Isolation and chromosomal localization of a germ line-specific highly repetitive DNA family in Acricotopus lucidus (Diptera, Chironomidae)

. In the chironomid Acricotopus lucidus, parts of the genome, the germ line-limited chromosomes, are eliminated from the future soma cells during early cleavage divisions. A highly repetitive, germ line-specific DNA sequence family was isolated, cloned and sequenced. The monomers of the tandemly repeated sequences range in size from 175 to 184 bp. Analysis of sequence variation allowed the further classification of the germ line-restricted repetitive DNA into two related subfamilies, A and B. Fluorescence in situ hybridization to gonial metaphases demonstrated that the sequence family is highly specific for the paracentromeric heterochromatin of the germ line-limited chromosomes. Restriction analysis of genomic soma DNA of A. lucidus revealed another tandem repetitive DNA sequence family with monomers of about 175 bp in length. These DNA elements are found only in the centromeric regions of all soma chromosomes and one exceptional germ line-limited chromosome by in situ hybridization to polytene soma chromosomes and gonial metaphase chromosomes. The sequences described here may be involved in recognition, distinction and behavior of soma and germ line-limited chromosomes during the complex chromosome cycle in A. lucidus and may be useful for the genetic and cytological analysis of the processes of elimination of the germ line-limited chromosomes in the soma and germ line. Received: 12 April 1997; in revised form 26 June 1997 / Accepted: 29 June 1997     

Developmentally regulated chromosome fragmentation linked to imprecise elimination of repeated sequences in paramecia

The chromosomes of ciliates are fragmented at reproducible sites during the development of the polyploid somatic macronucleus, but the mechanisms involved appear to be quite diverse in different species. In Paramecium aurelia, the process is imprecise and results in de novo telomere addition at locally heterogeneous positions. To search for possible determinants of chromosome fragmentation, we have studied an ~21-kb fragmentation region from the germ line genome of P. primaurelia. The mapping and sequencing of alternative macronuclear versions of the region show that two distinct multicopy elements, a minisatellite and a degenerate transposon copy, are eliminated by an imprecise mechanism leading either to chromosome fragmentation and the formation of new telomeres or to the rejoining of flanking sequences. Heterogeneous internal deletions occur between short direct repeats containing TA dinucleotides. The complex rearrangement patterns produced vary slightly among genetically identical cell lines, show non-Mendelian inheritance during sexual reproduction, and can be experimentally modified by transformation of the maternal macronucleus with homologous sequences. These results suggest that chromosome fragmentation in Paramecium is the consequence of imprecise DNA elimination events that are distinct from the precise excision of single-copy internal eliminated sequences and that target multicopy germ line sequences by homology-dependent epigenetic mechanisms.     

Genetic Evidence That the Ovo Locus Is Involved in Drosophila Germ Line Sex Determination

Zygotically contributed ovo gene product is required for the survival of female germ cells in Drosophila melanogaster. Trans-allelic combinations of weak and dominant ovo mutations (ovoD) result in viable germ cells that appear to be partially transformed from female to male sexual identity. The ovoD2 mutation is partially suppressed by many Sex-lethal alleles that affect the soma, while those that affect only the germ line fail to interact with ovoD2. One of two loss-of-function ovo alleles is suppressed by a loss-of-function Sex-lethal allele. Because ovo mutations are germ line dependent, it is likely that ovo is suppressed by way of communication between the somatic and germ lines. A loss-of-function allele of ovo is epistatic to germ line dependent mutations in Sex-lethal. The germ line dependent sex determination mutation, sans fille, and ovoD mutations show a dominant synergistic interaction resulting in partial transformation of germ line sexual identity. The ovo locus appears to be involved in germ line sex determination and is linked in some manner to sex determination in the soma.     

Sex determination in the germ line of Drosophila depends on genetic signals and inductive somatic factors

