Mutations in somePolycomb group genes ofDrosophila interfere with regulation of segmentation genes

Mutations in severalPolycomb (Pc) group genes cause maternal-effect or zygotic segmentation defects, suggesting thatPc group genes may regulate the segmentation genes ofDrosophila. We show that individuals doubly heterozygous for mutations inpolyhomeotic and six otherPc group genes show gap, pair rule, and segment polarity segmentation defects. We examined double heterozygous combinations ofPc group and segmentation mutations for enhancement of adult and embryonic segmentation defects.Posterior sex combs andpolyhomeotic interact withKrüppel ² and enhance embryonic phenotypes ofhunchback andknirps, andpolyhomeotic enhanceseven-skipped. Surprisingly, flies carrying duplications ofextra sex combs (esc), that were heterozygous for mutations ofeven-skipped (eve), were extremely subvital. Embryos and surviving adults of this genotype showed strong segmentation defects in even-numbered segments. Antibody studies confirm that expression ofeve is suppressed by duplications ofesc. However,esc duplications have no effect on other gap or pair rule genes tested. To our knowledge, this is only the second triplo-abnormal phenotype associated withPc group genes. Duplications of nine otherPc group genes have no detectable effect oneve. Expression ofengrailed (en) was abnormal in the central nervous systems of mostPc group mutants. These results support a role forPc genes in regulation of some segmentation genes, and suggest thatesc may act differently from otherPc group genes.     

Regulation of Krüppel expression in the anlage of the Malpighian tubules in the Drosophila embryo

The expression of most Drosophila segmentation genes is not limited to the early blastoderm stage, when the segmental anlagen are determined. Rather, these genes are often expressed in a variety of organs and tissues at later stages of development. In contrast to the early expression, little is known about the regulatory interactions that govern the later expression patterns. Among other tissues, the central gap gene Krüppel is expressed and required in the anlage of the Malpighian tubules at the posterior terminus of the embryo. We have studied the interaction of Krüppel with other terminal genes. The gap genes tailles and huckebein, which repress Krüppel in the central segmentation domain, activate Krüppel expression in the posterior Malpighian tubule domain. The opposite effect on the posterior Krüppel expression is achieved by the interposition of another factor, the homeotic gene fork head, which is not involved in the control of the central domain. In addition, Krüppel activates different genes in the Malpighian tubules than in the central domain. Thus, both the regulation and the function of Krüppel in the Malpighian tubules differ strikingly from its role in segmentation.     

The <Emphasis Type="Italic">Medicago truncatula SUNN</Emphasis> Gene Encodes a <Emphasis Type="Italic">CLV1</Emphasis>-like Leucine-rich Repeat Receptor Kinase that Regulates Nodule Number and Root Length

Four Medicago truncatula sunn mutants displayed shortened roots and hypernodulation under all conditions examined. The mutants, recovered in three independent genetic screens, all contained lesions in a leucine-rich repeat (LRR) receptor kinase. Although the molecular defects among alleles varied, root length and the extent of nodulation were not significantly different between the mutants. SUNN is expressed in shoots, flowers and roots. Although previously reported grafting experiments showed that the presence of the mutated SUNN gene in roots does not confer an obvious phenotype, expression levels of SUNN mRNA were reduced in sunn-1 roots. SUNN and the previously identified genes HAR1 (Lotus japonicus) and NARK (Glycine max) are orthologs based on gene sequence and synteny between flanking sequences. Comparison of related LRR receptor kinases determined that all nodulation autoregulation genes identified to date are the closest legume relatives of AtCLV1 by sequence, yet sunn, har and nark mutants do not display the fasciated clv phenotype. The M. truncatula region is syntenic with duplicated regions of Arabidopsis chromosomes 2 and 4, none of which harbor CLV1 or any other LRR receptor kinase genes. A novel truncated copy of the SUNN gene lacking a kinase domain, RLP1, is found immediately upstream of SUNN and like SUNN is expressed at a reduced level in sunn-1 roots.     

