See the embryonic expression pattern of brat at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site
The brat gene is expressed in the embryonic central and peripheral nervous systems including the embryonic brain. In third instar larva brat expression is detected in the larval central nervous system including the brain and the ventral ganglion, in two glands - the ring gland and the salivary gland, and in parts of the foregut - the gastric caecae and the proventriculus. Accumulated data suggests that Brat may regulate proliferation and differentiation by secretion/transport-mediated processes (Arama, 2000). Brat accumulates uniformly in the cytoplasm of cells in wing discs from third instar larvae (Sonoda, 2001).
The simple cellular composition and array of distally pointing hairs has made the Drosophila wing a favored system for studying planar polarity and the coordination of cellular and tissue level morphogenesis. A gene expression screen was carried out to identify candidate genes that functioned in wing and wing hair morphogenesis. Pupal wing RNA was isolated from tissue prior to, during and after hair growth and used to probe Affymetrix Drosophila gene chips. 435 genes were identified whose expression changed at least 5 fold during this period and 1335 whose expression changed at least 2 fold. As a functional validation, 10 genes were chosen where genetic reagents existed but where there was little or no evidence for a wing phenotype. New phenotypes were found for 9 of these genes providing functional validation for the collection of identified genes. Among the phenotypes seen were a delay in hair initiation, defects in hair maturation, defects in cuticle formation and pigmentation and abnormal wing hair polarity. The collection of identified genes should be a valuable data set for future studies on hair and bristle morphogenesis, cuticle synthesis and planar polarity (Ren, 2005).
The expression of the brain tumor (brat) decreased 5.5 fold from 24 to 40 hrs. This gene has been studied primarily due to the neural tumor phenotype seen in loss of function mutants. The wings of bratts/Df brat flies raised at semi-permissive conditions were examined. No hair phenotype was seen but the occasional loss of sensory bristle shaft cells (principally distally along the anterior margin) was seen and occasional duplicated bristle cells (principally in the costa). These phenotypes are suggestive of a role for brat in specifying cell fate or in Notch mediated lateral inhibition (Ren, 2005).
Inactivation of both alleles of the brat gene results in the production of a tumor-like neoplasm in the larval brain, and lethality in the larval third instar and pupal stages. Sequence analysis of four brat alleles reveal that all of them are mutated at the beta-propeller domain. The clustering of mutations in this domain strongly suggests that it has a crucial role in the normal function of Brat, and defines a novel protein motif involved in tumor suppression activity (Arama, 2000).
Many NHL domain proteins also share three other motifs: a Ring-finger, one or two B-box motifs, and a coiled-coil (RBCC) (Slack, 1998). All of the evidence of work with Brat points to the central role of the NHL domain in mediating Brat activity. Analysis of lin-41 alleles also suggests that the NHL domain plays an important role in Lin-41 function (Slack, 2000). However, another report shows that expression of the RBCC domain of human BERP (brain expressed Ring-finger protein) in PC12 cells blocks a response to nerve growth factor (El-Husseini, 1999), suggesting an essential role for this region of BERP and, by extension, other family members (Sonoda, 2001).
To test the role of these other motifs in HB mRNA regulation, transgenic flies that express wild-type Brat, the amino-terminal BCC domain (Brat lacking a Ring-finger), and the carboxy-terminal NHL domain were prepared. As controls, similar transgenic flies that express full-length Bratfs1 (NHLfs1 proteins have a single amino acid substitution at a conserved residue within the NHL domain) and NHLfs1 derivatives were also prepared. Expression of each transgene is controlled by the Gal4-binding site's upstream activating sequence (UAS); using appropriate genetic crosses, each Brat derivative was expressed during oogenesis under the control of a nos;GAL4-VP16 transgene in a bratfs1 mutant background (Sonoda, 2001).
Expression of full-length Brat+ but not Bratfs1 rescues the abdominal defects of bratfs1 embryos. For reasons that are not understood, overexpression of either protein severely disrupts oogenesis and females produce very few eggs. Although the basis of this phenotype is not understood, the segmentation pattern was analyzed among larvae derived from rare fertilized eggs. Expression of the amino-terminal BCC domain has no effect in wild-type females and does not rescue the defects of bratfs1 embryos, although the protein is stable in vivo and accumulates to a higher level than the endogenous mutant Brat protein. Somewhat surprisingly, expression of the wild-type NHL domain almost completely rescues the bratfs1 embryonic phenotype. In contrast, expression of the NHLfs1 mutant domain to essentially the same level, does not. Thus, these experiments suggest the Brat NHL domain is necessary and sufficient to regulate HB mRNA translation in the early embryo (Sonoda, 2001).
