Comparative analysis of Nerfin-1 and Pros expression in the developing nervous system revealed a marked, although not complete, overlap in expression. The nuclear co-localization of these proteins in neuronal precursor cells, and the fact that mutations in nerfin-1 and pros trigger axon guidance defects, raised the possibility that they may regulate the expression of one another. To determine the epistatic relationship between nerfin-1 and pros, their expression dynamics were studied in each other's loss-of-function mutant backgrounds. Pros immunostaining in nerfin-1null embryos did not identify any significant changes in Pros expression. In marked contrast, nerfin-1 mRNA and protein levels in embryos collected from two independent loss-of-function pros mutants revealed that pros is required for wild-type nerfin-1 expression levels. Nerfin-1 expression was significantly reduced throughout the CNS and PNS; however, its expression was not completely ablated in any of the prosnull alleles tested (Kuzin, 2005).
To determine if the axon guidance phenotype observed in prosnull (prosI13) mutant embryos could be explained by a requirement for wild-type nerfin-1 expression, the extent of CNS axon disorganization triggered by single and double mutant combinations were examined. Comparisons demonstrated that loss of pros function resulted in a more severe phenotype than that observed in the nerfin-1null embryos. Although both prosnull and nerfin-1null embryos exhibited disruptions in the longitudinal axon connectives, loss of pros function resulted in an overall greater disruption in commissure organization. In addition, the nerve cord in prosnull embryos was considerably wider when compared to nerfin-1 mutants or wild-type embryos. The axon scaffolding phenotype observed in nerfin-1null; prosI13 double mutant was more severe than that of either single mutant (Kuzin, 2005).
Analysis of two other transcription factor genes that are widely expressed in the developing CNS, lola and fru, has revealed that they too are required for proper axon guidance and both are required for proper longitudinal axon fasciculation. However, unlike the severe axon guidance phenotype observed in prosnull embryos, the disorganization of the CNS axon scaffolding is not as extensive in lola or fru loss-of-function mutants. To determine the epistatic relationship between nerfin-1 and lola or fru, the expression of each gene in each other's loss-of-function background was analyzed. In contrast to the marked reduction of nerfin-1 expression in pros mutants, no such reduction in nerfin-1 expression was found in lola or fru mutants, nor was the expression of lola or fru altered in nerfin-1null embryos (Kuzin, 2005).
Given the axon guidance defects in nerfin-1null embryos and the fact that Nerfin-1 is a Zn-finger nuclear protein, it was hypothesized that Nerfin-1 may be required for the correct expression of genes involved in axon guidance. Accordingly, the embryonic expression profiles of over 35 genes that have been shown to play important roles in axon guidance were examined. Included in the candidate screen were genes encoding transcription factors, RNA-binding proteins, cell surface receptor proteins, their ligands, signal transduction proteins, and components of the cytoskeleton. Homozygous nerfin-1null embryos were identified by the absence of Nerfin-1 immunoreactivity. Whole-mount in situ hybridization and/or protein immunostaining for altered spatial or temporal expression in nerfin-1null embryos identified six genes that require nerfin-1 function to achieve full wild-type expression levels (Kuzin, 2005).
Two genes involved in anterior vs. posterior commissure choice, those encoding the receptor tyrosine kinase Derailed, and its ligand Wnt5, both required nerfin-1 for full expression. In the absence of nerfin-1, ventral cord expression levels of Robo and Robo3 were unaffected; however, Robo2 expression levels were significantly reduced. Expression of Slit, the ligand for Robo receptors, and Commissureless, a factor responsible for clearing Robo receptors from commissural axons, was unaffected in nerfin-1null embryos (Kuzin, 2005).
Loss of nerfin-1 function also significantly delayed and/or reduced the early expression of the neuron-specific microtubule-associated MAP1B-like gene futsch. futsch expression is normally activated in newborn neurons starting at stage 11; however, in nerfin-1null embryos expression is first detected only at the stage 13. Not until embryonic stage 15 did the level of futsch expression in mutant embryos approach that of wild type. Reduced mRNA steady state levels for the genes encoding Leukocyte-antigen-related-like (Lar), another receptor tyrosine kinase, and G-oα47A gene, which encodes an alpha subunit of heterotrimeric G proteins, were also detected in nerfin-1null embryos. The reduced level of gene expression in mutant embryos was nervous system specific. For example, G-oα47A gene expression in mesodermal derived tissues was not altered in nerfin-1null embryos (Kuzin, 2005).