We have analyzed the mechanism of sex determination in the germ line of Drosophila by manipulating three parameters: (1) the ratio of X-chromosomes to sets of autosomes (X:A); (2) the state of activity of the gene Sex-lethal (Sxl), and (3) the sex of the gonadal soma. To this end, animals with a ratio of 2X:2A and 2X:3A were sexually transformed into pseudomales by mutations at the sex-determining genes Sxl (Sex-lethal), tra (transformer), tra-2 (transformer-2), or dsx (double-sex). Animals with the karyotype 2X;3A were also transformed into pseudofemales by the constitutive mutation SxlM1. The sexual phenotype of the gonads and of the germ cells was assessed by phase-contrast microscopy. Confirming the conclusions of Steinmann-Zwicky et al. (Cell 57, 157, 1989), we found that all three parameters affect sex determination in germ cells. In contrast to the soma in which sex determination is completely cell-autonomous, sex determination in the germ line has a non-autonomous component inasmuch as the sex of the soma can influence the sexual pathway of the germ cells. Somatic induction has a clear effect on 2X;2A germ cells that carry a Sxl+ allele. These cells, which form eggs in an ovary, can enter spermatogenesis in testes. Mutations that cause partial loss of function or gain of function of Sxl thwart somatic induction and, independently of the sex of the soma, dictate spermatogenesis or oogenesis, respectively. Somatic induction has a much weaker effect on 2X;3A germ cells. This ratio is essentially a male signal for germ cells which consistently enter spermatogenesis in testes, even when they carry SxlM1. In a female soma, however, SxlM1 enables the 2X;3A germ cells to form almost normal eggs. Our results show that sex determination in the germ line is more complex than in the soma. They provide further evidence that the state of Sxl, the key gene for sex determination and dosage compensation in the soma, also determines the sex of the germ cells, and that, in the germ line, the state of activity of Sxl is regulated not only by the X:A ratio, but also by somatic inductive stimuli.     

Satellite DNA properties of the germ line limited DNA and the organization of the somatic genomes in the nematodesAscaris suum andParascaris equorum

During the early cleavage divisions in some Ascarids, parts of the chromosomes are eliminated from the somatic blastomeres (chromatin diminution, Boveri, 1887) while the chromosomes in the germ line cells maintain their integrity. To characterize the germ line and soma genome, DNA was isolated from gametes and embryonic somatic cells of two Ascarid species,Parascaris equorum var. univalens andAscaris suum. It was shown that the germ line limited DNAs of these species have the same density and almost identical reassociation kinetics: in CsCl the predominant component of the germ line limited DNA ofP. equorum andA. suum has the buoyant density of 1.697g/cm³, while soma DNA of both species bands at 1.700 g/cm³. InP. equorum there is a small additional germ line limited satellite DNA component with the density of 1.690 g/cm³, identical to that of mitochondrial DNA of both organisms. Comparison of the reassociation kinetics of germ line and soma DNA demonstrates for both species that the eliminated DNA sequences are highly repetitive. In contrast to these similarities between the germ line limited DNAs ofP. equorum andA. suum the analysis of their base composition revealed differences (40% guanine plus cytosine inP. equorum and 36% inA. suum). The only very fast reassociating DNA sequences which we could isolate from soma DNA was demonstrated to be foldback DNA. The reassociation kinetics of totalA. suum soma DNA was investigated by hydroxylapatite chromatography. Least squares analysis of the data revealed about 10% of intermediate repetitive DNA sequences. Their interspersion between single copy DNA sequences was analyzed by comparing the reassociation kinetics of DNA fragments 0.35 and 7.2 kilobases long. Thus the DNA sequence arrangement ofAscaris does not follow the short period interspersion pattern observed in most organism.     

Processing of double-strand breaks is involved in the precise excision of paramecium internal eliminated sequences

In ciliates, the development of the somatic macronucleus involves the programmed excision of thousands of internal eliminated sequences (IES) scattered throughout the germ line genome. Previous work with Tetrahymena thermophila has suggested that excision is initiated by a staggered double-strand break (DSB) at one IES end. Nucleophilic attack of the other end by the 3'OH group carried by the firstly broken chromosome end leads to macronuclear junction closure. In this study, we mapped the 3'OH and 5'PO(4) groups that are developmentally released at Paramecium IES boundaries, which are marked by two conserved TA dinucleotides, one of which remains in the macronuclear genome after excision. We show that initiating DSBs at both ends generate 4-base 5' overhangs centered on the TA. Based on the observed processing of the 5'-terminal residue of each overhang, we present a new model for the precise closure of macronuclear chromosomes in Paramecium tetraurelia, different from that previously proposed for tetrahymena. In our model, macronucleus-destined broken ends are aligned through the partial pairing of their 5'-nTAn-3' extensions and joined after trimming of the 5' flaps.     

Germ line and oogenesis during paedogenetic reproduction inHeteropeza pygmaea Winnertz (Diptera: Cecidomyiidae)

The germ line during paedogenetic reproduction in the gall midgeHeteropeza pygmaea was followed cell generation by cell generation. There are altogether 8 or 9 successive divisions during one paedogenetic cycle, and all descendants of the primordial germ cell develop into occytes, while the trophocytes are of somatic origin. In the cytological race studied (from southern Finland) the germ-line cells possess 58 chromosomes, the somatic number being 10.     

