InteractiveFly: GeneBrief

nucleostemin3: Biological Overview | References

Growth and body size are regulated by the CNS, integrating the genetic developmental program with assessments of an animal's current energy state and environmental conditions. CNS decisions are transmitted to all cells of the animal by insulin/insulin-like signals. The molecular biology of the CNS growth control system has remained, for the most part, elusive. This study identifies NS3, a Drosophila nucleostemin family GTPase, as a powerful regulator of body size (Note: this gene is likely to be renamed by FlyBase to become ns1). ns3 mutants reach less than 60% of normal size and have fewer and smaller cells, but exhibit normal body proportions. NS3 does not act cell-autonomously, but instead acts at a distance to control growth. Rescue experiments were performed by expressing wild-type ns3 in many different cells of ns3 mutants. Restoring NS3 to only 106 serotonergic neurons rescues global growth defects. These neurons are closely apposed with those of insulin-producing neurons, suggesting possible communication between the two neuronal systems. In the brains of ns3 mutants, excess serotonin and insulin accumulate, while peripheral insulin pathway activation is low. Peripheral insulin pathway activation rescues the growth defects of ns3 mutants. The findings suggest that NS3 acts in serotonergic neurons to regulate insulin signaling and thus exert global growth control (Kaplan, 2008).

The overall size of an animal is determined by the number of cell divisions, the rate of destruction of cells, and the average size of cells. With some species, size is a relatively rigid outcome of the genetically controlled program of development. Other species, such as many fish and trees, can continue to grow through much of their lives. In both cases growth is influenced by continuing assessment of the energy state of the growing organism and the availability of nutrients, interpreted in the context of the organism's genetic program. The cell-intrinsic machinery that specifies whether or not a cell should divide and how large a cell should grow has been unveiled in elegant molecular detail (Conlon, 1999; Edgar 2006). Much remains to be learned about the cell-extrinsic mechanisms that coordinate the growth behaviors of individual cells. Global cell-extrinsic controls are needed to ensure that a properly proportioned animal is produced, with a size suited to its environment and genetic program (Kaplan, 2008).

Whole-animal growth control can be envisioned as having three components that operate in concert: (1) sensory and homeostatic inputs, (2) processing within the CNS, and (3) instructive outputs to the periphery. Sensory inputs to the CNS provide information regarding the energy status of the organism and the available nutrient status of the environment. The CNS assesses this information in light of the genetic program and instincts about future energy requirements; e.g., for growth, reproduction, migration, and hibernation. The CNS converts the processed information into output signals that alter feeding behavior and spread through the body to coordinate growth. The transmitted output signals instruct cells in peripheral tissues to grow, to cease growth, or to die, by influencing cell-intrinsic programs. Recent studies have shed light on the input and output signals involved in growth control. Much less is known about the central processing mechanisms (Kaplan, 2008).

A great deal of elegant work has identified signaling mechanisms by which energy states are sensed and the information is relayed to the CNS (Morton, 2006; Melcher, 2007). In mammals, dietary free fatty acids can act on the anterior pituitary gland to inhibit growth hormone secretion. In Drosophila, the fat body, a major metabolic organ that performs the combined functions of the liver and adipocytes of mammals, has been established as an important source of nutrient sensing. In response to high levels of amino acids in the fat body, this organ is thought to produce an as-yet-unknown signal that acts in the brain to suppress food intake (Zinke, 1999). Amino acid starvation results in strongly suppressed production of a fat body-derived glycoprotein known as Drosophila acid-labile subunit (dALS) (Colombani, 2003). When dALS is produced, it binds to and regulates the bioavailability of Drosophila insulin-like peptides (DILPs) secreted by the neurosecretory cells of the brain (Arquier, 2008), thus connecting sensed amino acid levels to the activity of a critical growth signal (Kaplan, 2008).

The Drosophila insulin system controls cell-intrinsic growth programs throughout the body. DILPs are secreted when nutrients are plentiful to stimulate growth of target tissues. DILPs are produced primarily by insulin-producing cells (IPCs), groups of seven neuroendocrine cells located in each of the two brain lobes, and then enter the circulation. The DILPs interact with insulin receptor tyrosine kinases (InRs) on target cells, activating a signaling cascade that ultimately stimulates growth through an increase in protein synthesis. Disruption of the insulin signaling pathway through either genetic ablation of the IPCs or mutation of intracellular components of the pathway leads to major developmental delay, growth retardation, and reduced adult size (Kaplan, 2008).

