InteractiveFly: GeneBrief

mushroom body miniature: Biological Overview | References

Proper cell growth is a prerequisite for maintaining repeated cell divisions. Cells need to translate information about intracellular nutrient availability and growth cues from energy sensing organs into growth promoting processes such as the sufficient supply with ribosomes for protein synthesis. Mutations in the mushroom body miniature (mbm) gene impair proliferation of neural progenitor cells (neuroblasts) in the central brain of Drosophila. Yet, the molecular function of Mbm has been unknown so far. This study shows that mbm does not affect the molecular machinery controlling asymmetric cell division of neuroblasts but instead decreases their cell size. Mbm is a nucleolar protein required for small ribosomal subunit biogenesis in neuroblasts. Accordingly, levels of protein synthesis are reduced in mbm neuroblasts. Mbm expression is transcriptionally regulated by Myc, which among other functions relays information from nutrient dependent signaling pathways to ribosomal gene expression. At the posttranslational level, Mbm becomes phosphorylated by protein kinase CK2, which has an impact on localization of the protein. It is concluded that Mbm is a new part of the Myc target network involved in ribosome biogenesis, which together with CK2-mediated signals enables neuroblasts to synthesize sufficient amounts of proteins required for proper cell growth (Hovhanyan, 2014).

A fundamental issue during development of a multicellular organism is to coordinate cell proliferation, the availability of nutrients, and cell growth. Prominent examples are neuroblasts, the progenitor cells of the Drosophila melanogaster central nervous system, which proliferate in a highly regulated manner during development. Upon selection and specification, central brain neuroblasts proliferate until the end of embryogenesis, when they enter a quiescent state until resuming proliferation with the beginning of larval development. Notable exceptions are the neuroblasts generating the mushroom bodies, a paired neuropil structure in the central brain involved in learning and memory processes, which proliferate throughout development. Depending on the neuroblast lineage, proliferation stops at late larval or pupal stages by terminal differentiation or apoptosis. The embryonic and larval waves of neurogenesis correlate with changes in neuroblast size. Embryonic neuroblasts decrease in size with each cell division until they enter quiescence; resumption of proliferation at the larval stage is preceded by cell growth. In contrast to embryonic neuroblasts, larval neuroblasts maintain their cell size until the end of the proliferation period, which is again accompanied by a decrease in cell size. Exit of neuroblasts from quiescence, and thereby activation of proliferation, depends on the nutritional status of the whole animal and is governed by the insulin receptor (InsR)-phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, triggered by insulin-like peptide-producing glia cells, which receive nutritional signals from the fat body (Britton, 1998; Chell, 2010; Sousa-Nunes, 2011). Maintaining InsR signaling in combination with blocking of apoptosis is sufficient for long-term survival and proliferation of neuroblasts even in the adult fly. On the other hand, cellular nutrient sensing is mediated by the target of rapamycin (TOR) pathway, which, together with the InsR pathway, regulates cell growth through a variety of effector proteins at the levels of gene expression, ribosome biogenesis, and protein synthesis. Whereas neuroblast reactivation requires the interconnected InsR-PI3K and TOR pathways, neuroblast growth at larval stages is maintained even under nutrient restriction, by anaplastic lymphoma kinase (Alk)-mediated but InsR-independent activation of the PI3K pathway in combination with a direct influence of Alk on TOR effector proteins (Cheng, 2011; Hovhanyan, 2014 and references therein).

Cell growth requires protein synthesis, which depends on a sufficient supply of functional ribosomes. Ribosome biogenesis takes place in the nucleolus and involves transcription of single rRNA units and their processing and modification into 18S, 28S, and 5.8S rRNAs, which assemble with multiple ribosomal proteins to separately form the small and large ribosomal subunits. Upon transport to the cytoplasm, both subunits mature before they build up functional ribosomes. In general, one key downstream effector of TOR signaling is the transcription factor Myc, which controls cell growth in part by regulating ribosome biogenesis through transcriptional control of rRNA, ribosomal proteins, and proteins required for processing and transport of ribosomal components. Genomewide analyses of Drosophila Myc transcriptional targets emphasized the role of Myc as a central regulator of growth control but also identified many target genes with unknown molecular functions of the corresponding proteins. One of the Myc-responsive genes with an unknown function was mushroom body miniature (mbm). The original hypomorphic mbm¹ allele was identified in a screen for viable structural brain mutants and showed a pronounced reduction in the size of the adult mushroom body neuropil, which was due at least in part to a reduction in the number of intrinsic mushroom body neurons (Heisenberg, 1985; Raabe, 2004). More severe allelic combinations indicated a general requirement for Mbm in brain development and uncovered a neuroblast proliferation defect as a major cause of the phenotype. However, which step requires Mbm for neuroblast proliferation remains elusive. Homology searches provided no clue about the molecular function of Mbm. Structural features of Mbm include several stretches enriched in certain amino acids, a putative nuclear localization signal, and two consecutive CCHC zinc knuckles (Raabe, 2004). This report describes Mbm as a new nucleolar protein. Mbm is highly expressed in neuroblasts and is required for proper cell growth but not for processes controlling asymmetric cell division. Corresponding to the observed cell size defect, evidence is provided that small but not large ribosomal subunit biogenesis is impaired in the mutant, which could be a consequence of defective rRNA processing. Mbm is a transcriptional target of Myc and requires posttranslational modification by casein kinase 2 (CK2) for full functionality, as revealed by mutation of identified CK2 phosphorylation sites. These results provide a new link between Myc and growth control of neuroblasts and also establish a function of CK2 in neuroblasts (Hovhanyan, 2014).

