Hot off the presses! Oct 25 PLoS Biol

The Oct 25 issue of the PLoS Biol is now up on Pubget (About PLoS Biol): if you're at a subscribing institution, just click the link in the latest link at the home page. (Note you'll only be able to get all the PDFs in the issue if your institution subscribes to Pubget.)

Latest Articles Include:

• How Symbiotic Bacteria Survive Host Defenses
  - PLoS Biol 9(10):e1001164 (2011)
  
• Australian Fish Stretching Their Legs
  - PLoS Biol 9(10):e1001167 (2011)
  
• Loss of Microtubule-to-Actin Linkage Disrupts Cortical Development
  - PLoS Biol 9(10):e1001175 (2011)
  
• Genetic Origins of Birth Defects Revealed by New Animal Model
  - PLoS Biol 9(10):e1001180 (2011)
  
• And Now There Are Five: A New Player in Intracellular Trafficking Pathways
  - PLoS Biol 9(10):e1001173 (2011)
  
• On the Nature of Scientific Progress: Anarchistic Theory Says "Anything Goes"—But I Don't Think So
  - PLoS Biol 9(10):e1001165 (2011)
  
• Rethinking Feyerabend: The "Worst Enemy of Science"?
  - PLoS Biol 9(10):e1001166 (2011)
  
• Toshisada Nishida (1941–2011): Chimpanzee Rapport
  - PLoS Biol 9(10):e1001185 (2011)
  
• A Holistic Approach to Marine Eco-Systems Biology
  - PLoS Biol 9(10):e1001177 (2011)
  
• Teaching How to Bridge Neuroscience, Society, and Culture
  - PLoS Biol 9(10):e1001178 (2011)
  
