Chromatin diminution in the copepod Mesocyclops edax: diminution of tandemly repeated DNA families from somatic cells.

Chromatin diminution, i.e., the loss of selected chromosomal regions during the differentiation of early embryonic cells into somatic cells, has been described in taxa as varied as ciliates, copepods, insects, nematodes, and hagfish. The nature of the eliminated DNA has been extensively studied in ciliate, nematode, and hagfish species. However, the small size of copepods, which makes it difficult to obtain enough DNA from early embryonic cells for cloning and sequencing, has limited such studies. Here, to identify the sequences eliminated from the somatic cells of a copepod species that undergoes chromatin diminution, we randomly amplified DNA fragments from germ line and somatic line cells of Mesocyclops edax, a freshwater cyclopoid copepod. Of 47 randomly amplified germ line clones, 45 (96%) contained short, tandemly repeated sequences composed of either 2 bp CA-repeats, 8 bp CAAATAGA-repeats, or 9 bp CAAATTAAA-repeats. In contrast, of 83 randomly amplified somatic line clones, only 47 (57%) contained such short, tandemly repeated sequences. As previously observed in some nematode species, our results therefore show that there is partial elimination of chromosomal regions containing (CAAATAGA and CAAATTAAA) repeated sequences during the chromatin diminution observed in the somatic cells of M. edax. We speculate that chromatin diminution might have evolved repeatedly by recruitment of RNAi-related mechanisms to eliminate nonfunctional tandemly repeated DNA sequences from the somatic genome of some species.     

Chromatin diminution in nematodes

The process of chromatin diminution in Parascaris and Ascaris is a developmentally controlled genome rearrangement, which results in quantitative and qualitative differences in DNA content between germ line and somatic cells. Chromatin diminution involves chromosomal breakage, new telomere formation and DNA degradation. The programmed elimination of chromatin in presomatic cells might serve as an alternative way of gene regulation. We put forward a new hypothesis of how an ancient partial genome duplication and chromatin diminution may have served to maintain the genetic balance in somatic cells and simultaneously endowed the germ line cells with a selective advantage.     

A novel UV-damaged DNA binding protein emerges during the chromatin-eliminating cleavage period in Ascaris suum.

During the early cleavage period of Ascaris suum , chromatin diminution takes place in the somatic founder cells. In the process of chromatin diminution numerous heterochromatic blocks, consisting predominantly of highly repeated DNA, are discarded during mitotic anaphase and are later on digested in the cytoplasm. Very little is known about proteins that are involved in chromatin diminution. We have detected a nuclear protein and purified it to near homogeneity by its preferential binding to UV-damaged DNA. We termed this protein chromatin diminution associated factor 1 (CDAF1), because maximum binding activity per nucleus was observed to develop in 4-8-cell stages, when chromatin diminution occurs for the first time. CDAF1 recognizes cyclobutane pyrimidine dimers in UV-damaged double-stranded DNA. Its binding properties identify CDAF1 as a novel kind of damaged-DNA binding protein. CDAF1 activity is almost not detectable in 1-celled embryos. It increases dramatically during formation of somatic founder cells and persists up to the first larval stage. However, CDAF1 is absent in tissues of adults. These findings led us to suggest that CDAF1 plays a dual role: during the early segregative cleavage period it might be involved in chromatin diminution as a transfactor and act in nucleotide excision repair as an accessory factor throughout embryogenesis.     

Chromatin diminution in early embryogenesis of Ascaris lumbricoides L. var. suum

The occurrence of chromatin diminution in early Ascaris lumbricoides L. embryos has been studied in detail, and it is shown that it is possible to preselect three characteristic types of mitoses: pre-diminution, diminution, and post-diminution mitosis. The first three embryonic mitotic divisions are of the pre-diminution type. Chromatin diminution occurs after the third mitosis, but there is a variation from embryo to embryo as to whether or not chromosomal diminution occurs during the fourth, fifth, and six divisions. However, the seventh embryonic division, which gives rise to an eight-cell embryo, always exhibits chromatin diminution. Subsequent mitoses of somatic cells already in the diminished state are of the post-diminution type of mitosis.     

