Coprinopsis cinerea dioxygenase is an oxygenase forming 10(S)-hydroperoxide of linoleic acid, essential for mushroom alcohol, 1-octen-3-ol, synthesis.

1-Octen-3-ol is a volatile oxylipin found ubiquitously in Basidiomycota and Ascomycota. The biosynthetic pathway forming 1-octen-3-ol from linoleic acid via the linoleic acid 10(S)-hydroperoxide was characterized 40 years ago in mushrooms, yet the enzymes involved are not identified. The dioxygenase 1 and 2 genes (Ccdox1 and Ccdox2) in the mushroom Coprinopsis cinerea contain an N-terminal cyclooxygenase-like heme peroxidase domain and a C-terminal cytochrome P450-related domain. Herein, we show that recombinant CcDOX1 is responsible for dioxygenation of linoleic acid to form the 10(S)-hydroperoxide, the first step in 1-octen-3-ol synthesis, whereas CcDOX2 conceivably forms linoleic acid 8-hydroperoxide. We demonstrate that KO of the Ccdox1 gene suppressed 1-octen-3-ol synthesis, although added linoleic acid 10(S)-hydroperoxide was still efficiently converted. The P450-related domain of CcDOX1 lacks the characteristic Cys heme ligand and the evidence indicates that a second uncharacterized enzyme converts the 10(S)-hydroperoxide to 1-octen-3-ol. Additionally, we determined the gene KO strain (ΔCcdox1) was less attractive to fruit fly larvae, while the feeding behavior of fungus gnats on ΔCcdox1 mycelia showed little difference from that on the mycelia of the WT strain. The proliferation of fungivorous nematodes on ΔCcdox1 mycelia was similar to or slightly worse than that on WT mycelia. Thus, 1-octen-3-ol seems to be an attractive compound involved in emitter-receiver ecological communication in mushrooms.

An unconventional myosin in Drosophila reverses the default handedness in visceral organs.

The internal organs of animals often have left-right asymmetry. Although the formation of the anterior-posterior and dorsal-ventral axes in Drosophila is well understood, left-right asymmetry has not been extensively studied. Here we find that the handedness of the embryonic gut and the adult gut and testes is reversed (not randomized) in viable and fertile homozygous Myo31DF mutants. Myo31DF encodes an unconventional myosin, Drosophila MyoIA (also referred to as MyoID in mammals; refs 3, 4), and is the first actin-based motor protein to be implicated in left-right patterning. We find that Myo31DF is required in the hindgut epithelium for normal embryonic handedness. Disruption of actin filaments in the hindgut epithelium randomizes the handedness of the embryonic gut, suggesting that Myo31DF function requires the actin cytoskeleton. Consistent with this, we find that Myo31DF colocalizes with the cytoskeleton. Overexpression of Myo61F, another myosin I (ref. 4), reverses the handedness of the embryonic gut, and its knockdown also causes a left-right patterning defect. These two unconventional myosin I proteins may have antagonistic functions in left-right patterning. We suggest that the actin cytoskeleton and myosin I proteins may be crucial for generating left-right asymmetry in invertebrates.

Analysis of Pattern Formation by Colored Petri Nets With Quantitative Regulation of Gene Expression Level.

Modeling and simulation are becoming indispensable tools for studying multicellular events such as pattern formation during embryonic development. In this paper, we propose a new approach for analyzing multicellular biological phenomena by combining colored hybrid Petri nets (ColHPNs) with newly devised biological experiments that can control level of a gene quantitatively. With this approach, we analyzed patterning of the boundary cells in the Drosophila large intestine, where one-cell-wide domain of boundary cells differentiate through Delta-Notch signaling. Biological experiments regulating the level of Delta resulted in six distinct patterns of boundary cells correlating with the level of Delta. All these patterns were successfully reproduced by simulation based on ColHPN modeling only by changing the parameter related to the level of Delta. By monitoring the concentration of the active form of Notch in each cell during simulation, it was revealed that these distinct modes of patterning correlate with the fluctuation range of active Notch. Combination of simulation and quantitative manipulation of a gene activity described here is a reliable and powerful approach for analyzing and understanding the patterning process regulated by Notch signaling. This approach can be easily adapted to address other similar pattern formation issues in the systems biology area.

