The Hippo signalling pathway maintains quiescence in Drosophila neural stem cells

Stem cells control their mitotic activity to decide whether to proliferate or to stay in quiescence. Drosophila neural stem cells (NSCs) are quiescent at early larval stages, when they are reactivated in response to metabolic changes. Here we report that cell-contact inhibition of growth through the canonical Hippo signalling pathway maintains NSC quiescence. Loss of the core kinases hippo or warts leads to premature nuclear localization of the transcriptional co-activator Yorkie and initiation of growth and proliferation in NSCs. Yorkie is necessary and sufficient for NSC reactivation, growth and proliferation. The Hippo pathway activity is modulated via inter-cellular transmembrane proteins Crumbs and Echinoid that are both expressed in a nutrient-dependent way in niche glial cells and NSCs. Loss of crumbs or echinoid in the niche only is sufficient to reactivate NSCs. Finally, we provide evidence that the Hippo pathway activity discriminates quiescent from non-quiescent NSCs in the Drosophila nervous system.

S tem cells are undifferentiated cells that have the unique ability to produce differentiating daughter cells and retain their identity by a process called self-renewal. Stem cells can exhibit a remarkable proliferative capacity, for example, during development or regenerative processes 1,2 . Deregulation of stem cell proliferation can lead to tumour formation or to a premature depletion of the progenitor pool 3 . Thus, stem cell proliferation has to be tightly regulated according to the cellular or organismal context. When proliferation is not required, stem cells are maintained in a state of quiescence in the G0-phase and need to be activated by systemic or local signals 3,4 . In Drosophila, neural stem cells (NSCs) proliferate in two phases 5 . The embryonic phase generates all cells of a functional larval central nervous system (CNS), while in the second proliferative phase cells forming the adult CNS are produced. In late embryonic stages NSCs enter quiescence, which requires intrinsic transcription factors 6,7 .
Changes in the physiological condition of the animal in response to feeding at early larval stages causes reactivation of NSCs 8 . The amino-acid sensing fat body releases systemic signals in response to the increase in dietary amino acids 8,9 and CNS glial cells translate these signals into a local activating signal. They produce and secrete insulin-like peptides that activate the insulin/ insulin-like growth factor signalling pathway in NSCs 10,11 . An initial step during reactivation is the drastic increase in NSC cell size from 4-5 mm during quiescence to 10-15 mm depending on the type of NSC 5,12 . Thus, growth in preparation for cell division is one of the initial hallmarks of NSC reactivation. The mechanisms regulating quiescence are less well understood. Glial cells secrete a glycoprotein (anachronism) that keeps NSCs in quiescence, but the precise molecular mechanism remains unknown 13 .
One of the major pathways that controls organ growth and cell proliferation in Drosophila and vertebrates is the conserved Salvador/Hippo/Warts signalling pathway (SHW) [14][15][16] . The SHW consists of a growth-repressive kinase cascade that modulates the activity of the transcriptional co-activator Yorkie (YAP/TAZ in vertebrates). The Hippo kinase activates the Warts kinase, which in turn directly phosphorylates Yorkie, creating a 14-3-3 binding site that restricts nuclear import and inactivates Yorkie 17,18 . If Hippo/Warts are inactive, non-phosphorylated Yorkie enters the nucleus and binds to transcription factors like Scalloped 19,20 and activates its transcriptional program promoting cell growth and proliferation 21,22 . Numerous upstream regulators of the SHW have been identified, including cell-cell contact, the actin cytoskeleton, G-protein coupled receptors or planar and apico-basal cell polarity 23 .
In the vertebrate skin or the liver, de-repression of YAP has been shown to promote stem cell proliferation 24 . However, whether this is true in NSCs and whether changes in Yorkie/YAP activity are causative for altering growth and proliferation during normal CNS development remains unclear. In Drosophila, the SHW has been implicated in CNS development of the neuroepithelium and of glial cells in the optic lobe, and in cell growth of a specific population of glial cells (subperineural glial cells) [25][26][27] . However, for central brain NSCs, no function has been attributed to the SHW.
