Regulation of axon regeneration by the RNA repair/splicing pathway

Abstract

Mechanisms governing a neuron’s regenerative ability are important but not well understood. We identified Rtca, RNA 3′-terminal phosphate cyclase, as an inhibitor for axon regeneration. Removal of dRtca cell-autonomously enhanced axon regrowth in the Drosophila central nervous system, whereas its overexpression reduced axon regeneration in the periphery. Rtca along with the RNA ligase Rtcb and its catalyst Archease operate in the RNA repair/splicing pathway important for stress induced mRNA splicing, including that of Xbp1, a cellular stress sensor. dRtca and dArchease had opposing effects on Xbp1 splicing, and deficiency of dArchease or Xbp1 impeded axon regeneration in Drosophila. Moreover, overexpressing mammalian Rtca in cultured rodent neurons reduced axonal complexity in vitro, whereas reducing its function promoted retinal ganglion cell axon regeneration after optic nerve crush in mice. Our study thus links axon regeneration to cellular stress and RNA metabolism, revealing new potential therapeutic targets for treating nervous system trauma.

INTRODUCTION

Failure of damaged axons to regenerate is the primary cause for permanent disabilities after central nervous system (CNS) injury and the non-reversible neurologic dysfunction in neurodegenerative diseases. The ability of a neuron to regenerate its axon after trauma is governed by the interaction between its intrinsic growth capacity and the local environment^1–4. Notwithstanding the discoveries of extracellular factors and intrinsic pathways that reduce the regenerative capacity of axons^5–11, effective therapies have not yet emerged because removing the known inhibitory cues only partially restores regeneration, thus indicating the presence of additional inhibitory machineries that remain to be discovered.

Studies using model organisms such as C. elegans have begun to identify new genes important for axon regeneration^{12, 13}, illustrating the power of the genetic approach. To identify additional factors essential for controlling axon regeneration, we have recently established a Drosophila sensory neuron injury model that exhibits class specific axon regeneration, and demonstrated that the class IV dendritic arborization (da) neuron is capable of regenerating its axon in the periphery but exhibits limited regrowth inside the CNS, resembling its mammalian counterpart at the phenotypic and molecular levels¹⁴. Utilizing this model, we have performed a candidate-based genetic screen focusing on axotomy-regulated genes from multiple organisms^15–20 and identified dRtca/CG4061 (RNA 3′-terminal phosphate cyclase), a cellular RNA processing enzyme with unknown biological function²¹, as an inhibitor for CNS axon regeneration. Furthermore, we find that dArchease, a RNA ligase co-factor, functions downstream of dRtca as a pro-regeneration factor. Rtca and Archease are components of the RNA repair/splicing pathway, and regulate the nonconventional mRNA splicing of Xbp1, a stress sensor. Thus, Xbp1 acts as a substrate, readout and downstream effector for the regulation of axon regeneration by the RNA repair/splicing pathway.

RESULTS

dRtca loss of function enhances axon regeneration

To assess axon regeneration, we used a previously described protocol¹⁴. Briefly, with a two-photon laser, we severed the axons of class IV da neurons (labeled with ppk-CD4tdGFP) within the ventral nerve cord (VNC) (Supplementary Fig. 1) of 2^nd instar larvae (at 48 hours after egg laying, h AEL), confirmed the degeneration of the remaining axons after one day (at 72 h AEL) and assessed their regeneration after two more days (120 h AEL) (Fig. 1a–g). Compared to wild types (WT), which showed limited regrowth (Fig. 1a, arrows), new axons regrew from the retracted axon stems and extended into the commissure region, forming elaborate branches and reconnected commissure segments in a loss of function (LOF) allele of dRtca – dRtca^NP5057 (Fig. 1b, arrowheads), an insertional allele with a P-element inserted in the 5′-UTR, disrupting mRNA splicing and reducing transcript expression (Supplementary Fig. 2a). Similar phenotypes were seen in trans-heterozygotes of dRtca^NP5057 over a deficiency line Df(1)BSC718 that lacks the dRtca locus (Fig. 1c), in a dRtca deletion allele dRtca^Δ generated from imprecise excision of dRtca^NP5057 (Fig. 1d), and even stronger phenotypes were seen in dRtca^Δmat in which both the zygotic and maternal transcripts were removed (Fig. 1e). dRtca^NP5057 is homozygous viable and fertile, so they were derived from homozygous mutant mothers. The mothers of dRtca^NP5057/ Df(1)BSC718 trans-heterozygotes and dRtca^Δ mutants were heterozygous for dRtca and may provide maternal wild type dRtca transcripts. The fact that dRtca^Δmat mutants, in which both the zygotic and maternal transcripts were removed, showed a stronger phenotype than dRtca^Δ zygotic mutants confirmed the maternal effect. Thus, the stronger phenotype of dRtca^NP5057 mutants compared to dRtca^NP5057/ Df(1)BSC718 trans-heterozygotes and dRtca^Δ mutants is likely because no wild type maternal transcripts were provided to dRtca^NP5057 mutants. The function of dRtca is cell-autonomous because its RNAi knockdown in class IV da neurons (ppk-Gal4>dRtcaRNAi) (Fig. 1f, arrowheads) but not in glial cells (repo-Gal4>dRtcaRNAi) (Fig. 1g, arrows) recapitulated the enhancement of regeneration. The regeneration phenotype was further quantified by assessing the “Regeneration percentage”, “Terminal branching” and “Commissure regrowth” (Fig. 1k, l, Supplementary Fig. 3a and Methods), as described previously¹⁴. The regenerating axons are illustrated in schematic diagrams with “Terminal branching” marked in red, “Commissure regrowth” in blue and other regrowing axons in black (Fig. 1a–g). The enhancement of regeneration is unlikely to be due to developmental defects of axon outgrowth because (1) The overall axon patterning of class IV da neurons in the uncut VNC is grossly normal (Supplementary Fig. 2b); (2) Reducing dRtca function in dRtca^Δ mutants or trans-heterozygotes of dRtca^NP5057 over Df(1)BSC718, or via dRtca RNAi in class IV da neurons did not result in obvious defects of axon terminal patterning in the VNC (Supplementary Fig. 2c).

Figure 1. dRtca loss of function enhances axon regeneration in the CNS and PNS.

(a–g) dRtca removal increases class IV da neuron axon regrowth in the VNC. The injury site is demarcated by the dashed circle. Limited regrowth is seen in WT (a, arrows). Increased regeneration is shown in dRtca LOF mutants (b, arrowheads), dRtca mutant over deficiency (c), dRtca zygotic deletion (d), dRtca zygotic and maternal deletion (e), class IV specific dRtca RNAi (f), but not in glia RNAi (g, arrows). The regenerating axons are illustrated in schematic diagrams with “Terminal branching” marked in red, “Commissure regrowth” in blue and other regrowing axons in black. (h–j) dRtca removal promotes class III da neuron axon regeneration in the periphery. Dashed circle marks the lesion site. Whereas WT class III da neurons fail to regenerate their axon in the PNS (h, arrow), dRtca zygotic and maternal deletion (i), and class III specific dRtca RNAi (j) elicit substantial regrowth (arrowheads). (k) dRtca LOF increases Regeneration percentage of class IV da neuron axons in the VNC (N = 46, 16, 18, 13, 14, 14, 14 lesioned segments for each genotype; **P < 0.01, ***P < 0.001, Fisher’s exact test, P < 0.0001, P < 0.0001, P = 0.0022, P < 0.0001, P < 0.0001, P = 1). (l) dRtca LOF increases Terminal branching and Commissure regrowth in the VNC (N = 29, 8, 9, 6, 7, 11, 6 larvae for each genotype; **P < 0.01, ***P < 0.001, one-way ANOVA followed by Dunnett’s multiple comparisons test). (m–o) dRtca LOF increases the Regeneration percentage (**P < 0.01, Fisher’s exact test, P = 0.0026, P = 0.0061), Regeneration index (*P < 0.05, **P < 0.01, one-way ANOVA followed by Dunnett’s multiple comparisons test) and Regeneration length (*P < 0.05, one-way ANOVA followed by Holm-Sidak’s multiple comparisons test; N = 19, 10, 17 neurons for each genotype) of class III da neuron axons in the PNS. Scale bar = 20 μm.