Gene expression suggests conserved mechanisms patterning the heads of insects and myriapods

Segmentation, i.e. the subdivision of the body into serially homologous units, is one of the hallmarks of the arthropods. Arthropod segmentation is best understood in the fly Drosophila melanogaster. But different from the situation in most arthropods in this species all segments are formed from the early blastoderm (so called long-germ developmental mode). In most other arthropods only the anterior segments are formed in a similar way (so called short-germ developmental mode). Posterior segments are added one at a time or in pairs of two from a posterior segment addition zone. The segmentation mechanisms are not universally conserved among arthropods and only little is known about the genetic patterning of the anterior segments. Here we present the expression patterns of the insect head patterning gene orthologs hunchback (hb), orthodenticle (otd), buttonhead-like (btdl), collier (col), cap-n-collar (cnc) and crocodile (croc), and the trunk gap gene Krüppel (Kr) in the myriapod Glomeris marginata. Conserved expression of these genes in insects and a myriapod suggests that the anterior segmentation system may be conserved in at least these two classes of arthropods. This finding implies that the anterior patterning mechanism already existed in the last common ancestor of insects and myriapods.     

Novel<Emphasis Type="Italic"> eceriferum</Emphasis> mutants in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

We conducted a novel non-visual screen for cuticular wax mutants in Arabidopsis thaliana (L.) Heynh. Using gas chromatography we screened over 1,200 ethyl methane sulfonate (EMS)-mutagenized lines for alterations in the major A. thaliana wild-type stem cuticular chemicals. Five lines showed distinct differences from the wild type and were further analyzed by gas chromatography and scanning electron microscopy. The five mutants were mapped to specific chromosome locations and tested for allelism with other wax mutant loci mapping to the same region. Toward this end, the mapping of the cuticular wax (cer) mutants cer10 to cer20 was conducted to allow more efficient allelism tests with newly identified lines. From these five lines, we have identified three mutants defining novel genes that have been designated CER22, CER23, and CER24. Detailed stem and leaf chemistry has allowed us to place these novel mutants in specific steps of the cuticular wax biosynthetic pathway and to make hypotheses about the function of their gene products.Abbreviations EMS Ethyl methane sulfonate - SEM Scanning electron microscopy - SSLP Simple sequence length polymorphism - WT Wild type     

aproctous,a locus that is necessary for the development of the proctodeum in Drosophila embryos,encodes a homolog of the vertebrate Brachyury gene

The proctodeum of the Drosophila embryo originates from the posterior end of the blastoderm and forms the hindgut. By enhancer-trap mutagenesis, using a P-element-lacZ vector, we identified a mutation that caused degeneration of the proctodeum during shortening of the germ band and named it aproctous (apro). Expression of the lacZ reporter gene, which was assumed to represent expression of the apro gene, began at the cellular blastoderm stage in a ring that encompassed about 10–15% of the egg's length (EL) and included the future proctodeum, anal pads, and posterior-most part of the visceral mesoderm. In later stages, strong expression of lacZ was detected in the developing hindgut and anal pads. Expression continued in the anal pads and epithelium of the hindgut of larvae; the epithelium of the hindgut of the adult fly also expressed lacZ. The spatial patterns of the expression of lacZ in various mutants suggested that the embryonic expression of apro was regulated predominantly by two gap genes, tailless (tll) and huckebein (hkb): tll is necessary for the activation of apro, while hkb suppressed the expression of apro in the region posterior to 10% EL. Cloning and sequencing of the apro cDNA revealed that apro was identical to the T-related gene (Trg) that is a Drosophila homolog of the vertebrate Brachyury gene. apro appears to play a key role in the development of tissues derived from the proctodeum.     

JAK-STAT signalling is required throughout telotrophic oogenesis and short-germ embryogenesis of the beetle Tribolium