Heterochromatin endoreduplication prior to gametogenesis and chromatin diminution during early embryogenesis in Mesocyclops edax (Copepoda: Crustacea)

The segregation of progenitor somatic cells from those of the primordial germ cells that sequester and retain elevated levels of DNA during subsequent developmental events, poses an interesting, alternative pathway of chromosome behavior during the reproductive cycle of certain species of cyclopoid copepods and several other organisms. Separation of maternal and paternal chromosome sets during very early cleavages (gonomery) is often a feature following marked elevations of DNA levels in germ cells for some of these species. Here, we report on the accumulation of large amounts of DNA in germ line nuclei of both female and male juveniles and adults of a freshwater copepod, Mesocyclops edax (Forbes, 1890). We also report the robust uptake of 3H-thymidine by germ cells prior to gametogenesis in this species. By using cytophotometric analysis of the DNA levels in both germ line cells and somatic cells from the same specimens we demonstrate that germ cell nuclei accumulate high levels of DNA prior to the onset of gametogenesis. These elevated amounts coincide with the levels of heterochromatic DNA discarded during chromatin diminution. A new model is proposed of major cytological events accompanying the process of chromatin diminution in M. edax.     

Evidence for endoreduplication: germ cell DNA levels prior to chromatin diminution in Mesocyclops edax.

We studied the functional significance of marked differences in the DNA content of somatic cells and germ line nuclei by static Feulgen-DNA cytophotometry for several species of microcrustaceans that exhibit chromatin diminution during very early stages of embryogenesis. Mature females and males showed many gonadal nuclei with elevated amounts of DNA that persist until dispersal of this "extra" DNA throughout the cytoplasm as fragments and coalescing droplets of chromatin during anaphase of the diminution division.     

Chromatin diminution is a key process explaining the eukaryotic genome size paradox and some mechanisms of genetic isolation

The functions of redundant (junk, selfish, parasitic, etc.) DNA in eukaryotes can be reliably inferred from chromatin diminution (programmed elimination of up to 94% of the genome from somatic germ cells in Ascaris and Cyclops). These functions should be sought in germ cells, where this DNA is preserved during the entire life time of the species. A possible biological role of redundant DNA as a factor disrupting meiotic chromosome synapsis is suggested. At the same time, chromatin diminution itself can act as a mechanism of postzygotic isolation. All stage of the complex diminution mechanism could not be fixed in the genetic program of the species via gradual accumulation of mutations. The "program" of diminution must have appeared at once and in the completed form.     

Chromatin diminution in the parasitic nematodes ascaris suum and parascaris univalens

Chromatin diminution in Parascaris univalens and Ascaris suum undoubtedly represents an interesting case of developmentally programmed DNA rearrangement in higher eukaryotes. It is a complex mechanism involving chromosomal breakage, new telomere addition and DNA degradation, and occurs in all presomatic cells. The process is rather specific with respect to its developmental timing and the chromosomal regions that are eliminated. The functional significance of chromatin diminution still remains an enigma. The fact, however, that single-copy, protein-coding genes are contained in the eliminated DNA demonstrates that in P. univalens and A. suum, there is a qualitative difference between germ-line and somatic genomes, and suggests that chromatin diminution may be used as a "throw-away" approach to gene regulation. We present a hypothesis as to how, during evolution, a partial genome duplication might have been linked to the process of chromatin diminution, in order to provide a selective advantage to parasitic DNA-eliminating nematodes.     

Spontaneous mutagenesis is enhanced in Apex heterozygous mice

Germ line DNA directs the development of the next generation and, as such, is profoundly different from somatic cell DNA. Spermatogenic cells obtained from young adult lacI transgenic mice display a lower spontaneous mutant frequency and greater in vitro base excision repair activity than somatic cells and tissues obtained from the same mice. However, spermatogenic cells from old lacI mice display a 10-fold higher mutant frequency. This increased spontaneous mutant frequency occurs coincidentally with decreased in vitro base excision repair activity for germ cell and testicular extracts that in turn corresponds to a decreased abundance of AP endonuclease. To directly test whether a genetic diminution of AP endonuclease results in increased spontaneous mutant frequencies in spermatogenic cell types, AP endonuclease heterozygous (Apex(+/-)) knockout mice were crossed with lacI transgenic mice. Spontaneous mutant frequencies were significantly elevated (approximately twofold) for liver and spleen obtained from 3-month-old Apex(+/-) lacI(+) mice compared to frequencies from Apex(+/+) lacI(+) littermates and were additionally elevated for somatic tissues from 9-month-old mice. Spermatogenic cells from 9-month-old Apex(+/-) lacI(+) mice were significantly elevated twofold compared to levels for 9-month-old Apex(+/+) lacI(+) control mice. These data indicate that diminution of AP endonuclease has a significant effect on spontaneous mutagenesis in somatic and germ line cells.     