The regulation of ribosome synthesis is likely to play an important role in the regulation of cell growth. The ncl-1 gene in C. elegans functions as an inhibitor of cell growth and ribosome synthesis. The Drosophila tumor suppressor brain tumor (brat) is an inhibitor of cell growth and is a functional homolog of the C. elegans gene ncl-1. The brat gene is able to rescue the large nucleolus phenotype of ncl-1 mutants. brat mutant cells are larger, have larger nucleoli, and have more ribosomal RNA than wild-type cells. Furthermore, brat overexpressing cells contain less ribosomal RNA than control cells. These results suggest that the tumorous phenotype of brat mutants may be due to excess cell growth and ribosome synthesis (Frank, 2002).
Because NCL-1 protein is most highly expressed in cells with low rates of rRNA and protein synthesis, such as cells of the early embryo and neurons in the adult, it was predicted that brat expression would be highest in cells with low levels of biosynthetic activity. The brat expression pattern was examined using RNA in situ hybridization. Using a BRAT RNA probe, high level brat expression was observed in brains from wild-type third instar larvae. This expression is quite uniform throughout the entirety of the brain hemispheres, including the optic lobe. Weaker but fairly uniform expression is also seen in virtually all cells of the imaginal discs. In the eye disc, higher brat expression levels were observed in small clusters of cells along the morphogenetic furrow. These are most likely the neuronal preclusters, the first cells in the eye disc to exit the cell cycle and differentiate into relatively metabolically inactive cells. This expression pattern is consistent with a hypothesis that brat, like ncl-1, functions as an inhibitor of cell growth (Frank, 2002).
Given that brat mutant cells are larger than wild-type cells, it was hypothesized that brat functions to inhibit cell growth, such that overexpression of brat would be expected to lead to a decrease in cell and organ size. Because ubiquitous overexpression of brat results in lethality, the Gal4-UAS system was used to overexpress a wild-type brat cDNA specifically in the developing eye using the eyeless-Gal4 line (ey-Gal4). The eyeless enhancer directs expression in actively proliferating cells of the eye disc. Expression of brat in the developing eye, using two different UAS-brat lines, results in a dramatic decrease in organ size (Frank, 2002).
The Gal4-UAS system to overexpress brat in the developing wing using the decapentaplegic-Gal4 line (dpp-Gal4). In this line, Gal4 is expressed between wing veins LIII and LIV. Overexpression of brat leads to an obvious decrease in the size of this intervein region using two different UAS-brat lines. To quantitate this growth inhibition, the wing blade area bounded by veins LIII and LIV was measured and compared with the area bound by veins LII and LIII, which serves as an internal control since it is affected only slightly. brat overexpression results in a 36% decrease in wing area relative to the control. The decrease in eye and wing size caused by brat overexpression is probably due to a combination of cell growth inhibition and cell death. To determine if there was an effect on cell size in the wing, the number of bristles was counted in a defined area. Since each cell in the wing is associated with a single bristle, the inverse of the number of bristles in a region of a defined area gives a relative estimate of cell size. Surprisingly, the UAS-brat line appears to have increased cell sizes in the wing (Frank, 2002).
Because overexpression of brat in the wing appears to cause an increase in cell size while inhibiting organ growth, the effect of brat overexpression in clones of cells was examined, thus allowing a comparison of overexpressing and control cells directly in the same tissue. The flip-out technique was used to overexpress brat and GFP in clones of cells. Wing discs were dissociated from staged larvae in which overexpression was induced and analyzed by flow cytometry. Overexpression of brat results in a slight increase in cell size with no effect on cell cycle phasing. Microscopic examination of clones reveals that overexpression of brat leads to cell death as evidenced by pycnotic nuclei visualized by DAPI staining of clones. To overcome this effect, the cell death inhibitor P35 was co-expressed with brat in the clones; an even larger increase in cell size was observed. P35 expression appears to be somewhat deleterious to cells, as on its own it causes a small but reproducible decrease in cell size. The increased cell size in the presence of P35 is probably due to the fact that P35 expression inhibits the cell death caused by brat overexpression, thus allowing a greater proportion of the brat overexpressing cells to be analyzed (Frank, 2002).
Although brat overexpression results in enlarged cells, analysis of clone areas shows that brat overexpression actually inhibits total clone growth. The areas of wing imaginal disc clones expressing brat, GFP and P35 was compared with control clones expressing only GFP and P35; brat overexpression leads to a significant decrease in clone area (Frank, 2002).
Because brat overexpression inhibits clone growth yet results in enlarged cells, it was hypothesized that brat might be causing a slowing of cell division. To address this possibility, clones were induced to express brat, P35 and GFP at 72 hours AED, and the number of cells per clone was counted 43 hours later. Clones expressing brat have significantly fewer cells than control clones expressing only P35 and GFP. These cells have 50% longer doubling times than control cells. Thus, overexpression of brat results in a slowing of cell division. Since cell size is controlled by the rates of both cell growth and cell division, the fact that brat overexpressing cells are larger than control cells is interpreted to mean that the inhibition of cell division rate is more severe than the inhibition of cell growth (Frank, 2002).
