We showed previously that wrapping glia induce L1-L4 neuronal differentiation in response to EGF from photoreceptors, but that L5 differentiation was regulated independently by an unknown mechanism (Fernandes et al., 2017). We speculated that another glial population may be involved in inducing L5 differentiation in response to EGF from photoreceptors. To test this hypothesis, we blocked EGF receptor (EGFR) signalling in all glia using a pan-glial driver to express a dominant negative form of EGFR (Repo>EGFR^DN). Although LPCs (Dac+ cells) still formed and assembled into columns, there was a complete block in lamina neuron differentiation as seen by the absence of the pan-neuronal marker, Embryonic lethal abnormal vision (Elav); that is, L5 differentiation was disrupted in addition to the differentiation of L1-L4 as expected (Figure 1C and D). Thus, EGFR activity in a glial population other than the wrapping glia is required for L5 neuronal differentiation.

Many glial types infiltrate the lamina (Figure 1—figure supplement 1A; Chotard and Salecker, 2007; Edwards et al., 2012). Therefore, we performed a screen using glia subtype-specific Gal4s to block EGFR signalling and determined what effect this manipulation had on L5s using Elav expression in the proximal lamina (Figure 1—figure supplement 1B-M; summarised in Supplementary file 1). Blocking EGFR signalling in the xg^O led to a dramatic reduction in the number of L5s (Figure 1E and F). To rule out early developmental defects, we used a temperature-sensitive Gal80 (Gal80^ts) and shifted animals from the permissive temperature to the restrictive temperature to limit EGFR^DN expression in xg^O to begin from the third larval instar, when lamina development initiates. This resulted in a similar loss of Elav positive cells in the proximal lamina as when EGFR^DN was expressed continuously in the xg^O, indicating that this phenotype is not due to an early defect in xg^O (Figure 1—figure supplement 1N). Xg^O are located below the lamina plexus, often with just one or two glial cells spanning the entire width of the lamina. While xg^O extend fine processes towards the lamina, they do not appear to contact LPCs or L5 neurons (Figure 1—figure supplement 1O). Importantly, blocking EGFR signalling in the xg^O did not affect xg^O numbers or morphology (Figure 1E and F, Figure 1—figure supplement 1O-R).

Since our screen used Elav expression in the proximal lamina to assess for the presence of L5s, we next examined lamina neuron-type markers to assess whether blocking EGFR activity in xg^O affected L5 neurons specifically. We used antibodies against Sloppy paired 2 (Slp2), Brain-specific homeobox (Bsh), and Seven-up (Svp) in combination to distinguish lamina neuron types: L2s and L3s express Slp2 alone, L1s co-express Svp and Slp2, L4s express Bsh alone, and L5s co-express Bsh and Slp2 (Figure 1G; Fernandes et al., 2017; Hasegawa et al., 2013; Tan et al., 2015). We found that the number of L5 neurons decreased specifically, while the number of all the other neuron types were unaffected (Figure 1G–I; p^L5 <0.0001, Mann-Whitney U-test). Finally, to test whether the absence of L5s simply reflected a developmental delay in differentiation, we examined adult optic lobes using a different L5 neuronal marker, POU domain motif 3 (Pdm3) (Tan et al., 2015). Similar to our results in the developing lamina, L5s were mostly absent in the adult lamina when EGFR was blocked in xg^O compared with controls (Figure 1—figure supplement 1S, T; N^exp = 10; N^ctrl = 11), indicating that the loss of L5s observed during development is not due to delayed induction. Thus, EGFR activity in xg^O is required for L5 neuronal differentiation.

The loss of L5 neurons when EGFR was blocked in xg^O could be explained either by a defect in neuronal differentiation or by an earlier defect in LPC formation or recruitment to columns. To distinguish between these possibilities, we counted the number of LPCs per column when EGFR signalling was blocked in xg^O compared to controls (Figure 2A–C). For these and later analyses we considered the youngest column located adjacent to the lamina furrow to be the first column, with column number (and age) increasing towards the posterior side (Figure 1A). In columns 1–4, there were no differences in the number of LPCs when EGFR was blocked in xg^O, indicating that LPC formation and column assembly occurred normally (Figure 2C), supporting the hypothesis that in response to EGFR activity, xg^O induce proximal LPCs to differentiate as L5s. Interestingly, the number of LPCs began to decrease in older columns (column 5 onwards) when EGFR signalling was blocked in xg^O (Figure 2C; *p<0.05, ***p<0.0002, Mann-Whitney U-test). This observation suggested that undifferentiated LPCs in older columns were being eliminated. We wondered whether LPCs that failed to differentiate into L5s underwent apoptosis, similar to the ‘extra’ LPCs that undergo apoptosis in controls. We used an antibody against the cleaved form of Death caspase-1 (Dcp-1), an effector caspase, to detect apoptotic cells (Akagawa et al., 2015) and, indeed, observed a significant increase in the number of Dcp-1 positive cells in the lamina when EGFR signalling was blocked in the xg^O (132.8 cells/unit volume±19.48 standard error of the mean) compared to controls (49.14 cells/unit volume±4.53) (Figure 2A–B and D, p<0.0005, Mann-Whitney U-test). Importantly, we observed Dcp-1 positive cells in the proximal row of the lamina (Figure 2B; N^exp = 20/20), which we never observed in controls (Figure 2A, N^ctrl = 19/19). Altogether these results showed that EGFR activity in xg^O induces the differentiation of L5 neurons, and proximal LPCs that fail to receive appropriate cues from xg^O die by apoptosis.

