Geographical clines in genetic polymorphisms are widely used as evidence of climatic selection and are expected to shift with climate change. We show that the classic latitudinal cline in the alcohol dehydrogenase polymorphism of Drosophila melanogaster has shifted over 20 years in eastern coastal Australia. Southern high-latitude populations now have the genetic constitution of more northerly populations, equivalent to a shift of 4° in latitude. A similar shift was detected for a genetically independent inversion polymorphism, whereas two other linked polymorphisms exhibiting weaker clinal patterns have remained relatively stable. These genetic changes are likely to reflect increasingly warmer and drier conditions and may serve as sensitive biomarkers for climate change.
Arginine methylation is now coming out of the shadows of protein phosphorylation and entering the mainstream, largely due to the identification of the family of enzymes that lay down this modification. In addition, modification-specific antibodies and proteomic approaches have facilitated the identification of an array of substrates for the protein arginine methyltransferases. This review describes recent insights into the molecular processes regulated by arginine methylation in normal and diseased cells
Drosophila hematopoiesis occurs in a specialized organ called the lymph gland. In this systematic analysis of lymph gland structure and gene expression, we define the developmental steps in the maturation of blood cells (hemocytes) from their precursors. In particular, distinct zones of hemocyte maturation, signaling and proliferation in the lymph gland during hematopoietic progression are described. Different stages of hemocyte development have been classified according to marker expression and placed within developmental niches: a medullary zone for quiescent prohemocytes, a cortical zone for maturing hemocytes and a zone called the posterior signaling center for specialized signaling hemocytes. This establishes a framework for the identification of Drosophila blood cells, at various stages of maturation, and provides a genetic basis for spatial and temporal events that govern hemocyte development. The cellular events identified in this analysis further establish Drosophila as a model system for hematopoiesis.
Differential gene regulation integrated in time and space drives developmental programs during embryogenesis. To understand how the program of gastrulation is regulated by Wnt/-catenin signaling, we have used genome-wide expression profiling of conditional -catenin mutant embryos. Known Wnt/-catenin target genes, known components of other signaling pathways, as well as a number of uncharacterized genes were downregulated in these mutants. To further narrow down the set of differentially expressed genes, we used whole-mount in situ screening to associate gene expression with putative domains of Wnt activity. Several potential novel target genes were identified by this means and two, Grsf1 and Fragilis2, were functionally analyzed by RNA interference (RNAi) in completely embryonic stem (ES) cell-derived embryos. We show that the gene encoding the RNA-binding factor Grsf1 is important for axial elongation, mid/hindbrain development and axial mesoderm specification, and that Fragilis2, encoding a transmembrane protein, regulates epithelialization of the somites and paraxial mesoderm formation. Intriguingly, the knock-down phenotypes recapitulate several aspects of Wnt pathway mutants, suggesting that these genes are components of the downstream Wnt response. This functional genomic approach allows the rapid identification of functionally important components of embryonic development from large datasets of putative targets.
The receptor Notch and its ligands of the Delta/Serrate/LAG2 (DSL) family are the central components in the Notch pathway, a fundamental cell signaling system that regulates pattern formation during animal development. Delta is directly ubiquitinated by Drosophila and Xenopus Neuralized, and by zebrafish Mind bomb, two unrelated RING-type E3 ubiquitin ligases with common abilities to promote Delta endocytosis and signaling activity. Although orthologs of both Neuralized and Mind bomb are found in most metazoan organisms, their relative contributions to Notch signaling in any single organism have not yet been assessed. We show here that a Drosophila ortholog of Mind bomb (D-mib) is a positive component of Notch signaling that is required for multiple Neuralized-independent, Notch-dependent developmental processes. Furthermore, we show that D-mib associates physically and functionally with both Serrate and Delta. We find that D-mib uses its ubiquitin ligase activity to promote DSL ligand activity, an activity that is correlated with its ability to induce the endocytosis and degradation of both Delta and Serrate (see also Le Borgne et al., 2005). We further demonstrate that D-mib can functionally replace Neuralized in multiple cell fate decisions that absolutely require endogenous Neuralized, a testament to the highly similar activities of these two unrelated ubiquitin ligases in regulating Notch signaling. We conclude that ubiquitination of Delta and Serrate by Neuralized and D-mib is an obligate feature of DSL ligand activation throughout Drosophila development.
