Drosophila ClC‐a is required in glia of the stem cell niche for proper neurogenesis and wiring of neural circuits

Abstract Glial cells form part of the neural stem cell niche and express a wide variety of ion channels; however, the contribution of these channels to nervous system development is poorly understood. We explored the function of the Drosophila ClC‐a chloride channel, since its mammalian ortholog CLCN2 is expressed in glial cells, and defective channel function results in leukodystrophies, which in humans are accompanied by cognitive impairment. We found that ClC‐a was expressed in the niche in cortex glia, which are closely associated with neurogenic tissues. Characterization of loss‐of‐function ClC‐a mutants revealed that these animals had smaller brains and widespread wiring defects. We showed that ClC‐a is required in cortex glia for neurogenesis in neuroepithelia and neuroblasts, and identified defects in a neuroblast lineage that generates guidepost glial cells essential for photoreceptor axon guidance. We propose that glia‐mediated ionic homeostasis could nonautonomously affect neurogenesis, and consequently, the correct assembly of neural circuits.

Among the ion channels expressed in glia, the vertebrate plasmamembrane ClC-2 chloride channel has been proposed as one of the channels involved in K + buffering, a key ionic homeostasis process in which glia are involved (Jentsch & Pusch, 2018;H. Wang et al., 2017). In the mature nervous system, increased neural activity leads to an increase in extracellular K + , which can alter neuronal excitability. To lower the concentration of K + , astrocytes take up the ion and distribute it to distant sites via the astrocytic syncytia. Uptake of K + occurs concomitantly with uptake of Cl − and water, producing transient astrocyte swelling (Bellot-Saez, Kékesi, Morley, & Buskila, 2017). Based on its expression in astrocytic glia, the ClC-2 channel has been proposed as one of the channels that might participate in this Cl − uptake (Blanz et al., 2007;Hoegg-Beiler et al., 2014;Sirisi et al., 2017). Mutations in CLCN2, which codes for ClC-2, are responsible for leukoencephalopathy with ataxia (LKPAT) (Depienne et al., 2013) and ClC-2 has been related to megalencephalic leukoencephalopathy with subcortical cysts (MLC) (Hoegg-Beiler et al., 2014;Jeworutzki et al., 2012;Sirisi et al., 2017). Both conditions are characterized by vacuolization of white matter and edema, most probably as a consequence of impaired K + buffering, but patients can also present learning disabilities and mild to moderate intellectual impairment. The finding that ClC-2 is expressed during development in glial precursors and is required for their differentiation (Hou et al., 2018), together with the fact that intellectual impairment can arise from connectivity defects, suggests that this channel may have additional functions during neural development. To explore this possibility, we leveraged the functional parallelisms between vertebrate and Drosophila glia (Chotard & Salecker, 2004;Corty & Freeman, 2013;Freeman & Doherty, 2006) and used the fly, where neurogenesis has been extensively studied, the niche is simpler than in vertebrates, and the ClC-a gene codes for the fly homolog of the vertebrate ClC-2 chloride channel.
The fly central nervous system contains three structures: the central brain (CB), the ventral nerve cord (VNC), and the optic lobe (OL).
The CB and VNC are generated by neural stem cells called neuroblasts that delaminate from the neuroectoderm during embryonic development and give rise to larval and adult brain through two rounds of neurogenesis (Doe, 2008). The OL originates from a group of neuroepithelial cells that proliferates and separates into two crescent shaped primordia, the outer and inner proliferation centers (OPC and IPC), which produce neuroblasts and precursor cells of the different visual processing centers (Apitz & Salecker, 2014). In addition, the OL has been extensively used as a model to explore neural circuit assembly (Plazaola-Sasieta, Fernández-Pineda, Zhu, & Morey, 2017), primarily because the modular nature and stereotyped development of the fly eye enable easy detection of wiring defects in photoreceptors and other visual system neurons.
The electrophysiological properties of Drosophila ClC-a are very similar to those of its mammalian counterpart (Flores, Niemeyer, Sepúlveda, & Cid, 2009;Jeworutzki et al., 2012). In addition, both ClC-2 and ClC-a are most abundant in epithelia and the brain. ClC-2 has been shown to play a role in transepithelial transport in enterocytes (Catalán, Niemeyer, Cid, & Sepúlveda, 2004). Similarly, ClC-a is also expressed in the epithelia of the fly digestive system, and is involved in transepithelial transport in stellate cells of the Malpighian tubules, the fly secretory system (Cabrero et al., 2014;Denholm et al., 2013).
In this study, we analyzed the expression pattern of Drosophila ClC-a in the brain, characterized the first loss-of-function mutant alleles of this chloride channel and investigated their effects on development of the nervous system. We found that ClC-a is expressed in several types of glia and uncovered a role for this channel in the niche. Its expression in cortex glia, which are in close contact with OPC and IPC neuroepithelial cells and neuroblasts, was necessary for the proper neurogenesis in these cell types, as well as for neuron survival. One of the secondary consequences of reduced neurogenesis was the significantly limited production of guidepost glial cells, which gave rise to nonautonomous neural circuit assembly phenotypes in photoreceptors. Both neurogenic and connectivity defects could be rescued by glial-specific expression of the rat ClC-2 vertebrate channel. We propose that the expression of ion channels in the glial niche can shape the development of the nervous system, controlling the number of glia and neurons generated, as well as the connectivity of the latter.