Since EGF from photoreceptors triggers EGFR activity in wrapping glia (Fernandes et al., 2017), we tested whether photoreceptor-derived EGF contributed to activating EGFR in xg^O also. Spi is initially produced as an inactive transmembrane precursor (mSpi) that needs to be cleaved into its active secreted form (sSpi) (Tsruya et al., 2002). This requires the intracellular trafficking protein Star and Rhomboid proteases (Tsruya et al., 2002; Urban et al., 2002; Yogev et al., 2008). We took advantage of a mutant for rhomboid 3 (rho3) in which photoreceptors are specified but cannot secrete EGF from their axons (Yogev et al., 2010), resulting in failure of L1-L4 neurons to differentiate along with a significant decrease in the number of L5s (Figure 3A and C; p^rho3 <0.0001; one-way ANOVA with Dunn’s multiple comparisons test) (Fernandes et al., 2017; Yogev et al., 2010). This result suggested that EGFR signalling in the xg^O could be activated by EGF secreted by photoreceptor axons. To test this hypothesis, we restored expression of wild-type Rho3 only in photoreceptors in rho3 mutant animals using a photoreceptor-specific driver (GMR-Gal4). Rho3 function in photoreceptors was sufficient to fully rescue not only L1-L4 neuronal differentiation, as previously reported (Yogev et al., 2010), but also L5 neuronal differentiation (Figure 3B and C; one-way ANOVA with Dunn’s multiple comparisons test). Since photoreceptor-derived EGF was insufficient to induce L5 neuronal differentiation when EGFR signalling was blocked in xg^O (Figure 1F and H), together these results suggest that xg^O likely respond to EGF from photoreceptors and relay these signals to induce differentiation of proximal LPCs into L5 neurons.

We next asked what signal(s) the xg^O secrete to induce L5 differentiation. Previously, we showed that MAPK signalling is necessary and sufficient for neuronal differentiation in the lamina (Fernandes et al., 2017). Therefore, we reasoned that xg^O-derived differentiation signal(s) must activate MAPK signalling through a receptor tyrosine kinase (RTK) in the proximal lamina. Indeed, blocking EGFR signalling in xg^O led to reduced levels of double phosphorylated MAPK (dpMAPK) specifically in the proximal lamina (Figure 3—figure supplement 1A, B). The Drosophila genome encodes 22 ligands which activate 10 RTKs upstream of MAPK signalling (Sopko and Perrimon, 2013). To identify the signal(s) secreted by xg^O, we misexpressed candidate ligands and screened for their ability to rescue the loss of L5s caused by blocking EGFR activity in the xg^O. To validate this approach, we tested whether autonomously restoring transcriptional activity downstream of MAPK in xg^O while blocking EGFR activity could rescue L5 differentiation. While blocking EGFR in xg^O resulted in laminas containing 0.063±0.014 L5s per column, co-expressing PntP1 with EGFR^DN in xg^O rescued the number of L5s per column to 0.213±0.025 (Figure 3F, Figure 3—figure supplement 1C ****p<0.0001 compared to EGFR^DN alone). We then screened 18 RTK ligands based on available reagents (Figure 3—figure supplement 1C). Four ligands, Spi, Branchless (Bnl), Thisbe (Ths), and Col4a1, produced statistically significant rescues when compared with the xg^O>EGFR^DN + CD8::GFP (Gal4 titration control) (Figure 3—figure supplement 1C; *p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001 one-way ANOVA with Dunn’s multiple comparisons test). To eliminate false positive hits, we determined whether these ligands were expressed in xg^O under physiological conditions. Using a previously validated bnl-Gal4 (Chen and Krasnow, 2014; Kamimura et al., 2006; Spéder and Brand, 2014; Tamamouna et al., 2021), we drove CD8::GFP expression and found that it was expressed in all cells of the optic lobe (Figure 3—figure supplement 1D), making it unlikely to be a viable hit. We found that a previously validated ths-Gal4 (Anllo and DiNardo, 2022; Wu et al., 2017) drove CD8::GFP expression in photoreceptors but not xg^O (Figure 3—figure supplement 1E) consistent with previous reports (Franzdóttir et al., 2009). However, when we examined Col4a1 expression using a previously validated Gal4 enhancer trap (Hennig et al., 2006), we found that it drove CD8::GFP expression in xg^O (Figure 3—figure supplement 1F). We also found that a spi-Gal4 (NP0289-Gal4; not previously validated) drove CD8::GFP expression in xg^O, but not photoreceptors or other cell types where spi is also known to be expressed, suggesting that this Gal4 line may report spi expression partially (Figure 3—figure supplement 1G). To further substantiate these results we performed fluorescence in situ hybridisation chain reaction (HCR), a form of fluorescent in situ hybridisation (Choi et al., 2018; Choi et al., 2016; Duckhorn et al., 2022), and confirmed that spi and Col4a1 mRNAs were present in the xg^O under physiological conditions (Figure 3—figure supplement 1H and I; see Materials and methods). This enabled us to narrow down our hits to two ligands: the EGF Spi and Col4a1, a type IV Collagen, which both rescued L5 differentiation resulting in laminas with 0.147±0.024 and 0.17±0.0197 L5s per column, respectively (Figure 3F–K and P p^spi-wt <0.01 and p^Col4a1 <0.0005, one-way ANOVA with Dunn’s multiple comparisons test; Figure 3—figure supplement 1C). Note that expressing either sSpi or wild-type (unprocessed) mSpi (referred to as Spi^wt) in xg^O rescued L5 numbers (Figure 3—figure supplement 1C), indicating that xg^O are capable of processing mSpi into the active form (sSpi).