We next tested whether reducing dRtca function would trigger a regenerative response in neurons normally incapable of regeneration by severing their axons in dRtca mutants (Fig. 1h–j). Indeed, dRtca removal in class III da neurons (labeled with 19-12-Gal4>CD4tdGFP, repo-Gal80), which unlike class IV da neurons did not regrow their axons that were severed in the periphery (Fig. 1h, arrow), elicited substantial regeneration in dRtca^Δmat mutants (Fig. 1i, arrowheads) and after RNAi knockdown of dRtca specifically in class III da neurons (19-12-Gal4>dRtcaRNAi) (Fig. 1j, arrowheads), leading to a significant increase in the “Regeneration percentage”, “Regeneration index” and “Regeneration length” (Fig. 1m–o, Supplementary Fig. 3b and Methods).

dRtca gain of function reduces axon regeneration

Conversely, overexpression of dRtca in class IV da neurons (ppk-Gal4>dRtca) mildly reduced their regenerative potential in the peripheral nervous system (PNS). In WT class IV da neurons, which regenerated about 74% of the time, new axons extended beyond the lesion site and followed along the axonal track (Fig. 2a, arrowheads). In contract, dRtca overexpression caused the incidence of regeneration to be reduced to 48% (Fig. 2b, arrow) and the length of the new axons to be significantly shortened as well (Fig. 2c–e). These data indicate that dRtca is an inhibitor of axon regeneration; not only does its removal cell-autonomously enhance axon regeneration in the CNS and enable regenerative incompetent neurons such as class III da neurons to regrow their axons in the PNS, its overexpression in regenerative competent neurons impedes axon regeneration in the periphery.

Figure 2. dRtca overexpression reduces axon regeneration in the PNS.

(a) WT class IV da neurons show robust axon regrowth in the periphery (arrowheads, dashed circle marks lesion site). (b) dRtca overexpression in class IV da neurons mildly reduces their regeneration potential (arrow). (c–e) dRtca overexpression reduces the Regeneration percentage (*P < 0.05, Fisher’s exact test, P = 0.0398) and Regeneration length (*P < 0.05, two-tailed unpaired Student’s t-test, P = 0.0458; N = 43, 27 neurons for each genotype) of class IV da neuron axons in the PNS. Scale bar = 20 μm.

Furthermore, the inhibitory function of dRtca is not limited to sensory neurons, because dRtca overexpression in motor neurons also suppressed motor axon regeneration after nerve crush, as demonstrated by the reduced elaboration of growth cones (Fig. 3 and Methods).

Figure 3. dRtca overexpression reduces motor axon regeneration.

Right after crush, the growth cones of the segmental motor nerves (labeled by m12-Gal4>CD4tdGFP) display a simple morphology with very few filopodia. 14.5 hours after crush, motor axons in WT larvae regenerates substantially with elaborated growth cones. dRtca overexpression reduces their regeneration. The total area of the growth cones at 14.5 hours post-crush is measured, and it is significantly reduced by dRtca overexpression (N = 34, 33 nerves; ***P < 0.001, two-tailed unpaired Student’s t-test). Scale bar = 20 μm.

The expression pattern of dRtca

We next examined the expression pattern of dRtca via two approaches. First, the P-element inserted in dRtca 5′-UTR (dRtca^NP5057) contains Gal4, which is in the same orientation as dRtca, thus can infer dRtca expression via a UAS reporter. We found that the dRtca-Gal4>CD4tdGFP reporter co-localized with the class IV da neuron marker ppk-CD4tdTomato (Fig. 4a), confirming its presence in class IV da neurons. Whereas dRtca-Gal4 expression was observed in other tissues in the PNS and VNC (Fig. 4a, b), our analyses indicate that dRtca functions cell autonomously in neurons to inhibit axon regeneration.

Figure 4. The expression pattern of dRtca in Drosphila.

(a) The P-element inserted in dRtca 5’-UTR (dRtca^NP5057) contains Gal4, which is in the same orientation as dRtca, thus can infer dRtca expression via a UAS reporter. dRtca-Gal4>CD4tdGFP reporter co-localizes with the class IV da neuron marker ppk-CD4tdTomato, confirming its presence in class IV da neurons. (b) dRtca-Gal4>CD4tdGFP shows dRtca is expressed in other tissues within the VNC. (c) dRtca immunostaining shows that dRtca protein is present in class IV (labeled with ppk-CD4tdGFP) and class III (labeled with 19-12-Gal4>CD4tdGFP, repo-Gal80) da neurons in WTs but not in dRtca^Δmat nulls, enriched in the nucleus. dRtca is also present in other types of multidendritic (md) neurons (labeled with 21-7-Gal4>CD4tdGFP). Dashed circles mark the nucleus of da neurons. The big patch-like staining is present in both WT and dRtca^Δmat nulls, and is likely nonspecific. Scale bar = 20 μm.

Second, we generated a polyclonal antibody against dRtca and showed that the dRtca protein was present in WT but not in dRtca^Δmat null class IV and class III da neurons, and was enriched in the nucleus (Fig. 4c, dashed circles). dRtca was also present in other types of multidendritic (md) neurons (Fig. 4c).

Interaction of dRtca with known regeneration regulators

To begin to understand the mechanisms underlying dRtca’s role in regeneration, we first sought to determine how it genetically interacts with the known axon regeneration regulators: Pten and Rac1. Deletion of Pten (phosphatase and tensin homolog), a negative regulator of the mammalian target of rapamycin (mTOR) pathway, has been shown to increase CNS axon regeneration in both mammals and flies^{8, 14, 22}. Overexpression of dRtca in the Pten mutant background (Pten^MGH6; ppk-Gal4>dRtca) or overexpression of Pten in dRtca null mutants (dRtca^Δ; ppk-Gal4>Pten) largely abolished the enhancement of axon regeneration as seen in the VNC in Pten^MGH6 or dRtca^Δ mutants (Fig. 5a, b). This suggests that dRtca and Pten likely function in parallel pathways. Interestingly, double mutants of dRtca and Pten (dRtca^NP5057; Pten^MGH6) did not further improve regeneration, as compared to dRtca mutants (Supplementary Fig. 4a, b), indicating the presence of additional brakes on regeneration. The regeneration phenotype in dRtca mutants appears to be comparable if not stronger than Pten mutants (Pten^MGH6, a hypomorphic allele) or Akt overexpression (ppk-Gal4>Akt) (Supplementary Fig. 4c, d).

