Caenorhabditis elegans polo-like kinase PLK-1 is required for merging parental genomes into a single nucleus

Before the first zygotic division, the nuclear envelopes of the maternal and paternal pronuclei disassemble, allowing both sets of chromosomes to be incorporated into a single nucleus in daughter cells after mitosis. We found that in Caenorhabditis elegans, partial inactivation of the polo-like kinase PLK-1 causes the formation of two nuclei, containing either the maternal or paternal chromosomes, in each daughter cell. These two nuclei gave rise to paired nuclei in all subsequent cell divisions. The paired-nuclei phenotype was caused by a defect in forming a gap in the nuclear envelopes at the interface between the two pronuclei during the first mitotic division. This was accompanied by defects in chromosome congression and alignment of the maternal and paternal metaphase plates relative to each other. Perturbing chromosome congression by other means also resulted in failure to disassemble the nuclear envelope between the two pronuclei. Our data further show that PLK-1 is needed for nuclear envelope breakdown during early embryogenesis. We propose that during the first zygotic division, PLK-1–dependent chromosome congression and metaphase plate alignment are necessary for the disassembly of the nuclear envelope between the two pronuclei, ultimately allowing intermingling of the maternal and paternal chromosomes.     

Paternal chromosome incorporation into the zygote nucleus is controlled by maternal haploid in Drosophila

maternal haploid (mh) is a strict maternal effect mutation that causes the production of haploid gynogenetic embryos (eggs are fertilized but only maternal chromosomes participate in development). We conducted a cytological analysis of fertilization and early development in mh eggs to elucidate the mechanism of paternal chromosome elimination. In mh eggs, as in wild-type eggs, male and female pronuclei migrate and appose, the first mitotic spindle forms, and both parental sets of chromosomes congress on the metaphase plate. In contrast to control eggs, mh paternal sister chromatids fail to separate in anaphase of the first division. As a consequence the paternal chromatin stretches and forms a bridge in telophase. During the first three embryonic divisions, damaged paternal chromosomes are progressively eliminated from the spindles that organize around maternal chromosomes. A majority of mh embryos do not survive the deleterious presence of aneuploid nuclei and rapidly arrest their development. The rest of mh embryos develop as haploid gynogenetic embryos and die before hatching. The mh phenotype is highly reminiscent of the early developmental defects observed in eggs fertilized by ms(3)K81 mutant males and in eggs produced in incompatible crosses of Drosophila harboring the endosymbiont bacteria Wolbachia.     

Parental source of chromosome imprinting and its relevance for X chromosome inactivation

In imprinting, homologous chromosomes behave differently during development according to their parental origin. Typically, paternally derived chromosomes are preferentially inactivated or eliminated. Examples of such phenomena include inactivation of the mammalian X chromosome, inactivation or elimination of one haploid chromosome set in male coccids, and elimination of paternal X chromosomes in the fly Sciara. It has generally been thought that the paternal chromosomes bear an imprint leading to their inactivation or elimination. However, alteration of the parental origin of chromosomes, as in the study of parthenogenotes in mammals and coccids, shows that passage of chromosomes through a male germ cell or fertilization is not essential for inactivation or elimination. It appears that neither chromosome set is programmed to resist or undergo inactivation. Instead the two sets differ in relative sensitivity, and the question is whether the maternal set have an imprint for resistance, or the paternal set one for susceptibility. Very early in development of mammals both X chromosomes are active. This makes it simpler to envisage the maternal X bearing an imprint for resistance to inactivation, which persists through the early developmental period. Similar considerations also apply in coccids and Sciara. Thus, imprinting should be regarded as a phenomenon conferred on the maternal chromosomes in the oocyte. This permits simpler models for the mechanism of X-inactivation, and weakens the case for evolution of X-inactivation from an earlier form of inactivation during male gametogenesis. One may speculate whether imprinting affects timing of gene action in development.     