In Drosophila, the JAK-STAT signalling pathway regulates a broad array of developmental functions including segmentation and oogenesis. Here we analysed the functions of Tribolium JAK-STAT signalling factors and of Suppressor Of Cytokine Signalling (SOCS) orthologues, which are known to function as negative regulators of JAK-STAT signalling, during telotrophic oogenesis and short-germ embryogenesis. The beetle Tribolium features telotrophic ovaries, which differ fundamentally from the polytrophic ovary of Drosophila. While we found the requirement for JAK-STAT signalling in specifying the interfollicular stalk to be principally conserved, we demonstrate that these genes also have early and presumably telotrophic specific functions. Moreover, we show that the SOCS genes crucially contribute to telotrophic Tribolium oogenesis, as their inactivation by RNAi results in compound follicles. During short-germ embryogenesis, JAK-STAT signalling is required in the maintenance of segment primordia, indicating that this signalling cascade acts in the framework of the segment-polarity network. In addition, we demonstrate that JAK-STAT signalling crucially contributes to early anterior patterning. We posit that this signalling cascade is involved in achieving accurate levels of expression of individual pair-rule and gap gene domains in early embryonic patterning.     

茄科作物青枯菌NADH脱氢酶F亚基在细胞运动和致病性中的功能研究

[目的]劳尔氏菌（Ralstonia solanacearum）在茄科作物上引起严重的细菌性青枯病,本研究旨在发掘青枯劳尔氏菌与致病相关的基因。[方法]利用Tn5转座子构建随机插入突变体,分析生物膜形成、细胞运动和致病性;对有表型变化的突变体,运用TAIL-PCR方法鉴定Tn5插入位点,确定所突变的基因。[结果]以模式菌株GMI000为出发菌,总共获得了400个突变体,其中2个突变体不能形成生物膜,在软琼脂平板上的运动能力下降;接种感病番茄植物,这2个突变体都不能引起萎焉症状。TAIL-PCR结果显示,2个突变体的Tn5插入位点都在NADH脱氢酶F亚基（nuoF）中,距离翻译起始位点分别为103-bp和225-bp。ripAY基因启动子推动的nuoF基因互补载体,完全恢复了2个突变体的表型。[结论]NADH脱氢酶复合物是微生物呼吸电子传递链中的第一步催化酶。我们的结果表明,NADH脱氢酶复合物对R.solanacearum生物膜形成、细胞运动和致病性也有重要作用。     

A requirement for <Emphasis Type="Italic">kit</Emphasis> in embryonic zebrafish melanocyte differentiation is revealed by melanoblast delay

Exploring differences in gene requirements between species can allow us to delineate basic developmental mechanisms, provide insight into patterns of evolution, and explain heterochronic differences in developmental processes. One example of differences in gene requirements between zebrafish and mammals is the requirement of the kit receptor tyrosine kinase in melanocyte development. kit is required for migration, survival and differentiation of all neural crest-derived melanocytes in mammals. In contrast, zebrafish kit is not required for differentiation of embryonic melanocytes during normal development. When melanoblast development in zebrafish embryos is delayed by injecting morpholinos targeted to the mitfa gene, we show that these delayed melanoblasts fail to differentiate in kit mutants. Thus, we show that there is a kit requirement for melanocyte differentiation in zebrafish when melanoblast development is delayed. Furthermore, we show that kit is not involved in maintaining melanocyte precursors through the developmental delay, but instead is required for differentiation of melanocytes after the block on their development is removed. Finally, we suggest there is a heterochronic shift in the onset of melanocyte differentiation between fish and mouse, and developmental delay of melanoblast development in zebrafish removes this heterochronic difference.Edited by D. Tautz     

Grainyhead-like 2 regulates neural tube closure and adhesion molecule expression during neural fold fusion

Defects in closure of embryonic tissues such as the neural tube, body wall, face and eye lead to severe birth defects. Cell adhesion is hypothesized to contribute to closure of the neural tube and body wall; however, potential molecular regulators of this process have not been identified. Here we identify an ENU-induced mutation in mice that reveals a molecular pathway of embryonic closure. Line2F homozygous mutant embryos fail to close the neural tube, body wall, face, and optic fissure, and they also display defects in lung and heart development. Using a new technology of genomic sequence capture and high-throughput sequencing of a 2.5 Mb region of the mouse genome, we discovered a mutation in the grainyhead-like 2 gene (Grhl2). Microarray analysis revealed Grhl2 affects the expression of a battery of genes involved in cell adhesion and E-cadherin protein is drastically reduced in tissues that require Grhl2 function. The tissue closure defects in Grhl2 mutants are similar to that of AP-2α null mutants and AP-2α has been shown to bind to the promoter of E-cadherin. Therefore, we tested for a possible interaction between these genes. However, we find that Grhl2 and AP-2α do not regulate each other's expression, E-cadherin expression is normal in AP-2α mutants during neural tube closure, and Grhl2;AP-2α trans-heterozygous embryos are morphologically normal. Taken together, our studies point to a complex regulation of neural tube fusion and highlight the importance of comparisons between these two models to understand more fully the molecular pathways of embryonic tissue closure.     