Figure 5. The interaction of dRtca with known regeneration regulators.

(a–c) dRtca likely functions in parallel with the Pten pathway, Rac1 mildly promotes CNS axon regeneration and functions downstream of dRtca. Overexpression of dRtca in Pten mutants or overexpression of Pten in dRtca nulls largely abolishes the enhancement of axon regeneration in the VNC in Pten^MGH6 or dRtca^Δ mutants, as reflected by Regeneration percentage (a), Terminal branching and Commissure regrowth (b). Overexpression of Rac1 in class IV da neurons increases the Regeneration percentage (a) without promoting Terminal branching or Commissure regrowth (b, c, arrowheads). Overexpressing the dominant negative Rac1 abolished the enhancement of CNS axon regeneration seen in dRtca nulls (a, b) (N= 46, 17, 18, 13, 17, 14, 14, 9 lesioned segments in a; N = 29, 9, 9, 6, 9, 7, 7, 4 larvae in b, *P < 0.05, **P < 0.01, ***P < 0.001, Fisher’s exact test in a, P = 0.0027, P = 0.3681, P = 0.0022, P = 0.7609, P = 0.0253, P = 0.0037, P < 0.0001, P = 0.4224, P = 0.0183; one-way ANOVA followed by Tukey’s or Dunnett’s multiple comparisons test in b). Scale bar = 20 μm.

Because the cytoskeleton regulator Rac GTPase is required for regenerative axon outgrowth in C. elegans²³, we overexpressed Rac1 in class IV da neurons (ppk-Gal4>Rac1) and found an increase in the number of axons initiating the regenerative response in the VNC but not Terminal branching or Commissure regrowth, a partial improvement of regeneration (Fig. 5a–c, arrowheads). Conversely, overexpressing the dominant negative form of Rac1 abolished the enhancement of CNS axon regeneration seen in dRtca null mutants (dRtca^Δmat; ppk-Gal4>Rac1DN) (Fig. 5a, b), whereas Rac1DN overexpression alone in class IV da neurons did not result in obvious axon regeneration defects in the PNS (Supplementary Fig. 4e–g). It thus seems likely that Rac1 functions downstream of dRtca in a pathway that converges onto the regulation of the cytoskeleton.

RNA repair/splicing pathway regulates axon regeneration

Rtca is a RNA processing enzyme that possesses the RNA-3′-phosphate cyclase activity and catalyzes the ATP-dependent conversion of a 3′ phosphate to a 2′,3′-cyclic phosphodiester at the end of RNA²¹. The RNA 2′,3′-cyclic phosphate ends play important roles in RNA metabolism, for example, as intermediates during RNA repair by ligases^{24, 25}. Rtcb (RNA 2′,3′-cyclic phosphate and 5′-OH ligase) represents a new type of RNA ligases that join 2′,3′-cyclic phosphate and 5′-OH RNA ends to yield a 3′-5′ phosphodiester splice junction^{24, 26–28}. Specifically, Rtcb is known to possess the cyclic phosphodiesterase activity, which hydrolyzes the 2′,3′-cyclic phosphate to a 3′-phosphate, as well as the ligase activity, which then joins the RNA 3′-phosphate to a 5′-OH RNA end. In addition, the specificity and efficacy of Rtcb’s ligase activity can be enhanced by Archease^{29, 30}, which is a small acidic protein conserved in eukarya, bacteria and archaea³¹. In E. coli, RtcA and RtcB are encoded in a single operon, suggesting that they might cooperate to provide the healing and sealing function in a RNA repair pathway²⁷. In one scenario, the healing refers to the restoration of ligatable 2′,3′-cyclic phosphate ends in the event that the inciting RNA damage directly generates RNA 3′-phosphates, or that the 2′,3′-cyclic phosphate products of RNA transesterification are further processed to a 3′-phosphate by a 2′,3′-cyclicphosphodiesterase³². However, this model cannot readily be reconciled by the subsequent finding that RtcB is adept at joining 3′-phosphate/5′-OH ends or 2′,3′-cyclic phosphate/5′-OH ends^{33, 34}. Therefore, the exact interrelationship between RtcA and RtcB remains undetermined. Interestingly, the RtcBA operon in E. coli is regulated by the σ54 coactivator RtcR, suggesting that the RNA repair functions are induced in response to cellular stress²¹. Whereas the biological role of Rtca remains unknown, the enzyme is speculated to function in some aspects of cellular RNA processing²¹. Taking into account these findings and our observations that Rtca loss of function enhances axon regeneration, we hypothesized that the Rtca-Archease dependent RNA repair/splicing pathway regulates axon regeneration. Specifically, we speculated that axon injury triggers a type of cellular stress leading to RNA damage/splicing, producing RNA 3′-phosphates that need to be processed and rejoined by the Rtcb ligase, which is catalyzed by Archease. Because Rtca converts RNA 3′-phosphate to 2′,3′-cyclic phosphate, it can slow down the ligation process and impede regeneration. Consequently, silencing Rtca promotes axon regeneration (Supplementary Fig. 5). Following this rationale, we then investigated the role of Archease in axon regeneration.

To determine the role of the Drosophila Archease (dArchease/CG6353) in axon regeneration, we first examined class IV da neuron axon regeneration in the periphery. To maximize the phenotype, we modified our PNS axon injury protocol as described previously¹⁴: axotomy was induced at 72 h AEL, degeneration was confirmed at 96 h AEL and regeneration was assayed at 120 h AEL. Different from WT neurons, which exhibited substantial regrowth of their severed axons, axon regeneration was significantly impaired in the dArchease LOF mutant allele dArchease^PBc01013, which is an insertional allele with a P-element inserted into the 5′-UTR thereby disrupting its mRNA splicing and eliminating dArchease transcripts (Supplementary Fig. 6), as revealed by a significant drop of the “Regeneration percentage”, “Regeneration index” and “Regeneration length” (Fig. 6a–c). This phenotype was confirmed in trans-heterozygotes of dArchease^PBc01013 over a deficiency line Df(3R)ED6076 or Df(3R)BSC678 that lacks the dArchease locus (Fig. 6a–c). dArchease^PBc01013 mutants are larval lethal. dArchease^PBc01013 mutants and dArchease^PBc01013/DfED6076, dArchease^PBc01013/DfBSC678 trans-heterozygotes showed a similar phenotype, suggesting that dArchease^PBc01013 mutants is likely an amorphic allele. The function of dArchease is required cell-autonomously as class IV da neuron specific knockdown of dArchease (ppk-Gal4>dArcheaseRNAi) but not glial cell RNAi (repo-Gal4>dArcheaseRNAi) was sufficient to phenocopy the regeneration failure (Fig. 6a–c). Moreover, loss of function of both dRtca and dArchease (dRtca^NP5057; dArchease^PBc01013) completely abolished the axon regeneration promoting effect in the VNC seen in dRtca^NP5057 mutants (Fig. 6d, e), producing many retracted or stalled axon stems (Fig. 6f, arrows). This epistasis analysis indicates that dArchease is a pro-regeneration factor downstream of dRtca, and they act in opposing ways to regulate axon regeneration (Supplementary Fig. 5).

Figure 6. The dRtca pathway in regulating axon regeneration.

