Prothoracicotropic hormone regulates developmental timing and body size in Drosophila - PubMed (original) (raw)

Prothoracicotropic hormone regulates developmental timing and body size in Drosophila

Zofeyah McBrayer et al. Dev Cell. 2007 Dec.

Abstract

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 overgrowth 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.

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Figures

Figure 1

Drosophila PTTH sequence, phylogeny and gene structure (A) Alignment of the D. melanogaster PTTH amino acid sequence to the indicated Lepidoptera species using the Clustal W alignment tool in MacVector ™. The dashed blue over line indicates a hydrophobic segment that likely serves as a signal peptide. The green arrowhead is positioned after a dibasic sequence that might serve as a maturation site for Drosophila PTTH. A green line is positioned at the maturation site of the Lepidoptera PTTH sequences. The red boxes indicated conserved cysteine residues. (B) Dendogram generated using the Clustal W tool of MacVector for several insect PTTH genes. Black lines Lepidoptera, green mosquito, and red Drosophilae. (C) The 5′ end gene structure of of various predicted and isolated Drosophila PTTH cDNAs. The numbers on top are base pairs starting at the first predicted methionine residue. The Flybase annotation of CG13687 starts at methionine 2 of the predicted sequence but the frame is open upstream for another 24 AA to a second potential start codon (M1). Line two shows the structure of a cDNA isolated by RT-PCR from third instar brain. It uses a different splice donor site such that the upstream sequence is in the -2 frame relative to the Flybase sequence and would use methionine 3 as a start. The third line indicates the structure of a cDNA isolated from a disc/brain library (Brown et al. 1988). In this case, the first intron is retained and the first in frame methionine (M4) is positioned downstream of the first intron splice acceptor site.

Figure 2

In situ hybridization of an antisense ptth RNA probe to a stage 17 embryo (A) and wandering third instar brain (B). The black circle in A indicates the position of two positive neurons (enlarged in the inset) that correspond to one of the neuron pairs shown in B. (C) No staining is seen in a third instar brain hybridized with a sense control probe. (D) The dendritic arbors and axon terminals (yellow arrow) of the PG neurons are highlighted using _ptth_-Gal4 to drive expression of a membrane-bound UAS- cd8GFP reporter. (E) Immunolocalization of PTTH using an HA-tagged genomic transgene. The yellow arrow indicates synaptic connections on the prothoracic gland. (F) Immunolocalization of PTTH-HA (green) and the ecdysone biosynthetic enzyme Phm (red) in the prothoracic gland. (G) Axons from the PDF-producing neurons (red) contact the dendritic arbors of PTTH-producing neurons.

Figure 3

Transcriptional profile of Drosophila ptth in wildtype y,w1118 and in pdf01 mutant as determined by semi-quantitative RT-PCR (A) Representative profile of ptth transcription in wild type animals. (B) Representative profile of ptth expression in pdf01 mutant. Top panels show the quantitative evaluation of ptth transcript levels using Scion Image software and the bottom panels the chemiluminescent detection... RPL17 transcription serves as an internal control. (C) Variations of ptth expression in wild type flies and pdf01 mutants. Mean values over three independent series of larvae were calculated. For each graph, the time of development is expressed in hours post ecolsion and intensity is given in arbitrary units. In C the dashed line and solid lines indicate the means of the pdf and wt measurements respectively.

Figure 4

Loss of PG neurons generates large flies while ectopic PTTH leads to small flies. (A) Expression of ptth > Gal4/UAS-cd8GFP in the PG neurons of a wandering third instar larva (B) Coexpression of UAS-Grim (2X) with ptth_-Gal4 (2X) results in loss of the PG neurons (no UAS-cd8GFP staining in a brain from a wandering third instar). (C–E) Crawling third instar larva, pupa and adult females, respectively, produced by ablation of PG neurons (left animal) or from control lines containing UAS_-grim alone (middle animal) or _ptth_-Gal4 alone (right animal). (F) Wing hair density in a 100 μ2 square section (n = 9 wings) located on the dorsal surface adjacent to the posterior crossvein (error bars SEM). (G) Overlay of wings produced from a PG neuron-ablated fly or a fly containing the UAS-Grim construct alone with no driver. (H) Over-expression of PTTH produces small flies compared to expression of Gal4 alone. (I) Overlay of a wing produced by overexpression of PTTH compared to a wing produced by the driver alone. (J) Pupal lengths and adult weights of animals with the indicated genotypes. Photographing individual pupa and measuring pixel number compared to a calibrated standard determined pupal lengths. Adult flies were weighed in batches of 10–30 flies and the average weight per fly determined (error is SEM).