Roles of single-minded in the left-right asymmetric development of the Drosophila embryonic gut.

Many animals have genetically determined left-right (LR) asymmetry of their internal organs. The midline structure of vertebrate embryos has important roles in LR asymmetric development both as the signaling center for LR asymmetry and as a barrier to inappropriate LR signaling across the midline. However, in invertebrates, the functions of the midline in LR asymmetric development are unknown. To elucidate these roles, we studied the involvement of single-minded (sim) in the LR asymmetry of the Drosophila embryonic gut, which develops in a stereotypic, asymmetric manner. sim encodes a bHLH/PAS transcription factor that is required for the development of the ventral midline structure. Here we report that sim was expressed in the midline of the foregut and hindgut primordia. The handedness of the embryonic gut was affected in sim mutant embryos and in embryos overexpressing sim. However, midline-derived events, which involve Slit/Robo and EGFr signaling and direct the development of the tissues adjacent to the midline, did not affect the laterality of this organ, suggesting a crucial role for the midline itself in LR asymmetry. In the sim mutants, the midline structures of the embryonic anal pad were deformed. The mis-expression of sim in the anal-pad primordium induced LR defects. We also found that different portions of the embryonic gut require sim functions at different times for normal LR asymmetry. Our results suggest that the midline structures are involved in the LR asymmetric development of the Drosophila embryonic gut.

GATAe-dependent and -independent expressions of genes in the differentiated endodermal midgut of Drosophila.

Two sequentially-expressed GATA factor genes, serpent (srp) and GATAe, are essential for development of the Drosophila endoderm. The earliest endodermal GATA gene, srp, has been thought to specify the endodermal fate, activating the second GATA gene GATAe, and the latter continues to be expressed in the endodermal midgut throughout life. Previously, we proposed that GATAe establishes and maintains the state of terminal differentiation of the midgut, since some functional genes in the midgut require GATAe activity for their expression. To obtain further evidence of the role of GATAe, we searched for additional genes that are expressed specifically in the midgut in late stages, and examined responses of a total of selected 15 genes to the depletion and overexpression of GATAe. Ten of the 15 genes failed to be expressed in the embryo deficient for GATAe activity, but, the other five genes did not require GATAe. Instead, srp is required for activating the five genes. These observations indicate that GATAe activates a major subset of genes in the midgut, and some other pathway(s) downstream of srp activates other genes.

Drosophila CTLA-2-like protein (D/CTLA-2) inhibits cysteine proteinase 1 (CP1), a cathepsin L-like enzyme.

In this study, we present a propeptide-like cysteine proteinase inhibitor, Drosophila CTLA-2-like protein (D/CTLA-2), a CG10460 (crammer) gene product, with an amino acid sequence significantly similar to the proregion of Drosophila cysteine proteinase 1 (CP1). Recombinant D/CTLA-2, expressed in E. coli, strongly inhibited Bombyx cysteine proteinase (BCP) with a Ki value of 4.7 nM. It also inhibited cathepsins L and H with Ki values of 3.9 (human liver) and 0.43 (rabbit liver) nM, and 7.8 nM (human liver), respectively. Recombinant D/CTLA-2 exhibited low but significant inhibitory activities to cathepsin B with Ki values of 15 nM (human liver) and 110 nM (rat liver), but hardly inhibited papain. We attempted to purify cysteine proteinases inhibited by D/CTLA-2 from total bodies of adult Drosophila. Recombinant D/CTLA-2 significantly inhibited CP1 with a Ki value of 12 nM, indicating that CP1, a cognate enzyme of D/CTLA-2, is a target enzyme of the inhibitor in Drosophila cells. These results indicate that D/CTLA-2 is a selective inhibitor of cathepsin L-like cysteine proteinases similar to other propeptide-like cysteine proteinase inhibitors such as Bombyx cysteine proteinase inhibitors (BCPI) and cytotoxic T-lymphocyte antigen-2 (CTLA-2). D/CTLA-2 was expressed over the whole life cycle of Drosophila. Strong expression was observed in the garland cells and prothoracic gland in the late stages of embryonic development. These results suggest that D/CTLA-2, implicated in intra- and extra-cellular digestive processes, functions in these tissues by suppressing uncontrolled enzymatic activities of CP1.

