In an extensive screen for Drosophila mutants with altered brain structure, a mutant, minibrain, was identified with a markedly reduced brain volume. The brains of adult mutant flies show no gross alterations in neuronal architecture. Major size reductions are seen in the optic lobes (50%-70%), most markedly in the lobula complex and in the central brain (40%-50%). The central brain hemispheres are reduced mainly in their ventral to dorsal and, respectively, anterior and posterior extensions. Axon bundles that project from the lobula complex to the lateral protocerebrum (optic stalk) are visibly thinner in such mutants (Tejedor, 1995).

A chronology of optic lobe development follows, before briefly describing the role of Minibrain in optic lobe cell proliferation.

A major part of the adult insect protocerebrum is the optic lobe, consisting of the first, second and third optic ganglia (known as the lamina, medulla and lobula/lobula plate, respectively). The optic lobes of the adult brain are derived from neuroblasts organized during the third instar larvae into two columnar epithelia: the inner proliferation center (ipc) and the outer proliferation center (opc).

First instar: A total of 30 to 40 precursor cells of the optic anlagen are found superficially in the lateral cell body layer of each brain hemisphere at the time of hatching (the transition from embryonic development to the first larval stage). These cells differ from the remaining cells of the hemisphere due to their somewhat smaller size. Within the early first instar, labelled nuclei appear in these smaller cells. The cells become larger, ellipsoid and epithelially arranged. From the second half of the first instar onwards two different epithelia can be distinguished; these develop into the opc and ipc.

Second instar: No obvious pattern of labeling is detectable until the end of the second instar, when the production of postmitotic neurons begins. At the end of the second instar, there are about 700 neuroblasts in the opc and about 400 neuroblasts in the ipc. Subsequently, a proliferation zone is formed at the medial edge of the opc. Mitotic figures and some small ganglion mother cells can be distinguished adjacent to the neuroblasts. The number of cells produced by this proliferation zone amounts to approximately 40,000, consisting of the medulla, the outermost cell layer of the optic lobe (Hofbauer, 1991).

Feeding behavior, one of the most essential activities in animals, is tightly regulated by neuroendocrine factors. Drosophila short neuropeptide F (sNPF) and the mammalian functional homolog neuropeptide Y (NPY) regulate food intake. Understanding the molecular mechanism of sNPF and NPY signaling is critical to elucidate feeding regulation. This study found that minibrain (mnb) and the mammalian ortholog Dyrk1a, target genes of sNPF and NPY signaling, regulate food intake in Drosophila and mice. In Drosophila neuronal cells and mouse hypothalamic cells, sNPF and NPY modulated the mnb and Dyrk1a expression through the PKA-CREB pathway. Increased Dyrk1a activated Sirt1 to regulate the deacetylation of FOXO, which potentiated FOXO-induced sNPF/NPY expression and in turn promoted food intake. Conversely, AKT-mediated insulin signaling suppressed FOXO-mediated sNPF/NPY expression, which resulted in decreasing food intake. Furthermore, human Dyrk1a transgenic mice exhibited decreased FOXO acetylation and increased NPY expression in the hypothalamus, and increased food intake. These findings demonstrate that Mnb/Dyrk1a regulates food intake through the evolutionary conserved Sir2-FOXO-sNPF/NPY pathway in Drosophila and mammals (Hong, 2012).

The production of sNPF and NPY in sNPFnergic and hypothalamic neurons of flies and mammals respectively, is increased during fasting. These neuropeptides are secreted to produce paracrine and endocrine effects but also feedback upon their synthesizing neurons where they respectively induce mnb and Dyrk1a gene expression through the PKA-CREB pathway. This Mnb/Dyrk1a kinase phosphorylates and activates the Sir2/Sirt1 deacetylase, which in turn deacetylates and activates the FOXO transcription factor. Among its many potential targets, FOXO then increases sNPF/NPY mRNA expression. Negative controls modulate the positive feedback of sNPF/NPY. Feeding activates the insulin receptor-PI3K-AKT pathway. FOXO becomes phosphorylated and transcriptionally inactivated by translocation to the cytoplasm. In this state the induction of sNPF/NPY by FOXO is decreased. Because sNPF and NPY are orexogenic, their positive feedback during fasting should reinforce the propensity for food intake whereas the negative regulation of sNPF and NPY mRNA during feeding condition would then contribute to satiety (Hong, 2012).

FOXO family transcriptional factors are involved in metabolism, longevity, and cell proliferation. FOXO is in part regulated in these processes by post-transcriptional modifications including phosphorylation and acetylation. In many model systems, the ligand activated Insulin-PI3K-AKT pathway phosphorylates FOXO to inactivate this transcription factor by moving it to the cytoplasm. The cytoplasmic localization of FOXO is mediated by 14-3-3 chaperone proteins in Drosophila and mammals. FOXO may also be acetylated, as is FoxO1 of mice, by the CREB-binding protein (CBP)/p300 acetylase and this inhibits FOXO transcriptional function by suppressing its DNA-binding affinity. Such FoxO1 acetylation can be reversed by SirT1 to help activate the FoxO1 transcription factor. This study describes for Drosophila how dFOXO in sNPFR1 neurons regulates the expression of sNPF and food intake. This mechanism parallels how hypothalamic FoxO1 regulates food intake through its control of orexigenic NPY and Agrp in rodents. Post-transcriptional modification of FOXO is central to these controls in both animals. sNPF and NPY expression is increased when FOXO is deacetylated by Sir2/Sirt1, while sNPF and NPY are decreased when FOXO is phosphorylated via the Insulin-PI3K-AKT pathway. Post-transcriptional modifications of FOXO proteins play a critical role for controlling food intake through the sNPF and NPY expression in flies and rodents (Hong, 2012).