We ruled out the trivial explanation that the rescue of L5 numbers by Spi was caused by autocrine EGFR reactivation in the xg^O, as Spi expression in xg^O did not autonomously rescue dpMAPK nuclear localisation in xg^O when EGFR signalling was blocked (Figure 3—figure supplement 1A, B, J, K). We then tested whether xg^O express spi and Col4a1 downstream of EGFR activity. We measured spi and Col4a1 transcript levels using in situ HCR in controls and when we blocked EGFR signalling in xg^O. Disrupting EGFR signalling in xg^O resulted in a significantly reduced fluorescence signal for spi and Col4a1 transcripts in xg^O compared with controls (Figure 3—figure supplement 1H and I, 1L-O; p^spi <0.01, p^Col4a1<0.005; Mann-Whitney U-test). Thus, xg^O express spi and Col4a1 in response to EGFR activity.

Col4a1 is thought to activate MAPK signalling through its putative receptor, the Discoidin domain receptor (Ddr) (Sopko and Perrimon, 2013). We used a Gal4 enhancer trap in the Ddr locus (not previously validated) to drive CD8::GFP expression and observed that GFP was expressed in all LPCs (Figure 3—figure supplement 1P). We confirmed these results using in situ HCR, which also detected Ddr expression throughout the lamina (Figure 3—figure supplement 1Q). Spi activates EGFR (Sopko and Perrimon, 2013), which was shown to be expressed in LPCs previously (Huang et al., 1998). Thus, LPCs express the RTKs that make them competent to respond to the EGF Spi and Col4a1 produced by xg^O. Moreover, expressing spi or Col4a1 in xg^O in which EGFR signalling was blocked rescued dpMAPK signal in L5s, indicating that, when expressed in xg^O, these ligands were sufficient to activate MAPK signalling in the proximal lamina (Figure 3—figure supplement 1R-T; **p<0.005, ****p<0.0001; one-way ANOVA with Dunn’s multiple comparisons test). Co-expressing Spi and Col4a1 in the xg^O>EGFR^DN background led to an enhanced and statistically significant rescue relative to individual ligand rescues alone, resulting in laminas with 0.267±0.025 L5s per column (Figure 3L–P; p<0.0001, Mann-Whitney U-test). We also tested whether these ligands could induce ectopic L5 differentiation when overexpressed in the xg^O. Overexpressing either Spi or Col4a1 resulted in a 19%±1.8 (p<0.05) and a 24%±4 (p<0.005) increase in the number of L5s per column relative to controls, respectively (Figure 3Q–S). Thus, Spi and Col4a1 from xg^O are sufficient to induce L5 differentiation.