Carbon dioxide (CO2) elicits different olfactory behaviors across species. In Drosophila, neurons that detect CO2 are located in the antenna, form connections in a ventral glomerulus in the antennal lobe, and mediate avoidance. By contrast, in the mosquito these neurons are in the maxillary palps (MPs), connect to medial sites, and promote attraction. In Drosophila loss of a microRNA, miR-279, leads to formation of CO2 neurons in the MPs. miR-279 acts through down-regulation of the transcription factor Nerfin-1. The ectopic neurons are hybrid cells. They express CO2 receptors and form connections characteristic of CO2 neurons, while exhibiting wiring and receptor characteristics of MP olfactory receptor neurons (ORNs). It is proposed that this hybrid ORN reveals a cellular intermediate in the evolution of species-specific behaviors elicited by CO2 (Cayirlioglu, 2008).
In insects, both the position of CO2 neurons and the behavior elicited by CO2 differ among species. For example, olfactory detection of CO2 through neurons positioned in or around the mouthparts of an insect, such as maxillary palps (MPs) and labial palps, correlates with feeding-related behaviors. Indeed, in some blood-feeding insects such as mosquitoes and tsetse flies, these neurons are harbored in the MPs and are important in locating hosts via plumes of CO2 that they emit. The hawkmoth, Manduca sexta, monitors nectar profitability of newly opened Datura wrightii flowers through CO2 receptor neurons located in their labial palps. In these examples, CO2 acts as an attractant. Conversely, in Drosophila CO2 is a component of a stress-induced odor that triggers avoidance behavior. This repellent response is driven by antennal neurons expressing the CO2 receptor complex Gr21a-Gr63a. How did these diverse behavioral responses to CO2 arise during insect evolution? It is proposed that this diversity emerged through multiple steps, including changes in cellular position (arising from elimination of CO2 neurons in one appendage and generation of these neurons in another) and changes in circuitry (Cayirlioglu, 2008).
In the course of a genetic screen for mutants disrupting the organization of the olfactory system, a mutant (S0962-07) was isolated that resulted in the formation of ectopic Gr21a-expressing neurons in the MPs. Some 22 ± 1.5 (mean ± SEM) green fluorescent protein (GFP)-positive cells were observed in the mutant MP, whereas the number of antennal Gr21a olfactory receptor neurons (ORNs) was unaffected. In the wild type, Gr21a cell bodies were restricted to the antenna. The ectopic MP cells expressed both CO2 receptors (Gr21a and Gr63a). Consistent with this finding, mutant cells conferred CO2 sensitivity to the MP. Staining the MP with an antibody to the pan-neuronal marker Elav revealed an increase of 21 ± 3.4 neurons in the mutant, which suggests that all ectopic neurons expressed Gr21a (Cayirlioglu, 2008).
In wild-type MPs, each sensillum contains two ORNs. By contrast, in the mutant MP sensilla, additional neurons expressing Elav and the general receptor Or83b were observed. This was also apparent when a MP ORN marker (MPS-GAL4) expressed in a subset of MP ORNs was used. This marker labels single cells within a subset of wild-type MP sensilla; however, in mutant MPs, two additional neurons were observed, bringing the total number of neurons within these sensilla to four. Thus, the generation of ectopic Gr21a-Gr63a neurons is due to an increase in the number of neurons within sensilla rather than transformation of MP ORNs (Cayirlioglu, 2008).
In the wild type, each class of adult ORNs sends projections from both antennae or MPs to the antennal lobe (AL). ORNs expressing same odorant receptors (ORs) typically form synapses in the same glomerulus within the AL. CO2 neurons in the antenna target the V-glomerulus. To specifically assess the targeting of ectopic MP CO2 neurons, flies were examined where the antennae were surgically removed. It was found that ectopic CO2 neurons targeted the V-glomerulus and other medial sites in the AL. The wiring specificity of antennal CO2 neurons in the mutants was identical to that in the wild type. Thus, the ectopic CO2 neurons in the MP target, at least in part, the same glomerulus innervated by the wild-type CO2 neurons in the antennae (Cayirlioglu, 2008).
