Hitoshi Ueda

Drosophila Blimp-1 Is a Transient Transcriptional Repressor That Controls Timing of the Ecdysone-Induced Developmental Pathway

ABSTRACT Regulatory mechanisms controlling the timing of developmental events are crucial for proper development to occur. ftz-f1 is expressed in a temporally regulated manner following pulses of ecdysteroid and this precise expression is necessary for the development of Drosophila melanogaster. To understand how insect hormone ecdysteroids regulate the timing of FTZ-F1 expression, we purified a DNA binding regulator of ftz-f1. Mass spectroscopy analysis revealed this protein to be a fly homolog of mammalian B lymphocyte-induced maturation protein 1 (Blimp-1). Drosophila Blimp-1 (dBlimp-1) is induced directly by 20-hydroxyecdysone, and its product exists during high-ecdysteroid periods and turns over rapidly. Forced expression of dBlimp-1 and RNA interference analysis indicate that dBlimp-1 acts as a repressor and controls the timing of FTZ-F1 expression. Furthermore, its prolonged expression results in delay of pupation timing. These results suggest that the transient transcriptional repressor dBlimp-1 is important for determining developmental timing in the ecdysone-induced pathway.

Midline governs axon pathfinding by coordinating expression of two major guidance systems.

Formation of the neural network requires concerted action of multiple axon guidance systems. How neurons orchestrate expression of multiple guidance genes is poorly understood. Here, we show that Drosophila T-box protein Midline controls expression of genes encoding components of two major guidance systems: Frazzled, ROBO, and Slit. In midline mutant, expression of all these molecules are reduced, resulting in severe axon guidance defects, whereas misexpression of Midline induces their expression. Midline is present on the promoter regions of these genes, indicating that Midline controls transcription directly. We propose that Midline controls axon pathfinding through coordinating the two guidance systems.

Autocrine regulation of ecdysone synthesis by β3-octopamine receptor in the prothoracic gland is essential for Drosophila metamorphosis

Significance Metamorphosis is an important biological process by which animals alter their body structures to become sexually mature adults. We discovered that tyramine signaling through the β3-octopamine receptor plays an essential role in producing the steroid hormone ecdysone, which is critical for metamorphosis. Based on our observations, we propose that monoamine signaling acts downstream of a body size checkpoint that allows metamorphosis to occur only when a critical body weight is attained during larval development and nutrients are sufficiently abundant. This work also provides a new perspective on an evolutionarily conserved monoaminergic regulation of steroid hormone production during developmental transitions such as metamorphosis. This study provides a new understanding of how metamorphosis is coordinately regulated by nutritional conditions and developmental timing. In Drosophila, pulsed production of the steroid hormone ecdysone plays a pivotal role in developmental transitions such as metamorphosis. Ecdysone production is regulated in the prothoracic gland (PG) by prothoracicotropic hormone (PTTH) and insulin-like peptides (Ilps). Here, we show that monoaminergic autocrine regulation of ecdysone biosynthesis in the PG is essential for metamorphosis. PG-specific knockdown of a monoamine G protein-coupled receptor, β3-octopamine receptor (Octβ3R), resulted in arrested metamorphosis due to lack of ecdysone. Knockdown of tyramine biosynthesis genes expressed in the PG caused similar defects in ecdysone production and metamorphosis. Moreover, PTTH and Ilps signaling were impaired by Octβ3R knockdown in the PG, and activation of these signaling pathways rescued the defect in metamorphosis. Thus, monoaminergic autocrine signaling in the PG regulates ecdysone biogenesis in a coordinated fashion on activation by PTTH and Ilps. We propose that monoaminergic autocrine signaling acts downstream of a body size checkpoint that allows metamorphosis to occur when nutrients are sufficiently abundant.