Probing spermatogenesis in Drosophila with P-element enhancer detectors.

Formation of motile sperm in Drosophila melanogaster requires the coordination of processes such as stem cell division, mitotic and meiotic control and structural reorganization of a cell. Proper execution of spermatogenesis entails the differentiation of cells derived from two distinct embryonic lineages, the germ line and the somatic mesoderm. Through an analysis of homozygous viable and fertile enhancer detector lines, we have identified molecular markers for the different cell types present in testes. Some lines label germ cells or somatic cyst cells in a stage-specific manner during their differentiation program. These expression patterns reveal transient identities for the cyst cells that had not been previously recognized by morphological criteria. A marker line labels early stages of male but not female germ cell differentiation and proves useful in the analysis of germ line sex-determination. Other lines label the hub of somatic cells around which germ line stem cells are anchored. By analyzing the fate of the somatic hub in an agametic background, we show that the germ line plays some role in directing its size and its position in the testis. We also describe how marker lines enable us to identify presumptive cells in the embryonic gonadal mesoderm before they give rise to morphologically distinct cell types. Finally, this collection of marker lines will allow the characterization of genes expressed either in the germ line or in the soma during spermatogenesis.     

Macronuclear genome sequence of the ciliate Tetrahymena thermophila, a model eukaryote

The ciliate Tetrahymena thermophila is a model organism for molecular and cellular biology. Like other ciliates, this species has separate germline and soma functions that are embodied by distinct nuclei within a single cell. The germline-like micronucleus (MIC) has its genome held in reserve for sexual reproduction. The soma-like macronucleus (MAC), which possesses a genome processed from that of the MIC, is the center of gene expression and does not directly contribute DNA to sexual progeny. We report here the shotgun sequencing, assembly, and analysis of the MAC genome of T. thermophila, which is approximately 104 Mb in length and composed of approximately 225 chromosomes. Overall, the gene set is robust, with more than 27,000 predicted protein-coding genes, 15,000 of which have strong matches to genes in other organisms. The functional diversity encoded by these genes is substantial and reflects the complexity of processes required for a free-living, predatory, single-celled organism. This is highlighted by the abundance of lineage-specific duplications of genes with predicted roles in sensing and responding to environmental conditions (e.g., kinases), using diverse resources (e.g., proteases and transporters), and generating structural complexity (e.g., kinesins and dyneins). In contrast to the other lineages of alveolates (apicomplexans and dinoflagellates), no compelling evidence could be found for plastid-derived genes in the genome. UGA, the only T. thermophila stop codon, is used in some genes to encode selenocysteine, thus making this organism the first known with the potential to translate all 64 codons in nuclear genes into amino acids. We present genomic evidence supporting the hypothesis that the excision of DNA from the MIC to generate the MAC specifically targets foreign DNA as a form of genome self-defense. The combination of the genome sequence, the functional diversity encoded therein, and the presence of some pathways missing from other model organisms makes T. thermophila an ideal model for functional genomic studies to address biological, biomedical, and biotechnological questions of fundamental importance.     

Macronuclear Genome Sequence of the Ciliate Tetrahymena thermophila, a Model Eukaryote

The ciliate Tetrahymena thermophila is a model organism for molecular and cellular biology. Like other ciliates, this species has separate germline and soma functions that are embodied by distinct nuclei within a single cell. The germline-like micronucleus (MIC) has its genome held in reserve for sexual reproduction. The soma-like macronucleus (MAC), which possesses a genome processed from that of the MIC, is the center of gene expression and does not directly contribute DNA to sexual progeny. We report here the shotgun sequencing, assembly, and analysis of the MAC genome of T. thermophila, which is approximately 104 Mb in length and composed of approximately 225 chromosomes. Overall, the gene set is robust, with more than 27,000 predicted protein-coding genes, 15,000 of which have strong matches to genes in other organisms. The functional diversity encoded by these genes is substantial and reflects the complexity of processes required for a free-living, predatory, single-celled organism. This is highlighted by the abundance of lineage-specific duplications of genes with predicted roles in sensing and responding to environmental conditions (e.g., kinases), using diverse resources (e.g., proteases and transporters), and generating structural complexity (e.g., kinesins and dyneins). In contrast to the other lineages of alveolates (apicomplexans and dinoflagellates), no compelling evidence could be found for plastid-derived genes in the genome. UGA, the only T. thermophila stop codon, is used in some genes to encode selenocysteine, thus making this organism the first known with the potential to translate all 64 codons in nuclear genes into amino acids. We present genomic evidence supporting the hypothesis that the excision of DNA from the MIC to generate the MAC specifically targets foreign DNA as a form of genome self-defense. The combination of the genome sequence, the functional diversity encoded therein, and the presence of some pathways missing from other model organisms makes T. thermophila an ideal model for functional genomic studies to address biological, biomedical, and biotechnological questions of fundamental importance.     