This study identified Mbm as a new component of the nucleolus which has no obvious homologue outside the Drosophilidae. In contrast to the tripartite organization of vertebrate nucleoli in a fibrillar center, a dense fibrillar component (DFC), and a granular component, nucleoli of Drosophila neuroblasts often appear as a homogenous structure at the ultrastructural level, sometimes with intermingled fibrillar and granular components. In neuroblasts, Mbm colocalizes with fibrillarin and Nop5. In vertebrates, the methyltransferase fibrillarin is associated with Nop56/58 (corresponding to Drosophila Nop5) as part of the C/D type of small nucleolar ribonucleoprotein (snoRNP) complex required for rRNA processing in the DFC (Matera, 2007). Indeed, this study observed an aberrant rRNA intermediate in mbm on Northern blots, implicating a requirement of Mbm in rRNA processing. More specifically, based on the retention of RpS6 in the nucleoli of mbm neuroblasts, a function of Mbm is proposed in small ribosomal subunit biogenesis. The complementary phenotype, failure of large ribosomal subunit nucleolar-to-cytoplasmic transport, was observed in Drosophila upon knockdown of nucleostemin 1 (NS1) (Rosby, 2009). Yet the molecular function of Mbm remains elusive at this point because of its unique domain composition, with two zinc knuckle structures, several clusters of acidic or basic amino acids, and arginine/glycine-rich sequence stretches. For example, proteins containing arginine/glycine (RGG) repeats are found in a variety of RNP complexes, including snoRNPs. For a detailed biochemical analysis of Mbm function in ribosome biogenesis, cellular systems that are more accessible than neuroblasts are required, as these represent only a minor fraction of all brain cells. However, despite expression of Mbm in tissue culture S2 cells, neither cell size nor proliferation defects were detected under knockdown conditions (Hovhanyan, 2014).

Metabolic labeling of mbm neuroblasts indicated lowered protein synthesis rates, which could have been due to the lack of sufficient numbers of functional ribosomes. mbm larval brains reach nearly wild-type size, with a delay of several days, indicating that protein synthesis is maintained to at least some degree in neuroblasts. Since the process of asymmetric cell division itself is not affected in mbm flies, this provides one likely explanation for impaired neuroblast growth and proliferation. The importance of sufficient cell growth for repeated division of neuroblasts has been documented for mutations in signaling components. The comparison of the relative protein expression levels of Mbm and other nucleolar proteins in different cell types showed a more pronounced expression of Mbm in neuroblasts. This is confirmed by a comparative transcriptome analysis between neuroblasts and neurons. Altogether, the data suggest a more neuroblast-specific function of Mbm in ribosome biogenesis. Indeed, whereas most ribosomal subunit components are required in all cells, different isoforms are expressed for some components, with one isoform being required in all cells and the other isoform being required more specifically in stem cell lineages. Whereas loss of the generally required components causes early lethality, loss of specifically expressed isoforms is associated with a decrease in neuroblast size and an underproliferation phenotype. This emphasizes the specific needs of neuroblasts in ribosome biogenesis and cell growth as rate-limiting steps for self-renewal (Hovhanyan, 2014 and references therein).

The identification of Mbm as a transcriptional target of Myc provides a potential link to systemic and cell-intrinsic growth control of neuroblasts mediated by the InsR-PI3K-Akt and TOR pathways. In contrast to other tissues, where Myc is a downstream effector of these pathways, information is still largely missing in the case of neuroblasts. Myc is expressed in neuroblasts, and upon knockdown, mild effects on neuroblast size but not neuroblast number were observed. Consistent with the role of Drosophila Myc in expression of many genes involved in ribosome biogenesis, removal of Myc function in single neuroblasts caused corresponding decreases in Mbm and Nop5 levels (Hovhanyan, 2014).

Mbm function is dependent on posttranslational modification by the protein kinase CK2. CK2 is a promiscuous kinase expressed in all eukaryotic cells, with a vast array of substrates with pleiotropic functions. However, CK2 not only acts as a heterotetrameric α₂β₂ holoenzyme but also exists as free populations of both subunits, with independent functions. Pronounced nuclear or nucleolar localization of CK2 subunits was observed in vertebrate cells. In the nucleolus, CK2 participates in rRNA transcription by phosphorylating different components of the RNA polymerase I transcription machinery (Filhol, 2009; St-Denis, 2009). Proteins involved in ribosome biogenesis, such as B23 (also known as nucleophosmin), are also CK2 phosphorylation targets. CK2 modulates the ability of B23 to act as a molecular chaperone, its mobility rate and compartmentalization in the nucleolus, and its shuttling between the nucleolus and the nucleoplasm. Mbm and Nopp140 are the only described nucleolar phosphorylation targets of CK2 in Drosophila. This correlates with the observed nucleolar accumulation of CK2α in neuroblasts and the copurification of Mbm and CK2α. Although Mbm proteins with mutated CK2 phosphorylation sites showed cytoplasmic accumulation, nucleolar localization was still evident. Yet they were largely unable to complement the loss of endogenous Mbm function, indicating that phosphorylation by CK2 not only is a localization determinant but also is important for proper functioning of Mbm in the nucleolus. CK2 is often considered a constitutively active kinase which is not regulated by second messenger signaling cascades. However, there is increasing evidence for regulation of CK2 at the levels of the holoenzyme, the dynamics of localization of individual subunits under different conditions, and interactions with small molecules such as polyamines. Overexpression of ornithine decarboxylase, the rate-limiting enzyme in polyamine biosynthesis and a known Myc target gene, increases CK2 phosphorylation activity toward nucleolar B23 in mouse keratinocytes. It would be interesting to test for a regulatory influence of CK2 on Mbm function under different conditions (Hovhanyan, 2014).

