The Halloween genes code for cytochrome P450 enzymes mediating synthesis of the insect moulting hormone.

The developmental events occurring during moulting and metamorphosis of insects are controlled by precisely timed changes in levels of ecdysteroids, the moulting hormones. The final four sequential hydroxylations of steroid precursors into the active ecdysteroid of insects, 20E (20-hydroxyecdysone), are mediated by four cytochrome P450 (P450) enzymes, encoded by genes in the Halloween family. Orthologues of the Drosophila Halloween genes phantom (phm; CYP306A1), disembodied (dib; CYP302A1), shadow (sad; CYP315A1) and shade (shd; CYP314A1) were obtained from the endocrinological model insect, the tobacco hornworm Manduca sexta. Expression of these genes was studied and compared with changes in the ecdysteroid titre that controls transition from the larval to pupal stage. phm, dib and sad, which encode P450s that mediate the final hydroxylations in the biosynthesis of ecdysone, were selectively expressed in the prothoracic gland, the primary source of ecdysone during larval and pupal development. Changes in their expression correlate with the haemolymph ecdysteroid titre during the fifth (final) larval instar. Shd, the 20-hydroxylase, which converts ecdysone into the more active 20E, is expressed in tissues peripheral to the prothoracic glands during the fifth instar. Transcript levels of shd in the fat body and midgut closely parallel the enzyme activity measured in vitro. The results indicate that these Halloween genes are transcriptionally regulated to support the high biosynthetic activity that produces the cyclic ecdysteroid pulses triggering moulting.

A role for betaFTZ-F1 in regulating ecdysteroid titers during post-embryonic development in Drosophila melanogaster.

Variations in ecdysteroid titers play crucial roles in arthropods by initiating and regulating molting and metamorphosis. The recent identification of genes coding for cytochrome P450 enzymes involved in Drosophila ecdysteroidogenesis provides new molecular tools to investigate the regulation of insect hormone production. In the present study, we used an enzyme immunoassay to show that the molting hormone titer is strictly correlated with the steroidogenic capacity of the ring gland. A temporal correlation between dynamics of ecdysone production and expression of genes encoding steroidogenic enzymes was observed during the third instar, suggesting that the timing of hormone production depends on transcriptional regulation of the biosynthetic enzymes. Using clonal analysis, levels of two steroidogenic enzymes, Phantom (PHM) and Disembodied (DIB), were shown to be very reduced in ftz transcription factor 1 (ftz-f1) mutant ring gland cells whereas there was no effect of the without children (woc) mutation, suggesting that FTZ-F1 regulates phm and dib expression. Since betaFTZ-F1 is the homolog of the vertebrate steroidogenic factor 1 (SF1), which plays a key role in the differentiation of vertebrate steroidogenic organs through transcriptional regulation of steroidogenic enzymes, this study emphasizes the strong parallels between insects and vertebrates with respect to the regulatory mechanisms of steroidogenesis.

A protein from the cabbage looper, Trichoplusia ni, regulated by a bacterial infection is homologous to 3-dehydroecdysone 3beta-reductase.

During the screening of immune-regulated genes from the cabbage looper, Trichoplusia ni, a 3-dehydroecdysone 3beta-reductase homologue (DERH) was cloned. In the course of development, 3-dehydroecdysone 3beta-reductase mediates the conversion of 3-dehydroecdysone (3dE) secreted from the prothoracic glands to ecdysone (E), which is subsequently converted to 20-hydroxyecdysone (20E), the major insect molting hormone. The cloned gene is upregulated in fat body during development and is strongly induced after the larva is challenged with bacteria. The gene codes for a 308 amino acid residue protein which shows 42.5% identity to Spodoptera littoralis 3-dehydroecdysone 3beta-reductase. Using the baculovirus expression system, the recombinant DERH was expressed. The purified protein mediates the reduction of 3-dehydromakisterone A to makisterone A, and requires NADPH as a cofactor. Western blots using an antiserum to T. ni DERH revealed the presence of the protein in larval hemolymph and integument. The data indicate that the protein is regulated developmentally and is induced after a challenge with bacteria. Immunohistochemical studies localized the enzyme exclusively in the epidermis and the cuticle.