Nonrandom chromosome arrangements in germ line nuclei of Sciara coprophila males: the basis for nonrandom chromosome segregation on the meiosis I spindle

Meiosis I in males of the Dipteran Sciara coprophila results in the nonrandom distribution of maternally and paternally derived chromosome sets to the two division products. Based on an earlier study (Kubai, D.F. 1982. J. Cell Biol. 93:655-669), I suggested that the meiosis I spindle does not play a direct role in the nonrandom sorting of chromosomes but that, instead, haploid sets are already separated in prophase nuclei well before the onset of spindle formation. Here I report more direct evidence that this hypothesis is true; this evidence was gained from ultrastructural reconstruction analyses of the arrangement of chromosomes in germ line nuclei (prophase nuclei in spermatogonia and spermatocytes) of males heterozygous for an X- autosome chromosome translocation. Because of this translocation, the maternal and paternal chromosome sets are distinguishable, so it is possible to demonstrate that (a) the two haploid chromosome sets occupy distinct maternal and paternal nuclear compartments and that (b) nuclei are oriented so that the two haploid chromosome sets have consistent relationships to a well-defined cellular axis. The consequences of such nonrandom aspects of nuclear structure for chromosome behavior on premeiotic and meiotic spindles are discussed.     

Parental source of chromosome imprinting and its relevance for X chromosome inactivation

Abstract. In imprinting, homologous chromosomes behave differently during development according to their parental origin. Typically, paternally derived chromosomes are preferentially inactivated or eliminated. Examples of such phenomena include inactivation of the mammalian X chromosome, inactivation or elimination of one haploid chromosome set in male coccids, and elimination of paternal X chromosomes in the fly Sciara . It has generally been thought that the paternal chromosomes bear an imprint leading to their inactivation or elimination. However, alteration of the parental origin of chromosomes, as in the study of parthenogenotes in mammals and coccids, shows that passage of chromosomes through a male germ cell or fertilization is not essential for inactivation or elimination. It appears that neither chromosome set is programmed to resist or undergo inactivation. Instead the two sets differ in relative sensitivity, and the question is whether the maternal set have an imprint for resistance, or the paternal set one for susceptibility. Very early in development of mammals both X chromosomes are active. This makes it simpler to envisage the maternal X bearing an imprint for resistance to inactivation, which persists through the early developmental period. Similar considerations also apply in coccids and Sciara . Thus, imprinting should be regarded as a phenomenon conferred on the maternal chromosomes in the oocyte. This permits simpler models for the mechanism of X-inactivation, and weakens the case for evolution of X-inactivation from an earlier form of inactivation during male gametogenesis. One may speculate whether imprinting affects timing of gene action in development.     

Nuclear transplantation in the mouse: heritable differences between parental genomes after activation of the embryonic genome

Paternal and maternal genomes apparently have complementary roles during embryogenesis in the mouse, and both are essential for development to term. However, there is no direct evidence to show that functional differences between parental genomes remain intact after activation of the embryonic genome at the 2-cell stage. In this study we demonstrate that transfer of paternal or maternal nuclei from early haploid preimplantation embryos back to fertilized eggs from which one pronucleus was removed resulted in development to term, but only if the remaining pronucleus was of the parental type opposite to the donor nucleus. Hence, functional differences between parental chromosomes are heritable and they survive activation of the embryonic genome and probable reprogramming of donor embryonic nuclei by epigenetic factors in the egg cytoplasm.     

The paternal sex ratio chromosome in the parasitic wasp Trichogramma kaykai condenses the paternal chromosomes into a dense chromatin mass.

A recently discovered B chromosome in the parasitoid wasp Trichogramma kaykai was found to be transmitted through males only. Shortly after fertilization, this chromosome eliminates the paternal chromosome set leaving the maternal chromosomes and itself intact. Consequently, the sex ratio in these wasps is changed in favour of males by modifying fertilized diploid eggs into male haploid offspring. In this study, we show that in fertilized eggs at the first mitosis the paternal sex ratio (PSR) chromosome condenses the paternal chromosomes into a so-called paternal chromatin mass (PCM). During this process, the PSR chromosome is morphologically unaffected and is incorporated into the nucleus containing the maternal chromosomes. In the first five mitotic divisions, 67% of the PCMs are associated with one of the nuclei in the embryo. Furthermore, in embryos with an unassociated PCM, all nuclei are at the same mitotic stage, whereas 68% of the PCM-associated nuclei are at a different mitotic phase than the other nuclei in the embryo. Our observations reveal an obvious similarity of the mode of action of the PSR chromosome in T. kaykai with that of the PSR-induced paternal genome loss in the unrelated wasp Nasonia vitripennis.     