In summary, Mbm is considered to be part of the Myc and CK2 regulatory networks for coordination of neuroblast growth and proliferation; however, more information about the molecular function of Mbm at the level of small ribosomal subunit biogenesis is still required (Hovhanyan, 2014).

Identification of Mushroom body miniature, a zinc-finger protein implicated in brain development of Drosophila

The mushroom bodies are bilaterally arranged structures in the protocerebrum of Drosophila and most other insect species. Mutants with altered mushroom body structure have been instrumental not only in establishing their role in distinct behavioral functions but also in identifying the molecular pathways that control mushroom body development. The mushroom body miniature¹ (mbm¹) mutation (Heisenberg, 1985; de Belle, 1996) results in grossly reduced mushroom bodies and odor learning deficits in females. With a survey of genomic rescue constructs, mbm¹ was pinpointed to a single transcription unit, and a single nucleotide exchange was identified in the 5' untranslated region of the corresponding transcript resulting in a reduced expression of the protein. The most obvious feature of the Mbm protein is a pair of C2HC zinc fingers, implicating a function of the protein in binding nucleic acids. The protein is poorly conserved and the nucleotide sequence is easily alignable only with the nucleotide sequence of seven Drosophila species closely related to Drosophila melanogaster. Protein alignment, however, shows conservation of the zinc finger regions in Drosophila pseudoobscura. Immunohistochemical analysis shows that expression of the Mbm protein is not restricted to the mushroom bodies. BrdUrd labeling experiments indicate a function of Mbm in neuronal precursor cell proliferation (Raabe, 2004).

Adaptive behavior of animals and humans requires functional neuronal circuits in the brain. The genetic programs that control the generation of these circuits by providing an adequate number of neurons, establishing neuronal connectivity, and remodeling them during development and in response to external stimuli during adulthood are just beginning to emerge. The mushroom bodies (MBs), a prominent neuropil structure of the insect brain, have become an attractive model system to study many aspects of this intricate network. Functional studies have established a role of the MBs in olfactory learning and memory, controlling locomotor activity, performing visual context generalization, and decision making. In contrast, the structural organization and the development of the MBs have been described in great detail. In the adult fly Drosophila melanogaster, ~2,500 intrinsic neurons (Kenyon cells) build up one MB. The Kenyon cell bodies are located in the dorsal cortex and extend their dendritic branches into the calyx, where prominent inputs from other brain regions are received. The Kenyon cell axons fasciculate in the peduncle and extend rostroventral, where most of them bifurcate to form a system of medially and dorsally projecting lobes. Each MB arises from a group of four apparently equipotent neuronal stem cells (neuroblasts), each of which generates in a sequential manner several types of Kenyon cells during larval and pupal stages. MB γ neurons are born before the mid-third-larval instar, then β' neurons are born, and finally the α/β neurons are added at pupal stages. The nomenclature of the Kenyon cells refers to the corresponding dorsally and medially projecting MB lobes in the adult fly. Immunohistochemical studies have identified additional subtypes of Kenyon cells. Yet, the anatomical description disregards the structural plasticity of the adult MBs as a consequence of changes in living conditions and experience. Furthermore, Kenyon cell axons and dendrites undergo massive remodeling during metamorphosis to establish adult-specific branching patterns. The axons of the γ neurons, which bifurcate in a dorsal and a medial branch in the larvae, degenerate and regrow only in the medial direction (Raabe, 2004).

What are the genetic programs controlling MB development? The first relevant mutants were identified nearly 25 years ago in screens for altered MB structure. More recently, mosaic techniques and gain-of-function systems have allowed searching for genes that control MB development among other (vital) processes outside of the MBs. Some of the genes identified so far control the number or proliferation pattern of MB neuroblasts; others regulate Kenyon cell axon growth and guidance or are required for remodeling of Kenyon cell axons during metamorphosis (Raabe, 2004).

This article describes the identification of the gene mushroom body miniature (mbm). Mutant mbm¹ females have grossly reduced MBs, which correlates with odor-learning deficits (Heisenberg, 1985; de Belle, 1996). The most prominent structural feature of the Mbm protein is a pair of zinc fingers of the C₂HC type, which indicates a function of the protein in binding nucleic acids. Expression of the Mbm protein is not restricted to the MBs, suggesting a function of Mbm in other aspects of brain development. In particular, when the hypomorphic mbm¹ allele was placed over a noncomplementing deficiency, the number of proliferating cells in larval brains is greatly reduced (Raabe, 2004).

