Range shifts of Potentilla fruticosa on the Qinghai-Tibetan Plateau during glacial and interglacial periods revealed by chloroplast DNA sequence variation.

The Qinghai-Tibetan Plateau is one of the most extensive habitats for alpine plants in the world. Therefore, the patterns of genetic variation in populations on the Plateau can reveal the detailed demographic history of alpine plants. We analysed the geographical structure of chloroplast matK sequence variation in Potentilla fruticosa L. (Rosaceae), a shrub currently found across the entire Plateau. We obtained sequence data from 508 individuals from 23 populations at sites ranging from the high-altitude interior to the relatively low-altitude northeastern Plateau. In the interior region, genetic diversity was high and included ancestral haplotypes. In contrast, northeastern populations were characterized by relatively low genetic diversity and recently derived haplotypes. The estimated expansion time in the interior population was 17 times that in the northeastern population. These data suggest that P. fruticosa expanded its range on the Plateau during periods of climatic cooling and contracted to the interior region during warmer periods. Thus, the interior region acted as a refugium and greatly contributed to the diversification of P. fruticosa.

Evolution of organic aerosols in the atmosphere.

Organic aerosol (OA) particles affect climate forcing and human health, but their sources and evolution remain poorly characterized. We present a unifying model framework describing the atmospheric evolution of OA that is constrained by high-time-resolution measurements of its composition, volatility, and oxidation state. OA and OA precursor gases evolve by becoming increasingly oxidized, less volatile, and more hygroscopic, leading to the formation of oxygenated organic aerosol (OOA), with concentrations comparable to those of sulfate aerosol throughout the Northern Hemisphere. Our model framework captures the dynamic aging behavior observed in both the atmosphere and laboratory: It can serve as a basis for improving parameterizations in regional and global models.

Mouse Lefty2 and zebrafish antivin are feedback inhibitors of nodal signaling during vertebrate gastrulation.

Mammalian lefty and zebrafish antivin form a subgroup of the TGF beta superfamily. We report that mouse mutants for lefty2 have an expanded primitive streak and form excess mesoderm, a phenotype opposite to that of mutants for the TGF beta gene nodal. Analogously, overexpression of Antivin or Lefty2 in zebrafish embryos blocks head and trunk mesoderm formation, a phenotype identical to that of mutants caused by loss of Nodal signaling. The lefty2 mutant phenotype is partially suppressed by heterozygosity for nodal. Similarly, the effects of Antivin and Lefty2 can be suppressed by overexpression of the nodal-related genes cyclops and squint or the extracellular domain of ActRIIB. Expression of antivin is dependent on Nodal signaling, revealing a feedback loop wherein Nodal signals induce their antagonists Lefty2 and Antivin to restrict Nodal signaling during gastrulation.

N-myc-dependent repression of ndr1, a gene identified by direct subtraction of whole mouse embryo cDNAs between wild type and N-myc mutant.

To identify genes regulated by N-myc, subtraction of whole embryo cDNA was carried out between wild type and N-myc-deficient mutant mice. Six cDNA clones were isolated as representing genes expressed higher in the mutant embryos and two as those expressed lower. One of them, Ndr1, coding for 43 kDa cytoplasmic protein was studied in detail. The Ndr1 gene was augmented 20-fold in the mutant embryos at 10.5 days post coitus which is indicative of repression by N-myc. An inverse relationship actually existed between the expression of N-myc and Ndr1 in various developing tissues of the wild type embryos. In the early stage of differentiation of these tissues when N-myc expression was high Ndr1 expression was low or undetectable, and later when N-myc activity diminished Ndr1 expression was augmented concomitantly with the occurrence of terminal differentiation. To establish the direct link between N-myc activity and the Ndr1 regulation, the Ndr1 gene was cloned and analyzed. The Ndr1 promoter activity was down-regulated by N-myc, and more strongly by the combination of N-myc and Max in the cotransfection assay. This repressive effect was mediated by the promoter region within 52 base pairs from the transcription start site but direct binding of N-myc:Max to the promoter sequence was not demonstrated, which is analogous to the cases recently reported for transcriptional repression by c-myc. c-myc also repressed Ndr1 promoter activity similarly to N-myc. The effect of N-myc:Max was sensitive to Trichostatin A, indicating involvement of histone deacetylase activity in repression of the Ndr1 promoter. The strategy we adopted in identifying target genes of a transcription factor should prove widely applicable when mutant animals are available.