Non-random chromosome arrangement in triploid endosperm nuclei

The endosperm is at the center of successful seed formation in flowering plants. Being itself a product of fertilization, it is devoted to nourish the developing embryo and typically possesses a triploid genome consisting of two maternal and one paternal genome complement. Interestingly, endosperm development is controlled by epigenetic mechanisms conferring parent-of-origin-dependent effects that influence seed development. In the model plant Arabidopsis thaliana, we have previously described an endosperm-specific heterochromatin fraction, which increases with higher maternal, but not paternal, genome dosage. Here, we report a detailed analysis of chromosomal arrangement and association frequency in endosperm nuclei. We found that centromeric FISH signals in isolated nuclei show a planar alignment that may results from a semi-rigid, connective structure between chromosomes. Importantly, we found frequent pairwise association of centromeres, chromosomal segments, and entire arms of chromosomes in 3C endosperm nuclei. These associations deviate from random expectations predicted by numerical simulations. Therefore, we suggest a non-random chromosomal organization in the triploid nuclei of Arabidopsis endosperm. This contrasts with the prevailing random arrangement of chromosome territories in somatic nuclei. Based on observations on a series of nuclei with varying parental genome ratios, we propose a model where chromosomes associate pairwise involving one maternal and one paternal complement. The functional implications of this predicted chromosomal arrangement are discussed.     

RanGTP and the actin cytoskeleton keep paternal and maternal chromosomes apart during fertilization

Zygotes require two accurate sets of parental chromosomes, one each from the mother and the father, to undergo normal embryogenesis. However, upon egg–sperm fusion in vertebrates, the zygote has three sets of chromosomes, one from the sperm and two from the egg. The zygote therefore eliminates one set of maternal chromosomes (but not the paternal chromosomes) into the polar body through meiosis, but how the paternal chromosomes are protected from maternal meiosis has been unclear. Here we report that RanGTP and F-actin dynamics prevent egg–sperm fusion in proximity to maternal chromosomes. RanGTP prevents the localization of Juno and CD9, egg membrane proteins that mediate sperm fusion, at the cell surface in proximity to maternal chromosomes. Following egg–sperm fusion, F-actin keeps paternal chromosomes away from maternal chromosomes. Disruption of these mechanisms causes the elimination of paternal chromosomes during maternal meiosis. This study reveals a novel critical mechanism that prevents aneuploidy in zygotes.     

Uniparental chromosome elimination at mitosis and interphase in wheat and pearl millet crosses involves micronucleus formation, progressive heterochromatinization, and DNA fragmentation

Complete uniparental chromosome elimination occurs in several interspecific hybrids of plants. We studied the mechanisms underlying selective elimination of the paternal chromosomes during the development of wheat (Triticum aestivum) x pearl millet (Pennisetum glaucum) hybrid embryos. All pearl millet chromosomes were eliminated in a random sequence between 6 and 23 d after pollination. Parental genomes were spatially separated within the hybrid nucleus, and pearl millet chromatin destined for elimination occupied peripheral interphase positions. Structural reorganization of the paternal chromosomes occurred, and mitotic behavior differed between the parental chromosomes. We provide evidence for a novel chromosome elimination pathway that involves the formation of nuclear extrusions during interphase in addition to postmitotically formed micronuclei. The chromatin structure of nuclei and micronuclei is different, and heterochromatinization and DNA fragmentation of micronucleated pearl millet chromatin is the final step during haploidization.     

