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Logo of hhmipaAbout Author manuscriptsSubmit a manuscriptHHMI Howard Hughes Medical Institute; Author Manuscript; Accepted for publication in peer reviewed journal
Nature. Author manuscript; available in PMC Aug 15, 2009.
Published in final edited form as:
PMCID: PMC2727603
HHMIMSID: HHMIMS116721

Coordinate control of synaptic-layer specificity and rhodopsins in photoreceptor neurons

Abstract

How neurons make specific synaptic connections is a central question in neurobiology. The targeting of the Drosophila R7 and R8 photoreceptor axons to different synaptic layers in the brain provides a model with which to explore the genetic programs regulating target specificity. In principle this can be accomplished by cell-type-specific molecules mediating the recognition between synaptic partners1. Alternatively, specificity could also be achieved through cell-type-specific repression of particular targeting molecules. Here we show that a key step in the targeting of the R7 neuron is the active repression of the R8 targeting program. Repression is dependent on NF-YC, a subunit of the NF-Y (nuclear factor Y) transcription factor2. In the absence of NF-YC, R7 axons terminate in the same layer as R8 axons. Genetic experiments indicate that this is due solely to the derepression of the R8-specific transcription factor Senseless3 (Sens) late in R7 differentiation. Sens is sufficient to control R8 targeting specificity and we demonstrate that Sens directly binds to an evolutionarily conserved DNA sequence upstream of the start of transcription of an R8-specific cell-surface protein, Capricious (Caps) that regulates R8 target specificity. We show that R7 targeting requires the R7-specific transcription factor Prospero4,5 (Pros) in parallel to repression of the R8targetingpathway by NF-YC. Previous studies demonstrated that Sens6,7 and Pros8 directly regulate the expression of specific rhodopsins in R8 and R7. We propose that the use of the same transcription factors to promote the cell-type-specific expression of sensory receptors and cell-surface proteins regulating synaptic target specificity provides a simple and general mechanism for ensuring that transmission of sensory information is processed by the appropriate specialized neural circuits.

The compound eye comprises about 750 simple eyes (ommatidia), each containing a cluster of eight photoreceptor neurons (R1–R8). These neurons form synaptic connections in two regions of the optic lobe, the lamina and the medulla (Fig. 1a). The R1–R6 neurons innervate the lamina; the R7 and R8 neurons form connections in the M6 and M3 medulla layers, respectively. Genetic studies have led to the identification of cell-surface proteins regulating R7 and R8 target specificity918. Notably, mis-targeting mutant R7 neurons terminate selectively in M3 (refs 914), the layer in which wild-type R8 axons terminate, suggesting a close relationship between the genetic programs controlling R7 and R8 target specificity. Here we describe transcriptional regulatory pathways that control the differential targeting specificity of these neurons.

Figure 1
NF-YC mutant R7 axons mis-target to the M3 layer

In a screen for R7 targeting mutants10, we identified a strong loss of function mutation in the NF-YC gene (Supplementary Fig. 1), which encodes a subunit of NF-Y, an evolutionarily conserved heterotrimeric transcription factor. Although NF-Y function has not been studied extensively in the fly19,20, it has been shown to act as both an activator21 and a repressor22 in other organisms. The targeting of visual-system neurons was assessed in mosaic animals9 to generate large patches of mutant retinal tissue early in development (Fig. 1b, b′). About 75% of NF-YC mutant R7 axons (n = 892 of 1,148) terminated in M3, the same layer as wild-type R8 axons (Fig. 1b′, b″). This phenotype was fully rescued by an NF-YC complementary DNA (Fig. 1b″). In contrast with the marked effect of NF-YC mutations on R7, targeting of R8 to the M3 layer and targeting of R1–R6 to the lamina were unaffected (Supplementary Fig. 2).

To assess whether NF-YC is required in a cell-autonomous fashion in R7 neurons, we generated mosaic flies in which a fraction of R7 neurons was rendered mutant and labelled with green fluorescent protein (GFP), whereas the remaining R7 neurons and all the R8 neurons were wild-type and unlabelled (Fig. 1c, c′). We observed that about 17% of the mutant R7 neurons (n = 144 of 807) mistargeted to M3 (Fig. 1c′, c″). The decrease in penetrance of the phenotype, in comparison with mutant R7 neurons generated by mitotic recombination induced earlier in the eye primordium (compare Fig. 1b″ with Fig. 1c″), probably reflects perdurance of NF-YC protein present in precursor cells. NF-YC is therefore required autonomously for R7 targeting but not for the targeting of other classes of photoreceptor neurons. As NF-YC is expressed in all R cells (Supplementary Fig. 3), NF-YC must function in combination with other factors or signals selectively activating NF-YC function in R7.

