(C) PLOS One This story was originally published by PLOS One and is unaltered. . . . . . . . . . . Serotonergic neuron ribosomal proteins regulate the neuroendocrine control of Drosophila development [1] ['Lisa Patricia Deliu', 'Clark H Smith Brain Tumour Centre', 'Arnie Charbonneau Cancer Institute', 'Alberta Children S Hospital Research Institute', 'Department Of Biochemistry', 'Molecular Biology Calgary', 'University Of Calgary', 'Alberta', 'Michael Turingan', 'Deeshpaul Jadir'] Date: 2022-11 The regulation of ribosome function is a conserved mechanism of growth control. While studies in single cell systems have defined how ribosomes contribute to cell growth, the mechanisms that link ribosome function to organismal growth are less clear. Here we explore this issue using Drosophila Minutes, a class of heterozygous mutants for ribosomal proteins. These animals exhibit a delay in larval development caused by decreased production of the steroid hormone ecdysone, the main regulator of larval maturation. We found that this developmental delay is not caused by decreases in either global ribosome numbers or translation rates. Instead, we show that they are due in part to loss of Rp function specifically in a subset of serotonin (5-HT) neurons that innervate the prothoracic gland to control ecdysone production. We find that these effects do not occur due to altered protein synthesis or proteostasis, but that Minute animals have reduced expression of synaptotagmin, a synaptic vesicle protein, and that the Minute developmental delay can be partially reversed by overexpression of synaptic vesicle proteins in 5-HTergic cells. These results identify a 5-HT cell-specific role for ribosomal function in the neuroendocrine control of animal growth and development. Ribosomes are essential cellular organelles required for production of new proteins. Extensive studies have shown how ribosome synthesis and function are important for growth at the cellular level, but less is known about how these processes control tissue and body growth. We have explored this issue by studying a class of Drosophila (fruit fly) mutants–the Minutes–which lack one functional copy of a ribosomal protein gene and, as a result, show a slowed rate of development from embryo to adulthood. An important phase in the life cycle of animals is the transition from the juvenile, growth stage to sexually mature adults. In fruit flies, this occurs as larvae mature into pupae and is controlled by a pulse of the steroid hormone ecdysone, which is secreted from an endocrine organ called the prothoracic gland (PG). We find that Minute animals have disrupted production of ecdysone and we pinpoint this defect to loss of ribosomal protein function within a subset of neurons that project from the brain to the PG and that control ecdysone production through the neurotransmitter, serotonin. Our results highlight how ribosomal protein function in a subset of neurons can control overall body growth and development. Funding: This work was supported by CIHR Project Grants (PJT-173517, PJT-152892) and an NSERC discovery grant to S.S.G. L.P.D. was supported by a Clark Smith Brain Tumour Centre graduate studentship. M.T. was supported by a Cumming School of Medicine Graduate Scholarship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Although much has been learned about cell competition, less is known about why rp/+ show a delay in development. Interestingly, some recent studies of the disc-intrinsic, mechanisms of rp/+ cell competition effects can also partially account for the organismal delay in development. For example, disc-specific Rp knockdown stimulates Xrp1 induction of dilp8 [ 49 ], and both loss of Xrp1 and disc-specific knockdown of dilp8 can each partially reverse the delay in development seen in rp/+ animals [ 47 , 50 , 51 ]. Loss of Rp function specifically in the PG can also explain the overall delay in development in rps6/+ animals [ 52 ]. These results suggest the overall delay in organismal development seen in rp/+ animals may result from specific tissue non-autonomous effects of Rps. However, the extent of these non-autonomous effects is not fully clear. An interesting class of mutants that exhibit alterations in larval development are the Minutes [ 39 , 40 ]. These are dominant mutants that are classically described by their developmental delay and short bristles. Almost all Minutes are rp/+ mutants and they have perhaps been best studied in context of cell competition, a process in which mosaic clones of rp/+ cells in imaginal disc epithelia are outcompeted and killed by surrounding wild-type (+/+) cells. Several mechanisms have been described to account why rp/+ cells are outcompeted including altered proteostasis [ 41 , 42 ], competition for dpp growth factor [ 43 ], induction of innate immune signaling [ 44 , 45 ], and induction of the transcription factor Xrp1 [ 46 , 47 ]. Given that rp genes are spread across the genome, cell competition may be a surveillance process to eliminate aneuploid cells, as marked by reduced rp gene copy number, to ensure proper tissue growth and homeostasis [ 48 ]. Drosophila larvae have provided an excellent model system in which to define the cell-, tissue- and body-level mechanisms that control developmental growth [ 6 , 24 , 25 ]. Larvae grow almost 200-fold in mass over 4–5 days before undergoing metamorphosis to the pupal stage. This developmental transition is controlled by a pulse of secretion of the steroid hormone, ecdysone, from the prothoracic gland (PG), which then acts on tissues to stimulate pupation at the end of the larval period [ 26 – 28 ]. The timing of this pulse is under control of two separate subsets of neurons expressing either the neuropeptide, PTTH, or the neuromodulator serotonin (5-HT), that each innervate the PG and stimulate ecdysone production [ 29 – 31 ]. This neuroendocrine network integrates signals from the environment and other tissues to ensure proper timing of the ecdysone pulse and the larval-pupal transition. For example, nutrient signals can act on both the 5-HT neurons and the PG to ensure proper coupling of development maturation with nutrients [ 31 , 32 ]. Epithelial disc damage also leads to a delay in larval development to allow time for proper tissue regeneration before transition to the pupal stage. One way that this delay is mediated is by suppression of PTTH signaling by dilp8, an insulin/relaxin-like peptide that signals from damaged discs to a subset of Lgr3 receptor expressing neurons that inhibit PTTH neuronal activity [ 33 – 37 ]. In addition, the inflammatory cytokine, Upd3 can signal directly from damaged discs to the PG to suppress ecdysone and delay development [ 38 ]. The complex links between ribosome function and animal development are exemplified by the organismal biology of ribosomal proteins (Rps) [ 8 , 9 ]. Metazoan ribosomes have 70–80 Rps, and mutants for almost all of these are homozygous lethal in animals, emphasising their essential role in ribosome synthesis and function. However, in many cases Rp mutants show dominant phenotypes as heterozygotes. These phenotypes are often specific to the affected Rp and can give rise to tissue-specific effects that cannot be explained simply by lowered overall protein synthesis and growth rates. For example, in zebrafish certain rp/+ mutants can develop peripheral nerve tumors [ 10 ]. Similarly, some Drosophila Rp mutants develop selective tissue overgrowth phenotypes [ 11 , 12 ]. Several Rp/+ mutants in mice have also been shown to each exhibit tissue specific developmental defects that differ based on the Rp affected. For example, rpl38/+ mice show specific skeletal segmentation defects [ 13 ], rps14/+ mice show defects in blood development [ 14 ], and rpl27a/+ mice show defects in cerebellar development [ 15 ]. The dominant effects of rp/+ mutations also extend to humans, where several pathologies, collectively termed ribosomopathies, are caused by heterozygosity for Rp mutations, and lead to tissue-specific effects such as blood disorders, congenital growth defects, and predisposition to cancer [ 16 – 18 ]. The mechanisms that determine these dominant effects of rp/+ mutations are not fully clear but are thought to involve selective alterations in mRNA translation. These alterations may occur either as a result of lowered ribosome numbers or due to ribosome heterogeneity, where ribosomes with different complements of Rps have been proposed to have different translational properties [ 19 – 23 ]. These studies emphasise the