Regulatory mechanisms of ecdysone-inducible Blimp-1 encoding a transcriptional repressor that is important for the prepupal development in Drosophila

Blimp‐1 is an ecdysone‐inducible transcription factor that is expressed in the early stage of the prepupal period. The timing of its disappearance determines expression timing of the FTZ‐F1 gene, whose temporally restricted expression is essential for the prepupal development. To elucidate the termination mechanism of Blimp‐1 gene expression, we examined the regulation of the Blimp‐1 gene using an organ culture system. The results showed that the Blimp‐1 gene is transcribed in cultured organs taken from a low ecdysteroid period even after extended exposure to 20‐hydroxyecdysone, while well‐known early genes such as E75A are repressed under the same conditions. Similar selective transcription was observed in the cultured organs obtained from a high ecdysteroid period. We further showed that Blimp‐1 transcripts quickly disappeared in the presence of actinomycin D. From these results, we concluded that the Blimp‐1 gene is transcribed when the ecdysteroid titer is high, but the expressed mRNA degrades rapidly; these unique regulations limit its expression to the high ecdysteroid stage.

Anterior epidermis-specific expression of the cuticle gene EDG84A is controlled by many cis-regulatory elements in Drosophila melanogaster

During insect metamorphosis, a pulse of ecdysteroids induces many different morphological changes depending on different parts of the body. In Drosophila, although a number of transcription factors are expressed in a stage-specific manner in response to an ecdysteroid pulse, little is known on the regulatory mechanism for space-specific gene expression during metamorphosis. The EDG84A gene encoding pupal cuticle protein is one of the targets of ecdysteroid-inducible transcription factor βFTZ-F1 and is expressed only in anterior epidermis of the body during mid- to late prepupal period, whereas βFTZ-F1 is expressed in almost all tissues. To address the regulatory mechanism of the tissue-specific expression of the EDG84A gene, we established transgenic fly lines which carry various upstream regions of the gene fused to the LacZ gene and examined the expression pattern of the reporter gene. Results of the transgenic fly reporter assays showed that the space-specific expression is controlled by at least four positive and two negative elements within a 263-bp region near the transcription start site, and at least three of them showed space-specific effects to the anterior body trunk. These results suggest that both high expression level and differential expression are achieved through many cis-regulatory elements.

Protease resistance of porcine acidic mammalian chitinase under gastrointestinal conditions implies that chitin-containing organisms can be sustainable dietary resources

Chitin, a polymer of N-acetyl-D-glucosamine (GlcNAc), is a major structural component in chitin-containing organism including crustaceans, insects and fungi. Mammals express two chitinases, chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase). Here, we report that pig AMCase is stable in the presence of other digestive proteases and functions as chitinolytic enzyme under the gastrointestinal conditions. Quantification of chitinases expression in pig tissues using quantitative real-time PCR showed that Chit1 mRNA was highly expressed in eyes, whereas the AMCase mRNA was predominantly expressed in stomach at even higher levels than the housekeeping genes. AMCase purified from pig stomach has highest activity at pH of around 2–4 and remains active at up to pH 7.0. It was resistant to robust proteolytic activities of pepsin at pH 2.0 and trypsin and chymotrypsin at pH 7.6. AMCase degraded polymeric chitin substrates including mealworm shells to GlcNAc dimers. Furthermore, we visualized chitin digestion of fly wings by endogenous AMCase and pepsin in stomach extract. Thus, pig AMCase can function as a protease resistant chitin digestive enzyme at broad pH range present in stomach as well as in the intestine. These results indicate that chitin-containing organisms may be a sustainable feed ingredient in pig diet.

