The equilibrium between antagonistic signaling pathways determines the number of synapses in Drosophila

Abstract

The number of synapses is a major determinant of behavior and many neural diseases exhibit deviations in that number. However, how signaling pathways control this number is still poorly understood. Using the Drosophila larval neuromuscular junction, we show here a PI3K-dependent pathway for synaptogenesis which is functionally connected with other previously known elements including the Wit receptor, its ligand Gbb, and the MAPkinases cascade. Based on epistasis assays, we determined the functional hierarchy within the pathway. Wit seems to trigger signaling through PI3K, and Ras85D also contributes to the initiation of synaptogenesis. However, contrary to other signaling pathways, PI3K does not require Ras85D binding in the context of synaptogenesis. In addition to the MAPK cascade, Bsk/JNK undergoes regulation by Puc and Ras85D which results in a narrow range of activity of this kinase to determine normalcy of synapse number. The transcriptional readout of the synaptogenesis pathway involves the Fos/Jun complex and the repressor Cic. In addition, we identified an antagonistic pathway that uses the transcription factors Mad and Medea and the microRNA bantam to down-regulate key elements of the pro-synaptogenesis pathway. Like its counterpart, the anti-synaptogenesis signaling uses small GTPases and MAPKs including Ras64B, Ras-like-a, p38a and Licorne. Bantam downregulates the pro-synaptogenesis factors PI3K, Hiw, Ras85D and Bsk, but not AKT. AKT, however, can suppress Mad which, in conjunction with the reported suppression of Mad by Hiw, closes the mutual regulation between both pathways. Thus, the number of synapses seems to result from the balanced output from these two pathways.

Fig 1. Ligand and receptor of the pro-synaptogenesis pathway.

A) The over- (↑) or down-expression (↓) of the ligand Gbb in the muscle elicits mild changes in the number of synapses as indicated by the number of active zones (AZ) viewed as nc82 spots. Genotypes: + = Mhc-Gal4/+. Gbb↑ = Mhc-Gal4/+;UAS-gbb/+. Gbb↓ = Mhc-Gal4/+;UAS-gbb^RNAi/+. B) The gbb mutant larvae, either in heterozygous (Gbb↓) or homozygous (Gbb↓↓) conditions show reduced number of synapses. These data support a role of this ligand in synaptogenesis. Note, however, that synapses have not been eliminated (see main text). The viability of homozygotes is very poor and only two LIII larvae could be obtained. This is evidence that Gbb plays additional developmental roles beyond synaptogenesis. The up-regulation of the native form of PI3K suppresses the synapse effect in the heterozygotes although the effect of PI3K on its own is somewhat reduced (compare with PI3K↑ in panel C). The constitutively active PI3K, PI3K*, improves the viability of the homozygous gbb null mutants but the mutant synapse number is only weakly recovered, and it remains clearly below normal (see main text). Genotypes: + = D42-Gal4/+. Gbb↓ = gbb¹/+; D42-Gal4/+. Gbb↓↓ = gbb¹/gbb²; D42-Gal4/+. PI3K↑Gbb↓ = UAS-PI3K/gbb¹; D42-Gal4/+. PI3K*↑ = UAS-PI3K*/+; D42-Gal4/+. PI3K*↑ Gbb↓↓ = UAS-PI3K*/+; gbb¹/gbb²; D42-Gal4/+. C) The up-regulation of the native form of PI3K (PI3K↑) increases the number of synapses while its down-regulation (PI3K↓) yields the opposite effect. The overexpression of the receptor Wit is ineffective for synapse formation while the mutant condition (Wit↓↓) strongly reduces synapses. Note, however, that neither Wit nor Gbb depletion can eliminate synapses fully. Combinations of Wit and PI3K show that the mutant condition for Wit prevails over the overexpression of PI3K suggesting that PI3K requires activation by Wit. Consistent with this notion, the downregulation of PI3K still maintains its phenotype of reduced synapse number even though Wit is over-expressed. In the same line, a constitutively active form of PI3K, PI3K*, suppresses the mutant condition for the receptor. These results support the conclusion that PI3K is functionally downstream from the receptor Wit. Genotypes: + = D42-Gal4/+ pooled with OK6-Gal4/+. PI3K↑ = UAS-PI3K/+; D42-Gal4/+. PI3K↓ = UAS-PI3K^DN/+; D42-Gal4/+. Wit↓↓ = OK6-Gal4/+; wit^A12/wit^B11. PI3K↑Wit↓↓ = OK6-Gal4/UAS-PI3K; wit^A12/wit^B11. Wit↑ = D42-Gal4/UAS-Wit. PI3K↓Wit↑ = UAS-PI3K^DN/+; D42-Gal4/UAS-Wit. PI3K*↑Wit↓↓ = UAS-PI3K*/+; OK6-Gal4/+; wit^A12/wit^B11. D-F) Representative images of motor neuron 6–7 from larval abdominal segment A3 in normal (+) (D), wit^A12/wit^B11 (↓) (E) and D42-Gal4>UAS-PI3K (↑) (F) genotypes. Number of NMJ analyzed in independent larvae is indicated in parenthesis adjacent to the genotype. Bar in F = 10μm.

