BIOLOGICAL OVERVIEW

Glial cells are the often overlooked wallflowers of the neural family. They are, however, just as important as neurons. Glia protect, nourish and respond to neurons in a very intimate way. Without glia, neurons could not be neurons. anachronism illustrates, on a biochemical level, just how important glial cells are to neural development.

Anachronism is a secreted glial glycoprotein that inhibits premature neuroblast proliferation. trol (terribly reduced optic lobes) acts downstream of ana to activate proliferation of quiescent neuroblasts in an ana-dependent pathway, possibly by inactivating or bypassing the ana repressor. Thus trol and ana are components of a novel developmental pathway, from glial cells to neurons via the secreted protein ANA, for the control of cell cycle activation in quiescent neuroblasts (Datta, 1995). This points to the important role of glia in negatively regulating proliferation of neuronal precursor cells, thereby controlling the timing of postembryonic neurogenesis (Ebens, 1993).

Examination of lacZ expression from an ana enhancer trap line as well as detection of the Ana protein show that ana is also expressed in the larval antennal-maxillary complex (AMC) at all larval stages. As has been reported for the central nervous system, ana expression in the AMC appears to be confined to glial cells. Larval olfactory system function in ana mutants was assayed in a behavioral paradigm. When tested with the three different chemoattractants, third instar ana⁹ mutant larvae showed diminished olfactory response compared to controls. Examination of a second ana allele revealed aberrant olfactory response to ethyl acetate, demonstrating that more than one mutation in ana can give rise to abnormal larval olfactory behavior. Assays of early first instar ana⁹ mutant larvae revealed defective olfactory behavior, implying that the olfactory phenotype stems from early larval AMC and/or embryonic origins. This is consistent with proliferation analysis in the early larval AMC region that uncovered a significantly higher number of S-phase cells in ana⁹ mutants (Park, 1997).

A mutation at the ana locus was originally identified by an insertion of a lacZ-containing P element that caused morphologic defects in the structure of the adult optic lobes. This is a strong hypomorph and possibly a null mutation by genetic criteria, although it shows variable expressivity of the adult morphologic abnormalities. The ana¹ P element insertion expresses ß-galactosidase in the CNS in a pattern similar to that seen by staining with an anti-Ana antibody. The cause of the aberrant optic lobe structure in ana mutants is due to precocious proliferation and differentiation of the optic lobe neuroblasts early in larval life. The initiation of proliferation of the central and thoracic neuroblasts is also premature in ana mutants. Molecular and biochemical analysis has revealed that ana encodes a novel glycoprotein secreted by glial cells neighboring neuroblasts that begin divisions prematurely in an ana mutant. These results led to the suggestion that ana encodes an inhibitor of premature neuroblast proliferation which acts to maintain quiescence of the optic lobe, central brain, and thoracic neuroblasts until the appropriate developmental stage (Park, 1997).

Additional alleles of ana have been generated by imprecise excision of the P-element insertion comprising the ana¹ mutation (Ebens, 1993). Of these, both ana⁸ and ana⁹ are deletion alleles with phenotypes statistically indistinguishable from ana¹, suggesting that they are minimally strong hypomorphic alleles and possibly null alleles. All three of these mutant alleles are homozygous viable and show no gross morphologic abnormalities. Homozygous mutant adults do not live long after eclosion under crowded conditions in competition with wild-type siblings. Previous studies of ana expression focused on the embryonic and larval CNS, where ana appears to be expressed in glial cells as early as stage 14 in embryogenesis and continuing throughout larval development. However, no phenotype in ana mutants has been described earlier than the first premature division of the optic lobe neuroblasts (onbs) at approximately 8-10 h post-hatch. To ask if ana is required for the development or function of other organs, the pattern of ana expression was examined to identify other systems within the developing fruitfly that may require ana function (Park, 1997).

The initial analysis of ana expression in other parts of the larva was carried out by monitoring ß-galactosidase activity produced from the P-element insertion in the ana¹ mutant allele. Larvae heterozygous for the ana¹ P[lacZ] insertion were harvested at first, second, or third instar and crudely dissected to assay for ß-galactosidase activity. Staining was observed in the anterior larval tip at all three developmental stages. Fine dissections of the mouth hooks and anterior tip allowed more detailed examination of the ß-galactosidase staining pattern. In newly hatched first instar animals, ß-galactosidase staining was seen in virtually all the cells in the anterior lobe of the larvae. This ubiquitous staining resolved by 4 h after larval hatching into a bilaterally symmetric pattern of nuclei that remained consistent throughout the remainder of larval life. In this latter pattern the stained nuclei appear to form a cuplike structure around the antennal or dome organ and the maxillary organ in the anterior lobe. From each cuplike structure, a single strand of stained nuclei meanders posteriorly reaching approximately to the dorsal bridge and vertical plate of the mouth hooks. This staining pattern is highly reminiscent of the glial sheath that surrounds the antennal and maxillary organs, ganglia, and larval nerve (Park, 1997).