Identifying the mechanisms operating within the CNS that serve to integrate environmental, nutritional, and physiological information to direct proper growth responses will require identifying components of these pathways and the neurons in which they act. This study identified NS3, a nucleostemin-family GTPase, as a powerful regulator of body size. Nucleostemins were discovered (Tsai, 2002) as genes highly expressed in human stem cells compared with differentiated cells, and their expression has been linked to certain types of cancer. NS3 is related to a yeast protein, Lsg1p, which functions in ribosome biogenesis; a role that is essential for growth of yeast cells (Kallstrom, 2003; Hedges, 2005). Surprisingly, this study finds that Drosophila NS3 is not required for growth in most cells, but rather acts specifically within serotonergic neurons to regulate insulin signaling and exert global control over cell size and number (Kaplan, 2008).

NS3 is part of a large family of YlqF-Related GTPase (YRG) proteins that are evolutionarily conserved in organisms as diverse as bacteria and mammals (Reynaud, 2005). Some nucleostemin proteins are located in nucleoli, and a yeast member of the family, Lsg1p, has been implicated in ribosome assembly. ns3 mutant animals grow slowly and reach an adult weight only ~60% that of controls. Based on the role of Lsg1p, it is suspected that NS3 would contribute to protein synthesis and thus cell growth. That view predicts a cell-autonomous function of NS3 within cells whose size and number are decreased in ns3 mutants, just as Minute mutations that cause lowered ribosome protein production reduce Drosophila body size (Kaplan, 2008).

Contrary to the cell-autonomous protein synthesis hypothesis, NS3 was found to play its major role in the nervous system. Clones of ns3 mutant cells in peripheral tissues such as eye and wing were normal in size, indicating that NS3 is not required for the growth of most cells, and suggesting instead that NS3 is important for signaling events that influence cell growth and division. To identify where in the animal NS3 acts to control growth, potentially rescuing ns3 activity was introduced into a large variety of cell types; rescue occurs only when the protein is expressed in the nervous system. From these findings it is also concluded that NS3 is unlikely to be secreted, since in that case ns3 expressed in tissues such as muscle, epidermis, or fat body would be expected to provide a rescuing effect (Kaplan, 2008).

The data suggest that NS3 functions in serotonergic neurons as part of the central neural regulation of growth. This conclusion is supported by experiments demonstrating that (1) in the absence of NS3, serotonin and insulin levels in the brain are elevated, while peripheral insulin pathway activation is reduced, and growth is severely disrupted. (2) Reintroduction of NS3 into serotonergic neurons of ns3 mutant animals relieves the buildup of insulin in IPCs and rescues the growth defects in these animals. Thus, a signal from serotonergic neurons controls insulin levels in IPCs. (3) Activation of insulin signaling in target tissues can bypass the growth defects in ns3 mutants. Thus, the action of NS3 in serotonergic neurons lies upstream of insulin signaling in the growth control hierarchy (Kaplan, 2008).

Human NUCLEOSTEMIN was cloned in a screen for genes that are highly expressed in stem cells and that are repressed upon stem cell differentiation (Tsai, 2002). NUCLEOSTEMIN is abundant in many undifferentiated cell types, is scarce in most differentiated cells, and is important for maintaining stem cells and cancer cells in a proliferative state (Tsai, 2002). Another member of this family, Lsg1p, is the yeast protein most closely related to Drosophila NS3. Lsg1p functions in ribosome biogenesis; a role that is essential for growth of yeast cells (Kallstrom, 2003; Hedges, 2005). Drosophila NS3 is a non-cell-autonomous regulator of growth that functions in serotonergic neurons to control body size. How could these three closely related proteins perform such different functions (Kaplan, 2008)?

One difference may be subcellular protein localization. In eukaryotes, members of the YRG protein family are found in the cytoplasm, mitochondria, nucleus, and nucleolus (Reynaud, 2005). NUCLEOSTEMIN is a nucleolar protein (Tsai, 2002), while Lsg1p has a somewhat punctate, cytoplasmic localization (Kallstrom, 2003). NS1 and NS2 are concentrated in the nucleolus, while NS3 is primarily located in the cytoplasm, in well-defined puncta. Thus, the localization of Drosophila NS3 is more similar to its yeast ortholog Lsg1p than to human NUCLEOSTEMIN or to the most closely related Drosophila orthologs, NS1 and NS2 (Kaplan, 2008).