Here, we show that the SHW maintains quiescence of NSCs at the transition from embryo to larval life in Drosophila. Loss of the core kinases hippo/warts, or upstream regulators kibra/Merlin/ expanded, leads to a premature initiation of cell growth and proliferation and thus reactivation from quiescence. Yorkie is inactive in quiescent NSCs and is necessary and sufficient for the reactivation and proliferation of NSCs. Cell-cell contact proteins Crumbs and Echinoid are expressed in both glial cells and NSCs and regulate the activity of Hippo and Warts, possibly via homophilic interactions in trans. The expression of Crumbs and Echinoid in glial cells and NSCs is nutrition-dependent, and their premature loss in glial cells is sufficient to initiate reactivation of NSCs. Moreover, the Yorkie activity discriminates between quiescent and non-quiescent NSCs, placing the SHW as a major regulator of growth in cellular quiescence in Drosophila NSCs.

Results
Loss of Hippo signalling causes premature NSCs reactivation.
To identify novel regulators of quiescence in NSCs, we depleted known growth regulators using RNAi-mediated gene knockdown in the insc-GAL4 pattern 28 . We scored NSCs (Deadpan-positive cells) cell size and proliferation rate (phosphohistone H3 (pH3)positive NSCs) 4 h after larval hatching (ALH), when all NSCs are quiescent-small in cell size (B4-5 mm) and non-proliferative ( Fig. 1a,g,h) 5,10,12 . Exceptions are four NSCs of the mushroom body (MBNBs) and one ventrolateral NSC (lNSC) that do not enter quiescence, have a large cell diameter and constantly proliferate (Fig. 1a) 29 . These 'non-quiescent NSCs' are quantified independently from all quiescent NSCs. First, we describe the fate of quiescent NSCs.
Knockdown of the core kinases of the SHW hippo or warts induces a marked premature increase in NSC cell size (Fig. 1b,c) from 4.5 mm (median, maximum 6.5 mm) in control brains 4 h ALH to 7 mm (median, maximum 13 mm; Fig. 1g). Since this suggests an early exit from quiescence, we tested for entry into S-phase using antibody staining for the S-phase cyclin CycE. We observed an increase in CycE-positive NSCs upon warts-RNAi ( Supplementary Fig. 1a,b). Consequently, the number of pH3-positive mitotic NSCs was also significantly increased (Fig. 1h). Next, we examined the known upstream regulators of the SHW expanded, kibra or Merlin for their function in NSCs. Indeed, RNAi showed similar albeit less-pronounced effects and caused premature cell growth at 4 h ALH ( Fig. 1d-g).
To ensure that this phenotype is not because of an impaired entry into quiescence, we analysed trans-heterozygous hpo JM1 /hpo KC202 mutants 30,31 at hatching (0-2 h ALH) and 4 h ALH ( Supplementary Fig. 1c,d), and stage-17 embryonic brains of wts-RNAi ( Supplementary Fig. 1e,f). In both situations NSCs did not show increased cell sizes or mitotic activity revealing a normal phase of quiescence (stage-17 at 0-2 h ALH). Interestingly, hpo mutant larvae exhibited a mild but significant increase in cell size at 4 h ALH mimicking the reactivation phenotype in wts-RNAi (Fig. 1i). Finally, we used the temperature-sensitive GAL4 repressor system GAL80ts to restrict the RNAi expression to only larval stages. Indeed, upon larval stage-restricted wts-or hpo-RNAi in NSCs we could monitor a similar increase in NSC diameter at 4 h ALH (Fig. 1j).
Thus, the SHW acts in Drosophila NSCs to maintain quiescence and cell-autonomous loss of pathway components leads to premature exit from quiescence.
phosphorylated Yorkie binds to 14-3-3 and stays inactive, we tested the loss of 14-3-3-zeta with RNAi and observed premature growth of NSCs at 4 h ALH (Fig. 2e,f), presumably owing to early activity of Yorkie. Thus, Yorkie is inactive in NSCs during quiescence, and is activated and localizes to the nucleus during reactivation or upon wts-RNAi.
Yorkie is necessary and sufficient for growth and proliferation.
To determine whether Yorkie is also necessary for NSC growth and proliferation, we analysed yki B5 null mutants 32 . Homozygous yki B5 mutants are embryonically semi-lethal and most larvae die at approximately 48 h ALH. Whereas, wild-type NSCs at 48 h ALH have been reactivated and are highly proliferative (Fig. 3a,c,d), no reactivation of quiescent NSCs can be observed in the yki B5 mutants (no cell growth and no pH3-positive NSCs; Fig. 3b-d). Moreover, NSCs cell size and their mitotic index at 48 h ALH revealed that yki B5 -mutant NSCs resemble quiescent NSCs (Fig. 3c,d). Next, we tested whether early expression of a constitutively active form of Yorkie (UAS-yki S168A ) 17 is sufficient to reactivate NSCs. Indeed, at 4 h ALH we observed a significant increase in NSCs cell diameter, which was also present when restricting the expression to only larval stages using the GAL80ts system ( Fig. 3e,f). We conclude that Yorkie function is necessary and sufficient for NSCs reactivation and initiation of growth and proliferation.