Loader Lite: a new software tool for the ABI PRISM 3700 DNA sequencer.

Here we describe the development of a novel software tool entitled Loader Lite that generates plate records or sample sheetsfor the ABI PRISMs 3700 DNA sequencer. The major advantage of this program is that it enables the ongoing operation of sequencing instruments without reference to external network(s). The autonomous operation of sequencing instruments is critical if sample throughput is to be maintained during periods of network outage. Loader Lite employs a deliberate strategy of inputting anonymous tray barcodes at run time. After sequencing, the barcodes are reconciled with relevant project details by reference to a database. This software takes advantage of barcode scanning technology by creating plate records directly on the local computer, serving an individual sequencer, immediately before importing and linking. This real-time synthesis of the plate records at the point of loading all but eliminates loading errors. Loader Lite is user-friendly, fully configurable, and permits the running of partial or full 384-well sample trays, using any standard combinations of run modules, dye sets, mobility files, analysis modules, etc. The 96-well format is not supported; however, this capability will appear in subsequent versions that are currently under development. This application is designed as an added value, adjunct program to the regular ABI PRISM 3700 Data Collection software. We have successfully used Loader Lite over the past six months to load approximately 7 million sequencing reactions and believe its utility and functionality will prove to be attractive to the wider sequencing community.

A quantitative analysis of the kinetics of Gal4 activator and effector gene expression in the zebrafish.

Using a temperature-inducible hsp70:Gal4 activator and UAS:myc-notch1a-intra as effector, we determined quantitatively the kinetics of expression of both transgenes and analysed the effects of varying their expressivity on several phenotypic traits in the developing zebrafish. hsp70:Gal4 is transcribed within 15 min after temperature-mediated induction, but Gal4 RNA decays rapidly. The Gal4 protein was found to be quite stable, as functional Gal4, which was detectable 1.5 h after heat shock (HS), persisted for at least 13 h. myc-notch1a-intra RNA is expressed approximately 1.5 h after HS, but unlike the Gal4 RNA, it was found to be very stable; it continues to accumulate during the succeeding 17 h after HS. Fully penetrant phenotypic effects are obtained after a relatively long activator induction with a 30-min HS.

Woc (without children) gene control of ecdysone biosynthesis in Drosophila melanogaster.

The first step in ecdysteroidogenesis, i.e. the 7,8-dehydrogenation of dietary cholesterol (C) to 7-dehydrocholesterol (7dC), is blocked in Drosophila melanogaster homozygous woc (without children) third instar larval ring glands (source of ecdysone). Unlike ring glands from wild-type D. melanogaster larvae, glands from woc mutants cannot convert radiolabelled C or 25-hydroxycholesterol (25C) to 7dC or 7-dehydro-25-hydroxycholesterol (7d25C) in vitro, nor to ecdysone (E). Yet, when these same glands are incubated with synthetic tracer 7d25C, the rate of metabolism of this polar Delta(5,7)-sterol into E is identical to that observed with glands from comparably staged wild-type larvae. The absence of this enzymatic activity in vivo is probably the direct cause of the observed low whole-body ecdysteroid titers in late third instar homozygous mutant larvae, the low ecdysteroid secretory activity in vitro of brain-ring gland complexes from these animals, and the failure of the larvae to pupariate (undergo metamorphosis). Oral administration of 7dC, but not C, results in a dramatic increase in ecdysteroid production both in vivo and in vitro by the woc mutant brain-ring gland complexes and affects a partial rescue to the beginning of pupal-adult development, but no further, despite elevated whole-body ecdysteroid titers. Data previously reported (Wismar et al., 2000) indicate that the woc gene encodes a zinc-finger protein that apparently modulates the activity of the 7,8-dehydrogenase.

Topographic restriction of TAG-1 expression in the developing retinotectal pathway and target dependent reexpression during axon regeneration.