Fig 2. Signaling factors downstream from the receptor Wit.

A) The over- or down-expression of Ras85D, either alone or in combination with manipulations of Wit, seem ineffective to change synapse number. Further experiments, however, proved the contrary (see below). Genotypes: + = D42-Gal4/+. Ras85D↑ = UAS-Ras85D/+; D42-Gal4/+. Ras85D↑Wit↓ = UAS-Ras85D/+; D42-Gal4/UAS-Wit^RNAi. Ras85D↓ = UAS-Ras85D^DN/+; +/+; D42-Gal4/+. Ras85D↓Wit↑ = UAS-Ras85D^DN/+; UAS-Wit /+; D42-Gal4/+. Ras85D↑Wit↑ = UAS-Wit/UAS-Ras85D; D42-Gal4/+. B) PI3K-Ras85D interactions. Both, the over- or down-expression of Ras85D, which seemed ineffective on the synapse number, suppress the synapse increase due to PI3K↑ when jointly expressed. In the case of Ras85D↓ the phenotype is converted into synapse decrease. These features demonstrate that Ras85D is functionally related to PI3K in the context of synaptogenesis. The up-regulation of a PI3K form that is deleted in the Ras phosphorylation site, myc-PI3K^ΔRBD, is still able of increase synapses even though its control, myc-PI3K, is somewhat less effective than PI3K. The PI3K^ΔRBD form, as the normal PI3K, transforms the Ras85D↓ phenotype into a severe synapse reduction. These data are consistent with Ras85D being functionally located somewhere down stream in the PI3K pathway. Genotypes: + = D42-Gal4/+. PI3K↑ = UAS-PI3K/+; D42-Gal4/+. PI3K↑Ras85D↑ = UAS-Ras85D/+; UAS-PI3K/+; D42-Gal4/+. PI3K↑Ras85D↓ = UAS-Ras85D^DN/+; UAS-PI3K/+; D42-Gal4/+. myc-PI3K↑ = D42-Gal4/UAS-myc-Dp110. myc-PI3K^ΔRBD↑ = D42-Gal4/UAS-myc-Dp110^RBD. myc-PI3K^ΔRBD↑Ras85D↓ = UAS-Ras85D^DN/+; D42-Gal4/UAS-myc-Dp110^RBD. C) Western blot analysis (triplicates) of phospho-AKT relative levels under Ras85D overexpression. No increase of pAKT is detected demonstrating that Ras85D is functionally downstream from AKT. Genotypes: + = D42-Gal4/+. Ras85D↑ = UAS-Ras85D/+; D42-Gal4/+.

Fig 3. Intermediate signaling in the pro-synaptogenesis pathway.

