Secretory competence in a gateway endocrine cell conferred by the nuclear receptor βFTZ-F1 enables stage-specific ecdysone responses throughout development in Drosophila

Hormone-induced changes in gene expression initiate periodic molts and metamorphosis during insect development. Successful execution of these developmental steps depends upon successive phases of rising and falling 20-hydroxyecdysone (20E) levels, leading to a cascade of nuclear receptor-driven transcriptional activity that enables stage- and tissue-specific responses to the steroid. Among the cellular processes associated with declining steroids is acquisition of secretory competence in endocrine Inka cells, the source of ecdysis triggering hormones (ETHs). We show here that Inka cell secretory competence is conferred by the orphan nuclear receptor βFTZ-F1. Selective RNA silencing of βftz-f1 in Inka cells prevents ETH release, causing developmental arrest at all stages. Affected larvae display buttoned-up, the ETH-null phenotype characterized by double mouthparts, absence of ecdysis behaviors, and failure to shed the old cuticle. During the mid-prepupal period, individuals fail to translocate the air bubble, execute head eversion and elongate incipient wings and legs. Those that escape to the adult stage are defective in wing expansion and cuticle sclerotization. Failure to release ETH in βftz-f1 silenced animals is indicated by persistent ETH immunoreactivity in Inka cells. Arrested larvae are rescued by precisely-timed ETH injection or Inka cell-targeted βFTZ-F1 expression. Moreover, premature βftz-f1 expression in these cells also results in developmental arrest. The Inka cell therefore functions as a "gateway cell", whose secretion of ETH serves as a key downstream physiological output enabling stage-specific responses to 20E that are required to advance through critical developmental steps. This secretory function depends on transient and precisely timed βFTZ-F1 expression late in the molt as steroids decline

Enteroendocrine peptides regulate feeding behavior via controlling intestinal contraction of the silkworm Bombyx mori.

Our previous study demonstrated that predominant feeding inhibitory effects were found in the crude extracts of foregut and midgut of the silkworm Bombyx mori larvae. To address the entero-intestinal control crucial for the regulation of insect feeding behavior, the present study identified and functionally characterized feeding inhibitory peptides from the midgut of B. mori larvae. Purification and structural analyses revealed that the predominant inhibitory factors in the crude extracts were allatotropin (AT) and GSRYamide after its C-terminal sequence. In situ hybridization revealed that AT and GSRYamide were expressed in enteroendocrine cells in the posterior and anterior midgut, respectively. Receptor screening using Ca2+-imaging technique showed that the B. mori neuropeptide G protein-coupled receptor (BNGR)-A19 and -A22 acted as GSRYamide receptors and BNGR-A5 acted as an additional AT receptor. Expression analyses of these receptors and the results of the peristaltic motion assay indicated that these peptides participated in the regulation of intestinal contraction. Exposure of pharynx and ileum to AT and GSRYamide inhibited spontaneous contraction in ad libitum-fed larvae, while exposure of pharynx to GSRYamide did not inhibit contraction in non-fed larvae, indicating that the feeding state changed their sensitivity to inhibitory peptides. These different responses corresponded to different expression levels of their receptors in the pharynx. In addition, injection of AT and GSRYamide decreased esophageal contraction frequencies in the melamine-treated transparent larvae. These findings strongly suggest that these peptides exert feeding inhibitory effects by modulating intestinal contraction in response to their feeding state transition, eventually causing feeding termination

A Homeostatic Sleep-Stabilizing Pathway in <i>Drosophila</i> Composed of the Sex Peptide Receptor and Its Ligand, the Myoinhibitory Peptide

