Prothoracicotropic Hormone Regulates Developmental Timing and Body Size in Drosophila

In insects, control of body size is intimately linked to nutritional quality as well as environmental and genetic cues that regulate the timing of developmental transitions. Prothoracicotropic hormone (PTTH) has been proposed to play an essential role in regulating the production and/or release of ecdysone, a steroid hormone that stimulates molting and metamorphosis. In this report we examine the consequences on Drosophila development of ablating the PTTH-producing neurons. Surprisingly, PTTH production is not essential for molting or metamorphosis. Instead, loss of PTTH results in delayed larval development and eclosion of larger flies with more cells. Prolonged feeding, without changing the rate of growth, causes the developmental delay and is a consequence of low ecdysteroid titers. These results indicate that final body size in insects is determined by a balance between growth rate regulators such as insulin and developmental timing cues such as PTTH that set the duration of the feeding interval

Prothoracicotropic Hormone Regulates Developmental Timing and Body Size in Drosophila

In insects, control of body size is intimately linked to nutritional quality as well as environmental and genetic cues that regulate the timing of developmental transitions. Prothoracicotropic hormone (PTTH) has been proposed to play an essential role in regulating the production and/or release of ecdysone, a steroid hormone that stimulates molting and metamorphosis. In this report we examine the consequences on Drosophila development of ablating the PTTH-producing neurons. Surprisingly, PTTH production is not essential for molting or metamorphosis. Instead, loss of PTTH results in delayed larval development and eclosion of larger flies with more cells. Prolonged feeding, without changing the rate of growth, causes the developmental delay and is a consequence of low ecdysteroid titers. These results indicate that final body size in insects is determined by a balance between growth rate regulators such as insulin and developmental timing cues such as PTTH that set the duration of the feeding interval

R-Smad Competition Controls Activin Receptor Output in Drosophila

Animals use TGF-β superfamily signal transduction pathways during development and tissue maintenance. The superfamily has traditionally been divided into TGF-β/Activin and BMP branches based on relationships between ligands, receptors, and R-Smads. Several previous reports have shown that, in cell culture systems, “BMP-specific” Smads can be phosphorylated in response to TGF-β/Activin pathway activation. Using Drosophila cell culture as well as in vivo assays, we find that Baboon, the Drosophila TGF-β/Activin-specific Type I receptor, can phosphorylate Mad, the BMP-specific R-Smad, in addition to its normal substrate, dSmad2. The Baboon-Mad activation appears direct because it occurs in the absence of canonical BMP Type I receptors. Wing phenotypes generated by Baboon gain-of-function require Mad, and are partially suppressed by over-expression of dSmad2. In the larval wing disc, activated Baboon cell-autonomously causes C-terminal Mad phosphorylation, but only when endogenous dSmad2 protein is depleted. The Baboon-Mad relationship is thus controlled by dSmad2 levels. Elevated P-Mad is seen in several tissues of dSmad2 protein-null mutant larvae, and these levels are normalized in dSmad2; baboon double mutants, indicating that the cross-talk reaction and Smad competition occur with endogenous levels of signaling components in vivo. In addition, we find that high levels of Activin signaling cause substantial turnover in dSmad2 protein, providing a potential cross-pathway signal-switching mechanism. We propose that the dual activity of TGF-β/Activin receptors is an ancient feature, and we discuss several ways this activity can modulate TGF-β signaling output

The NDNF-like factor Nord is a Hedgehog-induced extracellular BMP modulator that regulates wing patterning and growth

Hedgehog (Hh) and Bone Morphogenetic Proteins (BMPs) pattern the developing wing by functioning as short- and long-range morphogens, respectively. Here, we show that a previously unknown Hh-dependent mechanism fine-tunes the activity of BMPs. Through genome-wide expression profiling of the wing imaginal discs, we identify as a novel target gene of the Hh signaling pathway. Nord is related to the vertebrate Neuron-Derived Neurotrophic Factor (NDNF) involved in congenital hypogonadotropic hypogonadism and several types of cancer. Loss- and gain-of-function analyses implicate Nord in the regulation of wing growth and proper crossvein patterning. At the molecular level, we present biochemical evidence that Nord is a secreted BMP-binding protein and localizes to the extracellular matrix. Nord binds to Decapentaplegic (Dpp) or the heterodimer Dpp-Glass-bottom boat (Gbb) to modulate their release and activity. Furthermore, we demonstrate that Nord is a dosage-dependent BMP modulator, where low levels of Nord promote and high levels inhibit BMP signaling. Taken together, we propose that Hh-induced Nord expression fine-tunes both the range and strength of BMP signaling in the developing wing

All isoforms of Baboon and mammalian Activin receptors can initiate cross-talk.