Next, to test whether xg^O-derived Spi and Col4a1 are normally required to induce L5 neuronal differentiation, we disrupted their expression specifically in xg^O. We used RNA interference (RNAi) to knock down spi and Col4a1 expression both individually and simultaneously in xg^O using previously validated lines (Chen et al., 2016; Csordás et al., 2020; Louradour et al., 2017; Morante et al., 2013; Pastor-Pareja and Xu, 2011). While knocking down spi led to a mild decrease in L5 numbers, which was not statistically significant, knocking down Col4a1 in the xg^O led to a statistically significant decrease in L5s (0.78±0.03 L5s per column) relative to controls (0.92±0.02 L5s per column) (Figure 3T, U and W; *p<0.05 one-way ANOVA with Dunn’s multiple comparisons test). However, knocking down both spi and Col4a1 simultaneously in xg^O led to a strong decrease in L5s (0.61±0.02 L5s per column; Figure 3V–W; ****p<0.0001, one-way ANOVA with Dunn’s multiple comparisons test). Under these conditions we also observed Dcp-1 positive apoptotic cells in the most proximal row of the lamina, which were never observed in controls (Figure 3—figure supplement 2) but were observed when L5 differentiation was blocked above (Figure 2B). Thus, xg^O-derived Spi and Col4a1 are both necessary and sufficient to induce L5 differentiation. Altogether, we found that xg^O secrete multiple factors that lead to activation of the MAPK cascade in the proximal lamina to induce differentiation of L5s.

We recently showed that a gradient of Hh signalling activity in lamina columns specifies L1-L5 identities such that high levels specify L2 and L3 (distal cell) identities, intermediate levels specify L1 and L4 (intermediate cell) identities, and low levels specify L5 (proximal cell) identity (Bostock et al., 2022). Since overexpressing spi and Col4a1 in the xg^O resulted in ectopic L5 neurons, we wondered what the source of these ectopic cells was. We quantified other lamina neuron types when either spi or Col4a1 was overexpressed in xg^O and found no decrease in the number of L1-L3s (Slp2-only expressing cells) or L4s (Bsh-only expressing cells) per column compared to controls (Figure 3S). Thus, ectopic L5s were not produced at the expense of other lamina neuron types. In wild-type optic lobes, each lamina column contains an ‘extra’ LPC, which is located immediately distal to the LPC fated to differentiate as an L5. These ‘extra’ LPCs do not differentiate but instead undergo apoptosis and are eliminated (Figures 2A and 4A). We hypothesised that though fated to die, ‘extra’ LPCs are specified with L5 identity through low Hh signalling activity in the proximal lamina, and that the overexpression of Spi and Col4a1 in xg^O generated ectopic L5s by inducing differentiation and survival of the ‘extra’ LPCs. To test this hypothesis, we forced neuronal differentiation throughout the lamina by expressing an activated form of MAPK (MAPK^ACT) (Figure 4—figure supplement 1A-D) or by overexpressing the MAPK transcriptional effector, Pointed P1 (PntP1), in the lamina (Figure 4A–D) . As reported previously, hyperactivating MAPK signalling in the lamina led to premature neuronal differentiation: instead of sequential differentiation of L1-L4, seen as a triangular front, most lamina columns differentiated simultaneously ( (Figure 4) , Figure 4—figure supplement 1A, C; Fernandes et al., 2017). We observed no LPCs that remained undifferentiated (Dac+ and Elav-) past lamina column 5, including the row of cells that normally correspond to the ‘extra’ LPCs (Figure 4C, Figure 4—figure supplement 1A, C). Importantly, we also observed a concomitant decrease in cleaved Dcp-1 positive cells (Figure 4C and E ; p<0.0001, Mann-Whitney U-test), suggesting that forcing the ‘extra’ LPCs to differentiate blocked their death.

Next, we examined the distribution of lamina neuron types when we forced neuronal differentiation. We often observed two rows of cells co-expressing Slp2 and Bsh in the proximal lamina (Figure 4B and D, Figure 4—figure supplement 1B, D), indicating the presence of ectopic L5s. To distinguish between premature and ectopic differentiation, we quantified the number of lamina neuron types (L1-L3, L4, and L5) per column in older columns (column 7 onwards, once mature columns were observed in controls, Figure 4—figure supplement 1E). While there was no significant difference between the average number of L1-L3s or L4s per column, the average number of L5s per column was ~1.4-fold higher in laminas in which differentiation was ectopically induced compared with controls, that is, they contained 1.4±0.08 L5s per column compared to 1.00±0.05 L5s per column in controls (Figure 4B, D and F–G; p<0.0001, Mann-Whitney U-test). Thus, hyperactivating MAPK signalling in the lamina drove ectopic differentiation of L5 neurons. Importantly, ectopic L5s were only observed in the proximal but never in the distal lamina (Figure 4D, N=18/18; Figure 4—figure supplement 1D, N=9/9). Taken together, the absence of cell death in the row distal to L5s and the presence of ectopic L5s in this row indicate that hyperactivating MAPK signalling induces the ‘extra’ LPCs to differentiate into L5s. Thus, the ‘extra’ LPCs are specified as L5s though fated to die normally. These data are consistent with our work showing that lamina precursors are specified by Hh signalling prior to differentiation and that the most proximal cells, which experience the lowest levels of Hh pathway activity and are specified as L5s (Bostock et al., 2022). Importantly, the presence of ectopic L5s when differentiation is induced demonstrates that more LPCs are specified as L5s than differentiate normally.