Genetics
Flies were grown in standard medium at 25 C except for RNAi experiments, which were performed at 29 C. All genotypes analyzed are specified in the Supplementary Information. To label membranes and nucleus, we used UAS-mCD8-GFP (BDSC 5137), UAS-mCD8-RFP.LG (BDSC 27398), UAS-mCD8GFP, lexAop-CD2-RFP (BDSC 67093), UAS-H2B-RFP (Mayer, Emery, Berdnik, Wirtz-Peitz, & Knoblich, 2005), and UAS-H2B-YFP (Bellaïche, Gho, Kaltschmidt, Brand, & Schweisguth, 2001), as specified in the genotype list. In experiments where nuclear labeling was used for quantification, the same transgene was employed for control and mutant  (Evans et al., 2009)  intersectional genetic strategy to generate a specific driver. In addition to surface-associated cortex glia on the OPC, mir8-GAL4, labels cortex glia and neurons in the brain, as well as other cells in the animal. To restrict mir-8 expression exclusively to surface-associated cortex glia and cortex glia, we combined the following transgenes: repo-FLP6.2 (Stork, Sheehan, Tasdemir-Yilmaz, & Freeman, 2014), tub > GAL80 > (BDSC 38879), and mir8-GAL4. In this combination, GAL4 is only expressed in cortex glia since the GAL80 repressor has only been flipped out in this cell type but persists in non-glial mir-8 expressing cells (Supplementary Figure 9). For the sake of simplicity, we refer to this combination as the mir-8 cxg driver.

| DNA constructs
For UAS-ClC-a and UAS-CLCN2 transgenes, we used the Gateway  (Flores, Niemeyer, Sepúlveda, & Cid, 2009;Jeworutzki et al., 2012), and it is known to be expressed in Drosophila head and body (Flores et al., 2009). The final constructs were injected into the attp40 (25C6) landing site on the second chromosome.

| Immunohistochemistry
Fly brains were dissected in Schneider medium and fixed in 4% PFA

| Measurements and quantifications
To assess adult OL and CB size, we took two confocal images of each brain in the appropriate orientations to measure the antero-posterior and dorso-ventral axis of each structure at their widest part. We multiplied those two measurements to obtain a relative value in arbitrary units (a.u). The number of brains analyzed ranged from 6 to 44 for OL (to obtain fully independent measurements, only one OL per brain was quantified), and from 3 to 22 for CB when assessing phenotype (Figures 2c,l,m and 4h,i). In rescue experiments (Figures 2d and 4j,k), the number of brains analyzed ranged from 12 to 32. To assess brain size at different larval stages (Figure 4g), the diameter of one larval brain hemisphere per animal was measured in the antero-posterior axis. The n ranged from 23 to 37 animals analyzed.
To measure neuroepithelia volume, the tissue was stained with anti-E-cadherin antibody and manually segmented using the "SUR-FACE" tool included in Imaris software. This tool provides the volume in μm 3 of the surfaces generated ( Figure 4b). The n for this experiment was between eight and nine brains.
To quantify the number of cells in OPC, IPC, and CB neuroblast clones ( Figure 4d,e), we performed manual counting in confocal stacks. Cells in the clone were identified as TOPRO+ nuclei surrounded by labeled membrane. We counted as many clones as possible per brain provided that they were identifiable as individual clones. The number of clones analyzed was 52 (control) and 31(mutant) in the OPC and 18 (control and mutant) in the IPC, and the number of type I neuroblast clones was 39 (control) and 22 (mutant).
To assess cell death in developing OLs (Figure 4f), we manually counted Dcp-1 + /TOPRO + puncta per brain hemisphere. This value was divided by the hemisphere volume obtained through manual segmentation of the structure and using the "SURFACE" tool included in Imaris software. The n for this experiment was six brains.
To quantify the subset of medulla glia among glial cells in the chiasm region at different stages, we manually counted ClC-a + /Repo + nuclei ( Figure 5m). The n for this experiment was five brains.
To quantify the DL1 lineages nuclear green G-TRACE signal was manually counted in confocal stacks and glial cells identified by coexpression of the glial marker Repo (Figure 6g,h). The n for this quantification was between 11-13 clones for the control and 9-10 clones for the mutant.
To quantify mature INPs in the DL1 and DL2 lineages (Figure 6l), we manually counted Dpn + positive nuclei surrounded by tdtom + membranes of the R9D11-tdtomato marker. To differentiate DL1 from DL2, we used gcm-lacZ, which specifically labels the DL2 lineage (Supplementary Figure 10). The n for this experiment was between 11 and 12 brains.
