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Biomedical subjects

R A Pedersen

Publications and source records attributed to R A Pedersen.

At least 19 recordsLinked to original sources
PubMed2006

Molecular cytogenetic studies towards the full karyotype analysis of human blastocysts and cytotrophoblasts.

Numerical chromosome aberrations in gametes typically lead to failed fertilization, spontaneous abortion or a chromosomally abnormal fetus. By means of preimplantation genetic diagnosis (PGD), we now can screen human embryos in vitro for aneuploidy before transferring the embryos to the uterus. PGD allows us to select unaffected embryos for transfer and increases the implantation rate in in vitro fertilization programs. Molecular cytogenetic analyses using multi-color fluorescence in situ hybridization (FISH) of blastomeres have become the major tool for preimplantation genetic screening of aneuploidy. However, current FISH technology can test for only a small number of chromosome abnormalities and hitherto failed to increase the pregnancy rates as expected. We are in the process of developing multi-color FISH-based technologies to score all 24 chromosomes in single cells within a three-day time limit, which we believe is vital to the clinical setting. Also, human placental cytotrophoblasts (CTBs) at the fetal-maternal interface acquire aneuploidies as they differentiate to an invasive phenotype. About 20-50% of invasive CTB cells from uncomplicated pregnancies were found to be aneuploid, suggesting that the acquisition of aneuploidy is an important component of normal placentation, perhaps limiting the proliferative and invasive potential of CTBs. Since most invasive CTBs are interphase cells and possess extreme heterogeneity, we applied multi-color FISH and repeated hybridizations to investigate the feasibility of a full karyotype analysis of individual CTBs. In summary, this study demonstrates the strength of Spectral Imaging analysis and repeated hybridizations, which provides a basis for full karyotype analysis of single interphase cells.

Blastocyst↗
PubMed2001

Towards a full karyotype screening of interphase cells: 'FISH and chip' technology.

Numerical chromosome aberrations are incompatible with normal human development. Our laboratories develop hybridization-based screening tools that generate a maximum of cytogenetic information for each polar body or blastomere analyzed. The methods are developed considering that the abnormality might require preparation of case-specific probes and that only one or two cells will be available for diagnosis, most of which might be in the interphase stage. Furthermore, assay efficiencies have to be high, since there is typically not enough time to repeat an experiment or reconfirm a result prior to fertilization or embryo transfer. Structural alterations are delineated with breakpoint-spanning probes. When screening for numerical abnormalities, we apply a Spectral Imaging-based approach to simultaneously score as many as ten different chromosome types in individual interphase cells. Finally, DNA micro-arrays are under development to score all of the human chromosomes in a single experiment and to increase the resolution with which micro-deletions can be delineated.

Blastomeres↗
PubMed2001

Detection of structural and numerical chromosome abnormalities in interphase cells using spectral imaging.

Chromosome abnormalities are common causes of congenital malformations and spontaneous abortions. They include structural abnormalities, polyploidy, trisomy, and mosaicism. In in vitro fertilization (IVF) programs, preimplantation genetic diagnosis (PGD) of oocytes and embryos has become the technique of choice to select against abnormal embryos before embryo transfer. For diagnosis of structural abnormalities, we developed case-specific breakpoint-spanning DNA probes. Screening of an in-house yeast artificial chromosome (YAC) library is facilitated by information from publicly available databases and published articles. Most numerical chromosome abnormalities, on the other hand, are detrimental to early embryonic development and increase with maternal age. We therefore developed a multichromosome screening technique based on spectral imaging to simultaneously detect and score as many as 10 different chromosome types. The probe set was chosen to detect more than 70% of all numerical chromosome aberrations responsible for spontaneous abortions. Detecting structural and numerical abnormalities in single interphase cells using spectral imaging is a powerful technique for multilocus genetic screening.

Chromosome Aberrations↗
PubMed2001

Blastomeres arising from the first cleavage division have distinguishable fates in normal mouse development.

