minibrain: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - minibrain

Synonyms -

Cytological map position - 16F1--16F2

Function - serine/threonine kinase

Keywords - optic lobes, brain

Symbol - mnb

FlyBase ID: FBgn0259168

Genetic map position - 1-58.2

Classification - minibrain family

Cellular location - potentially nuclear



NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Shaikh, M. N., Gutierrez-Avino, F., Colonques, J., Ceron, J., Hammerle, B. and Tejedor, F. J. (2016). Minibrain drives the Dacapo dependent cell cycle exit of neurons in the Drosophila brain by promoting asense and prospero expression. Development [Epub ahead of print]. PubMed ID: 27510975
Summary:
A key issue in neurodevelopment is to understand how precursor cells decide to stop dividing and commence their terminal differentiation at the correct time and place. This study shows that minibrain (mnb), the Drosophila ortholog of the Down syndrome candidate gene MNB/DYRK1A, is transiently expressed in newborn neuronal precursors known as ganglion cells (GCs). Mnb promotes the cell cycle exit of GCs through a dual mechanism that regulates the expression of the cyclin-dependent kinase inhibitor Dacapo, the homolog of vertebrate p27kip1. On the one hand, Mnb upregulates the expression of the proneural transcription factor (TF) Asense, which promotes Dacapo expression. On the other, Mnb induces the expression of Prospero, a homeodomain TF that in turn inhibits the expression of Deadpan, a pan-neural TF that represses dacapo. In addition to its effects on Asense and Prospero, Mnb also promotes the expression of the neuronal-specific RNA regulator Elav, strongly suggesting that Mnb facilitates neuronal differentiation. These actions of Mnb ensure the precise timing of neuronal birth, coupling the mechanisms that regulate neurogenesis, cell cycle control and terminal differentiation of neurons.
Shaikh, M. N. and Tejedor, F. J. (2018). Mnb/Dyrk1A orchestrates a transcriptional network at the transition from self-renewing neurogenic progenitors to postmitotic neuronal precursors. J Neurogenet 32(1): 37-50. PubMed ID: 29495936
Summary:
The Down syndrome and microcephaly related gene Mnb/Dyrk1A encodes an evolutionary conserved protein kinase subfamily that plays important roles in neurodevelopment. minibrain (mnb) mutants of Drosophila melanogaster (Dm) exhibit reduced adult brains due to neuronal deficits generated during larval development. These deficits are the consequence of the apoptotic cell death of numerous neuronal precursors that fail to properly exit the cell cycle and differentiate. Recent studies have found that in both the Dm larval brain and the embryonic vertebrate central nervous system (CNS), a transient expression of Mnb/Dyrk1A promotes the cell cycle exit of newborn neuronal precursors by upregulating the expression of the cyclin-dependent kinase inhibitor p27kip1 (called Dacapo in Dm). In the larval brain, Mnb performs this action by regulating the expression of three transcription factors, Asense (Ase), Deadpan (Dpn) and Prospero (Pros), which are key regulators of the self-renewal, proliferation, and terminal differentiation of neural progenitor cells. The cellular/temporal expression pattern of Ase, Dpn, Pros and Mnb was studied in detail, and possible regulatory effects were analyzed among them at the transitions from neurogenic progenitors to postmitotic neuronal precursors in the Dm larval brain. The emerging picture of this analysis reveals an intricate regulatory network in which Mnb appears to play a pivotal role helping to delineate the dynamics of the expression patterns of Ase, Dpn and Pros, as well as their specific functions in the aforementioned transitions. The results also show that Ase, Dpn and Pros perform several cross-regulatory actions and contribute to shape the precise cellular/temporal expression pattern of Mnb. It is proposed that Mnb/Dyrk1A plays a central role in CNS neurogenesis by integrating molecular mechanisms that regulate progenitor self-renewal, cell cycle progression and neuronal differentiation.
BIOLOGICAL OVERVIEW

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).

Third instar: During the middle of the third instar, a second proliferation zone (still part of the opc) develops at the lateral rim of the opc. This zone consists of neuroblasts that have become separated from the main anlage by a deep furrow. Ganglion mother cells and ganglion cells are produced at the inner edge of this crescent. However, this zone yields many fewer cells than found in the medulla anlagen (about 6000 at 25 hours after puparium formation). These cells form the lamina (Hofbauer, 1991). Since mitotically active lamina precursor cells, which normally produce lamina neurons, are absent in mutants that lack retinal innervation, it is concluded that the arrival of photoreceptor axons in the larval brain initiates cell division to produce lamina neurons (Selleck, 1991). For more information about the effect of retinal innervation, see the development of the lamina visual center of the brain.