SMARCAD1 is an ATP-dependent stimulator of nucleosomal H2A acetylation via CBP, resulting in transcriptional regulation

Histone acetylation plays a pivotal role in transcriptional regulation, and ATP-dependent nucleosome remodeling activity is required for optimal transcription from chromatin. While these two activities have been well characterized, how they are coordinated remains to be determined. We discovered ATP-dependent histone H2A acetylation activity in Drosophila nuclear extracts. This activity was column purified and demonstrated to be composed of the enzymatic activities of CREB-binding protein (CBP) and SMARCAD1, which belongs to the Etl1 subfamily of the Snf2 family of helicase-related proteins. SMARCAD1 enhanced acetylation by CBP of H2A K5 and K8 in nucleosomes in an ATP-dependent fashion. Expression array analysis of S2 cells having ectopically expressed SMARCAD1 revealed up-regulated genes. Using native genome templates of these up-regulated genes, we found that SMARCAD1 activates their transcription in vitro. Knockdown analysis of SMARCAD1 and CBP indicated overlapping gene control, and ChIP-seq analysis of these commonly controlled genes showed that CBP is recruited to the promoter prior to SMARCAD1. Moreover, Drosophila genetic experiments demonstrated interaction between SMARCAD1/Etl1 and CBP/nej during development. The interplay between the remodeling activity of SMARCAD1 and histone acetylation by CBP sheds light on the function of chromatin and the genome-integrity network.

A biological timer in the fat body comprising Blimp-1, βFtz-f1 and Shade regulates pupation timing in Drosophila melanogaster.

During the development of multicellular organisms, many events occur with precise timing. In Drosophila melanogaster, pupation occurs about 12 h after puparium formation and its timing is believed to be determined by the release of a steroid hormone, ecdysone (E), from the prothoracic gland. Here, we demonstrate that the ecdysone-20-monooxygenase Shade determines pupation timing by converting E to 20-hydroxyecdysone (20E) in the fat body, which is the organ that senses nutritional status. The timing of shade expression is determined by its transcriptional activator βFtz-f1. The βftz-f1 gene is activated after a decline in the expression of its transcriptional repressor Blimp-1, which is temporally expressed around puparium formation in response to a high titer of 20E. The expression level and stability of Blimp-1 is critical for the precise timing of pupation. Thus, we propose that Blimp-1 molecules function like sand in an hourglass in this precise developmental timer system. Furthermore, our data suggest that a biological advantage results from both the use of a transcriptional repressor for time determination and the association of developmental timing with nutritional status of the organism. Summary: Pupation timing in Drosophila is determined in the fat body by regulating 20-HE production. The unstable transcriptional repressor Blimp-1 is a key component of this system.

The Binding of Multiple Nuclear Receptors to a Single Regulatory Region Is Important for the Proper Expression of EDG84A in Drosophila melanogaster

Nuclear receptor transcription factor family members share target sequence similarity; however, little is known about how these factors exert their specific regulatory control. Here, we examine the mechanism regulating the expression of the Drosophila EDG84A gene, a target gene of the orphan nuclear receptor βFTZ-F1, as a model to study the cooperative behavior among nuclear receptors. We show that the three nuclear receptors βFTZ-F1, DHR3, and DHR39 bind to a common element in the EDG84A promoter. The expression level of the EDG84A promoter-lacZ reporter genes in DHR39-induced and mutant animals, respectively, suggests that DHR39 works as a repressor. The activity of a reporter gene carrying a mutation preventing DHR3 binding was reduced in ftz-f1 mutants and rescued by the induced expression of βFTZ-F1, suggesting that DHR3 and βFTZ-F1 activate the EDG84A gene in a redundant manner. A reporter gene carrying a mutation that abolishes DHR39 and FTZ-F1 binding was prematurely expressed, and the expression level of the reporter gene carrying a mutation preventing DHR3 binding was reduced. These findings suggest that the temporal expression of this gene is mainly controlled by βFTZ-F1 but that the binding of DHR3 is also important. Comparison of the binding site sequence among Drosophila species suggests that DHR3 binding ability was gained after the melanogaster subgroup evolved, and this ability may contribute to the robust expression of this gene. These results show the complicated regulatory mechanisms utilized by multiple nuclear receptors to properly regulate the expression of their target gene through a single target site.