<p>(A) Overexpression of wildtype Babo_a or Babo_b in S2 cells led to phosphorylation of dSmad2 and Mad. In this experiment there was a detectable background level of P-dSmad2 well below the Babo-induced levels. Arrowhead indicates that the displayed image contains two portions of one blot. (B) Constitutively active versions of mammalian Activin receptors Alk4 and Alk7 (Alk4* and Alk7*), like Babo*, induce phosphorylation of <i>Drosophila</i> Mad in S2 cells.</p

The <i>Drosophila</i> Zinc Finger Transcription Factor Ouija Board Controls Ecdysteroid Biosynthesis through Specific Regulation of <i>spookier</i>

<div><p>Steroid hormones are crucial for many biological events in multicellular organisms. In insects, the principal steroid hormones are ecdysteroids, which play essential roles in regulating molting and metamorphosis. During larval and pupal development, ecdysteroids are synthesized in the prothoracic gland (PG) from dietary cholesterol via a series of hydroxylation and oxidation steps. The expression of all but one of the known ecdysteroid biosynthetic enzymes is restricted to the PG, but the transcriptional regulatory networks responsible for generating such exquisite tissue-specific regulation is only beginning to be elucidated. Here, we report identification and characterization of the C₂H₂-type zinc finger transcription factor Ouija board (Ouib) necessary for ecdysteroid production in the PG in the fruit fly <i>Drosophila melanogaster</i>. Expression of <i>ouib</i> is predominantly limited to the PG, and genetic null mutants of <i>ouib</i> result in larval developmental arrest that can be rescued by administrating an active ecdysteroid. Interestingly, <i>ouib</i> mutant animals exhibit a strong reduction in the expression of one ecdysteroid biosynthetic enzyme, <i>spookier</i>. Using a cell culture-based luciferase reporter assay, Ouib protein stimulates transcription of <i>spok</i> by binding to a specific ~15 bp response element in the <i>spok</i> PG enhancer element. Most remarkable, the developmental arrest phenotype of <i>ouib</i> mutants is rescued by over-expression of a functionally-equivalent paralog of <i>spookier</i>. These observations imply that the main biological function of Ouib is to specifically regulate <i>spookier</i> transcription during <i>Drosophila</i> development.</p></div

Baboon phosphorylation of Mad is inhibited by dSmad2.

<p>(A) Phospho-Smad accumulation upon exposure to Daw ligand. S2 cells transfected with Flag-Mad were treated with dsRNA for <i>dSmad2</i> or GFP, and transfected with Flag-dSmad2 as indicated. Phospho-Smad and Flag signals were assayed on samples before Daw exposure and after 1 and 3 hours of incubation. Note that there is a gel artifact affecting the appearance of the Flag bands in several lanes. Quantified P-Mad band intensities are plotted to illustrate that accumulation of P-Mad depends on the level of dSmad2. The experiment was repeated with several batches of reporter cells, and the relative signals for the 3 hour time point were always observed in the same order. The 1 hour time points were near background detection and are less reliable due to noise. (B) Mutated dSmad2 proteins with varying phosphorylation efficiency modulate the rate of P-Mad accumulation. Babo* stimulation revealed the steady-state levels of P-dSmad2 and P-Mad. Daw exposure for 1 or 4 hours showed the difference in response to short-term signaling between the WT and HD forms of dSmad2. In both conditions, P-Mad levels were inversely correlated to P-dSmad2 levels. (C–K) Wing imaginal discs from third instar larvae were stained to detect P-Mad and imaged by confocal microscopy. For each condition at least three discs were imaged, and a representative Maximal Intensity Projection encompassing the wing blade is shown (6 sections @ 3 micron interval for C,D,F–H; all sections @ 3 micron interval for E; 5 sections @ 2 micron interval for I–K). Anterior is to the left and the scale bar shown in panel C applies to C–K. The normal P-Mad staining pattern is shown for <i>vg</i>-GAL4 alone (C; scale bar = 100 microns). Expression of a UAS-<i>dSmad2</i> RNAi construct did not alter the P-Mad pattern or the shape of the wing disc (D). Wing discs from <i>babo^fd4/fd4</i> homozygotes showed nearly normal pMad gradient (E). Expression of Babo* altered P-Mad staining, obliterating the normal gradient in the pouch (F). Note the normal P-Mad staining outside of the pouch where <i>vg</i>-Gal4 is not expressed. Babo* and <i>dSmad2</i> RNAi together generated ectopic P-Mad in the entire wing pouch (G). Providing Babo* with additional Punt also produced ectopic P-Mad (H). <i>tkv</i> RNAi prevented P-Mad accumulation in the middle of the wing pouch (I), and addition of Babo* did not counteract this P-Mad pattern (J). Additional knockdown of <i>dSmad2</i> led to ectopic P-Mad (K), which paralleled the results without <i>tkv</i> RNAi. Data in panels C, D, F, and G were from the same experiment and were stained in parallel. Panel H is from a different experiment, but the pouch signals can be compared to the others because the endogenous P-Mad along the posterior margin has similar staining. Samples in panels I–K were stained and processed in parallel.</p

Stimulation of the Baboon receptor leads to phosphorylation of both R-Smads independently of BMP Type I receptors.