To quantify the number of total glial cells in the future chiasm region in mid L3 (Figure 8b), we manually counted Repo + nuclei in confocal stacks. The following criteria were used to limit the area where we performed the counting: distally, we avoided counting flattened nuclei characteristic of surface glia and big round nuclei characteristic of surface-associated cortex glia; the proximal limit was set the signal gap generated by the presence of the IPC. The n for this experiment was between 5 and 8 brains.

| Statistics
Statistical analysis was carried out using Prism 6 (GraphPad Software Inc, San Diego, CA). When data did not follow a normal distribution or resulted from a previous mathematical computation (i.e., ratio to volume), we used nonparametric tests. For group comparisons, we used To evaluate the statistical significance of enhancements in qualitatively categorized photoreceptor phenotypes (i.e., strong, medium, weak, no phenotype) (Figure 7l,m), we performed a Chi-squared test of independence between phenotype categories and genotypes to obtain P-values.

| RESULTS
3.1 | ClC-a is expressed in various glial types in the developing brain: Surface-associated cortex glia, cortex glia and ensheathing glia Additional experiments indicated that ClC-a + nuclei present on the surface of the CB and in cortical areas belonged to cortex glia. The membrane and nuclear patterns of ClC-a + cells were consistent with the nuclear patterns and the membrane scaffold, also known as the trophospongium (Hoyle, Williams, & Phillips, 1986), observed with the recently described cortex glia driver wrapper (Coutinho-Budd et al., 2017) (compare Figure 1g with Figure 1l). In fact, there was extensive colocalization between ClC-a + and wrapper + membranes in the CB and OL (Figure 1m,n), including surface-associated cortex glia on the OPC (Figure 1n,n'').
In order to assess the presence of glial types other than surfaceassociated cortex glia and cortex glia, we used an intersectional strategy whereby only ClC-a + /wrapper − cells (i.e., noncortex glia cells) were labeled. This revealed that ClC-a was also expressed in different subtypes of ensheathing glia such as neuropil-and tract-ensheathing glia. ClC-a was expressed in neuropil-ensheathing glia surrounding CB neuropils, including the mushroom body calyx ( With regards to ClC-a subcellular distribution, similar to the vertebrate channel and as previously described in stellate cells (Cabrero et al., 2014), the channel is localized in the plasma membrane of glia as shown by co-localization of the antibody signal with a membrane marker ( Figure 1k).
Together, these data indicate that ClC-a is already expressed in early development in the plasma membrane of surface-associated cortex glia and cortex glia cells, which are in direct contact with and wrap proliferative tissues such as the neuroepithelia of the OL (OPC, IPC) and neuroblasts in the CB, forming part of the niche. ClC-a is also expressed in different types of ensheathing glia whose processes contribute to compartmentalization of the brain by demarcating different neuropils and neuronal tracts.

| MiMIC insertions in the ClC-a locus generate strong loss-of-function alleles
To explore the role of ClC-a in glia, we characterized a set of Minos- In summary, here we have characterized the first ClC-a mutants, which are strong loss-of-function alleles.

| Mutations in ClC-a result in smaller brains with photoreceptor guidance defects
To explore the effect of ClC-a mutations on brain development, we started by dissecting adult brains and searching for defects that could have a developmental origin based on ClC-a expression patterns in the larval brain. The observation that ClC-a was expressed in glia on proliferative tissues in the brain (i.e., neuroepithelia and neuroblasts) led us to hypothesize that mutant brains might be smaller than control ones, and to test this idea we measured OLs from control and mutant animals. We did indeed observe a reduction in OL size in mutants, which was particularly evident in 05423 ClC-a-GAL4 /Df, the strongest allelic combination, and was also present in 05423 ClC-a-GAL4 /14007 ( Figure 2c) and 14,007/Df (Figure 4h,i).
F I G U R E 1 ClC-a is expressed in cortex glia and ensheathing glia during brain development. (a-i) Analysis of ClC-a expression in the developing brain. Brain illustrations show the orientation of imaging planes for the indicated panels at different larval stages. ClC-a specific GAL4 driver (ClCa-GAL4) was used to label cellular membranes (green) and nuclei (red) of ClC-a + cells. Glial nuclei were labeled with anti-Repo antibody (blue).
Anti-E-cadherin (E-cad, magenta) was used to identify neuroepithelial cells. Neuroblasts and photoreceptors were labeled with anti-Deadpan (Dpn, gray) and anti-Chaoptin (gray), respectively. In order to confirm the requirement of ClC-a in glia, we performed cell-type-specific knock down and rescue experiments. In addition to the ClC-a driver, we also used the general glial driver Repo-GAL4 to directly support the conclusion that the channel is required in glia.
Using these two drivers, ClC-a knockdown by RNAi phenocopied the photoreceptor phenotypes seen in the mutant (Figure 2h,j,k). Moreover, expression with both drivers of ClC-a and rat CLCN2 cDNA res- Remarkably, mutant brains with no phenotype were already smaller than controls. We also observed that for the OL, brains with the medium or strong photoreceptor phenotype tend to be slightly smaller. However, despite the qualitative jump in the severity of guidance defects between the medium and strong photoreceptor phenotype classification, the OL size of these animals is not significantly different. Thus, taken together these results suggest that the brain size phenotype is independent of the photoreceptor phenotype.