If the two most proximal cells in each lamina column are both specified as L5s, how then is L5 differentiation limited to only the most proximal row in response to diffusible signals secreted by xg^O? We tested whether the ‘extra’ LPCs differentiated as L5s when apoptosis was blocked in animals mutant for Death regulator Nedd2-like caspase (Dronc), an initiator caspase essential for caspase-dependent cell death (Fuchs and Steller, 2011). Cleaved Dcp-1 was absent in homozygous Dronc^I24 animals confirming that apoptosis was blocked (Figure 5A; N=26/26; with full penetrance). Indeed, we detected cells that were positive for the lamina marker Dachshund (Dac) but negative for the pan-neuronal marker Elav between L1-L4 and L5 neurons past column 5, which were never observed in controls (Figure 5A compared to Figure 4A; N=13/13; with full penetrance). These cells did not express lamina neuron-type markers Slp2 or Bsh, which L5s co-express and which individually label L1-L3s and L4s, respectively (Figure 5B and C). Thus, although the ‘extra’ LPCs were retained when apoptosis was blocked, they did not differentiate into neurons.

We observed ectopic L5s only when all LPCs were forced to differentiate, bypassing the need for differentiation signals from glia, but not when apoptosis was blocked (Figure 4, Figure 4—figure supplement 1A-D, and Figure 5A–C). This suggests that the ‘extra’ LPCs, though specified as L5s, did not receive differentiation signals from xg^O in Dronc mutants or in the wild-type, where failing to differentiate they were eliminated by apoptosis. How are only half of the LPCs specified as L5s chosen to differentiate in an invariant manner? The most proximal row of LPCs fated to differentiate into L5s is the row nearest to xg^O, and therefore, the first to receive differentiation signals. We speculated that newly induced L5s may then limit the ability of more distal LPCs to differentiate, by preventing MAPK activation in neighbouring cells. Aos is a transcriptional target of MAPK signalling and a secreted antagonist of the EGF Spi (Freeman et al., 1992; Golembo et al., 1996). We wondered if newly induced L5s secrete Aos to limit differentiation signals from xg^O. To test this hypothesis, we examined argos (aos) expression with an enhancer trap in the aos locus, aos^W11. aos-lacZ (aos^W11/+) was expressed in xg^O and differentiating lamina neurons, with the highest levels detected in L5s (Figure 5D–E). Interestingly, we also noted ectopic L5s in the laminas of aos^W11 heterozygotes, which could be the result of decreased Aos expression, as aos^W11 is a hypomorphic loss-of-function allele (Figure 5E). These observations suggested that Aos could act in L5s as a feedback-induced sink for Spi to limit further differentiation in columns. To test this hypothesis, we knocked down aos by RNAi using a driver expressed specifically in developing L5s (Jenett et al., 2012; Figure 5G). We observed a statistically significant ~1.2-fold increase in the number of L5s relative to controls, that is, 0.99±0.02 L5s per column compared to 0.83±0.01 L5s per column in controls (Figure 5F–H; p<0.0005, Mann-Whitney U-test). Altogether, our data indicate a model in which xg^O induce MAPK activity in the most proximal LPCs, resulting in their differentiation and in the production of the feedback inhibitor Aos. In turn, Aos limits further differentiation in the column by fine-tuning the availability of the differentiation signal Spi, which ensures that only one L5 differentiates per column, and determines the final number of neurons in each lamina column.

We have shown that in addition to the wrapping glia (Fernandes et al., 2017), another population of glia, the xg^O, also receive and relay signals from photoreceptors to induce neuronal differentiation in the lamina (Figure 1E–F). This is the first functional role ascribed to xg^O. Remarkably, xg^O are born from central brain DL1 type II neuroblasts and migrate into the optic lobes to positions below the developing lamina (Ren et al., 2018; Viktorin et al., 2013). This underscores an extraordinary degree of coordination and interdependence between the compound eye, optic lobe, and central brain. Photoreceptor signals drive wrapping glial morphogenesis and infiltration into the lamina (Franzdóttir et al., 2009), thus setting the pace of L1-L4 neuronal differentiation (Fernandes et al., 2017). Defining the signals that enable xg^O to navigate the central brain and optic lobe will be a critical contribution to our understanding of how development is coordinated across brain regions.

