent protein negative (GFP-) embryo was aggregated with three tetraploid blastomeres from 4-cell GFP-positive (GFP+) embryos. The developmental rate of rat 2n(GFP-) <--> 4n(GFP+) embryos to the morula or blastocyst stages was 93% (109/117) and was similar to that found for 2n(GFP-) <--> 2n(GFP+) embryos (100%, 51/51). After embryo transfer, 2n(GFP-) <--> 4n(GFP+) conceptuses were examined on day 14 of pregnancy, the developmental rate to fetus was quite low (4%, 4/109) and they were all aberrant and smaller than 2n(GFP-) <--> 2n(GFP+) conceptuses, whereas immunohistochemical analysis showed no staining for GFP in fetuses. Our results suggest that rat tetraploid embryos are able to prolong the development of diploid blastomeres that cannot develop independently, although postimplantation development was incomplete.

Reproductive Behavior of Induced Allotetraploid ×Chitalpa and In Vitro Embryo Culture of Polyploid Progeny

×Chitalpa tashkentensis Elias &amp; Wisura is a sterile intergeneric hybrid [Catalpa bignonioides Walt. × Chilopsis linearis (Cav.) Sweet]. To restore fertility in ×Chitalpa the following were evaluated: 1) oryzalin as a polyploidization agent, 2) fertility of induced polyploids, and 3) in vitro culture methods for embryo rescue of interploid crosses. Meristems of ×Chitalpa `Pink Dawn' were submerged in an aqueous solution of 150 μm oryzalin for 0, 6, 12, or 24 hours and ploidy analyzed via flow cytometry. As treatment duration increased, recovery of diploids decreased as mixoploids and shoot mortality increased. Two tetraploid shoots occurred in the 24-hour treatment. Four tetraploids and two cytochimeras were stabilized in total. Tetraploids flowered sparsely; however, cytochimeras flowered profusely and these were used to study fertility at the tetraploid level. Diploid ×Chitalpa `Pink Dawn' pollen was essentially nonviable, but cytochimera pollen stained and germinated equal to or greater than pollen of C. bignonioides and C. linearis `Bubba'. Cytochimera ×Chitalpa were selfed yielding tetraploid seedlings, crossed with C. bignonioides to yield triploids, but failed in reciprocal crosses with C. linearis `Bubba' and `Burgundy Lace'. To increase recovery of triploids, germination of triploid and tetraploid embryos was investigated, as either intact ovules or excised embryos, on Schenk and Hildebrandt (SH) basal salts supplemented with sucrose at 20, 40, and 80 g·L-1, presence or absence of 2% coconut-water, and gibberellic acid (GA3) at 0, 1, 2, or 4 μm, and harvested weekly beginning 2 weeks after pollination (WAP). Germination of triploids (cytochimera ×Chitalpa × diploid C. bignonioides) and tetraploids (selfed cytochimera ×Chitalpa) were greatest with excised embryos at 7 WAP on SH supplemented with sucrose at 20 g·L-1 and ≥1 μm GA3. Germination of triploids (diploid C. linearis × cytochimera ×Chitalpa) was &lt;5% at 4, 5, or 6 WAP on the same medium as above. Oryzalin effectively induced polyploidy and restored fertility in ×Chitalpa `Pink Dawn'. Successful crosses between hybrid and parental taxa of different ploidy levels, coupled with embryo culture will facilitate a ×Chitalpa breeding program. Chemical names used: 4(dipropylamino)-3,5-dinitrobenzenesulfonamide (oryzalin).

Tetraploid embryo complementation法による遺伝子改変マウスの作出法の確立

Tetraploid embryo complementation法による遺伝子改変マウスの作出法の確立

Diploid porcine parthenotes produced by inhibition of first polar body extrusion during in vitro maturation of follicular oocytes