A) Interactions between Hiw and PI3K. The overexpression (Hiw↑) increases the number of synapses, while the mutant hiw^ND8 or a form deleted in the E3 domain, Hiw^ΔE3, show a non-significant tendency towards reduction only. However, while the over-expression of PI3K (PI3K↑) increases the number of synapses and its downregulation with a dominant negative form (PI3K↓) reduces it, both effects are suppressed by manipulating Hiw in the opposite directions. These data place Hiw functionally downstream from PI3K in the pathway. Genotypes: + = D42-Gal4/+. Hiw↑ = UAS-Hiw/+; D42-Gal4/+. Hiw↓ = males hiw^ND8; +/+; D42-Gal4/+. Hiw^ΔE3↑ = D42-Gal4/UAS-NT-Hiw^ΔRING. PI3K↑ = UAS-PI3K/+; D42-Gal4/+. PI3K↓ = UAS-PI3K^DN/+; D42-Gal4/+. PI3K↓Hiw↑ = UAS-Hiw/UAS-PI3K^DN; D42-Gal4/+. PI3K↑Hiw↓ = males hiw^ND8; UAS-PI3K/+; D42-Gal4/+. B) The MAPKs of the pathway. Wnd shows the expected pro-synaptogenesis effect while Hep fails to reach statistical significance. However, their target, Bsk/JNK, does show the expected synapse increase. Apparently, the Bsk↓ construct is ineffective and there is no evidence of synergy or antagonism with PI3K in the PI3K↑Bsk↑ or PI3K↑ Bsk↓ conditions (however, see below). Genotypes: + = D42-Gal4/+. Wnd↑ = UAS-Wnd/+; D42-Gal4/+. Hep↑ = UAS-Hep/+; D42-Gal4/+. Bsk↑ = UAS-Bsk/+; D42-Gal4/+. Bsk↓ = D42-Gal4/UAS-Bsk^DN. PI3K↑Bsk↑ = UAS-Bsk/UAS-PI3K; D42-Gal4/+. PI3K↑Bsk↓ = UAS-PI3K/+; D42-Gal4/UAS-Bsk^DN. C) The possible regulation of Bsk by Ras85D. Although the up-regulation of Bsk had proven pro-synaptogenic, its concomitant over-expression with the seemingly ineffective Ras85D transforms the pro- into an anti-synaptogenesis effect. Also, the apparently ineffective Ras85D↓ suppresses the pro-synaptogenic effect of Bsk↑. These data are indicative of a regulatory interaction between Ras85D and Bsk. The Bsk↓ condition, which proved ineffective, remained unchanged irrespective of the two possible alterations of Ras85D. Genotypes: + = D42-Gal4/+. Bsk↑Ras^85D↑ = UAS-Bsk/UAS-Ras85D; D42-Gal4/+. Bsk↑Ras^85D↓ = UAS-Ras85D^DN/+; UAS-Bsk/+; D42-Gal4/+. Bsk↓Ras^85D↑ = UAS-Ras85D/+; D42-Gal4/UAS-Bsk^DN. Bsk↓Ras^85D↓ = UAS-Ras85D^DN/+; +/+; D42-Gal4/UAS-Bsk^DN. D) The Bsk regulator Puc and the convergence with Ras85D in a tripartite interaction. Whereas the downregulation of Puc or Bsk separately yields no effect on synaptogenesis, their joint co-downregulation results in a strong decrease of the number of synapses. Also, the synapse increase elicited by Bsk↑ is rendered non-significant because of the wide dispersion of values elicited by the joint down regulation of Puc. The two triple combinations tested result in a notable reduction of the variability in the number of synapses (see text). Together, the data in C and D reveal a complex regulatory network at the final step of the MAPK cascade. Genotypes: + = D42-Gal4/+. Puc↓ = UAS-puc^RNAi/+; D42-Gal4/+. Puc↓Bsk↓ = UAS-puc^RNAi/+; D42-Gal4/UAS-Bsk^DN. Puc↓Bsk↑ = UAS-Bsk/UAS-puc^RNAi; D42-Gal4/+. Puc↓Bsk↑Ras^85D↓ = UAS-Ras85D^DN/+; UAS-puc^RNAi/UAS-Bsk; D42-Gal4/+. Puc↓Bsk↓Ras^85D↑ = UAS-Ras85D, UAS-puc^RNAi/+; D42-Gal4/ UAS-Bsk^DN.

Fig 4. Effectors of the pathway.