TAG-1, a glycosylphosphatidyl inositol (GPI)-anchored protein of the immunoglobulin (Ig) superfamily, exhibits an unusual spatiotemporal expression pattern in the fish visual pathway. Using in situ hybridization and new antibodies (Abs) against fish TAG-1 we show that TAG-1 mRNA and anti-TAG-1 staining is restricted to nasal retinal ganglion cells (RGCs) in 24- to 72-h-old zebrafish embryos and in the adult, continuously growing goldfish retina. Anti-TAG-1 Abs selectively label nasal RGC axons in the nerve, optic tract, and tectum. Axotomized RGCs reexpress TAG-1, which occurs as late as 12 days after optic nerve lesion, when regenerating RGC axons arrive in the tectum, suggesting TAG-1 reexpression is target contact-dependent. Accordingly, TAG-1 reexpression ceases upon interruption of the regenerating projection by a second lesion. The topographic restriction of TAG-1 expression and its target dependency during regeneration suggests that TAG-1 might play a role in the retinotopic organization and restoration of the retinotectal pathway.

The mutation without children(rgl) causes ecdysteroid deficiency in third-instar larvae of Drosophila melanogaster.

Larvae homozygous for the recessive lethal allele without children(rgl) (woc(rgl)) fail to pupariate. Application of exogenous 20-hydroxyecdysone elicits puparium formation and pupation. Ecdysteroid titer measurements on mutant larvae show an endocrine deficiency in the brain-ring gland complex, which normally synthesizes ecdysone, resulting in a failure of the larvae to achieve a threshold whole body hormone titer necessary for molting. Ultrastructural investigation revealed extensive degeneration of the prothoracic cells of the ring gland in older larvae. The woc gene, located in polytene chromosomal region 97F, consists of 11 exons. A 6.8-kb transcript is expressed throughout development but is absent in the mutant woc(rgl) larvae. The woc gene encodes a protein of 187 kDa. Eight zinc fingers of the C2-C2 type point to a possible function as a transcription factor. The woc protein shows considerable homology to human proteins which have been implicated in both mental retardation and a leukemia/lymphoma syndrome.

Transcriptional activation of the Drosophila ecdysone receptor by insect and plant ecdysteroids.

A number of insect ecdysteroids, plant ecdysteroids and juvenoids were assayed for their ability to activate Drosophila nuclear receptors in transfected tissue culture cells. Discrete modifications to 20-hydroxyecdysone, the apparent natural ligand for the ecdysone receptor (EcR), conferred dramatic changes on the transcriptional activity of this receptor, suggesting that other biologically relevant EcR ligands may exist. Conversely, none of the compounds tested had a significant effect on the activity of three Drosophila orphan nuclear receptors: DHR38, DHR78 or DHR96. Taken together, these results demonstrate the selectivity of EcR for a series of natural and synthetic ecdysone agonists and suggest that as yet untested compounds may be responsible for activating DHR38, DHR78 and DHR96.

Laser-induced gene expression in specific cells of transgenic zebrafish.

Over the past few years, a number of studies have described the generation of transgenic lines of zebrafish in which expression of reporters was driven by a variety of promoters. These lines opened up the real possibility that transgenics could be used to complement the genetic analysis of zebrafish development. Transgenic lines in which the expression of genes can be regulated both in space and time would be especially useful. Therefore, we have cloned the zebrafish promoter for the inducible hsp70 gene and made stable transgenic lines of zebrafish that express the reporter green fluorescent protein gene under the control of a hsp70 promoter. At normal temperatures, green fluorescent protein is not detectable in transgenic embryos with the exception of the lens, but is robustly expressed throughout the embryo following an increase in ambient temperature. Furthermore, we have taken advantage of the accessibility and optical clarity of the embryos to express green fluorescent protein in individual cells by focussing a sublethal laser microbeam onto them. The targeted cells appear to develop normally: cells migrate normally, neurons project axons that follow normal pathways, and progenitor cells divide and give rise to normal progeny cells. By generating other transgenic lines in which the hsp70 promoter regulates genes of interest, it should be possible to examine the in vivo activity of the gene products by laser-inducing specific cells to express them in zebrafish embryos. As a first test, we laser-induced single muscle cells to make zebrafish Sema3A1, a semaphorin that is repulsive for specific growth cones, in a hsp70-sema3A1 transgenic line of zebrafish and found that extension by the motor axons was retarded by the induced muscle.