Isolation of novel cDNAs by subtractions between the anterior mesendoderm of single mouse gastrula stage embryos.

The anterior mesendoderm of mid- to late primitive streak stage mouse embryos has the ability to induce anterior neuroectodermal fate in naive epiblast [S.-L. Ang and J. Rossant (1993) Development 118, 139-149]. A number of genes have been found to be expressed in this tissue, notably the transcription factor Lim1. Lim1-null mice have anterior mesendoderm defects that result in a lack of head formation. Thus, the anterior mesendoderm of gastrula stage mouse embryos should express Lim1-regulated genes that are essential for head development. To identify Lim1-regulated genes, a differential screen with subtraction was developed, using cDNA pools that were amplified from the anterior mesendoderm of single wild-type and Lim1-null gastrula stage embryos. This novel screen strategy has yielded 22 cDNAs that show differential expression between anterior mesendoderm cells of wild-type and Lim1-null embryos. The expression of one novel cDNA SII6 initially colocalizes with Lim1 in the anterior mesendoderm of gastrula stage embryos. Moreover, SII6 expression is undetectable in the anterior mesendoderm of Lim1-null embryos. This screen identifies a set of putative Lim1 target genes that may have important roles in vertebrate head formation. Furthermore, this differential screen strategy should provide a broadly applicable approach to identify differences in gene expression between embryonic tissues of limiting quantity.

lefty-1 is required for left-right determination as a regulator of lefty-2 and nodal.

lefty-1, lefty-2, and nodal are expressed on the left side of developing mouse embryos and are implicated in left-right (L-R) determination. The role of lefty-1 was examined by analyzing mutant mice lacking this gene. The lefty-1-deficient mice showed a variety of L-R positional defects in visceral organs. Unexpectedly, however, the most common feature of lefty-1-/- mice was thoracic left isomerism (rather than right isomerism). The lack of lefty-1 resulted in bilateral expression of nodal, lefty-2, and Pitx2 (a homeobox gene normally expressed on the left side). These observations suggest that the role of lefty-1 is to restrict the expression of lefty-2 and nodal to the left side, and that lefty-2 or nodal encodes a signal for "leftness."

Overexpression of a membrane protein, neuropilin, in chimeric mice causes anomalies in the cardiovascular system, nervous system and limbs.

Neuropilin is a type 1 membrane protein, which is highly conserved among Xenopus frog, chicken and mouse. The extracellular part of the neuropilin protein is composed of three unique domains, each of which is thought to be involved in molecular and/or cellular interactions. In mice, neuropilin is expressed in the cardiovascular system, nervous system and limbs at particular developmental stages. To clarify the roles of neuropilin in morphogenesis in vivo, we generated mouse embryonic stem (ES) cell clones that constitutively expressed exogenous neuropilin, then produced chimeras using these ES cell clones. The chimeras overexpressed neuropilin and were embryonic lethal. The chimeric embryos exhibited several morphological abnormalities; excess capillaries and blood vessels, dilation of blood vessels, malformed hearts, ectopic sprouting and defasciculation of nerve fibers, and extra digits. All of these abnormalities occurred in the organs in which neuropilin is expressed in normal development. The variety of abnormalities occurring in these chimeric embryos suggested diverse functions of neuropilin in embryonic morphogenesis, which may be ascribed to multiple interaction domains identified in the molecule. Correct spatiotemporal expression of neuropilin seems to be essential for normal development of the cardiovascular system, nervous system and limbs.

bcl-2 deficiency in mice leads to pleiotropic abnormalities: accelerated lymphoid cell death in thymus and spleen, polycystic kidney, hair hypopigmentation, and distorted small intestine.