Modeling and analysis of the Delta-Notch dependent boundary formation in the Drosophila large intestine.

BACKGROUND: The boundary formation in the Drosophila large intestine is widely studied as an important biological problem. It has been shown that the Delta-Notch signaling pathway plays an essential role in the formation of boundary cells. RESULTS: In this paper, we propose a mathematical model for the Delta-Notch dependent boundary formation in the Drosophila large intestine in order to better interpret related experimental findings of this biological phenomenon. To achieve this, we not only perform stability analysis on the model from a theoretical point of view, but also perform numerical simulations to analyze the model with and without noises, the phenotype change with the change of Delta or Notch expression, and the perturbation influences of binding and inhibition parameters on the boundary formation. CONCLUSIONS: By doing all these work, we can assure that our model can better interpret the biological findings related to the boundary formation in the Drosophila large intestine.

Histogenesis of the Os Penis and Os Clitoridis in Rats: (chondrogenesis/bone formation/testosterone/genital tubercle).

The development of the os penis in normal male rats and the os clitoridis in the females treated with testosterone were studied histologically and histochemically. Alcian blue-positive materials, alkaline phosphatase activities and calcium deposit were detected. The os penis of the rats was composed of a proximal segment and a distal one. The proximal segment of the os penis was formed by the fusion of a membrane bone in its distal half and an ossifying hyaline cartilage in its proximal half. The distal segment of the os penis developed as a fibrocartilage bone. Two steps were demonstrated in the development of the os penis: the formation of the rudiments, and the differentiation of the proximal and distal segments. The treatment of newborn females with testosterone induced an os clitoridis homologous to the proximal segment of the os penis. The development of the os clitoridis was compared with that of the os penis.

Organization of Actin Filaments and Zonula Adherens during Somitogenesis in the Chick Embryo: (somitogenesis/actin/zonula adherens/chick embryo).

The organization of F-actin during somitogenesis in the chick embryo was studied by use of rhodamine-conjugated phalloidin and transmission electron microscopy (TEM). Separation of a somite from the segmental plate proceeded simultaneously with the organization of segmental plate cells into a hemispherical epithelial sheet whose open side was directed antero-laterally. At the same time, intense staining of F-actin appeared in the apical surface of the epithelial sheet. Observations by TEM showed that zonulae adherentes associated with many actin filaments increased in the apical region of cells being organized into an epithelial sheet while this junctional apparatus was only sparsely distributed in the segmental plate cells. The hemispherical sheet subsequently closed to form an epithelial vesicle, with increase in curvature of its apical surface, and narrowing of cellular apices. At the same time, the zonulae adherentes and actin filaments in the cellular apices further increased, and many cellular processes formed on the apical surface of the epithelial somites. These findings suggest that segmentation involves organization of zonulae adherentes and a contractile process caused by acin filaments anchored to the zonulae adherentes.

Adenylate kinase 2 deficiency limits survival and regulates various genes during larval stages of Drosophila melanogaster.