The implied expression of ana in the anterior lobe was verified by staining with an anti-Ana monoclonal antibody. The ana¹ mutation blocks secretion of the ana repressor, which then builds up in the cytoplasm of cells expressing ana. First instar larvae heterozygous for ana¹ were dissected and stained for ß-galactosidase to mark the lacZ-expressing cells. The samples were then stained with the anti-Ana antibody to localize cells expressing ana. In all cases examined, the anti-Ana antibody staining appears to mark two nerve tracts adjacent to the curve of ß-galactosidase stained nuclei. To control for the possibility that the ana¹ mutation had altered the pattern of ana expression in the anterior lobe, ana expression was also examined in wild-type animals at all three larval stages with the anti-Ana antibody. In all samples examined, the ana expression pattern in the anterior lobe was similar to that seen in ana¹mutant animals (Park, 1997).

The ß-galactosidase staining pattern suggested that ana might be expressed in the glial sheath surrounding the antennal and maxillary organs, antennal and maxillary ganglia, and larval antennal nerve. This possibility would be consistent with the pattern of expression previously described in the larval central nervous system (Ebens, 1993). To test this hypothesis, double labeling was done with ß- galactosidase and a cell type-specific marker. The enhancer trap lines 3-101 and 3-109 label distinct subsets of glial cells in the embryonic central nervous system. The pattern of lacZ expression for each enhancer trap line was analyzed in the anterior lobe of first, second, and third instar larvae. Line 3-101 showed no apparent lacZ expression in the anterior lobe at any stage of larval life. Line 3-109, however, expressed lacZ in a pattern very similar to that seen in ana¹ mutant animals which carry an insertion of a lacZ-bearing P element at the ana locus. This result suggests that the cells expressing ana in the anterior lobe are glial in nature. Because of the difficulty of obtaining reliable antibody penetration for double-labeling studies, further evidence of the glial identity of the ana expressing cells was obtained by analysis of the number of cells expressing lacZ in animals heterozygous for ana¹ and 3-109. If ana¹ and 3-109 result in ß-galactosidase expression in a similar but not overlapping set of cells, one would expect the double ana⁹ heterozygote to contain both sets of labeled cells (Park, 1997).

This should result in a total number of labeled cells equivalent to the sum of the number of cells labeled in an ana¹ heterozygote and a 3-109 animal. Alternatively, if ana¹ and 3-109 label the same population of cells, the double heterozygote should result in the same number of labeled cells as ana¹ or 3-109 animals. Cell counts of the number of labeled cells in ana¹heterozygotes, 3-109 animals, and ana1/+;+/3-109 transheterozygotes revealed that approximately the same number of cells were labeled in the transheterozygote as were by either ana¹or 3-109 alone (Park, 1997).

The early onset of the larval olfactory phenotype coupled with expression of ana in glial cells of larval AMC raised the question of whether ana also acts as a proliferation repressor in the AMC region. Therefore, 5-bromodeoxyuridine (BrdU) incorporation was used to examine the relative numbers of S-phase cells in ana mutant and control AMCs early in first instar. A 2 h BrdU labeling of newly hatched first instar larvae shows that ana⁹ mutant larvae have a significantly higher number of BrdU stained cells in each side of the anterior lobe than do Oregon R or P[ana+];ana¹ animals (Park, 1997).

How might mutations in ana result in olfactory defects? In wild-type embryos the primordia of the AMC become visible in the epidermis by stage 13 of embryogenesis, and by early stage 14 the axons of the antennal organ have already grown along the larval antennal nerve into the subesophageal ganglion. Initial detection of ana expression in the embryonic CNS using ß-galactosidase staining occurs at stages 14-16 (Ebens, 1993), after development of the larval antennal lobe and connectivity with the antennal organ. However, staining with the ana antibody has been observed in embryonic glial cells of CNS glial cells as early as stage 11. In addition, ana could be influencing early development through maternally supplied gene products although maternally supplied ana message or protein has not been described. Thus, it is possible that the larval olfactory phenotype may stem from an earlier embryonic CNS defect, or from abnormal development of the larval AMC during embryogenesis due to previously undetected expression of ana in the developing AMC. If ana is not required for the initial development of the larval olfactory system during embryogenesis, how might mutations in ana cause aberrant olfactory behavior? Lack of ana function in the larval CNS is known to result in premature proliferation of the central brain neuroblasts as early as first instar (Ebens, 1993). Mutations in ana result in supernumerary S-phase cells in the larval AMC area in newly hatched first instar and the organs they may contribute to are as yet unknown. It has been documented in vertebrate and mammalian systems that the olfactory epithelium continues to add new neurons throughout much of the life of the system rather than only once during the initial development and assembly of the olfactory system. The same may hold true for the larval olfactory system central nervous system in Drosophila. If this is true, and if ana function in glial cells of the AMC results in inhibition of neuroblast division as it does in the CNS, then mutations in ana may result in premature addition of cells to the larval AMC and aberrant wiring of antennal ganglion or abnormal innervation of the larval antennal lobe. Interestingly enough, trials of ana⁹ and Oregon R adults with ethyl acetate at several concentrations in a T-maze choice paradigm revealed no significant olfactory phenotype (Park, 1997).