Transgenic mice homozygously lacking in the bcl-2 gene were generated using homologous recombination in embryonal stem cells. The complete absence of Bcl-2 alpha and -beta proteins did not interfere with normal embryonic development. Abnormalities became evident after birth, although the severity varied among homozygous null mice, bcl-2-/- mice displayed pleiotropic abnormalities similar to those in the previously described bcl-2-/- mice, including growth retardation, smaller ears, short lives, polycystic kidney, atrophic thymus and spleen with accelerated apoptotic cell death of lymphocytes, and hair hypopigmentation in the second hair follicle cycle. Our bcl-2-/- mice also revealed novel defects in the small intestine, characterized by retarded development, accelerated exfoliation of epithelial cells, and very few mitotic progenitor cells.

Determination of pyrimidine nucleoside diphosphates by use of combined reactions of pyruvate kinase and lactate dehydrogenase.

ADP is known to be easily determined spectrophotometrically after it is utilized to produce the corresponding amount of NAD by combined reactions of pyruvate kinase and lactate dehydrogenase. We studied whether CDP and UDP can be also determined in a similar manner if they were incubated for a longer period with an increased amount of pyruvate kinase. It was shown that CDP and UDP could be utilized to produce the corresponding amount of NADH oxidized after an incubation of at least 25 min and that 0 to 300 nmols of these nucleotides were able to be determined spectrophotometrically.

Defects of embryonic organogenesis resulting from targeted disruption of the N-myc gene in the mouse.

The highest expression of the N-myc gene occurs during embryonic organogenesis in the mouse ontogeny, with the peak of expression around embryonic day 9.5. Homozygous N-myc-deficient mice, produced by germline transmission of a disrupted allele in ES cells, developed normally to day 10.5, indicating dispensability of N-myc expression in the earlier period, but later accumulated organogenic abnormalities and died around day 11.5. The most notable abnormalities were found in the limb bud, visceral organs (lung, stomach, liver and heart) and the central/peripheral nervous systems, and were highly correlated with the site of N-myc expression. The limb buds and the lungs excised from N-myc-deficient mutant embryos were placed in culture to allow their development to stages beyond the point of death of the embryos. Analyses indicated that the mutant limbs failed to develop distal structures and the development of bronchi from the trachea was defective in the lungs. The latter defect was largely corrected by addition of fetal calf serum to the culture medium, suggesting that an activity missing in the mutant lung was replenished by a component of the serum. The phenotype of N-myc-deficient mutant embryos indicated requirement of the N-myc function in many instances of tissue interactions in organogenesis and also in cell-autonomous regulation of tissue maturation.

Transition of localization of the N-Myc protein from nucleus to cytoplasm in differentiating neurons.

N-myc is a developmentally regulated proto-oncogene encoding a putative sequence-specific DNA-binding protein. Previous studies on tissue distribution of N-myc transcripts indicated that one of the major sites of N-myc expression is the CNS and neural crest derivatives in developing embryos. We investigated N-Myc protein expression in embryonic neural tissues and found that the protein was usually localized in the nucleus, but accumulated in the cytoplasm upon differentiation of specific classes of neurons, e.g., retinal ganglion cells, neurons of spinal ganglia, and Purkinje cells of the cerebellum. The change of localization of N-Myc from the nucleus to the cytoplasm indicates a novel feature of regulation of myc family proteins and suggests functions of N-myc in the cytoplasm of maturing neurons.

Embryonic lethality resulting from disruption of both N-myc alleles in mouse zygotes.

The N-myc gene is considered to play a major regulatory role in embryogenesis of the mouse because of its high expression in the organogenesis period and its encoding of nuclear proteins with DNA binding motifs. To elucidate the putative regulatory function of N-myc in embryogenesis, we undertook to inactivate this gene in ES cells. The N-myc alleles were disrupted in ES cells, line E14, by means of homologous recombination of targeting vectors that carry neomycin or hygromycin resistant genes. Homologous recombinants were obtained at the frequency of one in 6 x 10(5) electroporated cells. The inactivated N-myc alleles were transmitted through mouse germ lines. Crosses of heterozygous mice resulted in production of wild-type, heterozygous, and N-myc-null pups and fetuses at a ratio of 1:2:0, indicating embryonic lethality of the homozygotes. ES cells totally deficient in N-myc expression were also obtained by consecutive gene disruption with the use of the targeting vectors, demonstrating the non-essentiality of N-myc expression in the stem-cell state. N-myc-null ES cells offer a valuable tool in chimera analysis to elucidate the requirement for N-myc function in embryogenesis.