• Protection of Sinorhizobium against Host Cysteine-Rich Antimicrobial Peptides Is Critical for Symbiosis
  - PLoS Biol 9(10):e1001169 (2011)
  Sinorhizobium meliloti differentiates into persisting, nitrogen-fixing bacteroids within root nodules of the legume Medicago truncatula. Nodule-specific cysteine-rich antimicrobial peptides (NCR AMPs) and the bacterial BacA protein are essential for bacteroid development. However, the bacterial factors central to the NCR AMP response and the in planta role of BacA are unknown. We investigated the hypothesis that BacA is critical for the bacterial response towards NCR AMPs. We found that BacA was not essential for NCR AMPs to induce features of S. meliloti bacteroids in vitro. Instead, BacA was critical to reduce the amount of NCR AMP-induced membrane permeabilization and bacterial killing in vitro. Within M. truncatula, both wild-type and BacA-deficient mutant bacteria were challenged with NCR AMPs, but this resulted in persistence of the wild-type bacteria and rapid cell death of the mutant bacteria. In contrast, BacA was dispensable for bacterial survival in an M. tr! uncatula dnf1 mutant defective in NCR AMP transport to the bacterial compartment. Therefore, BacA is critical for the legume symbiosis by protecting S. meliloti against the bactericidal effects of NCR AMPs. Host AMPs are ubiquitous in nature and BacA proteins are essential for other chronic host infections by symbiotic and pathogenic bacteria. Hence, our findings suggest that BacA-mediated protection of bacteria against host AMPs is a critical stage in the establishment of different prolonged host infections.
• Structure and Evolution of Streptomyces Interaction Networks in Soil and In Silico
  - PLoS Biol 9(10):e1001184 (2011)
  Soil grains harbor an astonishing diversity of Streptomyces strains producing diverse secondary metabolites. However, it is not understood how this genotypic and chemical diversity is ecologically maintained. While secondary metabolites are known to mediate signaling and warfare among strains, no systematic measurement of the resulting interaction networks has been available. We developed a high-throughput platform to measure all pairwise interactions among 64 Streptomyces strains isolated from several individual grains of soil. We acquired more than 10,000 time-lapse movies of colony development of each isolate on media containing compounds produced by each of the other isolates. We observed a rich set of such sender-receiver interactions, including inhibition and promotion of growth and aerial mycelium formation. The probability that two random isolates interact is balanced; it is neither close to zero nor one. The interactions are not random: the distribution of the! number of interactions per sender is bimodal and there is enrichment for reciprocity—if strain A inhibits or promotes B, it is likely that B also inhibits or promotes A. Such reciprocity is further enriched in strains derived from the same soil grain, suggesting that it may be a property of coexisting communities. Interactions appear to evolve rapidly: isolates with identical 16S rRNA sequences can have very different interaction patterns. A simple eco-evolutionary model of bacteria interacting through antibiotic production shows how fast evolution of production and resistance can lead to the observed statistical properties of the network. In the model, communities are evolutionarily unstable—they are constantly being invaded by strains with new sets of interactions. This combination of experimental and theoretical observations suggests that diverse Streptomyces communities do not represent a stable ecological state but an intrinsically dynamic eco-evolutionary phenome! non.
• Development and Evolution of the Muscles of the Pelvic Fin
  - PLoS Biol 9(10):e1001168 (2011)
  Locomotor strategies in terrestrial tetrapods have evolved from the utilisation of sinusoidal contractions of axial musculature, evident in ancestral fish species, to the reliance on powerful and complex limb muscles to provide propulsive force. Within tetrapods, a hindlimb-dominant locomotor strategy predominates, and its evolution is considered critical for the evident success of the tetrapod transition onto land. Here, we determine the developmental mechanisms of pelvic fin muscle formation in living fish species at critical points within the vertebrate phylogeny and reveal a stepwise modification from a primitive to a more derived mode of pelvic fin muscle formation. A distinct process generates pelvic fin muscle in bony fishes that incorporates both primitive and derived characteristics of vertebrate appendicular muscle formation. We propose that the adoption of the fully derived mode of hindlimb muscle formation from this bimodal character state is an evolutionar! y innovation that was critical to the success of the tetrapod transition.
• Accelerated Recruitment of New Brain Development Genes into the Human Genome
  - PLoS Biol 9(10):e1001179 (2011)
  How the human brain evolved has attracted tremendous interests for decades. Motivated by case studies of primate-specific genes implicated in brain function, we examined whether or not the young genes, those emerging genome-wide in the lineages specific to the primates or rodents, showed distinct spatial and temporal patterns of transcription compared to old genes, which had existed before primate and rodent split. We found consistent patterns across different sources of expression data: there is a significantly larger proportion of young genes expressed in the fetal or infant brain of humans than in mouse, and more young genes in humans have expression biased toward early developing brains than old genes. Most of these young genes are expressed in the evolutionarily newest part of human brain, the neocortex. Remarkably, we also identified a number of human-specific genes which are expressed in the prefrontal cortex, which is implicated in complex cognitive behaviors. ! The young genes upregulated in the early developing human brain play diverse functional roles, with a significant enrichment of transcription factors. Genes originating from different mechanisms show a similar expression bias in the developing brain. Moreover, we found that the young genes upregulated in early brain development showed rapid protein evolution compared to old genes also expressed in the fetal brain. Strikingly, genes expressed in the neocortex arose soon after its morphological origin. These four lines of evidence suggest that positive selection for brain function may have contributed to the origination of young genes expressed in the developing brain. These data demonstrate a striking recruitment of new genes into the early development of the human brain.
• Three-Dimensional Regulation of Radial Glial Functions by Lis1-Nde1 and Dystrophin Glycoprotein Complexes
  - PLoS Biol 9(10):e1001172 (2011)
  Radial glial cells (RGCs) are distinctive neural stem cells with an extraordinary slender bipolar morphology and dual functions as precursors and migration scaffolds for cortical neurons. Here we show a novel mechanism by which the Lis1-Nde1 complex maintains RGC functions through stabilizing the dystrophin/dystroglycan glycoprotein complex (DGC). A direct interaction between Nde1 and utrophin/dystrophin allows for the assembly of a multi-protein complex that links the cytoskeleton to the extracellular matrix of RGCs to stabilize their lateral membrane, cell-cell adhesion, and radial morphology. Lis1-Nde1 mutations destabilized the DGC and resulted in deformed, disjointed RGCs and disrupted basal lamina. Besides impaired RGC self-renewal and neuronal migration arrests, Lis1-Nde1 deficiencies also led to neuronal over-migration. Additional to phenotypic resemblances of Lis1-Nde1 with DGC, strong synergistic interactions were found between Nde1 and dystroglycan in RGCs. ! As functional insufficiencies of LIS1, NDE1, and dystroglycan all cause lissencephaly syndromes, our data demonstrated that a three-dimensional regulation of RGC's cytoarchitecture by the Lis1-Nde1-DGC complex determines the number and spatial organization of cortical neurons as well as the size and shape of the cerebral cortex.
• Multifactorial Origins of Heart and Gut Defects in nipbl-Deficient Zebrafish, a Model of Cornelia de Lange Syndrome
  - PLoS Biol 9(10):e1001181 (2011)
  Cornelia de Lange Syndrome (CdLS) is the founding member of a class of multi-organ system birth defect syndromes termed cohesinopathies, named for the chromatin-associated protein complex cohesin, which mediates sister chromatid cohesion. Most cases of CdLS are caused by haploinsufficiency for Nipped-B-like (Nipbl), a highly conserved protein that facilitates cohesin loading. Consistent with recent evidence implicating cohesin and Nipbl in transcriptional regulation, both CdLS cell lines and tissues of Nipbl-deficient mice show changes in the expression of hundreds of genes. Nearly all such changes are modest, however—usually less than 1.5-fold—raising the intriguing possibility that, in CdLS, severe developmental defects result from the collective action of many otherwise innocuous perturbations. As a step toward testing this hypothesis, we developed a model of nipbl-deficiency in zebrafish, an organism in which we can quantitatively investigate the combinatorial ! effects of gene expression changes. After characterizing the structure and embryonic expression of the two zebrafish nipbl genes, we showed that morpholino knockdown of these genes produces a spectrum of specific heart and gut/visceral organ defects with similarities to those in CdLS. Analysis of nipbl morphants further revealed that, as early as gastrulation, expression of genes involved in endodermal differentiation (sox32, sox17, foxa2, and gata5) and left-right patterning (spaw, lefty2, and dnah9) is altered. Experimental manipulation of the levels of several such genes—using RNA injection or morpholino knockdown—implicated both additive and synergistic interactions in causing observed developmental defects. These findings support the view that birth defects in CdLS arise from collective effects of quantitative changes in gene expression. Interestingly, both the phenotypes and gene expression changes in nipbl morphants differed from those in mutants or morphants for! genes encoding cohesin subunits, suggesting that the transcri! ptional functions of Nipbl cannot be ascribed simply to its role in cohesin loading.
• Dpp Signaling Activity Requires Pentagone to Scale with Tissue Size in the Growing Drosophila Wing Imaginal Disc
  - PLoS Biol 9(10):e1001182 (2011)
  The wing of the fruit fly, Drosophila melanogaster, with its simple, two-dimensional structure, is a model organ well suited for a systems biology approach. The wing arises from an epithelial sac referred to as the wing imaginal disc, which undergoes a phase of massive growth and concomitant patterning during larval stages. The Decapentaplegic (Dpp) morphogen plays a central role in wing formation with its ability to co-coordinately regulate patterning and growth. Here, we asked whether the Dpp signaling activity scales, i.e. expands proportionally, with the growing wing imaginal disc. Using new methods for spatial and temporal quantification of Dpp activity and its scaling properties, we found that the Dpp response scales with the size of the growing tissue. Notably, scaling is not perfect at all positions in the field and the scaling of target gene domains is ensured specifically where they define vein positions. We also found that the target gene domains are not def! ined at constant concentration thresholds of the downstream Dpp activity gradients P-Mad and Brinker. Most interestingly, Pentagone, an important secreted feedback regulator of the pathway, plays a central role in scaling and acts as an expander of the Dpp gradient during disc growth.
• Memory Phenotype CD4 T Cells Undergoing Rapid, Nonburst-Like, Cytokine-Driven Proliferation Can Be Distinguished from Antigen-Experienced Memory Cells
  - PLoS Biol 9(10):e1001171 (2011)
  Memory phenotype (CD44bright, CD25negative) CD4 spleen and lymph node T cells (MP cells) proliferate rapidly in normal or germ-free donors, with BrdU uptake rates of 6% to 10% per day and Ki-67 positivity of 18% to 35%. The rapid proliferation of MP cells stands in contrast to the much slower proliferation of lymphocytic choriomeningitis virus (LCMV)-specific memory cells that divide at rates ranging from <1% to 2% per day over the period from 15 to 60 days after LCMV infection. Anti-MHC class II antibodies fail to inhibit the in situ proliferation of MP cells, implying a non–T-cell receptor (TCR)-driven proliferation. Such proliferation is partially inhibited by anti–IL-7Rα antibody. The sequence diversity of TCRβ CDR3 gene segments is comparable among the proliferating and quiescent MP cells from conventional and germ-free mice, implying that the majority of proliferating MP cells have not recently derived from a small cohort of cells that expand through multip! le continuous rounds of cell division. We propose that MP cells constitute a diverse cell population, containing a subpopulation of slowly dividing authentic antigen-primed memory cells and a majority population of rapidly proliferating cells that did not arise from naïve cells through conventional antigen-driven clonal expansion.
• Mouse PRDM9 DNA-Binding Specificity Determines Sites of Histone H3 Lysine 4 Trimethylation for Initiation of Meiotic Recombination
  - PLoS Biol 9(10):e1001176 (2011)
  Meiotic recombination generates reciprocal exchanges between homologous chromosomes (also called crossovers, COs) that are essential for proper chromosome segregation during meiosis and are a major source of genome diversity by generating new allele combinations. COs have two striking properties: they occur at specific sites, called hotspots, and these sites evolve rapidly. In mammals, the Prdm9 gene, which encodes a meiosis-specific histone H3 methyltransferase, has recently been identified as a determinant of CO hotspots. Here, using transgenic mice, we show that the sole modification of PRDM9 zinc fingers leads to changes in hotspot activity, histone H3 lysine 4 trimethylation (H3K4me3) levels, and chromosome-wide distribution of COs. We further demonstrate by an in vitro assay that the PRDM9 variant associated with hotspot activity binds specifically to DNA sequences located at the center of the three hotspots tested. Remarkably, we show that mutations in cis locat! ed at hotspot centers and associated with a decrease of hotspot activity affect PRDM9 binding. Taken together, these results provide the direct demonstration that Prdm9 is a master regulator of hotspot localization through the DNA binding specificity of its zinc finger array and that binding of PRDM9 at hotspots promotes local H3K4me3 enrichment.
• The Fifth Adaptor Protein Complex
  - PLoS Biol 9(10):e1001170 (2011)
  Adaptor protein (AP) complexes sort cargo into vesicles for transport from one membrane compartment of the cell to another. Four distinct AP complexes have been identified, which are present in most eukaryotes. We report the existence of a fifth AP complex, AP-5. Tagged AP-5 localises to a late endosomal compartment in HeLa cells. AP-5 does not associate with clathrin and is insensitive to brefeldin A. Knocking down AP-5 subunits interferes with the trafficking of the cation-independent mannose 6-phosphate receptor and causes the cell to form swollen endosomal structures with emanating tubules. AP-5 subunits can be found in all five eukaryotic supergroups, but they have been co-ordinately lost in many organisms. Concatenated phylogenetic analysis provides robust resolution, for the first time, into the evolutionary order of emergence of the adaptor subunit families, showing AP-3 as the basal complex, followed by AP-5, AP-4, and AP-1 and AP-2. Thus, AP-5 is an evolution! arily ancient complex, which is involved in endosomal sorting, and which has links with hereditary spastic paraplegia.
• Regulation of Nucleotide Excision Repair by UV-DDB: Prioritization of Damage Recognition to Internucleosomal DNA
  - PLoS Biol 9(10):e1001183 (2011)
  How tightly packed chromatin is thoroughly inspected for DNA damage is one of the fundamental unanswered questions in biology. In particular, the effective excision of carcinogenic lesions caused by the ultraviolet (UV) radiation of sunlight depends on UV-damaged DNA-binding protein (UV-DDB), but the mechanism by which this DDB1-DDB2 heterodimer stimulates DNA repair remained enigmatic. We hypothesized that a distinctive function of this unique sensor is to coordinate damage recognition in the nucleosome repeat landscape of chromatin. Therefore, the nucleosomes of human cells have been dissected by micrococcal nuclease, thus revealing, to our knowledge for the first time, that UV-DDB associates preferentially with lesions in hypersensitive, hence, highly accessible internucleosomal sites joining the core particles. Surprisingly, the accompanying CUL4A ubiquitin ligase activity is necessary to retain the xeroderma pigmentosum group C (XPC) partner at such internucleosom! al repair hotspots that undergo very fast excision kinetics. This CUL4A complex thereby counteracts an unexpected affinity of XPC for core particles that are less permissive than hypersensitive sites to downstream repair subunits. That UV-DDB also adopts a ubiquitin-independent function is evidenced by domain mapping and in situ protein dynamics studies, revealing direct but transient interactions that promote a thermodynamically unfavorable β-hairpin insertion of XPC into substrate DNA. We conclude that the evolutionary advent of UV-DDB correlates with the need for a spatiotemporal organizer of XPC positioning in higher eukaryotic chromatin.

