Postsynaptic glutamate receptor production depends on contact between pre and postsynaptic cells. It is thus a contact-dependent form of differentiation. Previous studies used immunohistochemistry and electrophysiology to show that this differentiation involves clustering of functional glutamate receptors at the synapse.1,3
But where did these receptors come from? Cell-cell contact could trigger assembly of pre-synthesized protein subunits, translation of pre-transcribed mRNAs, or synthesis and subsequent translation of appropriate subunit mRNAs. To determine which, we used quantitative real-time RT-PCR and immunoblots ().
Figure 1 Expression of Drosophila GluRIIA and GluRIIB mRNA and protein through embryonic and larval development. (A) Amount of GluRIIA mRNA in wildtype (Oregon R) embryos, measured using quantitative real time RT-PC R. Quantity is presented relative to the amount (more ...)
GluRIIA and GluRIIB are rate-limiting for formation of glutamate receptors in the embryonic/larval NMJ; their rate of production and abundance determine the timing and types of glutamate receptors that are produced. Furthermore, quantitative real time RT-PCR can be used to measure abundance of GluRIIA and GluRIIB mRNA because GluRIIA and GluRIIB are expressed only in muscle.6,7
We used this method to quantify GluRIIA and GluRIIB mRNA abundance throughout embryonic and larval development ().
As shown in , GluRIIA mRNA is expressed at very low but detectable levels before suddenly increasing approximately twenty-fold around 12 hours AEL. Surprisingly, GluRIIA mRNA abundance then falls back to the initial basal level within three hours and remains at that low level until the animal hatches (). GluRIIB mRNA abundance is also initially very low, but like GluRIIA mRNA rises approximately twenty-fold before falling again to basal levels for the rest of embryogenesis (). However, the peak of GluRIIB mRNA expression during embryogenesis is slightly delayed (to 16–18 h AEL), compared to that of GluRIIA.
To ensure the reliability of our measurements, actin 5C mRNA was isolated and amplified simultaneously for every GluRIIA or GluRIIB measurement, and used to correct for possible sample-to-sample variation in mRNA isolation and/or amplification.11
As shown in (inset), this variation was minimal, as actin 5C C(t) values were not statistically different across samples. The dramatic changes in GluRIIA and GluRIIB mRNA abundance therefore appear to be real. These mRNA spikes are also visible in data from the modENCODE Drosophila transcriptome project, which uses high-density whole genome tiling microarrays to identify all the transcriptionally active regions of the genome throughout embryogenesis.12
In agreement with our quantitative real-time PCR results, the MODENCODE data show qualitative peaks in GluRIIA and GluRIIB exon expression in mid embryogenesis, with GluRIIB exon expression peaking slightly later than GluRIIA (www.modencode.org/
Although production of protein depends on the existence of mRNA, the abundance of GluRIIA and GluRIIB mRNA might not necessarily correlate with abundance of GluRIIA and GluRIIB protein. We therefore measured the amount of GluRIIA and GluRIIB protein during embryogenesis by quantitative immunoblotting ( and D
). In contrast to the spikes of mRNA abundance, GluRIIA and GluRIIB protein increased gradually through embryogenesis, consistent with the gradual increases in abundance of clustered and functional NMJ glutamate receptors measured in previous studies.3,13
GluR mRNA and protein abundance are apparently disconnected during embryonic development, given that GluR mRNA abundance spikes midway through embryogenesis while protein abundance gradually increases. Is GluR mRNA abundance also disconnected from protein abundance during larval development? Drosophila larval NMJ development has been extensively studied, and it is well established that increasing numbers of glutamate receptors are added to the growing NMJ during larval development. Quantitative real time PCR, however, shows that GluRIIA and GluRIIB mRNA abundance falls gradually through larval development ( and F). This result is also supported by MODENCODE data.