(a–c) dArchease and Xbp1 are required for class IV da neuron axon regeneration in the PNS. dArchease or Xbp1 deficiency, shown in LOF mutants, mutant over deficiency, class IV da neuron specific RNAi, but not glia RNAi, impedes class IV da neuron axon regeneration in the periphery, as quantified by Regeneration percentage (a), Regeneration index (b) and Regeneration length (c) (N= 26, 16, 26, 18, 25, 20, 18, 15, 16, 17 neurons). (d–f) Epistasis analysis of dRtca, dArchease and Xbp1 indicates that dArchease and Xbp1 function downstream of dRtca. Double mutants of dRtca and dArchease completely diminish the enhancement of CNS axon regeneration in dRtca single mutants, while double mutants of dRtca and Xbp1 show a milder attenuation. A representative image of the stalled axon regeneration in the VNC of dRtca and dArchease double mutants is shown in (f) (N = 46, 16, 15, 16, 18 lesioned segments in d; N = 29, 8, 7, 8, 9 larvae in e). (g, h) Heat-shock induces the splicing of the active form of Xbp1 (Xbp1s), which is enhanced by dRtca LOF, but inhibited in dArchease LOF mutants, and double mutants of dRtca and dArchease (g). Blots were cropped and full-length images are presented in Supplementary Figure 12. The ratio of Xbp1s/Xbp1μ is quantified in (h) (N = 3 experiments; *P < 0.05, **P < 0.01, ***P < 0.001, Fisher’s exact test in a, d, P = 0.004, P = 0.0023, P = 0.0049, P = 0.0217, P = 0.7406, P = 0.0581, P = 0.0025, P = 0.004, P = 0.7244 in a, P = 0.0001, P = 0.4831, P = 0.2178, P < 0.0001, P = 0.0008, P = 0.004 in d; one-way ANOVA followed by Dunnett’s, Tukey’s, Holm-Sidak’s or Dunn’s multiple comparisons test in b, c, e, h). Scale bar = 20 μm.

Xbp1 is a substrate of dRtca/dArchease in regeneration

What might be the RNA substrates processed by this dRtca-dArchease dependent RNA repair/splicing pathway for the regulation of axon regeneration? We investigated the X-box binding protein 1 (Xbp1) as a candidate substrate for the following reasons: (1) Cellular stress such as ER stress triggers an adaptive intracellular signaling cascade known as the unfolded protein response (UPR)³⁵. One main branch of the UPR is the activation of Ire1, which cleaves Xbp1 pre-mRNA in the cytoplasm, converting the unspliced Xbp1μ, a putative transcriptional repressor, into the unconventionally spliced Xbp1s, by eliminating a 26-nucleotide intron (23 in flies) that changes the open reading frame of the 3^rd exon, resulting in a new protein with transcriptional activity³⁶. Xbp1s directly activates ER stress target genes to facilitate refolding and also degradation of misfolded proteins³⁵. (2) The RNA ligase Rtcb and its cofactor Archease are involved in the non-conventional splicing induced by the UPR, and Archease is required for the splicing of the Xbp1 mRNA³⁷. (3) Loss of xbp1 function in C. elegans results in severely reduced axon regeneration¹³. To determine the function of Xbp1 in axon regeneration in the PNS and CNS, we used a mutant allele of Xbp1^k13803, an allele with a P-element inserted into its 5′-UTR thus reducing transcripts³⁸, and showed that axon regeneration in the periphery was mildly reduced (Fig. 6a–c). This defect was stronger in trans-heterozygotes of Xbp1^k13803 over a deficiency line Df(2R)BSC484 that lacks the Xbp1 locus (Fig. 6a–c), suggesting that Xbp1^k13803 is likely a hypomorphic allele. Class IV da neuron specific (ppk-Gal4>Xbp1RNAi) but not glia specific (repo-Gal4>Xbp1RNAi) RNAi of Xbp1 reproduced the impairment of regeneration (Fig. 6a–c), indicating it functions cell-autonomously. Moreover, double mutants of dRtca and Xbp1 (dRtca^NP5057; Xbp1^k13803) dampened the enhancement of CNS axon regeneration seen in dRtca^NP5057 mutants (Fig. 6d, e), indicating that Xbp1 is indeed a pro-regeneration factor downstream of dRtca. Consistent with these lines of evidence, overexpression of the spliced form Xbp1s in class IV da neurons significantly enhanced axon regeneration in the VNC (Fig. 6d, e), and it also promoted axon regeneration in the periphery when overexpressed in class III da neurons (Supplementary Fig. 7, arrowheads). The observations that dRtca Xbp1 double mutants did not completely eliminate the enhanced regeneration phenotype in dRtca mutants, and that Xbp1s overexpression led to milder enhancement of regeneration compared to dRtca LOF, suggest that additional substrates contribute to regeneration regulation.

In order to directly assess the nonconventional splicing of Xbp1 mRNA in vivo, we resorted to the heat-shock paradigm. Fly larvae of various genotypes underwent a 40°C heat-shock and the abundance of Xbp1 splice variants was assessed using semi-quantitative RT-PCR. The ratio of Xbp1s/Xbp1μ was then quantified. Heat-shock induced the expression of the spliced form Xbp1s. In contrast to the enhanced expression of Xbp1s in dRtca mutants, Xbp1s level was greatly reduced in dArchease LOF mutants. Double mutants of dRtca and dArchease resembled dArchease mutants in the reduction of Xbp1 splicing (Fig. 6g, h). Taken together, these data indicate that dRtca and dArchease in the RNA repair/splicing pathway negatively and positively regulate the stress induced Xbp1 mRNA splicing, respectively, so that Xbp1 acts as a readout and effector for the regulation of axon regeneration (Supplementary Fig. 5).

Mammalian Rtca inhibits CNS axon regeneration

Having established the role of dRtca in axon regeneration in Drosophila, we went on to determine whether its function is evolutionarily conserved in mammals. We started by examining the expression pattern of the mammalian ortholog of dRtca in vitro. Antibodies raised against human RTCA recognized rodent Rtca in the cell bodies and processes of cultured hippocampal neurons (Fig. 7a). Moreover, the expression of Rtca transcripts in the DRG in vivo increased progressively throughout development, reaching the highest level in adults (Fig. 7b). Remarkably, using quantitative RT-PCR, we found that Rtca transcript level in the DRG was significantly reduced following lesion of the sciatic nerve peripherally, but not lesion of the central axon branch of DRG neurons with a spinal cord hemisection (Fig. 7c). Since the peripheral processes of DRG neurons are capable of regeneration, whereas their central axons that project into the spinal cord fail to regrow after injury³⁹, the selective suppression of Rtca following peripheral injury supports the hypothesis that the persisting expression of Rtca is inhibitory to axon regeneration in the CNS. Furthermore, in agreement with the overexpression phenotype in flies, overexpression of Rtca in cultured hippocampal neurons reduced axon complexity, and dramatically reduced proximal axonal branching (Fig. 7d, e) without affecting total axon length, indicative of the inhibitory function of Rtca.

Figure 7. Rtca is an inhibitory factor for axon growth in vitro and RGC regeneration in vivo in rodents.