Two independent studies have recently suggested similar models in which the embryonic and abembryonic parts of the mouse blastocyst become separated already by the first cleavage division. However, no lineage tracing studies carried out so far on early embryos provide the support for such a hypothesis. Thus, to re-examine the fate of blastomeres of the two-cell mouse embryo, we have undertaken lineage tracing studies using a non-perturbing method. We show that two-cell stage blastomeres have a strong tendency to develop into cells that comprise either the embryonic or the abembryonic parts of the blastocyst. Moreover, the two-cell stage blastomere that is first to divide will preferentially contribute its progeny to the embryonic part. Nevertheless, we find that the blastocyst embryonic-abembryonic axis is not perfectly orthogonal to the first cleavage plane, but often shows some angular displacement from it. Consequently, there is a boundary zone adjacent to the interior margin of the blastocoel that is populated by cells derived from both earlier and later dividing blastomeres. The majority of cells that inhabit this boundary region are, however, derived from the later dividing two-cell stage blastomere that contributes predominantly to the abembryonic part of the blastocyst. Thus, at the two-cell stage it is already possible to predict which cell will contribute a greater proportion of its progeny to the abembryonic part of the blastocyst (region including the blastocyst cavity) and which to the embryonic part (region containing the inner cell mass) that will give rise to the embryo proper.

Animals↗
PubMed2000

Mouse numb is an essential gene involved in cortical neurogenesis.

During neurogenesis of the mammalian neocortex, neural progenitor cells divide to generate daughter cells that either become neurons or remain as progenitor cells. The mouse numb (m-numb) gene encodes a membrane-associated protein that is asymmetrically localized to the apical cell membrane of dividing cortical progenitor cells and may be segregated to only the apical daughter cell that has been suggested to remain as a progenitor cell. To examine m-numb function during neural development, we generated a loss-of-function mutant allele of m-numb. Mice homozygous for this mutation exhibit severe defects in cranial neural tube closure and precocious neuron production in the forebrain and die around embryonic day 11.5 (E11. 5). These findings suggest that m-numb is an essential gene that plays a role in promoting progenitor cell fate during cortical neurogenesis.

Animals↗
PubMed2000

Multilocus genetic analysis of single interphase cells by spectral imaging.

Numerical chromosome aberrations are detrimental to early embryonic, fetal and perinatal development of mammals. When fetuses carrying a chromosomal imbalance survive to term, an aberrant gene dosage typically leads to stillbirth or causes a severely altered phenotype. Aneuploidy of any of the 24 chromosomes will negatively impact on human development, and a preimplantation and prenatal genetic diagnosis test should thus score as many chromosomes as possible. Since cells available for analysis are likely to be in interphase, we set out to develop a rapid enumeration procedure based on hybridization of chromosome-specific probes and spectral imaging detection. The probe set was chosen to allow the simultaneous enumeration of ten chromosome types and was expected to detect more than 70% of all numerical chromosome aberrations responsible for spontaneous abortions, i.e., human chromosomes 9, 13, 14, 15, 16, 18, 21, 22, X, and Y. Cell fixation protocols were optimized to achieve the desired detection sensitivity and reproducibility. We were able to resolve and identify ten separate chromosomal signals in interphase nuclei from different types of cells, including lymphocytes, uncultured amniocytes, and blastomeres. In summary, this study demonstrates the strength of spectral imaging, allowing us to construct partial spectral imaging karyotypes for individual interphase cells by assessing the number of each of the target chromosome types.

Blastomeres↗
PubMed2000

Case-specific, breakpoint-spanning DNA probes for analysis of single interphase cells.

Balanced reciprocal translocations are known to interfere with homolog pairing in meiosis. Many individuals carrying such chromosomal abnormalities suffer from reduced fertility or spontaneous abortions and seek help in the form of assisted reproductive technology. Although most translocations are relatively easy to detect in metaphase cells, the majority of embryonic cells biopsied in the course of in vitro fertilization (IVF) procedures are in interphase. These nuclei are, thus, unsuitable for analysis by chromosome banding or painting using fluorescence in situ hybridization (FISH). Our assay, based on FISH detection of breakpoint-spanning DNA probes, identifies translocations in interphase nuclei by microscopic inspection of hybridization domains. Probes are selected that span the breakpoint regions on normal homologs. The probes should hybridize to several hundred kilobases of DNA flanking the breakpoint. The two breakpoint-spanning DNA probes for the translocation chromosomes are labeled in separate colors (e.g., red and green). The translocation event producing two fused red/green hybridization domains can then be detected in interphase cell nuclei using a fluorescence microscope. We applied this scheme to analyze somatic and germ cells from 21 translocation patients, each with distinct breakpoints. Here, we summarize our experience and provide a description of strategies, cost estimates, as well as typical time frames.