How can GluR mRNA and protein abundance be disconnected? Glutamate receptor protein perdurance is very high: greater than 24 h in larvae.9,14
Therefore, it does not require much mRNA for protein to accumulate—even less as NMJ growth slows in larvae. The only time that a lot of GluR mRNA would be required is at the very start of synaptogenesis, when a large number of receptors must be synthesized de novo. Consistent with this, GluRIIA and GluRIIB mRNA abundance peaks during embryogenesis at about the same time that presynaptic nerves contact postsynaptic muscles, when the need for GluR protein production per unit time is highest. In other words, contact by the presynaptic neuron appears to trigger a burst of transcription such that the postsynaptic muscle is quickly flooded with glutamate receptor mRNA. Presumably, only a subset of the mRNA is subsequently required for translation. This subset is preserved, while the rest of the mRNA is degraded.
The first step in testing this hypothesis is determining whether GluRIIA and GluRIIB mRNA production in postsynaptic muscle does in fact depend on contact from the presynaptic nerve. To test this, we genetically manipulated pre and postsynaptic cell contact using scratch
) mutants. Scratch is a predicted transcription factor expressed in neuronal precursor cells and required for proper neuronal differentiation.15
In a previous study,16
we showed that scrt[KG02164]
mutants extend neuronal axons into the body wall musculature, but the neurons completely fail to form neuromuscular junctions onto muscles. Muscles in scrt[KG02164]
mutants therefore develop without significant contact by presynaptic neurons, and there is no detectable expression of GluRIIA or GluRIIB in immunoblots (data not shown). If our hypothesis that postsynaptic GluRIIA and GluRIIB mRNA production is triggered by contact by the presynaptic nerve is correct, then the spikes in GluRIIA and GluRIIB mRNA production shown in and B
should be absent from scrt[KG02164]
shows a confocal micrograph of NMJs formed by intersegmental nerve branch B (ISNb) on ventral muscles 15, 6, 7, 13 and 12 in a single embryonic (~22 h AEL) hemisegment. shows the same muscle field in a scrt[KG02164] mutant embryo. Note the presence of ISNb, but no NMJs. shows GluRIIA mRNA abundance at various time points during embryogenesis, as in , except in scrt[KG02164] embryos. shows the same thing for GluRIIB mRNA. The spikes in GluRIIA and GluRIIB mRNA abundance measured from wildtype embryos are completely absent.
Figure 2 GluRIIA and GluRIIB mRNA expression in scratch mutant embryos. (A) Confocal micrograph of motor neuron terminals from intersegmental (ISN) nerve branch ISNb innervating ventral longitudinal muscles 15, 6/7, 13 & 12 in control (w) embryos (more ...)
We conclude based on these results, that GluRIIA and GluRIIB mRNA transcription is triggered in postsynaptic muscles by contact from the presynaptic nerve. GluRIIA and GluRIIB mRNA abundance rises quickly after cell-cell contact. GluR mRNA abundance drops off rapidly within a few hours of initial synapse formation, then more gradually throughout the rest of embryogenesis and larval development. Despite this drop off in mRNA abundance, GluRIIA and GluRIIB protein production proceeds steadily, leading to gradual accumulation of GluR protein. Presumably, a subset of the initially produced mRNA is preserved and preferentially used for translation. But where is this mRNA?
shows confocal micrographs of anti-GluRIIA mRNA Fluorescence In Situ Hybridization (FISH) in combination with immunohistochemistry in muscles of first instar larvae (~40 h AEL). Specifically, the figure shows FISH and immunohistochemistry in ventral longitudinal muscles 6 and 7, and the NMJ formed in the cleft between the two muscles. Presynaptic axons and nerve terminals have been visualized using anti-HRP antibodies (left column and right column, blue). GluRIIA mRNA has been visualized using fluorescently-labeled GluRIIA mRNA antisense probes and FISH (middle column and right column, green). As shown, the GluRIIA mRNA appeared as small puncta distributed throughout the muscle.