Thus, ana is expressed by glial cells of the larval AMC and ana mutants show diminished olfactory sensitivity to several odorants. Evidence has been presented that the larval olfactory phenotype is probably not due to preture division of the larval CNS neuroblasts, but may be due to supernumerary S-phase cells in the early first instar AMC or lack of ana function in the embryo. Future studies will be aimed at determining whether the extra BrdU-labeled cells in the AMC are neuroblasts, and whether the progeny of those labeled cells are incorporated into the larval AMC. These investigations will enhance understanding of glial involvement in nervous system develment and function (Park, 1997).

cDNA clone length - 2.4kb

Bases in 5' UTR - 230

Exons - five

Bases in 3' UTR - 764

PROTEIN STRUCTURE

Amino Acids 474

Structural Domains

The protein has a high histidine concentration at the C-terminus. There are six potential N-linked glycosylation sites (Ebens, 1993).

Drosophila miR-124 regulates neuroblast proliferation through its target anachronism

MicroRNAs (miRNAs) have been implicated as regulators of central nervous system (CNS) development and function. miR-124 is an evolutionarily ancient, CNS-specific miRNA. On the basis of the evolutionary conservation of its expression in the CNS, miR-124 is expected to have an ancient conserved function. Intriguingly, investigation of miR-124 function using antisense-mediated miRNA depletion has produced divergent and in some cases contradictory findings in a variety of model systems. This study investigated miR-124 function using a targeted knockout mutant and evidence is presented for a role during central brain neurogenesis in Drosophila. miR-124 activity in the larval neuroblast lineage is required to support normal levels of neuronal progenitor proliferation. anachronism (ana), which encodes a secreted inhibitor of neuroblast proliferation, was functionally identified as an important target of miR-124 acting in the neuroblast lineage. ana has previously been thought to be glial specific in its expression and to act from the cortex glia to control the exit of neuroblasts from quiescence into the proliferative phase that generates the neurons of the adult CNS during larval development. Evidence is provided that ana is expressed in miR-124-expressing neuroblast lineages and that ana activity must be limited by the action of miR-124 during neuronal progenitor proliferation. The possibility is discussed that the apparent divergence of function of miR-124 in different model systems might reflect functional divergence through target site evolution (Weng, 2012).

The finding that miR-124 activity is required in the NB lineage to support proliferation contrasts with findings from vertebrate systems that have suggested a role for miR-124 in limiting neuronal progenitor proliferation and in promoting neuronal differentiation. Several independent studies have reported that miR-124 promotes neuronal differentiation in mouse neural progenitor cells in culture by downregulating inhibitors of differentiation including the RNA splicing regulator PTB1, the SCP1 phosphatase, the transcription factor SOX9 and the ephrin B1 receptor. Depletion of miR-124 using antisense oligonucleotides has been reported to promote proliferation and reduce differentiation of neuronal progenitors isolated from the subventricular zone (SVZ) of the mouse embryonic CNS. Comparable results were obtained by injection of a pump to deliver antisense oligonucleotides to deplete miR-124 in vivo in the SVZ of the mouse brain. Taken together, the analysis of vertebrate miR-124 function mainly lends support to the idea that its expression in differentiating neurons acts to turn off negative regulators of differentiation and that loss of the miRNA supports proliferation of neuronal precursors (Weng, 2012).

A possible basis for the difference between these findings and those in vertebrate systems is that the expression of miR-124, although broadly CNS specific, differs in detail between insects and vertebrates. This study observed expression in neural progenitors, which has not been generally reported in the vertebrate, although the presence of the miR-124 primary transcript in the NB and GMC is consistent with the expression of the miR-124-GFP reporter. In situ hybridization does not reveal significant levels of mature miR-124 in vertebrate neural progenitors, but one report using an in vivo sensor for miRNA activity has indicated that miR-124 activity can be detected in mouse neural progenitor cells. Taken together, these findings prompt the question of whether there might be a corresponding function in the neural progenitors of the vertebrate CNS that might have been overlooked owing to low-level expression of the mature miRNA (Weng, 2012).