(a) A RTCA antibody (HPA027982) recognizes endogenous Rtca in cultured hippocampal neurons. Rtca is present in the soma and neuronal processes (arrowheads), which are labeled with anti-Tau staining. Scale bar = 20 μm. (b) The expression of Rtca transcripts in the whole DRGs in vivo is upregulated throughout development, with the highest level in adults. (N = 3 experiments; the expression level of each data point was normalized to the average value of E18; *P < 0.05, one-way ANOVA followed by Dunnett’s multiple comparisons test). (c) Rtca transcript level in the DRG is significantly reduced after a peripheral sciatic nerve lesion, whereas lesioning the central axon branch of DRG neurons with a spinal cord hemisection does not alter its expression (N = 3 experiments; for peripheral lesion, expression level of the lesioned DRG is normalized to the corresponding control DRG in pairs; for central lesion, expression level of each data point was normalized to the average value of Control; *P < 0.05, two-tailed unpaired Student’s t-test, P = 0.0237, P = 0.8544). (d, e) Ectopic expression of Rtca in cultured hippocampal neurons reduces axon complexity when transfected from div (days in vitro) 0 to div 3, and dramatically reduces proximal axonal branching (N = 18, 18 neurons; ***P < 0.001, two-way ANOVA). Scale bar = 20 μm. (f–h) Two weeks after optic nerve crush in P35 adult siblings (Rtca^+/+ and Rtca^Ins/+), and Rtca^Ins/Ins mutant mice, regenerating fibers were labeled by intravitreal injection of Alexa 555-labeled cholera toxin B (CtB555). (f, g) Tissue sections through the optic nerve from a Rtca^Ins/Ins mutant mice show regenerating axons more than 800 μm distal to the lesion site, whereas no obvious axon regrowth beyond the crush site is seen in similar sections from a sibling control (Rtca^Ins/+, asterisk marks the crush site). (h) Regenerating fibers were counted at specified distances from the lesion site. More fibers regenerate in Rtca^Ins/Ins mice compared to siblings (N = 5 Rtca^+/+, 5 Rtca^Ins/+ and 6 Rtca^Ins/Ins mice; ***P < 0.001, two-way ANOVA). Scale bar = 200 μm.

We next asked if knocking out Rtca during development enhances axon regeneration of adult retinal ganglion cells (RGCs) in vivo. For this purpose, we generated a mutant allele with a LacZ cassette inserted after the 3^rd exon (Supplementary Fig. 8a) to disrupt splicing and reduce transcription (reduced to ~19%, Supplementary Fig. 8b), thereby generating Rtca^LacZ_loxP (Rtca^Ins/Ins) mutant mice. Rtca protein level was also reduced to ~18% in the mutants (Supplementary Fig. 8c), suggesting this is likely a hypomorphic allele. Homozygous Rtca^Ins/Ins mice were born, although fewer in number than expected from the mendelian ratio. By adulthood, there were no obvious differences in RGC number or RGC axon morphology among mutant (Rtca^Ins/Ins), het (Rtca^Ins/+) and WT (Rtca^+/+) animals (Supplementary Fig. 9a, c). Since the LacZ cassette is inserted into the Rtca locus, it can be used as a reporter for examining Rtca expression. LacZ staining was observed in the RGC layer of Rtca^Ins/+ mice (Supplementary Fig. 10a–c) but not in Rtca^+/+ littermates (Supplementary Fig. 10d–f). Moreover, β-Galactosidase immunostaining in Rtca^Ins/+ mice showed distinct expression of the LacZ reporter within NeuN⁺ neurons in the retina, which was absent in Rtca^+/+ littermates (Supplementary Fig. 10g), indicating that Rtca is indeed expressed in RGCs. To assess RGC axon regeneration, we performed optic nerve crush in Rtca^Ins/Ins, Rtca^Ins/+ and Rtca^+/+ littermate mice at two developmental time points: postnatal day 35 (P35) and two–three months old, and the extent of axon regeneration was measured in the optic nerve after two weeks or three weeks, respectively. In mice injured at P35, compared to siblings (Rtca^Ins/+ and Rtca^+/+), which did not exhibit substantial axon regrowth beyond the crush site (Fig. 7f, asterisk indicates the crush site), Rtca^Ins/Ins mutant mice showed a substantial increase in the number of regenerating axons at various distances from the injury site with some regenerating axons extending over 1.5 mm beyond the crush site (Fig. 7g, h). The regeneration enhancement in mice operated on at two-three months old was milder but still significant (Supplementary Fig. 11a–d). In these animals, curving, turning and looping of axons were observed, indicative of new axon growth (Supplementary Fig. 11b, arrowheads), and the furthest distance that axons travelled beyond the injury site was about 3.5 times longer in the mutants (Supplementary Fig. 11c). The crush site was further marked by the presence of ED1 staining, which labeled the infiltrating macrophages⁴⁰. Whereas axons rarely penetrated beyond the ED1⁺ region in sibling controls, a large number of axons were seen hundreds of microns beyond the ED1⁺ region in Rtca^Ins/Ins mutants (marked with asterisk in Supplementary Fig. 11e). Reducing Rtca function did not affect RGC survival after injury (Supplementary Fig. 9b), confirming that this increase in regenerating axons was not secondary to an increased RGC number. Our finding that reducing Rtca expression increased the regenerative potential of adult RGCs thus provides evidence for a potentially conserved role of Rtca as an anti-regeneration factor.

DISCUSSION

Our findings reveal an important role of the RNA repair/splicing pathway in regulating the intrinsic axon regeneration potential in response to PNS and CNS injury in Drosophila. Rtca and Archease integrate the injury signals triggered by axotomy and lead to the activation of downstream effectors such as the stress response cascade involving Xbp1 splicing, and affect the ability of a neuron to regenerate. Axon injury has been suggested as a cellular stress and the mTOR pathway, a potential determinant of neuronal regeneration competence, could be inactivated under stress conditions such as hypoxia or DNA damage⁴¹. Interestingly, Xbp1 splicing has been observed in RGCs after optic nerve injury and forced activation of Xbp1 promotes RGC survival⁴². Our work implicates that proper splicing of Xbp1 is also important for axon regeneration in Drosophila.

Moreover, recent work in C. elegans also suggests the involvement of stress response pathways, such as heat-shock, hypoxia and UPR, in axon regeneration¹³. However, how the injury signal is relayed to the stress response is unclear. Our work identifies a missing link and implicates the Rtca-Archease dependent RNA metabolism machinery as a regeneration regulator. A priori, axonal injury could either signal directly to the stress pathways, which then recruit Rtca-Archease, or alternatively, Rtca and Archease may represent injury response elements upstream of the stress pathways. Our results showed that the Xbp1 dependent UPR pathway acts downstream of Rtca-Archease in controlling axon regeneration, and the remaining question is whether and how it impinges on other stress pathways, such as hypoxia or DNA damage. It will be important in future studies to identify other substrates in addition to Xbp1 that are modified by Rtca-Archease, and to search for response genes downstream of Xbp1. Our study raises the prospect of manipulating Rtca, Archease and Xbp1 as potential therapeutic interventions for treating nervous system injury.

As a first step to determine whether the Rtca pathway may have an evolutionarily conserved function in axon regeneration, we have examined CNS axon regeneration after optic nerve crush in a hypomorphic mouse mutant allele of Rtca and have suggestive evidence showing that may be indeed the case. The enhancement of RGC axon regeneration phenotype in the Rtca mutant is modest as compared to that seen in PTEN, KLF4 or SOCS3 knockouts. This may be due to the residual Rtca function in this hypomorphic allele or developmental compensation. Future experiments using mammalian injury models to examine the Rtca null allele, and to assess other components of the RNA repair/splicing pathway are therefore warranted to further define its potential role in axon regeneration.