How might brat and ncl-1 affect ribosome synthesis? It has been proposed that in E. coli and in Drosophila rRNA synthesis is regulated by the polysome to free ribosomal subunit ratio. When this ratio is high, rRNA synthesis is upregulated. Conversely, when translation, and therefore this ratio, are low, rRNA synthesis is inhibited. Since NCL-1 and BRAT are both cytoplasmic proteins, one possibility is that brat and ncl-1 serve as sensors of this ratio. Alternatively, they could directly affect this ratio by serving as translational repressors. Future work should provide insight into the specific mechanism of brat and ncl-1 action (Frank, 2002).
How stem cells generate both differentiating and self-renewing daughter cells is unclear. This study shows that Drosophila larval neuroblasts - stem cell-like precursors of the adult brain - regulate proliferation by segregating the growth inhibitor Brat and the transcription factor Prospero into only one daughter cell. Like Prospero, Brat binds and cosegregates with the adaptor protein Miranda. In larval neuroblasts, both Brat and Prospero are required to inhibit self-renewal in one of the two daughter cells. While Prospero regulates cell-cycle gene transcription, Brat acts as a posttranscriptional inhibitor of dMyc. In brat or prospero mutants, both daughter cells grow and behave like neuroblasts leading to the formation of larval brain tumors. Similar defects are seen in lethal giant larvae (lgl) mutants where Brat and Prospero are not asymmetric. This study has identified a molecular mechanism that may control self-renewal and prevent tumor formation in other stem cells as well (Betschinger, 2006).
These data reveal a molecular mechanism that controls self-renewal in Drosophila larval neuroblasts. The growth regulator Brat segregates asymmetrically during neuroblast division and inhibits self-renewal in one of the two daughter cells. Together with the asymmetrically segregating transcription factor Prospero, Brat ensures that this daughter cell will stop growing, exit the cell cycle, and differentiate into neurons. In brat or prospero mutants, or in lgl mutants, where Brat and Prospero are not asymmetrically segregated, both daughter cells proliferate leading to the formation of a brain tumor and death of the animal. These tumors are neoplastic and can be transplanted into the abdomen of wild-type flies where they overgrow, invade other tissues, and eventually kill the host (Betschinger, 2006).
Asymmetric cell division has been studied in the Drosophila central and peripheral nervous systems. In the peripheral nervous system, the determinants Numb and Neuralized segregate into one of the two daughter cells, and in their absence, this cell is transformed into its sister cell. In the embryonic central nervous system, Prospero acts as a segregating determinant, but in prospero mutants, many GMCs are still correctly specified. The data suggest that this is because Prospero acts partially redundant with Brat. In embryos double mutant for prospero and brat, most GMCs expressing the marker Eve are missing and neuronal differentiation in the embryonic CNS is greatly impaired. These observations suggest that Brat and Prospero act together to specify GMC fate in Drosophila embryos (Betschinger, 2006).
Although cell-cycle markers are expressed longer and stronger in prospero and brat, prospero mutant embryos, uncontrolled overproliferation has not been described in Drosophila embryos so far. In larvae, however, both brat and prospero mutant neuroblasts can initiate tumor formation. It is proposed that this difference is due to distinct mechanisms of cell growth during the two stages. During embryogenesis, cell number increases dramatically but the total volume of the embryo remains constant. Embryonic neuroblasts therefore shrink with each division and they might exit the cell cycle simply because they become too small. Support for this model comes from mutations affecting cell size asymmetry during neuroblast divisions, like Gβ13F (Fuse, 2003) or Ric-8 (Hampoelz, 2005): in these mutants, GMCs are larger, neuroblasts shrink faster and, as a consequence, divide less often. In larval neuroblasts, the situation is quite different. Several results indicate that larval neuroblasts grow significantly while cell growth is inhibited in GMCs. First, the total volume of clones generated from individual neuroblasts is several times more than the initial volume of the neuroblast. Second, the size of 'old' and 'young' (earlier and more recently generated) GMCs is approximately the same, indicating that GMCs do not grow significantly during clone formation. Third, larval neuroblasts do not become progressively smaller during development indicating that the loss of cytoplasm from each division must be compensated for by growth. Taken together, these results suggest that larval neuroblasts might be more appropriate as a model for the control of self-renewal in stem cells (Betschinger, 2006).
