Yoshiki Hotta

Tetrodotoxin and Manganese Ions: Effects on Electrical Activity and Tension in Taenia Coli of Guinea Pig

Tetrodotoxin, at concentrations up to 5 x 10-6 gram per milliliter, has no effect on the spontaneous discharge in the smooth muscle of taenia coli. However, the spontaneous discharge is abolished by Mn++ at a concentration of 0.5 millimole per liter. The contraction induced by immersing the muscle in isotonic KCl solution is also suppressed in the presence of Mn++. Because Mn++ is a specific suppressor of the spike induced by Ca++ and tetrodotoxin is an inhibitor of the spike induced by Na+, we suggest that Ca++ is a charge carrier in the production of spike potential in the smooth muscle and that the entry of intervening Ca++ through the membrane acts as a trigger for the contraction of smooth muscle.

Isolation and characterization of a gene for a ryanodine receptor/calcium release channel in Drosophila melanogaster

The nucleotide sequence of a 25.7 kilobase Drosophila melanogaster genomic DNA segment containing a gene for a ryanodine receptor/calcium release channel homologue has been determined. Computer analysis and partial cDNA cloning revealed 26 exons comprising the protein‐coding sequence in this gene. The predicted protein is homologous in amino acid sequence and shares characteristic structural features with the mammalian ryanodine receptors. In blot hybridization analysis, a ~16 kilobase RNA species was identified abundantly in a 6–12 h embryo as the transcript from this gene. In situ hybridization to polytene chromosomes indicated that this gene locates at band position 44F on the second chromosome.

Genetic and behavioral studies of female sex appeal inDrosophila

The sex appeal of aDrosophila melanogaster female is defined here as the stimulus (or set of stimuli) which induces wing vibration in courting males. A quantitative measure of sex appeal is the cumulative duration of wing vibration induced by a given female averaged over several consecutive test intervals using different standardized male testers (sex appeal parameter, SAP). By use of SAP, both males and females are found to have the same amount of sex appeal on the first day after eclosion. However, males rapidly lose it by the next day, so that mature males become distinct from females. We report the ontogeny of the males response to sex appeal. By the SAP method, we also demonstrate that the males response is dependent on his previous encounter with females. The sex appeal of 287 gynandromorphs was examined in order to localize the sex appeal focus by means of blastoderm fate mapping. Most mosaic flies were classified as either positive (femalelike, with high SAPs) or negative (malelike, with SAPs of zero). Sixteen percent of the gynandromorphs had intermediate levels of SAP, inducing only short vibrations, a response which males rarely give to normal females. Assuming that the gynanders with such intermediate sex appeal must have both female and male foci, distances to the foci from external landmarks were calculated. The center of the focus seems to be an internal structure mapping to the ventroposterior region of the blastoderm fate map, close to the primordia of the anterior sternites. The focus might include a large mesodermal area, but only part of it must have a female genotype for the sex appeal to be expressed. A possible involvement of the fat bodies in production of the sex appeal stimulus is discussed in relation to these findings. Consistent with this conclusion is the fact that females whose abdomens were amputated still retain enough sex appeal to induce male wing vibrations.

Actin gene mutations in Drosophila; heat shock activation in the indirect flight muscles.

We have identified four mutations affecting the actin III isoform in the indirect flight muscles (IFM) of Drosophila. One mutation does not produce any protein product, and three direct the synthesis of electrophoretic variants of actin. Complementation tests and recombination mapping indicate that all mutations are alleles of an actin gene at chromosomal band 88F (act88F gene). The effect of these mutations is restricted to the IFM. We conclude that the act88F gene is expressed only in the IFM to encode actin III, which is its major isoform. In two of the actin mutants, heat shock proteins are constitutively expressed in the IFM. Genetic evidence strongly suggest that this anomaly is primarily caused by the mutations in the act88F structural gene.

Germline transformation with Drosophila mutant actin genes induces constitutive expression of heat shock genes.

Two Drosophila mutants KM75 and HH5, which are mutated in the act88F actin gene specific for the indirect flight muscles (IFM), synthesize heat shock proteins (hsps) constitutively in a tissue-specific manner. We have introduced cloned mutant act88F genes into a strain containing the wild-type act88F allele by P-element-mediated transformation. Flies transformed with a 4.05 kb KM75 act88F gene fragment encoding the p42 actin variant express both p42 and hsps specifically in the IFM. Using normal/mutant chimeric genes, the mutation sites of KM75 and HH5 were mapped within the sequence encoding the last 72 amino acids of actin. An in vitro mutated gene encoding a protein that lacks the 72 carboxy-terminal amino acids also induces constitutive hsp synthesis.

