Ryutaro Murakami

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.

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

SUMMARY 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.

Regulation of pattern formation in the Drosophila hindgut by wg, hh, dpp, and en.

The hindgut of the Drosophila embryo is subdivided into three major domains, the small intestine, large intestine, and rectum, each of which is characterized by specific gene expression. Here we show that the expression of wingless (wg), hedgehog (hh), decapentaplegic (dpp), and engrailed (en) corresponds to the generation or growth of particular domains of the hindgut. wg, expressed in the prospective anal pads, is necessary for activation of hh in the adjacent prospective rectum. hh is expressed in the prospective rectum, which forms anteriorly to the anal pads, and necessary for the expression of dpp at the posterior end of the adjacent large intestine. wg and hh are also necessary for the development of their own expression domains, anal pads, and rectum, respectively. dpp, in turn, causes the growth of the large intestine, promoting DNA replication. en defines the dorsal domain of the large intestine, repressing dpp in this domain. A one-cell-wide domain, which delineates the anterior and posterior borders of the large intestine and its internal border between the dorsal and ventral domains, is induced by the activity of en. We propose a model for the gene regulatory pathways leading to the subdivision of the hindgut into domains.

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.

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.

Left-right asymmetry in Drosophila melanogaster gut development

While left–right (LR) asymmetric morphogenesis is common to various animal species, there have been no systematic studies of the LR asymmetry of body structures of Drosophila melanogaster. In the present paper the LR asymmetric development of the Drosophila gut is described, in which three major parts, the foregut, midgut and hindgut, show almost invariant LR asymmetry. The asymmetry is generated by a twist of each part in particular orientations, resulting in a left‐handed (sinistral) convolution as a whole. The frequency of spontaneous reversal of LR orientations is very low (< 0.6%) and reversal of each part of the gut occurs independently. The bicoid mutation causes duplication of the posterior half of the gut, essentially keeping the left‐handed twist, suggesting that the LR asymmetry may depend on some intrinsic nature of the cells or tissues rather than a graded distribution of morphogens in the egg. The handedness of particular gut parts was randomized or became symmetric in mutants of brachyenteron, huckebein and patched, suggesting that different gene pathways can interfere in determining LR asymmetry of the gut. It is noteworthy that all of these genes are expressed LR symmetrically.

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 eggs 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.

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.

Morphology of the Digestive System in the Wood-Feeding Termite Nasutitermes takasagoensis (Shiraki) (Isoptera: Termitidae)

Abstract The morphologies of epithelial cells throughout the alimentary canal of the wood-feeding termite Nasutitermes takasagoensis (Shiraki) were examined. The digestive tract consists of four principal portions, which are the foregut, the midgut, the mixed segment and the hindgut. The midgut epithelium is primarily composed of columnar cells and degenerative cells. Most columnar cells have one or more autophagic vacuoles at cell apexes, suggesting a rapid turnover of the midgut cells. In the mixed segment, the mesenteric epithelium occupies half of the gut wall and the proctodeal epithelium covers the remaining wall. Extensive invaginations of the basal membrane are characteristic of the mesenteric columnar cells, suggesting active transport of an ionic fluid. The hindgut can be divided into five segments, the first of which is a simple tube lined with a thick cuticle, termed the first proctodeal segment. The epithelium of the third segment, the paunch, consists of cuboidal cells, which are covered by multiple cuticular layers. The apical membrane of these epithelial cells forms regular invaginations, suggesting that they have an absorptive function. In the anterior paunch, numerous spirochetes are found adhered to the gut wall. Our observations indicate that termites such as N. takasagoensis appear to have developed structures that enable more efficient interactions with intestinal microorganisms, particularly by the elongation and differentiation of the hindgut and the creation of the mixed segment.

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.