Porcupine homolog is required for canonical Wnt signaling and gastrulation in mouse embryos

Wnt signaling plays important roles in development and disease. The X-chromosomal Porcupine homolog gene (Porcn) encodes an evolutionary conserved member of the membrane bound O-acyl transferase (MBOAT) superfamily that has been shown to be required for the palmitoylation and secretion of Wnt3a, a mechanism that has been suggested to be conserved for all mammalian Wnt ligands. PORCN mutations in humans cause Focal Dermal Hypoplasia (FDH), a disorder causing developmental defects in heterozygous females and embryonic lethality in hemizygous males. In this study, Porcn mutant mouse embryonic stem (ES) cells were used to analyze the role of Porcn in mammalian embryonic development. In vitro, we show an exclusive requirement for Porcn in Wnt secreting cells and further, that any of the four Porcn isoforms is sufficient to allow for the secretion of functional Wnt3a. Embryos generated by aggregation of Porcn mutant ES cells with wildtype embryos fail to complete gastrulation in vivo, but remain in an epiblast-like state, similar to Wnt3 and Gpr177/Wls mutants. Consistent with this phenotype, in vitro differentiated mutant ES cells fail to generate endoderm and mesoderm derivatives. Taken together, these data confirm the importance of Porcn for Wnt secretion and gastrulation and suggest that disruption of early development underlies the male lethality of human PORCN mutants.     

Cloning and characterization of the histidine kinase gene<Emphasis Type="Italic"> Dic1</Emphasis> from<Emphasis Type="Italic"> Cochliobolus heterostrophus</Emphasis> that confers dicarboximide resistance and osmotic adaptation

A gene for a putative two-component histidine kinase, which is homologous to os-1 from Neurospora crassa, was cloned and sequenced from the plant-pathogenic fungus Cochliobolus heterostrophus. The predicted protein possessed the conserved histidine kinase domain, the response regulator domain, and six tandem repeats of 92-amino-acids at the N-terminal end that are found in histidine kinases from other filamentous fungi. Introduction of the histidine kinase gene complemented the deficiency of the C. heterostrophus dic1 mutant, suggesting that the Dic1 gene product is a histidine kinase. Dic1 mutants are resistant to dicarboximide and phenylpyrrole fungicides, and they are sensitive to osmotic stress. We previously classified dic1 alleles into three types, based on their phenotypes. To explain the phenotypic differences among the dic1 mutant alleles, we cloned and sequenced the mutant dic1 genes and compared their sequences with that of the wild-type strain. Null mutants for Dic1, and mutants with a deletion or point mutation in the N-terminal repeat region, were highly sensitive to osmotic stress and highly resistant to both fungicides. A single amino acid change within the kinase domain or the regulator domain altered the sensitivity to osmotic stress and conferred moderate resistance to the fungicides. These results suggest that this predicted protein, especially its repeat region, has an important function in osmotic adaptation and fungicide resistance.Communicated by C. A. M. J. J. van den Hondel     

The white-phase-specific gene<Emphasis Type="Italic"> WH11</Emphasis> is not required for white-opaque switching in<Emphasis Type="Italic"> Candida albicans</Emphasis>