The possibililty has also been considered the possibility that the difference between the current findings and those reported using vertebrate models reflect methodological differences, i.e. the use of a genetic null mutant versus miRNA depletion using injected or transfected antisense oligonucleotides to reduce miRNA activity. Previous analysis of genetic mutants has not supported the conclusions of antisense injections to deplete miRNA function in Drosophila embryos. Antisense methods allow partial reduction of function and might introduce a degree of experimental variability. It will be of interest to learn whether mouse knockouts of miR-124 support the findings reported using antisense methods (Weng, 2012).

A third and perhaps more interesting possibility is that the different effects of miR-124 on neuronal progenitor proliferation reflect evolutionary divergence of miR-124 function. miR-124 has hundreds of potential targets as identified by computational prediction, by expression profiling of miRNA overexpression and depletion and by immunopurification of miRNA-containing ribonucleoproteins. There is little evidence of conservation of the identified or predicted targets between insects, nematodes and vertebrates. Furthermore, each of the reports on miR-124 function in vertebrates has attributed its role to the regulation of different targets. This might reflect subtly different roles for the miRNA in different neuronal progenitor cell models in vitro, in different regions of the developing CNS or at different stages of development. Evidence has been presented for different miR-124 functions at different stages in Xenopus eye development. It is also possible that miR-124 acts via several functionally significant targets that each serve as repressors of neuronal differentiation in vivo (Weng, 2012).

In Drosophila, this study has identified ana as a target of miR-124 in vivo and provided direct genetic evidence that downregulation of ana expression in neuronal progenitors is required to support a normal level of proliferation within the larval central brain. The ana gene is not conserved beyond the Drosophila family. This leads to the intriguing proposal that miR-124 might have acquired a novel target in Drosophila, which has led to an entirely distinct function in the control of CNS proliferation from that found in vertebrates. It might also suggest that an evolutionarily ancient and presumably conserved role of miR-124 awaits discovery (Weng, 2012).

Perlecan participates in proliferation activation of quiescent Drosophila neuroblasts

Drosophila neuroblasts act as stem cells. Their proliferation is controlled through cell cycle arrest and activation in a spatiotemporal pattern. Several genes have been identified that control the pattern of neuroblast quiescence and proliferation in the central nervous system (CNS), including anachronism (ana), even skipped (eve) and terribly reduced optic lobes (trol). eve acts in a non-cell-autonomous manner to produce a transacting factor in the larval body that stimulates cell division in the population of quiescent optic lobe neuroblasts. ana encodes a secreted glial glycoprotein proposed to repress premature proliferation of optic lobe and thoracic neuroblasts. trol was shown to act downstream of ana to activate proliferation of quiescent neuroblasts either by inactivating or bypassing ana-dependent repression. This study shows that trol codes for Drosophila Perlecan, a large multidomain heparan sulfate proteoglycan originally identified in extracellular matrix structures of mammals. The results suggest that trol acts in the extracellular matrix and binds, stores, and sequesters external signals and, thereby, participates in the stage- and region-specific control of neuroblast proliferation (Voigt, 2002).

Targets of Activity

trol (terribly reduced optic lobes), acting downstream of ana in neuroblasts in the larval brain, may be part of a developmental switch that controls activation of neuroblast proliferation (Datta, 1995).

DEVELOPMENTAL BIOLOGY

Embryonic

In certain ana mutants, ANA protein accumulates to high levels in ana expressing cells, facilitating their identification. ANA glycoprotein is not expressed in affected neuroblasts, but in a subclass of neighboring glial cells (Ebens, 1993).

Effects of mutation or deletion

In ana mutants, quiescent postembryonic central brain and optic lobe cells precociously enter S phase (DNA synthesis) and persist into adulthood (Ebens, 1993). ana mutants exhibit a variable phenotype, ranging from misrouting of fiber tracts to massive disorganization of the adult optic lobes (Ebens, 1993).

REFERENCES

Datta, S. (1995). Control of proliferation activation in quiescent neuroblasts of the Drosophila central nervous system. Development 121: 1173-1182. PubMed Citation: 7743929

Ebens, A. J., Garren, B., Cheyette, N.R. and Zipursky, S.L. (1993). The Drosophila anachronism locus: a glycoprotein secreted by glia inhibits neuroblast proliferation. Cell 74: 15-27. PubMed Citation: 7916657

Park, Y., Caldwell, M. C. and Datta, S. (1997). Mutation of the central nervous system neuroblast proliferation repressor ana leads to defects in larval olfactory behavior. J. Neurobiol. 33(2): 199-211. 9240375

Voigt, A., Pflanz, R., Schäfer, U. and Jäckle, H. (2002). Perlecan participates in proliferation activation of quiescent Drosophila neuroblasts. Dev. Dyn. 224(4): 403-12. PubMed Citation: 12203732

Weng, R. and Cohen, S. M. (2012). Drosophila miR-124 regulates neuroblast proliferation through its target anachronism. Development 139(8): 1427-34. PubMed Citation: 22378639

date revised: 25 September 2012

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