We investigated nuclear progression and in vitro embryonic development after parthenogenetic activation of porcine oocytes exposed to cytochalasin B (CB) during in vitro maturation (IVM). Nuclear progression was similar in control oocytes and oocytes matured in the presence of 1 microg/ml CB (IVM-CB group) by 37 h IVM; at this time the proportion of oocytes that had reached or passed through the anaphase-I stage did not differ significantly between the IVM-CB and the control groups (61.3 and 69.9% respectively; P < 0.05). After IVM for 37 h, no polar body extrusion was observed in the IVM-CB group. In these oocytes, the two lumps of homologous chromosomes remained in the ooplasm after their segregation and turned into two irregular sets of condensed chromosomes. By 41 h IVM, the double sets of chromosomes had reunited in 89.5% IVM-CB oocytes and formed a single large metaphase plate, whereas 68.8% of the control oocytes had reached the metaphase-II stage by this time. When IVM-CB oocytes cultured for 46 h were stimulated with an electrical pulse and subsequently cultured for 8 h without CB, 39.0% of them extruded a polar body and 82.9% of them had a female pronucleus. Chromosome analysis revealed that the majority of oocytes that extruded a polar body were diploid in both the control and the IVM-CB groups. However, the incidence of polyploidy in the IVM-CB group was higher than that in the control group (P < 0.05). In vitro development of diploid parthenotes in the control and the IVM-CB groups was similar in terms of blastocyst formation rates (45.8 and 42.8% respectively), number of blastomeres (39.9 and 44.4 respectively), the percentage of dead cells (4.3 and 2.9% respectively), and the frequency of apoptotic cells (7.3 and 6.3% respectively). Tetraploid embryos had a lower blastocyst formation rate (25.5%) and number of cells (26.2); however, the proportion of apoptotic nuclei (7.0%) was similar to that in diploid parthenotes. These results suggest that the proportion of homozygous and heterozygous genes does not affect in vitro embryo development to the blastocyst stage.

THE EFFECTIVENESS OF HEAT, COLD AND 6-DIMETHYLAMINOPURINE SHOCKS FOR INDUCING TETRAPLOIDY IN THE KURUMA SHRIMP, MARSUPENAEUS JAPONICUS (BATE)

Abstract In this study tetraploid Marsupenaeus japonicus (Bate) embryos were produced by preventing the first division in mitosis. The effectiveness of temperature and chemical shocks for producing tetraploid M. japonicus were assessed when applied at different times postspawning and for different durations. Tetraploid M. japonicus embryos (spawned at 27°C) were produced by heat shocks at 35°C and 36°C in three and eight spawning samples respectively, and a cold shock at 5°C in a single spawning sample. All temperature shocks inducing tetraploidy were applied 18–23 min postspawning for a 5–10 min duration. The percentage of spawnings successfully inducing tetraploid embryos (i.e., frequency of induction) ranged from 33.33% to 66.67% for the 21, 22 and 23 min postspawning heat shock treatment regimes. The percentage of tetraploid embryos within an induction (i.e., induction rate), as determined by flow cytometry, ranged from 8.82% to 98.12% (ave. ± S.E.) (34.4 ± 21.4%) for the 35°C shock treatments, from 1...

Assembling Aggregates between Embryonic Stem (ES) Cells and Tetraploid Embryos

Assembling Aggregates between Embryonic Stem (ES) Cells and Tetraploid Embryos

Assembling Aggregates between Diploid and Tetraploid Embryos

Assembling Aggregates between Diploid and Tetraploid Embryos

Production of chimeras by aggregation of embryonic stem cells with diploid or tetraploid mouse embryos

The production of mouse chimeras is a common step in the establishment of genetically modified animal strains. Chimeras also provide a powerful experimental tool for following cell behavior during both prenatal and postnatal development. This protocol outlines a simple and economical technique for the production of large numbers of mouse chimeras using traditional diploid morula<-->diploid embryonic stem (ES) cell aggregations. Additional steps are included to describe the procedures necessary to produce specialized tetraploid chimeras using tetraploid morula<-->diploid ES cell aggregations. This increasingly popular form of chimera produces embryos of nearly complete ES cell derivation that can be used to speed transgenic production or ask developmental questions. Using this protocol, mouse chimeras can be generated and transferred to pseudopregnant surrogate mothers in a 5-d period.

Nuclear transfer reprogramming does not improve the low developmental potency of embryonic stem cells induced by long-term culture

Epigenetic states of embryonic stem (ES) cells are easily altered by long-term cultivation and lose their developmental potential. To rescue this reduced developmental capacity, nuclear transfer (NT) of ES cells was carried out, and original ES and ES cells from cloned blastocysts (ntES) cells established after NT were compared with in vitro differentiation ability and developmental potential by embryoid body formation and tetraploid aggregation respectively. In the establishment of ntES cell lines, the oocytes fused with the ES cell were activated, and further cultured to cloned blastocysts. When in vitro differentiation ability was examined between original and ntES cell lines derived from ES cells with extensive passages (ES-ep), the day of appearance of simple embryoid body, cystic embryoid body, and spontaneous beating was almost similar. The developmental rates of ES-ep cells, that aggregated with tetraploid embryos to term, ranged from 3 to 6%. Moreover, the majority of live pups died soon after birth. In the ntES cell lines derived from ES-ep cells, developmental rates ranged from 0 to 5%. Those pups also died soon after birth, similar to the ES-ep-derived pups. These results suggest that profound epigenetic modifications of ES cells were retained in the re-established cell lines by NT.