Hot off the presses! Jul 26 PLoS Biol

The Jul 26 issue of the PLoS Biol is now up on Pubget (About PLoS Biol): if you're at a subscribing institution, just click the link in the latest link at the home page. (Note you'll only be able to get all the PDFs in the issue if your institution subscribes to Pubget.)

Latest Articles Include:

• Why Biotrophs Can't Live Alone
  - PLoS Biol 9(7):e1001097 (2011)
  
• Different Paths, Same Structure: "Developmental Systems Drift" at Work
  - PLoS Biol 9(7):e1001113 (2011)
  
• Genetic Switch Underlies C. albicans Quick Change Act
  - PLoS Biol 9(7):e1001108 (2011)
  
• SRP and NAC: Guiding a Nascent Protein's First Steps
  - PLoS Biol 9(7):e1001103 (2011)
  
• Darwin in Mind: New Opportunities for Evolutionary Psychology
  - PLoS Biol 9(7):e1001109 (2011)
  Evolutionary Psychology (EP) views the human mind as organized into many modules, each underpinned by psychological adaptations designed to solve problems faced by our Pleistocene ancestors. We argue that the key tenets of the established EP paradigm require modification in the light of recent findings from a number of disciplines, including human genetics, evolutionary biology, cognitive neuroscience, developmental psychology, and paleoecology. For instance, many human genes have been subject to recent selective sweeps; humans play an active, constructive role in co-directing their own development and evolution; and experimental evidence often favours a general process, rather than a modular account, of cognition. A redefined EP could use the theoretical insights of modern evolutionary biology as a rich source of hypotheses concerning the human mind, and could exploit novel methods from a variety of adjacent research fields.
• The Reality of Pervasive Transcription
  - PLoS Biol 9(7):e1000625 (2011)
  
• Response to "The Reality of Pervasive Transcription"
  - PLoS Biol 9(7):e1001102 (2011)
  
• Darwin's Theory of Descent with Modification, versus the Biblical Tree of Life
  - PLoS Biol 9(7):e1001096 (2011)
  
• Horace Judson (1931–2011)
  - PLoS Biol 9(7):e1001104 (2011)
  
• Invertebrate Post-Segregation Distorters: A New Embryo-Killing Gene
  - PLoS Biol 9(7):e1001114 (2011)
  Cytoplasmic incompatibility induced by inherited intracellular bacteria of arthropods, and Medea elements found in flour beetles, are both forms of postsegregation distortion involving the killing of embryos in order to increase the ratio of progeny that inherit them. The recently described peel-zeel element of Caenorhabditis elegans also uses this mechanism; like Medea the genes responsible are in the nuclear genome but it shares a paternal mode of action with the bacteria. The peel-1 gene has now been shown to encode a potent toxin that is delivered by sperm, and rescued by zygotic transcription of the linked zeel-1. The predominance of self-fertilization in C. elegans has produced an unusual distribution pattern for a selfish genetic element; further population and functional studies will shed light on its evolution. The element might also have potential for use in disease control.
• Nothing Iffy about HIF in the Hypothalamus
  - PLoS Biol 9(7):e1001116 (2011)
  Two crucial biological processes are (1) the sensing and coordination of responses to low oxygen levels and (2) the control of food intake and energy expenditure. The hypoxia-inducible factor (HIF) family of proteins is known to regulate responses to low oxygen, whereas neuropeptides derived from proopiomelanocortin (POMC) are implicated in the control of food intake and energy expenditure. It is now becoming apparent that these two apparently disparate processes may be linked, with the exciting discovery that HIF proteins can act in the brain to regulate food intake and energy expenditure as reported in the current issue of PLoS Biology. This primer discusses the traditional role of HIF proteins in terms of responding to oxygen levels in the periphery and also their new role in coordinating responses to nutrients in the brain through regulation of POMC.
• Modernizing Reference Genome Assemblies
  - PLoS Biol 9(7):e1001091 (2011)
  