The other members of the family, in order of increasing divergence from human NS, have been designating NS2 (CG6501 also termed Ngp), NS3 (CG14788), and NS4 (CG9320). These proteins share a common domain structure, consisting of a basic domain at their N termini, coiled-coil domains, GTP-binding motifs, a putative RNA-binding domain, and an acidic domain near their C termini. The GTP-binding motifs are circularly permuted compared with their arrangement in most GTPases. The G1–G4-binding domains are ordered G4–G1–G2–G3 from the N to C termini, rather than the canonical G1–G2–G3–G4 orientation observed in Ras family GTPases. NS1 and NS2 expressed as YFP-fusion proteins in cultured Drosophila S2 cells share the nucleolar localization of human NS, but NS3 has a punctate, cytoplasmic distribution. The same punctate, mostly cytoplasmic localization pattern of NS3-YFP was observed when it was produced in larval salivary glands and neurons. The NS3-YFP fusion protein is functional. The N-terminal basic domain has been shown to be required for the nucleolar localization of human NS (Tsai, 2002). The number and arrangement of N-terminal basic residues varies between members of the Drosophila Nucleostemin family and is lowest in NS3, which may explain its distinct localization (Kaplan, 2008).

How, then, can the striking difference in function between Lsg1p and NS3 be explained? Although NS3 and Lsg1p regulate growth in very different ways, they may be acting similarly at a molecular level. In addition to a function in ribosome biogenesis, Lsg1p may play a role in translation (Kallstrom, 2003). It is possible that NS3 also regulates translation, but to a different end. Lsg1p and NS3 share a conserved RNA-binding domain. Using this domain, NS3 could, for example, regulate the translation of a factor involved in serotonin metabolism or release. Identifying proteins and/or RNAs bound to NS3 in serotonergic neurons may be a means of addressing this question (Kaplan, 2008).

Serotonin modulates the development and function of a number of different neuronal signaling systems. Most serotonergic cells are interneurons acting entirely within the CNS, where they serve an important processing role-integrating informational inputs and converting them into instructive outputs. It is proposed that signals from serotonergic neurons modulate insulin secretion, control growth, and thus coordinate the formation of proportional sizes of tissues and organs (Kaplan, 2008).

Reintroduction of NS3 into only 106 serotonergic neurons substantially rescued the growth defects seen in ns3 mutant animals. Complete rescue might be achieved through additional expression in certain other, as yet unknown cells, or might depend on precise times or amounts of production in the serotonergic neurons. Even relatively imprecisely controlled NS3 production in serotonergic neurons provides as much rescue as expression in the entire nervous system and restores close-to-normal body size, so it is thought that the main location of NS3 action with respect to body size control has been found. The DDC-Gal4 driver that was used to drive expression in serotonergic neurons also drives expression in dopaminergic neurons. Because expression in dopaminergic neurons alone gave no rescue, it is concluded that NS3 acts specifically in serotonergic neurons to control growth. However, the possibility exists that NS3 activity is required simultaneously in both serotonergic and dopaminergic neurons. Once a Gal4 transgene that drives expression only in serotonergic neurons is available, this question can be addressed (Kaplan, 2008).

Projections from serotonergic neurons are closely apposed with those of insulin-producing neurons, so IPCs are favorably positioned to receive direct input from serotonergic neurons and indeed to send signals back. In accordance with control of insulin secretion by serotonergic neurons, it was observed that the brains of ns3 mutants contain higher than normal serotonin and insulin, while peripheral insulin pathway activation was reduced. Local, peripheral activation of Akt, a kinase that functions as a key transducer of the insulin signaling pathway, rescued the growth defects of ns3 mutants. These data show that the ability of insulin pathway transduction to drive growth is preserved in the mutants. NS3 acts within the CNS to control growth signaling via the insulin pathway, and thus exerts global control over cell size and number (Kaplan, 2008).

While it is suggested that the buildup of insulin observed in ns3 mutants is due to a defect in insulin secretion, the tools necessary to address this question directly do not yet exist. Once an assay is developed that is sensitive enough to detect circulating levels of Drosophila insulins, it will be possible to measure changes in insulin levels in response to acute manipulation of serotonin function or genetic perturbations that affect the serotonin or insulin signaling pathways, such as ns3 mutation. Methods for control of protein production in subsets of serotonergic neurons would allow the identification of the specific serotonergic neurons that contribute to insulin regulation. At that time it will be useful to do electron microscope reconstruction experiments to trace the connections of the relevant neurons with target neurons (Kaplan, 2008).

The results are consistent with either positive or negative regulation of insulin secretion by serotonin. If serotonin negatively regulates insulin secretion, the elevated serotonin levels in ns3 mutants would be expected to inhibit insulin secretion, which could explain the high levels of insulin in the IPCs and the defect in peripheral insulin signaling. Alternatively, if serotonin is a positive regulator of insulin secretion, the results could be explained by a defect in serotonin release in the ns3 mutants. In such a scenario, the defect in serotonin release would deprive IPCs of stimulation. The consequent failure to secrete insulin would block peripheral insulin pathway activation (Kaplan, 2008).