S0962-07 was mapped to a P-element insertion some 1 kb upstream of a microRNA, miR-279. MicroRNAs (miRNAs) are small noncoding RNAs of about 22 nucleotides that bind to specific sequences of the 3'-untranslated region (3'UTR) of target genes and thereby repress gene expression posttranscriptionally. In recent years, miRNAs were implied in a variety of functions in the nervous system of different organisms. To assess whether miR-279 is responsible for the observed phenotype, three small deletions were generated that uncovered the miR-279 genomic region. These deletion mutants exhibited phenotypes indistinguishable from S0962-07. The ectopic CO2 phenotype was rescued by a 3-kb fragment of genomic DNA encoding only miR-279. Thus, miR-279 is the gene disrupted in S0962-07 and must repress targets in the MP to inhibit ectopic CO2 neuron development (Cayirlioglu, 2008).
To assess whether miR-279 is expressed in the developing MPs, transgenic flies were generated carrying a transcriptional reporter construct (miR-279-GAL4). Expression was monitored in flies carrying this GAL4 construct and the reporter UAS-mCD8GFP. Around 40 to 50 hours after puparium formation (APF), large cells reminiscent of sensory organ precursors in other epithelia expressed miR-279. At later stages, miR-279-expressing cells were found in clusters with smaller cells, some of which expressed neuronal markers. As ORNs matured, miR-279 expression was lost (Cayirlioglu, 2008).
Attempts were identify the target gene(s) responsible for the miR-279 mutant phenotype. About 205 potential target mRNAs of miR-279 were previously predicted. One of the strongest candidates for miR-279 regulation is Nerfin-1. The Nerfin-1 3'UTR contains multiple miR-279 binding sites and encodes a transcription factor expressed in neuronal precursors and transiently in nascent neurons in the embryonic central nervous system. Nerfin-1 protein appeared in miR-279-positive cells between 50 and 60 hours APF. Nerfin-1 and miR-279 gradually redistributed, generating complementary expression patterns. Cells with high levels of Nerfin-1 expressed low levels of miR-279 and vice versa (Cayirlioglu, 2008).
To test whether Nerfin-1 is up-regulated in miR-279 mutants, mutant MPs were stained with antibodies to Nerfin-1. 22 ± 4.8 additional Nerfin-1-expressing cells were found in miR-279 mutant MPs relative to controls. This is similar to the number of ectopic CO2 neurons in the MP. The vast majority of CO2 ORNs in the MP expressed Nerfin-1. Thus, the expression pattern of Nerfin-1 protein in the wild type and in mutant MPs is consistent with nerfin-1 mRNA being a target for miR-279 in vivo (Cayirlioglu, 2008).
To determine whether miR-279 directly binds to nerfin-1 3'UTR and inhibits its expression, a luciferase reporter assay was used in cultured cells. The luciferase-coding region was fused to the full-length nerfin-1 3'UTR, which contains four conserved 8-nucleotide oligomer target sites for miR-279, as well as to a subregion containing three of these sites. Luciferase activity of both nerfin-1 sensor constructs was strongly repressed when cells were cotransfected with miR-279. By contrast, the activity of either nerfin-1 sensor was unaffected by noncognate miR-315. Antisense oligomers directed against the miR-279 core sequence specifically relieved nerfin-1 reporter repression. Thus, it is concluded that nerfin-1 is a direct target of miR-279 (Cayirlioglu, 2008).
Next whether Nerfin-1 down-regulation by miR-279 inhibits the development of CO2 neurons in the MPs was assessed. To do this, the level of nerfin-1 was reduced by half genetically in a miR-279 mutant background. This decreased the number of CO2 neurons in the MP relative to miR-279 mutants, providing strong in vivo evidence that miR-279 is necessary to down-regulate Nerfin-1 in MPs during normal development. Nerfin-1 up-regulation alone was not sufficient to generate a miR-279-like phenotype. Taken together, these findings suggest that miR-279 down-regulates Nerfin-1 and other targets to prevent CO2 neuron development in the MPs (Cayirlioglu, 2008).
When analyzing the axonal projections of the CO2 neurons in the MPs, it was observed that these neurons targeted one or more medial glomeruli in addition to the V-glomerulus, the target of antennal CO2 neurons. These medial glomeruli are normally innervated by MP Or42a and Or59c ORNs. Double-labeling experiments revealed that mutant neurons also coexpressed Or42a and Or59c, but not other MP ORs. Analysis of subsets of MP ORNs also revealed that Or42a and Or59c classes each showed an approximate increase of 10 cells in the MPs, whereas others were unaffected. These results indicate that the ectopic CO2 neurons are formed as additional cells within Or42a and Or59c sensilla and are hybrid in identity. They express ORs and exhibit wiring characteristics of two classes of neurons (Cayirlioglu, 2008).