3.4 | Expression of ClC-a in surface-associated cortex glia and cortex glia is required for neuroepithelial expansion and the generation of neuroblast lineages, and is sufficient to restore brain size In order to unravel how mutations in ClC-a resulted in smaller brains, we first assessed the status of glia in ClC-a mutants. We used the 05423 ClC-a-GAL4 /14007 allelic combination to visualize glia membranes and nuclei in the mutant background. Our analysis showed that the distribution pattern of glial cell bodies on the brain surface and deep in the cortex was similar in control and mutant animals. Although the number of nuclei/hemisphere volume ratio in the mutant was slightly reduced compared to control (Supplementary Figure 6A), importantly, the membrane scaffold appeared indistinguishable from the one observed in controls covering the whole hemisphere. As in control animals, ClC-a mutant surface-associated cortex glia and cortex glia processes were in close contact with the OPC and IPC neuroepithelia respectively (Figure 3a,b,e,f). In addition, in the OL and the CB alike, cortex glia processes formed the trophospongium. Thus, individual neuroblasts were enclosed in chambers that enlarged to adapt to their lineage expansion (Figure 3c,g), and mature neuronal cell bodies were progressively enwrapped by cortex glia processes (Figure 3d,d',h,h'). From these observations, we conclude that mutations in the channel do not result in major morphological changes in the trophospongium formed by cortex glia.
In turn, these results suggested that ClC-a was instead required for the proper physiology of surface-associated cortex glia and cortex glia.
Cortex glia have been shown to be essential for neurogenesis (Dumstrei et al., 2003), and since surface-associated cortex glia processes are tightly associated with the OPC (Morante et al., 2013) and cortex glia to the IPC, we set out to examine whether the small OLs in mutant adult brains (Figure 2c,l) were a consequence of defects in these neuroepithelia. Neuroepithelia in the OL start as sheets of cells that divide symmetrically and expand until mid L3 (Ngo et al., 2010). As they do so, they bend along the dorso-ventral axis, creating a crescent shaped structure with the opening pointing posteriorly (Nassif, Noveen, & Hartenstein, 2003  Given that neuroblasts originated from the OPC and the central brain are close contact with ClC-a expressing glia, we also used clonal analysis to assess how neuroblasts generated their lineages in ClC-a mutants. For this analysis, we focused on neuroblasts of the CB since it allowed us to address the origin of CB size reduction in mutants. Importantly, both control and mutant animals showed the same number of neuroblasts; thus, CB size reduction in mutants was not due to a decrease in neuroblasts (Supplementary Figure 6B). Using a similar clone induction protocol as for neuroepithelial clones, the median size of type I neuroblast clones in the control background was 34 cells, whereas the median size for clones in the mutant background was reduced to 26 cells (Figure 4e). In addition, at this same mid L3 stage, we also detected more dispersed cell death in mutant than control brains (Figure 4f) in regions other than the neuroepithelia, which we had shown were death free (Figure 4c). This result is consistent with the described trophic role of cortex glia processes that wrap the cell Hence, although evenly distributed in the brain, we cannot rule out that some of this cell death contributes to the reduction in size of type I neuroblast clones in the ClC-a mutant background.
Together, these data suggest that the lack of ClC-a in surface- 3.5 | Defects are also observed in the neuroblast lineage that gives rise to ClC-a + ensheathing glia, which are necessary guideposts for photoreceptor axons innervating the medulla In an attempt to understand how the nonautonomous photoreceptor guidance phenotype is related to ClC-a expression in the OL, we performed a detailed developmental expression analysis in the region where photoreceptor innervation takes place. In control L2 brains, horizontal views showed that the OPC and IPC were still juxtaposed and that ClC-a + cell bodies were present on the surface of the brain and in the CB (Figure 1d). In L3 frontal views, we observed that a population of glia, which preceded the arrival of photoreceptor axons in the lamina (Dearborn, 2004;Perez & Steller, 1996), progressively positioned amid the expanding region between the OPC and IPC during the early to mid L3 stages (Figure 5a,b) and ended up forming a barrier between the developing lamina and the lopn (Fan et al., 2005). Taking advantage of the recent availability of markers for different glial cell types we have been able to accurately characterize the ClC-a + cells forming the barrier.
The aforementioned glial population could be divided into two sets of nuclei, the ClC-a − nuclei of satellite glia (Supplementary Figure 10A, B) and a population of ClC-a + nuclei, with lower expression than cortex glia, known as medulla glial cells (Chotard & Salecker, 2008) (Figure 5b).