During embryonic development, morphogens act as graded positional cues to dictate cell fate specification and tissue patterning. Recent findings indicate that morphogen gradients also serve to guide axonal pathfinding during development of the nervous system. These findings challenge our previous notions about morphogens and axon guidance molecules, and suggest that these proteins, rather than having sharply divergent functions, act more globally to provide graded positional information that can be interpreted by responding cells either to specify cell fate or to direct axonal pathfinding. This review presents the roles identified for members of three prominent morphogen families – the Hedgehog, Wnt and TGFß/BMP families – in axon guidance, and discusses potential implications for the molecular mechanisms underlying their guidance functions.
"Work, finish, publish!" Faraday's (1) advice to a young scientist is still good advice for all scientists today. But where should an author publish? Authors face the twin problems of obtaining the best exposure and fairest review of their work. PNAS has a big plus in these two areas; quality control is provided by the members and foreign associates of a national academy of unusual depth and breadth, and PNAS is the first choice of many of them for publishing their own best work. But PNAS is not just a house or U.S. journal; it is open to all scientists, and currently some 30% of its published articles come from abroad. Last year, authors from 70 countries published in PNAS (Table 1), and the editors of PNAS want to increase further the international input to the journal. I have
A type of retroviral gene trap vectors has been developed that can induce conditional mutations in most genes expressed in mouse embryonic stem (ES) cells. The vectors rely on directional site-specific recombination systems that can repair and reinduce gene trap mutations when activated in succession. After the gene traps are inserted into the mouse genome, genetic mutations can be produced at a particular time and place in somatic cells. In addition to their conditional features, the vectors create multipurpose alleles amenable to a wide range of postinsertional modifications. Here we have used these directional recombination vectors to assemble the largest library of ES cell lines with conditional mutations in single genes yet assembled, presently totaling 1,000 unique genes. The trapped ES cell lines, which can be ordered from the German Gene Trap Consortium, are freely available to the scientific community.
The fundamental question of how sequence defines conformation is explicitly answered if the structures of all possible sequences of a macromolecule are determined. We present here a crystallographic screen of all permutations of the inverted repeat DNA sequence d(CCnnnN6N7N8GG), where N6, N7, and N8 are any of the four naturally occurring nucleotides. At this point, 63 of the 64 possible permutations have been crystallized from a defined set of solutions. When combined with previous work, we have assembled a data set of 37 single-crystal structures from 29 of the sequences in this motif, representing three structural classes of DNA (B-DNA, A-DNA, and four-stranded Holliday junctions). This data set includes a unique set of amphimorphic sequence, those that crystallize in two different conformations and serve to bridge the three structural phases. We have thus constructed a map of DNA structures that can be walked through in single nucleotide steps. Finally, the resulting data set allows us to dissect in detail the stabilization of and conformational variations within structural classes and identify significant conformational deviations within a particular structural class that result from sequence rather than crystal or crystallization effects.
When Sertoli and germ cells were cocultured in vitro, anchoring junctions, such as desmosome-like junctions and adherens junctions (AJ) (e.g., ectoplasmic specialization, ES), are formed. This event was marked by an induction of several kinases, such as PI 3-kinase (phosphatidylinositol 3-kinase), phosphorylated protein kinase B (p-PKB), PAK-2 (p21-activated kinase-2), and their downstream effector, ERK as well as an increase in PKB intrinsic activity. Studies by immunohistochemistry indeed localized PI 3-kinase, p-PKB, and PAK to the apical ES in the seminiferous epithelium of the rat testis. Furthermore, PI 3-kinase was also co-localized with p-PKB to the same site at the ES. These kinases were also shown to associate with ES-associated proteins, such as ß1 integrin, p-FAK and c-Src, by co-immunoprecipitation, suggesting that the integrin/laminin protein complex at the apical ES is likely utilized these protein kinases as regulatory proteins to modulate Sertoli-germ cell AJ dynamics via the ERK signaling pathway. To further validate this hypothesis, an in vivo model using AF-2364 [1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide] to perturb Sertoli-germ cell anchoring junction function, inducing germ cell loss from the epithelium in adult rats, was used in conjunction with specific inhibitors. Similar to the in vitro results, the event of germ cell loss induced by AF-2364 in vivo was associated with an induction of PI 3-kinase, p-PKB, PAK-2, and p-ERK; as well as a surge in intrinsic PAK activity in lysates of testes when spermatids/spermatocytes began to dislodge from the epithelium. Perhaps the most important of all, it was shown that pre-treatment of rats with wortmannin, a PI 3-kinase inhibitor, or an anti-ß1-integrin antibody indeed delayed the AF-2364-induced spermatid loss from the epithelium. In summary, these results have unequivocally demonstrated that the ß1 integrin-PI 3-kinase-PKB-ERK is a putative signaling pathway that regulates Sertoli-germ cell anchoring junction dynamics in the testis.