Blastomeres↗
PubMed2000

goosecoid expression represses Brachyury in embryonic stem cells and affects craniofacial development in chimeric mice.

The homeobox gene goosecoid, originally identified in Xenopus, is expressed in the organizer or its equivalent during gastrulation in the frog, chick, zebrafish and mouse. To investigate the role of goosecoid in mouse development, we have generated embryonic stem cells that stably overexpress the murine homolog of goosecoid. These cells show a repression of the gastrulation-associated gene Brachyury. Interestingly, repression of Brachyury is conserved between Xenopus and mouse despite the lack of conservation of the Brachyury promoter. Further characterization of the goosecoid-overexpressing ES cells revealed that they maintain the expression of stage-specific embryonic antigen-1, and teratomas derived from goosecoid-overexpressing cells show the presence of cell types derived from all three germ layers. Some highly chimeric mice derived from goosecoid-overexpressing cells displayed skull defects. These observations suggest that goosecoid may play a role in specification of anterior mesendodermal fates and specifically in mouse craniofacial development.

Animals↗
PubMed1999

Requirement for the Xrcc1 DNA base excision repair gene during early mouse development.

Surveillance and repair of DNA damage are essential for maintaining the integrity of the genetic information that is needed for normal development. Several multienzyme pathways, including the excision repair of damaged or missing bases, carry out DNA repair in mammals. We determined the developmental role of the X-ray cross-complementing (Xrcc)-1 gene, which is central to base excision repair, by generating a targeted mutation in mice. Heterozygous matings produced Xrcc1-/- embryos at early developmental stages, but not Xrcc1-/- late-stage fetuses or pups. Histology showed that mutant (Xrcc1-/-) embryos arrested at embryonic day (E) 6.5 and by E7.5 were morphologically abnormal. The most severe abnormalities observed in mutant embryos were in embryonic tissues, which showed increased cell death in the epiblast and an altered morphology in the visceral embryonic endoderm. Extraembryonic tissues appeared relatively normal at E6.5-7.5. Even without exposure to DNA-damaging agents, mutant embryos showed increased levels of unrepaired DNA strand breaks in the egg cylinder compared with normal embryos. Xrcc1-/- cell lines derived from mutant embryos were hypersensitive to mutagen-induced DNA damage. Xrcc1 mutant embryos that were also made homozygous for a null mutation in Trp53 underwent developmental arrest after only slightly further development, thus revealing a Trp53-independent mechanism of embryo lethality. These results show that an intact base excision repair pathway is essential for normal early postimplantation mouse development and implicate an endogenous source of DNA damage in the lethal phenotype of embryos lacking this repair capacity.

Animals↗
PubMed1999

Preimplantation diagnosis of the beta1 integrin knockout mutation as a model for aneuploid gene testing.

The autosomal beta1 integrin knockout mouse mutation was selected as a model to experimentally determine preimplantation diagnosis test reliability for autosomal gene deletions and duplications. In experiment 1, which analyzed 198 individually disaggregated single blastomeres, the observed test frequencies matched the mathematically predicted frequencies calculated from the independently derived values of 90% normal allele amplification, 92% mutant allele amplification, 4% alternate allele contamination, and 4% failure to transfer amplifiable target DNA into the PCR reaction mix. This experiment correctly predicted a normal embryonic phenotype in 143 (99.3%) of the 144 phenotypically normal autosomal recessive results. Experiment 2 compared single biopsied blastomere test results to test results on the remaining embryonic cells cultured 1 week until trophoblast outgrowth. Single biopsied blastomere analysis correctly predicted a normal autosomal recessive phenotype in 87 (98%) of the 89 embryos that would have been selected for implantation. Experiment 3 compared the PCR results of two biopsied blastomeres tested independently to the PCR result from the remaining cultured blastomeres to improve test reliability. Given that embryos would have been implanted only when two normal results were obtained, 17 of 17 phenotypically normal embryos would have been implanted from among the 44 embryos tested. These experiment 3 results are consistent with the mathematical prediction that about 99.9% of embryos implanted with two unaffected biopsied blastomere results would have had a phenotypically normal genotype.