Changes in nuclear morphology associated with elevated DNA levels during gametogenesis in cyclopoid copepods with chromatin diminution

Abstract. Most species of freshwater cyclopoid copepods follow a conventional course of DNA replication during gametogenesis, but certain species regularly undergo chromatin diminution during early embryogenesis, a process that is accompanied by the exclusion of large amounts of heterochromatic DNA from progenitor somatic cells and selective retention of this DNA by primordial germ cells after their segregation from the soma. We have used scanning microdensitometry and image analysis cytometry of individual Feulgen-stained nuclei to determine the DNA levels of individual somatic cell nuclei, oocytes, spermatocytes, and sperm for seven species, including Acanthocyclops brevispinosus, Acanthocyclops vernalis, Ectocyclops phaleratus, Eucyclops agilis, Eucyclops ensifer, Macrocyclops albidus , and Thermocyclops decipiens . The oocyte nuclei of these species have twice the DNA content of their diploid somatic cell nuclei. In specimens of Cyclops strenuus, Mesocyclops edax, Mesocyclops longisetus, Mesocyclops longisetus curvatus , and Metacyclops mendocinus , marked increases in DNA levels were noted in both female and male germ cells before meiosis. The appearance of enlarged nuclei with densely stained chromocenters is a distinguishing feature of oocytes and spermatocytes of cyclopoid species that exhibit excessive accumulations of DNA during gametogenesis and subsequently undergo chromatin diminution. The net increase in DNA content of the prediminution nuclei is 6–10 times the DNA level of their somatic cell nuclei and is largely attributable to increases in the amount of DNA associated with their heterochromatic chromocenters. The identification of a morphologically distinctive type of germ cell and its dramatic accumulation of large amounts of DNA before meiosis are discussed in terms of the selective elimination of heterochromatin during early cleavage stages in these cyclopoid species.     

Chromosome diminution in Ascaris suum. Two-fold increase of nucleosomal histone to DNA ratios during development

The swine intestinal nematode, Ascaris suum, eliminates chromatin material from its primordial somatic cells during early embryogenesis. A technique for isolation of nuclei from pre- and post-diminution stage embryos has been developed and these isolated nuclei were used in investigations of nuclear events during diminution. The amount of DNA per nucleus determined by diphenylamine assays and isotope dilutions was 0.66 pg and 0.29 pg in pre- and post-diminution nuclei, respectively. Thus, A. suum loses 56% of its nuclear DNA during diminution. The loss of nuclear DNA enabled in vivo examination of histone to DNA ratios as a function of changes in DNA quantities. Ascaris histones were identified by acid extractability and tryptic fingerprint comparison with rat liver histones. Measurement of histone quantities was accomplished using linearity of Coomassie blue binding to histones separated in dodecyl sulfate gels. Ascaris nucleosomal histones levels were relatively constant in pre- and post-diminution nuclei. However, nucleosomal histone to DNA ratios approximately doubled during diminution.     

Nucleolar cycle and localization of NORs in early embryos of Parascaris univalens

During early embryogenesis of the nematode Parascaris univalens (2n=2) the processes of chromatin diminution and segregation of the germ and somatic cell lineages take place simultaneously. In this study we analyzed the nucleolar cycle in early embryos, both in germinal and somatic blastomeres, by means of silver staining and antibodies against the nucleolar protein fibrillarin. We observed an identical nucleolar cycle in both types of blastomeres, hence, the chromatin diminution process has no effect on the nucleolar cycle of somatic blastomeres. We report the existence of outstanding differences between this cycle and those previously reported during early embryogenesis of other species. There is a true nucleolar cycle in early embryos that shows a peculiar nucleolar disorganization at prophase, and a preferential localization of prenucleolar bodies only on the euchromatic regions during nucleologenesis. Moreover, fibrillarin does not form a perichromosomal sheath in metaphase or anaphase holocentric chromosomes, probably owing to their special centromeric organization. The number and location of nucleolus organizer regions (NORs) in the chromosomal complement have been determined using silver impregnation, chromomycin A₃/distamycin A staining, and fluorescent in situ hybridization using an rDNA probe. There are only two NORs, one per chromosome, and these are lost in blastomeres after chromatin diminution. Moreover, the constant presence of two nucleoli in somatic blastomeres suggests that NORs are not affected during the fragmentation of euchromatic regions when this process occurs.     