Adenylate kinase isozyme 2 (AK2) is located in mitochondrial intermembrane space and regulates energy metabolism by reversibly converting ATP and AMP to 2 ADPs. We previously demonstrated that disruption of the Drosophila melanogaster AK2 gene (Dak2) resulted in growth arrest during the larval stage and subsequent death. Two other groups found that human AK2 mutations cause reticular dysgenesis, a form of severe combined immunodeficiency (SCID) that is associated with severe hematopoietic defects and sensorineural deafness. However, the mechanisms underlying differential outcomes of AK2 deficiency in Drosophila and human systems remain unknown. In this study, effects of tissue-specific inactivation of the Dak2 gene on Drosophila development were analyzed using RNAi-mediated gene knockdown. In addition, to investigate the roles of AK2 in the regulation of gene expression during development, microarray analysis was performed using RNA from first and second instar larvae of Dak2-deficient mutant and wild-type D. melanogaster. Knockdown of Dak2 in all germ layers caused cessation of growth and subsequent death of flies. Microarray analysis revealed that Dak2 deficiency downregulates various genes, particularly those involved in the proteasomal function and in mitochondrial translation machinery. These data indicate that adenine nucleotide interconversion by Dak2 is crucial for developmental processes of Drosophila melanogaster.

Dorsoventral patterning of the Drosophila hindgut is determined by interaction of genes under the control of two independent gene regulatory systems, the dorsal and terminal systems.

Dorsoventral (DV) patterning in the trunk region of Drosophila embryo is established through intricate molecular interactions that regulate Dpp/Scw signaling during the early blastoderm stages. The hindgut of Drosophila, which derives from posterior region of the cellular blastoderm, also shows dorsoventral patterning, being subdivided into distinct dorsal and ventral domains. engrailed (en) is expressed in the dorsal domain, which determines dorsal fate of the hindgut. Here we show that a repressor Brk restricts en expression to the dorsal domain of the hindgut. Expression domain of brk during early blastdermal stages is defined through antagonistic interaction with dpp, and expression domains of dpp and brk in the early blastoderm include prospective hindgut domain. After stage 9, dpp expression in the dorsal domain of the hindgut primordium disappears, but, the brk expression in the ventral domain continues. It was found that Dorsocross (Doc), which is a targe gene of Dpp, is responsible for restricting brk expression to the ventral domain of the hindgut. On the other hand, activation of en is under the control of brachyenteron (byn) that is regulated independently of dpp, brk, and Doc. The cooperative interaction of common DV positional cues with byn during hindgut development represents another aspect of mechanisms of DV patterning in the Drosophila embryo.

The drumstick gene acts cell-non-autonomously and triggers specification of the small intestine in the Drosophila hindgut.

An odd family gene drumstick (drm) encodes a zinc finger protein, and is necessary for the development of the small intestine, an anterior domain of the ectodermal hindgut of Drosophila melanogaster. However, mechanisms that specify the small intestine, as well as gene regulatory pathways leading to transcriptional activation of drm, are still unclear. We found that drm is expressed in two different tissues abutting the anterior end of the hindgut primordium, that is, the posterior-most region of the midgut (endoderm) and basal portion of the Malpighian tubules. A small intestine marker gene, unpaired (upd), begins to be expressed at the anterior-most region of the hindgut primordium that abuts the basal portion of Malpighian tubules, and the upd-positive region expands, resulting in a short tube during stages 11-13. The small intestine develops in both of the mutant embryos, serpent (srp) and Krüppel (Kr), that lack the drm-positive midgut or Malpighian tubules, respectively, while it fails to develop in the Kr srp double-mutant embryos that lack both of the drm-positive tissues. These results demonstrate that drm expressed in the abutting tissues cell-non-autonomously induces development of the small intestine in the hindgut primordium, probably by deploying some extracellular signaling factor. drm expression in the posterior gut region disappears and the small intestine fails to form in tailless (tll) mutant embryos, whereas over-expression of tll causes expansion of drm expression throughout the midgut, inducing a longer small intestine. These results indicate that drm is activated under the control of tll and triggers development of the small intestine cell-non-autonomously through some extracellular signaling.

Adenylate kinase isozyme 2 is essential for growth and development of Drosophila melanogaster.