Aneuploidy↗
PubMed1999

Testis sperm extraction and intracytoplasmic sperm injection guided by prior fine-needle aspiration mapping in patients with nonobstructive azoospermia.

OBJECTIVE: To evaluate intracytoplasmic sperm injection (ICSI) outcomes in a cohort of men with nonobstructive azoospermia who underwent prior fine-needle aspiration (FNA) "maps" to localize sperm and guide testis sperm extraction (TESE). DESIGN: Retrospective clinical study. SETTING: University-based infertility practice. PATIENT(S): A consecutive cohort of 19 infertile, azoospermic men. INTERVENTION(S): Couples underwent IVF-ET in which TESE procedures were informed and directed by prior FNA maps of the testis. MAIN OUTCOME MEASURE(S): Sperm retrieval and pregnancy rates. RESULT(S): In 21 IVF-ET and ICSI cycles, sufficient sperm for all oocytes were retrieved in 20 TESE attempts (95%). A mean of 3.1 biopsies per patient were required, with an average size of 72 mg. Mean operative time for the TESE procedure was 88 minutes. Overall, the two-pronuclear fertilization rate was 66%; ongoing clinical pregnancies were obtained in 10 of 21 initiated cycles (48%). CONCLUSION(S): In an effort to reduce IVF-ET cancellation rates in cases of nonobstructive azoospermia, diagnostic testis FNA can define those patients who are good candidates for TESE. It also directs sperm retrieval and minimizes tissue removal from nonobstructed testes.

Adult↗
PubMed1999

Status of genomic imprinting in mouse spermatids.

The advent of human round spermatid microinjection (ROSI) into oocytes as a treatment for severe male infertility raises the question of whether spermatids have undergone all of the maturation processes necessary for normal development. It is particularly important to know whether spermatids have undergone correct genomic imprinting, which results in the parent-of-origin-specific expression of only one allele of a gene. We assessed the imprinting status of three maternally and three paternally expressed genes in interspecific hybrid embryos generated by injecting Mus castaneus spermatids into Mus musculus oocytes. We used the single nucleotide primer extension (SNuPE) assay to measure the relative expression of maternal and paternal alleles on the basis of sequence polymorphisms in the transcripts. Expression of imprinted genes in mouse embryos derived by ROSI did not differ from controls, indicating that paternal genes have undergone proper imprinting by the round spermatid stage.

Animals↗
PubMed1999

Modification of the Comet assay for the detection of DNA strand breaks in extremely small tissue samples.

We modified the Comet assay to enable the quantification of DNA strand breakage in individual cells of extremely small tissue samples. This modification was used to analyze cells isolated from the ectoplacental cone and egg cylinder of mouse embryos at embryonic day 7.5. We detected more naturally occurring DNA strand breaks and a higher number of apoptotic cells in the ectoplacental cone compared with the egg cylinder.

Animals↗
PubMed1999

Dlx5 regulates regional development of the branchial arches and sensory capsules.

We report the generation and analysis of mice homozygous for a targeted deletion of the Dlx5 homeobox gene. Dlx5 mutant mice have multiple defects in craniofacial structures, including their ears, noses, mandibles and calvaria, and die shortly after birth. A subset (28%) exhibit exencephaly. Ectodermal expression of Dlx5 is required for the development of olfactory and otic placode-derived epithelia and surrounding capsules. The nasal capsules are hypoplastic (e.g. lacking turbinates) and, in most cases, the right side is more severely affected than the left. Dorsal otic vesicle derivatives (e. g. semicircular canals and endolymphatic duct) and the surrounding capsule, are more severely affected than ventral (cochlear) structures. Dlx5 is also required in mandibular arch ectomesenchyme, as the proximal mandibular arch skeleton is dysmorphic. Dlx5 may control craniofacial development in part through the regulation of the goosecoid homeobox gene. goosecoid expression is greatly reduced in Dlx5 mutants, and both goosecoid and Dlx5 mutants share a number of similar craniofacial malformations. Dlx5 may perform a general role in skeletal differentiation, as exemplified by hypomineralization within the calvaria. The distinct focal defects within the branchial arches of the Dlx1, Dlx2 and Dlx5 mutants, along with the nested expression of their RNAs, support a model in which these genes have both redundant and unique functions in the regulation of regional patterning of the craniofacial ectomesenchyme.