Tracking germinal center B cells expressing germ-line immunoglobulin γ1 transcripts by conditional gene targeting

Germinal centers (GCs) represent the main sites for the generation of high-affinity, class-switched antibodies during T cell-dependent antibody responses. To study gene function specifically in GC B cells, we generated Cgamma1-cre mice in which the expression of Cre recombinase is induced by transcription of the Ig gamma1 constant region gene segment (Cgamma1). In these mice, Cre-mediated recombination at the fas, Igbeta, IgH, and Rosa26 loci occurred in GC B cells as early as 4 days after immunization with T cell-dependent antigens and involved >85% of GC B cells at the peak of the GC reaction. Less than 2% of IgM(+) B cells showed Cre-mediated recombination. These cells carried few Ig somatic mutations, expressed germ-line Cgamma1- and activation-induced cytidine deaminase-specific transcripts and likely include GC B cell founders and/or plasma cell precursors. Cre-mediated recombination involved most IgG1, but also a fraction of IgG3-, IgG2a-, IgG2b-, and IgA-expressing GC and post-GC B cells. This result indicates that a GC B cell can transcribe more than one downstream C(H) gene before undergoing class switch recombination. The efficient induction of Cre expression in GC B cells makes the Cgamma1-cre allele a powerful tool for the genetic analysis of these cells, as well as, in combination with a suitable marker for Cre-mediated recombination, the tracking of class-switched memory B and plasma cells in vivo. To expedite the genetic analysis of GC B cells, we have established Cgamma1-cre F(1) embryonic stem cells, allowing further rounds of gene targeting and the cloning of compound mutants by tetraploid embryo complementation.

Developmental abnormalities of NT mouse embryos appear early after implantation

In mammals, cloning by nuclear transfer (NT) into an enucleated oocyte is a very inefficient process, even if it can generate healthy adults. We show that blastocysts derived from embryonic stem (ES) donor cells develop at a high rate, correctly express the pluripotential marker gene Oct4 in ICM cells and display normal growth in vitro. Moreover, the majority of them implant in the uterus of recipient females. We combine embryological studies, gene expression analysis during gastrulation and generation of chimaeric embryos to identify the developmental origin (stage and tissue affected) of NT embryo mortality. The majority died before mid-gestation from defects arising early, either at peri-implantation stages or during the gastrulation period. The first type of defect is a non-cell autonomous defect of the epiblast cells and is rescued by complementation of NT blastocysts with normal ES or ICM cells. The second type of defect affects growth regulation and the shape of the embryo but does not directly impair the initial establishment of the patterning of the embryo. Only chimaeras formed by the aggregation of NT and tetraploid embryos reveal no growth abnormalities at gastrulation. These studies indicate that the trophoblast cell lineage is the primary source of these defects. These embryological studies provide a solid basis for understanding reprogramming errors in NT embryos. In addition, they unveil new aspects of growth regulation while increasing our knowledge on the role of crosstalk between the extra-embryonic and the embryonic regions of the conceptus in the control of growth and morphogenesis.

An alternative method of deriving embryonic stem cell-like clones by aggregation of diploid cells with tetraploid embryos

To assess whether embryonic stem (ES) cells could be derived from the aggregation of diploid cells with tetraploid embryos. Randomized, prospective study. University embryology and gamete biotechnology laboratory. F1 (C57BL6/DBA2) mice. Four- to eight-cell F1 tetraploid embryos were aggregated with 10 to 15 donor E14 ES cells. Embryogenesis and ES cell establishment. No difference (78% to 89%) in blastocyst formation was detected between the aggregated tetraploid and the control diploid embryos. In a total of 27 transfers, pregnancy was detected in three tetraploid (23.1%) and five diploid (35.7%) cases, and three live births developed from the aggregated tetraploid embryos. The tetraploid blastocysts without aggregation were plated, but no ES cell-like colony was formed. Six of eight aggregated blastocysts derived well-proliferated colonies, which were positive for anti-stage-specific embryonic antigen (SSEA)-1 antibody, Oct-4, and alkaline phosphatase. The microsatellite assay confirmed the homogenous makeup among the donor E14 cells and live-birth and ES-like cells derived from the E14-aggregated, tetraploid embryo. The aggregation of pluripotent diploid cells with tetraploid embryos yielded live births and ES-like cells that were homogenous to the donor diploid cells.