Work with both C. elegans and mammals supports the existence of a connection between serotonin and insulin signaling, although the nature of this connection remains unclear. In C. elegans, mutation of the insulin receptor gene daf-2 results in increased reproductive longevity that is dependent on DAF-16, a forkhead transcription factor that carries out a program of gene expression that is inhibited in response to insulin signaling. Worms defective in serotonin synthesis exhibit increased reproductive longevity, an effect that can be reversed by mutation of daf-16, which mimics aspects of insulin pathway activation. Studies in mammals have produced contradictory reports about the effect of serotonin on insulin secretion. Studies of rabbit pancreas slices and rat islets of Langerhans found that exposure to serotonin resulted in increased insulin secretion. In contrast, work using golden hamster pancreases found that serotonin inhibited insulin secretion (Kaplan, 2008).

In mammals, insulin acts via the insulin receptor to regulate blood glucose, while the related insulin-like growth factors (IGFs) act through IGF receptors to stimulate cell growth. However, in Drosophila, regulation of both hemolymph glucose and cell growth are controlled by the DILPs, acting via a single insulin receptor (Baker, 2007). Interestingly, serotonin increases IGF-I release from human granulosa cells. Thus, despite the separation of glucose regulation from growth control in mammals, serotonin influences both. The serotonin-insulin connection may be an example of evolutionarily conserved physiology (Kaplan, 2008).

Search PubMed for articles about Drosophila Nucleostemin3

Arquier, N., Geminard, C., Bourouis, M., Jarretou, G., Honegger, B., Paix, A. and Leopold, P. (2008). Drosophila ALS regulates growth and metabolism through functional interaction with insulin-like peptides. Cell Metab. 7: 333–338. PubMed ID: 18396139

Baker, K.D. and Thummel, C.S. 2007. Diabetic larvae and obese flies-emerging studies of metabolism in Drosophila. Cell Metab. 6: 257–266. PubMed ID: 17908555

Colombani, J., Raisin, S., Pantalacci, S., Radimerski, T., Montagne, J. and Leopold, P. (2003). A nutrient sensor mechanism controls Drosophila growth. Cell 114: 739–749. PubMed ID: 14505573

Conlon, I. and Raff, M. (1999). Size control in animal development. Cell 96: 235–244. PubMed ID: 9988218

Edgar, B. A. (2006). How flies get their size: Genetics meets physiology. Nat. Rev. Genet. 7: 907–916. PubMed ID: 17139322

Hedges, J., West, M. and Johnson, A. W. (2005). Release of the export adapter, Nmd3p, from the 60S ribosomal subunit requires Rpl10p and the cytoplasmic GTPase Lsg1p. EMBO J. 24: 567–579. PubMed ID: 15660131

Kallstrom, G., Hedges, J. and Johnson, A. (2003). The putative GTPases Nog1p and Lsg1p are required for 60S ribosomal subunit biogenesis and are localized to the nucleus and cytoplasm, respectively. Mol. Cell. Biol. 23: 4344–4355. PubMed ID: 12773575

Kaplan, D. D., Zimmermann, G., Suyama, K., Meyer, T. and Scott, M. P. (2008). A nucleostemin family GTPase, NS3, acts in serotonergic neurons to regulate insulin signaling and control body size. Genes Dev. 22(14): 1877-93. PubMed ID: 18628395

Melcher, C., Bader, R., and Pankratz, M. J. (2007). Amino acids, taste circuits, and feeding behavior in Drosophila: Towards understanding the psychology of feeding in flies and man. J. Endocrinol. 192: 467–472. PubMed ID: 17332516

Morton, G. J., Cummings, D. E., Baskin, D. G., Barsh, G. S. and Schwartz, M. W. (2006). Central nervous system control of food intake and body weight. Nature 443: 289–295. PubMed ID: 16988703

Reynaud, E. G., Andrade, M. A., Bonneau, F., Ly, T. B., Knop, M., Scheffzek, K. and Pepperkok, R. (2005). Human Lsg1 defines a family of essential GTPases that correlates with the evolution of compartmentalization. BMC Biol. 3: 21. PubMed ID: 16209721

Tsai, R. Y. and McKay, R. D. (2002). A nucleolar mechanism controlling cell proliferation in stem cells and cancer cells. Genes Dev. 16: 2991–3003. PubMed ID: 12464630

Zinke, I., Kirchner, C., Chao, L. C., Tetzlaff, M. T. and Pankratz, M. J. (1999). Suppression of food intake and growth by amino acids in Drosophila: The role of pumpless, a fat body expressed gene with homology to vertebrate glycine cleavage system. Development 126: 5275–5284. PubMed ID: 10556053

date revised: 25 January 2009

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