In both vertebrate and invertebrate developing nervous systems, programmed cell death is thought to come in two broad flavours: first as an intrinsically programmed fate whereby specific lineages or identifiable progenitors, neurons, or glia undergo stereotyped clearance (Hidalgo and ffrench-Constant, 2003; Miguel-Aliaga and Thor, 2009; Pinto-Teixeira et al., 2016; Yamaguchi and Miura, 2015) and second as an extrinsically controlled outcome of competition among neurons for limited target-derived trophic factors, which adjust overall cell numbers through stochastic clearance (also known as the neurotrophic theory) (Davies, 2003; Hidalgo and ffrench-Constant, 2003; Miguel-Aliaga and Thor, 2009; Yamaguchi and Miura, 2015). In the lamina, although the LPCs eliminated by programmed cell death are identifiable and the process stereotyped, it does not appear to be linked to an intrinsic programme. Rather, the predictable and stereotyped nature of apoptosis and differentiation are a consequence of stereotyped responses to extrinsic signalling determined by the architecture of the tissue. Thus, our work highlights that stereotyped patterns of apoptosis can arise from extrinsic signalling, suggesting a new mode to reliably pattern development of the nervous system.

In many contexts, neurotrophic factors promote cell survival by activating MAPK signalling (Ballif and Blenis, 2001; Park and Poo, 2013). In the lamina, MAPK-induced neuronal differentiation and cell survival appear intimately linked. LPCs that do not activate MAPK signalling sufficiently do not differentiate and are eliminated by apoptosis, likely through regulation of the proapoptotic factor Head involution defective, which has been described extensively in flies (Bergmann et al., 2002; Bergmann et al., 1998; Kurada and White, 1998). Thus, here the xg^O-secreted ligands Spi and Col4a1, which activate MAPK, appear to be functioning as differentiation signals as well as trophic factors. Col4a1, in particular, may perform dual roles by promoting MAPK activity directly through its receptor Ddr, and perhaps also by limiting Spi diffusivity to aid in localising MAPK activation.

It will be interesting to determine whether the processes described here represent conserved strategies for regulating neuronal number. Certainly, given the diversity of cell types and structural complexity of vertebrate nervous systems, exploiting tissue architecture would appear to be an effective and elegant strategy to regulate cell numbers reliably and precisely.

Drosophila melanogaster strains and crosses were reared on standard cornmeal medium and raised at 25°C or 29°C or shifted from 18°C to 29°C for genotypes with temperature-sensitive Gal80, as indicated in Supplementary file 2.

We used the following mutant and transgenic flies in combination or recombined in this study (see Supporting File 2 for more details; {} enclose individual genotypes, separated by commas).

{y,w,hsflp¹²²; sp/Cyo; TM2/TM6B}, {y,w; sp/Cyo, Bacc-GFP; Dr/TM6C}, (from BDSC: 36349).