Two different populations of ganglion cells originate from the lateral proliferation zone of the ipc. Most of the cells differentiate to become the cell body layer of the lobula plate. The other cells participate in the formation of the inner medulla neuropil. About 15000 cells are generated from the lateral proliferation zone. An additional small dorsal proliferation zone develops from the ipc and these cells become part of the lobula cell body layer (Hofbauer, 1991).

Ganglion cells begin to grow axons shortly after their final mitosis: corresponding to the gradient of cell proliferation, there is also a gradient of differentiation in each developing neuropil. The axons of the lamina cells grow centrally, forming a fine fiber sheet at the inner margin of the lamina cell body area at right angles to the medulla neuropil. When differentiation of the lamina starts during the middle of the third instar, the youngest part of the lamina neuropil is in contact with the youngest, most lateral part of the medulla neuropil. The cells of the lobula complex send their axons centrally and form a neuropil opposite and almost parallel to the medulla neuropil. Lobula and lobula plate will develop from this fiber mass. The first fibers connecting the different visual neuropils appear very early, at a time when only the minor part of the ganglion cells are postmitotic. Photoreceptor cell axons start growing into the brain during the middle of the third instar, beginning at the posterior edge of the developing eye and progressing towards the anterior. At the same time, the proliferation of lamina ganglion cell progenitors begins and the newly generated lamina cells become connected with newly ingrowing photoreceptor cells (Hofbauer, 1991).

When the development of optic lobes in minibrain mutants is compared with that of wild type, it is found that the outer proliferation centers (opcs) in mutants attain an irregular structure. The opcs are distinguishable in third instar larvae, appearing in tightly packed, ribbon-like layers of cells, demarcated from the hemisphere. The proliferating neuroblasts appear as a very regular dome-like pattern in wild-type brain. One thick and two thin ribbons of bromodeoxuridine (BrdU) labeled cells (indicating cells that have gone through the DNA synthetic S phase) can be distinguished in the distal brain hemispheres. This regular spatial distribution of BrdU labeled neuroblasts is disturbed in the opcs of mnb mutants. In strong alleles, this ribbon of opc neuroblasts is condensed, and the BrdU-labelled nuclei are not distributed evenly. The regular structure of the thin ribbons disappears, resulting in a scattered distribution of labeled nuclei in these areas. It is important to note that in most cases the altered opc structure does not apparently change the number of labeled cells in comparison with wild type. Nevertheless, abnormally large cells with dark nuclei (probably degenerating cells) are frequently seen near to neuroblast-like cells. In comparison with wild type, mutants are missing a clear demarcation between neuroblasts and gangion cell bodies. In contrast to mutant opcs, no detectable structural alteration is seen in mutant ipcs. From observations of pupal brains, it is concluded that the minibrain phenotype is primarily caused by the inability of mutants to generate a sufficient number of optic lobe and central brain neurons during postembryonic development (Tejedor, 1995).

Minibrain/Dyrk1a regulates food intake through the Sir2-FOXO-sNPF/NPY pathway in Drosophila and mammals

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).


GENE STRUCTURE

The mnb open reading frame starts at an ATG codon within exon 3. There are three alternatively spliced transcripts (A, B and C). The transcripts share exons 1 through 8 and have divergent 3' ends. The last 307 amino acids of protein A, the last 4 amino acids of protein B, and the last 7 amino acids of protein C are generated by alternative splicing. The last 307 amino acids of protein A come from exon 10, while the last amino acids of protein B and C come from 9b and 9a respectively (Tejedor, 1995).
Genomic length - 25 kb

Bases in 5' UTR - 2030

Exons - 10

Bases in 3' UTR - 940 for 3' UTR of exon 10


PROTEIN STRUCTURE

Amino Acids - 843 (protein A), 539 (protein B) and 543 (protein C)

Structural Domains

The Mnb protein kinases share extensive sequence similarity with kinases involved in the regulation of cell division. This includes three domains similar to shaggy kinases. The proteins have a distinct amino-terminal domain, a kinase core domain, and a characteristic but variable C-terminal. The amino-terminal domain harbors a potential nuclear translocation signal. The core domain extends approximately from amino acid 97 to 425. Several amino acids residues are conserved in all serine-threonine kinases. The Mnb protein kinases share the most extensive sequence similarity with YAK1 protein kinase, with a sequence identity of 33.7%. The C-terminal domains of Minibrain related kinases contains a so-called GAS sequence, rich in glycine, alanine, and serine. The alignment of Shaggy and Minibrain kinases shows that both proteins have an overall identity of 28.8% over 420 amino acids (Tejedor, 1995).


minibrain: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 7 Sept 97 

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