Mutant mbm¹ flies display a sexually dimorphic phenotype. The MBs of mbm¹ females develop normally until the beginning of the third larval instar, when the axons of the Kenyon cells start to degenerate inappropriately. Kenyon cell perikarya apparently survive, but no regeneration of the axons is seen during metamorphosis, leading to a grossly reduced MB structure in the adult. In the WT, degeneration of the γ-neuron axons becomes evident no earlier than the early pupal stage. Antifasciclin II staining revealed the appearance of hole-like structures in the lobe system, which become infiltrated by glia cell processes to engulf degenerating axons. In mbm¹, hole-like structures are already seen at late third-larval instar, indicating a premature degeneration of axons. At early pupal stage, only residual anti-fasciclin II staining can be detected in mbm¹. In addition, a general reduction in the size of the lobe system was observed. To determine whether the reduced MB neuropil size seen in adult mbm¹ females results from a selective loss of γ-neuron axons or whether other Kenyon cell subtypes are also affected by the mutation, frontal sections from heads of WT and mbm¹ flies were stained with an antibody against the Drosophila 14-3-3 homologue Leonardo. Leonardo protein can be detected in the perikarya, dendrites, and axonal projections of the α/β, β', and γ neurons of WT flies. The MB phenotype of mbm¹ is variable. In mbm¹ females displaying a moderate phenotype, the structural subdivision of the MBs is maintained. In particular, the α/α', β/β', and γ-lobes can be distinguished, but there is an overall reduction in size. Even in animals with a strong mbm¹ phenotype, rudimentary calyx, peduncle, and lobe structures are formed. Thus, mbm¹ affects all Kenyon cell subtypes in the adult fly. The anti-Leonardo stainings also revealed that the reduction in MB neuropil size always correlates with a reduction in the number of Leonardo-positive Kenyon cell bodies. This finding appears to contradict the previous notion that the Kenyon cell bodies in mbm¹ might survive into adulthood despite deprivation of dendritic and axonal branches at late larval development. However, the remaining Leonardo-positive Kenyon cell bodies are surrounded by a large number of densely packed, unstained cells. These cells might represent former Kenyon cells, which have lost their projections and concomitantly no longer express Kenyon-cell-specific proteins. Alternatively, but not mutually exclusive to the former explanation, mutations in mbm might also interfere with the generation of Kenyon cells. The expression pattern of the Mbm protein and the defects observed in cell proliferation assays indicate such a function (Raabe, 2004).

The mbm¹ mutation was initially mapped by complementation analysis with the deficiencies Df(2L)net-PMF and Df(2L)al to the distal region of chromosome 2L. It was noticed that the MB phenotype of transheterozygous Df(2L)al/mbm¹, and in particular of Df(2L)net-PMF/mbm¹ flies, was even more variable than in homozygous mbm¹ flies. Mapping of the breakpoints of these deficiencies by Southern blotting showed that localization of the mbm gene was not straightforward. This conclusion is because Df(2L)net-PMF and Df(2L)al proved to be nonoverlapping deficiencies that are separated by more than 30 kb, a stretch of DNA that encompasses 12 known or predicted transcription units. The complementation and mapping results could be explained if mbm mapped under one deficiency and the other contained an additional mutation. Alternatively, the mbm gene might reside within the 30-kb genomic interval with both deficiencies exerting a silencing effect on the expression of the mbm gene. In the case of the terminal deficiency Df(2L)net-PMF, genes near the breakpoint come in close vicinity to telomeric heterochromatin, which could lead to silencing of gene expression (telomeric position effect). Although Df(2L)al is not adjacent to a telomere, based on the variability of the MB phenotype seen in Df(2L)al/mbm¹ animals, it is suspected that it too depresses mbm expression through an indirect mechanism. To test the hypothesis that the mbm gene is deleted by neither deficiency but is localized to the 30-kb interval, a set of overlapping deficiencies were generated by jump-out mutagenesis of a P-element strain (P[lacW]185/319), which carries two P-element insertions in the vicinity of the distal breakpoint of Df(2L)al. Classification of these deficiencies by complementation analysis verified that only deficiencies that remove genomic material distal to the smoothened (smo) gene did not complement the mbm¹ mutation. Indeed, the mbm¹ phenotype became more pronounced in these transheterozygous animals. Only very rarely, females were recovered and, in contrast to the original mbm¹ mutation, also the surviving males had reduced MBs. Mapping of the distal breakpoint of one deficiency, Df(2L)A1, narrowed the mbm gene to a genomic interval of 10 kb distal to smo. To identify the mbm transcription unit within this genomic segment, a set of overlapping genomic rescue constructs was generated. Only constructs encompassing the predicted CG11604 transcription unit were able to rescue the semilethality and the MB phenotype of mbm¹/Df(2L)A1 animals. As an independent verification that CG11604 indeed corresponds to the mbm gene, genomic DNA isolated from mbm¹ flies was sequenced, as was the parental cn, bw, sp strain, which was used for mutagenesis. One C to T nucleotide exchange was detected in the 5' untranslated region of the CG11604 transcript, thereby introducing an additional translational start codon. This ATG codon is followed after 33 nucleotides by an in-frame TAA stop codon, thus creating a short ORF just in front of the predicted ORF of the CG11604 transcription unit. Because of the disassembly of the eukaryotic ribosomal complex after translation of an ORF, it can be assumed that this short additional ORF blocks at least to some degree the translation of the CG11604 ORF in mbm¹ flies (Raabe, 2004).

To validate the expression of the Mbm protein in flies, an antiserum against the Mbm protein was generated and used in Western blot analysis. The CG11604 transcription unit is predicted to encode a polypeptide of 539 aa with a calculated molecular mass of 62 kDa. On Western blots of brain homogenates of male and female third-instar larvae, the anti-serum recognizes a major protein band of ~83 kDa and a slightly faster migrating protein isoform. Only very low levels of Mbm protein are detected in adult flies. Despite the sexually dimorphic MB phenotype of mbm¹ flies, the expression level of the Mbm protein is equally reduced in male and female larvae. These results show that mbm¹ is a hypomorphic allele. The discrepancy between the predicted molecular mass of 62 kDa of the Mbm protein and the observed doublet of protein bands at 83 kDa on Western blots prompted a verification of the specificity of the antiserum. Transgenic flies, which allowed expression of a cDNA corresponding to the mbm gene under Gal4/UAS control, were generated. High levels of Mbm protein accumulate in adult flies upon heat shock-induced expression of the mbm transgene. Again, the same doublet of protein bands was detected by the anti-Mbm antiserum. The appearance of two protein isoforms, which are derived from a single cDNA, also suggests that the Mbm protein is subject to posttranslational modification (Raabe, 2004).

In summary, genetic and molecular evidence is provided that the CG11604 transcription unit corresponds to the mbm gene. Furthermore, a single point mutation in the 5' untranslated region of the mbm transcript leads to reduction in Mbm protein expression (Raabe, 2004).

