Somatic polyploidization and cellular proliferation drive body size evolution in nematodes

Most of the hypodermis of a rhabditid nematode such as Caenorhabditis elegans is a single syncytium. The size of this syncytium (as measured by body size) has evolved repeatedly in the rhabditid nematodes. Two cellular mechanisms are important in the evolution of body size: changes in the numbers of cells that fuse with the syncytium, and the extent of its acellular growth. Thus nematodes differ from mammals and other invertebrates in which body size evolution is caused by changes in cell number alone. The evolution of acellular syncytial growth in nematodes is also associated with changes in the ploidy of hypodermal nuclei. These nuclei are polyploid as a consequence of iterative rounds of endoreduplication, and this endocycle has evolved repeatedly. The association between acellular growth and endoreduplication is also seen in C. elegans mutations that interrupt transforming growth factor-β signaling and that result in dwarfism and deficiencies in hypodermal ploidy. The transforming growth factor-β pathway is a candidate for being involved in nematode body size evolution.

Inverse radiation dose-rate effects on somatic and germ-line mutations and DNA damage rates

The mutagenic effect of low linear energy transfer ionizing radiation is reduced for a given dose as the dose rate (DR) is reduced to a low level, a phenomenon known as the direct DR effect. Our reanalysis of published data shows that for both somatic and germ-line mutations there is an opposite, inverse DR effect, with reduction from low to very low DR, the overall dependence of induced mutations being parabolically related to DR, with a minimum in the range of 0.1 to 1.0 cGy/min (rule 1). This general pattern can be attributed to an optimal induction of error-free DNA repair in a DR region of minimal mutability (MMDR region). The diminished activation of repair at very low DRs may reflect a low ratio of induced (“signal”) to spontaneous background DNA damage (“noise”). Because two common DNA lesions, 8-oxoguanine and thymine glycol, were already known to activate repair in irradiated mammalian cells, we estimated how their rates of production are altered upon radiation exposure in the MMDR region. For these and other abundant lesions (abasic sites and single-strand breaks), the DNA damage rate increment in the MMDR region is in the range of 10% to 100% (rule 2). These estimates suggest a genetically programmed optimatization of response to radiation in the MMDR region.

Somatic and germ cell parasitism in a colonial ascidian: Possible role for a highly polymorphic allorecognition system

A colonial protochordate, Botryllus schlosseri, undergoes a natural transplantation reaction in the wild that results alternatively in colony fusion (chimera formation) or inflammatory rejection. A single, highly polymorphic histocompatibility locus (called Fu/HC) is responsible for rejection versus fusion. Gonads are seeded and gametogenesis can occur in colonies well after fusion, and involves circulating germ-line progenitors. Buss proposed that colonial organisms might develop self/non-self histocompatibility systems to limit the possibility of interindividual germ cell “parasitism” (GCP) to histocompatible kin [Buss, L. W. (1982) Proc. Natl. Acad. Sci. USA 79, 5337–5341 and Buss, L. W. (1987) The Evolution of Individuality (Princeton Univ. Press, Princeton]. Here we demonstrate in laboratory and field experiments that both somatic cell and (more importantly) germ-line parasitism are a common occurrence in fused chimeras. These experiments support the tenet in Buss’s hypothesis that germ cell and somatic cell parasitism can occur in fused chimeras and that a somatic appearance may mask the winner of a gametic war. They also provide an interesting challenge to develop formulas that describe the inheritance of competing germ lines rather than competing individuals. The fact that fused B. schlosseri have higher rates of GCP than unfused colonies additionally provides a rational explanation for the generation and maintenance of a high degree of Fu/HC polymorphism, largely limiting GCP to sibling offspring.

Distinct spectra of somatic mutations accumulated with age in mouse heart and small intestine

Somatic mutation accumulation has been implicated as a major cause of cancer and aging. By using a transgenic mouse model with a chromosomally integrated lacZ reporter gene, mutational spectra were characterized at young and old age in two organs greatly differing in proliferative activity, i.e., the heart and small intestine. At young age the spectra were nearly identical, mainly consisting of G·C to A·T transitions and 1-bp deletions. At old age, however, distinct patterns of mutations had developed. In small intestine, only point mutations were found to accumulate, including G·C to T·A, G·C to C·G, and A·T to C·G transversions and G·C to A·T transitions. In contrast, in heart about half of the accumulated mutations appeared to be large genome rearrangements, involving up to 34 centimorgans of chromosomal DNA. Virtually all other mutations accumulating in the heart appeared to be G·C to A·T transitions at CpG sites. These results suggest that distinct mechanisms lead to organ-specific genome deterioration and dysfunction at old age.