Can the insect nervous system synthesize ecdysteroids?

The term "neurosteroid" refers to both classic and unique steroid molecules that are synthesized from cholesterol (C) by the central and peripheral nervous systems of higher vertebrates. Therein, they accumulate and modulate nervous activity by a variety of mechanisms other than the classic steroid receptor-mediated modulation of genomic activity, although such may also be involved. Since the insect nervous system expresses ecdysteroid receptors and responds both directly and developmentally to ecdysteroids, the possibility of ecdysteroidogenesis in the pupal and adult central and peripheral nervous system of Manduca sexta and the nervous system of Drosophila melanogaster larvae was investigated. The endogenous concentrations of the critical, dietary-derived delta 5,7-sterols ergosterol and 7-dehydrocholesterol (7dC) remained 10 to 20-fold higher in the Manduca pupal and adult nervous tissues than was found in the larval hemolymph at the cessation of feeding. In addition, it was determined that the Manduca pupal nervous system, but not that of the adult, could synthesize 3H/14C-7dC or 3H-7-dehydro-25-hydroxycholesterol (3H-7d25C) from 3H/14C-cholesterol (3H/14C-C) or the polar sterol substrate 3H-25-hydroxycholesterol (3H-25C), respectively. However, none of the nervous system samples from the two species and the several stages analyzed, a small window of neural development in these insects, were capable of incorporating any of the above tracer precursor sterols into a radiolabelled ecdysteroid, i.e. less than 0.0005%. Thus, the absence of neurosteroidogenesis by the insect nervous system stands in sharp contrast to previously described nervous system steroid hormone biosynthesis by the mammalian nervous system.

Molecular cloning, expression, and activity of zebrafish semaphorin Z1a.

Semaphorins/collapsins are a large family of secreted and cell surface molecules that are thought to guide growth cones to their targets. Although some members are clearly repulsive to specific growth cones in vitro, the in vivo role of many of these molecules in vertebrate embryos is still unclear. As a first step towards clarifying the in vivo role of semaphorins/collapsins, we analyzed semaZ1a in the simple and well-characterized zebrafish embryo. SemaZ1a is a secreted molecule that is highly homologous to Sema III/D/collapsin-1, and it can collapse chick dorsal root ganglion growth cones in vitro. It is expressed in highly specific patterns within the developing embryo, which suggests that it influences outgrowth by a variety of growth cones including those of the posterior lateral line ganglion. Consistent with this hypothesis, the peripherally extending growth cones of posterior lateral line neurons retract and partially collapse during normal outgrowth.

Molecular cloning and developmental expression of a zebrafish axonal glycoprotein similar to TAG-1.

TAG-1 is a mammalian cell adhesion molecule of the immunoglobulin superfamily that is expressed transiently by a subset of neurons and serves as a fertile substrate for neurite outgrowth in vitro (Furley, A.H., Morton, S.B., Manalo, D., Karagogeos, S., Dodd, H., Jessell, T.M., 1990 The axonal glycoprotein TAG-1 is an immunoglobulin superfamily member with neurite outgrowth promoting activity. Cell 61, 157-170). In order to examine the in vivo function of this molecule, we have cloned a zebrafish tag1-like cDNA and analyzed its expression patterns. tag1 Is expressed transiently by specific subsets of neurons when they are projecting their axons or when they are migrating. The specific and dynamic pattern of expression of zebrafish tag1 is consistent with its proposed role in axon guidance and cell migration.

Role of sonic hedgehog in branchiomotor neuron induction in zebrafish.