Adenylate kinases are phylogenetically widespread, highly conserved, and involved in energy metabolism and energy transfer. Of these, adenylate kinase (AK) isozyme 2 is uniquely localized in the mitochondrial intermembrane space and its physiological role remains largely unknown. In this study, we selected Drosophila melanogaster to analyze its role in vivo. AK isozyme cDNAs were cloned and their gene expressions were characterized in D. melanogaster. The deduced amino acid sequences contain highly conserved motifs for P-loop, NMP binding, and LID domains of AKs. In addition, the effects of AK2 gene knockout on phenotype of AK2 mutants were examined using P-element technology. Although homozygous AK2 mutated embryos developed without any visible defects, their growth ceased and they died before reaching the third instar larval stage. Maternally provided AK2 mRNA was detected in fertilized eggs, and weak AK2 activity was observed in first and second instar larvae of the homozygous AK2 mutants, suggesting that maternally provided AK2 is sufficient for embryonic development. Disappearance of AK2 activity during larval stages resulted in growth arrest and eventual death. These results demonstrate that AK2 plays a critical role in adenine nucleotide metabolism in the mitochondrial intermembrane space and is essential for growth in D. melanogaster.

GATA factors as key regulatory molecules in the development of Drosophila endoderm.

Essential roles for GATA factors in the development of endoderm have been reported in various animals. A Drosophila GATA factor gene, serpent (srp, dGATAb, ABF), is expressed in the prospective endoderm, and loss of srp activity causes transformation of the prospective endoderm into ectodermal foregut and hindgut, indicating that srp acts as a selector gene to specify the developmental fate of the endoderm. While srp is expressed in the endoderm only during early stages, it activates a subsequent GATA factor gene, dGATAe, and the latter continues to be expressed specifically in the endoderm throughout life. dGATAe activates various functional genes in the differentiated endodermal midgut. An analogous mode of regulation has been reported in Caenorhabditis elegans, in which a pair of GATA genes, end-1/3, specifies endodermal fate, and a downstream pair of GATA genes, elt-2/7, activates genes in the differentiated endoderm. Functional homology of GATA genes in nature is apparently extendable to vertebrates, because endodermal GATA genes of C. elegans and Drosophila induce endoderm development in Xenopus ectoderm. These findings strongly imply evolutionary conservation of the roles of GATA factors in the endoderm across the protostomes and the deuterostomes.

An endoderm-specific GATA factor gene, dGATAe, is required for the terminal differentiation of the Drosophila endoderm.

GATA factors play an essential role in endodermal specification in both protostomes and deuterostomes. In Drosophila, the GATA factor gene serpent (srp) is critical for differentiation of the endoderm. However, the expression of srp disappears around stage 11, which is much earlier than overt differentiation occurs in the midgut, an entirely endodermal organ. We have identified another endoderm-specific Drosophila GATA factor gene, dGATAe. Expression of dGATAe is first detected at stage 8 in the endoderm, and its expression continues in the endodermal midgut throughout the life cycle. srp is required for expression of dGATAe, and misexpression of srp resulted in ectopic dGATAe expression. Embryos that either lacked dGATAe or were injected with double-stranded RNA (dsRNA) corresponding to dGATAe failed to express marker genes that are characteristic of differentiated midgut. Conversely, overexpression of dGATAe induced ectopic expression of endodermal markers even in the absence of srp activity. Transfection of the dGATAe cDNA also induced endodermal markers in Drosophila S2 cells. These studies provide an outline of the genetic pathway that establishes the endoderm in Drosophila. This pathway is triggered by sequential signaling through the maternal torso gene, a terminal gap gene, huckebein (hkb), and finally, two GATA factor genes, srp and dGATAe.

Drosophila Tbx6-related gene, Dorsocross, mediates high levels of Dpp and Scw signal required for the development of amnioserosa and wing disc primordium.