It is interesting that the loss of miR-279 generates a CO2 neuron within a sensillum harboring four neurons in the MP, given that the antennal CO2 sensilla in Drosophila are the only sensilla in the olfactory system to harbor four ORNs. Because miR-279 acts within the precursor cells in the MP to prevent Nerfin-dependent formation of olfactory neurons, this observation raises the intriguing possibility that positioning of CO2 neurons on different olfactory appendages might have evolved through changes at the level of precursor cell development. Thus, the evolutionary elimination of CO2 neurons from MP sensilla might have required decreasing the number of cells with neuronal identities through down-regulation of Nerfin-1 by miR-279 (Cayirlioglu, 2008).
Although it was hypothesized that relocation of CO2 ORNs to different appendages was important in the evolution of differences in CO2 sensing, additional mechanisms must have evolved to modify the neural circuitry to alter species-specific behaviors in response to CO2. The ectopic CO2 neurons are hybrid cells, which express additional receptors (Or59c or Or42a) and also target medial glomeruli, typically innervated by wild-type ORNs expressing these ORs. This is particularly interesting given that CO2 neurons in mosquitoes connect to medial glomeruli, driving an attractive response. It is speculated that this hybrid cell represents an evolutionary intermediate on a path leading to species-specific CO2 behavior. Perhaps suppressing the expression of Or59c or Or42a ORs could convert this hybrid cell to one dedicated only to CO2 reception. The nature of the behavioral output to CO2 (i.e., attraction versus repulsion) by this cell, however, may be dictated by altering the wiring specificity to one site or the other (medial versus ventral, respectively). More generally, it is proposed that natural selection can work on such an evolutionary intermediate to generate different combinations of OR, wiring, and cellular positional specificities, depending on the insects' environmental needs. This may in turn lead to novel olfactory responses to different odorants, or to the same odorant in different species (Cayirlioglu, 2008).
The mRNA encoding the Drosophila Zn-finger transcription factor Nerfin-1, required for CNS axon pathfinding events, is subject to post-transcriptional silencing. Although nerfin-1 mRNA is expressed in many neural precursor cells including all early delaminating CNS neuroblasts, the encoded Nerfin-1 protein is detected only in the nuclei of neural precursors that divide just once to generate neurons and then only transiently in nascent neurons. Using a nerfin-1 promoter controlled reporter transgene, replacement of the nerfin-1 3' UTR with the viral SV-40 3' UTR releases the neuroblast translational block and prolongs reporter protein expression in neurons. Comparative genomics analysis reveals that the nerfin-1 mRNA 3' UTR contains multiple highly conserved sequence blocks that either harbor and/or overlap 21 predicted binding sites for 18 different microRNAs. To determine the functional significance of these microRNA-binding sites and less conserved microRNA target sites, their ability to block or limit the expression of reporter protein was studied in nerfin-1 expressing cells during embryonic development. The results indicate that no single microRNA is sufficient to fully inhibit protein expression but rather multiple microRNAs that target different binding sites are required to block ectopic protein expression in neural precursor cells and temporally restrict expression in neurons. Taken together, these results suggest that multiple microRNAs play a cooperative role in the post-transcriptional regulation of nerfin-1 mRNA, and the high degree of microRNA-binding site evolutionary conservation indicates that all members of the Drosophila genus employ a similar strategy to regulate the onset and extinction dynamics of Nerfin-1 expression (Kuzin, 2007).
During embryonic stage 10, nerfin-1 mRNA is detected in all early delaminating CNS NBs albeit at differing levels; and within many of the NBs the message appears to be asymmetrically distributed. Although nerfin-1 mRNA expression is pan-neural during this early stage in CNS development, immunostains using different Nerfin-1 specific polyclonal antibodies detect significant levels of Nerfin-1 protein only in the ventral cord MP2 NBs. The punctate/irregular distribution of the nerfin-1 mRNA in NBs lacking detectable levels of Nerfin-1 protein is reminiscent of that observed for mRNAs targeted for miRNA mediated cleavage in mammals, suggesting that nerfin-1 message in many of the NBs may likewise be targeted for degradation (Kuzin, 2007).