From this seemingly homogenous mid L3 medulla glia population the Xg o and a glial type that had been classified before as satellite glia based on position (Fan et al., 2005). Based on cell type specific drivers F I G U R E 4 Legend on next page. and co-localization experiments (Supplementary Figure 10A-C) we concluded that the latter glial type did not belong to the satellite glia population and we named it palisade glia (pag). They were positioned on the same plane as the cortex glia projection and the Xg o , forming a continuous ClC-a + glial barrier between the developing lamina and the lopn. We do not know if pag persist or which type they are in the adult (Figure 5e).  Figure 10D-F).
Hence, our data support the idea that medulla glial cells are DL1 prog- To study the cause of this marked reduction, we first used the earmuff R09D11 genomic enhancer-fragment driven reporter CD4-tdtomato (Han, Jan, & Jan, 2011) to selectively label all type II neuroblast lineages and assess DL1. Type II neuroblast lineages are characterized by the generation of intermediate neural progenitors (INP) that can undergo several rounds of additional asymmetric divisions before they disappear (Boone & Doe, 2008). Within an INP sublineage, which is temporally patterned, gliogenesis is most likely taking place in progeny of the last INP divisions (Bayraktar & Doe, 2013). In control brains, there are eight type II neuroblasts, six of which are positioned medially (DM1-6) and two laterally (DL1/2), closer to the OL (Figure 6a,b). In mutants, although we observed some brains with instances of DM mispositioning, the DL1/2 cluster was found together and laterally located with respect to the rest of the DM neuroblasts (Figure 6c,d). However, its position with respect to the OL was sometimes changed. To assess proliferation defects in the lineage, our initial approach was to compare control to mutant DL1 clones.
However, even though the clonal analysis protocol used in our study was very similar to those employed in other studies analyzing type II clones, which are identified by the presence of INPs (Dpn positive cells in the lineage), we obtained hardly any type II clones (2 out of 116 analyzed clones) and none in the DL1/2 cluster. We thus opted to perform lineage tracing to compare the DL1 glial progeny in control and mutant animals (Figure 6e,f). This analysis showed that in the mutant background there was strong reduction of Repo + /DL1 + cells (Figure 6f,g). Interestingly, the DL1 neuronal lineage was slightly increased in mutants compared to controls (Figure 6h). In an attempt to understand how these results came about we reasoned that we could use the number of INPs in the lineage as a readout (Figure 6i).
Since DL1  and that the IPC, which is the region where these cells enter the OL in normal conditions, is defective in mutants, medulla glia could be F I G U R E 4 ClC-a is required for neuroepithelial expansion, neuroblast lineages, as well as neuronal viability, and is sufficient to rescue brain size.  Figures 7, 8 and 9). OPC, outer proliferation center; IPC, inner proliferation center hindered from reaching their final destination. However, during the DL1 G-TRACE lineage analysis in mutant animals we have not detected glial cells in regions other than the optic lobe, which suggests that migrations defects would not be the major contributing factor to the reduced number of medulla glial cells in ClC-a mutant optic lobes.
At this point, the question arises of how the marked reduction in medulla glia affects photoreceptor guidance. Since the presence of medulla glia in mid L3 coincides with the beginning of photoreceptor innervation, we next explored the spatiotemporal relationship between these two cell types in control flies. As rows of ommatidia form in the F I G U R E 5 Legend on next page. eye disc, photoreceptors extend axons that reach the OL through the optic stalk. In mid L3 stages, R8s from the first rows of ommatidia pro- Removal of one copy of slit slightly enhanced the ClC-a photoreceptor guidance phenotype when assessed in an allelic combination with few brains showing only medium and weak photoreceptor phenotypes (and presumably with just a slight reduction in medulla glia) (Figure 7l). In addition, knocking down slit in ClC-a + glia in the barrier recapitulated photoreceptor guidance defects (Figure 7m).
Based on our results and previously published studies (Fan et al., 2005;Pappu et al., 2011;Suzuki et al., 2016;Tayler et al., 2004), we propose that the substantial reduction in medulla glia is most probably due to a combination of DL1 lineage proliferation and/or temporal specification defects, which results in a significant reduction in Slit protein in the region. As a consequence, photoreceptors that innervate the OL close to the glial boundary fasciculate with the axons of distal cells (C2, C3, T2 and T3) which derive from the IPC and are known to innervate the medulla from its proximal site (Hofbauer & Campos-Ortega, 1990;Meinertzhagen & Hansen, 1993).