Ongoing immunoglobulin somatic mutation in germinal center B cell-like but not in activated B cell-like diffuse large cell lymphomas

B cell diffuse large cell lymphoma (B-DLCL) is a heterogeneous group of tumors, based on significant variations in morphology, clinical presentation, and response to treatment. Gene expression profiling has revealed two distinct tumor subtypes of B-DLCL: germinal center B cell-like DLCL and activated B cell-like DLCL. In a separate study, we determined that B-DLCL can also be subdivided into two groups based on the presence or absence of ongoing Ig gene hypermutation. Here, we evaluated the correlation between these B-DLCL subtypes established by the two different methods. Fourteen primary B-DLCL cases were studied by gene expression profiling using DNA microarrays and for the presence of ongoing mutations in their Ig heavy chain gene. All seven cases classified as germinal center B cell-like DLCL by gene expression showed the presence of ongoing mutations in the Ig genes. Five of the seven cases classified by gene expression as activated B cell-like DLCL had no ongoing somatic mutations, whereas, in the remaining two cases, a single point mutation was observed in only 2 of 15 and 21 examined molecular clones of variable heavy (VH) chain gene, respectively. These two cases were distantly related to the rest of the activated B cell-like DLCL tumors by gene expression. Our findings validate the concept that lymphoid malignancies are derived from cells at discrete stages of normal lymphocyte maturation and that the malignant cells retain the genetic program of those normal cells.

Somatic mosaicism in Fanconi anemia: Evidence of genotypic reversion in lymphohematopoietic stem cells

Somatic mosaicism has been observed previously in the lymphocyte population of patients with Fanconi anemia (FA). To identify the cellular origin of the genotypic reversion, we examined each lymphohematopoietic and stromal cell lineage in an FA patient with a 2815–2816ins19 mutation in FANCA and known lymphocyte somatic mosaicism. DNA extracted from individually plucked peripheral blood T cell colonies and marrow colony-forming unit granulocyte–macrophage and burst-forming unit erythroid cells revealed absence of the maternal FANCA exon 29 mutation in 74.0%, 80.3%, and 86.2% of colonies, respectively. These data, together with the absence of the FANCA exon 29 mutation in Epstein–Barr virus-transformed B cells and its presence in fibroblasts, indicate that genotypic reversion, most likely because of back mutation, originated in a lymphohematopoietic stem cell and not solely in a lymphocyte population. Contrary to a predicted increase in marrow cellularity resulting from reversion in a hematopoietic stem cell, pancytopenia was progressive. Additional evaluations revealed a partial deletion of 11q in 3 of 20 bone marrow metaphase cells. By using interphase fluorescence in situ hybridization with an MLL gene probe mapped to band 11q23 to identify colony-forming unit granulocyte–macrophage and burst-forming unit erythroid cells with the 11q deletion, the abnormal clone was exclusive to colonies with the FANCA exon 29 mutation. Thus, we demonstrate the spontaneous genotypic reversion in a lymphohematopoietic stem cell. The subsequent development of a clonal cytogenetic abnormality in nonrevertant cells suggests that ex vivo correction of hematopoietic stem cells by gene transfer may not be sufficient for providing life-long stable hematopoiesis in patients with FA.

SQV-7, a protein involved in Caenorhabditis elegans epithelial invagination and early embryogenesis, transports UDP-glucuronic acid, UDP-N- acetylgalactosamine, and UDP-galactose

Caenorhabditis elegans sqv mutants are defective in vulval epithelial invagination and have a severe reduction in hermaphrodite fertility. The gene sqv-7 encodes a multitransmembrane hydrophobic protein resembling nucleotide sugar transporters of the Golgi membrane. A Golgi vesicle enriched fraction of Saccharomyces cerevisiae expressing SQV-7 transported UDP-glucuronic acid, UDP-N-acetylgalactosamine, and UDP-galactose (Gal) in a temperature-dependent and saturable manner. These nucleotide sugars are competitive, alternate, noncooperative substrates. The two mutant sqv-7 missense alleles resulted in a severe reduction of these three transport activities. SQV-7 did not transport CMP-sialic acid, GDP-fucose, UDP-N-acetylglucosamine, UDP-glucose, or GDP-mannose. SQV-7 is able to transport UDP-Gal in vivo, as shown by its ability to complement the phenotype of Madin-Darby canine kidney ricin resistant cells, a mammalian cell line deficient in UDP-Gal transport into the Golgi. These results demonstrate that unlike most nucleotide sugar transporters, SQV-7 can transport multiple distinct nucleotide sugars. We propose that SQV-7 translocates multiple nucleotide sugars into the Golgi lumen for the biosynthesis of glycoconjugates that play a pivotal role in development.