The role of zebrafish hedgehog genes in branchiomotor neuron development was analyzed by examining mutations that affect the expression of the hedgehog genes and by overexpressing these genes in embryos. In cyclops mutants, reduction in sonic hedgehog (shh) expression, and elimination of tiggy-winkle hedgehog (twhh) expression, correlated with reductions in branchiomotor neuron populations. Furthermore, branchiomotor neurons were restored in cyclops mutants when shh or twhh was overexpressed. These results suggest that Shh and/or Twhh play an important role in the induction of branchiomotor neurons in vivo. In sonic-you (syu) mutants, where Shh activity was reduced or eliminated due to mutations in shh, branchiomotor neurons were reduced in number in a rhombomere-specific fashion, but never eliminated. Similarly, spinal motor neurons were reduced, but not eliminated, in syu mutants. These results demonstrate that Shh is not solely responsible for inducing branchiomotor and spinal motor neurons, and suggest that Shh and Twhh may function as partially redundant signals for motor neuron induction in zebrafish.

Development of branchiomotor neurons in zebrafish.

The mechanisms underlying neuronal specification and axonogenesis in the vertebrate hindbrain are poorly understood. To address these questions, we have employed anatomical methods and mutational analysis to characterize the branchiomotor neurons in the zebrafish embryo. The zebrafish branchiomotor system is similar to those in the chick and mouse, except for the location of the nVII and nIX branchiomotor neurons. Developmental analyses of genes expressed by branchiomotor neurons suggest that the different location of the nVII neurons in the zebrafish may result from cell migration. To gain insight into the mechanisms underlying the organization and axonogenesis of these neurons, we examined the development of the branchiomotor pathways in neuronal mutants. The valentino b337 mutation blocks the formation of rhombomeres 5 and 6, and severely affects the development of the nVII and nIX motor nuclei. The cyclops b16 mutation deletes ventral midline cells in the neural tube, and leads to a severe disruption of most branchiomotor nuclei and axon pathways. These results demonstrate that rhombomere-specific cues and ventral midline cells play important roles in the development of the branchiomotor pathways.

Differential incorporation of cholesterol and cholesterol derivatives into ecdysteroids by the larval ring glands and adult ovaries of Drosophila melanogaster: a putative explanation for the l(3)ecd1 mutation.

Studies in vitro revealed that intact ring glands of Drosophila melanogaster convert tritiated cholesterol (C) and 25-hydroxycholesterol (25C) via 7-dehydrocholesterol (7dC) and 7-dehydro-25-hydroxycholesterol (7d25C), respectively, to ecdysone (E) and 2-deoxyecdysone (2dE), while both intact and homogenized ovaries synthesize only 2dE from these precursors. Emulsified 7d25C was incorporated directly into ecdysteroids by these tissue preparations at a much greater rate than was 7d25C made in situ from 25C. To probe the basis of the biochemical defect in the ecdysteroid deficient conditional mutant ecdysoneless (ecd1), the differential incorporation into ecdysteroids of C (via 7dC), and particularly of 25C (via 7d25C), was measured relative to that observed after the incubation of 7d25C directly with both wild type and mutant tissues in vitro at 30 degrees C, the restrictive temperature. Both C and 25C were equally 7,8-dehydrogenated in situ to 7dC or 7d25C, respectively, by both wild type and mutant tissues at 30 degrees C. However, the rate of subsequent conversion of either of these delta 5,7-sterol intermediates synthesized in situ to ecdysteroids was reduced an average of 50% in the mutant tissues relative to the wild type. Yet, when emulsified 7d25C was incubated directly with either the wild type or mutant tissues at the restrictive temperature, the amplified rate of conversion of the freely available 7d25C to ecdysteroid by these tissues was identical. These data suggest that the defect in ecd1 tissue-mediated ecdysteroidogenesis does not involve a "hit" on any of the enzymes involved in either the 7,8-dehydrogenation of C or 25C or in the subsequent oxidation of 7d25C or 7dC to ecdysteroid. Rather, the mutation appears to affect the expression of a gene governing the translocation of delta 5,7-sterol intermediates from the subcellular compartment where they are synthesized and/or stored to the site of subsequent oxidation to ecdysteroid.