GATA4 and the Two Sides of Gene Expression Reprogramming

See related article, pages 837–845 During embryonic development as well as in postnatal life, cardiac cells are continuously exposed to a host of extracellular signals to which they adapt and respond by activating intracellular signaling pathways and modulating the expression of nuclear genes. In the adult heart, the most common and easily recognized response to stressful stimuli is hypertrophy of cardiomyocytes and remodeling of the entire organ.1 This reactive growth response requires de novo synthesis of contractile and structural proteins, obtained through a complex series of events culminating in gene expression reprogramming.2 GATA4 is a zinc-finger transcription factor highly expressed in cardiomyocytes at different developmental stages where it regulates the transcription of several structural and regulatory genes, including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), α- and β-myosin heavy chain (MHC).3 In human hearts, GATA4 has also been demonstrated to be a critical regulator of cardiac development, as shown by the association between GATA4 mutations and the presence of congenital cardiac malformations.4 The specific effects of GATA4 deletion during embryonic age have been extensively investigated in genetically modified mouse models.5–7 Mice homozygous for a GATA4 null allele6 or homozygous GATA4-deficient mice (GATA4−/−)5 die early in development because of abnormal …

231 HISTOCOMPATIBLE PARTHENOGENETIC EMBRYONIC STEM CELLS

Organ or tissue transplantation is the preferred treatment for numerous diseases but is hindered by immunologic barriers. Genetically matched pluripotent embryonic stem cells generated via nuclear transfer (ntES cells) or parthenogenesis (pES cells) are possible sources of histocompatible cells and tissues. We have developed two ways of isolating pES cells that carry the full complement of major histocompatibility complex (MHC) antigens of the oocyte donors. One method entails activation of oocytes after blockade of karyokinesis in meiosis II, followed by selection of predominantly homozygous pES cells that have undergone recombination in their MHC antigen region to restore the heterozygous maternal MHC genotype (parthenote recombinant, or prES cells). The second method involves activation of immature oocytes after blockade of karyokinesis of meiosis I, followed by selection of predominantly heterozygous pES lines that retain the MHC genotype of the oocyte donor (parthenote clone recombinant, or pcrES cells). The cells are pluripotent by several criteria: teratoma formation, in vitro differentiation into hematopoietic elements, and high-level skin chimerism in blastocyst chimeras. Breeding of 8 founder females and examination of over 700 progeny failed to demonstrate germ line transmission of the pES cells. Injection of over 50 tetraploid embryos with these lines and embryo transfer have failed to support full gestational development. However, differentiated tissues from these pluripotent ES cells engraft when transplanted into genetically matched immunocompetent recipients, demonstrating that selected pES cells can serve as a source of histocompatible tissues for transplantation.

Generation of embryos directly from embryonic stem cells by tetraploid embryo complementation reveals a role for GATA factors in organogenesis.

Gene targeting in ES (embryonic stem) cells has been used extensively to study the role of proteins during embryonic development. In the traditional procedure, this requires the generation of chimaeric mice by introducing ES cells into blastocysts and allowing them to develop to term. Once chimaeric mice are produced, they are bred into a recipient mouse strain to establish germline transmission of the allele of interest. Although this approach has been used very successfully, the breeding cycles involved are time consuming. In addition, genes that are essential for organogenesis often have roles in the formation of extra-embryonic tissues that are essential for early stages of post-implantation development. For example, mice lacking the GATA transcription factors, GATA4 or GATA6, arrest during gastrulation due to an essential role for these factors in differentiation of extra-embryonic endoderm. This lethality has frustrated the study of these factors during the development of organs such as the liver and heart. Extraembryonic defects can, however, be circumvented by generating clonal mouse embryos directly from ES cells by tetraploid complementation. Here, we describe the usefulness and efficacy of this approach using GATA factors as an example.

Generation of embryos directly from embryonic stem cells by tetraploid embryo complementation reveals a role for GATA factors in organogenesis

Gene targeting in ES (embryonic stem) cells has been used extensively to study the role of proteins during embryonic development. In the traditional procedure, this requires the generation of chimaeric mice by introducing ES cells into blastocysts and allowing them to develop to term. Once chimaeric mice are produced, they are bred into a recipient mouse strain to establish germline transmission of the allele of interest. Although this approach has been used very successfully, the breeding cycles involved are time consuming. In addition, genes that are essential for organogenesis often have roles in the formation of extra-embryonic tissues that are essential for early stages of post-implantation development. For example, mice lacking the GATA transcription factors, GATA4 or GATA6, arrest during gastrulation due to an essential role for these factors in differentiation of extra-embryonic endoderm. This lethality has frustrated the study of these factors during the development of organs such as the liver and heart. Extraembryonic defects can, however, be circumvented by generating clonal mouse embryos directly from ES cells by tetraploid complementation. Here, we describe the usefulness and efficacy of this approach using GATA factors as an example.