Figure 3 GluRIIA mRNA aggregates in first instar larval muscles. Parts show confocal micrographs of ventral longitudinal muscles 6 and 7 in one hemisegment of a first instar larvae. Left column shows NMJs in cleft of muscles 6 and 7, visualized using fluorescently-conjugated (more ...)
Several types of experiments confirmed that the puncta revealed by FISH really represented GluRIIA mRNA. First, we performed FISH using GluRIIA sense probes, which should not hybridize to the GluRIIA mRNA. As expected, FISH using sense probes showed no GluRIIA puncta (data not shown). Next, we used FISH to look for GluRIIA mRNA puncta in GluRIIA[AD9]/Df(2L) Exel8016 mutants (GluRIIA-/-), which contain deletions that completely remove the GluRIIA gene. As expected, GluRIIA puncta were not visible in GluRIIA-/- mutants ().
We also performed FISH under conditions where GluRIIA expression is increased. For example, we measured GluRIIA mRNA aggregate abundance after knockdown of Dicer1. Dicer1 is required for production of microRNAs, including microRNAs that suppress GluRIIA and GluRIIB mRNA abundance.10
When Dicer1 levels are reduced, GluRIIA expression is dramatically increased.10
Consistent with the idea that GluRIIA FISH puncta represent GluRIIA mRNA, we observed a large increase in the number of GluRIIA puncta after RNAi-mediated knockdown of muscle Dicer1.10
More directly, we overexpressed GluRIIA in third instar larvae (~100 h AEL; ) using a muscle-specific Gal4 driver (24B Gal4) in combination with a UAS-GluRIIA full-length genomic transgene.14
The GluRIIA mRNA puncta in third instar larvae appeared similar to those in first instar larvae, and were also absent in GluRIIA null mutants (). But when GluRIIA was overexpressed, the density of GluRIIA mRNA aggregates increased approximately 7-fold, compared to wildtype larvae or 24B-Gal4/+ controls (24B-Gal4 = 1.0 ± 0.4, N = 11; 24B-Gal4; UAS-GluRIIA = 6.9 ± 1.3, N = 13; p = 0.0005).
Figure 4 GluRIIA mRNA aggregates in third instar larval muscles. (A) Top parts: Confocal micrographs of ventral longitudinal muscles 6 and 7 in one hemisegment of a wild type third instar larvae. Left column shows NMJs, visualized using fluorescently-conjugated (more ...)
Based on these results, we conclude that the GluRIIA FISH signal represents GluRIIA, and that the density of GluRIIA FISH puncta correlates with GluRIIA mRNA abundance.
, we provided evidence that GluRIIA gene expression depends on muscle innervation. The presence of GluRIIA FISH puncta must therefore also be dependent on muscle innervation. To test this, we simultaneously visualized GluRIIA using FISH and motor axon terminals using anti-HRP antibodies, in homozygous prospero
mutant embryos. Prospero
mutants show delayed, highly variable, and/or absent body wall muscle innervation,1,3
and are therefore ideal for microscopically comparing GluRIIA puncta and degree of innervation (). We quantified the GluRIIA FISH signal two ways. First (), we measured total GluRIIA FISH fluorescence signal intensity (average pixel value over the entire visible muscle surface). This was compared to the NMJ size (area, measured in pixels). Consistent with the idea that muscle innervation triggers GluRIIA mRNA expression and GluRIIA mRNA expression correlates with GluRIIA FISH signal intensity, we observed a weakly linear (R2
= 0.29) but highly statistically significant (slope different from 0 at p = 0.0005) correlation between GluRIIA FISH signal intensity and NMJ size (). We also measured GluRIIA mRNA puncta density by manually counting puncta and dividing by muscle area to calculate mRNA aggregate density (). Similar to the results obtained by measuring FISH signal intensity, we observed a weakly linear (R2
= 0.15) but statistically significant (slope different from 0 at p = 0.02) correlation between GluRIIA mRNA aggregate density and NMJ size ().