Mnb/Dyrk1a participate in olfactory learning, circadian rhythm, and the development of the nervous system and brain. Mnb and Dyrk1a proteins contain a nuclear targeting signal sequence, a protein kinase domain, a PEST domain, and a serine/threonine rich domain. The kinase domains are evolutionary well-conserved from flies to humans. In Down syndrome (DS), chromosome 21 trisomy gives patients three copies of a critical region that includes the Mnb/Dyrk1a; trisomy of this region is associated with anomalies of both the nervous and endocrine systems. DS patients often show high Body Mass Index due to the increased fat mass. Children with DS have elevated serum leptin coupled with leptin resistance, both of which contribute to the obesity risk common to DS patients. This study found a novel function of Mnb/Dyrk1a that may underlay this metabolic condition of DS patients. Mnb/Dyrk1a regulates food intake in flies and mice. This is controlled by sNPF/NPY-PKA-CREB upstream signaling and thus produces downstream affects upon Sir2/Sirt1-FOXO-sNPF/NPY. Fasting not only increases the expression of mnb, but also of sNPF, suggesting that Mnb kinase activates a positive feedback loop where Sir2-dFOXO induces sNPF gene expression. Notably, fasting increases Sirt1 deacetylase activity and localizes FoxO1 to the nucleus in the orexogenic AgRP neurons of the mouse hypothalamus. Increased dosage of Dyrk1a in DS patients may reinforce the positive feedback by NPY and disrupt the balance between hunger and satiety required to maintain a healthy body mass (Hong, 2012).

Insulin produced in the pancreas affects the hypothalamus to regulate feeding in mammals. Insulin injected into the intracerebroventrical of the hypothalamus reduces food intake while inhibiting insulin receptors of the hypothalamic ARC nucleus causes hyperphasia and obesity in rodent models. This study showed a similar pattern for Drosophila where overexpression of insulin-like peptide (Dilp2) at insulin producing neurons decreased food intake while food intake was increased by inhibiting the insulin receptor in sNPFR1 expressing neurons. Likewise, during fasting, serum insulin and leptin levels are decreased in mammals, as is mRNA for insulin-like peptides of Drosophila. Thus, the mechanism by which insulin and insulin receptor signaling suppresses food intake is conserved from fly to mammals in at least some important ways (Hong, 2012).

Previous work has shown how sNPF signaling regulates Dilp expression through ERK in IPCs and controls growth in Drosophila (Lee, 2008). This study shows that sNPF signaling regulates mnb expression through the PKA-CREB pathway in non-IPC neurons and controls food intake. Since sNPF works through the sNPFR1 receptor, sNPFR1 in IPCs and non-IPCs neurons might transduce different signals and thereby modulate different phenotypes. Four Dilps (Dilp1, 2, 3, and 5) are expressed in the IPCs of the brain. Interestingly, levels of Dilp1 and 2 mRNA are reduced in the sNPF mutant, which has small body size, but this study finds only Dilp3 and 5 mRNA levels are reduced upon 24 h fasting. Likewise, only Dilp5 is reduced when adult flies are maintained on yeast-limited diets. In addition, Dilp1 and 2 null mutants show slight reduced body weights but Dilp3 and Dilp5 null mutants do not. These results suggest that Dilp1 and 2 behave like a mammalian insulin growth factor for size regulation while Dilp3 and 5 act like a mammalian insulin for the regulation of metabolism. However, in the long term starvation, Dilp2 and Dilp5 mRNA levels are reduced and Dilp3 mRNA expression is increased (Hong, 2012).

During fasting, sNPF but not sNPFR1 mRNA expression was increased in samples prepared from fly heads increasing food intake. In contrast, in feeding, the high level of insulin signaling reduced sNPF but not sNPFR1 mRNA expression and suppresses food intake. Interestingly, in the antenna of starved flies, sNPFR1 but not sNPF mRNA expression is increased and induces presynaptic facilitation, which results in effective odor-driven food search. However, high insulin signaling suppresses sNPFR1 mRNA expression and prevents presynaptic facilitation in DM1 glomerulus. These results indicate that starvation-mediated or insulin signaling-mediated sNPF-sNPFR1 signaling plays a critical role in Drosophila feeding behavior including food intake and food search even though the fine tuning is different (Hong, 2012).

This study presents a molecular mechanism for how sNPF and NPY regulate food intake in Drosophila and mice. A system of positive feedback regulation for sNPF and NPY signaling is described that increases food intake and a mode of negative regulation for sNPF and NPY by the insulin signaling that suppresses food intake. Modifications of the FOXO protein play a critical role for regulating sNPF and NPY expression, resulting in the control of food intake (Hong, 2012).