Metabolism in vitro of cholesterol and 25-hydroxycholesterol by the larval prothoracic glands of Manduca sexta.

The prothoracic glands in vitro convert 25-hydroxycholesterol (25C) to 25-hydroxy-7-dehydrocholesterol (7d25C) and to ecdysteroids at a greater rate than cholesterol (C) is converted to ecdysteroids via 7-dehydrocholesterol (7dC). Mediated via a cytochrome P450 most probably located in the endoplasmic reticulum (ER), both intact and extensively homogenized prothoracic glands, as well as crude subcellular fractions, were able to 7,8-dehydrogenate 25C to 7d25C eight-fold more efficiently than they could convert C to 7dC. However, less than a two-fold difference was observed in the subsequent monooxygenase mediated conversion of these two intermediates formed in situ into ecdysteroids, mainly ecdysone (E) and 2-deoxyecdysone (2dE) and/or their 3-dehydroderivatives. When 7dC, and particularly 7d25C, were made directly available to these tissue preparations, their conversion to ecdysteroids greatly exceeded that of the in situ conversion of either C or 25C, via 7dC or 7d25C, respectively. Indeed, there was an eight-fold increase in the VMAX for 25C dehydrogenation by homogenized glands relative to the dehydrogenation of C. Most important, however, was the 1000-fold increase in the VMAX observed for the direct production of E from emulsified 7d25C by gland homogenates relative to E production from 25C via 7d25C synthesized in situ. Thus, it is apparent that even after the rapid and efficient conversion of 25C to 7d25C within the ER, the subsequent rate of conversion of this intermediate to E is greatly retarded relative to that observed following the direct incubation of emulsified 7d25C with gland homogenates. These differential kinetics of direct and indirect 7d25C incorporation into E are interpreted as evidence for the existence of a barrier to the efficient translocation of the delta 5,7-sterol intermediates from the ER to another site where the subsequent, uncharacterized initial conversions leading to ecdysteroids take place. On the basis of studies on mammalian adrenal cortical steroidogenesis, this site is postulated to be the inner membrane/matrix of the mitochondria. The present data support the hypothesis that the translocation of both 7dC and 7d25C, first from the site of their probable synthesis within the ER membranes, next through the cytosol to the outer mitochondrial membrane, and then across the intramitochondrial aqueous space to the inner membrane/matrix compartment, may be analogous to the translocation in the adrenal cortex of ER-derived C, first to the plasma membrane and/or to the outer mitochondrial membrane and then to the inner mitochondrial membrane/matrix for P450scc-mediated conversion into pregnenolone.

Bryostatins trigger human polymorphonuclear neutrophil and monocyte oxidative metabolism: association with in vitro antineoplastic activity.

Bryostatin-1-but not bryostatin-13-a macrocyclic lactone isolated from the marine bryozoan Bugula neritina, triggered human polymorphonuclear neutrophil (PMN) and monocyte release of reactive oxygen radicals, as measured by the generation of lucigenin chemiluminescence and by the ferricytochrome c reduction assay. The release of oxygen radicals by bryostatins was sensitive to inhibitors of protein kinases, but resistant to the inhibition of phospholipase A2 activity and arachidonic acid metabolism (prior treatment with mepacrine or indomethacin). Comparison of the effect of protein kinase (PK) inhibitors H-8, H-7 and staurosporine on bryostatin-1-induced neutrophil oxygen radical release further suggested a requirement for activation of phospholipid-dependent PKC, but not for cGMP- or cAMP-dependent PK. In cytostatic assays, PMNs treated with bryostatin-1 inhibited the growth of the erythroleukaemic cell line K562 in a concentration-dependent manner. These findings suggest that the reported antineoplastic effect of bryostatins may result at least in part from activation of PMNs and monocytes.