Given that NF-YC is part of a transcription factor complex20 and is expressed in the nucleus of R7 neurons, it is likely that the change in targeting specificity reflects a change in gene expression. Wild-type R7 neurons initially target to the temporary R7 layer in the medulla and then, during mid-pupal development, extend to their final target layer11. Targeting of NF-YC mutant R7 neurons to the temporary layer is indistinguishable from the wild type (Fig. 1d, e). Extension to the final target layer at 70% after puparium formation (APF) is frequently disrupted, with many R7 neurons terminating in the layer within which R8 terminates (Fig. 1f, g). Consistent with this finding was our observation that NF-YC mutant R7 neurons expressed all five early R7 markers tested in wild-type patterns (Table 1). We reasoned, then, that NF-YC might repress a subset of R8-specific genes in the R7 neuron that later in development control final target layer selection. Indeed, the R8-specific transcription factor Sens was expressed ectopically in NF-YC mutant R7 neurons (Table 1 and Fig. 2).

Figure 2
Mis-expression of Sens in NF-YC mutant R7s is necessary and sufficient for the R7 mis-targeting phenotype
Table 1
Marker expression in NF-YC mutant eye discs

sens is a key regulator of R8 development23. In wild-type larval eye discs, Sens is expressed in two or three cells that have the potential to become R8 before becoming restricted to a single differentiating R8 neuron23 (Fig. 2a–b″). Sens remains expressed in R8 into the adult6,7 (see Supplementary Fig. 6b). It is required at a very early stage of eye development to regulate R8 specification23 and, much later during pupal development and in the adult, to regulate the transcription of R8-specific rhodopsins directly7. In NF-YC mutant larval eye discs, Sens expression in R8 begins before overt R8 differentiation as in the wild type. By contrast, Sens mis-expression in mutant R7 neurons (Fig. 2c) was first observed 15–20 h after the onset of differentiation as assessed by the expression of the R7-specific marker pros (Table 1, and compare Fig. 2c′ with Fig. 2c″). Expression of Sens in mutant R7 neurons persists throughout pupal development (Fig. 2d–e″) and into the adult (see Supplementary Fig. 6d) and is cell-autonomous (Fig. 2f–f″). As Sens mis-expression occurs after the onset of R7 differentiation and NF-YC mutant R7 neurons mis-target to the M3 layer during the late phase of R7 targeting, sens may promote an R8 targeting program that is distinct from the role of sens in cell fate earlier in development.

If upregulation of Sens in NF-YC mutant R7 neurons is responsible for targeting to the M3 layer, removal of Sens from NF-YC mutant cells should suppress the targeting defect. To test this, we induced mitotic recombination on two different chromosomes (namely chromosomes X and 3) to generate R7 neurons that were simultaneously mutant for both NF-YC and sens, and we assessed their targeting in an otherwise wild-type background (Supplementary Fig. 4a). Removing sens from NF-YC mutant R7 neurons completely suppressed the mistargeting phenotype (Fig. 2g). Thus, during wild-type development the NF-YC mediated repression of sens in R7 is necessary to prevent inappropriate targeting to M3.

To test whether Sens is sufficient to implement an R8 targeting program, we mis-expressed sens in R7 neurons (Supplementary Fig. 4b; see Methods). Under these conditions about 25% (n = 104 of 418) of the R7 neurons were redirected to the M3 layer, thus pheno-copying NF-YC loss-of-function mutations (Fig. 2h). Additional experiments using the method7 in which Sens was provided conditionally early in development to promote R8 cell fate, but removed later, support the view that Sens functions at later stages of R8 development to promote targeting (Supplementary Fig. 5). Taken together, these data raise the possibility that Sens could directly control the expression of cell-surface proteins regulating R8 target specificity.

Caps is the only cell-surface molecule that has been shown to be both specifically expressed in the R8 neuron and required for R8 targeting18 and it is therefore an excellent candidate for direct regulation by Sens. Indeed, like Sens, Caps is expressed ectopically in R7 in NF-YC mutants (Fig. 3a–f). Expression of Caps, as detected with an enhancer trap, is specifically activated in NF-YC mutant R7 neurons about 9h after the onset of Sens expression. Furthermore, a previous study18 showed that ectopic expression of Caps in R7 respecified their connections to the R8 layer. Both NF-YC mutant R7 neurons and R7 neurons mis-expressing Caps initially target correctly but then select the inappropriate M3 layer during mid-pupal development. Taken together, these observations indicate that caps could be a downstream target of Sens.