Phenotypic switching between white and opaque cells is important for adaptation to different host environments and for mating in the opportunistic fungal pathogen Candida albicans. Genes that are specifically activated in one of the two cell types are likely to be important for their phenotypic characteristics. The WH11 gene is a white-phase-specific gene that has been suggested to be involved in the maintenance of the white-phase phenotype. To elucidate the role of WH11 in white-opaque switching, we constructed mutants of the C. albicans strain WO-1 in which the WH11 gene was deleted. The wh11 mutants were still able to form both white and opaque cells whose cellular and colony phenotypes were indistinguishable from those of the wild type. Deletion of WH11 also did not affect the activation and deactivation of the white-phase-specific WH11 promoter and the opaque-phase-specific OP4 and SAP1 promoters in the appropriate cell type. Finally, switching from the white to the opaque phase and vice versa occurred with the same frequency in wild-type and wh11 mutants. Therefore, the WH11 gene is not required for phenotypic switching, and its protein product seems to have other roles in white cells, which are dispensable after the switch to the opaque phase.Communicated by E. Cerdá-Olmedo     

Tracking down the "head blob": comparative analysis of wingless expression in the developing insect procephalon reveals progressive reduction of embryonic visual system patterning in higher insects

The evolution of larval head morphology in holometabolous insects is characterized by reduction of antennal appendages and the visual system components. Little insight has been gained into molecular developmental changes underlying this morphological diversification. Here we compare the expression of the segment polarity gene wingless (wg) in the pregnathal head of fruit fly, flour beetle and grasshopper embryos. We provide evidence that wg activity contributes to segment border formation, and, subsequently, the separation of the visual system and protocerebrum anlagen in the anterior procephalon. In directly developing insects like grasshopper, seven expression domains are formed during this process. The activation of four of these, which correspond to polar expression pairs in the optic lobe anlagen and the protocerebral ectoderm, has shifted to postembryonic stages in flour beetle and Drosophila. The remaining three domains map to the protocerebral neuroectoderm, and form by disintegration of a large precursor domain in flour beetle and grasshopper. In Drosophila, the precursor domain remains intact, constituting the previously described “head blob”. These data document major changes in the expression of an early patterning gene correlated with the dramatic evolution of embryonic visual system development in the Holometabola.     

Involvement of the nadA gene in formation of G-group aflatoxins in Aspergillus parasiticus

The nadA gene is present at the end of the aflatoxin gene cluster in the genome of Aspergillus parasiticus as well as in Aspergillus flavus. RT-PCR analyses showed that the nadA gene was expressed in an aflatoxin-inducible YES medium, but not in an aflatoxin-non-inducible YEP medium. The nadA gene was not expressed in the aflR gene-deletion mutant, irrespective of the culture medium used. To clarify the nadA gene’s function, we disrupted the gene in aflatoxigenic A. parasiticus. The four nadA-deletion mutants that were isolated commonly accumulated a novel yellow-fluorescent pigment (named NADA) in mycelia as well as in culture medium. When the mutants and the wild-type strain were cultured for 3 days in YES medium, the mutants each produced about 50% of the amounts of G-group aflatoxins that the wild-type strain produced. In contrast, the amounts of B-group aflatoxins did not significantly differ between the mutants and the wild-type strain. The NADA pigment was so unstable that it could non-enzymatically change to aflatoxin G₁ (AFG₁). LC–MS measurement showed that the molecular mass of NADA was 360, which is 32 higher than that of AFG₁. We previously reported that at least one cytosol enzyme, together with two other microsome enzymes, is necessary for the formation of AFG₁ from O-methylsterigmatocystin (OMST) in the cell-free system of A. parasiticus. The present study confirmed that the cytosol fraction of the wild-type A. parasiticus strain significantly enhanced the AFG₁ formation from OMST, whereas the cytosol fraction of the nadA-deletion mutant did not show the same activity. Furthermore, the cytosol fraction of the wild-type strain showed the enzyme activity catalyzing the reaction from NADA to AFG₁, which required NADPH or NADH, indicating that NADA is a precursor of AFG₁; in contrast, the cytosol fraction of the nadA-deletion mutant did not show the same enzyme activity. These results demonstrated that the NadA protein is the cytosol enzyme required for G-aflatoxin biosynthesis from OMST, and that it catalyzes the reaction from NADA to AFG₁, the last step in G-aflatoxin biosynthesis.     