<div><p>Sleep, a reversible quiescent state found in both invertebrate and vertebrate animals, disconnects animals from their environment and is highly regulated for coordination with wakeful activities, such as reproduction. The fruit fly, <i>Drosophila melanogaster</i>, has proven to be a valuable model for studying the regulation of sleep by circadian clock and homeostatic mechanisms. Here, we demonstrate that the sex peptide receptor (SPR) of <i>Drosophila</i>, known for its role in female reproduction, is also important in stabilizing sleep in both males and females. Mutants lacking either the SPR or its central ligand, myoinhibitory peptide (MIP), fall asleep normally, but have difficulty in maintaining a sleep-like state. Our analyses have mapped the SPR sleep function to <i>pigment dispersing factor</i> (<i>pdf</i>) neurons, an arousal center in the insect brain. MIP downregulates intracellular cAMP levels in <i>pdf</i> neurons through the SPR. MIP is released centrally before and during night-time sleep, when the sleep drive is elevated. Sleep deprivation during the night facilitates MIP secretion from specific brain neurons innervating <i>pdf</i> neurons. Moreover, flies lacking either SPR or MIP cannot recover sleep after the night-time sleep deprivation. These results delineate a central neuropeptide circuit that stabilizes the sleep state by feeding a slow-acting inhibitory input into the arousal system and plays an important role in sleep homeostasis.</p></div

<i>MIP</i> encoding SPR ligands is required to stabilize sleep.

<p>(A) Standard sleep plots of pan-neural <i>MIP-RNAi</i> (<i>elav-Gal4, UAS-MIP-IR1</i>) and its control virgin females in a 12-h∶12-h light∶dark cycle (L∶D). Shaded boxes depict dark periods. (B–K) Sleep parameter of virgin females (B–F) and males (G–K) of indicated genotypes. (B, G) Total sleep duration per day. (C, H) Waking activity. (D, I) Sleep bout number per day. (E, J) Mean sleep-bout duration. (F, K) Total sleep duration per day under 12-h∶12-h D∶D condition. Flies used in these assays formed a separate cohort to those in (A–E) and (G–J) (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001974#pbio.1001974.s002" target="_blank">Figure S2</a> for complete data). Number in bars indicates <i>n</i> of the tested flies. Data are shown as means ± SEM. <i>**</i>, <i>p</i><0.01; <i>***</i>, <i>p</i><0.001 for the comparison to both <i>Gal4</i> and <i>UAS</i> controls by Student's <i>t</i> test (all except E, J) and Mann-Whitney U test (E, J).</p

The sleep function of SPR is mapped to <i>pdf</i> neurons.

<p>(A–B) Total sleep duration per day of virgin females of the indicated genotypes. Data are shown as means ± SEM. In (A), <i>n</i> = 16–64 for each bar. <i>***</i>, <i>p</i><0.001 for the comparison both <i>Gal4</i> and <i>UAS</i> controls by Student's <i>t</i> test. In (B), number in bars indicates <i>n</i> of the tested flies. <i>**</i>, <i>p</i><0.01, <i>***</i>, <i>p</i><0.001 for the comparison to <i>w¹¹¹⁸</i> controls by Student's <i>t</i> test. (C) <i>pdf</i> neurons express SPR. Confocal sections of the female brain of indicated genotypes stained by anti-SPR (magenta) and anti-GFP (green). Magenta and green channels are shown separately. Each brain hemisphere has four to five l-LNvs and four s-LNvs, most of which are labeled by anti-SPR (<i>n</i> = 6). Note that SPR expression is broad and not restricted to <i>pdf</i> neurons. Scale bar, 10 µm.</p

Comparisons between MIP protein and mRNA levels suggest synchronized and massive secretion of MIP occurring in the brain between ZT 8 and ZT 20.

<p>(A) Representative confocal images of anti-MIP staining of the <i>w¹¹¹⁸</i> male brain (left) and the ventral nerve cord (VNC, right), isolated at six different time points (ZT 0, 4, 8, 12, 16, and 20). (B) Normalized MIP-immunoreactivity of the indicated CNS areas. <i>n</i> = 15–24 (for the brain regions); <i>n</i> = 5–6 (for the VNC). The letters from “a” to “d” indicate significant differences between ZT groups (<i>p</i><0.05), determined by one-way ANOVA with Tukey's post hoc test. (C) Bright-field images of the male brain stained using <i>in situ</i> hybridization for MIP mRNA transcripts. Scale bars, 50 µm. (D) MIP (red) and per (blue) mRNA transcript levels in the male heads, measured by quantitative reverse transcription PCR and normalized to ZT 0. <i>n</i> = 3. Data are shown as means ± SEM.</p

SPR expression in the CNS is essential for sleep maintenance.