Yorkie activates the bantam microRNA during reactivation.
Next we tested if the expression of the Yorkie target genes four-jointed (fj-lacZ) 33 , expanded 34 and the microRNA bantam 35 correlates with the subcellular translocation of Yorkie during reactivation. Fj-lacZ and expanded showed only weak expression in quiescent NSCs (4 h ALH) but clear upregulation at 48 h ALH ( Supplementary Fig. 3a,b). We analysed the expression and activity of bantam that is known to regulate proliferation and growth [35][36][37][38] by using a GFP-sensor system 36 . The loss of GFP expression and thus the activity of bantam coincides with the activation of Yorkie, as quiescent NSCs (4 h ALH) show strong GFP staining (Fig. 4a, upper panels) and reactivated NSCs (24 h ALH) have markedly reduced GFP signals monitoring bantam activity (Fig. 4a, lower panels). We combined the bantam sensor with wts-RNAi and examined an early activity of bantam at 4 h ALH (Fig. 4b), suggesting that premature reactivation by wts-RNAi expression causes early activation of the Yorkie downstream target bantam. To test if bantam is also necessary for NSC reactivation, we analysed bantamD1 deletion mutants at 24 h ALH and observed NSC reactivation in the brain, but markedly reduced cell size and proliferative capacity ( Fig. 4c-f). This effect seemed stronger in NSCs of the ventral nerve cord (VNC) at 24 h ALH, which in bantamD1 mutants were indistinguishable from quiescent NSCs in control VNCs at 4 h ALH, with severely reduced cell sizes and nearly no pH3-positive NSCs (Fig. 4e,f). The difference between brain and VNC NSCs can be attributed to the spatiotemporal progression of NSC reactivation from anterior to posterior, which is also reflected in the size distribution of the wild-type control (Fig. 4e). Thus, we conclude that bantam is an important target of Yorkie during reactivation of NSCs, yet other unknown targets are likely involved in growth and proliferation of NSCs.
SHW regulates reactivation depending on nutritional status. Because reactivation of NSCs is dependent on a nutritional stimulus and insulin signalling from CNS glia 8,10,11 , we tested if premature reactivation upon wts-RNAi depends on nutrition. Gene knockdown of wts in starved larvae resulted in a minor but still significant increase in cell size compared with wts knockdown in well-fed larvae ( Supplementary Fig. 3c,d and Fig. 1c,g) but we could not detect pH3-positive NSCs. Thus, the SHW might regulate growth initiation of NSCs in parallel to the nutritional response model. This shows, that sensing of new nutritional resources occurs within the first 4 h ALH and SHW can initiate cell growth, but reactivation even in loss of the SHW depends on the nutritional status of the organism.
Crumbs and Echinoid activate SHW during NSC quiescence. We sought to investigate how external signals regulated the cell-intrinsic, growth-repressing activity of the SHW during reactivation. Since a number of inter-and extracellular SHW regulators are known, we tested the transmembrane proteins Crumbs [39][40][41][42] and Echinoid 43,44 for their role in NSCs quiescence. When targeting crb or ed by RNAi in NSCs, a significant increase in NSCs cell size can be measured ( Fig. 5a and Supplementary  Fig. 4a,b) and in response to crb/ed-RNAi reactivated NSCs showed nuclear localization of Yorkie (Fig. 5b). For ed we analysed a embryonic/larval non-lethal hypomorphic allele (ed F72 ) that also exhibited premature reactivation revealed by increased NSC cell diameters ( Fig. 5a) and incorporation of EdU monitoring S-phases ( Supplementary Fig. 4c).