| Expression of ClC-a in cortex glia is sufficient to restore ensheathing glia guidepost cells and rescue photoreceptor guidance defects
To test whether ClC-a expression in cortex glia was sufficient to regulate DL1 proliferation, and assess if ClC-a expression in medulla glia (cell type classified as ensheathing glia) played any role in photoreceptor guidance, we performed a cell-type-specific rescue experiment. We carried out a surface-associated cortex glia and cortex glia-specific rescue. We reasoned that with such a specific driver, we could rescue the generation of medulla glia from DL1 and at the same time avoid ClC-a expression in medulla glia (Figure 8a). Since it was not possible to specifically label medulla glia in this experiment, we used Repo to mark and count glial nuclei in the region in mid L3, when the first photoreceptors begin to innervate the brain. At this time, the glial population is compact and easy to identify, whereas in late L3, additional ClC-a − glia such as epithelial and marginal glia appear in high numbers and complicate counting. In control animals, mid L3 glia nuclei included ClC-a − satellite glia and medulla glia (Figure 8a,b). In mutants, the number of glial cells was reduced to half due to the marked reduction in medulla glial cells ( Figure 8a,b), but expression of ClC-a exclusively in surface-associated cortex glia and cortex glia resulted in an almost complete rescue in the F I G U R E 5 Strong reduction in a subset of ClC-a + ensheathing glial cells is observed in ClC-a mutants. Developmental analysis of cells that express ClC-a in the OL region in control animals (05423 ClC-a-GAL4 /+) and those same cells in ClC-a mutant animals (05423 ClC-a-GAL4 /14007). Surface-associated cortex glia and cortex glia membranes are shown in green and nuclei in red. All glial nuclei were labeled with anti-Repo antibody (blue). (a-d) Images of the ClC-a + glial barrier from early to late L3 control OLs with the corresponding schematics, in frontal views (a-c) and horizontal view ( Figure 10). OPC, outer proliferation center; IPC, inner proliferation center; sa-cxg, surface-associated cortex glia; cxg, cortex glia; sg, satellite glia; meg, medulla glia; pag, palisade glia; Xg o , outer chiasm glia; eg, epithelial glia; mg, marginal glia; me, medulla number of glial cells present in the barrier region in mid L3 (Figure 8a,b).
More importantly, this medulla glia rescue also rescued the photoreceptor guidance phenotype (Figure 8c). Surprisingly, autonomous ClC-a expression in medulla glia was not necessary for their viability, for migration from their point of origin in the CB to position themselves in the OL, or for Slit secretion, since photoreceptor guidance defects were fully rescued when medulla glia were in their position but did not express ClC-a. Thus, we conclude that the strong reduction in medulla glia and the photoreceptor guidance phenotypes are a secondary consequence of the ClC-a requirement in cortex glia and its none autonomous role in neurogenesis.

| Mutations in ClC-a result in widespread wiring defects
Although animals indicate that the wiring of many other neurons in this system is also probably affected (compare Figure 5e with Figure 5k). Moreover, we also observed defects in CB structures such as mushroom bodies (MBs). Each hemisphere contains one MB, which is formed by the neurons derived from four special type I neuroblasts that never enter quiescence. These neurons extend dendrites forming the calyx, and axons project into a fascicle called the peduncle that splits into two branches called lobes (Figure 9a). Similar to photoreceptors, mushroom bodies are neural structures that are highly dependent on glia-neuron interactions.
It has been shown that glia wrap the peduncle and the lobes during development (Spindler, Ortiz, Fung, Takashima, & Hartenstein, 2009) and in the adult (Kremer et al., 2017), and that different type II DM neuroblasts contribute glia that associate with the mushroom body (Ren et al., 2018). In control animals, ClC-a + glia surrounded the MB calyx splaying of axons and misguidance, and a misshapen superficial calyx due to premature fusion of the four MB lineages in the cortical region (Spindler et al., 2009). Thus, as observed for photoreceptor guidance phenotypes, MB defects in ClC-a mutants may be due to reduced production of glia associated with MB circuitry, whether that glia is ClC-a + or not. In summary, since guidance defects in the ClC-a mutant seem to be widespread, we propose that the ClC-a requirement for proper circuit assembly is not restricted to the OL but is general to the brain.

| DISCUSSION
In this study, we have shown that the ClC-a chloride channel function in the glial niche has a nonautonomous but profound effect on two key aspects of neural development: the generation of neurons and glia in the appropriate numbers, time, and place, and in consequence, the correct assembly of neural circuits. Importantly, the fact that the fly (ClC-a) and rat (CLC-2) chloride channels rescue brain size and guidance defects suggests that both can perform the same physiological function.
The reduced neurogenesis observed in ClC-a mutants could have several origins. Our cell death analysis, clonal study and EdU experiment suggest that in the OL, one of the causes could be defective neuroepithelium expansion. However, another possibility could be that ClC-a function in surface-associated cortex glia covering the neuroepithelium regulated the proneural wave progression and hence the neurepithelium to neuroblast transition. A premature start of this transition could prevent the completion of neuroepithelial expansion, and hence result in a reduced number of OPC neuroblasts. Alternatively, it is formally possible that the reduced neurogenesis observed is a consequence of both defective neuroepithelium expansion and premature neurepithelium to neuroblast transition. Indeed, glia covering the OPC neuroepithelium has been shown to regulate both processes (Morante et al., 2013;Perez-Gomez et al., 2013).

Concomitant defects in neuroblast proliferation and photoreceptor
targeting have been observed in other studies (González, Romani, Cubas, Modolell, & Campuzano, 1989;Kanai et al., 2018;Zhu et al., 2008), and it has been proposed that the Activin signaling pathway is . Although ClC-a expression is downregulated in (k''), we have shown that they express ClC-a in other panels (Figures 1h and 5c,d).