• Gene Gain and Loss during Evolution of Obligate Parasitism in the White Rust Pathogen of Arabidopsis thaliana
  - PLoS Biol 9(7):e1001094 (2011)
  Biotrophic eukaryotic plant pathogens require a living host for their growth and form an intimate haustorial interface with parasitized cells. Evolution to biotrophy occurred independently in fungal rusts and powdery mildews, and in oomycete white rusts and downy mildews. Biotroph evolution and molecular mechanisms of biotrophy are poorly understood. It has been proposed, but not shown, that obligate biotrophy results from (i) reduced selection for maintenance of biosynthetic pathways and (ii) gain of mechanisms to evade host recognition or suppress host defence. Here we use Illumina sequencing to define the genome, transcriptome, and gene models for the obligate biotroph oomycete and Arabidopsis parasite, Albugo laibachii. A. laibachii is a member of the Chromalveolata, which incorporates Heterokonts (containing the oomycetes), Apicomplexa (which includes human parasites like Plasmodium falciparum and Toxoplasma gondii), and four other taxa. From comparisons with othe! r oomycete plant pathogens and other chromalveolates, we reveal independent loss of molybdenum-cofactor-requiring enzymes in downy mildews, white rusts, and the malaria parasite P. falciparum. Biotrophy also requires "effectors" to suppress host defence; we reveal RXLR and Crinkler effectors shared with other oomycetes, and also discover and verify a novel class of effectors, the "CHXCs", by showing effector delivery and effector functionality. Our findings suggest that evolution to progressively more intimate association between host and parasite results in reduced selection for retention of certain biosynthetic pathways, and particularly reduced selection for retention of molybdopterin-requiring biosynthetic pathways. These mechanisms are not only relevant to plant pathogenic oomycetes but also to human pathogens within the Chromalveolata.
• A Novel Sperm-Delivered Toxin Causes Late-Stage Embryo Lethality and Transmission Ratio Distortion in C. elegans
  - PLoS Biol 9(7):e1001115 (2011)
  The evolutionary fate of an allele ordinarily depends on its contribution to host fitness. Occasionally, however, genetic elements arise that are able to gain a transmission advantage while simultaneously imposing a fitness cost on their hosts. We previously discovered one such element in C. elegans that gains a transmission advantage through a combination of paternal-effect killing and zygotic self-rescue. Here we demonstrate that this element is composed of a sperm-delivered toxin, peel-1, and an embryo-expressed antidote, zeel-1. peel-1 and zeel-1 are located adjacent to one another in the genome and co-occur in an insertion/deletion polymorphism. peel-1 encodes a novel four-pass transmembrane protein that is expressed in sperm and delivered to the embryo via specialized, sperm-specific vesicles. In the absence of zeel-1, sperm-delivered PEEL-1 causes lethal defects in muscle and epidermal tissue at the 2-fold stage of embryogenesis. zeel-1 is expressed transiently ! in the embryo and encodes a novel six-pass transmembrane domain fused to a domain with sequence similarity to zyg-11, a substrate-recognition subunit of an E3 ubiquitin ligase. zeel-1 appears to have arisen recently, during an expansion of the zyg-11 family, and the transmembrane domain of zeel-1 is required and partially sufficient for antidote activity. Although PEEL-1 and ZEEL-1 normally function in embryos, these proteins can act at other stages as well. When expressed ectopically in adults, PEEL-1 kills a variety of cell types, and ectopic expression of ZEEL-1 rescues these effects. Our results demonstrate that the tight physical linkage between two novel transmembrane proteins has facilitated their co-evolution into an element capable of promoting its own transmission to the detriment of organisms carrying it.
• Antagonism of LIN-17/Frizzled and LIN-18/Ryk in Nematode Vulva Induction Reveals Evolutionary Alterations in Core Developmental Pathways
  - PLoS Biol 9(7):e1001110 (2011)
  Most diversity in animals and plants results from the modification of already existing structures. Many organ systems, for example, are permanently modified during evolution to create developmental and morphological diversity, but little is known about the evolution of the underlying developmental mechanisms. The theory of developmental systems drift proposes that the development of conserved morphological structures can involve large-scale modifications in their regulatory mechanisms. We test this hypothesis by comparing vulva induction in two genetically tractable nematodes, Caenorhabditis elegans and Pristionchus pacificus. Previous work indicated that the vulva is induced by epidermal growth factor (EGF)/RAS and WNT signaling in Caenorhabditis and Pristionchus, respectively. Here, we show that the evolution of vulva induction involves major molecular alterations and that this shift of signaling pathways involves a novel wiring of WNT signaling and the acquisition o! f novel domains in otherwise conserved receptors in Pristionchus vulva induction. First, Ppa-LIN-17/Frizzled acts as an antagonist of WNT signaling and suppresses the ligand Ppa-EGL-20 by ligand sequestration. Second, Ppa-LIN-18/Ryk transmits WNT signaling and requires inhibitory SH3 domain binding motifs, unknown from Cel-LIN-18/Ryk. Third, Ppa-LIN-18/Ryk signaling involves Axin and β-catenin and Ppa-axl-1/Axin is epistatic to Ppa-lin-18/Ryk. These results confirm developmental system drift as an important theory for the evolution of organ systems and they highlight the significance of protein modularity in signal transduction and the dynamics of signaling networks.
• Generation of Healthy Mice from Gene-Corrected Disease-Specific Induced Pluripotent Stem Cells
  - PLoS Biol 9(7):e1001099 (2011)
  Using the murine model of tyrosinemia type 1 (fumarylacetoacetate hydrolase [FAH] deficiency; FAH−/− mice) as a paradigm for orphan disorders, such as hereditary metabolic liver diseases, we evaluated fibroblast-derived FAH−/−-induced pluripotent stem cells (iPS cells) as targets for gene correction in combination with the tetraploid embryo complementation method. First, after characterizing the FAH−/− iPS cell lines, we aggregated FAH−/−-iPS cells with tetraploid embryos and obtained entirely FAH−/−-iPS cell–derived mice that were viable and exhibited the phenotype of the founding FAH−/− mice. Then, we transduced FAH cDNA into the FAH−/−-iPS cells using a third-generation lentiviral vector to generate gene-corrected iPS cells. We could not detect any chromosomal alterations in these cells by high-resolution array CGH analysis, and after their aggregation with tetraploid embryos, we obtained fully iPS cell–derived healthy mice with an as! tonishing high efficiency for full-term development of up to 63.3%. The gene correction was validated functionally by the long-term survival and expansion of FAH-positive cells of these mice after withdrawal of the rescuing drug NTBC (2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione). Furthermore, our results demonstrate that both a liver-specific promoter (transthyretin, TTR)-driven FAH transgene and a strong viral promoter (from spleen focus-forming virus, SFFV)-driven FAH transgene rescued the FAH-deficiency phenotypes in the mice derived from the respective gene-corrected iPS cells. In conclusion, our data demonstrate that a lentiviral gene repair strategy does not abrogate the full pluripotent potential of fibroblast-derived iPS cells, and genetic manipulation of iPS cells in combination with tetraploid embryo aggregation provides a practical and rapid approach to evaluate the efficacy of gene correction of human diseases in mouse models.
• Hypoxia-Inducible Factor Directs POMC Gene to Mediate Hypothalamic Glucose Sensing and Energy Balance Regulation
  - PLoS Biol 9(7):e1001112 (2011)
  Hypoxia-inducible factor (HIF) is a nuclear transcription factor that responds to environmental and pathological hypoxia to induce metabolic adaptation, vascular growth, and cell survival. Here we found that HIF subunits and HIF2α in particular were normally expressed in the mediobasal hypothalamus of mice. Hypothalamic HIF was up-regulated by glucose to mediate the feeding control of hypothalamic glucose sensing. Two underlying molecular pathways were identified, including suppression of PHDs by glucose metabolites to prevent HIF2α degradation and the recruitment of AMPK and mTOR/S6K to regulate HIF2α protein synthesis. HIF activation was found to directly control the transcription of POMC gene. Genetic approach was then employed to develop conditional knockout mice with HIF inhibition in POMC neurons, revealing that HIF loss-of-function in POMC neurons impaired hypothalamic glucose sensing and caused energy imbalance to promote obesity development. The metabolic e! ffects of HIF in hypothalamic POMC neurons were independent of leptin signaling or pituitary ACTH pathway. Hypothalamic gene delivery of HIF counteracted overeating and obesity under conditions of nutritional excess. In conclusion, HIF controls hypothalamic POMC gene to direct the central nutrient sensing in regulation of energy and body weight balance.
• Synaptic Transmission from Horizontal Cells to Cones Is Impaired by Loss of Connexin Hemichannels
  - PLoS Biol 9(7):e1001107 (2011)