METHODS

Fly stocks

ppk-CD4-tdGFP⁴³, ppk-CD4-tdTomato⁴³, ppk-Gal4⁴⁴, repo-Gal4⁴⁵, 19-12-Gal4⁴⁶, reop-Gal80⁴⁷, 21-7-Gal4⁴⁸, hs-FLP; ppk-Gal4; UAS>CD2>mCD8-GFP⁴⁹, UAS-Pten (Tian Xu lab), Pten^MGH6 (Iswar Hariharan lab) and UAS-Xbp1s (UAS-Xbp1-RB)³⁸ (Hyung Don Ryoo lab) have been previously described. UAS-Dcr-2, Df(1)BSC718, Df(3R)Exel6186, Df(2R)BSC484, Dp(1;3)DC413, UAS-Rac1, UAS-Rac1DN, Xbp1^k13803, UAS-Xbp1RNAi, m12-Gal4⁵⁰ (P(Gal4)tey^5053A) and hs-FLPD5 were from Bloomington stock center. dRtca^NP5057 was from Kyoto DGRC. UAS-dRtcaRNAi and UAS-dArcheaseRNAi were from VDRC. dArchease^PBc01013 was from Exelixis. dRtca^Δ was generated from imprecise excision of dRtca^NP5057, in which the entire coding sequence of dRtca 3′ to the NP5057 insertion site together with the neighboring gene CG4045 were replaced by a 3Kb fragment from the P element. To generate dRtca^Δmat, because dRtca^Δ males were sterile, a duplication stock Dp(1;3)DC413, which has a duplication of the region covering dRtca on the 3^rd chromosome, was used. Specifically, dRtca^Δ;; Dp(1;3)DC413/TM6B males were mated with dRtca^Δ/FM6 females to get the dRtca^Δ homozygous females. To generate the UAS-dRtca stock, the entire coding sequence of dRtca was cloned into the PUAST vector using the 5′-tgctccgattgttctgacac-3′ and 5′-attttccggcgcctttag-3′ primers. For experiments in Drosophila, both male and female larvae were used.

Mice

We used an embryonic stem (ES) cell from the European Conditional Mouse Mutagenesis Program (EUCOMM Project 82885) – a knockout-first line with a LacZ cassette inserted into the Rtca locus, to generate the Rtca mutant mouse (UCSF ES Cell Targeting Core). The resulting mouse Rtca^LacZ_loxP (Rtca^Ins/Ins) has a lacZ cassette inserted in-between the 3^rd and 4^th exons of Rtca, meanwhile, the 4^th exon is flanked by loxP sites (Supplementary Fig. 8a). The lacZ insertion disrupted Rtca splicing and dramatically reduced transcript and protein levels (Supplementary Fig. 8b, c), and thus resulted in a hypomorphic allele. All mice were housed in an animal facility and maintained in a temperature-controlled and light-controlled environment with an alternating 12-hour light/dark cycle. Up to 5 mice of the same sex and similar age were housed in a cage. The animals had no prior history of drug administration, surgery or behavioral testing. All protocols have been approved by the UCSF Institutional Animal Care and Use Committee.

Sensory axon lesion in Drosophila

da neuron axon lesion and imaging in the PNS or within the VNC were performed in live fly larvae as previously described¹⁴.

Quantitative analyses of sensory axon regeneration

Quantification was performed as previously described¹⁴. Briefly, for axon regeneration in the VNC, we used “Regeneration percentage”, which depicts the percent of regenerating commissure segments among all the segments that were severed; “Terminal branching”, which counts the number of axons that regenerated, reached the commissure bundle and appeared to form elaborate branches; “Commissure regrowth”, which counts the number of axons that had regenerated to connect the boundaries of commissure segments, longitudinally or laterally (Supplementary Fig. 3a). For axon regeneration in the PNS, we used “Regeneration percentage”, which depicts the percent of regenerating axons among all the axons that were lesioned; “Regeneration length”, which measures the increase of axon length; “Regeneration index”, which is calculated as an increase of “axon length”/“distance between the cell body and the axon converging point (DCAC)” (Supplementary Fig. 3b).

Live imaging

Live imaging was performed as described^{51, 52}. Embryos were collected for 2 hours on yeasted grape juice agar plates and were aged at 25°C or room temperature. At the appropriate time, a single larvae was mounted in 90% glycerol under coverslips sealed with grease, imaged using a Leica SP5 microscope, and returned to grape juice agar plates between imaging sessions.

Generation of clones

FLP-out clones were produced as previously described⁴⁹.

Drosophila motor nerve crush assay and quantification

As described previously⁵³, the segmental nerves of 3^rd instar larvae were pinched tightly through the cuticle for 5 s with Dumostar # 5 forceps, when the larvae were under CO₂ anesthesia. Larvae were then transferred to a grape plate and kept alive for varying periods of time at 25°C. For quantification of motor axon regeneration, the total area of the growth cone at 14.5 hours after crush was measured.

Neuronal cell culture, transfection and analysis

Hippocampal neurons were cultured from E19 Long-Evans rats at 150,000/ coverslip and maintained at serum-free B27-containing media. Plasmid transfections were done using Lipofectamine2000 (Invitrogen) at the indicated dates (div 0–3). For overexpression experiments, neurons were transfected with 0.75 μg of the Rtca expression construct (Open Biosystems) together with 0.5 μg GFP expressing plasmid (pLentilox 3.7). For axon analysis, we restricted all our morphological analysis to CTIP2 negative CA3 pyramidal neurons⁵⁴, and used Neurolucide (MBF Bioscience).

Optic nerve crush

For all in vivo experiments, optic nerve crush, tissue processing and imaging were performed in a double blind manner. P35 or 8–12 weeks old adult mice (male and female) were used, and the surgery was done by crushing the optic nerve behind the globe with forceps. The mice were then kept for 2 or 3 weeks respectively until perfusion. For the procedure performed at P35, RGC axons were anterogradely labeled through intravitreal injections of 1 μl cholera toxin subunit B (CtB555, 10μg/μl; Molecular Probes) two days before perfusion. Longitudinal sections (20 μm) were prepared and stained with anti-NF200 (labeling the neurofilament in axons) and anti-ED1 (labeling the infiltrating macrophages). The crush site was determined by the presence of ED1 staining, the clustering of NF200 staining, and/or the physical pinching/narrowing of the nerve. For quantification, the distance between the crush site and the tip of the longest axon was measured – Longest axon, and the number of axon fibers 50, 100, 150, 200, 250, 300, 350, 500, 750, 1000 and 1500 μm away from the crush site was counted for each section.

Quantification of RGC survival

Retina sections were immunostained with anti-Tuj1 (beta-III tubulin) to visualize RGCs and DAPI to detect nuclei. The number of RGCs were counted and normalized to the length of the corresponding retina region to calculate the RGC density. The RGC density from each sample was then normalized to the average value of the sibling controls.

Sciatic nerve lesion and spinal cord hemisection for expression analysis

For sciatic nerve lesion, mice were anesthetized (isoflurane) and the sciatic nerve was exposed at mid-thigh level, then sutured and transected distal to the ligature and the wound was closed. To lesion central processes of peripheral neurons, mice were anaesthetized (Ketamine (100 mg/kg)/ Xylazine (10 mg/kg)), then a laminectomy was performed at the thoracic level. The T6-T7 dorsal columns were then lesioned bilaterally using Dumont #5 Fine Forceps. In the cases of both peripheral and central nerve injury, L4 – L6 DRGs were collected 7 days after lesion for expression analysis. For each condition, DRGs from sham-operated mice (exposed but not lesioned sciatic nerve and thoracic laminectomy without dorsal column transaction) were used as controls.