(i) Frontal view of an early L3 OL. (j) Horizontal view of a mid L3 OL. (k) Horizontal view of a late L3 OL. Phenotype analysis for slit/ClC-a genetic interaction (m) and slit knockdown (l). Phenotype penetrance and expressivity for each condition is depicted as the percentage of brains with no phenotype, weak, medium, and strong phenotypes. Scale bars represent 10 μm. * p < .05, (See also Supplementary Figure 12). sa-cxg, surfaceassociated cortex glia; meg, medulla glia; Bn, Bolwig's nerve; Lp, lamina plexus; Xg o , outer chiasm glia; sg, satellite glia; OPC, outer proliferation center; IPC, inner proliferation center; eg, epithelial glia; mg, marginal glia; pag, palisade glia; me, medulla required to produce the proper number of neurons to enable proper connection of incoming photoreceptor axons to their targets (Zhu et al., 2008). Interestingly, mutations in the proneural gene asense, which is expressed in type I neuroblasts, GMCs and INPs, has adult targeting phenotypes that are extremely similar to the ones observed in ClC-a mutants (González et al., 1989 (Blanz et al., 2007;Bösl et al., 2001 ;Edwards et al., 2010). However, CLCN2 is expressed in astrocytes and oligodendrocytes early in development (Makara et al., 2003) and has been detected in Bergman glia (Jeworutzki et al., 2012), which are important for neuronal migration in the formation of cortical structures. Together with our findings, these observations suggest that it would be worth exploring the role of this channel in the vertebrate neural stem cell niche. Interestingly, expression of CLCN2 has been found outside the brain in an unrelated stem cell niche. It is expressed in Sertoli cells (Bösl et al., 2001), which are the primary somatic cells of the seminiferous epithelium that form the spermatogonial stem cell niche through physical support and expression of paracrine factors (Chen et al., 2005;Oatley, Racicot, & Oatley, 2011).
CLCN2 mutant mice showed disorganized distribution of germ cells in tubules at 3 weeks, germ cells did not pass beyond meiosis I, and were completely lost at later stages (Bösl et al., 2001;Edwards et al., 2010).
Hence, similarly to the possible role of ClC-a regulating neurogenesis in the neural stem cell niche, CLC-2 could be regulating spermatogenesis in the spermatogonial stem cell niche.
Although the Sertoli CLCN2 expression/germ cell depletion correlation in mouse is in accordance with our data suggesting an important F I G U R E 8 Legend on next page.
F I G U R E 8 ClC-a expression exclusively in surface-associated cortex glia and cortex glia rescues the formation of medulla glia and photoreceptor guidance defects. (a) Schematics depicting the cortex glia-specific rescue experiment and representative confocal sections for each condition. The DL1 lineage and frontal view schematics of mid L3 OLs show ClC-a expression (green) and glial cells (blue). Outlined in a solid red line is the medulla glia population. Outlined in a dashed orange line is the glial population that has been assessed in this experiment. Representative sections of the confocal stacks used for quantification show in a dashed orange line the glial population that was quantified. In controls (14007/+) ClC-a is expressed in cortex glia surrounding the DL1 neuroblast and its progeny in the CB, and in surface-associated cortex glia and cortex glia over the OPC and IPC respectively, and in medulla glial cells in the OL. The ClC-a mutant (14,007/Df ) shows the absence of ClC-a expression and a strong reduction of medulla glial cells; and in an animal where ClC-a expression has been exclusively restored in surface-associated cortex glia and cortex glia (mir-8 cxg ), medulla glial cells are recovered but do not express ClC-a since they are a subtype of ensheathing glia. (b) Quantification and comparisons of glial nuclei (Repo, blue) in control, mutant, and rescue animals. (c) Quantification of photoreceptor guidance phenotypes in control and rescue brains. Control brains represent genotypes for both the GAL4 driver and the UAS transgene since they could not be distinguished in the genetic scheme of the experiment (mir-8 cxg control and UAS-ClC-a control). Scale bars represent 10 μm. * p < .05, ** p < .01, *** p < .001. GMC, ganglion mother cell; sa-cxg, surface-associated cortex glia; sg, satellite glia; meg, medulla glia; cxg, cortex glia; OPC, outer proliferation center; IPC, inner proliferation center role of the ClC-a/CLC-2 chloride channel in stem cell niches, it remains unclear how a chloride ion channel could nonautonomously modulate neurogenesis. ClC-a function in Malpighian tubules has been associated with the movement of Cl − ions (Cabrero et al., 2014), but it is possible that its function in glia of the stem cell niche is unrelated to ion exchange. For example, it might recruit signaling molecules to modulate neuroblast proliferation. Conceptually, one way to test whether the channel function is related to the movement of ions would be to perform rescue experiments of ClC-a mutant phenotypes with a channel defective for the pore function. In practice, however, this type of experiment is not that straightforward since