  In the vertebrate retina, horizontal cells generate the inhibitory surround of bipolar cells, an essential step in contrast enhancement. For the last decades, the mechanism involved in this inhibitory synaptic pathway has been a major controversy in retinal research. One hypothesis suggests that connexin hemichannels mediate this negative feedback signal; another suggests that feedback is mediated by protons. Mutant zebrafish were generated that lack connexin 55.5 hemichannels in horizontal cells. Whole cell voltage clamp recordings were made from isolated horizontal cells and cones in flat mount retinas. Light-induced feedback from horizontal cells to cones was reduced in mutants. A reduction of feedback was also found when horizontal cells were pharmacologically hyperpolarized but was absent when they were pharmacologically depolarized. Hemichannel currents in isolated horizontal cells showed a similar behavior. The hyperpolarization-induced hemichannel current was s! trongly reduced in the mutants while the depolarization-induced hemichannel current was not. Intracellular recordings were made from horizontal cells. Consistent with impaired feedback in the mutant, spectral opponent responses in horizontal cells were diminished in these animals. A behavioral assay revealed a lower contrast-sensitivity, illustrating the role of the horizontal cell to cone feedback pathway in contrast enhancement. Model simulations showed that the observed modifications of feedback can be accounted for by an ephaptic mechanism. A model for feedback, in which the number of connexin hemichannels is reduced to about 40%, fully predicts the specific asymmetric modification of feedback. To our knowledge, this is the first successful genetic interference in the feedback pathway from horizontal cells to cones. It provides direct evidence for an unconventional role of connexin hemichannels in the inhibitory synapse between horizontal cells and cones. This is an imp! ortant step in resolving a long-standing debate about the unus! ual form of (ephaptic) synaptic transmission between horizontal cells and cones in the vertebrate retina.
• Formation of the Long Range Dpp Morphogen Gradient
  - PLoS Biol 9(7):e1001111 (2011)
  The TGF-β homolog Decapentaplegic (Dpp) acts as a secreted morphogen in the Drosophila wing disc, and spreads through the target tissue in order to form a long range concentration gradient. Despite extensive studies, the mechanism by which the Dpp gradient is formed remains controversial. Two opposing mechanisms have been proposed: receptor-mediated transcytosis (RMT) and restricted extracellular diffusion (RED). In these scenarios the receptor for Dpp plays different roles. In the RMT model it is essential for endocytosis, re-secretion, and thus transport of Dpp, whereas in the RED model it merely modulates Dpp distribution by binding it at the cell surface for internalization and subsequent degradation. Here we analyzed the effect of receptor mutant clones on the Dpp profile in quantitative mathematical models representing transport by either RMT or RED. We then, using novel genetic tools, experimentally monitored the actual Dpp gradient in wing discs containing rec! eptor gain-of-function and loss-of-function clones. Gain-of-function clones reveal that Dpp binds in vivo strongly to the type I receptor Thick veins, but not to the type II receptor Punt. Importantly, results with the loss-of-function clones then refute the RMT model for Dpp gradient formation, while supporting the RED model in which the majority of Dpp is not bound to Thick veins. Together our results show that receptor-mediated transcytosis cannot account for Dpp gradient formation, and support restricted extracellular diffusion as the main mechanism for Dpp dispersal. The properties of this mechanism, in which only a minority of Dpp is receptor-bound, may facilitate long-range distribution.
• Hyphal Development in Candida albicans Requires Two Temporally Linked Changes in Promoter Chromatin for Initiation and Maintenance
  - PLoS Biol 9(7):e1001105 (2011)
  Phenotypic plasticity is common in development. For Candida albicans, the most common cause of invasive fungal infections in humans, morphological plasticity is its defining feature and is critical for its pathogenesis. Unlike other fungal pathogens that exist primarily in either yeast or hyphal forms, C. albicans is able to switch reversibly between yeast and hyphal growth forms in response to environmental cues. Although many regulators have been found involved in hyphal development, the mechanisms of regulating hyphal development and plasticity of dimorphism remain unclear. Here we show that hyphal development involves two sequential regulations of the promoter chromatin of hypha-specific genes. Initiation requires a rapid but temporary disappearance of the Nrg1 transcriptional repressor of hyphal morphogenesis via activation of the cAMP-PKA pathway. Maintenance requires promoter recruitment of Hda1 histone deacetylase under reduced Tor1 (target of rapamycin) signal! ing. Hda1 deacetylates a subunit of the NuA4 histone acetyltransferase module, leading to eviction of the NuA4 acetyltransferase module and blockage of Nrg1 access to promoters of hypha-specific genes. Promoter recruitment of Hda1 for hyphal maintenance happens only during the period when Nrg1 is gone. The sequential regulation of hyphal development by the activation of the cAMP-PKA pathway and reduced Tor1 signaling provides a molecular mechanism for plasticity of dimorphism and how C. albicans adapts to the varied host environments in pathogenesis. Such temporally linked regulation of promoter chromatin by different signaling pathways provides a unique mechanism for integrating multiple signals during development and cell fate specification.
• Coupled Evolution of Transcription and mRNA Degradation
  - PLoS Biol 9(7):e1001106 (2011)
  mRNA levels are determined by the balance between transcription and mRNA degradation, and while transcription has been extensively studied, very little is known regarding the regulation of mRNA degradation and its coordination with transcription. Here we examine the evolution of mRNA degradation rates between two closely related yeast species. Surprisingly, we find that around half of the evolutionary changes in mRNA degradation were coupled to transcriptional changes that exert opposite effects on mRNA levels. Analysis of mRNA degradation rates in an interspecific hybrid further suggests that opposite evolutionary changes in transcription and in mRNA degradation are mechanistically coupled and were generated by the same individual mutations. Coupled changes are associated with divergence of two complexes that were previously implicated both in transcription and in mRNA degradation (Rpb4/7 and Ccr4-Not), as well as with sequence divergence of transcription factor bindi! ng motifs. These results suggest that an opposite coupling between the regulation of transcription and that of mRNA degradation has shaped the evolution of gene regulation in yeast.
• Defining the Specificity of Cotranslationally Acting Chaperones by Systematic Analysis of mRNAs Associated with Ribosome-Nascent Chain Complexes
  - PLoS Biol 9(7):e1001100 (2011)
  Polypeptides exiting the ribosome must fold and assemble in the crowded environment of the cell. Chaperones and other protein homeostasis factors interact with newly translated polypeptides to facilitate their folding and correct localization. Despite the extensive efforts, little is known about the specificity of the chaperones and other factors that bind nascent polypeptides. To address this question we present an approach that systematically identifies cotranslational chaperone substrates through the mRNAs associated with ribosome-nascent chain-chaperone complexes. We here focused on two Saccharomyces cerevisiae chaperones: the Signal Recognition Particle (SRP), which acts cotranslationally to target proteins to the ER, and the Nascent chain Associated Complex (NAC), whose function has been elusive. Our results provide new insights into SRP selectivity and reveal that NAC is a general cotranslational chaperone. We found surprising differential substrate specificity ! for the three subunits of NAC, which appear to recognize distinct features within nascent chains. Our results also revealed a partial overlap between the sets of nascent polypeptides that interact with NAC and SRP, respectively, and showed that NAC modulates SRP specificity and fidelity in vivo. These findings give us new insight into the dynamic interplay of chaperones acting on nascent chains. The strategy we used should be generally applicable to mapping the specificity, interplay, and dynamics of the cotranslational protein homeostasis network.
• The Cryo-EM Structure of a Complete 30S Translation Initiation Complex from Escherichia coli
  - PLoS Biol 9(7):e1001095 (2011)
  Formation of the 30S initiation complex (30S IC) is an important checkpoint in regulation of gene expression. The selection of mRNA, correct start codon, and the initiator fMet-tRNAfMet requires the presence of three initiation factors (IF1, IF2, IF3) of which IF3 and IF1 control the fidelity of the process, while IF2 recruits fMet-tRNAfMet. Here we present a cryo-EM reconstruction of the complete 30S IC, containing mRNA, fMet-tRNAfMet, IF1, IF2, and IF3. In the 30S IC, IF2 contacts IF1, the 30S subunit shoulder, and the CCA end of fMet-tRNAfMet, which occupies a novel P/I position (P/I1). The N-terminal domain of IF3 contacts the tRNA, whereas the C-terminal domain is bound to the platform of the 30S subunit. Binding of initiation factors and fMet-tRNAfMet induces a rotation of the head relative to the body of the 30S subunit, which is likely to prevail through 50S subunit joining until GTP hydrolysis and dissociation of IF2 take place. The structure provides insights! into the mechanism of mRNA selection during translation initiation.