The Mbm protein does not display a significant overall homology to other proteins. Characteristic features of the protein are several domains that are highly enriched in certain amino acids. At the N terminus, an arginine/glycine-rich region is found, followed by a proline-rich region and several clusters of acidic or basic amino acids. The most prominent structural feature of Mbm is a pair of zinc fingers of the Cys-Xaa₂-Cys-Xaa₄-His-Xaa₄-Cys type located in the C-terminal half of the protein (amino acid positions 354–367 and 371–386). This type of zinc finger (sometimes referred to as zinc knuckle because of the short intervening sequences between the ion-contacting cysteine and histidine residues) was originally identified in the nucleocapsid proteins of retroviruses but has now been recognized as a general structural motif present in proteins of many eukaryotes. In general, it is thought that the Cys-Xaa₂-Cys-Xaa₄-His-Xaa₄-Cys motif mediates binding to RNA or DNA. Binding studies with the two Cys-Xaa₂-Cys-Xaa₄-His-Xaa₄-Cys fingers present in the nucleocapsid protein p7 of HIV have shown that they function concomitantly in binding RNA. Positively charged residues present within the zinc-finger structure and in the flanking sequences contribute to establish contacts with RNA. Mbm has a similar tandem arrangement of two Cys-Xaa₂-Cys-Xaa₄-His-Xaa₄-Cys fingers, which are separated by only three amino acids. Several basic amino acids are present N-terminal to and within both zinc-finger structures (Raabe, 2004).

Besides mbm, few other genes in the Drosophila genome encode proteins with two or more consecutive Cys-Xaa₂-Cys-Xaa₄-His-Xaa₄-Cys fingers. A protein with two zinc fingers but unknown biological function is encoded by CG9715. The Drosophila homologue of the cellular nucleic acid binding protein (CNBP) contains six zinc-finger domains. Vertebrate members of the CNBP family have been shown to promote expression of the c-myc and the colony stimulating factor-1 genes. In contrast, CNBP has been reported to bind to the 5' untranslated sequences of ribosomal mRNAs. The first evidence that Mbm is able to bind to nucleic acids comes from an in vitro assay with a recombinant Mbm protein, which binds in a zinc-finger-dependent manner to DNA-coated cellulose beads (Raabe, 2004).

By using the anti-Mbm antiserum, the expression pattern of the Mbm protein in the developing brain was determined. The finding that CNBP is able to bind to both RNA and DNA and can be found in the nucleus or the cytoplasm also raises the question of the subcellular localization of the Mbm protein (Raabe, 2004).

Mbm shows a widespread expression in third-instar larval brains with no apparent difference between males and females. It can be detected at low levels in the MB neuropil. Most prominently, Mbm protein is found in the nuclei of many cells outside of the MBs. In mbm¹, a global reduction in staining is seen in the brains of male and female larvae. This result corresponds to the Western blot analysis, but cannot explain the sexually dimorphic phenotype of mbm¹. In a group of large cells, which, based on their large size, are neuroblasts, Mbm can be found either in the cell nucleus or in the cytoplasm. The change in subcellular localization correlates with the cell cycle. This result becomes evident by costaining with an antibody directed against phosphorylated histone H3, which marks chromosomes from late G₂ phase throughout mitosis. Cells in anaphase are devoid of detectable levels of Mbm protein. Direct evidence for a possible function of Mbm in cell proliferation was provided by BrdUrd pulse-labeling experiments. Dissected third-instar larval brains from WT and mbm¹/Df(2L)A1 females were analyzed for incorporation of BrdUrd into newly synthesized DNA within a time window of 90 min. To identify the region in the dorsal brain where the MB neuroblasts are localized, costaining was performed with an antiserum against D-Mef2, which selectively labels Kenyon cells. Two to four BrdUrd-labeled cells are derived from a single, BrdUrd-positive neuroblast in WT brains. Beneath several neuroblasts, large clusters of D-Mef2-stained Kenyon cells can be recognized. In mbm¹/Df(2L)A1 animals, there is a dramatic reduction in the number of BrdUrd- and D-Mef2-positive cells. In many cases, only single cells or two-cell clusters are labeled with BrdUrd, indicating a general defect in the proliferation pattern of central brain neuroblasts (Raabe, 2004).

Again, these results parallel findings with the CNBP protein. Knockout experiments in mice have shown that CNBP is essential for forebrain formation (Chen, 2003). In conjunction with other studies, which have demonstrated an increase of c-myc promotor activity and cell proliferation upon overexpression of CNBP (Shimizu, 2003), it has been proposed that CNBP regulates forebrain formation through induction of c-Myc expression, which in turn stimulates cell proliferation and differentiation (Chen, 2003; Raabe, 2004 and references therein).

Therefore, the mbm gene codes for a protein with two consecutive zinc-finger domains and nucleic acid binding properties. The Mbm protein cycles in larval neuroblasts in amount and subcellular localization. The protein is also found in low concentration in the larval MB neuropil. Mutational analysis shows that it is an essential gene involved in CNS development. In the original mutant mbm¹ in which the developmental defects are largely confined to the female MBs, less Mbm protein is synthesized in both sexes. The molecular and systemic functions of Mbm are still poorly understood. The described binding of zinc-finger proteins, such as CNBP to both RNA and DNA, together with the finding that Mbm and CNBP can be localized to the cytoplasm and the nucleus, allow for a function in transcriptional and translational control. Mbm appears to regulate proliferation of neuronal precursor cells but also structural plasticity of Kenyon cells in the third-instar larva and during metamorphosis. Verification of Mbm as a DNA- or RNA-binding protein, together with the identification of the nucleotide sequences recognized by Mbm, should help to identify putative target genes and to understand the biological functions of Mbm in more detail (Raabe, 2004).