{ey-Gal80; sp/Cyo;} (BDSC: 35822), {;Gal80^ts; TM2/TM6B} (BDSC: 7108), {w¹¹¹⁸;; R27G05-Gal4} (BDSC: 48073), {w¹¹¹⁸;;25A01-Gal4} (BDSC: 49102), {y,w; R64B07-Gal4;} (larval L5-Gal4), {y,w; hh-Gal4/TM3} (BDSC: 67493), {;tub-Gal80^ts; repo-Gal4/TM6B}, {w¹¹¹⁸;GMR-Gal4/Cyo;} (BDSC: 9146), {y,w;Pin/Cyo;repo-QF/TM6B} (BDSC: 66477), {y,w; NP6293-Gal4/Cyo,UAS-lacZ;} (perineurial glia; Kyoto Stock Center: 105188), {w; NP2276-Gal4/Cyo; } (subperineurial glia; Kyoto Stock Center: 112853), {w¹¹¹⁸;; R54H02-Gal4} (cortex glia; BDSC: 45784), {w¹¹¹⁸;; R10C12-Gal4} (epithelial and marginal glia; BDSC: 47841), {w;Mz97-Gal4, UAS-Stinger/Cyo;} (wrapping and xg^O; BDSC: 9488), {w¹¹¹⁸;; R53H12-Gal4} (chiasm glia; BDSC: 50456), {y,w; spi^NP0289-Gal4/Cyo, UAS-lacZ;} (Kyoto Stock Center: 112128), {w¹¹¹⁸; Cg-Gal4;} (BDSC: 7011), {w;; bnl^NP2211-Gal4} (Kyoto Stock Center: 112825), {w; ths^MI07139-Gal4/Cyo; MKRS/TM6B} (BDSC: 77475), {;;rho3^PLLb, UAS-CD8::GFP/TM6B}, {;UAS-rho3-3xHA;} (gifts from B Shilo), {;;aos^w11/TM6B} (aos-lacZ; BDSC: 2513), {y,w; sp/Cyo, Bacc-GFP; 10xQUAS-6xmCherry-HA} (BDSC: 52270), {y,w;;10xUAS-myrGFP} (BDSC: 32197), {;UAS-CD8::GFP;}, {;;UAS-CD8::GFP} (gifts from C Desplan), {y,w;;UAS-nls.lacZ}, (BDSC: 3956), {y,w; UAS-LifeAct-GFP/Cyo;} (BDSC: 35544), {w¹¹¹⁸;UAS-Dcr-2;} (BDSC: 24650), {w¹¹¹⁸;;UAS-Dcr-2} (BDSC: 24651), {;UAS-EGFR^DN; UAS-EGFR^DN} (BDSC: 5364), {;UAS-aop^ACT;} (Kyoto Stock Center: 108425), {y,w;UAS-rl^sem;} (rl^sem = MAPK^ACT; BDSC: 59006), {w¹¹¹⁸;;UAS-PntP1} (BDSC: 869), {w¹¹¹⁸;UAS-aos^RNAi;} (VDRC47181), {w;UAS-jeb;} (a gift from A Gould), {y,w, UAS-Col4a1^EY11094/(Cyo);} (BDSC: 20661), {;;UAS-Cg25c-RFP} (Zang et al., 2015) (Col4a1=Cg25c), {;UAS-Wnt5;} (BDSC: 64298), {;;UAS-s.spi} (a gift from B Shilo), {;UAS-m.spi::GFP-myc;} (a gift from B Shilo), {;;UAS-m.spi::GFP-myc} (a gift from B Shilo), {w, UAS-grk.sec/Cyo;} (BDSC: 58417), {;UAS-vn^EPgy/Cyo;} (BDSC: 58498), {;;UAS-krn-3xHA} (FlyORF: F002754), {;UAS-bnl/Cyo; MKRS/TM6C} (BDSC: 64232), {;UAS-Ilp1;}, {;UAS-Ilp6;} (gifts from P Leopold), {w¹¹¹⁸, UAS-Pvf1^XP;;} (BDSC: 19632), {w¹¹¹⁸; UAS-Pvf2^XP;} (BDSC: 19631), {;UAS-Wnt4^EPgy2/Cyo;} (BDSC: 20162), {;;UAS-boss-3xHA} (FlyORF: F001365), {y,sev; SAM.dCas9.Trk;} (BDSC: 81322), {y,sev; SAM.dCas9.Pvf3;} (BDSC: 81346), {y,sev; SAM.dCas9.ths;} (BDSC: 81347), {y,sev; SAM.dCas9.pyr;} (BDSC: 81330), {w¹¹¹⁸; Ddr^CR01018-Gal4;} (BDSC: 81157).

We dissected eye-optic lobe complexes from early pupae (0–5 hr after puparium formation) in ×1 phosphate-buffered saline (PBS), fixed in 4% formaldehyde for 20 min, blocked in 5% normal donkey serum, and incubated in primary antibodies diluted in block for two nights at 4°C. Samples were then washed in ×1 PBS with 0.5% Triton-X (PBSTx), incubated in secondary antibodies diluted in block, washed in PBSTx and mounted in SlowFade (Life Technologies).

When performing phospho-MAPK stains, dissections were performed in a phosphatase inhibitor buffer as detailed in Amoyel et al., 2016.

We used the following primary antibodies in this study: mouse anti-Dac^2-3 (1:20, Developmental Studies Hybridoma Bank [DSHB]), mouse anti-Repo (1:20, DSHB), rat anti-Elav (1:100, DSHB), mouse anti-Elav (1:20, DSHB), rabbit anti-Dcp-1 (1:100; Cell Signalling #9578), chicken anti-GFP (1:400; EMD Millipore), mouse anti-Svp (1:50, DSHB), rabbit anti-Slp2 (1:100; a gift from C Desplan), rabbit-Bsh (1:500; a gift from C Desplan), Rat anti-Pdm3 (1:1000; a gift from C Desplan), guinea pig anti-Brp (1:100; a gift from C Desplan), rabbit anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (1:100, Cell Signaling #9101), chicken anti-RFP (1:500; Rockland #600-901-379s), mouse anti-β-galactosidase (1:500; Promega #Z3781), chicken anti-β-galactosidase (1:500; abcam #9361), rabbit-anti-GFP (1:500; Thermo Fisher Scientific #A6455), AlexaFluor405 conjugated Goat Anti-HRP (1:100; Jackson Immunolabs), AlexaFluor405-, Cy3-, or AlexaFluor647-conjugated Goat Anti-HRP (1:200; Jackson Immunolabs). Secondary antibodies were obtained from Jackson Immunolabs or Invitrogen and used at 1:800. Images were acquired using Zeiss 800 and 880 confocal microscopes with ×40 objectives.