Hot off the presses! Jul 26 PLoS Biol

The Jul 26 issue of the PLoS Biol is now up on Pubget (About PLoS Biol): if you're at a subscribing institution, just click the link in the latest link at the home page. (Note you'll only be able to get all the PDFs in the issue if your institution subscribes to Pubget.)

Latest Articles Include:

• Why Biotrophs Can't Live Alone
  - PLoS Biol 9(7):e1001097 (2011)
  
• Different Paths, Same Structure: "Developmental Systems Drift" at Work
  - PLoS Biol 9(7):e1001113 (2011)
  
• Genetic Switch Underlies C. albicans Quick Change Act
  - PLoS Biol 9(7):e1001108 (2011)
  
• SRP and NAC: Guiding a Nascent Protein's First Steps
  - PLoS Biol 9(7):e1001103 (2011)
  
• Darwin in Mind: New Opportunities for Evolutionary Psychology
  - PLoS Biol 9(7):e1001109 (2011)
  Evolutionary Psychology (EP) views the human mind as organized into many modules, each underpinned by psychological adaptations designed to solve problems faced by our Pleistocene ancestors. We argue that the key tenets of the established EP paradigm require modification in the light of recent findings from a number of disciplines, including human genetics, evolutionary biology, cognitive neuroscience, developmental psychology, and paleoecology. For instance, many human genes have been subject to recent selective sweeps; humans play an active, constructive role in co-directing their own development and evolution; and experimental evidence often favours a general process, rather than a modular account, of cognition. A redefined EP could use the theoretical insights of modern evolutionary biology as a rich source of hypotheses concerning the human mind, and could exploit novel methods from a variety of adjacent research fields.
• The Reality of Pervasive Transcription
  - PLoS Biol 9(7):e1000625 (2011)
  
• Response to "The Reality of Pervasive Transcription"
  - PLoS Biol 9(7):e1001102 (2011)
  
• Darwin's Theory of Descent with Modification, versus the Biblical Tree of Life
  - PLoS Biol 9(7):e1001096 (2011)
  
• Horace Judson (1931–2011)
  - PLoS Biol 9(7):e1001104 (2011)
  
• Invertebrate Post-Segregation Distorters: A New Embryo-Killing Gene
  - PLoS Biol 9(7):e1001114 (2011)
  Cytoplasmic incompatibility induced by inherited intracellular bacteria of arthropods, and Medea elements found in flour beetles, are both forms of postsegregation distortion involving the killing of embryos in order to increase the ratio of progeny that inherit them. The recently described peel-zeel element of Caenorhabditis elegans also uses this mechanism; like Medea the genes responsible are in the nuclear genome but it shares a paternal mode of action with the bacteria. The peel-1 gene has now been shown to encode a potent toxin that is delivered by sperm, and rescued by zygotic transcription of the linked zeel-1. The predominance of self-fertilization in C. elegans has produced an unusual distribution pattern for a selfish genetic element; further population and functional studies will shed light on its evolution. The element might also have potential for use in disease control.
• Nothing Iffy about HIF in the Hypothalamus
  - PLoS Biol 9(7):e1001116 (2011)
  Two crucial biological processes are (1) the sensing and coordination of responses to low oxygen levels and (2) the control of food intake and energy expenditure. The hypoxia-inducible factor (HIF) family of proteins is known to regulate responses to low oxygen, whereas neuropeptides derived from proopiomelanocortin (POMC) are implicated in the control of food intake and energy expenditure. It is now becoming apparent that these two apparently disparate processes may be linked, with the exciting discovery that HIF proteins can act in the brain to regulate food intake and energy expenditure as reported in the current issue of PLoS Biology. This primer discusses the traditional role of HIF proteins in terms of responding to oxygen levels in the periphery and also their new role in coordinating responses to nutrients in the brain through regulation of POMC.
• Modernizing Reference Genome Assemblies
  - PLoS Biol 9(7):e1001091 (2011)
  