Dopamine-Mushroom Body Circuit Regulates Saliency-Based Decision-Making in Drosophila

Fruit flies can make appropriate choices among alternative flight options on the basis of the relative salience (prominence) of competing visual cues. This choice behavior consists of early and late phases; the former requires activation of the dopaminergic system and mushroom bodies, whereas the latter is independent of these activities. Immunohistological analysis showed that mushroom bodies are densely innervated by dopaminergic axons. Thus, the circuit from the dopamine system to mushroom bodies is crucial for choice behavior in Drosophila (Zhang, 2007).

Value-based decision-making is a complex behavior controlled, in part, by the dopamine system. Primates make choices among many available options to produce an advantageous response. The complexity of the mammalian brain has made it difficult to fully understand the neural circuits underlying value-based decision-making. To discern these circuits, this phenomenon was studied in Drosophila, because the functions of dopamine neurons are largely conserved evolutionarily. For example, forming aversive olfactory memories in Drosophila requires dopamine, allows punishment prediction, and involves neural activities that are similar to primates and rodents during conditioning (Schultz, 2006; Zhang, 2007 and references therein).

To explore the circuitry mediating value-based choice behavior of fruit flies, a novel paradigm was developed involving relative saliency evaluation of contradictory cues. Flies were trained in a flight simulator to associate heat punishment with one of two bars with compound cues, position (upper and lower) and color (blue and green). After training with one bar (e.g., upper and blue), flies were confronted with conflicting cues (e.g. upper-green and lower-blue) and had to decide whether to follow the position or color cue depending on their relative saliency. Position and color saliency were quantified by vertical separation between the bar center of gravity (DeltaCOG) and color intensity (CI), respectively. Amount of time spent in the conditioned quadrants was quantified as a preference index (PI) over 2-min blocks. Wild-type Berlin (WTB), Canton-S (CS), and mutant mushroom body miniature¹ (mbm¹) flies were trained with an upper-blue bar (CI = 1.0 and DeltaCOG = 60°). They were then tested for choice behavior by changing both the color (blue to green; CI unchanged) and position cue saliency (DeltaCOG from 60° to 40°). Wild types preferentially chose the position cue and followed the upper-green bar, whereas mutants could not decide which bar to follow, as evidenced by substantially reduced PIs (Zhang, 2007).

To further characterize choice behavior, WTB, CS and mbm¹ flies were tested by using a wide range of position cue saliencies (DeltaCOG: 0° to 60° in 5° or 10° increments) without changing the color cue CI. The choice curve of wild types (WTB and CS) exhibited a distinct transition in the preference for position cues, as a function of relative salience (position versus color), at DeltaCOG = 30° and could be fit by a sigmoid function (Boltzmann fit, r² = 0.97 for WTB and CS). In contrast, position cue preference in mbm¹ flies climbed up progressively (linear fit, r² = 0.92). Mushroom bodies (MBs) are essential for olfactory but not visual reinforcement learning, and, in the visual choice paradigm, mbm¹ flies could not distinguish pertinent position or color cues when their saliencies varied. This is consistent with previous findings that MBs participate in decision-making when Drosophila confronts a shape-color dilemma (Tang, 2001). Flies could interpret cue saliency as a representation of punishment probability and alter their choice strategy accordingly. Along these lines, without prior training wild types randomly chose all saliency cues (Zhang, 2007).

Primate studies suggest two general categories of decision-making: simple perceptual and value-based. The former is based on simple linear subtraction of alternative sensory inputs, and the latter on nonlinear calculation of the relative values of stimuli. This stud investigated which decision-making type fruit flies used when faced with conflicting visual cues. For this purpose, flies were trained with both color and position cues (CI = 1.0 and DeltaCOG = 60°), and then their preference for a single cue (each tested separately) was assessed during the posttraining session. When position cue saliency was varied (DeltaCOG from 0° to 60°), a sigmoid retrieval curve was not evident. Wild-type and mbm¹ flies performed similarly under these conditions indicating that retrieval of single visual cues is not MB dependent. It was then asked how visual perception of separated cues after compound training contributes to decision-making. The choice curve predicted by subtracting the PI at CI = 1.0 from the PIs of position cues (DeltaCOG from 0° to 60°) was linear and similar to the performance of mbm¹ flies in the position-color dilemma. Thus, mbm¹ flies make perceptual decisions in conflict situations by a simple subtraction mechanism, which is thought a general mechanism for perceptual decision-making in the human brain. In contrast to mbm¹, wild-type flies performed according to a sigmoid choice curve, and the mechanism underlying should be beyond simple comparison of the different cues perception (Zhang, 2007).

How and when MBs contribute to the decision-making process was investigated by selectively disrupting their function at different stages of choice behavior with shibire^ts1 (shi^ts1), a temperature-sensitive mutant form of dynamin. In shi^ts1 mutants, synaptic transmission is normal at permissive temperature (PT, below 30°C) and blocked at restrictive temperature (RT, above 30°C). Transgenic 247/upstream activation sequence (UAS)-shi^ts1 flies, with restricted shi^ts1 expression in MBs, were trained to follow bars with compound cues (CI = 1.0 and DeltaCOG = 60°) at PT (24°C) then tested at RT (30°C) for 6 min of choice performance with conflicting cues. They showed a sigmoid choice curve at PT, but a linear one at RT, which is similar to mbm¹ flies; wild types were unaffected by the temperature shift (Zhang, 2007).