To determine if spi, Col4a1, and Ddr transcripts were present in the xg^O, we performed HCR as detailed in Duckhorn et al., 2022. We designed 20–21 probe pairs against target genes, excluding regions of strong similarity to other transcripts, with corresponding initiator sequences for amplifiers B3 (Choi et al., 2018). HCR probes (sequences included as source data; see Figure 3—source data 1) were purchased as DNA Oligos from Thermo Fisher Scientific (100 µm in water and frozen).

Eye-optic lobe complexes were dissected, fixed, and washed as detailed above. Samples were incubated in Probe Hybridisation Buffer for 30 min at 37°C followed by incubation with probes (0.01 µM) at 37°C overnight. The samples were then washed four times for 15 min each with probe wash buffer at 37°C followed by two washes for 5 min each with ×5 saline sodium citrate solution (20XSSCT solution in distilled water – 58.44 g/mol sodium chloride, 294.10 g/mol 560 sodium citrate, pH adjusted to 7 with 14 N hydrochloric acid, with 0.001% Tween 20) at room temperature. Samples were then incubated with amplification buffer for 10 min at room temperature. 12 pmol of hairpins H1 and H2 were snap-cooled (95°C for 90 s and then cooled to room temperature for 20 min) separately to avoid oligomerisation. The snap-cooled hairpins were then added to the samples in the amplification buffer (protected from light) and incubated overnight at room temperature. The samples were then washed with 5XSSCT for 15 min before being incubated in darkness with 1:15 dilution of DAPI (Sigma D9542) for 90 min. Samples were washed with ×1 PBS for 30 min and then mounted as detailed above.

We used Fiji-ImageJ (Schindelin et al., 2012) or Imaris (version x64-9.5.1) to process and quantify confocal images as described below. We used Adobe Photoshop and Adobe Illustrator software to prepare figures. We used GraphPad Prism 8 to perform statistical tests. In all graphs, whiskers indicate the standard error of the mean (SEM).

We used the surfaces tool in Imaris to manually select the lamina region (based on Dac expression). We then used the spots tool to identify Dcp-1 positive cells (cell diameter = 5 μm) within the selected region using the default thresholding settings, and plotted these values normalised to the volume of the selected lamina region in GraphPad Prism 8.

We quantified the number of L-neuron types per column using Elav, Bsh, and Slp2. We quantified 30 optical slices (step size = 1 μm) located centrally in the lamina. Column number was identified by counting HRP-labelled photoreceptor axon bundles.

In Fiji-ImageJ we used the free hand selection tool to draw a region of interest (ROI) around the xg^O (marked by the xg^O>CD8::GFP). We then measured the mean fluorescence intensity (MFI) of spi and Col4a1 transcripts labelled by HCR within each ROI. We quantified 30 optical slices (step size = 1 μm) located centrally in the lamina and then plotted the average for each optic lobe.

We quantified the number of xg^O (Figure 1—figure supplement 1Q) by manually counting the number of Repo positive nuclei within LifeAct-GFP positive xg^O per 40 μm optical section in Fiji-ImageJ. We used a step size of 1 μm while acquiring the z-stacks and centred each 40 μm optical section in the middle of the lamina using photoreceptor axons (HRP), and the lobula plug (Dac expression) as landmarks. Quantifications were performed blind.

We quantified the lengths of the fine glial processes that extend distally from the xg^O towards the lamina plexus (Figure 1—figure supplement 1O,P,R) by using the straight-line selection and measuring tools in Fiji-ImageJ to measure xg^O process lengths in a 10 μm optical section centred in the middle of the lamina. Quantifications were performed blind.

Using Fiji we manually drew ROIs with the free hand selection tool around the xg^O nucleus (based on Repo) and LPCs in the most proximal row of the lamina (based on Dac expression) and added these to the ROI manager. We then enlarged the ROIs (Edit > Selection > Enlarge) by 3.00 pixel units to include the cytoplasm. We then used the XOR function in the ROI Manager to only select the cytoplasm of the xg^O. We then measured the MFI of dpMAPK in the nucleus and the cytoplasm of the xg^O in 20 centrally located optical slices (corresponding to 20 μm) for each optic lobe. We plotted the nuclear:cytoplasmic ratios of dpMAPK MFI in GraphPad Prism 8.

Using Fiji we manually drew regions of interest around L5s (based on Slp2+Bsh co-expression) in each column. We then measured the MFI of β-Galactosidase in the ROIs. We quantified 10 optical slices (step size = 1 μm) for each optic lobe and plotted the average values as a function of column (age).