• Gene Gain and Loss during Evolution of Obligate Parasitism in the White Rust Pathogen of Arabidopsis thaliana
  - PLoS Biol 9(7):e1001094 (2011)
  Biotrophic eukaryotic plant pathogens require a living host for their growth and form an intimate haustorial interface with parasitized cells. Evolution to biotrophy occurred independently in fungal rusts and powdery mildews, and in oomycete white rusts and downy mildews. Biotroph evolution and molecular mechanisms of biotrophy are poorly understood. It has been proposed, but not shown, that obligate biotrophy results from (i) reduced selection for maintenance of biosynthetic pathways and (ii) gain of mechanisms to evade host recognition or suppress host defence. Here we use Illumina sequencing to define the genome, transcriptome, and gene models for the obligate biotroph oomycete and Arabidopsis parasite, Albugo laibachii. A. laibachii is a member of the Chromalveolata, which incorporates Heterokonts (containing the oomycetes), Apicomplexa (which includes human parasites like Plasmodium falciparum and Toxoplasma gondii), and four other taxa. From comparisons with othe! r oomycete plant pathogens and other chromalveolates, we reveal independent loss of molybdenum-cofactor-requiring enzymes in downy mildews, white rusts, and the malaria parasite P. falciparum. Biotrophy also requires "effectors" to suppress host defence; we reveal RXLR and Crinkler effectors shared with other oomycetes, and also discover and verify a novel class of effectors, the "CHXCs", by showing effector delivery and effector functionality. Our findings suggest that evolution to progressively more intimate association between host and parasite results in reduced selection for retention of certain biosynthetic pathways, and particularly reduced selection for retention of molybdopterin-requiring biosynthetic pathways. These mechanisms are not only relevant to plant pathogenic oomycetes but also to human pathogens within the Chromalveolata.
• A Novel Sperm-Delivered Toxin Causes Late-Stage Embryo Lethality and Transmission Ratio Distortion in C. elegans
  - PLoS Biol 9(7):e1001115 (2011)
  The evolutionary fate of an allele ordinarily depends on its contribution to host fitness. Occasionally, however, genetic elements arise that are able to gain a transmission advantage while simultaneously imposing a fitness cost on their hosts. We previously discovered one such element in C. elegans that gains a transmission advantage through a combination of paternal-effect killing and zygotic self-rescue. Here we demonstrate that this element is composed of a sperm-delivered toxin, peel-1, and an embryo-expressed antidote, zeel-1. peel-1 and zeel-1 are located adjacent to one another in the genome and co-occur in an insertion/deletion polymorphism. peel-1 encodes a novel four-pass transmembrane protein that is expressed in sperm and delivered to the embryo via specialized, sperm-specific vesicles. In the absence of zeel-1, sperm-delivered PEEL-1 causes lethal defects in muscle and epidermal tissue at the 2-fold stage of embryogenesis. zeel-1 is expressed transiently ! in the embryo and encodes a novel six-pass transmembrane domain fused to a domain with sequence similarity to zyg-11, a substrate-recognition subunit of an E3 ubiquitin ligase. zeel-1 appears to have arisen recently, during an expansion of the zyg-11 family, and the transmembrane domain of zeel-1 is required and partially sufficient for antidote activity. Although PEEL-1 and ZEEL-1 normally function in embryos, these proteins can act at other stages as well. When expressed ectopically in adults, PEEL-1 kills a variety of cell types, and ectopic expression of ZEEL-1 rescues these effects. Our results demonstrate that the tight physical linkage between two novel transmembrane proteins has facilitated their co-evolution into an element capable of promoting its own transmission to the detriment of organisms carrying it.
• Antagonism of LIN-17/Frizzled and LIN-18/Ryk in Nematode Vulva Induction Reveals Evolutionary Alterations in Core Developmental Pathways
  - PLoS Biol 9(7):e1001110 (2011)
  Most diversity in animals and plants results from the modification of already existing structures. Many organ systems, for example, are permanently modified during evolution to create developmental and morphological diversity, but little is known about the evolution of the underlying developmental mechanisms. The theory of developmental systems drift proposes that the development of conserved morphological structures can involve large-scale modifications in their regulatory mechanisms. We test this hypothesis by comparing vulva induction in two genetically tractable nematodes, Caenorhabditis elegans and Pristionchus pacificus. Previous work indicated that the vulva is induced by epidermal growth factor (EGF)/RAS and WNT signaling in Caenorhabditis and Pristionchus, respectively. Here, we show that the evolution of vulva induction involves major molecular alterations and that this shift of signaling pathways involves a novel wiring of WNT signaling and the acquisition o! f novel domains in otherwise conserved receptors in Pristionchus vulva induction. First, Ppa-LIN-17/Frizzled acts as an antagonist of WNT signaling and suppresses the ligand Ppa-EGL-20 by ligand sequestration. Second, Ppa-LIN-18/Ryk transmits WNT signaling and requires inhibitory SH3 domain binding motifs, unknown from Cel-LIN-18/Ryk. Third, Ppa-LIN-18/Ryk signaling involves Axin and β-catenin and Ppa-axl-1/Axin is epistatic to Ppa-lin-18/Ryk. These results confirm developmental system drift as an important theory for the evolution of organ systems and they highlight the significance of protein modularity in signal transduction and the dynamics of signaling networks.
• Generation of Healthy Mice from Gene-Corrected Disease-Specific Induced Pluripotent Stem Cells
  - PLoS Biol 9(7):e1001099 (2011)
  Using the murine model of tyrosinemia type 1 (fumarylacetoacetate hydrolase [FAH] deficiency; FAH−/− mice) as a paradigm for orphan disorders, such as hereditary metabolic liver diseases, we evaluated fibroblast-derived FAH−/−-induced pluripotent stem cells (iPS cells) as targets for gene correction in combination with the tetraploid embryo complementation method. First, after characterizing the FAH−/− iPS cell lines, we aggregated FAH−/−-iPS cells with tetraploid embryos and obtained entirely FAH−/−-iPS cell–derived mice that were viable and exhibited the phenotype of the founding FAH−/− mice. Then, we transduced FAH cDNA into the FAH−/−-iPS cells using a third-generation lentiviral vector to generate gene-corrected iPS cells. We could not detect any chromosomal alterations in these cells by high-resolution array CGH analysis, and after their aggregation with tetraploid embryos, we obtained fully iPS cell–derived healthy mice with an as! tonishing high efficiency for full-term development of up to 63.3%. The gene correction was validated functionally by the long-term survival and expansion of FAH-positive cells of these mice after withdrawal of the rescuing drug NTBC (2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione). Furthermore, our results demonstrate that both a liver-specific promoter (transthyretin, TTR)-driven FAH transgene and a strong viral promoter (from spleen focus-forming virus, SFFV)-driven FAH transgene rescued the FAH-deficiency phenotypes in the mice derived from the respective gene-corrected iPS cells. In conclusion, our data demonstrate that a lentiviral gene repair strategy does not abrogate the full pluripotent potential of fibroblast-derived iPS cells, and genetic manipulation of iPS cells in combination with tetraploid embryo aggregation provides a practical and rapid approach to evaluate the efficacy of gene correction of human diseases in mouse models.
• Hypoxia-Inducible Factor Directs POMC Gene to Mediate Hypothalamic Glucose Sensing and Energy Balance Regulation
  - PLoS Biol 9(7):e1001112 (2011)
  Hypoxia-inducible factor (HIF) is a nuclear transcription factor that responds to environmental and pathological hypoxia to induce metabolic adaptation, vascular growth, and cell survival. Here we found that HIF subunits and HIF2α in particular were normally expressed in the mediobasal hypothalamus of mice. Hypothalamic HIF was up-regulated by glucose to mediate the feeding control of hypothalamic glucose sensing. Two underlying molecular pathways were identified, including suppression of PHDs by glucose metabolites to prevent HIF2α degradation and the recruitment of AMPK and mTOR/S6K to regulate HIF2α protein synthesis. HIF activation was found to directly control the transcription of POMC gene. Genetic approach was then employed to develop conditional knockout mice with HIF inhibition in POMC neurons, revealing that HIF loss-of-function in POMC neurons impaired hypothalamic glucose sensing and caused energy imbalance to promote obesity development. The metabolic e! ffects of HIF in hypothalamic POMC neurons were independent of leptin signaling or pituitary ACTH pathway. Hypothalamic gene delivery of HIF counteracted overeating and obesity under conditions of nutritional excess. In conclusion, HIF controls hypothalamic POMC gene to direct the central nutrient sensing in regulation of energy and body weight balance.
• Synaptic Transmission from Horizontal Cells to Cones Is Impaired by Loss of Connexin Hemichannels
  - PLoS Biol 9(7):e1001107 (2011)