These experiments show that the restriction of cell growth in the GMC requires the genes lgl, brat, and prospero. While lgl seems to be required indirectly due to its role in asymmetric protein segregation, Prospero and Brat act in the GMC to regulate several important events: They repress neuroblast fate, inhibit cell-cycle progression, and prevent cell growth. Prospero is a homeodomain transcription factor, and the cell-cycle genes Cyclin A, Cyclin E, and Dacapo (the fly homolog of the CDK inhibitor p21) were shown to be among its transcriptional targets. Similar to Drosophila Prospero, its vertebrate homolog Prox-1 has been shown to regulate cell-cycle genes, and loss of prox-1 leads to increased proliferation of retinal progenitor cells (Betschinger, 2006).
For Brat, two different functions have been described: First, it acts as a translational regulator of the gap-gene hunchback. Hunchback is expressed in the embryonic nervous system but is not present in wild-type or brat mutant larval neuroblasts and is unlikely to be relevant for the growth regulatory activity of Brat. More likely, Brat acts through its well-described inhibitory activity on ribosomal RNA synthesis. Cells mutant for brat or its C. elegans homolog ncl-1 have larger nucleoli, more ribosomal RNA, and higher rates of protein synthesis, and these activities have been made responsible for the cell size increase that is observed in C. elegans and Drosophila brat mutant cells. These data suggest that this second function of Brat is also linked to posttranscriptional gene regulation. It is proposed that Brat downregulates dMyc in one of the two daughter cells and thereby inhibits protein synthesis and cell growth. Whether Brat controls dMyc translation, protein stability, or RNA stability is unclear. Interestingly, the C. elegans Brat homolog ncl-1 has been identified as one of the genes required for RNAi (Kim, 2005). Since the microRNA pathway was shown to be involved in regulation of Drosophila stem cell proliferation (Hatfield, 2005), differential regulation of this pathway in neuroblasts and GMCs by Brat could provide another explanation for its mutant phenotype (Betschinger, 2006).
Brat is part of a protein family that is characterized by a C-terminal NHL domain, several zinc-finger like B boxes, and a coiled-coil region. While the vertebrate members of this family (TRIM-2, TRIM-3, and TRIM-32) are not well characterized, the mutant phenotype of the two other Drosophila members (Dappled and Mei-P26) suggests a common function as tumor suppressors. Mutations in dappled cause melanomic tumors of the fat body, and mei-P26 mutations lead to ovarian tumors. While dappled tumors have not been well characterized, the mei-P26 phenotype has been attributed to overproliferation of undifferentiated germ cells. It is similar to-and genetically interacts with-bag of marbles, a well-characterized repressor of proliferation in the daughter cells of germline stem cells. Thus, it is conceivable that proliferation control in stem cells is a common activity of NHL domain proteins (Betschinger, 2006).
Recent evidence suggests that some human brain tumors contain stem cell-like neural progenitors that are responsible for tumor formation. Together with the identification of stem cell-like subpopulations in leukaemia, multiple myeloma, and breast cancer, this has led to the so-called cancer stem cell hypothesis which proposes that only a small population of cells in a tumor have the ability to proliferate and self-renew. This discovery suggests mechanisms for tumorigenesis other than the simple loss of proliferation control, in particular dedifferentiation of cells into additional stem cells and symmetric division of stem cells. Animal models for tumor stem cells are essential for developing new therapeutic approaches that target these mechanisms. Although Drosophila can only mimic some aspects of tumorigenesis, it might contribute to the identification of the molecular pathways operating in tumor stem cells. Human Lgl has already been implicated in tumor progression, and the characterization of Brat homologs will verify the relevance of Drosophila as a tumor stem cell model (Betschinger, 2006).
Brain development in Drosophila is characterized by two neurogenic periods, one during embryogenesis and a second during larval life. Although much is known about embryonic neurogenesis, little is known about the genetic control of postembryonic brain development. This study used mosaic analysis with a repressible cell marker (MARCM) to study the role of the brain tumor (brat) gene in neural proliferation control and tumour suppression in postembryonic brain development of Drosophila. The findings indicate that overproliferation in brat mutants is due to loss of proliferation control in the larval central brain and not in the optic lobe. Clonal analysis indicates that the brat mutation affects cell proliferation in a cell-autonomous manner and cell cycle marker expression shows that cells of brat mutant clones show uncontrolled proliferation, which persists into adulthood. Analysis of the expression of molecular markers, which characterize cell types in wild-type neural lineages, indicates that brat mutant clones comprise an excessive number of cells, which have molecular features of undifferentiated progenitor cells that lack nuclear Prospero (Pros). pros mutant clones phenocopy brat mutant clones in the larval central brain, and targeted expression of wild-type pros in brat mutant clones promotes cell cycle exit and differentiation of brat mutant cells, thereby abrogating brain tumour formation. Taken together, these results provide evidence that the tumour suppressor brat negatively regulates cell proliferation during larval central brain development of Drosophila, and suggest that Prospero acts as a key downstream effector of brat in cell fate specification and proliferation control (Bello, 2006).