Although the dynamics of nerfin-1 mRNA and protein expression differ considerably during the early stages of nervous system development, by stage 13 the pattern of Nerfin-1 expression closely matches that of its mRNA and close inspection of nerfin-1 message distribution in the Nerfin-1 protein expressing cells revealed an even cytoplasmic distribution. Expression of both the message and protein in the new born CNS and PNS neurons is short lived; levels of both rapidly decline such that by late stage 14 both message and protein levels are significantly lower throughout the nervous system (Kuzin, 2007).
miRNA target prediction programs have identified multiple putative miRNA binding sites within the nerfin-1 1,622 bp 3' UTR and many of these sites are conserved in other nerfin-1 Drosophila orthologues. For example, a Drosophila EvoPrint (Odenwald, 2005) of the nerfin-1 locus (using D. melanogaster as the reference sequence and D. sechellia, D. yakuba, D. erecta, D. ananassae, D. persimilis, D. pseudoobscura, D. willistoni, D. mojavensis and D. grimshawi as test sequences) revealed conserved sequence blocks within the 3' UTR that contain or overlap 21 predicted miRNA binding sites for 18 different miRNAs. The conserved sequences are present in all, or all but one, species used in the analysis and represent over 100 million years of collective evolutionary divergence. The partial and/or interrupted conservation within the predicted miRNA binding sites may reflect the fact that initial base-pairing of an miRNA and its mRNA target sequence requires only eight bases to initiate translational regulation. EvoPrint analysis of the nerfin-1 3' UTR also identified additional conserved sequence blocks that do not contain or overlap predicted miRNA binding sites and their role(s) in gene function are currently unknown. In vivo cis-regulatory analysis of the nerfin-1 3' UTR failed to detect any transcriptional enhancer activity. In addition to the conserved miRNA target sites, less conserved predicted binding sites have been identified within the 3' UTR. For example, the central miR-279/miR-286 and miR-279 target sites are present in the species that are evolutionarily close to D. melanogaster but not conserved in the more distant D. persimilis, D. pseudoobscura, D. willistoni, D. mojavensis and D. grimshawi species (Kuzin, 2007).
The conservation of nerfin-1 miRNA sites suggests that miRNA mediated post-transcriptional regulation of nerfin-1 occurs in all members of this genus. MicroRNAs most likely regulate other EIN-domain containing zinc finger genes. For example, multiple miRNA binding sites have also been detected in the vertebrate IA-1 3' UTR, and EvoPrint analysis reveals that one of sites within the human IA-1 gene is highly conserved (Kuzin, 2007).
To determine if the conserved nerfin-1 3' UTR sequences are required for the embryonic NB post-transcriptional regulation, a series of reporter transgene constructs were generated that tested the silencing activity of different regions of its 3' UTR. The starting construct was prepared by replacing the nerfin-1 ORF and 3' UTR in an 11 kb nerfin-1 genomic rescue construct with a sequence that contains the ORF for a nuclear targeted Green Fluorescent Protein (GFP-NLS) linked to the viral SV-40 3' trailer that lacks any predicted miRNA binding sites. As expected, transformants that contain the P[nerfin-1.GFP-NLS.SV-40] construct expressed GFP in all early delaminating CNS NBs and no translational block of GFP expression was detected when compared to nerfin-1 mRNA expression. The full-length or different sub-regions of the nerfin-1 3' UTR containing the conserved sequence blocks were then inserted into a unique restriction site within the vector's SV-40 3' UTR. Embryo GFP-immunostains were performed on multiple independent transformant lines for each construct. As controls, multiple independent transformant lines that contain the nerfin-1 3' UTR sequences in the opposite orientation were also generated for each construct and embryo GFP-immunostains revealed that in all cases the translational block in GFP expression was orientation dependent (Kuzin, 2007).