Dopamine plays a crucial role in the motivation to acquire a reward or avoid a punishment. In Drosophila, dopaminergic transmission also mediates punishment prediction and associates punishment with a conditioned stimulus (Riemensperger, 2005). Expression of shi^ts1 in dopaminergic neurons is triggered by tyrosinse hydroxylase (TH)-Gal4 and dopa decarboxylase (Ddc)-Gal4. Ddc/UAS-shi^ts1 flies express shi^ts1 in both dopaminergic and serotoninergic neurons, whereas TH-Gal4/UAS-shi^ts1 flies express it only in the former. Both types of transgenic flies were tested for choice behavior and exhibited a sigmoid choice curve at PT, similar to wild types. However, their choice behavior was severely impaired at RT, as evidenced by a linear choice curve, indicating that dopamine deprivation was sufficient to disturb decision-making based on relative cue saliency (Zhang, 2007).

Because dopaminergic synaptic activity is necessary for memory acquisition in aversive olfactory conditioning (Schwaerzel, 2003), blocking it could impair visual memory required for decision-making rather than the process itself. To address this issue, flies were trained at PT and their preference tested for conditioned cues at RT, which required memory retrieval. Flies of all genotypes (CS, 247/UAS-shi^ts1, Ddc/UAS-shi^ts1, and TH/UAS-shi^ts1) performed similarly at both temperatures. Therefore, reduced dopaminergic transmission specifically disrupts saliency-based decision-making (Zhang, 2007).

In addition to MBs, the ellipsoid body (EB) in the Drosophila central complex was examined for its potential contribution to decision-making. Transgenic flies c507/UAS-shi^ts1 expressing shi^ts1 specifically in the EB showed normal sigmoid choice behavior at both temperatures, indicating that the EB is not critical for this behavior (Zhang, 2007).

Both dopamine and MBs are involved in saliency-based decision-making, and D1-type dopamine receptors are densely distributed in MB lobes. To determine how dopamine and MBs interact, the anatomical relation between them was examined by simultaneously expressing a red fluorescent protein (RFP), driven by 247-Gal4, specifically in MB neurons and visualizing dopaminergic neurons with immunostaining for TH, an enzyme specifically used in dopamine synthesis. Dopaminergic fibers were broadly distributed in Drosophila brain, with the highest density around MBs. Higher magnification showed that TH staining was concentrated in MB lobes rather than calyces or peduncles; thus, dopaminergic processes occupy MB lobes containing Kenyon cell axons, as confirmed by labeling dopaminergic neurons with green fluorescent protein (GFP)–tagged synaptic vesicle protein Synaptotagmin I (Syt I). Furthermore, dopaminergic axons, not dendrites, invade MB lobes, because the dendrite-specific Drosophila Down Syndrome Cell Adhesion Molecule conjugated to GFP (Dscam[17.1]-GFP) in dopaminergic neurons did not colocalize with immunostaining for the MB marker Fasciclin II (Fas II). Dopaminergic axons specifically innervate MB lobes, because the prominent lobe-like profile of dopaminergic fibers was largely abolished in flies treated with hydroxyurea (HU) to ablate MBs. Their absence in calyces suggests that dopamine regulates MBs by acting on Kenyon cell output (Zhang, 2007).

To determine whether choice behavior is time dependent, decision-making was examined at different times after flies encountered conflicting visual cues. During the first 30 s of conflict cues presentation, wild types (WTB and CS) showed linear choice performance according to position cue saliency; however, sigmoid choice behavior was evident at 90 to 120 and 330 to 360 s. These results suggest that decisive choices are time dependent and that the early test phase likely involved simple perceptual decision-making. To explore the circuits involved, MBs and dopaminergic function were selectively disrupted at varying times after choice behavior testing began with temperature-sensitive 247/UAS-shi^ts1 and TH/UAS-shi^ts1 flies. Flies were given a choice test using a DeltaCOG shift of 60° to 40° with CI = 1.0 because these parameters caused the largest difference in choice behavior between mutants and wild types. After testing started, flies were kept at PT for 1, 2, or 4 min before exposure to RT. Both 247/UAS-shi^ts1 and TH/UAS-shi^ts1 flies executed clear choices at PT; however, those kept at PT for 1 or 2 min, but not 4 min, performed worse at RT. These findings indicate that MB dopaminergic activity is only required during the first 4 min after encountering conflicting cues and not after stable choice behavior is established (Zhang, 2007).

The above results suggest that choice behavior of flies requires two phases: an initial involving dopaminergic and MBs activities and a later executing phase that is independent of these activities. Accordingly, it was hypothesized that, if flies were presented with a second set of conflicting cues, then dopamine system and MB would be reactivated. This hypothesis was tested by first determining whether wild types correctly discern the salient cue after sequential transition of cue positions (DeltaCOG shift from 60° to 40° then to 20°, at CI = 1.0); their choice was not significantly different from that seen after a direct transition (DeltaCOG shift from 60° to 20°). Next, TH/UAS-shi^ts1 and 247/UAS-shi^ts1 flies were exposed to two sequential sets of conflicting position-color cues and exhibited normal choice behavior with notable PIs for the first choice test at PT (DeltaCOG = 40°, upper-green bar, and CI = 1.0). However, when these flies were tested for the second cue set (DeltaCOG = 20°, upper-green bar, and CI = 1.0), they followed the color rather than the position cue, resulting in negative PIs. Both transgenic fly strains performed correctly at PT but incorrectly (PIs near zero) when the second choice test was performed at RT, whereas their visual perception to DeltaCOG = 20° was still normal. Flies were also tested with a shape-color dilemma as the second choice and acted similarly to the performance in position-color dilemma. Thus, the dopamine- and MB-independent execution of a decision is specific for an established choice condition; a new conflicting set again requires dopamine and MB activities for decision-making (Zhang, 2007).