Figure 5

Ablation of PG neurons produces developmental delay. (A) The percentage of larvae of the indicated genotype that had ecdysed to the 2nd instar stage (A) third instar stage (B) or undergone pupariation (C) are plotted relative to the time in hours after egg laying (AEL). Dark blue is UAS-Grim, turquoise is _ptth_-gal4 and red is ablated larvae. N = number of repetitions containing 8–10 larvae per sample. Results are expressed as mean ±-standard error of the mean.

Figure 6

Ablation of PG neurons results in a greater critical weight but no change in growth rates. (A) Percent of larvae that underwent pupariation after starvation at a given size after ecdysis to third instar (L3) for PTTH ablated larvae (ptth > Grim), ptth > Gal4 larvae and UAS-Grim larvae (N = 15–36 individuals at each data point). The critical weights that correspond to 50% threshold for pupariation were 2.5 mg, 0.72 and 0.71 for PTTH ablated larvae, ptth > Gal4 larvae and UAS-Grim larvae, respectively. (B) Plot of weight at a given time after L3 ecdysis for PTTH ablated larvae (ptth > Grim), ptth > Gal4 larvae and UAS-Grim larvae (N = 15–36 individuals at each data point).

Figure 7

Ablation of PG neurons results in a delayed rise in ecdysteroid titers (A) that is rescued by feeding larvae 20E (B), and produces an asynchronous developmental transcriptional profile (C) and low the transcription of several ecdysone biosynthetic enzymes prior to metamorphosis (D,E). (A) Ecdysteroid titer in pg/10 larvae plotted against time after ecdysis to third instar. (B) Larvae in which the PG neurons were ablated were fed food containing 0.33mg/ml 20E or control food and the time to pupariation measured. (C) Temporal profiles of 20E-regulated transcription in UAS-grim, ptth-GAL4, and ptth-GAL4;UAS-grim animals. Total RNA was isolated from animals staged in hours after the L2 to L3 molt. UAS-grim 48 hour, ptth-GAL4 96 hour, and ptth-GAL4; UAS-grim 192 hour animals were isolated as newly formed prepupae. Hybridizations were performed to detect the EcR, E74, and BR-C early 20E-inducible mRNAs, as well as tissue-specific genes, IMP-L1, Fbp-1, ng-1, and Sgs-4. rp49 was included as a control for loading and transfer. (D-I) Q-PCR analysis of the transcriptional levels of the indicated genes involved in ecdysteroid biosynthesis for PTTH ablated larvae (ptth > Grim), ptth-Gal4 larvae and UAS-Grim larvae (mean ± SEM, n = 3). The levels were normalized to ribosomal protein rpL23 transcriptional levels in the same samples.

References

1. Agui N, Granger NA, Gilbert LI, Bollenbacher WE. Cellular localization of the insect prothoracicotropic hormone: In vitro assay of a single neurosecretory cell. Proc Natl Acad Sci U S A. 1979;76:5694–5698. - PMC - PubMed
1. Andres AJ, Fletcher JC, Karim FD, Thummel CS. Molecular analysis of the initiation of insect metamorphosis: a comparative study of Drosophila ecdysteroid-regulated transcription. Dev Biol. 1993;160:388–404. - PubMed
1. Bollenbacher WE, Vedeckis WV, Gilbert LI. Ecdysone titers and prothoracic gland activity during the larval-pupal development of Manduca sexta. Dev Biol. 1975;44:46–53. - PubMed
1. Brand AH, Perrimon N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development. 1993;118:401–415. - PubMed
1. Caldwell PE, Walkiewicz M, Stern M. Ras activity in the Drosophila prothoracic gland regulates body size and developmental rate via ecdysone release. Curr Biol. 2005;15:1785–1795. - PubMed

Prothoracicotropic hormone regulates developmental timing and body size in Drosophila - PubMed (original) (raw)