<p>S2 cells were transiently transfected with FLAG-tagged Smad expression constructs and analyzed by Western blot for C-terminal phosphorylation (P-dSmad2 and P-Mad). The FLAG-Mad band is shown as a loading control (FLAG-dSmad2 is not a useful loading control because of signaling-induced degradation as described later in the text). For all Western blot figures, a thin horizontal line indicates different infrared channels from the same blot. Co-expression of a constitutively active form of Baboon (Babo*) led to phosphorylation of dSmad2 and Mad (A, left blot). Exposure of cells expressing endogenous Baboon to the Dawdle ligand (Daw) had the same effect (A, right blot). RNAi treatment was used to determine receptor requirement for ligand activity (B). Controls confirmed that Dpp ligand treatment caused Mad phosphorylation independently of Baboon (B, left half). Dpp activity required the Punt Type II receptor and the Tkv and Sax BMP Type I receptors (B, right half). In contrast, Daw signaling to Mad required Baboon and Punt, but not Tkv and Sax.</p

Excessive Baboon signaling perturbs wing development in a Mad-dependent manner.

<p><i>vestigial-</i>GAL4 (<i>vg</i>) was used to express combinations of Baboon, dSmad2, and RNAi for <i>mad</i> and <i>dSmad2.</i> Normal wing development (A; one copy of <i>vg</i>-GAL4) was disrupted by Babo* expression (B, C; Babo*mod and Babo*strong are UAS insertions with varying activity as characterized in other assays). The Babo* phenotype was abrogated by simultaneous <i>mad</i> RNAi (H) and resembled <i>mad</i> RNAi alone (E). In contrast, the crumpling defect of Babo* wings was enhanced in conjunction with <i>dSmad2</i> RNAi (I) and was more severe than <i>dSmad2</i> RNAi alone (F). Overexpression of FLAG-dSmad2 did not affect wing formation (D), but partially rescued the Babo* overexpression phenotype, producing normal sized flat wings with residual peripheral vein defects (G). For each genotype, a representative wing is shown out of 4–7 wings photographed. Within a genotype there was only slight variation in appearance, except that 3/6 dSmad2 RNAi wings had vein defects and a blister (shown) and 3/6 had vein defects without a blister (not shown).</p

P-Mad elevation in <i>dSmad2</i> null mutant tissues depends on <i>baboon</i>.

<p>The schematic depicts the location of the l(X)G0348 <i>P</i> element insertion in relation to the <i>dSmad2</i> locus (A). The F4 excision product removed the entire coding region of <i>dSmad2</i> and portions of the <i>P</i>-element. The genomic breakpoints are indicated above the <i>dSmad2</i> mRNA; they were determined by sequencing PCR products, indicated by dotted lines. (B–D) P-Mad was detected by IHC of fixed larval tissues from several genotypes. For each image, a merged DAPI (blue) and P-Mad (red) panel is displayed above the isolated P-Mad channel. (B) Single confocal sections of P-Mad staining in the fat body. Under the staining conditions employed, endogenous nuclear P-Mad in a control fat body was barely detected (B1), but was increased in a <i>dSmad2</i> null mutant animal (B2). <i>Baboon</i> single mutants and <i>dSmad2</i>; <i>baboon</i> double mutants had normal P-Mad staining (B3,4). (C, D) P-Mad staining at two representative positions along the digestive tract. Images are Maximal Intensity Projections of 3 micron interval confocal sections through the entire sample. The P-Mad primary antibody was omitted from “No Ab Control” samples to convey any background staining and auto-fluorescence in the red channel. (C) In the gastric caeca near the proventriculus, <i>dSmad2</i> mutants showed elevated P-Mad (C3) compared to wildtype control males (C2). <i>baboon</i> single mutants and <i>dSmad2</i>; <i>baboon</i> double mutants showed wildtype levels (C4 and C5). Distal Malpighian tubule staining (lumpy tubes marked with asterisks) showed the same pattern, with the <i>dSmad2</i> mutant displaying the strongest P-Mad staining (D3 compared to D2, D4 and D5).</p