Spatial and temporal emergence of high proliferative potential hematopoietic precursors during murine embryogenesis

During mouse embryogenesis, two waves of hematopoietic progenitors originate in the yolk sac. The first wave consists of primitive erythroid progenitors that arise at embryonic day 7.0 (E7.0), whereas the second wave consists of definitive erythroid progenitors that arise at E8.25. To determine whether these unilineage hematopoietic progenitors arise from multipotential precursors, we investigated the kinetics of high proliferative potential colony-forming cells (HPP-CFC), multipotent precursors that give rise to macroscopic colonies when cultured in vitro. No HPP-CFC were found at presomite stages (E6.5–E7.5). Rather, HPP-CFC were detected first at early somite stages (E8.25), exclusively in the yolk sac. HPP-CFC were found subsequently in the bloodstream at higher levels than the remainder of the embryo proper. However, the yolk sac remains the predominant site of HPP-CFC expansion (>100-fold) until the liver begins to serve as the major hematopoietic organ at E11.5. On secondary replating, embryonic HPP-CFC give rise to definitive erythroid and macrophage (but not primitive erythroid) progenitors. Our findings support the hypothesis that definitive but not primitive hematopoietic progenitors originate from yolk sac-derived HPP-CFC during late gastrulation.

Abscisic Acid-Dependent and -Independent Expression of the Carrot Late-Embryogenesis-Abundant-Class Gene Dc3 in Transgenic Tobacco Seedlings1

We studied the expression of three promoter 5′ deletion constructs (−218, −599, and −1312) of the LEA (late embryogenesis abundant)-class gene Dc3 fused to β-glucuronidase (GUS), where each construct value refers to the number of base pairs upstream of the transcription start site at which the deletion occurred. The Dc3 gene is noted for its induction by abscisic acid (ABA), but its response to other plant hormones and various environmental stresses has not been reported previously for vegetative cells. Fourteen-day-old transgenic tobacco (Nicotiana tabacum L.) seedlings were exposed to dehydration, hypoxia, salinity, exogenous ethylene, or exogenous methyl jasmonate (MeJa). GUS activity was quantified fluorimetrically and expression was observed by histochemical staining of the seedlings. An increase in GUS activity was observed in plants with constructs −599 and −1312 in response to dehydration and salinity within 6 h of stress, and at 12 h in response to hypoxia. No increase in endogenous ABA was found in any of the three lines, even after 72 h of hypoxia. An ABA-independent increase in GUS activity was observed when endogenous ABA biosynthesis was blocked by fluridone and plants were exposed to 5 μL L−1 ethylene in air or 100 μm MeJa. Virtually no expression was observed in construct −218 in response to dehydration, salinity, or MeJa, but there was a moderate response to ethylene and hypoxia. This suggests that the region between −218 and −599 is necessary for ABA (dehydration and salinity)- and MeJa-dependent expression, whereas ethylene-mediated expression does not require this region of the promoter.

Expression of error-prone polymerases in BL2 cells activated for Ig somatic hypermutation

High affinity antibodies are generated in mice and humans by means of somatic hypermutation (SHM) of variable (V) regions of Ig genes. Mutations with rates of 10−5–10−3 per base pair per generation, about 106-fold above normal, are targeted primarily at V-region hot spots by unknown mechanisms. We have measured mRNA expression of DNA polymerases ι, η, and ζ by using cultured Burkitt's lymphoma (BL)2 cells. These cells exhibit 5–10-fold increases in heavy-chain V-region mutations targeted only predominantly to RGYW (R = A or G, Y = C or T, W = T or A) hot spots if costimulated with T cells and IgM crosslinking, the presumed in vivo requirements for SHM. An ∼4-fold increase pol ι mRNA occurs within 12 h when cocultured with T cells and surface IgM crosslinking. Induction of pols η and ζ occur with T cells, IgM crosslinking, or both stimuli. The fidelity of pol ι was measured at RGYW hot- and non-hot-spot sequences situated at nicks, gaps, and double-strand breaks. Pol ι formed T⋅G mispairs at a frequency of 10−2, consistent with SHM-generated C to T transitions, with a 3-fold increased error rate in hot- vs. non-hot-spot sequences for the single-nucleotide overhang. The T cell and IgM crosslinking-dependent induction of pol ι at 12 h may indicate an SHM “triggering” event has occurred. However, pols ι, η, and ζ are present under all conditions, suggesting that their presence is not sufficient to generate mutations because both T cell and IgM stimuli are required for SHM induction.