A) Epistasis analysis of the AP-1 components, Fos and Jun. Single manipulations of these two transcription factors in either direction yields the expected synapse increase in the case of Jun↑ only, which could be interpreted as if the other three constructs were ineffective. However, Fos↓ transforms the Jun↑ synapse increase effect into a decrease. This and other results throughout this study demonstrate that the seemingly ineffective constructs are functional. Likely, Jun and Fos play their role through interactions with other anti-synaptogenesis transcription factors (see below). Genotypes: + = D42-Gal4/+. Jun↑ = D42-Gal4/UAS-Jun. Jun↓ = UAS-Jun^DN/+; D42-Gal4/+. Fos↑ = UAS-Fos/+; D42-Gal4/+. Fos↓ = UAS-Fos^DN/+; D42-Gal4/+. Jun↑Fos↑ = UAS-Fos/+; D42-Gal4/ UAS-Jun. Jun↓Fos↓ = UAS-Fos^DN/UAS-Jun^DN; D42-Gal4/+. Jun↑Fos↓ = UAS-Fos^DN/+; D42-Gal4/UAS-Jun. B) The Med-Mad components. The up- or down-regulation of these two transcription factors yield effects consistent with a role as negative regulators of synaptogenesis which implies the existence of an anti-synaptogenesis pathway. Genotypes: + = D42-Gal4/+. Medea↑ = D42-Gal4/UAS-Medea^#5.13A3. Medea↓ = D42-Gal4/UAS-TRiP.JF02218attP. Mad↑ = UAS-Mad^#2A2/D42-Gal4/+. Mad↓ = UAS-Mad^RNAi /+; D42-Gal4/+. C) Mad interactions with AP-1. The apparently ineffective Jun↓ and Fos↓ constructs suppress the Mad↓ phenotype when jointly expressed. This set of data proves that the constructs are effective and that Fos and Jun interact with the anti-synaptogenesis factor Mad. Genotypes: + = D42-Gal4/+. Mad↓Jun↓ = UAS-Mad^RNAi /UAS-Jun^DN; D42-Gal4/+. Mad↓Fos↓ = UAS-Fos^DN/UAS-Mad^RNAi; D42-Gal4/+. D) In a separate experiment, hence the reason for a separate panel, two conditions that had shown pro-synaptogenesis effects, Jun↑ and Mad↓, although they still show synapse increase, the magnitude of the effect is lower than each element separately. This is suggestive of an antagonistic interaction between these two transcription factors. Genotypes: + = D42-Gal4/+. Jun↑Mad↓ = UAS-Mad^RNAi /+; D42-Gal4/UAS-Jun.

Fig 5. The anti-synaptogenesis pathway.

A) The microRNA bantam shows a tendency towards synapse reduction which becomes evident by suppressing the synapse increase of PI3K↑, Hiw↑ and Bsk↑. By contrast, AKT escapes the repression by Ban since it maintains its synapse increase phenotype reported previously. Although below statistical significance, the pro-synaptogenesis tendency exhibited by Ras85D↑ seems also reverted by Ban. Also, the apparently ineffective Bsk↓ or the pro-synaptogenesis Bsk↑, both become anti-synaptogenic by Ban. These data justify the inclusion of Ban in the anti-synaptogenesis pathway and indicate additional targets for this microRNA. Genotypes: + = D42-Gal4/+. Ban↑ = D42-Gal4/UAS-bantam. Ban↑PI3K↑ = UAS-PI3K/+; D42-Gal4/UAS-bantam. Ban↑Hiw↑ = UAS-Hiw/+; D42-Gal4/UAS-bantam. Ban↑Ras85D↑ = UAS-Ras85D/+; D42-Gal4/UAS-bantam. Ban↑AKT↑ = D42-Gal4/UAS-bantam,UAS-Akt. Ban↑Bsk↑ = UAS-Bsk/+; D42-Gal4/UAS-bantam. Ban↑Bsk↓ = UAS-Bsk^DN/+; +/+; D42-Gal4/UAS-bantam. B) Assays of selected MAPK and GTPases candidates for the anti-synaptogenesis signaling. Genotypes: + = D42-Gal4/+. Put↓ = UAS-put^RNAi/+; D42-Gal4/+. Rala↓ = UAS-Rala^DN/+; D42-Gal4/+. Slpr↓ = D42-Gal4/UAS-Slpr^RNAi. Lic↑ = UAS-Lic/+; +/+; D42-Gal4/+. p38↓ = UAS-p38a^RNAi/+; D42-Gal4/+. Ras64B↑ = UAS-Ras64B^V14/+; D42-Gal4/+. Ask1↓ = D42-Gal4/UAS-Ask1^RNAi. AKT↑Mad↑ = UAS-Akt/+; +/+; D42-Gal4/UAS-Mad^#2A2.

Fig 6. Summary diagram of antagonistic signaling pathways for synaptogenesis and their interactions.

The pro-synaptogenesis pathway is shown in green while the anti-synaptogenesis one is shown in red. Arrows indicate activation and blunt lines indicate repression. Full lines correspond to interactions tested in this study and dotted lines refer to studies in other cellular contexts. The factor Yki was not included in this study and it is shown to illustrate interactions reported with the anti-synaptogenesis factors tested here. The vertebrate orthologues are shown between parentheses.