Immunohistochemistry and LacZ staining

Immunohistochemistry analyses were performed on whole mount fly larvae, cultured neurons and on mouse tissue sections collected from OCT-embedded tissues. Primary antibodies used were anti-Tuj1 (Covance, MMS-435P, 1:1,000), anti-Tau (Millipore, MAB3420, 1:1,000), anti-RTCA (Sigma, SAB2102059 and HPA027982, 1:200), anti-CTIP2 (Abcam, ab18465, 1:2,000), anti-NF200 (Sigma, N4142, 1:200), anti-ED1 (Abcam, ab31630, 1:200), anti-β-Galactosidase (MP Biomedicals cappel, 55976, 1:5,000) and anti-NeuN (Millipore, MAB377, 1:200). A custom-made antibody against the entire dRtca protein was generated (Thermo, 1:200). LacZ staining was performed using the Senescence β-Galactosidase Staining Kit (Cell signaling) according to manufacturer’s instructions.

Heat-shock paradigm

In order to assess the stress induced splicing of Xbp1, 3^rd instar larvae were heat-shocked at 40°C for 2 hours.

Quantitative RT-PCR

Semi-quantitative RT-PCR was done for dRtca, α-tubulin, dArchease and Xbp1 in flies, and Rtca and β-actin in mice according to the manufacturer’s protocols. The sequences were as follows: two regions of the dRtca transcripts were amplified with primers 5′-gaggaccaagctagcgattg-3′ and 5′-caagtaggagccgtcgatct-3′; 5′-caaacaggtctgcgttgatg -3′ and 5′-ctgtcatttgctcggctaca-3′. α-tubulin was amplified with 5′-acaacgaggctatctacgaca-3′ and 5′-ttttcagtgttgcagtgaattt-3′. Two regions of the dArchease transcripts were amplified with primers 5′-acactaagcggttgctggtt-3′ and 5′-ggtgatctccagcttcttgc-3′; 5′-cgagatttcctgccattgtt-3′ and 5′-gcaaaggggttgaaacagaa-3′. Xbp1 was amplified with primers 5′-aggccatcaacgagtcactg-3′ and 5′-ctttccagagtgaggccagg-3′. Two regions of the mouse Rtca transcripts were amplified with primers 5′-tggatcatcacttggcaaaa-3′ and 5′-ggctagcgccatgaaaataa-3′; 5′-aacctttacccccgagaaga-3′ and 5′-aggacacagggcattgaaac-3′.

Real-time quantitative RT-PCR was done for mouse Rtca. 2μg of RNA was reverse transcribed to cDNA using iScript cDNA Synthesis Kit (Bio-Rad). All RNA concentrations were measured by NanoDrop 1000 Spectrophotometer. Real-time detection and quantification of cDNAs were performed with the iCycler instrument (Bio-Rad). Quantitative PCR was performed in a 20 μL reaction mixture using SYBR@GreenER qPCR Supermixes (Invitrogen). 50 cycles of amplification was performed according to manufacturer’s instructions. Fluorescence data were collected at annealing stages and real-time analysis performed with iCycler^™ iQ Optical System Software V3.0a. Serial dilutions of cDNAs were used for construction of the standard curve. Ct values were determined with automatically set baseline and manually adjusted fluorescence threshold. Gene expressions were normalized with that of β-actin.

Statistical analysis

No statistical methods were used to pre-determine sample sizes but our sample sizes are similar to those reported in previous publications^{7–9, 14, 22} and the statistical analyses were done afterwards without interim data analysis. Data are expressed as mean ± SEM in bar and dot plots, or as median with min/max values in box-and-whisker plots. No data points were excluded. For the experiments done in Drosophila, data collection and analyses were not performed blind to the conditions of the experiments. For in vivo experiments in mice, optic nerve crush, tissue processing and imaging were performed in a double blind manner. Two-tailed unpaired Student’s t-test was performed for comparison between two groups of samples. One-way ANOVA followed by multiple comparison test was performed for comparisons among three or more groups of samples. Two-way ANOVA analyses were used to assess significance of multiple data points. Fisher’s exact test was used to compare the percentage. The data meet the assumptions of the tests. The variance has been tested in each group of the data and the variance is similar among genotypes. Data distribution was assumed to be normal but this was not formally tested. The data were collected and processed randomly. Each experiment has been successfully reproduced at least three times and was performed on multiple days. Statistical significance was assigned, *P < 0.05, **P < 0.01, ***P < 0.001.

A supplementary methods checklist is available.

Supplementary Material

2

Supplementary Figure 1. A schematic diagram depicting the axotomy protocol of da neuron axons in the PNS and CNS. The axons in the PNS and CNS are labeled. Dashed line indicates VNC boundary. The axon terminal within the VNC of one representative class IV da neuron is shown in green.

Supplementary Figure 2. dRtca loss of function as in dRtca^NP5057, which alters transcript expression, enhances axon regeneration in the VNC, without affecting axon terminal patterning. (a) P element insertion in dRtca^NP5057 disrupts mRNA splicing and reduces transcript level. Arrows indicate the primer locations. Two pairs of primers were used and are color coded in green and turquoise. The PCR products are color coded in the same way according to the primer position. Blots were cropped and full-length images are presented in Supplementary Figure 13. (b) The overall patterning of class IV da neuron axons in the VNC (labeled by ppk-CD4tdGFP) is not affected after dRtca LOF. (c) Single clone analysis shows that dRtca LOF does not affect gross patterning of the nerve terminals. The axon terminal of a single class IV da neuron is shown in whole animal dRtca LOF mutants (upper panels), or in animals with dRtca RNAi in all class IV da neurons (lower panels). Scale bar = 20 μm.

Supplementary Figure 3. Quntification of sensory axon regeneration. (a) For axon regeneration in the VNC, “Terminal branching” counts the number of axons that regenerate, reach the commissure bundle and appear to form elaborate branches, shown in red. “Commissure regrowth” counts the number of axons that have regenerated to connect the boundaries of commissure segments, longitudinally or laterally, shown in blue. (b) For axon regeneration in the PNS, “Regeneration index” is calculated as an increase of “axon length”/“distance between the cell body and the axon converging point (DCAC)”.