Figure 3
Sens regulates caps expression

Examination of the DNA sequences 1 kilobase upstream of caps and within the first large intron led to the identification of four and three putative Sens-binding sites, respectively. We identified an evolutionarily conserved Sens-binding site 500 base pairs upstream of the caps transcriptional start site (Fig. 3g; see Supplementary Methods). Sens protein binds specifically to this site in gel-shift assays, making it likely that caps is a direct target of Sens (Fig. 3h, i). However, Sens must regulate R8 target specificity by controlling the expression of other genes in addition to caps, because loss of caps does not suppress the NF-YC mutant phenotype (NF-YC caps 20.3 ± 11.0%, 23 brains, 441 R7 neurons; NF-YC 24.1 ± 10.5%, 15 brains, 281 R7 neurons; mean s ± s.d.). This is consistent with the finding that loss of caps, in an otherwise wild-type background, results in targeting defects in about 50% of the R8 neurons18. Together, these data suggest that Sens directly regulates the expression of Caps, a cell-surface protein controlling R8 target specificity, and must also regulate the expression of other genes involved in this process.

Specific repression of sens in R7 neurons could arise through interactions between NF-YC and the R7-specific transcription factor Pros. Like NF-YC, Pros is also required for R7 target specificity24,25. It is expressed in R7 from an early stage of its development through to the adult8,24 in a similar fashion to Sens expression in R8. About 20% of the pros-null mutant R7 neurons terminate in M3 (Fig. 4a). Two lines of evidence support the view that Pros works in parallel with NF-YC: first, the loss of pros in R7 neurons does not lead to ectopic expression of Sens (Fig. 4b, c), and second, the frequency of mis-targeting R7 axons in single pros-null mutant cells is markedly increased by removing NF-YC (Fig. 4a; see Methods). Thus, Pros could either promote R7 targeting directly or, like NF-YC, act to repress an R8 targeting program, or both.

Figure 4
Pros and NF-YC regulate R7 targeting in parallel pathways

Thus, R7 targeting requires NF-YC and, in parallel, Pros, whereas R8 targeting relies on Sens-dependent regulation of caps and other genes. Mutations in many other genes required for R7 targeting cause R7 neurons to mis-target to the M3 layer specifically rather than terminating promiscuously in the medulla. This underscores a tight inter-relationship between the mechanisms regulating targeting to these two layers. On the basis of the strong M3 mis-targeting phenotype of NF-YC mutant R7 neurons and complete suppression of the phenotype by the removal of sens, a key mechanism regulating R7 layer specificity is repression of an R8 targeting program. More generally, repression of inappropriate pathways may promote differential targeting in closely related neurons26.

The roles of Pros and Sens in target layer selection are analogous to their function in controlling the expression of R7-specific and R8-specific rhodopsins. R7 and R8 neurons express different rhodopsins and hence detect different wavelengths of light. In R8, Sens directly represses the transcription of R7 rhodopsins and directly activates the transcription of an R8 rhodopsin7. In the R7neuron, Pros binds to an upstream regulatory sequence in the R8 rhodopsin genes and represses their expression7,8. NF-YC mutant R7 neurons no longer express R7 rhodopsins, and all express R8 rhodopsins (Supplementary Fig. 6). This is consistent with the finding that NF-YC mutant R7 neurons in adults express Sens but no longer express Pros. Thus, transcription of both R8-specific rhodopsins and, as we have shown here, an R8-specific targeting protein Caps is directly regulated by Sens (Fig. 4d).

These observations suggest a simple solution to the mechanisms by which sensory neurons connect to the neural circuits specialized for the reception of different sensory stimuli (for example, different wavelengths of light or different odours). Although the molecular basis of this coupling is understood in considerable detail for vertebrate olfactory neurons, in which odorant receptors have a direct function in controlling target specificity27, little is known about the coupling in other sensory systems. Coupling is likely to be regulated in a different fashion in other neurons, because even in the fly olfactory system, for example, targeting is independent of sensory receptor expression28. On the basis of our studies on Sens we propose that the same transcription factors directly control both rhodopsin expression and the cell-surface proteins that control target layer specificity. More generally, we speculate that in many sensory neurons a common set of transcription factors may directly control, and thereby coordinate, the expression of cell-surface proteins regulating target specificity and the receptors detecting specific sensory stimuli.