DNA-Feulgen cytophotometric determination of genome size for the freshwater-invading copepod Eurytemora affinis.

Variation in nuclear DNA content within some eukaryotic species is well documented, but causes and consequences of such variation remain unclear. Here we report genome size of an estuarine and salt-marsh calanoid copepod, Eurytemora affinis, which has recently invaded inland freshwater habitats independently and repeatedly in North America, Europe, and Asia. Adults and embryos of E. affinis from the St. Lawrence River drainage were examined for somatic cell DNA content and the presence or absence of embryonic chromatin diminution, using Feulgen-DNA cytophotometry to determine a diploid or 2C genome size of 0.6-0.7 pg DNA/cell. The majority of somatic cell nuclei, however, have twice this DNA content (1.3 pg/nucleus) in all of the adults examined and possibly represent a population of cells arrested at the G2 stage of the cell cycle or associated with some degree of endopolyploidy. Both suggestions contradict assumptions that DNA replication does not occur in adult tissues during the determinate growth characteristic of copepods. Absence of germ cell nuclei with markedly elevated DNA values, commonly found for species of cyclopoid copepods that show chromatin diminution, indicates that E. affinis lacks this trait. The small genome size and presumed absence of chromatin diminution increase the potential utility of E. affinis as a model for genomic studies on mechanisms of adaptation during freshwater invasions.     

Cytophotometric determination of genome size in two species of Cyclops of Lake Baikal (Crustacea: Copepoda, Cyclopoida) in ontogenetic development

The genome size of Cyclops in cells at early stages of cleavage (up to the fifth division) and in somatic cells was estimated by static digital Feulgen cytophotometry in order to study quantitative changes in DNA content during chromatin diminution. Described here cytophotometric method was approbated on five different digital-imaging systems in blood cells of four vertebrate species. In all cases, we observed a direct correlation between the data obtained with known from the literature on genome size and high reproducibility, which will allow these systems to be used in future work. We also optimized the conditions for DNA hydrolysis of both blood smears and for two species of Cyclops from the Moscow population as 30 min in 5 N HCl at 24°C. Here, we first revealed chromatin diminution in two endemic Baikal species of Cyclopoida: Acanthocyclops incolotaenia and Diacyclops galbinus. We estimated the extent of chromatin diminution in Diacyclops galbinus as 95.5–96.2%. Cytometric analysis of the third species, Mesocyclops leuckarti, did not reveal obvious chromatin diminution.     

Chromatin diminution and early cleavage in Parascaris univalens (Nematoda)

Summary In Parascaris developmental commitment to the germ line and somatic lineages is indicated by the orientation of the mitotic spindle in blastomeres, the topology of cells in the embryo, and chromatin diminution in presomatic blastomeres. Using three different experimental techniques: transient pressure treatment, application of cytochalasin B, and isolation of blastomeres, we have succeeded in uncoupling several developmental processes during cleavage of P. univalens. The following results were obtained: (1) Following mitotic nondisjunction we observed identical behavior of all chromatids in each blastomere. Thus chromosome differentiation by differential replication does not occur. (2) Chromosome fragments obtained by pressure treatment of egg cells underwent chromatin diminution. Thus this process does not require an intact germ-line chromosome. However, chromosomes immobilized on a monopolar spindle did not undergo chromatin diminution. Thus diminution appears to require segregation of chromatids. (3) Blastomeres that completely lacked chromosomes as a result of mitotic nondisjunction underwent normal early cleavage divisions. (4) Pressure treatment or prolonged treatment with cytochalasin B caused egg cells or germ line blastomeres to lose their germ line quality, as deduced from the coincident occurrence of symmetrical (presomatic-like) cleavage and chromatin diminution. (5) Isolated blastomeres from 2-cell embryos, i.e. 1/2 blastomeres, usually cleaved according to their prospective fates in the whole embryo. However, in some partial embryos derived from such blastomeres, chromatin diminution was delayed for either one or two cleavage mitoses. An activation model as an alternative to a prelocalization model is presented, which can account for early blastomere topogenesis and chromatin diminution.     

Chromatin diminution in Ascaris suum: nucleotide sequence of the eliminated satellite DNA.