Supplementary Figure 4. Analyses of the CNS regeneration phenotype in dRtca and Pten pathway mutants, as well as PNS axon regeneration after Rac1DN overexpression. (a, b) Double mutants of dRtca and Pten do not further promote axon regeneration in the VNC. (a) Regeneration percentage is not significantly different between dRtca single mutants and dRtca Pten double mutants (N = 46, 16, 12 lesioned segments; ***P < 0.001, Fisher’s exact test, P < 0.0001, P = 0.0005, P = 0.1746). (b) Terminal branching and Commissure regrowth are comparable between dRtca single mutants and dRtca Pten double mutants (N = 28, 8, 6 larvae; *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA followed by Holm-Sidak’s multiple comparisons test and Tukey’s multiple comparisons test, respectively). (c, d) The regeneration phenotype in dRtca LOF mutants appears to be comparable if not stronger than Pten mutants (Pten^MGH6, a hypomorphic allele) or Akt overexpression (ppk-Gal4>Akt) (N = 46, 17, 13, 16, 14 lesioned segments; **P < 0.01, ***P < 0.001, Fisher’s exact test in c, P = 0.0027, P = 0.0002, P = 0.0001, P < 0.0001; N = 29, 9, 7, 8, 7 larvae; *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA followed by Holm-Sidak’s multiple comparisons test and Dunn’s multiple comparisons test, respectively, in d). (e–g) Rac1DN overexpression in class IV da neurons does not affect axon regeneration in the periphery (N = 27, 20 neurons; P > 0.05, Fisher’s exact test in e, P = 0.7365, and two-tailed unpaired Student’s t-test in f and g, P = 0.3533, P = 0.6708).

Supplementary Figure 5. The proposed model of the Rtca-Archease-Xbp1 pathway in the regulation of regeneration. Axon damage results in some type of cellular stress and the repair/splicing of mRNA substrates, such as Xbp1. The 3′-phosphate of the spliced RNA is converted into 2′,3′-cyclic phosphate by Rtca, whereas the RNA ligase Rtcb reverses this reaction and re-ligates the processed RNA, with the help of its catalyst Archease. Thus Rtca and Archease may have opposing functions on RNA repair/splicing and on regeneration.

Supplementary Figure 6. dArchease^PBc01013 mutants show altered mRNA splicing and reduced transcript expression, and is thus a loss of function allele. Arrows indicate the primer locations. Two pairs of primers were used and are color coded in green and turquoise. The PCR products are color coded in the same way according to the primer position. Blots were cropped and full-length images are presented in Supplementary Figure 14.

Supplementary Figure 7. Xbp1s overexpression increases class III da neuron axon regeneration in the periphery. Dashed circle marks the injury site and arrowheads show the regenerating axon (N = 19, 24 neurons; *P < 0.05, Fisher’s exact test, P = 0.0261 and two-tailed unpaired Student’s t-test, P = 0.0384). Scale bar = 20 μm.

Supplementary Figure 8. The Rtca mutant mouse. (a) A knockout-first line with a LacZ cassette inserted into the Rtca locus was used to generate the Rtca mutant mouse. The resulting mouse Rtca^LacZ_loxP (Rtca^Ins/Ins) has a lacZ cassette inserted in-between the 3^rd and 4^th exons of Rtca, meanwhile, the 4^th exon is flanked by loxP sites. (b) The lacZ insertion disrupts Rtca splicing and dramatically reduces transcript level, and thus results in a hypomorphic allele. Arrows indicate the primer locations. Two pairs of primers were used and are color coded in green and turquoise. The PCR products are color coded in the same way according to the primer position. Blots were cropped and full-length images are presented in Supplementary Figure 15. Quantification of Rtca mRNA shows about 81% reduction (N = 4 experiments; the expression level of each data point was normalized to the average value of WT; **P < 0.01, two-tailed unpaired Student’s t-test, P = 0.0035). (c) Western blot analysis using retina tissue was performed. Anti-Rtca HPA027982 recognizes amino acids 111–193. The antibody cleanly labels the Rtca protein in WT, which is ~39 KD, and shows a dramatic reduction of the protein after the insertion site in Rtca^Ins/Ins mutants, confirming the reduction of the Rtca full-length protein. The green line indicates the antigen region. Quantification of Rtca protein shows about 82% reduction (N = 3 experiments; the expression level of each data point was normalized to the average value of WT; *P < 0.05, two-tailed unpaired Student’s t-test, P = 0.0252).

Supplementary Figure 9. Rtca LOF during development does not affect adult RGC number or survival after injury. (a–c) Three weeks after optic nerve crush of sibling controls (Rtca^+/+ and Rtca^Ins/+) and Rtca^Ins/Ins mutant mice, retina sections from both the control eye (uninjured nerve) and injured eye (crushed nerve) were immunostained for Tuj1 (βIII tubulin) to label RGC and DAPI to label nuclei. Confocal imaging of retina sections from mutant animals, normalized to siblings, shows no differences in basal RGC number in the contralateral uninjured retinas in (a, N = 3 siblings and 3 Rtca^Ins/Ins mice) or in RGC survival three weeks after optic nerve crush (b, N = 4 siblings and 5 Rtca^Ins/Ins mice; P > 0.05, two-tailed unpaired Student’s t-test, P = 0.6776, P = 0.9249). Representative confocal images of the retina sections immunostained for Tuj1 and DAPI are shown in (c). Scale bar = 20 μm.

Supplementary Figure 10. Rtca is expressed in retinal ganglion cells. (a–f) Positive LacZ staining was observed in the RGC layer of Rtca^Ins/+ mice (a–c, arrow) but not in WT littermates (d–f). (g) β-Galactosidase staining in Rtca^Ins/+ mice shows distinct expression of the LacZ reporter within NeuN⁺ neurons in the retinal ganglion cell layer of the retina, which is absent in WT littermates, indicating that Rtca is expressed by RGCs. Scale bar = 20 μm.

Supplementary Figure 11. Rtca reduction enhances RGC axon regeneration after optic nerve crush. (a–d) Three weeks after optic nerve crush in 8–12 weeks old adult siblings (Rtca^+/+ and Rtca^Ins/+), and Rtca^Ins/Ins mutant mice, regenerating fibers were labeled by anti-NF200 immunostaining. (a, b) Tissue sections through the optic nerve of a Rtca^Ins/Ins mutant mice show regenerating axons up to 300 μm distal to the lesion site, whereas no obvious axon regrowth beyond the crush site is seen in similar sections from a sibling control (Rtca^Ins/+, asterisk marks the crush site). Boxed regions are shown at higher magnification. Curving, turning and looping of axons were observed, indicative of new axon growth (arrowheads). (c) The furthest distance that axons travelled beyond the injury site is about 3.5 times longer in the mutants compared to siblings. (d) Regenerating fibers were counted at specified distances from the lesion site. More fibers regenerate in Rtca^Ins/Ins mice compared to sibling controls (N = 7 siblings and 4 Rtca^Ins/Ins mice; ***P < 0.001, two-tailed unpaired Student’s t-test in c; **P < 0.01, two-way ANOVA in d, P = 0.0012). (e) The optic nerve crush site is marked by the presence of ED1 staining, which labels the infiltrating macrophages. Whereas axons (labeled by NF200) rarely grow through the ED1⁺ region in the sibling controls (Rtca^Ins/+), a large number of axons are seen hundreds of microns beyond the ED1⁺ region in Rtca^Ins/Ins mutants. Asterisks mark the crush site. Scale bar = 100 μm.

Supplementary Figure 12. Full length DNA agarose gel for the analysis of Xbp1 splicing. Full length DNA agarose gel (corresponding to Figure 6g).

Supplementary Figure 13. Full length DNA agarose gel for the analysis of dRtca expression. Full length DNA agarose gel (corresponding to Supplementary Figure 2a).

Supplementary Figure 14. Full length DNA agarose gel for the analysis of dArchease expression. Full length DNA agarose gel (corresponding to Supplementary Figure 6).

Supplementary Figure 15. Full length DNA agarose gel for the analysis of Rtca expression. Full length DNA agarose gel (corresponding to Supplementary Figure 8b).