Theodore R. F. Wright

The genetics of biogenic amine metabolism, sclerotization, and melanization in Drosophila melanogaster.

Publisher Summary The genetics of Drosophila provides a number of different approaches toward initiating the investigation of the areas of biogenic-amine metabolism that have previously not been studied or are completely novel to any organism. Biogenic amines are particularly important compounds in insects, for they not only serve as neurotransmitters and modulaters of adenylate cyclase activity in their neurophysiology but also are essential for cross-linking proteins and chitin during sclerotization in the insect cuticle throughout development and especially at times of eclosion (hatching) and molting. A thorough understanding of the regulation of the synthesis, inactivation, and degradation of biogenic amines in the nervous system is mandatory for the elucidation of their roles in the neurophysiology and behavior of any organism. The availability of mutations that interrupt biogenic-amine metabolism in an experimentally suitable organism such as Drosophila should provide powerful analytical tools for neurochemists, neurophysiologists, and behavioral biologists. To facilitate and stimulate research on biogenic amines and sclerotization, this chapter discusses some known facts about the genetics and biochemistry of biogenic amines and sclerotization in Drosophila .

The genetics of embryogenesis in Drosophila.

Publisher Summary This chapter describes that any study of genetic variations modify embryogenesis can be approached with two objectives in mind. On the one hand, one might emphasize the embryological aspects of such studies by concentrating on how mutation-produced modifications of particular embryological events can help to elucidate the developmental interactions involved, and aid in the search for the cellular and biochemical mechanisms that are fundamental to them. On the other hand, one might emphasize the genetic aspects of investigating such mutations. The purpose of this chapter to examine how the study of genetic variations that affect embryogenesis in drosophila has in the past contributed, and may in the future contribute, to an understanding of the precise nature of gene action in development. The ultimate goal is, of course, an understanding of the mechanisms that integrate the activities of both the genome and the cytoplasm such that development regularly proceeds from one state of determination and differentiation to the next.

Temporal and spatial expression of the yellow gene in correlation with cuticle formation and DOPA decarboxylase activity in drosophila development

The yellow (y) gene of Drosophila is required for the formation of black melanin and its deposition in the cuticle. We have studied by immunohistochemical methods the temporal and spatial distribution of the protein product of the y gene during embryonic and pupal development and have correlated its expression with events of cuticle synthesis by the epidermal cells and with cuticle sclerotization. Except for expression in early embryos, the y protein is only found in the epidermal cells and may be secreted into the cuticle as it is being deposited. The amount of y protein in various regions of the embryo and pupa correlates directly with the intensity of melanization over any section of the epidermis. Expression of the y gene begins in the epidermal cells at 48 hr after pupariation and is well correlated with the beginning deposition of the adult cuticle. At this stage the adult cuticle is unsclerotized and unpigmented and dopa decarboxylase levels, a key enzyme in catecholamine metabolism which provides the crosslinking agents as well as the precursors for melanin, is low. As a separate event 26 hr after the onset of y gene expression, the first melanin deposition occurs in the head bristles and pigmentation continues in an anterior to posterior progression until eclosion. This melanization wave is correlated with elevated dopa decarboxylase activity. Crosslinking of the adult cuticle also occurs in a similar anterior to posterior progression at about the same time. We have shown by imaginal disc transplantation that timing of cuticle sclerotization depends on the position of the tissue along the anterior-posterior axis and that it is not an inherent feature of the discs themselves. We suggest that actual melanization and sclerotization of the cuticle by crosslinking are initiated at this time in pupal development by the availability of the catecholamine substrates which diffuse into the cuticle. Intensity of melanization and position of melanin pigment is determined by the presence or absence of the y protein in the cuticle, thus converting the y protein prepattern into the melanization pattern.