Figure 5 GluRIIA mRNA aggregate density is proportional to NMJ size. (A) Confocal micrographs showing muscles 6 and 7 in three different homozygous prospero mutant embryos, in which muscle innervation is delayed and variable. The examples are arranged with (more ...)
FISH using whole undissected embryos showed GluRIIA mRNA punctae similar to those visible in dissected larval muscle (–), but it was more difficult to determine the distribution and density of the mRNPs under these conditions. Qualitiatively, few/no GluRIIA mRNPs were visible in embryonic body wall muscle 6–9 h or 9–12 h AEL (before innervation), while mRNPs were clearly abundant at 12–15 h AEL, consistent with the idea that innervation triggers GluRIIA expression and mRNA aggregate formation. Sense controls, as expected, showed little/no FISH signal (data not shown).
What about other GluR mRNAs? GluRIIA subunits are present in only a subset of Drosophila embryonic/larval body wall NMJ glutamate receptors. Other receptors contain GluRIIB, and both GluRIIA and GluRIIB-containing receptors contain GluRIIC. We therefore visualized GluRIIB and GluRIIC mRNA using FISH. shows confocal micrographs of GluRIIB FISH in ventral longitudinal muscles of first instar (top six parts) and third instar (bottom six parts) larvae. shows confocal micrographs of GluRIIC FISH in ventral longitudinal muscles of first instar (top six parts) and third instar (bottom six parts) larvae. As with GluRIIA, FISH against GluRIIB and GluRIIC revealed what appear to be small mRNA aggregates distributed throughout the muscle cells. Sense probe controls also showed no signal.
Figure 6 GluRIIB mRNA aggregates in first and third instar larval muscles. Top six parts: Confocal micrographs of first instar NMJs and muscles as in , except using antisense probes against GluRIIB instead of GluRIIA. Bottom parts show (lack of) FISH signal (more ...)
GluRIIC mRNA aggregates in first and third instar larval muscles. Parts are as described for , except probes for GluRIIC mRNA were used. Scale bars: 15 um.
However, close inspection and comparison of GluRIIA, GluRIIB and GluRIIC mRNA aggregates revealed some differences. Primarily, the density of GluRIIC mRNA aggregates appeared to be noticeably increased, relative to GluRIIA or GluRIIB. This is interesting because Drosophila embryonic/larval body wall muscles require approximately double the amount of GluRIIC protein compared to GluRIIA or GluRIIB protein. In other words, the density of GluR mRNA aggregates appears to be proportional to the cells ‘need’ for that particular GluR protein. We quantified this by manually counting the number of mRNA aggregates for each GluR in first instar ventral longitudinal muscles (as described for GluRIIA in ), and plotting the relative mRNA aggregate density (). The number of GluRIIC mRNA aggregates was approximately the same as the number of GluRIIA and GluRIIB mRNA aggregates combined (), consistent with the fact that all NMJ glutamate receptors contain GluRIIC, plus either GluRIIA or GluRIIC. The number of GluRIIA mRNA aggregates was also approximately double the number of GluRIIB aggregates (), consistent with the fact that receptors with GluRIIA subunits predominate during embryogenesis and early larval development.10
Figure 8 Different GluR mRNAs are associated with different mRNA aggregates. (A) mRNA aggregate density in third instar larval muscles 6 and 7, for GluRIIA, GluRIIB and GluRIIC. Values are ‘background-subtracted’ such that the number of punctae (more ...)
If the density of GluR mRNA aggregates differs between GluRIIA, GluRIIB and GluRIIC, then GluRIIA, GluRIIB and GluRIIC mRNAs cannot all be found in the same aggregates. There must be separate aggregates for each GluR mRNA. To test this explicitly, we performed multiplex FISH to simultaneously visualize GluRIIA and GluRIIC mRNA aggregates, in combination with immunohistochemistry (). As expected given the quantitative differences in mRNA aggregate density, GluRIIA and GluRIIC mRNA aggregates were physically segregated ().