<p>(A) Standard sleep plots of <i>CS</i> control and cantonized <i>SPR</i>-deficient mutant females (<i>CS,SPR</i>^−/−) under 12-h∶12-h light∶dark (L∶D). Shaded boxes depict dark periods. (v) and (m) indicate virgin and mated females, respectively. (B–F, L) Sleep parameter of females of indicated genotypes. Females used in (L) are virgin. (G–K, M) Sleep parameter of males of indicated genotypes. (B, G, L, M) Total sleep duration per day. (C, H) Waking activity. (D, I) Sleep bout number per day. (E, J) Mean sleep-bout duration. (F, K) Total sleep duration per day under 12-h∶12-h D∶D condition. Flies used in these assays formed a separate cohort to those in (A–E) and (G–J) (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001974#pbio.1001974.s002" target="_blank">Figure S2</a> for complete data). Number in bars indicates <i>n</i> of the tested flies. Data are shown as means ± standard error of the mean (SEM). **, <i>p</i><0.01; <i>***</i>, <i>p</i><0.001 for the comparison to <i>CS</i> or <i>w¹¹¹⁸</i> by Student's <i>t</i> test (B–D, F–I, K–M) and Mann-Whitney U test (E, J).</p

MIP and SPR are required for maintaining sleep homeostasis.

<p>(A) Experimental protocol used to quantify sleep homeostasis. (B–E) The percentage change in sleep (% Δsleep) after 12 h of mechanical sleep deprivation (green), 6 h (blue), and 12 h of sleep recovery (red) in females of indicated genotypes, measured the next morning. Number in bars indicates <i>n</i>. Data are shown as means ± SEM. <i>*</i>, <i>p</i><0.05, **, <i>p</i><0.01 ***, <i>p</i><0.001 for the comparison to each controls by Student's <i>t</i> test.</p

A model depicting an operating principle of three major metabotropic pathways that regulate sleep.

<p>During the day, light and modulatory inputs from other brain areas activate l-LNvs to secrete PDF, a major arousal-promoting factor. PDF from l-LNvs in turn activates the motor control center directly or indirectly through s-LNvs, and keeps flies awake. During the day, the sleep pressure gradually builds up and stimulates <i>MIP-LMIo</i> neurons to secrete MIP, which attenuates PDF release by modulating s-LNvs and allows the daytime sleep to ensue. Released MIP is expected to act on l-LNvs as well, but its effects are probably cancelled by direct excitatory inputs from light, octopamine, and dopamine. At the beginning of the night, waning excitatory inputs and elevating GABAergic input to l-LNvs (mainly through ionotropic GABA_A receptor) drive flies fall asleep (not shown in the model). Thereafter, three metabotropic pathways (sNPF, MIP, and GABA) stabilize and maintain sleep state during the night by supplying inhibitory modulations to l-LNvs, and keeping them from releasing PDF. <i>MIP-LMIos</i> function as a sleep-pressure sensor and adjust their secretory activities accordingly. As the night approaches the end, sleep pressure declines and metabotropic GABA input is needed.</p

Axonal processes of <i>MIP-LMIo</i> neurons innervate the dendritic field of <i>pdf</i> neurons.

<p>(A) A confocal image of <i>pdf-Gal4/UAS-EGFP</i> fly brain stained with anti-MIP (green) and anti-EGFP (magenta) antibodies. The brain is oriented with dorsal up. (B) A high magnification confocal image of the optic lobe of <i>pdf-Gal4/UAS-DenMark</i> fly brain stained with anti-MIP (green) and anti-RFP (magenta) antibodies. Anti-RFP labels a dendrite marker, DenMark, which visualizes the dendritic field of <i>pdf</i> neurons. Asterisks indicate somata of <i>MIP-LMIo</i> neurons. Sections perpendicular to the dotted yellow lines are shown separately in (B′) and (B″). (B′) shows MIP-immunoreactive processes (yellow arrows) innervate the dendritic field of <i>pdf</i> neurons (yellow arrowheads). (B″) shows some MIP-labeling (arrows) occurs near the dendritic field of <i>pdf</i> neurons (arrowheads), indicating that MIP can also function as a paracrine factor. White arrows labeled with d, a, m indicate dorsal, anterior, and medial orientations, respectively. Scale bars, 50 µm.</p