低H~+_-ATPase活性植物乳杆菌突变菌筛选及基因表达的相对定量分析

【目的】筛选H~+_-ATPase活性降低的植物乳杆菌突变菌,比较其与亲本菌基因表达水平的差异,进一步探索H~+_-ATPase的调控机制。【方法】利用硫酸新霉素诱变、筛选突变菌,并对亲本菌(ZUST)和突变菌(ZUST-1、ZUST-2)进行生长、产酸能力及H~+_-ATPase活性的测定。分别提取亲本菌和突变菌的基因组DNA,扩增H~+_-ATPase全部编码基因并测序。通过荧光定量PCR对H~+_-ATPase全部编码基因进行相对定量分析。【结果】突变菌的生长和产酸能力均低于亲本菌,突变菌ZUST-1和ZUST-2的H~+_-ATPase活性比亲本菌分别降低了10.1%和28.8%。突变菌ZUST-1和ZUST-2的atp A基因均有22个位点发生突变,而ZUST-2的atp C基因有6个位点发生突变。突变菌ZUST-1和ZUST-2的atp A在对数期基因表达水平分别比亲本菌ZUST下调了41.1%和35.7%,在稳定期分别下调了43.6%和14.2%;ZUST-1的atp C基因在对数期的表达水平比ZUST略高,在稳定期比ZUST上调了30%,而ZUST-2的atp C基因未表达。【结论】突变菌H~+_-ATPase活性减弱会导致其全部编码基因在稳定期表达水平上调(除ZUST-2的atp C不表达外),而且atp A和atp C基因突变导致的基因表达水平的差异是影响H~+_-ATPase活性的主要因素,此研究结果为进一步研究植物乳杆菌中H~+_-ATPase的调控机制奠定了基础。     

The<Emphasis Type="Italic"> GAOLAOZHUANGREN2</Emphasis> gene is required for normal glucose response and development of<Emphasis Type="Italic"> Arabidopsis</Emphasis>

Recent studies of glucose (Glc) sensing and signaling have revealed that Glc acts as a critical signaling molecule in higher plants. Several Glc sensing-defective Arabidopsis mutants have been characterized in detail, and the corresponding genes encoding Glc-signaling proteins have been isolated. However, the full complexity of Glc signaling in higher plants is not yet fully understood. Here, we report the identification and characterization of a new Glc-insensitive mutant, gaolaozhuangren2 (glz2), which was isolated from transposon mutagenesis experiments in Arabidopsis. In addition to its insensitivity to Glc, the glz2 plant exhibits several developmental defects such as short stature with reduced apical dominance, short roots, small and dark-green leaves, late flowering and female sterility. Treatment with 4% Glc blocked expression of the OE33 gene in wild-type plants, whereas expression of this gene was unchanged in the glz2 mutant plants. Taken together, our results suggest that the GLZ2 gene is required for normal glucose response and development of Arabidopsis.Mingjie Chen and Xiaoxiang Xia contributed equally to this work.     

Ectopic expression of Nkx2.5 suppresses the formation of the sinoatrial node in mice

The sinoatrial node (SAN), functionally known as the pacemaker, regulates the cardiac rhythm or heartbeat. Several genes are expressed in the developing SAN and form a genetic network regulating the fate of the SAN cells. The short stature homeobox gene Shox2 is an important player in the SAN genetic network by regulating the expression of different cardiac conduction molecular markers including the early cardiac differentiation marker Nkx2.5. Here we report that the expression patterns of Shox2 and Nkx2.5 are mutually exclusive from the earliest stages of the venous pole and the SAN formation. We show that tissue specific ectopic expression of Shox2 in the developing mouse heart downregulates the expression of Nkx2.5 and causes cardiac malformations; however, it is not sufficient to induce a SAN cell fate switch in the working myocardium. On the other hand, tissue specific overexpression of Nkx2.5 in the heart leads to severe hypoplasia of the SAN and the venous valves, dis-regulation of the SAN genetic network, and change of the SAN cell fate into working myocardium, and causes embryonic lethality, recapitulating the phenotypes including bradycardia observed in Shox2^−/− mutants. These results indicate that Nkx2.5 activity is detrimental to the normal formation of the SAN. Taken together, our results demonstrate that Shox2 downregulation of Nkx2.5 is essential for the proper development of the SAN and that Shox2 functions to shield the SAN from becoming working myocardium by acting upstream of Nkx2.5.