Chromosome spatial order in human cells: evidence for early origin and faithful propagation

We have investigated the origin and nature of chromosome spatial order in human cells by analyzing and comparing chromosome distribution patterns of normal cells with cells showing specific chromosome numerical anomalies known to arise early in development. Results show that all chromosomes in normal diploid cells, triploid cells and in cells exhibiting nondisjunction trisomy 21 are incorporated into a single, radial array (rosette) throughout mitosis. Analysis of cells using fluorescence in situ hybridization, digital imaging and computer-assisted image analysis suggests that chromosomes within rosettes are segregated into tandemly linked “haploid sets” containing 23 chromosomes each. In cells exhibiting nondisjunction trisomy 21, the distribution of chromosome 21 homologs in rosettes was such that two of the three homologs were closely juxtaposed, a pattern consistent with our current understanding of the mechanism of chromosomal nondisjunction. Rosettes of cells derived from triploid individuals contained chromosomes segregated into three, tandemly linked haploid sets in which chromosome spatial order was preserved, but with chromosome positional order in one haploid set inverted with respect to the other two sets. The spatial separation of homologs in triploid cells was chromosome specific, providing evidence that chromosomes occupy preferred positions within the haploid sets. Since both triploidy and nondisjunction trisomy 21 are chromosome numerical anomalies that arise extremely early in development (e.g., during meiosis or during the first few mitoses), our results support the idea that normal and abnormal chromosome distribution patterns in mitotic human cells are established early in development, and are propagated faithfully by mitosis throughout development and into adult life. Furthermore, our observations suggest that segregation of chromosome homologs into two haploid sets in normal diploid cells is a remnant of fertilization and, in normal diploid cells, reflects segregation of maternal and paternal chromosomes. Received: 19 January 1998; in revised form: 28 May 1998 / Accepted: 30 June 1998     

Epigenetic discrimination by mouse metaphase II oocytes mediates asymmetric chromatin remodeling independently of meiotic exit

In mammalian fertilization, paternal chromatin is exhaustively remodeled, yet the maternal contribution to this process is unknown. To address this, we prevented the induction of meiotic exit by spermatozoa and examined sperm chromatin remodeling in metaphase II (mII) oocytes. Methylation of paternal H3-K4 and H3-K9 remained low, unlike maternal H3, although paternal H3-K4 methylation increased in zygotes. Thus, mII cytoplasm can sustain epigenetic asymmetry in a cell-cycle dependent manner. Paternal genomic DNA underwent oocyte-mediated cytosine demethylation and acquired maternally-derived K12-acetylated H4 (AcH4-K12) independently of microtubule assembly and maternal chromatin. AcH4-K12 persisted without typical maturation-associated deacetylation, irrespective of paternal pan-genomic cytosine methylation. Contrastingly, somatic cell nuclei underwent rapid H4 deacetylation; sperm and somatic chromatin exhibited asymmetric AcH4-K12 dynamics simultaneously within the same mII oocyte. Inhibition of somatic histone deacetylation revealed endogenous histone acetyl transferase activity. Oocytes thus specify the histone acetylation status of given nuclei by differentially targeting histone deacetylase and acetyl transferase activities. Asymmetric H4 acetylation during and immediately after fertilization was dispensable for development when both parental chromatin sets were hyperacetylated. These studies delineate non-zygotic chromatin remodeling and suggest a powerful model with which to study de novo genomic reprogramming.     

Timing of mitotic chromosome loss caused by the ncd mutation of Drosophila melanogaster.

We studied the timing of mitotic loss of maternally and paternally derived chromosomes among the progeny of Drosophila melanogaster females homozygous for an amorphic mutation in ncd, a gene encoding a kinesin-like protein. In order to determine the division at which chromosome loss occurs, we estimated the fraction of XO nuclei resulting from X chromosome loss by scoring the phenotype of 47 adult cuticular landmarks in 160 XX-XO mosaics (gynandromorphs) derived from maternal X chromosome loss, and 33 gynandromorphs derived from paternal X chromosome loss. The results show that while most of the mitotic loss of maternally derived chromosomes occurs at the first cleavage division, the mitotic loss of paternally derived chromosomes occurs only at the second and later divisions. This means that paternally derived chromosomes are immune from the effects of ncd prior to karyogamy, which occurs after the first cleavage division. We discuss the implications of these results for the function of the ncd gene product and for other kinesin-like proteins in Drosophila.     

Molecular studies of trisomy 18.