importance of further work to understand how Rp function contributes to organismal growth and development. The regulation of ribosome and protein synthesis are conserved mechanisms of growth control. Several decades of studies in unicellular systems such as E. coli, yeast and cultured mammalian cells have defined both the signaling pathways that couple growth cues to ribosome synthesis and function, and the mechanisms by which changes in mRNA translation drive cell growth and proliferation [ 1 – 4 ]. However, the mechanisms that operate in whole animals during developmental growth are less clear. In these contexts, body growth is not determined solely by processes that govern cell-autonomous growth, but also by inter-organ communication to ensure coordinated growth and development across all tissues and organs [ 5 – 7 ]. Hence, tissue specific changes in ribosome function have the potential to mediate non-autonomous effects on whole-body physiology to control organismal development. Results Rps13/+ animals do not show a global decrease in ribosome levels or protein synthesis For our study we used flies heterozygous for a previously characterized allele of ribosomal protein S13, P[lacW]M (2)32A (hereafter referred to as rpS13/+ animals), which have decreased expression of rpS13 mRNA (S1A Fig) and have been observed to have the classic Minute phenotype of shorter and thinner bristles and a delay in larval development [53]. We quantified the delay in development of rpS13/+ and controls (w1118) by measuring the time it took for animals to reach the pupal stage after egg laying. We found that rpS13/+ animals were delayed in development by about 40 hours, which corresponds to a delay of approximately 20% compared to control animals (Fig 1A). We also measured body size as the larvae developed, and we saw that rpS13/+ larvae had a smaller size compared to age-matched control animals at different stages of larval development (S1B Fig). However, due to their prolonged larval period, the rpS13/+ animals grew for a longer time. Hence, when we measured both final larval and pupal size, we found that, in both cases, rpS13/+ animals were about 12% larger than controls (Figs 1B and 1C and S1C). We measured mouth hook movements as measure of feeding rate and saw a small, but significant, increase in rpS13/+ larvae when compared to controls (S1D Fig). This indicates that the growth and developmental phenotypes of rpS13/+ animals do not result simply from reduced feeding. These data suggest that rpS13/+ animals exhibit a reduced growth and developmental rate. PPT PowerPoint slide PNG larger image TIFF original image Download: Fig 1. Developmental delay of rpS13/+ animals is not due to a decrease in ribosome numbers or reduced translation rates. (A) Developmental timing from larval hatching to pupation of w1118 and rpS13/+ animals, n = 147 and 170 respectively. Data are presented as +/- SEM. *p < 0.05, Mann-Whitney U test. (B) Representative images of w1118 and rpS13/+ pupae. Scale bar, 1000 μm. (C) Pupal volume of rpS13/+ (n = 212) and w1118 controls (n = 229). Data are presented as +/- SEM. *p < 0.05, Mann-Whitney U test. (D) Transcript levels of 18S and 28S rRNA in whole-body RNA samples from w1118 and rpsS13/+ third instar wandering larvae. n = 4 independent samples per genotype. Data are presented as +/- SEM. ns = not significant, Student’s t-test. (E) Relative protein concentration levels from third instar wandering w1118 and rpsS13/+ larvae. Absorbance was measured at 465nm using the Bradford assay, n = 5 independent samples per genotype. Individual data points are plotted, and the bars represent mean +/- SEM. ns = not significant, Student’s t-test. (F) Whole-body puromycin labelling of w1118 and rpsS13/+ third instar wandering larvae. Left, Ponceau S staining showing total protein. Right, anti—puromycin and anti—eIF2∝ (loading control) immunoblots. (G) Quantification of puromycin staining, n = 6 independent samples per genotype. Data are presented as +/- SEM. ns = not significant, Student’s t-test. https://doi.org/10.1371/journal.pgen.1010371.g001 Studies in different model systems have shown that the phenotypes seen in rp/+ animals are often associated with lowered ribosome numbers and reduced protein synthesis [21]. We therefore