CLC-2 pore gating is quite complex. Channels of the CLC family are thought to function as a homodimers, with each subunit forming a pore and presenting both independent and common pore gating mechanisms (Jentsch & Pusch, 2018). Given the many studies supporting the function of CLC-2 as a channel, we next discuss different ways in which ionic imbalance caused by mutations in ClC-a could result in the phenotypes described. One of the possibilities we considered was whether ionic imbalance in ClC-a mutants affected secretion. Glial insulin-like peptides (dILPs) (Chell & Brand, 2010;Sousa-Nunes et al., 2011). In vertebrates, an increase in intracellular Ca 2+ in astrocytes, which is caused by activation of G protein-coupled receptors and release of calcium from intracellular stores or calcium entry from the extracellular space through different types of channel, has been reported to evoke the release of gliotransmitters (Bazargani & Attwell, 2016;Khakh & McCarthy, 2015;Shigetomi, Patel, & Khakh, 2016). In this scenario, membrane potential changes mediated by Cl − channel activity could modulate activation of GPCR or voltage dependent Ca 2 + channels, mediating an increase in the Ca 2+ intracellular concentration and resulting in secretion. In fact, the opening of voltage Compare panels (f) to (b), (g) to (c), (h) to (d), and (i) to (e). Schematic (j) and volume-rendering 3D reconstructions and confocal sections of mushroom body neuroblast clones in control (k, l) and ClC-a mutant (m-o) brains labeled in green. (k) Control clone, (l) cross section of a control clone at the level of the peduncle, (m) mutant clone, (n) cross section at the level of the peduncle of a mutant clone in (m), and (o) Mutant clone with a strong phenotype. Scale bars represent 10 μm. Ca, calyx; Pe, peduncle; mL, medial lobe; vL, vertical lobe dependent Ca 2+ channels has been proposed as the mechanism behind the increase in aldosterone production and secretion (Fernandes-Rosa et al., 2018) resulting from gain-of-function mutations of CLCN2, which are behind primary aldosteronism and cause sustained depolarization of glomerulosa adrenal cells (Fernandes-Rosa et al., 2018;Scholl et al., 2018). To test whether loss of function of ClC-a/CLC-2 channels also affected secretion, we performed glia-  (Bösl et al., 2001). Assays in Xenopus oocytes have shown that ClC-a activity is also sensitive to pH (H. G-P. and R. E., unpublished results). Thus, it may be that the lack of ClC-a in cortex glia leads to a more acidic extracellular pH due to deficient Cl − recycling for HCO 3 − /Cl − exchangers. Since changes in extracellular and intracellular pH have been shown to affect the proliferative capacity of both wild type and cancer cells (Carswell & Papoutsakis, 2000;Ciapa & Philippe, 2013;Flinck, Kramer, & Pedersen, 2018;Persi et al., 2018;White, Grillo-Hill, & Barber, 2017), ClC-a function in pH regulation could explain the proliferation defects observed in the mutant.
Regardless of the molecular mechanism that mediates the effect of ClC-a on neurogenesis, our findings support the notion that gliamediated ionic balance could be important for brain development. Our results are in accordance with those of recent studies suggesting a link between several ion channels and the development of the nervous system, with channels being important both in stem cells (Li, 2011;Liebau, Kleger, Levin, & Yu, 2013) and glia (Olsen et al., 2015). A recent example of a channel function in stem cells is the gene SCN3A, which codes for the NaV1.3 sodium channel. This channel is mainly expressed during development and is highly enriched in basal/outer radial glia progenitors and migrating newborn neurons (Smith et al., 2018). The appearance of this type of progenitor and defined neuronal migration has been associated with the establishment of gyri in the cortex (Fietz et al., 2010;Hansen, Lui, Parker, & Kriegstein, 2010;Reillo, De Juan Romero, García-Cabezas, & Borrell, 2011). Intriguingly, mutations in the SCN3A gene result in structural malformations of gyri in the cortex (Smith et al., 2018). Another example is the glial-specific Kir4.1 channel, which is related to neurodevelopmental disorders with associated cognitive defects. Mutations in KCNJ10, which codes for the glial-specific Kir4.1 channel, underlie SeSAME/EAST syndrome (seizures, sensorineural deafness, ataxia, intellectual disability and electrolyte imbalance/ epilepsy, ataxia, sensorineural deafness, and tubulopathy) (Bockenhauer et al., 2009;Scholl et al., 2009) and have been detected in patients diagnosed with autism spectrum disorder and epilepsy (Sicca et al., 2011(Sicca et al., , 2016. Reduced Kir4.1 expression in astrocytes significantly contributes to the etiology of Rett syndrome (Kahanovitch et al., 2018;Lioy et al., 2011), which shares many pathophysiological traits with SeSAME/EAST. Moreover, Kir4.1 protein is detected as early as embryonic day 20 in glial cells in the developing cortex and hippocampus (Moroni, Inverardi, Regondi, Pennacchio, & Frassoni, 2015), suggesting that it could influence neural development in these regions. Together with our findings, these observa-