Previous studies suggested that brat loss-of-function mutants lead to massive cellular overgrowth and tumour formation in larval optic lobes of Drosophila. These studies also indicated an embryonic requirement for brat to suppress tumour formation. By contrast, the current analysis showed that the brat overproliferation phenotype is due to loss of proliferation control in the larval central brain; the optic lobes initially appear wild-type-like but subsequently are overgrown by neoplastic central brain brat mutant tissue. This conclusion is further supported by MARCM clonal analysis which demonstrated that loss of brat function causes overproliferation in the larval central brain only (Bello, 2006).
In vivo mosaic analysis reveals a cell-autonomous, larval requirement for brat to limit cell proliferation in the brain. Although brat is expressed in all parts of the nervous system both in the embryo, induction of brat mutant clones in the first larval instar is sufficient to cause massive overproliferation in the central brain but not the ventral ganglia. This may suggest that either unknown compensatory mechanisms actively suppress a brat mutant phenotype in the larval ventral ganglia, or that this reflects region-specific differences in cell cycle control. Indeed, transcriptional activity of the mitotic regulator string/Cdc25 is regulated by a plethora of cis-acting elements, most of which are devoted to differential control of cell proliferation during embryonic and larval neurogenesis (Bello, 2006).
During postembryonic neurogenesis, intense proliferation takes place in the brain. This analysis shows that central brain brat mutant clones display sustained cell cycle marker expression, indicating that mutant cells are unable to withdraw from the cell cycle. This is further supported by the presence of enormous brat mutant clones with pronounced proliferative activity even in 3-week-old adult brains, an observation that contrasts with the postmitotic adult wild-type brain. Previous studies have shown that cessation of proliferation in the developing Drosophila brain occurs during metamorphosis, although the underlying genetic mechanisms are currently unknown. The elevated and aberrant cell cycle activity of central brain brat mutant cells suggests that these cells are either able to escape or that they lack cell cycle termination signals (Bello, 2006).
Mosaic analysis demonstrates that enlarged brat mutant clones comprise cells that display sustained expression of neural progenitor cell markers, and simultaneously lack marker gene expression specific for differentiating ganglion cells. Indeed, lack of axonal processes suggests that brat mutant clones comprise an excessive number of mutant cells that are unable to exit the cell cycle and hence do not differentiate into ganglion cells but rather continue to proliferate. These data indicate that brat mutation impairs proliferation control of neural progenitor cells, namely either neuroblasts and GMCs or only one of these progenitors, since in the wild-type central brain only these two cell types are actively engaged in the cell cycle. Based on this analysis it is not possible to distinguish unambiguously between the two possibilities and the underlying mechanisms. The possibility that differentiating ganglion cells de-differentiate due to brat mutation was excluded, because lack of differentiation was consistently observed right after clone induction and also at any later stages of mutant clone development. This was especially exemplified by the lack of nuclear Pros expression, which in the wild type is unambiguously detectable in differentiating progeny of larval neuroblast lineages, namely GMCs as well as ganglion cells (Bello, 2006).
Moreover, loss-of-function analysis indicates that brat mutant MARCM clones lack Pros and also phenocopy pros mutant clones. Thus, enlarged pros mutant clones consist of cells that are devoid of Elav expression, that lack axonal processes but display sustained expression of Grh and Mira as well as cell cycle markers such as CycE, CycB and PH3. These data suggest that mutant clones are essentially devoid of terminally differentiating postmitotic ganglion cells, indicating that Pros functions like Brat in terminating neural progenitor cell proliferation and inducing ganglion cell differentiation. In the embryonic CNS, Pros functions to terminate cell proliferation by repression of cell-cycle activators and simultaneously to induce a differentiation program, effectively coupling the two events. This Pros function appears to be warranted by its localization in the basal cortex of asymmetrically dividing neuroblasts and hence its distribution to only one daughter cell, the GMC. Upon completion of mitosis, Pros translocates from cytoplasm into the nucleus where it executes its transcriptional program ensuring both terminal division of the GMC and cell differentiation of its progeny. In the larval CNS nuclear localisation of Pros is observed in GMCs and ganglion cells but not in the neuroblast, suggesting that Pros has comparable functional features in larval central brain neurogenesis (Bello, 2006).