Finally, we quantified mRNA aggregate density relative to the NMJ (). Many important postsynaptic proteins (including CaMKII, calmodulin, PCP4, dendrin, neurogranin, TrkA, TrkB, NMDAR1, GluR2, GluR5 and GlyR A2) are thought to be locally translated in dendrites,17–21
and there is evidence that GluRIIA may be preferentially translated near NMJs.22,23
However, it's unclear whether GluR mRNAs are preferentially localized near NMJs. A previous study by Currie et al. (1995) suggests that GluRIIA is not localized.24
However, the methods utilized in that study were not suitable for quantification, and did not permit visualization of mRNA aggregates or NMJs. We therefore quantified GluRIIA, GluRIIB and GluRIIC mRNA aggregate density relative to the NMJ, using simultaneous FISH and immunohistochemistry. Specifically, we used the anti-HRP signal (which delineates the NMJ) to quantify GluR mRNP density within three muscle regions: “NMJ” (defined by HRP signal), “Peri-NMJ” (within 10 um of any part of the NMJ) and “extra-NMJ” (muscle area outside the area defined by Peri-NMJ region). As shown (), and consistent with the results described for each individual GluR mRNA (–
), there was no increase in mRNA aggregate density near NMJs for any of the three GluR mRNAs examined. If anything, there was a slight tendency for GluR mRNP density to be highest farther away from the NMJ, although this trend was not statistically significant. Qualitatively, most GluRIIA mRNA aggregates tended to surround nuclei when GluRIIA was overexpressed ().
Increasing evidence suggests that mRNA in vivo is continuously associated with a shifting cast of proteins that control mRNA editing, trafficking, translation and stability. These mRNA and proteins often aggregate as so-called ‘messenger ribonucleoprotein (mRNP) particles. The GluR mRNA aggregates that we observed in confocal micrographs using FISH are similar to previously described mRNP particles.25
Furthermore, the density of GluR mRNA aggregates appears proportional to protein need, suggesting that the GluR mRNA aggregates we describe here might represent translating mRNPs. Indeed, GluRIIA mRNA has previously been suggested to be associated with the eukaryotic translation initiation factor eIF4E near Drosophila third instar NMJs.22
The increased resolution of FISH, along with the ability to perform simultaneous immunohistochemistry to visualize eIF4E and NMJs, allows us to test directly whether GluR mRNA is colocalized with eIF4E near NMJs. As shown in , eIF4E is, like GluRIIA mRNA, distributed in a punctate pattern throughout muscle cells. However, the nanoscale resolution of FISH and confocal microscopy shows clearly that eIF4E is not significantly colocalized with GluRIIA mRNA aggregates (). We confirmed the lack of colocalization quantitatively by calculating two different background-corrected colocalization measures: Pearson's correlation coefficient and Mander's overlap coefficient.26
Pearson's coefficient for GluRIIA and eIF4E was −0.635 ± 0.038 (N = 7), and the Mander's overlap coefficient was 0.033 ± 0.006 (N = 7). Both values are very low, suggesting no significant overlap. However, to confirm this we rotated the eIF4E channel 90 degrees clockwise relative to the GluRIIA FISH channel using Photoshop and re-measured the GluRIIA/eIF4E overlap coefficients. Pearson's coefficient for GluRIIA and rotated eIF4E was −0.659 ± 0.035 (N = 7), and the Mander's overlap coefficient was 0.016 ± 0.004 (n = 7). The lack of any significant difference confirms that any overlap is essentially coincidental. However, this does not mean that GluRIIA mRNA does not associate with eIF4E at all (see discussion).
Figure 9 GluRIIA mRNA aggregates do not colocalize with eIF4E protein. Shown is a confocal micrograph of ventral longitudinal muscles 6 and 7 in a hemisegment of a third instar larva. The NMJ has been labeled with anti-HRP antibodies (blue). GluRIIA mRNA aggregates (more ...)