A histological and ultrastructural analysis of developmental defects produced by the mutation, lethal(1)myospheroid, in Drosophila melanogaster

Abstract The recessive embryonic lethal, lethal(1)myospheroid, is located at 21.7 map units on the X chromosome in Drosophila melanogaster. Embryos hemizygous for this mutation appear to develop normally until the time of the first muscular contractions. Due to the physical stress of these initial contractions, dramatic tissue separations occur, which characterize the phenotype of this mutation. The dorsal suture separates with the herniation of midgut and nervous tissue, and the somatic and visceral muscles retract from their sites of attachment. An ultrastructural examination of the development of the muscle attachment sites in these embryos indicates that in 1(1)mys embryos there is a delay in the formation of normal cell-cell attachments. At the muscle-tendon cell junction, the deposition of the apparently normal extracellular matrix of this desmosomal attachment occurs considerably later in development than normal. The 1(1)mys locus apparently makes a product which is either defective, made more slowly, or produced in smaller amounts than normal. The 1(1)mys product is probably necessary for the production of a component of the extracellular matrix of cell-cell attachments.

Developmental relationship between dopa decar☐ylase, dopamine acetyltransferase, and ecdysone inDrosophila

The developmental profiles of dopa decar☐ylase (DDC) and dopamine acetyltransferase (DAT) were determined and compared with that of ecdysone. Five peaks of DDC activity are observed corresponding to the five major transition points inDrosophila development, embryonic hatching, first and second molts, puparium formation, and adult eclosion. Marked changes in DAT activity are also observed, although the pattern does not correlate well with changes in developmental stages. DDC expression is correlated with increasing ecdysone concentration at puparium formation, while the peaks of DDC at embryonic hatching and adult eclosion are not temporally correlated with ecdysone. The temperature-sensitive ecdysoneless mutant (ecd1) which is reversibly blocked in ecdysone production at restrictive temperatures [Garen, A., Kauvar, L., and Lepesant, J. A. (1977).Proc. Nat. Acad. Sci. USA74, 5099–5133] was used to show that the 20-fold increase in DDC activity in pupating larvae is hormone dependent. It was found that theecd1 mutant failed to block the midpupal increase in ecdysone concentration.

The genetics of dopa decarboxylase in Drosophila melanogaster

Of 204 mutations located in the 8–12 band Df(2L)130 region, 37B9-C1,2;37D1-2, 199 have been assigned to twelve lethal genes and one visible gene (hook). The 13 genes are not evenly distributed. Twelve, (possibly all thirteen) are in the seven band region 37B10-C4 giving a gene-to-band ratio of almost two. Only one gene, 1(2)37Cf, may be in the four band region 37C5-7, and none are localized in band 37D1. In situ hybridization places the dopa decarboxylase structural gene, Ddc, in or very close to band 37C1,2 (Hirsh and Davidson, 1981). The χ methyl dopa hypersensitive gene, 1(2) amd, is 0.002 map units distal to Ddc. Df(2L)VA17, 37C1,2; 37F5-38A1 may actually break in the 37C1,2 singlet. It places six genes, hook, 1(2)amd, and four lethal genes, in a maximum of five bands, 37B10, 11, 12, 13 and perhaps part of the 37C1,2 singlet and localizes six genes, Ddc plus five lethal genes, in a maximum of three bands; probably part of the 37C1,2 singlet plus bands, C3, and C4. Wild type activity of five of twelve lethal genes is necessary for female fertility. — Band 37C5 puffs at the time of pupariation; Puff Stages 8–10. Twelve of eighteen alleles of 1(2)37Cf havs been examined as heterozygotes over CyO and none affect the appearance of a homozygous 37C5 puff. — Of the 204 mutations considered here only one Ddcp1, affects the function of more than one gene. It eliminates Ddc+ and l(2) 37Ca+ function and at 30 ° C reduces l(2)37Ce+ function. It is not a deficiency but could be a polar mutant.

Catecholamine metabolism and in vitro induction of premature cuticle melanization in wild type and pigmentation mutants of Drosophila melanogaster.

The major pathway leading to adult cuticle melanization in Drosophila melanogaster has been investigated by a combination of biochemical and genetic approaches. By comparing catecholamine pools in newly emerged flies and in frass (excreta) collected 1 to 4 days after eclosion from wild type with those obtained from several pigmentation mutants, the major flow of catecholamines through the pathway to an unidentified final catabolite was determined. We also demonstrate that incubation with dopamine in vitro induces premature melanization in wild type unpigmented pharate adults several hours before the developmentally programmed onset of melanization, supporting the hypothesis that the availability of catecholamines may be the limiting factor determining the onset of melanization and that the major enzymatic activities that act downstream of dopa decarboxylase in the pathway are deposited into the cuticle before pigmentation begins. In vitro melanization studies with various pigmentation mutants that are associated with critical enzymatic steps in Drosophila catecholamine metabolism are consistent with their proposed function and suggest a central role of N-beta-alanyldopamine in adult cuticle pigmentation.