In addition, the results provide evidence that Pros acts downstream of Brat in neural proliferation control. The following points support this notion: (1) brat mutant clones lack nuclear Pros; (2) brat and pros mutant clones are indistinguishable both at the morphological and at the molecular level; (3) Brat expression is unaltered in pros mutant clones, which together with point no. 1 strongly suggests that Brat is epistatic over Pros; and (4) trans-activation of wild-type pros in brat mutant clones is sufficient to promote both cell cycle exit and differentiation. The experiments, however, do not provide any evidence about the direct or indirect nature of their interaction. Since overexpressed Pros is detected specifically in brat mutant clones in a wild-type-like pattern, the possibility that brat acts as a translational repressor of Pros, comparable to its role in hunchback repression during embryonic abdominal segmentation, is excluded. In addition, brat mutation apparently does not affect pros transcription, since pros RNA in situ hybridization in zygotic brat mutants produced a pattern indistinguishable from wild-type controls. Thus, Brat and Pros may act indirectly in the same pathway, regulating progenitor cell proliferation control in the brain. Alternatively, Brat may act in a process required to cargo Pros, comparable to the function of its mammalian homolog BERP (Bello, 2006).
In vivo mosaic analysis demonstrates that a single mutation in either brat or pros is sufficient to cause brain tumour formation in a cell-autonomous manner, suggesting that indefinite proliferation of brat and pros mutant cells is a cell intrinsic property. GFP-labelled MARCM cells each derive from a common precursor cell, implying that brat and pros mutant cells all descend from individual tumour cells of origin and hence lead to brain tumour formation in a clonally related manner. Moreover, the data indicate that pros and brat mutant clones in the larval central brain are composed of an excessive number of mutant progenitor cells that are unable to differentiate into ganglion cells but rather continue to proliferate. In this sense the results provide in vivo support for the notion that the initiating event in the formation of a malignant tumour is an error in the process of normal differentiation (Bello, 2006).
In addition, the unlimited capacity to generate undifferentiated, proliferating progeny suggests that cells mutant for brat or pros retain self-renewing capacities. In human, brain cancers are thought to arise either from normal stem cells or from progenitor cells in which self-renewal pathways have become activated, however the underlying mechanisms are elusive. The results in Drosophila may therefore provide a rationale and genetic model for the origin of brain cancer stem cells. Although parallels to human tumour formation are speculative, it is noteworthy that TRIM3, a human homolog of brat is located on chromosome 11p15, a region frequently deleted in brain tumours. Moreover, functional studies have shown that the pros homologue Prox1 regulates proliferation and differentiation of neural progenitor cells in the mammalian retina. These data may indicate that brat and pros function in cell differentiation and tumour suppression in an evolutionarily conserved manner (Bello, 2006).
Homeostatic regulation of ionic currents is of paramount importance during periods of synaptic growth or remodeling. The translational repressor Pumilio (Pum) is a regulator of sodium current [I(Na)] and excitability in Drosophila motoneurons. This study shows that Pum is able to bind directly the mRNA encoding the Drosophila voltage-gated sodium channel Paralytic (Para). A putative binding site for Pum was identified in the 3' end of the para open reading frame (ORF). Characterization of the mechanism of action of Pum, using whole-cell patch clamp and real-time reverse transcription-PCR, reveals that the full-length protein is required for translational repression of para mRNA. Additionally, the cofactor Nanos is essential for Pum-dependent para repression, whereas the requirement for Brain Tumor (Brat) is cell type specific. Thus, Pum-dependent regulation of I(Na) in motoneurons requires both Nanos and Brat, whereas regulation in other neuronal types seemingly requires only Nanos but not Brat. Pum is able to reduce the level of nanos mRNA and as such a potential negative-feedback mechanism has been identified that protects neurons from overactivity of Pum. Finally, coupling was shown between I(Na) (para) and I(K) (Shal) such that Pum-mediated change in para results in a compensatory change in Shal. The identification of para as a direct target of Pum represents the first ion channel to be translationally regulated by this repressor and the location of the binding motif is the first example in an ORF rather than in the canonical 3'-untranslated region of target transcripts (Muraro, 2008).
Identification of the molecular components that underlie homeostasis of membrane excitability in neurons remains a key challenge. This study shows that the translational repressor Pum binds para mRNA, which encodes the Drosophila voltage-gated Na+ channel. This observation provides a mechanistic understanding for the previously documented ability of Pum to regulate INa and membrane excitability in Drosophila motoneurons (Mee, 2004). Thus, alteration in activity of Pum, in response to changing exposure to synaptic excitation, enables neurons to continually reset membrane excitability through the translational control of a voltage-gated Na+ channel (Muraro, 2008).