Since NSC reactivation involves niche glia cells 10,11 , we explored the role of niche signalling during quiescence. Using repo-GAL4 for glial-RNAi, we targeted crb or ed in glial cells only, which was sufficient to observe a similar significant but lesspronounced increase in NSC cell size compared with knockdown of crb or ed in NSCs ( Fig. 5c and Supplementary Fig. 4d,e). To exclude that this is a consequence of altered SHW in glial cells, we analysed NSCs behaviour in glial wts-RNAi. In contrast to crb-or ed-RNAi we could observe a premature growth initiation in subperineural glial cells as described before ( Supplementary  Fig. 4f) 27 . The effect on reactivation of NSCs was stronger compared with glial crb or ed RNAi ( Supplementary Fig. 4g). To test whether wts-RNAi and therefore premature glial growth can bypass the nutritional stimulus, we analysed the effect of nutritional deprivation. Similar to wts-RNAi in NSCs in nutrition-deprived conditions, wts-RNAi in glial cells in starved larvae leads to a minor reactivation phenotype and a less pronounced but still significant increase in NSCs cell diameter ( Supplementary Fig. 4g). Thus, premature glial growth in wts-RNAi can initiate growth in NSCs but full reactivation is dependent on nutrition. Knockdown of crb or ed in glial cells did not result in a premature growth initiation in glial cells (compare Supplementary Fig. 4d,e with f). To show that the premature growth initiation in NSCs upon glial-RNAi of crb and/or ed is NSC diameter in yki S168A

h ALH
inscG4>UAS-yki S168A through altering the SHW pathway in NSCs, we made use of the bantam activity sensor. Indeed, we could monitor a premature activation of bantam (loss of GFP) in NSCs upon glial crb/ed RNAi (Fig. 5d). Therefore, knockdown of crb or ed in niche glial cells leads to premature reactivation of NSCs through altering Hippo activity in trans.
To test potential homophilic interactions of Crumbs 45 , we targeted crb simultaneously in both NSCs and glial cells by RNAi (insc-GAL4/ repo-GAL4) and observed an increase in the strength of premature reactivation of NSCs compared with glial knockdown alone (Fig. 5c,e and Supplementary Fig. 4h,i). Since the phenotype was similar to the NSC-specific RNAi of crb or ed alone we conclude that crb and ed act both in trans and in cis. Knockdown in glial cells removes the interaction in trans (between glial cells and NSCs) but leaves the interaction in cis (on NSCs), which causes a less-pronounced phenotype. Conversely, knockdown of crb or ed in NSC or in NSC and glial cells simultaneously interrupts both interactions in cis and in trans and causes stronger phenotypes. 24   were calculated from two biological replicates. Control brain n ¼ 109 NSCs (3 brain lobes); banD 1 brain n ¼ 319 NSCs (7 brain lobes); control VNC n ¼ 109 NSCs (3 VNC); banD 1 VNC n ¼ 253 NSCs (4 VNC). (f) Average number of NSCs in mitosis (pH3-positive) at 24 h ALH in wild-type of 3 brain lobes and 3 VNCs and banD 1 7 brain lobes and 4 VNCs. All images are single focal planes, anterior up. Scale bar, 10 mm. See also Supplementary Fig. 3.
Simultaneous glial-and NSC-specific knockdown of crb caused Yorkie nuclear localization in reactivated NSCs ( Supplementary  Fig. 4h) connecting crb function to the SHW. Finally, we assessed redundancy and performed double RNAi gene knockdowns of crb and ed simultaneously with the double driver GAL4 line (NSCs and glial cells concurrently), which caused NSC reactivation in the same degree as knockdown of ed alone (Fig. 5c,e and Supplementary Fig. 4i). Thus, other factors might compensate or influence the Hippo activity in NSCs along with crb or ed.
In conclusion, cell-contact inhibition of growth by niche glial cells through the SHW maintains quiescence in Drosophila NSCs. Interestingly, loss of Hippo signalling in the niche alone is able to initiate cell growth in NSC even during starvation.

Glial Crumbs and Echinoid expression depends on nutrition.