We have determined the parental origin of 50 cases of trisomy 18. In 48 cases the additional chromosome was maternal in origin, and in 2 cases it was paternal in origin. Seven cases, including both those with an additional paternal chromosome, appeared to be the result of postzygotic error. In contrast to the situation in nondisjunction involving chromosomes 21 and X, there was no evidence for nullochiasmate nondisjunction.     

A major imprinted gene involved in hydatidiform mole is not located in 2q31.2-qter or 5q34-qter

Introduction

Hydatidiform mole is an abnormal human pregnancy, characterised by absent or abnormal embryonic differentiation, vesicular chorionic villi and trophoblastic hyperplasia. Although the mole phenotype has hereto not been correlated to mutations in the molar genome, the aetiology for hydatidiform moles clearly is genetic: Most molar genomes analysed either have had a relative excess of paternal genome sets relative to maternal genome sets, or a global error in maternally imprinted genes, giving them a “paternal pattern”. However it remains yet to be specified which gene(s) in the molar genome actually causes the molar phenotype when present in a state of “paternal excess” or “maternal deficiency”.

Material and methods

A molar pregnancy in a woman with a balanced translocation (t(2;5) was subjected to histopathological evaluation and genetic analyses of ploidy and parental origin of the genome.

Results

Morphology: Partial hydatidiform mole. Karyotyping of metaphase chromosomes: 69,XXY,der(5)t(2;5)(q23;q33)mat. SNP array analysis mapped the breakpoints to 2q31.2 (genome position 179 Mb) and 5q34 (genome position 165 Mb). DNA microsatellite marker analysis showed that for the regions not involved in the translocation, the conceptus had two paternal and one maternal allele(s). Telomeric to the breakpoint on chromosome 2, the mole had two paternal and two maternal alleles and telomeric to the breakpoint on chromosome 5 the mole had paternal alleles, exclusively.

Conclusions

If the molar phenotype is caused by paternal excess of one gene, only, it is unlikely that this gene is located telomeric to genome position 179 Mb on chromosome 2. And similarly, if the phenotype complete mole is caused by the presence of exclusively paternally imprinted alleles of one gene, this gene is not located telomeric to genome position 165 Mb on chromosome 5.     

Ploidy barrier to endosperm development in maize

Maize kernels inheriting the indeterminate gametophyte mutant (ig) on the female side had endosperms that ranged in ploidy level from diploid (2x) to nonaploid (9x). In crosses with diploid males, only kernels of the triploid endosperm class developed normally. Kernels of the tetraploid endosperm class were half-sized but with well-developed embryos that regularly germinated. Kernels of endosperm composition other than triploid or tetraploid were abortive.-Endosperm ploidy level resulting from mating ig/ig x tetraploid Ig similarly was variable. Most endosperms started to degenerate soon after pollination and remained in an arrested state. Hexaploid endosperm was exceptional; it developed normally during the sequence of stages studied and accounted for plump kernels on mature ears. Since such kernels have diploid maternal tissues (pericarp) but triploid embryos, the present finding favors the view that endosperm failure or success in such circumstances is governed by conditions within the endosperm itself.-Whereas tetraploid endosperm consisting of three maternal genomes and one paternal genome is slightly reduced in size but supports viable seed development, that endosperm having two maternal and two paternal chromosome sets was highly defective and conditioned abortion. Thus, development of maize endosperm evidently is affected by the parental source of its sets of chromosomes.     

Genome Desertification in Eutherians: Can Gene Deserts Explain the Uneven Distribution of Genes in Placental Mammalian Genomes?

The evolution of genome size as well as structure and organization of genomes belongs among the key questions of genome biology. Here we show, based on a comparative analysis of 30 genomes, that there is generally a tight correlation between the number of genes per chromosome and the length of the respective chromosome in eukaryotic genomes. The surprising exceptions to this pattern are placental mammalian genomes. We identify the number and, more importantly, the uneven distribution of gene deserts among chromosomes, i.e., long (>500 kb) stretches of DNA that do not encode for genes, as the main contributing factor for the observed anomaly of eutherian genomes. Gene-rich placental mammalian chromosomes have smaller proportions of gene deserts and vice versa. We show that the uneven distribution of gene deserts is a derived character state of eutherians. The functional and evolutionary significance of this particular feature of eutherian genomes remains to be explained.