Previous studies report several mRNAs subject to direct Pum regulation including hb, bicoid (bcd), CycB, eIF4E, and possibly the transcript destabilization factor smaug (smg). The majority of these identified transcripts concentrate the roles of Pum to the establishment of the embryonic anterior-posterior axis (hb and bcd) and germ-line function/oogenesis (CycB). However, in the last few years, new findings have expanded the role of Pum to encompass predicted roles in memory formation, neuron dendrite morphology, and glutamate receptor expression in muscle. Indeed, the role of Pum is likely to be very much more widespread given that Pum pull-down assays followed by microarray analysis of bound mRNAs have now identified a plethora of possible additional targets of translational regulation (Gerber, 2006). The ~1000 or so genes identified are implicated to be involved in various cellular functions, suggesting that Pum-dependent translational repression might be a mechanism used in different stages of development and in diverse tissue function. To date, para is the first confirmed Pum target encoding a voltage-gated ion channel (Muraro, 2008).
Pum-binding motifs have been identified in the 3'-UTRs of many mRNAs known to bind to this protein. Analysis of 113 such genes expressed in adult Drosophila ovaries has identified a consensus 8 nt binding motif [UGUAHAUA]. This sequence contains the UGUA tetranucleotide that is a defining characteristic of the NRE-like motif described in the 3'-UTR of hb mRNA. Such an 8 nt motif has been identified within the ORF of para at the 3' end of the transcript. The biochemical binding data support the notion that this motif is indeed sufficient to bind Pum and as such represents the first such site to be localized to an ORF of any transcript. However, to translationally repress para mRNA, the data also show a requirement for regions of Pum in addition to the RBD. Interestingly, this kind of requirement has also been shown for another Pum target, eIF4E. The translational silencing of mRNAs is a complex mechanism on which only little information is available. It could involve deadenylation and degradation of the mRNA and/or the circularization of the mRNA and the recruitment of factors that would preclude translation. The fact that different Pum targets may require only the RBD (hb) or the full-length protein (eIF4E and para) suggests that Pum-mediated translational repression may follow complex target mRNA-specific mechanisms, most probably involving the interaction of other domains of Pum with additional, so far unknown, factors. In this regard, it is interesting to note that the N terminus of Pum has regions of low complexity including prion-like domains rich in Q/R. These domains may provide a platform for other proteins that influence the fate of Pum targets (Muraro, 2008).
The putative Pum binding motif lies within an exon that is common to all para splice variants identified (at least in the embryo) but is possibly subject to editing by adenosine deamination. Thus, in an analysis of splicing of para, a number of individual cDNA clones were sequenced and one splice variant was recovered that shows A-to-I editing in this motif. Together with a differential requirement for specific cofactors, editing of this motif might serve to influence how para is affected by Pum and, as such, further increase diversity in level of expression of INa in differing neurons or disease states (Muraro, 2008).
The known mechanism of action of Pum-dependent translational repression is absolutely dependent on additional cofactors. The most studied example, that of hb mRNA during early embryogenesis, requires the presence of both Nanos and Brat. However, the requirement for these two cofactors is seemingly transcript dependent. Thus, Pum-mediated repression of CycB mRNA requires Nanos but not Brat. However, Pum-dependent repression of bcd is apparently Nanos independent, because levels of Nanos in the anterior of the early embryo are undetectable. Although it was clearly shown that Pum-dependent repression of para mRNA in the Drosophila CNS requires Nanos, the requirement for Brat is less clear and seems to be neuronal cell type specific. A requirement for a different combination of cofactors for Pum-dependent translational regulation of a single gene transcript has not been reported previously, but clearly might represent an additional level of regulation. Such differential regulation might be required to spatially restrict the effect of Pum to certain cell types within the CNS. Voltage-gated Na+ currents are responsible for the initiation and propagation of the action potential and determine, together with other voltage-gated ion conductances, the membrane excitability of a neuron. Despite para being the sole voltage-gated sodium channel gene in Drosophila [compared with at least nine different genes in mammals, neuronal subpopulations nevertheless exhibit distinctive INa characteristics. To achieve this, para is known to undergo extensive alternative splicing and, additionally, RNA editing. It is highly likely that both alternative splicing and RNA editing generate mRNAs that encode channels with differing electrophysiological properties. It is also conceivable that these mechanisms might yield para transcripts that contain differing arrangements of Pum/Nanos binding sites, which may, or may not, recruit Brat. Indeed, it has been proposed that variations of the NRE consensus sequence may result in Pum-NRE-Nanos complexes with different topographies, resulting in altered recruitment abilities for additional cofactors such as Brat. Additional work is necessary to clarify where, in para mRNA, the binding sites for the Pum/Nanos complex are localized and how the recruitment of Brat is facilitated in only some neurons. In the hb repression complex, Brat has been shown to interact with the cap-binding protein d4EHP. Therefore, additional cofactors might be necessary for Pum-dependent para repression in the Brat-independent neuronal cell subtypes (Muraro, 2008).