Regional differentiation along the dorsoventral (DV) axis of the Drosophila embryo primarily depends on a graded BMP signaling activity generated by Decapentaplegic (Dpp) and Screw (Scw). We have identified triplicated Dpp and Scw target genes Dorsocross1, 2 and 3 (Doc1, 2, 3) that have a conserved T-box domain related to the vertebrate Tbx6 subfamily and act redundantly to induce dorsal structures. Doc genes are expressed in the dorsal region in the early blastoderm. After gastrulation, newly expressed Doc appears in a segmental pattern in the ectoderm. This expression correlates spatially with the second phase of Dpp expression in the ectoderm. Doc expression in the early blastoderm is abolished in either dpp or scw mutant embryos, whereas the ectodermal segmented expression depends only on Dpp. Inactivation of Doc genes with RNAi dramatically affected the development of amnioserosa and wing disc primordia, both of which depend on high levels of BMP signaling, although leg disc primordium, which depends on low levels of BMP, remained intact. Doc1 mRNA expressed in Xenopus embryos induced ventral mesoderm, suppressed activin-induced events and induced Xvent genes, which are analogous to the effects of native Tbx6 and its upstream regulator, BMP-4. These results suggest that the Tbx6 subfamily act in the BMP signaling pathway required for embryonic patterning in both animals.

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.

Embryonic development of mouse external genitalia: insights into a unique mode of organogenesis.

The mammalian external genitalia are specialized appendages for efficient copulation, internal fertilization and display marked morphological variation among species. In this paper, we described the embryonic development of mouse genital tubercle (GT), an anlage of the external genitalia utilizing the scanning electron microscope (SEM) analysis. It has been shown that the Distal Urethral Epithelium (DUE) may fulfill an essential role in the outgrowth control of the GT. Our present SEM analysis revealed a small distal protrusion at the tip of the GT of normal embryos as well as some morphological differences between male and female embryonic external genitalia. Previous analysis shows that the teratogenic dose of Retinoic Acid (RA) induces a drastic marformation of the urethral plate, but not gross abnormalities for GT outgrowth. Interestingly, a small distal protrusion at the tip of GT was clearly observed also after RA treatement. Furthermore, we showed that treatment with anti-androgen flutamide resulted in the demasculinization of the GT in males. The unique character of GT development and the sexual dimorphism are discussed.

Boundary formation by notch signaling in Drosophila multicellular systems: experimental observations and gene network modeling by Genomic Object Net.

The Delta-Notch signaling system plays an essential role in various morphogenetic systems of multicellular animal development. Here we analyzed the mechanism of Notch-dependent boundary formation in the Drosophila large intestine, by experimental manipulation of Delta expression and computational modeling and simulation by Genomic Object Net. Boundary formation representing the situation in normal large intestine was shown by the simulation. By manipulating Delta expression in the large intestine, a few types of disorder in boundary cell differentiation were observed, and similar abnormal patterns were generated by the simulation. Simulation results suggest that parameter values representing the strength of cell-autonomous suppression of Notch signaling by Delta are essential for generating two different modes of patterning: lateral inhibition and boundary formation, which could explain how a common gene regulatory network results in two different patterning modes in vivo. Genomic Object Net proved to be a useful and flexible biosimulation system that is suitable for analyzing complex biological phenomena such as patternings of multicellular systems as well as intracellular changes in cell states including metabolic activities, gene regulation, and enzyme reactions.

Novel tissue units of regional differentiation in the gut epithelium of Drosopbila, as revealed by P-element-mediated detection of enhancer.

We analysed spatial patterns of expression of a lacZ reporter gene in the gut of Drosophila larvae that had been transformed with a P-element-lacZ vector to identify regional differences in gene expression. lacZ-positive epithelial cells formed distinct domains with discrete transverse and longitudinal boundaries along the gut tube. Boundaries were often found at sites at which morphological boundaries were not obvious. The gut epithelium was subdivided into 36 compartments by the boundaries. We refer to these novel compartments as "tissue compartments". The lacZ-positive domain of each strain appeared as a single tissue compartment or as a combination of several tissue compartments. The tissue compartment is considered to be a unit of regional differentiation. The spatial organization of the tissue compartments may represent the "floor plan", determined by genes that control the regional differentiation of this nonsegmental organ.