Cheats as first propagules: A new hypothesis for the evolution of individuality during the transition from single cells to multicellularity

The emergence of individuality during the evolutionary transition from single cells to multicellularity poses a range of problems. A key issue is how variation in lower‐level individuals generates a corporate (collective) entity with Darwinian characteristics. Of central importance to this process is the evolution of a means of collective reproduction, however, the evolution of a means of collective reproduction is not a trivial issue, requiring careful consideration of mechanistic details. Calling upon observations from experiments, we draw attention to proto‐life cycles that emerge via unconventional routes and that transition, in single steps, individuality to higher levels. One such life cycle arises from conflicts among levels of selection and invokes cheats as a primitive germ line: it lays the foundation for collective reproduction, the basis of a self‐policing system, the selective environment for the emergence of development, and hints at a plausible origin for a soma/germ line distinction.     

Mammalian X Chromosome Dosage Compensation: Perspectives From the Germ Line

Sex chromosomes are advantageous to mammals, allowing them to adopt a genetic rather than environmental sex determination system. However, sex chromosome evolution also carries a burden, because it results in an imbalance in gene dosage between females (XX) and males (XY). This imbalance is resolved by X dosage compensation, which comprises both X chromosome inactivation and X chromosome upregulation. X dosage compensation has been well characterized in the soma, but not in the germ line. Germ cells face a special challenge, because genome wide reprogramming erases epigenetic marks responsible for maintaining the X dosage compensated state. Here we explain how evolution has influenced the gene content and germ line specialization of the mammalian sex chromosomes. We discuss new research uncovering unusual X dosage compensation states in germ cells, which we postulate influence sexual dimorphisms in germ line development and cause infertility in individuals with sex chromosome aneuploidy.     

Chromatin elimination--an oddity or a common mechanism in differentiation and development?

Over many decades, a great number of exceptions from the rule of equal segregation of the chromosomes during cell division have been found in different animal species. The most diversified is the process of chromosome re-arrangement that takes place during the specification of soma versus germ-line cell fate in the embryos from the whole spectrum of animal phyla. In nematodes, copepodes, insects, hagfish, and marsupials, the chromatin/chromosome elimination is a common path of normal cell differentiation and development. This also raises the question of the mechanisms and factors that promote elimination in pre-somatic cell lines and/or inhibit the elimination in the prospective germ cells. We will discuss the possible role of the germ plasm in this process.     

Evolution of programmed DNA rearrangements in a scrambled gene

Gene unscrambling in spirotrichous ciliates involves massive genome-wide DNA deletion and rearrangement events during development. During each sexual cycle, the somatic nucleus (macronucleus) regenerates from the germ line nucleus (micronucleus). Development of the polyploid somatic genome requires programmed DNA deletion of micronuclear-limited intragenic noncoding sequences and permutation and amplification of the protein-coding regions. Recent studies suggest that, despite novel insertions of endogenous transposon or foreign DNA into the germ line genome, ciliates possess a whole-genome surveillance system that guides the recapitulation of a functional somatic genome. This renders the germ line genome an extremely dynamic structure over evolutionary time. Here we describe the germ line and somatic architectures of the gene encoding alpha-telomere-binding protein in three early-diverging species (Holosticha sp., Uroleptus sp., and Paraurostyla weissei) and trace the natural history of DNA rearrangements in this gene in six species, including three previously studied oxytrichids. Comparisons of homologous coding regions between earlier and later diverging species provide evidence for fusion of scrambled germ line fragments as small as 24 bp during evolution, as well as simultaneous fragmentation and scrambling of the germ line locus and shifting of the boundaries between coding and noncoding DNA, leading to distinct gene architectures in each species. We infer an evolutionary recombination pathway that passes through identified intermediate species and gives rise to the observed patterns in all known species, capitalizing on their unique DNA rearrangement machinery and germ line flexibility.     