Both Crumbs 46 and Echinoid 47 are expressed in epithelial cells and their role in glial cells and NSCs was surprising. Using a functional Crumbs::GFP fusion protein 48 , crb-mRNA in situ ( Supplementary Fig. 5b) or antibody staining for Echinoid we observed expression of both in glial cells and NSCs during quiescence (Fig. 6a,b and Supplementary Fig. 5a-c). Since our data so far suggests a cell-contact inhibition of growth by niche glial cells we analysed whether Crumbs::GFP localizes to contact sites of glial cells and NSCs. Indeed, we could observe a slight accumulation in NSCs towards the contact site with glial cells ( Supplementary Fig. 5d). To prove that NSC and glial cells indeed form cell-cell contacts, we stained for E-Cadherin and could monitor adherens junctions between glial cells and NSCs ( Supplementary Fig. 5e). Crumbs::GFP expression in glial cells and NSCs was lost over time (8 and 24 h ALH, respectively) whereas Echinoid was downregulated mainly in glial cells (24 h , Fig. 6a,b and Supplementary Fig. 5a,c). Nutritional deprivation prolongs quiescence and Crumbs::GFP, crb-mRNA or Echinoid expression was maintained in glial cells and NSCs at 24 h ALH (Fig. 6a,b and Supplementary Fig. 5a,c,f), whereas early activation of the Insulin-like receptor signalling leads to premature loss of Crumbs::GFP in NSCs ( Supplementary  Fig. 5g). Thus, Crumbs and Echinoid are expressed in nonepithelial niche glial cells and NSCs during the phase of quiescence and are developmentally downregulated in response to nutrition.
Ectopic crb causes decreased NSC growth and proliferation. Next we assessed whether ectopic expression of crb or ed would prolong quiescence. Expression of crb in glial cells leads to embryonic lethality, and its expression in brain NSCs results in cell clustering and an increase in NSCs of the MBNB (Supplementary Fig. 5h) complicating the analysis. These phenotypes were not apparent in NSCs of the VNC and we were able to analyse their growth behaviour and their were calculated from two biological replicates. Control brain n ¼ 355 NSCs (7 brain lobes); inscG44crb-RNAi, n ¼ 313 NSCs (7 brain lobes); inscG44ed-RNAi, n ¼ 300 NSCs (6 brain lobes); ed F72 n ¼ 1,169 (20 brain lobes). ARTICLE mitotic index. Expression of crb was not able to suppress reactivation, but was sufficient to strongly reduce growth and proliferation at 24 h ALH (Fig. 6c-g). Co-expression of crb and ed had no additive effect on growth suppression, but the mitotic index was further reduced (Fig. 6e-g). Thus, although we observed a strong reduction in growth and proliferation, ectopic crb and ed did not extend quiescence, which might be owing to the nutritional reactivatory signal overcoming crb input.
SHW discriminates between quiescent and non-quiescent NSCs.
Next we sought to investigate whether the SHW discriminates quiescent from non-quiescent NSCs in Drosophila. Analysing the growth behaviour and mitotic index of non-quiescent NSCs of the mushroom bodies (MBNBs) at 4, 24 and 48 h ALH and in nutritional deprivation unravelled that their growth and proliferation depends on nutrition and requires active regulation (Fig. 7a,b). Testing the SHW we observed constant nuclear Yorkie levels in MBNBs (Fig. 7c) and expression of Fj-lacZ, expanded and activity  of bantam (Fig. 4a and Supplementary Fig. 3a,b) confirming a continuous Yorkie activity. Indeed, in yki B5 mutants we could observe a lack of MBNBs growth and a marked reduction in the proliferative capacity of MBNBs at 48 h ALH (Fig. 7a,b,d) compared with wild-type MBNBs (Fig. 7e). We conclude that continuous Yorkie activity is necessary for MBNBs growth and proliferation and Yorkie activity discriminates between quiescent and non-quiescent NSCs in Drosophila.