A genetic study of inositol trisphosphate involvement in phototransduction using Drosophila mutants

Phosphatidylinositol 4,5-bisphosphate phosphodiesterase activity was found to be almost absent in the compound eyes of Drosophila visual mutant, norpA (no receptor potentials A). We compared the enzyme activities among independently isolated norpA alleles, each having a different degree of the vision defect. A close correlation was found between the size of receptor potentials (electroretinogram), phototactic behavior and the enzyme activity. The correlation exists not only among alleles, but also in a single, temperature-dependent allele under different temperature; the enzyme activity of flies kept at 18 degrees C (phototactic) was about five times higher than that of the ones kept at 28 degrees C (blind). These results suggest that hydrolysis of phosphatidylinositol 4,5-bisphosphate is involved in phototransduction process in Drosophila eyes.

Ocular and Cerebellar Defects in Zebrafish Induced by Overexpression of the LIM Domains of the Islet-3 LIM/Homeodomain Protein

Islet-3 is an LIM/homeodomain protein that is expressed specifically in the eyes and the presumptive tectum in the central nervous system of zebrafish (Danio rerio) embryos. Overexpression of the protein (LIM(Isl-3)) consisting only of the Islet-3 LIM domains in embryos specifically prevented formation of the optic vesicles; caused abnormal termination of the expression of wnt1, engrailed2, and pax2 in the mesencephalic and metencephalic region between 14 hr and 20 hr postfertilization; and severely impaired morphogenetic movement in this region between 20 hr and 26 hr, which should normally lead to formation of the cerebellar primordium. Such defects were all rescued by simultaneous overexpression of Islet-3, suggesting that LIM(Isl-3) acted as a specific dominant-negative variant of Islet-3. These data, combined with the results of mosaic analyses, suggest that Islet-3 is activated by putative LIM-binding cofactors and functions to promote evagination of the optic vesicles and to maintain reciprocal interaction between the mesencephalon and the mesencephalic-metencephalic boundary essential for normal development of this region.

Mammalian Gcm genes induce Hes5 expression by active DNA demethylation and induce neural stem cells

Signaling mediated by Notch receptors is crucial for the development of many organs and the maintenance of various stem cell populations. The activation of Notch signaling is first detectable by the expression of an effector gene, Hes5, in the neuroepithelium of mouse embryos at embryonic day (E) 8.0–8.5, and this activation is indispensable for the generation of neural stem cells. However, the molecular mechanism by which Hes5 expression is initiated in stem-producing cells remains unknown. We found that mammalian Gcm1 and Gcm2 (glial cells missing 1 and 2) are involved in the epigenetic regulation of Hes5 transcription by DNA demethylation independently of DNA replication. Loss of both Gcm genes and subsequent lack of Hes5 upregulation in the neuroepithelium of E7.5–8.5 Gcm1−/−; Gcm2−/− mice resulted in the impaired induction of neural stem cells. Our data suggest that Hes5 expression is serially activated first by Gcms and later by the canonical Notch pathway.

Alteration of cell fate by ectopic expression of Drosophila glial cells missing in non-neural cells

Abstract The glial cells missing (gcm) gene encodes an essential transcription factor that converts neuronal precursor cells to glial fate in the Drosophila nervous system. In this study, we tested effects of gcm ectopic expression on fate of non-neural cells. When gcm expression was continuously induced in epidermal cells from around stage 9, these cells started to exhibit mesenchymal cell morphology at stage 13, which was preceded by the onset of expression of Repo, a glial marker. The morphological change was coincident with loss of expression of an epidermal cell-adhesion molecule. In addition to the epidermis, fate of mesodermal cells was also affected by gcm ectopic expression. These findings suggest that gcm can convert gene expression and cell morphology even outside the neuroectoderm.

Molecular characterization of mutant actin genes which induce heat-shock proteins in Drosophila flight muscles.

Heat‐shock proteins (hsps) are constitutively induced by the mutant actins in the Drosophila indirect flight muscles (IFM). We compared primary structures of the mutant actin genes (KM75 and HH5) which induce hsps and of the non‐inducing alleles (KM129 and KM88). The KM75 actin has lost 20 amino acids at the C‐terminus. The HH5 actin has only one amino acid substitution, from Gly‐336 to Ser. In KM129, the C‐terminal part of actin is replaced by novel amino acids. KM88 is a null allele, with an amber mutation early in the coding region of the mutated actin gene. Although all of the KM75, HH5 and KM129 actins have defects near the C‐terminus, only hsp‐inducing mutant actins cause enlargement of the IFM nuclei as well as a disruption of myofibrils even in the presence of two copies of the normal genes. We further consider the underlying mechanisms linking these features of the hsp‐inducing alleles.