Insertion of the full-length 3' UTR into the P[nerfin-1.GFP-NLS.SV-40] reporter recapitulated the silencing of nerfin-1 mRNA translation. Similar to the endogenous Nerfin-1 protein expression during embryonic stages 10 and 11, significant levels of GFP expression in the ventral cord were observed only in the MP2 NBs. However, reporter transgenes that contained sub-regions of the 3' UTR gave only partial or no block in NB GFP expression. For example, although the iB and iH constructs, consisting respectively of the conserved 5' and 3' multiple miRNA binding site sub-regions, significantly reduced GFP expression in stage 11 NBs, both of these sub-regions only partially blocked expression during stage 10. Further sub-division of the 5' conserved miRNA binding site cluster (constructs iC, iD and iE) revealed that the overlapping miR-9A, miR-9B and miR-9C binding sites and the miR-279/mir-286 both contributed to the partial inhibition observed with the iB construct, but the conserved Bantam miRNA-binding site did not. It is worth noting that the 5' predicted miR-279/mir-286 target site within the nerfin-1 rescue construct contains the sequence TCTAGTCA that agrees with the predicted miR-279/mir-286 binding site. This sequence differs in the second to last base from that of the D. melanogaster genomic sequence (FlyBase BLAST), in which there is a T in place of C. cDNA sequence analysis of all ESTs in the database reveals a C instead of a T at this position (Kuzin, 2007).
Given that only the full-length insert recapitulates the silencing of endogenous expression in the CNS, it is concluded the miRNAs act in a cooperative fashion to regulate the onset of Nerfin-1 protein expression. The block in translation by sub-regions of the 3' UTR was more effective at stage 11 than at stage 10; this could reflect time of onset of miRNA expression or the possibility that the level of mRNA expression is too high for a complete block at the earlier stage. Previous studies have shown that ectopic expression of nerfin-1 outside the wild-type temporal/spatial boundaries during CNS development results in axon guidance defects. The requirement for multiple miRNA binding sites may reflect the need for tight spatial control of Nerfin-1 expression (Kuzin, 2007).
Dissection of the sub-regions reveals that the miR-9A, miR-9B and miR-9C combined site, as well as the miR-279/mir-286 site, contribute to silencing, but the Bantam site did not show an effect. The conservation of the Bantam site suggests that it is functionally important, but no effect on embryonic CNS expression of nerfin-1 was observed. Consistent with this, no effect on Nerfin-1 protein expression was detected in bantam minus embryos. Bantam has been shown to have developmental roles in post-embryonic development. Analysis of the 3' sub-region sites indicates that in the CNS, the combined miR-279/miR-286 site exhibits partial silencing, with no effect observed for the other miRNA binding sites. Interestingly, the less conserved centrally located miR-279/miR-286 and miR-279 sites did not promote silencing. These two sites share less homology to the miR-279 and miR-286 binding sites than the other conserved miR-279/mir-286 target sites. Construct iG, which contains the overlapping miR-92A, miR-92B, and miR-310-313 sites revealed no detectable miRNA silencing. In addition, construct iK that contains a predicted miR-5 binding site did not affect the reporter mRNA translation in the embryonic CNS and PNS. The other sites in the 3' sub-region exhibited an effect in PNS silencing, suggesting spatial specificity for microRNA effects on nerfin-1 expression (Kuzin, 2007).
During embryonic PNS development, nerfin-1 mRNA and protein are transiently expressed in secondary precursor cells that divide once to generate neurons, and then both its transcript and encoded protein are only transiently detected in nascent neurons. Unlike the post-transcriptional regulation observed in the developing CNS, when the full-length nerfin-1 3' UTR was included in the reporter transgene the onset of GFP expression was not blocked in precursor cells but the duration of GFP expression in the nascent neuron was significantly reduced. The rapid extinction of detectable GFP expression mirrored that of the endogenous Nerfin-1 transient expression; the short-lived expression was observed throughout the PNS in the ventral, lateral and dorsal neurons such that by stage 15 little or no GFP immunostaining was detected. Similar to the reporter results obtained in the CNS for the different 3' UTR sub-regions, no one sub-region or single miRNA binding site was able to fully limit GFP expression in older stage 14 and 15 neurons. However, except for the predicted Bantam miRNA-binding site that showed no detectable effect on silencing GFP expression, all of the other 3' UTR sub-regions exhibited different degrees of silencing. Each of the constructs had differential effects on reporter expression in different cells of the PNS, suggesting an involvement of miRNAs in cell-type regulation of Nerfin-1 expression. For example, construct iB, containing the 5' end of the 3' UTR, exhibited a higher levels of silencing in individual cells of the dorsal and lateral clusters; a construct containing the 3' end of the 3' UTR, exhibited a higher level of silencing in the chordotonal neurons in the lateral cluster than in other cells of the lateral and ventral clusters; another construct containing a subset of sites in the 3' UTR, exhibited a higher level of silencing in a subset of cells in the dorsal and lateral clusters than in other cells of the same clusters (Kuzin, 2007).