METHODS SUMMARY

To generate NF-YC or pros mutant R7s, we used the MARCM (mosaic analysis with a repressible cell marker) technique as described previously10. To generate R7 mosaics (that is, mutant R7 neurons surrounded by wild-type cells) GMR-FLP was used as a source of recombinase. For larger patches of mutant tissue in the eye, including R7 neurons (eye mosaics), the ey3.5-FLP construct was used. A similar version of the MARCM technique with GMR-FLP was used to generate double-mutant R7 neurons. Mutant R7 neurons were marked only when flipping occurred on both chromosomes X and 3. GMR-FLP was used with MARCM to generate R7 neurons expressing Sens (UAS-sens), in addition to the inducible marker. For assessing expression of R7 and R8 markers, hemizygous NF-YC40 mutant third-instar eye discs and late pupae were analysed.

Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature.

METHODS

The stocks used to assess the targeting of NF-YC mutant R7 neurons in large clones were ey3.5-FLP, tubP-GAL80, FRT19; act-GAL4, UAS-lacZ/CyO and NF-YC40, FRT19/FM7, Kr-GFP. To assess whether NF-YC was autonomously required in R7 neurons we used GMR-FLP, which induces mitotic recombination in R7, but not R8 cell precursors. The stocks used were GMR-FLP, tubP-GAL80, FRT19; act-GAL4, UAS-syt-GFP/CyO and NF-YC40, FRT19/FM7, Kr-GFP. FRT19/FRT19 was used as a control. Because NF-YC40 mutant animals die before eclosion, analysis of R7 and R8 markers was performed on hemizygous mutant NF-YC40 FRT19/Y and wild-type FRT19 eye discs and pupal retinas. To assess the possibility that mis-expression of sens in R7 clones was autonomous we used the same stocks used to assess the NF-YC requirement in R7. The stocks used to generate flies with R7 neurons double mutant for NF-YC and sens or NF-YC and caps were NF-YC40, FRT19/FM7, Kr-GFP; +; sensE1, FRT79/TM6B or NF-YC40, FRT19/FM7, Kr-GFP; +; capsC18fs, FRT79/TM6B crossed to GMRFLP, tubP-GAL80, FRT19; act-GAL4, UAS-syt-GFP/CyO; tubP-GAL80, FRT79/TM6B. A similar scheme was used to generate single R7 neurons mutant for both NF-YC and pros (FRT82B, pros17.17). As a control, to generate animals with R7 neurons mutant for NF-YC only, NF-YC40, FRT19/FM7, Kr-GFP; +; FRT79 or FRT82B flies were crossed to GMRFLP, tubP-GAL80, FRT19; act-GAL4, UAS-syt-GFP/ CyO; tubP-GAL80, FRT79 or tubP-GAL80 FRT82B/TM6B. The stocks used to mis-express sens in clones of wild-type R7 neurons were FRT40/FRT40; UAS-sensC5/TM6B and GMR-FLP, tubP-GAL80, FRT40/CyO; tub-GAL4, UAS-Nsyb-GFP/TM6B. Similar results were obtained with independent insertions on chromosomes X (UAS-sensC1) and 3 (UAS-sensC6). We confirmed that wild-type R7 neurons mis-expressing sens also expressed the R7-specific marker Pros at 40% APF. As a control we generated clones of wild-type R7 neurons by crossing FRT40/FRT40 to GMR-FLP, tubP-GAL80, FRT40/CyO; tub-GAL4, UAS-Nsyb-GFP/TM6B.

Supplementary Material

supplemental figures

Supplementary Information is linked to the online version of the paper at www.nature.com/nature.

Acknowledgements

We thank Y. N. Jan, H. J. Bellen, A. Nose, S. Britt, C. Desplan, T. Cook, U. Banerjee, the Developmental Studies Hybridoma Bank and Bloomington Stock Center for reagents; H. Schjerven, S. T. Smale and M. Carey for advice and assistance with gel-shift assays; and members of the Zipursky laboratory for comments on the manuscript. S.L.Z. is an investigator of the Howard Hughes Medical Institute. M.M. was supported by the Howard Hughes Medical Institute. S.K.Y. was supported by the Ruth L.Kirschstein National Research Service Award GM7185. T.H. was supported by the Burroughs Wellcome Fund for Biomedical Research. A.N. was supported by the European Molecular Biology Organization and the Human Frontiers Science Program. E.B. was supported by Juan de la Cierva postdoctoral contract and the grant BMC2006-07334 from the Ministerio de Educación y Ciencia (MEC), Spain.

Footnotes

Author Information Reprints and permissions information is available at www.nature.com/reprints.

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