This study demonstrated two distinct decision-making processes in Drosophila: one that is nonlinear and saliency-based and the other that is linear, simple perceptual. The latter process could be performed in the absence of dopaminergic-MB circuits by subtracting the saliency of conflicting cues, but the ability to amplify the difference at crucial points was compromised. Thus, linear choice performance was displayed instead of the sigmoid pattern of wild types. It is proposed that changing from linear to nonlinear decision-making depends on a gating mechanism of the dopaminergic-MB circuit whereby only the stronger 'winner' signal is transmitted to the MB while other weaker inputs are inhibited. Thus, flies implementing the gating function in MBs and the amplification effects of dopamine can accomplish a winner-takes-all decision. Two different phases, namely formation and execution, are involved in saliency-based decision-making in Drosophila, and a dynamic balance must be established between maintaining an existing choice and switching to a new decision (Zhang, 2007).

Search PubMed for articles about Drosophila Mbm

Britton, J. S. and Edgar, B. A. (1998). Environmental control of the cell cycle in Drosophila: nutrition activates mitotic and endoreplicative cells by distinct mechanisms. Development 125: 2149-2158. PubMed ID: 9570778

Chell, J. M. and Brand, A. H. (2010). Nutrition-responsive glia control exit of neural stem cells from quiescence. Cell 143: 1161-1173. PubMed ID: 21183078

Chen, W., Liang, Y., Deng, W., Shimizu, K., Ashique, A. M., Li, E. and Li, Y. P. (2003) Development 130: 1367-1379. PubMed ID; Online text

Cheng, L. Y., Bailey, A. P., Leevers, S. J., Ragan, T. J., Driscoll, P. C. and Gould, A. P. (2011). Anaplastic lymphoma kinase spares organ growth during nutrient restriction in Drosophila. Cell 146: 435-447. PubMed ID: 21816278

de Belle, J. S. and Heisenberg, M. (1996). Expression of Drosophila mushroom body mutations in alternative genetic backgrounds: a case study of the mushroom body miniature gene (mbm). Proc. Natl. Acad. Sci. 93: 9875-9880. PubMed ID; Online text

Filhol, O. and Cochet, C. (2009). Protein kinase CK2 in health and disease: Cellular functions of protein kinase CK2: a dynamic affair. Cell Mol Life Sci 66: 1830-1839. PubMed ID: 19387551

Heisenberg, M., Borst, A., Wagner, S. and Byers, D. (1985). Drosophila mushroom body mutants are deficient in olfactory learning. J Neurogenet 2: 1-30. PubMed ID: 4020527

Hovhanyan, A., Herter, E. K., Pfannstiel, J., Gallant, P. and Raabe, T. (2014). Drosophila Mbm is a nucleolar Myc and CK2 target required for ribosome biogenesis and cell growth of central brain neuroblasts. Mol Cell Biol 34(10):1878-91. PubMed ID: 24615015

Matera, A. G., Terns, R. M. and Terns, M. P. (2007). Non-coding RNAs: lessons from the small nuclear and small nucleolar RNAs. Nat Rev Mol Cell Biol 8: 209-220. PubMed ID: 17318225

Raabe, T., Clemens-Richter, S., Twardzik, T., Ebert, A., Gramlich, G. and Heisenberg, M. (2004). Identification of mushroom body miniature, a zinc-finger protein implicated in brain development of Drosophila. Proc Natl Acad Sci U S A 101: 14276-14281. PubMed ID: 15375215

Riemensperger, T., Voller, T. Stock, P., Buchner, E. and Fiala, A. (2005). Punishment prediction by dopaminergic neurons in Drosophila. Curr. Biol. 15: 1953-60. PubMed ID: 16271874

Rosby, R., Cui, Z., Rogers, E., deLivron, M. A., Robinson, V. L. and DiMario, P. J. (2009). Knockdown of the Drosophila GTPase nucleostemin 1 impairs large ribosomal subunit biogenesis, cell growth, and midgut precursor cell maintenance. Mol Biol Cell 20: 4424-4434. PubMed ID: 19710426

Schultz, W. (2006). Behavioral theories and the neurophysiology of reward. Annu. Rev. Psychol. 57: 87-115. PubMed ID: 16318590

Schwaerzel, M., et al. (2003). Dopamine and octopamine differentiate between aversive and appetitive olfactory memories in Drosophila. J. Neurosci. 23: 10495-502. PubMed ID: 14627633

Shimizu, K., Chen, W., Ashique, A. M., Moroi, R. and Li, Y. P. (2003). Molecular cloning, developmental expression, promoter analysis and functional characterization of the mouse CNBP gene. Gene 307: 51-62. PubMed ID: 12706888

Sousa-Nunes, R., Yee, L. L. and Gould, A. P. (2011). Fat cells reactivate quiescent neuroblasts via TOR and glial insulin relays in Drosophila. Nature 471: 508-512. PubMed ID: 21346761

St-Denis, N. A. and Litchfield, D. W. (2009). Protein kinase CK2 in health and disease: From birth to death: the role of protein kinase CK2 in the regulation of cell proliferation and survival. Cell Mol Life Sci 66: 1817-1829. PubMed ID: 19387552

Tang, S. and Guo, A. (2001). Choice behavior of Drosophila facing contradictory visual cues. Science 294(5546): 1543-7. PubMed ID: 11711680

Zhang, K., Guo, J. Z., Peng, Y., Xi, W. and Guo, A. (2007). Dopamine-mushroom body circuit regulates saliency-based decision-making in Drosophila. Science 316(5833): 1901-4. PubMed ID: 17600217

date revised: 8 March 2016

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