Animals↗
PubMed1999

Polarity of the mouse embryo is anticipated before implantation.

In most species, the polarity of an embryo underlies the future body plan and is determined from that of the zygote. However, mammals are thought to be an exception to this; in the mouse, polarity is generally thought to develop significantly later, only after implantation. It has not been possible, however, to relate the polarity of the preimplantation mouse embryo to that of the later conceptus due to the lack of markers that endure long enough to follow lineages through implantation. To test whether early developmental events could provide cues that predict the axes of the postimplantation embryo, we have used the strategy of injecting mRNA encoding an enduring marker to trace the progeny of inner cell mass cells into the postimplantation visceral endoderm. This tissue, although it has an extraembryonic fate, plays a role in axis determination in adjacent embryonic tissue. We found that visceral endoderm cells that originated near the polar body (a marker of the blastocyst axis of symmetry) generally became distal as the egg cylinder formed, while those that originated opposite the polar body tended to become proximal. It follows that, in normal development, bilateral symmetry of the mouse blastocyst anticipates the polarity of the later conceptus. Moreover, our results show that transformation of the blastocyst axis of symmetry into the axes of the postimplantation conceptus involves asymmetric visceral endoderm cell movement. Therefore, even if the definitive axes of the mouse embryo become irreversibly established only after implantation, this polarity can be traced back to events before implantation.

Animals↗
PubMed1998

A murine AP-endonuclease gene-targeted deficiency with post-implantation embryonic progression and ionizing radiation sensitivity.

Apurinic/apyrimidinic endonuclease (here designated APE/REF) carries out repair incision at abasic or single-strand break damages in mammals. This multifunctional protein also has putative role(s) as a cysteine 'reducing factor' (REF) in cell-stress transcriptional responses. To assess the significance of APE/REF for embryonic teratogenesis we constructed a more precisely targeted Ape/Ref-deficient genotype in mice. Ape/Ref gene replacement in ES cells eliminated the potential of APE/REF protein synthesis while retaining the Ape/Ref bi-directional promoter that avoided potential inactivation of an upstream gene. Chimeric animals crossed into Tac:N:NIHS-BC produced germline transmission. Homozygous null Ape/Ref-embryos exhibited successful implantation and nearly normal developmental progression until embryonic day 7.5 followed by morphogenetic failure and adsorption of embryos by day 9.5. We characterized the cellular events proceeding to embryonic lethality and examined ionizing radiation sensitivity of pre-implantation Ape/Ref-null embryos. After intermating of heterozygotes, Mendelian numbers of putative Ape/Ref-null progeny embryos at day 6.5 displayed a several-fold elevation of pycnotic, fragmenting cell nuclei within the embryo proper-the epiblast. Increased cell-nucleus degeneration occurred within epiblast cells while mitosis continued and before obvious morphogenetic disruption. Mitogenic response to epiblast cell death, if any, was ineffective for replacement of lost cells. Extra-embryonic yolk sac, a trophectoderm derived lineage retained normal appearance to day 9. Explanted homozygous Ape/Ref-null blastocysts displayed increased sensitivity to gamma-irradiation, most likely a manifestation of APE/REF incision defect. Our study establishes that this new Ape/Ref deficiency genotype is definitely capable of post-implantation developmental progression to the onset of gastrulation. Function(s) of APE/REF in base damage incision and also conceivably in mitogenic responses towards epiblast cell death are critical for transit through the gastrulation stage of embryonic growth and development.

Alleles↗