investigated ribosome levels and protein synthesis in rpS13/+ animals. In order to measure ribosome numbers, we measured mature 18S and 28S rRNA in wandering L3 whole larval lysates. We saw no significant difference in rRNA levels between rpS13/+ and control larvae (Fig 1D). Total protein content in wandering L3 larval lysates also showed no significant difference in rpS13/+ larvae compared to control larvae (Fig 1E). Finally, we investigated whether rpS13/+ animals show a decrease in protein synthesis rate. To do this we used a puromycin labelling assay [54]. We first quantified the levels of puromycin incorporation of rpS13/+ and control animals at the wandering larval stage in order to developmentally match control and rpS13/+ animals and found no significant difference in protein synthesis rates (Fig 1F and 1G). We repeated this assay at two other earlier time points with aged-matched larvae, and once again found no decrease in translation rates in rpS13/+ larvae compared to control larvae (S2A and S2B Fig). This suggests that the Minute delayed development is not due to a global loss of ribosome numbers or translational capacity. rpS13/+ animals show a defect in ecdysone signalling The duration of the larval period is controlled in large part by the steroid hormone, ecdysone [27]. In particular, at the end of larval development, a neuro-endocrine circuit stimulates a pulse of ecdysone production and secretion from the prothoracic gland (PG). This circulating ecdysone then acts on larval tissues to trigger the larval to pupal transition. Any defects in this neuro-endocrine circuit leads to a delay in larval development to the pupal stage. Given their delayed development, we examined whether rpS13/+ animals show a defect in ecdysone signaling. We did this by measuring the transcript levels of phantom and spookier both of which encode enzymes for PG ecdysone production. As previously described, both showed maximal expression peaks at 120 hours AEL in control animals, consistent with the ecdysone pulse that triggers pupation (Fig 2A). However, in rpS13/+ animals these peaks were delayed by about one day (144 hours) and continued to show expression even at 168 hours for larvae that were still wandering (Fig 2A), suggesting a delay in ecdysone signalling. We also found that feeding larvae 20-hydroxyecdysone (20-HE) was able to partially reverse the development timing delay seen in the rpS13/+ by about one third of the total delay (Fig 2B). Ecdysone synthesis in the PG can be stimulated by several different signaling pathways, including the Ras/ERK and TOR kinase pathways [32,55,56]. When we overexpressed the TOR activator, Rheb in the PG, we found that while it had no effect on developmental timing in control animals, it was sufficient to partially reverse the development timing delay seen in the rpS13/+ animals again by about one third of the total delay (Fig 2C). Together, these data indicate that rpS13/+ animals exhibit a delay in development that can be explained in part due to blunted ecdysone signaling. PPT PowerPoint slide PNG larger image TIFF original image Download: Fig 2. Delayed development in rpS13/+ animals is due to impaired ecdysone production. (A) qRT-PCR measurements of phantom and spookier mRNA levels in w1118 and rpS13/+ larvae. n = 4 independent samples per genotype. Data are presented as +/- SEM. *p < 0.05, two-way ANOVA and post-hoc Student’s t-test. (B) Time to pupation of w1118 and rpS13/+ larvae grown in control food or food supplemented with ecdysone (20HE). Data are presented as +/- SEM. *p < 0.05, Mann-Whitney U test. n = 142 (+/+), 92 (+/+ with 20HE), 148 (rpS13/+), 116 (rpS13/+ with 20HE). (C) Time to pupation of +/+ and rpS13/+ larvae with or without UAS-rheb expression in the prothoracic gland using P0206-Gal4. Data are presented as +/- SEM. ns = not significant, *p < 0.05, Mann-Whitney U test. n = 152 (+/+), 157 (UAS-rheb/+), 151 (rpS13/+), 144 (UAS-rheb/rpS13). https://doi.org/10.1371/journal.pgen.1010371.g002 [END] --- [1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1010371 Published and (C) by PLOS One Content appears here under this condition or license: Creative Commons - Attribution BY 4.0. via Magical.Fish Gopher News Feeds: gopher://magical.fish/1/feeds/news/plosone/