In contrast to translational repression of hb, the data show that Nanos is unlikely to be a limiting factor of Pum-dependent repression of para translation. Consistent with this finding is the observation that overexpression of pum is sufficient to downregulate (and probably translationally repress) nanos mRNA. However, the opposite is not true; overexpression of nanos does not affect levels of pum mRNA. These data suggest that Pum is at least a principal orchestrating factor (if not the prime factor) in regulation of para translation. Moreover, the demonstration that overexpression of pum is sufficient to greatly downregulate nanos mRNA (relative to para mRNA), together with a requirement of Nanos for Pum-dependent para mRNA repression, implicates the existence of a protective negative-feedback mechanism that prevents overrepression of para mRNA. In the absence of such feedback, it is conceivable that excessive overrepression of para mRNA might lead to neurons falling silent as their membrane excitability drops below a critical threshold. Were this to happen, then signaling in the affected neuronal circuit would be severely compromised (Muraro, 2008).
Overexpression of full-length Pum in aCC/RP2 motoneurons not only causes a decrease in INa but also a significant decrease in IKfast. Additionally, pan-neuronal overexpression of Pum causes a significant decrease in Shal mRNA, a gene encoding a potassium channel known to contribute to IKfast. This result was surprising given that Shal was not identified as a Pum target from microarray analysis. That this mechanism might, therefore, be indirect is corroborated by the finding that IKfast and Shal mRNA remain at wild-type levels when Pum is overexpressed in a para-null background. It is, perhaps, counterintuitive that a reduction in INa, to achieve a reduction in membrane excitability, should be accompanied by a similar decrease in outward IKfast. However, changes in ionic conductances should not be considered in isolation and such a relationship might serve to maintain action potential kinetics within physiological constraints. Covariation of INa and IK as a mechanism for changing neuronal excitability has been described in these motoneurons previously. Moreover, there is precedent for coupling between transcripts: injection of Shal mRNA into lobster PD (pyloric dilator) neurons results in an expected increase in IA but also an unexpected linearly correlated increase in Ih, an effect that acts to preserve membrane excitability. Injection of a mutated, nonfunctional, Shal mRNA is also sufficient to increase Ih indicative that this coregulation is activity independent (MacLean, 2003). It remains to be shown whether genetic manipulation of para mRNA levels in Drosophila motoneurons will similarly evoke compensatory changes in Shal expression (Muraro, 2008).
In a previous study, it was shown that blockade of synaptic release, through pan-neuronal expression of tetanus toxin light chain, is sufficient to evoke a compensatory increase in membrane excitability in aCC/RP2 that was accompanied by increases in INa, IKfast, and also IKslow (Baines, 2001). In contrast, the current study showed that overexpression of pum is sufficient to decrease INa and IKfast but does not significantly affect IKslow (although there is a small nonsignificant reduction in this current). Clearly, the complete absence of synaptic input is a more severe change that likely elicits a greater compensatory change in these neurons than when Pum is overexpressed. However, whether removal of synaptic excitation also invokes additional compensatory mechanisms that act preferentially on IKslow remains to be determined. What is consistent, however, is that change in synaptic excitation of these motoneurons is countered by Pum-dependent regulation of both para mRNA translation and magnitude of INa (Muraro, 2008).
A key question remains as to what the mechanism is that transduces changes in synaptic excitation to altered Pum activity. Perhaps the most parsimonious mechanism will be one linked to influx of extracellular Ca2+. Indeed, experimental evidence supports a role for Ca2+, because blocking its entry can preclude changes in neuronal excitability observed as a result of activity manipulation. In addition, changes of gene expression resulting from activity-mediated Ca2+ entry have been described both in vitro and in vivo after plasticity changes such as long-term potentiation. Whether Ca2+ influx influences translation and/or transcription of Pum remains to be shown. Stimulation of mammalian neurons in culture with glutamate, after a preconditioning period of forced quiescence, results in an increase of Pum2 protein levels after just 10 min. The rapidity of this response suggests that it is mediated by a posttranscriptional mechanism. This study examined the role of Pum on Ca2+ channel activity. Neither IBa(Ca) nor levels of the voltage-gated calcium channel coded by Dmca1A (cacophony, Calcium channel α1 subunit, type A) are affected in aCC/RP2 motoneurons in which pum [full length (FL)] is overexpressed. The fact that Pum does not affect Ca2+channel activity directly could reinforce the idea of its serving as a primary sensor of activity changes (Muraro, 2008).
In summary, this study has shown that Pum is able to bind to para mRNA, an effect that is sufficient to regulate both INa and membrane excitability in Drosophila motoneurons. This mechanism requires the cofactor Nanos but does not obligatorily require Brat. Given that mammals express two Pum genes, Pum1 and Pum2, it will be of importance to determine whether this protein is also able to regulate sodium channel translation in the mammalian CNS (Muraro, 2008).
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date revised: 10 April 2009
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