Somatic mosaicism in Wiskott–Aldrich syndrome suggests in vivo reversion by a DNA slippage mechanism

Somatic mosaicism caused by in vivo reversion of inherited mutations has been described in several human genetic disorders. Back mutations resulting in restoration of wild-type sequences and second-site mutations leading to compensatory changes have been shown in mosaic individuals. In most cases, however, the precise genetic mechanisms underlying the reversion events have remained unclear, except for the few instances where crossing over or gene conversion have been demonstrated. Here, we report a patient affected with Wiskott–Aldrich syndrome (WAS) caused by a 6-bp insertion (ACGAGG) in the WAS protein gene, which abrogates protein expression. Somatic mosaicism was documented in this patient whose majority of T lymphocytes expressed nearly normal levels of WAS protein. These lymphocytes were found to lack the deleterious mutation and showed a selective growth advantage in vivo. Analysis of the sequence surrounding the mutation site showed that the 6-bp insertion followed a tandem repeat of the same six nucleotides. These findings strongly suggest that DNA polymerase slippage was the cause of the original germ-line insertion mutation in this family and that the same mechanism was responsible for its deletion in one of the propositus T cell progenitors, thus leading to reversion mosaicism.

A rice homeobox gene, OSH1, is expressed before organ differentiation in a specific region during early embryogenesis.

Homeobox genes encode a large family of homeodomain proteins that play a key role in the pattern formation of animal embryos. By analogy, homeobox genes in plants are thought to mediate important processes in their embryogenesis, but there is very little evidence to support this notion. Here we described the temporal and spatial expression patterns of a rice homeobox gene, OSH1, during rice embryogenesis. In situ hybridization analysis revealed that in the wild-type embryo, OSH1 was first expressed at the globular stage, much earlier than organogenesis started, in a ventral region where shoot apical meristem and epiblast would later develop. This localized expression of OSH1 indicates that the cellular differentiation has already occurred at this stage. At later stages after organogenesis had initiated, OSH1 expression was observed in shoot apical meristem [except in the L1 (tunica) layer], epiblast, radicle, and their intervening tissues in descending strength of expression level with embryonic maturation. We also performed in situ hybridization analysis with a rice organless embryo mutant, orl1, that develops no embryonic organs. In the orl1 embryo, the expression pattern of OSH1 was the same as that in the wild-type embryo in spite of the lack of embryonic organs. This shows that OSH1 is not directly associated with organ differentiation, but may be related to a regulatory process before or independent of the organ determination. The results described here strongly suggest that, like animal homeobox genes, OSH1 plays an important role in regionalization of cell identity during early embryogenesis.

Development of Valpha4+ NK T cells in the early stages of embryogenesis.

The majority of T lymphocytes start to develop at around day 15 of gestation (d15)-d17 in the thymus and comprise the peripheral repertoire characterized by the expression of polymorphic T-cell antigen receptors (TCRs). Contrary to these conventional T cells, a subset of T cells, called natural killer (NK) T cells (most of them expressing an invariant TCR encoded by the Valpha14Jalpha281 gene with a 1-nt N-region), preferentially differentiates extrathymically and dominates the peripheral T-cell population at a high frequency (5% in splenic T cells and 40% in bone marrow T cells). Here, we investigated the development of NK T cells and found that the invariant Valpha14+ TCR transcripts and the circular DNA created by Valpha14 and Jalpha281 gene rearrangements can be detected in the embryo body at d9.5 of gestation and in the yolk sac and the fetal liver at d11.5-d13.5 of gestation, but not in the thymus, whereas T cells with Valpha1+ TCR expression, a major population in the thymus, were not observed at these early stages of gestation. Fluorescence-activated cell sorter analysis also demonstrated that there exist CD3+ alpha beta+ T cells, almost all of which are Valpha14/Vbeta8+ NK+ T cells, during early embryogenesis. To our knowledge, this demonstrates for the first time that a T lymphocyte subset develops in extrathymic tissues during the early stages of embryogenesis.