We have determined the prototype sequence of the DNA which is eliminated in the course of chromatin diminution in Ascaris suum. This DNA which is virtually absent from somatic cells but retained in the germ line consists predominantly of highly repetitive sequences which are variants of an AT rich 123 base pair repeat unit. Both major and minor variants have been sequenced. The overall structure of this germ line limited DNA corresponds to the segmental organization characteristic of satellite DNAs. Possible correlations between the mechanism of chromatin diminution and some properties of the satellite sequence are discussed.     

Neither a germ line-specific nor several somatically expressed genes are lost or rearranged during embryonic chromatin diminution in the nematode Ascaris lumbricoides var. suum

Ascaris lumbricoides var. suum is a parasitic nematode of pigs. Its embryos undergo chromatin diminution between the third and fifth cleavages, resulting in the loss of about 30% of the DNA from all somatic precursor cells while the germ line DNA stays intact. Most of the eliminated DNA has been shown to be satellite sequences. Theodor Boveri [(1910) In "Festschrift fur R. Hertwig, III," Vol. 3, pp. 131-214, Fischer] proposed that functions essential only to the germ line might be lost from the soma. We have examined this proposal by cloning a gene encoding the major sperm protein (MSP) using a cloned MSP gene from Caenorhabditis elegans as a probe. The MSP appears to be expressed only in the testis of Ascaris, as it is in Caenorhabditis. Actin and alpha tubulin were also cloned to serve as somatically expressed gene controls. By probing Southern blots of somatic and germ line DNA with these cloned genes, it was found that none of them was lost or rearranged during chromatin diminution. Thus at least one germ line-specific gene is neither lost nor rearranged during chromatin diminution. We also found that the two nematode species differ widely in their numbers of both MSP and actin genes. Caenorhabditis has greater than 30 MSP genes, but Ascaris has no more than three; whereas Ascaris has many more actin genes than Caenorhabditis.     

Temporal control of DNA replication and the adaptive value of chromatin diminution in copepods.

Chromatin diminution is a precisely controlled, highly repeatable, genome-wide deletion of noncoding heterochromatic segments from the presomatic line. The somatic line is reduced in size and reorganized; the germ line remains unaltered. Little is understood about its mechanistic underpinnings and adaptive significance in the nematodes, copepods, and hagfish in which it occurs. Here, we propose that microcrustacean copepods, whose cytology, development, and evolutionary ecology are well understood from an adaptationist point of view, provide the vehicle to test how chromatin diminution might orchestrate certain cell cycle dynamics, with the consequence of influencing the evolution of nuclear DNA contents, organismal development rates, and body size.     

Somatic PI3K activity regulates transition to the spermatocyte stages in <Emphasis Type="Italic">Drosophila</Emphasis> testis

Spermatogenesis, involving multiple transit amplification divisions and meiosis, occurs within an enclosure formed by two somatic cells. As the cohort of germline cells divide and grow, the surface areas of the somatic cells expand maintaining a tight encapsulation throughout the developmental period. Correlation between the somatic cell growth and germline development is unclear. Here, we report standardization of a quantitative assay developed for estimating the somatic roles of target molecules on germline division and differentiation in Drosophila testis. Using the assay, we studied the somatic roles of phosphatidylinositol-3-kinase (PI3K). It revealed that the expression of PI3K^DN is likely to facilitate the early germline development at all stages, and an increase in the somatic PI3K activity during the early stages delays the transition to spermatocyte stage. Together, these results suggest that somatic cell growth plays an important role in regulating the rate of germline development.     

Ultrastructure of developing Ascaris larvae undergoing lipid to carbohydrate interconversion.

Triglyceride utilization is correlated with glycogen resynthesis in developing Ascaris suum larvae. Glycogen content, determined by amyloglucosidase hydrolysis at 2-day intervals, decreases sharply during embryonation from 15% (per mg dry weight) at day 4 to 2% at day 12. Thereafter glycogen increases 3-fold through day 20, marking the resynthesis period. Triglyceride lipid droplets are confirmed primarily to the posterior half of the larvae and mark the site of the interconversion. Although they serve as precursors to glycogen, only a small diminution was observed ultrastructurally. Glycogen accumulation, on the other hand, correlates well with increases determined biochemically. alpha-glycogen builds up among lipid droplets, while beta-glycogen concentrates in the cytoplasm of somatic muscle cells paralleling myofibril development. Dense granules, restricted to the lipid body region, are considered as the possible subcellular site for the enzymatic conversion.