Discussion
NSCs need to tightly control the balance between proliferation and quiescence, since deregulation can lead to tumour formation or premature depletion of the progenitor pool 1,3 . In order to orchestrate their behaviour according to the status of the organism they need to communicate with their surrounding microenvironment. Similar to the neurogenic niche in vertebrates 49 , processes of glial cells in insects enwrap NSCs to form an enclosed chamber known as the trophospongium 50 . It is still debated whether Drosophila NSCs are independent of niche signalling and indeed NSCs in culture undergo asymmetric cell division to self-renew and produce differentiating progeny 51,52 , exhibiting very similar behaviour as their counterparts in vivo, like progressing through an intrinsically regulated series of transcription factors (temporal transcription factor cascade) 53 and generating diverse cell types and lineages in vitro resembling the in vivo lineages in cell number and identity 54 . Conversely, loss of contact to the surrounding epithelium leads to a randomization of the mitotic spindle in isolated embryonic NSCs 55 and compromising Drosophila E-Cadherin function in niche glial cells impairs the mitotic activity of NSCs 56 . During quiescence and reactivation NSCs show a strong dependency on extrinsic signals from glial cells. Glial cells express and secrete quiescence promoting factors 13 , or in response to nutrition, activating factors 10,11 . We now show that niche glial cells express transmembrane proteins Crumbs and Echinoid that act in trans to activate the SHW in NSCs to maintain quiescence and suppress cell growth. Thus, like vertebrate adult NSCs, Drosophila NSCs show different degrees of dependency on niche signalling. During quiescence both NSC populations need extrinsic cues from the niche to maintain quiescence (for example, this work 10,11,57,58 ), whereas during the active-phase lineage progression maybe more cell-intrinsic, pre-programed and to a less degree depending on the niche 54,59 . The SHW and its effector Yorkie/YAP have been widely implicated in stem cell biology and organ size restriction. In the vertebrate liver progenitors, the hepatocytes, the SHW controls quiescence of these stem cells 60 . Combined loss of Mst1/2 (homologues of Drosophila Hippo) resulted in loss of YAP phosphorylation, leading to a massive overgrowth and hepatocellular carcinoma. Other regenerative tissues, like the skin or the intestine, also harbour stem cells and an involvement of the SHW was likewise shown [61][62][63][64][65]   inappropriate growth and proliferation of NSC in the Drosophila larvae. Similar to Drosophila, mouse adult quiescent NSCs (aNSCs) also show a prominent cell growth before the initiation of proliferation 59,66 . Thus, a similar mechanism of promoting quiescence by growth restriction might exist in adult vertebrate NSCs. Indeed, a recent molecular study on NSC quiescence showed that multiple SHW members like Lats2 (Warts homologue) or WWC2 (Kibra homologue) are upregulated in aNSCs, which after BMP4 exposure enter into a quiescence-like status in cell culture 57 . Moreover, our study demonstrates that a crosstalk between the niche glial cells and the stem cells via Crumbs and Echinoid activates the SHW to repress growth during quiescence. This might also be conserved in mouse aNSCs since Martynoga et al. 57 showed that upon BMP4-induced quiescence expression of crumbs2 (CRB2, Drosophila crumbs homologue) is also upregulated in aNSCs. Whether CRB2 is expressed in the vertebrate niche and activates the SHW in NSCs during quiescence still has to be shown. Nevertheless, given these similarities it is attractive to speculate that the Hippo pathway might act as a general regulator of NSC quiescence in vertebrates and invertebrates. In Drosophila two different populations of NSCs can be discriminated: NSCs that become significantly smaller and enter quiescence at the end of embryonic stages, and NSCs that constantly proliferate and do not go into quiescence. How this difference is established is not known up to now. Here we show that the activity of the transcriptional regulator Yorkie seems to be a major difference between these two populations of NSCs. Although quiescent NSCs have active Hippo signalling and thus no active Yorkie, the non-quiescent NSCs show constant nuclear Yorkie and constant expression of the known Yorkie-targets bantam and Four-jointed. We did not observe failure to enter into quiescence upon wts-RNAi or in trans-heterozygous hpo mutants and thus it seems less likely that SHW activation is needed to initiate quiescence.