The genetics of dopa decarboxylase in Drosophila melanogaster. IV. The genetics and cytology of the 37B10-37D1 region.

Of 204 mutations located in the 8-12 band Df(2L)130 region, 37B9-C1,2;37D1-2, 199 have been assigned to twelve lethal genes and one visible gene (hook). The 13 genes are not evenly distributed. Twelve, (possibly all thirteen) are in the seven band region 37B10-C4 giving a gene-to-band ratio of almost two. Only one gene, 1(2)37Cf, may be in the four band region 37C5-7, and none are localized in band 37D1. In situ hybridization places the dopa decarboxylase structural gene, Ddc, in or very close to band 37C1,2 (Hirsh and Davidson, 1981). The chi methyl dopa hypersensitive gene, 1(2) and, is 0.002 map units distal to Ddc. Df(2L)VA17, 37C1,2; 37F5-38A1 may actually break in the 37C1,2 singlet. It places six genes, hook, 1(2) and, and four lethal genes, in a maximum of five bands, 37B10, 11, 12, 13 and perhaps part of the 37C1,2 singlet and localizes six genes, Ddc plus five lethal genes, in a maximum of three bands; probably part of the 37C1,2 singlet plus bands, C3, and C4. Wild type activity of five of twelve lethal genes is necessary for female fertility. --Band 37C5 puffs at the time of pupariation; Puff Stages 8-10. Twelve of eighteen alleles of 1(2)37Cf have been examined as heterozygotes over CyO and none affect the appearance of a homozygous 37C5 puff. --Of the 204 mutations considered here only one Ddcpl, affects the function of more than one gene. It eliminates Ddc+ and 1(2)37Ca+ function and at 30 degrees C reduces 1(2)37Ce+ function. It is not a deficiency but could be a polar mutant.

The selection for mutants in Drosophila melanogaster hypersensitive to ?-methyl dopa, a dopa decarboxylase inhibitor

SummaryThe use of dietary α-methyl dopa, an inhibitor of Drosophila melanogaster dopa decarboxylase, to screen for mutants defective in cuticle development was investigated. The results show that dietary α-methyl dopa is toxic to larvae and that death always occurs at the larval molts. This and other evidence suggests that α-methyl dopa is a specific inhibitor of some aspect of cuticle development. From a screen of 2748 EMS-treated lethal-free second chromosomes, 7 dominant α-methyl dopa hypersensitive mutants were obtained. All these mutants are recessive lethal alleles on standard food and map to one locus, 53.1±, designated as l(2)amd. Assays of dopa decarboxylase activities do not reveal any differences between mutant heterozygotes (l(2)amd/+) and wild type (+/+).

The alpha methyl dopa hypersensitive gene, l(2)amd, and two adjacent genes in Drosophila melanogaster: Physical location and direct effects of amd on catecholamine metabolism

SummaryThe dopa decarboxylase gene (Ddc) is located in a very dense cluster of genes many of whose functions appear to be related to the physiological role of dopa decarboxylase (DDC) in catecholamine metabolism. In Drosophila melanogaster catecholamine metabolism is involved in the production of neurotransmitters and in the synthesis of cross-linking agents for cuticular sclerotization. In this report we consider three loci near Ddc that affect cuticle formation. The alpha methyl dopa hypersensitive gene, l(2)amd, is definitively assigned to a transcriptional unit 2 kb distal to Ddc. The assignment of l(2)37 Bd and l(2)37 Cc to coding regions in the immediate vicinity of amd and Ddc is examined. amd+ gene activity performs a vital function essential for the formation of insect cuticle and also determines the level of sensitivity to the DDC analogue inhibitor, alpha methyl dopa. We present data that provide direct evidence that the amd+ gene product is required for a step in the metabolism of dopa to one or more novel catecholamines involved in the colorless sclerotization of cuticle.