Taken together, the data suggest that the miRNA binding sites in the 3' UTR are required to restrict the onset (CNS) and extinction (PNS) dynamics of Nerfin-1 protein expression. The limited expression of Nerfin-1 protein may be the result of translational inhibition and/or enhanced miRNA mediated degradation of the nerfin-1 mRNA. To determine whether mRNA expression dynamics were different for different constructs and thus were affected by the presence of different combinations of nerfin-1 miRNA binding sites, the mRNA expression dynamics of the nerfin-1.GFP-NLS.SV-40 transgene was compared to mRNA expression dynamics of this transgene containing the various nerfin-1 3'UTR fragments. The in situ hybridization mRNA study of embryos containing these different nerfin-1 3' UTR transgene constructs revealed that none of the nerfin-1 miRNA binding site constructs exhibited a marked alteration of the PNS or CNS expression dynamics of the reporter transgene during embryonic development. However, because the in situ hybridizations only reveal relative steady state mRNA levels, the possibility that the miRNAs may be promoting nerfin-1 mRNA degradation cannot definitely be ruled out(Kuzin, 2007).
Whereas the overlapping miR-9A, miR-9B, and miR-9C target sites showed partial silencing of nerfin-1 expression in the CNS, no effect was observed in the PNS. Interestingly, mutational analysis of a miR-9a mutant reveals that it is required for embryonic PNS development, and it has been shown to silence expression of senseless mRNA. However, the current studies show that miR-9A is unlikely to be a dominant regulator of embryonic Nerfin-1 protein expression; analysis with a number of cell fate markers reveal that nerfin-1 mutation is not likely to effect embryonic PNS cell fate and staining miR-9a mutants with antibody to Nerfin-1 reveals no alteration in the number or positions of Nerfin-1 positive cells. In contrast, the miR-305 and miR-13B sites partially reduced reporter expression in the PNS but not in the CNS, and the combined miR-34/315/305, miR-307 sites also exhibited partial silencing in the PNS but not in the CNS. This observation suggests that part of the reason for the complexity of miRNA binding sites in the nerfin-1 3'UTR could be due to tissue specificity of miRNA expression (Kuzin, 2007).
This study has examined the ability of the predicted miRNA binding sites within the Drosophila nerfin-1 3' UTR to silence mRNA translation in vivo. The principle finding of this study is that multiple miRNAs act cooperatively to regulate the spatial and temporal expression of Nerfin-1 in the developing embryonic nervous system. Indeed, no single miRNA-binding site is sufficient to recapitulate the endogenous post-transcriptional regulation in either the embryonic CNS or PNS. In the CNS, mRNA binding sites for multiple miRNAs are required to regulate the spatial expression of Nerfin-1 by silencing expression in all but the MP NBs. In the developing PNS, these studies indicate that miRNA mediated regulation does not restrict the onset of Nerfin-1 expression but rather it helps accelerate the rate of disappearance of Nerfin-1 in nascent neurons (Kuzin, 2007).
Whereas the whole 3' UTR was required for wild-type expression of nerfin-1, three individual sites had a partial effect of silencing in the CNS and four individual sites had only a partial effect in silencing in the PNS. The incomplete silencing in the CNS was stronger at a later stage of development than at an earlier stage, pointing to temporal effects of individual miRNAs. In two instances, partial silencing of nerfin-1 expression is accomplished by different sites in the CNS and PNS pointing to a potential tissue specificity of miRNA effects. miR-9A, miR-9B and miR-9C showed an effect in the CNS but not in the PNS, and, in contrast, the combined miR-34/315/305, miR-307 sites exhibited partial silencing in the PNS but not in the CNS. The same differential effect was observed for combined miR-305 and miR-13B binding sites. In addition, in the PNS, partial effects exhibited a degree of cell type specificity, suggesting that individual miRNAs exhibit cellular specificity even within a single tissue. The results suggest that the high number of conserved miRNA binding sites in the nerfin-1 3' RNA are likely to reflect differential temporal and spatial specificity of miRNA function. Further confirmation of this awaits in depth studies of the tissue specificity of miRNA expression (Kuzin, 2007).
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