The Hippo Pathway Regulates Homeostatic Growth of Stem Cell Niche Precursors in the Drosophila Ovary

The Hippo pathway regulates organ size, stem cell proliferation and tumorigenesis in adult organs. Whether the Hippo pathway influences establishment of stem cell niche size to accommodate changes in organ size, however, has received little attention. Here, we ask whether Hippo signaling influences the number of stem cell niches that are established during development of the Drosophila larval ovary, and whether it interacts with the same or different effector signaling pathways in different cell types. We demonstrate that canonical Hippo signaling regulates autonomous proliferation of the soma, while a novel hippo-independent activity of Yorkie regulates autonomous proliferation of the germ line. Moreover, we demonstrate that Hippo signaling mediates non-autonomous proliferation signals between germ cells and somatic cells, and contributes to maintaining the correct proportion of these niche precursors. Finally, we show that the Hippo pathway interacts with different growth pathways in distinct somatic cell types, and interacts with EGFR and JAK/STAT pathways to regulate non-autonomous proliferation of germ cells. We thus provide evidence for novel roles of the Hippo pathway in establishing the precise balance of soma and germ line, the appropriate number of stem cell niches, and ultimately regulating adult female reproductive capacity.     

Immunocytological and FISH analysis of pole cell formation and soma elimination of germ line-limited chromosomes in the chironomid Acricotopus lucidus

In the chironomid Acricotopus lucidus, germ line-soma differentiation becomes evident with the formation of the pole cells and the elimination of the germ line-limited chromosomes (Ks) from the future somatic nuclei of the embryo. Unlike in Drosophila, the early nuclear divisions do not proceed synchronously in A. lucidus. Usually, only one nucleus, the future pole nucleus, penetrates into the pole plasm, always at a telophase stage in the course of a regular mitosis. This happens by chance, depending on the orientation of the mitotic spindles of the early syncytial nuclei. Consequently, the time and the cell cycle at which a nucleus reaches the pole plasm, and pole cells arise, vary between embryos of the same oviposition. When entering the first germ line mitosis, while polar plasm and syncytial plasm are still not separated, some future somatic nuclei begin to eliminate their Ks. While the soma chromosomes (Ss) undergo normal anaphasic migration to the opposite poles, the K chromatids do not separate and remain in the equatorial plane, as demonstrated by fluorescence in situ hybridization using germ line-specific DNA probes. The elimination of the Ks does not occur at the same time in all future somatic nuclei. Nondisjunction of Ks was observed in the first mitosis of the pole nucleus, leading to primordial germ cells with different compositions of their K complements. The pattern and timing of elimination mitoses in the embryos indicate that each of the future somatic nuclei seems to regulate the elimination of the Ks autonomously.     

Control of development of the oral apparatus of Paramecium during sexual reproduction: an embryological perspective

This study shows that development of the new soma during sexual reproduction in ciliates can be conceptualized on the same basis as embryogenesis in multicellular organisms. In conjugating Paramecium, development of a new oral apparatus takes place during fertilization and the first three divisions of the zygotic nucleus and completes well before the postsexual cell undergoes the first cell fission. The control of oral development is analyzed by microsurgical removal of the zygotic nucleus or the postzygotic nuclei from conjugants. The enucleated exconjugants can pass through an early hurdle in oral development (the initiation of oral membranelle assembly) and subsequently develop an oral apparatus. Such oral apparatuses nevertheless exhibit structural and functional abnormalities including fragmentation and misalignment of oral membranelles, absence of the postoral microtubular bundle, reduction in the length of buccal cavity, and impaired phagocytosis. Other stomatogenic aspects, such as the arrangement of basal bodies in the oral membranelles, remain unaffected. The two groups of exconjugants, one derived from cells enucleated at the zygotic stage, and the other at the postzygotic stage, exhibit the same types of oral abnormality. We conclude that (i) the zygotic nucleus is not essential for the initiation of oral membranelle assembly. The existence of zygotic signals for subsequent oral development is not ruled out, but these are insufficient. (ii) Postzygotic nuclei, as well as maternal nuclei (the old somatic nucleus and meiotic derivatives of the germ nucleus), control oral development. This reveals a parallelism between postsexual development in ciliates and the early embryology of multicellular organisms, in their reliance on information provided by maternal, as well as early postzygotic nuclei. (iii) The activity of the old somatic nucleus alone is not sufficient for the later stages of oral development. Probably, some stomatogenic functions of the old somatic nucleus normally utilized for the later stages of oral development in binary fission are inactivated during sexual reproduction. Alternatively, the old somatic nucleus may rely on some critical conditions prescribed by the postzygotic nuclei in order to act.     

Germ-line sex determination in Drosophila melanogaster

In Drosophila melanogaster, the mechanism of sex determination is substantially different in the germ line and in the soma. In the germ line, the process is not completely cell-autonomous, but requires some signals from the soma. Only some of the genes involved in somatic sex determination are also needed for germ cell development. Recent genetic studies have identified loci required for germ-line sex determination.