Non-quiescent NSCs increase in size upon larval hatching and like in quiescent NSCs, this growth depends on nutrition and on Yorkie function. This also influences the proliferative capacity, since both populations of NSCs either showed no proliferation or exited proliferation prematurely. A similar function of YAP was described during vertebrate neurogenesis in the developing chick neural tube. YAP is highly expressed in NSCs and co-localizes with Sox2 a neural progenitor marker. Loss of YAP leads to premature differentiation, whereas overexpression leads to an increase in the progenitor pool and accelerated cell cycle progression 67 . More recently YAP expression was also found in the mouse ventricular zone in Sox2-positive progenitors 68 and, importantly, simultaneous depletion of YAP and FAT4 or Dachs1 rescued a prolonged neuroprogenitor cell proliferation phenotype in a mouse model for Van Maldergem syndrome 69 . Therefore, Yorkie/YAP emerges as an important regulator of NSC biology and it is therefore of great importance to identify the precise molecular mechanisms and target genes by which Yorkie/YAP promote growth, proliferation and stem cell identity in NSCs. Interestingly, in Drosophila we could uncover an essential difference between NSCs in the brain and the VNC. In both populations of stem cells Yorkie activates its well-established target bantam. Yet, loss of bantam severely impaired growth and proliferation of the VNC NSCs, whereas the effects were milder in brain NSCs. Thus, to fully understand the function of Yorkie in NSCs it will be important in the future to unravel the stem cell-specific target genes that are regulated by Yorkie in NSCs.  (Stock number:  33614), warts-RNAi (34064), crumbs-RNAi (38373), 14-3-3zeta-RNAi (31498). NSC-specific RNAi was performed with insc-GAL4 (w 1118 ; P{GawB}inscMZ1407), glial-specific RNAi was performed with repo-GAL4 (w 1118 ; P{GAL4}repo/TM6b, iab-lacZ). Both GAL4 driver lines carried UAS-CD8::gfp or UAS-CD4td::gfp and UAS-dicer2. For larval-restricted RNAi we combined the insc-GAL4, UAS-CD8::gfp with the tubP-GAL80[ts] (Bloomington Stock 7108) and embryonic phases were cultured at 18°C before shifting to 29°C just ALH. All other RNAi experiments were conducted at 29°C during larval life-embryonic phases were cultured at 25°C. Mutants alleles used were, ed F72 , hpo JM1 , hpo KC202 , yki B5 and banD 1 balanced over CyO, Pw[ þ mC] ¼ Dfd-EYFP2, CyO, P(GAL4-twi.G)2.2, P(UAS-2xEGFP)AH2.2 or TM6B, Pw[ þ mC] ¼ Dfd-EYFP3, Sb 1 Tb 1 ca 1 . UAS-yki S168A (Stock number: 28818), UAS-crb (5544) and fj-LacZ (6370) were obtained from the BSC. The fly stock carrying the crumbs-extracellular domain tagged with GFP (Crumbs::GFP-A/TM3) was a kind gift from Yang Hong 48 . For nutritional deprivation egg collection were made on and hatched larvae transferred to agar plates prepared with 1 l PBS, 10 g agar, 50 g sucrose. For well-fed larvae collection, flies were reared on standard Drosophila fly food and egg collections were made on and hatched larvae transferred to agar plates prepared with 1 l apple juice, 27 g agar supplemented with dry yeast.

Methods
Antibodies, in situ probes and immunohistochemistry. Immunohistochemistry experiments were performed as previously described 70 . In brief, larval CNSs were dissected in PBS, fixed in 4% formaldehyde. Washing steps were performed using PBS with 0.3% Triton X-100 (3 Â 5 min) and primary antibodies were incubated overnight at 4°C. Antibodies used were guinea pig anti-Deadpan In situ probe for crumbs was PCR-generated using the following forward primers: 5 0 -CGTTGGTGGCCAGAAATTGG-3 0 and 5 0 -CACAGTGCTGACCCT CGAAT-3 0 (5 0 -TAATACGACTCACTATAGGAGACCAC-3 0 ) as reverse primer ((XX) ¼ T7 sequence). After purification of the PCR product the in vitro transcription using T7 and the Dig RNA Labelling Mix (Roche) was used to generate the RNA in situ probe.
EdU incorporation assay. To detect mitotic activity using EdU incorporation we dissected larval CNS at appropriate time points and incubated them for 1 h in 10 mM EdU/PBS. CNSs were fixed for 15 min in 4% formaldehyde/PBS and Alexa Fluor azide was detected according to the manufacturer's instructions (Click-iT EdU Imaging Kit, Invitrogen).
Image acquisition and processing. Confocal images were acquired on a Leica SP5 confocal laser-scanning microscope using Â 63 glycine immersion objective lens or a Leica SP8 confocal laser-scanning microscope using Â 40 objective lens. All images represent single confocal sections except for images of EdU incorporation in ed F72 mutants that are projections of z-stacks. Images were processed using the Leica LAS, Adobe Photoshop and assembled in Adobe Illustrator. Cell size measurements and pH3-cell counts were done using the Leica LAS, statistical analysis were conducted using SigmaPlot. The medial cell body of NSCs were measured for cell diameter evaluation excluding the longest and the shortest axis of the cell.
Intensity measurements for Yorkie localization. The Leica LAS AF software was used to determine the pixel intensities of each channel across a cell. The values for each channel were exported to Excel and the whole cell and nuclear areas were determined by the signal intensity of the Deadpan staining (nucleus) and the Phalloidin staining (outer cell membrane). The intersection was defined as cytoplasm. According to this definition the Yorkie signal intensity values were separated into nuclear and cytoplasmic fraction and pixel intensities were averaged for each fraction. The ratio between the means was calculated as a measure for the relative amount of protein per sub-compartment. SigmaPlot was used for statistical analysis.