  In the vertebrate retina, horizontal cells generate the inhibitory surround of bipolar cells, an essential step in contrast enhancement. For the last decades, the mechanism involved in this inhibitory synaptic pathway has been a major controversy in retinal research. One hypothesis suggests that connexin hemichannels mediate this negative feedback signal; another suggests that feedback is mediated by protons. Mutant zebrafish were generated that lack connexin 55.5 hemichannels in horizontal cells. Whole cell voltage clamp recordings were made from isolated horizontal cells and cones in flat mount retinas. Light-induced feedback from horizontal cells to cones was reduced in mutants. A reduction of feedback was also found when horizontal cells were pharmacologically hyperpolarized but was absent when they were pharmacologically depolarized. Hemichannel currents in isolated horizontal cells showed a similar behavior. The hyperpolarization-induced hemichannel current was s! trongly reduced in the mutants while the depolarization-induced hemichannel current was not. Intracellular recordings were made from horizontal cells. Consistent with impaired feedback in the mutant, spectral opponent responses in horizontal cells were diminished in these animals. A behavioral assay revealed a lower contrast-sensitivity, illustrating the role of the horizontal cell to cone feedback pathway in contrast enhancement. Model simulations showed that the observed modifications of feedback can be accounted for by an ephaptic mechanism. A model for feedback, in which the number of connexin hemichannels is reduced to about 40%, fully predicts the specific asymmetric modification of feedback. To our knowledge, this is the first successful genetic interference in the feedback pathway from horizontal cells to cones. It provides direct evidence for an unconventional role of connexin hemichannels in the inhibitory synapse between horizontal cells and cones. This is an imp! ortant step in resolving a long-standing debate about the unus! ual form of (ephaptic) synaptic transmission between horizontal cells and cones in the vertebrate retina.
• Formation of the Long Range Dpp Morphogen Gradient
  - PLoS Biol 9(7):e1001111 (2011)
  The TGF-β homolog Decapentaplegic (Dpp) acts as a secreted morphogen in the Drosophila wing disc, and spreads through the target tissue in order to form a long range concentration gradient. Despite extensive studies, the mechanism by which the Dpp gradient is formed remains controversial. Two opposing mechanisms have been proposed: receptor-mediated transcytosis (RMT) and restricted extracellular diffusion (RED). In these scenarios the receptor for Dpp plays different roles. In the RMT model it is essential for endocytosis, re-secretion, and thus transport of Dpp, whereas in the RED model it merely modulates Dpp distribution by binding it at the cell surface for internalization and subsequent degradation. Here we analyzed the effect of receptor mutant clones on the Dpp profile in quantitative mathematical models representing transport by either RMT or RED. We then, using novel genetic tools, experimentally monitored the actual Dpp gradient in wing discs containing rec! eptor gain-of-function and loss-of-function clones. Gain-of-function clones reveal that Dpp binds in vivo strongly to the type I receptor Thick veins, but not to the type II receptor Punt. Importantly, results with the loss-of-function clones then refute the RMT model for Dpp gradient formation, while supporting the RED model in which the majority of Dpp is not bound to Thick veins. Together our results show that receptor-mediated transcytosis cannot account for Dpp gradient formation, and support restricted extracellular diffusion as the main mechanism for Dpp dispersal. The properties of this mechanism, in which only a minority of Dpp is receptor-bound, may facilitate long-range distribution.
• Hyphal Development in Candida albicans Requires Two Temporally Linked Changes in Promoter Chromatin for Initiation and Maintenance
  - PLoS Biol 9(7):e1001105 (2011)
  Phenotypic plasticity is common in development. For Candida albicans, the most common cause of invasive fungal infections in humans, morphological plasticity is its defining feature and is critical for its pathogenesis. Unlike other fungal pathogens that exist primarily in either yeast or hyphal forms, C. albicans is able to switch reversibly between yeast and hyphal growth forms in response to environmental cues. Although many regulators have been found involved in hyphal development, the mechanisms of regulating hyphal development and plasticity of dimorphism remain unclear. Here we show that hyphal development involves two sequential regulations of the promoter chromatin of hypha-specific genes. Initiation requires a rapid but temporary disappearance of the Nrg1 transcriptional repressor of hyphal morphogenesis via activation of the cAMP-PKA pathway. Maintenance requires promoter recruitment of Hda1 histone deacetylase under reduced Tor1 (target of rapamycin) signal! ing. Hda1 deacetylates a subunit of the NuA4 histone acetyltransferase module, leading to eviction of the NuA4 acetyltransferase module and blockage of Nrg1 access to promoters of hypha-specific genes. Promoter recruitment of Hda1 for hyphal maintenance happens only during the period when Nrg1 is gone. The sequential regulation of hyphal development by the activation of the cAMP-PKA pathway and reduced Tor1 signaling provides a molecular mechanism for plasticity of dimorphism and how C. albicans adapts to the varied host environments in pathogenesis. Such temporally linked regulation of promoter chromatin by different signaling pathways provides a unique mechanism for integrating multiple signals during development and cell fate specification.
• Coupled Evolution of Transcription and mRNA Degradation
  - PLoS Biol 9(7):e1001106 (2011)
  mRNA levels are determined by the balance between transcription and mRNA degradation, and while transcription has been extensively studied, very little is known regarding the regulation of mRNA degradation and its coordination with transcription. Here we examine the evolution of mRNA degradation rates between two closely related yeast species. Surprisingly, we find that around half of the evolutionary changes in mRNA degradation were coupled to transcriptional changes that exert opposite effects on mRNA levels. Analysis of mRNA degradation rates in an interspecific hybrid further suggests that opposite evolutionary changes in transcription and in mRNA degradation are mechanistically coupled and were generated by the same individual mutations. Coupled changes are associated with divergence of two complexes that were previously implicated both in transcription and in mRNA degradation (Rpb4/7 and Ccr4-Not), as well as with sequence divergence of transcription factor bindi! ng motifs. These results suggest that an opposite coupling between the regulation of transcription and that of mRNA degradation has shaped the evolution of gene regulation in yeast.
• Defining the Specificity of Cotranslationally Acting Chaperones by Systematic Analysis of mRNAs Associated with Ribosome-Nascent Chain Complexes
  - PLoS Biol 9(7):e1001100 (2011)
  Polypeptides exiting the ribosome must fold and assemble in the crowded environment of the cell. Chaperones and other protein homeostasis factors interact with newly translated polypeptides to facilitate their folding and correct localization. Despite the extensive efforts, little is known about the specificity of the chaperones and other factors that bind nascent polypeptides. To address this question we present an approach that systematically identifies cotranslational chaperone substrates through the mRNAs associated with ribosome-nascent chain-chaperone complexes. We here focused on two Saccharomyces cerevisiae chaperones: the Signal Recognition Particle (SRP), which acts cotranslationally to target proteins to the ER, and the Nascent chain Associated Complex (NAC), whose function has been elusive. Our results provide new insights into SRP selectivity and reveal that NAC is a general cotranslational chaperone. We found surprising differential substrate specificity ! for the three subunits of NAC, which appear to recognize distinct features within nascent chains. Our results also revealed a partial overlap between the sets of nascent polypeptides that interact with NAC and SRP, respectively, and showed that NAC modulates SRP specificity and fidelity in vivo. These findings give us new insight into the dynamic interplay of chaperones acting on nascent chains. The strategy we used should be generally applicable to mapping the specificity, interplay, and dynamics of the cotranslational protein homeostasis network.
• The Cryo-EM Structure of a Complete 30S Translation Initiation Complex from Escherichia coli
  - PLoS Biol 9(7):e1001095 (2011)
  Formation of the 30S initiation complex (30S IC) is an important checkpoint in regulation of gene expression. The selection of mRNA, correct start codon, and the initiator fMet-tRNAfMet requires the presence of three initiation factors (IF1, IF2, IF3) of which IF3 and IF1 control the fidelity of the process, while IF2 recruits fMet-tRNAfMet. Here we present a cryo-EM reconstruction of the complete 30S IC, containing mRNA, fMet-tRNAfMet, IF1, IF2, and IF3. In the 30S IC, IF2 contacts IF1, the 30S subunit shoulder, and the CCA end of fMet-tRNAfMet, which occupies a novel P/I position (P/I1). The N-terminal domain of IF3 contacts the tRNA, whereas the C-terminal domain is bound to the platform of the 30S subunit. Binding of initiation factors and fMet-tRNAfMet induces a rotation of the head relative to the body of the 30S subunit, which is likely to prevail through 50S subunit joining until GTP hydrolysis and dissociation of IF2 take place. The structure provides insights! into the mechanism of mRNA selection during translation initiation.