DEVELOPMENT AND STEM CELLS RESEARCH ARTICLE 1893

Development 138, 1893-1902 (2011) doi:10.1242/dev.064204 © 2011. Published by The Company of Biologists Ltd FatJ acts via the Hippo mediator Yap1 to restrict the size of neural progenitor cell pools Nick J. Van Hateren1,2,3,*, Raman M. Das2,3,*,†, Guillaume M. Hautbergue3, Anne-Gaëlle Borycki2, Marysia Placzek1,2,‡ and Stuart A. Wilson3,‡

SUMMARY The size, composition and functioning of the spinal cord is likely to depend on appropriate numbers of progenitor and differentiated cells of a particular class, but little is known about how cell numbers are controlled in specific cell cohorts along the dorsoventral axis of the neural tube. Here, we show that FatJ cadherin, identified in a large-scale RNA interference (RNAi) screen of cadherin genes expressed in the neural tube, is localised to progenitors in intermediate regions of the neural tube. Loss of function of FatJ promotes an increase in dp4-vp1 progenitors and a concomitant increase in differentiated Lim1+/Lim2+ neurons. Our studies reveal that FatJ mediates its action via the Hippo pathway mediator Yap1: loss of downstream Hippo components can rescue the defect caused by loss of FatJ. Together, our data demonstrate that RNAi screens are feasible in the chick embryonic neural tube, and show that FatJ acts through the Hippo pathway to regulate cell numbers in specific subsets of neural progenitor pools and their differentiated progeny.

KEY WORDS: Cadherin, Neural tube, Chick, RNAi, Screen, FatJ, Progenitor cells, Interneurons, Phenotype, Hippo pathway, Notch signalling, Morphogen

INTRODUCTION the greatest homology to dFat (Matakatsu and Blair, 2006) and Neural progenitor cells are patterned along the dorsoventral axis is expressed in a variety of tissues with active PCP signalling of the neural tube, proliferate, then migrate laterally and (Rock et al., 2005), including the kidney, where loss of Fat4 differentiate into defined neuronal classes (Kintner, 2002; alters orientated cell divisions and leads to kidney dysfunction. Bylund et al., 2003). The tight regulation of patterning, Fat4 additionally plays a role within the CNS: loss of Fat4 proliferation and differentiation ensures an appropriate balance expression in the cerebellum reduces the apical membrane of progenitor versus differentiated cells that is key to tissue compartment, suggesting a role in apical-basal polarity (Ishiuchi homeostasis. Signalling pathways mediating these events are et al., 2009). Furthermore, mouse Fat4–/– embryos display a therefore crucial to the proper size, composition and functioning wider spinal cord than wild-type littermates, suggesting that of the nervous system (Dahmane et al., 2001). However, an FatJ/Fat4 might play a role in spinal cord development (Saburi outstanding question is that of how appropriate progenitor and et al., 2008). differentiated cell numbers are exacted. Recent evidence has We have previously developed a system for robust knockdown implicated the Hippo pathway in such regulation, showing that of chicken genes (Das et al., 2006). In the present study, we employ components of the Hippo signalling pathway govern neural this system to knockdown the expression of all cadherin domain- progenitor cell number (Cao et al., 2008). containing genes expressed within the chicken neural tube and Cadherin proteins are extracellular proteins that play important thereby analyse cadherin gene function during neural development. roles in cell adhesion and cell signalling (Halbleib and Nelson, Our unbiased approach defines a number of cadherin genes that 2006). The Fat cadherins comprise a subfamily containing the may play a role in cell patterning, proliferation and differentiation largest proteins in the superfamily (Tanoue and Takeichi, 2005). within the developing spinal cord. Of these, we have studied one, Drosophila Fat (dFat) was first identified as a putative tumour FatJ, in detail. FatJ is localised to progenitor cells within suppressor gene involved in planar cell polarity and tissue size intermediate regions along the dorsoventral axis of the neural tube. regulation via the Hippo signalling pathway (Mahoney et al., Our studies reveal that FatJ acts via the Hippo pathway mediator 1991; Matakatsu and Blair, 2004; Willecke et al., 2006). Of the Yap1 to regulate the size of the dp4-vp1 progenitor cell pools and four vertebrate orthologues of dFat, Fat4 (also called FatJ) shows hence differentiated Lim1+/Lim2+ interneuron numbers. This study is the first large-scale RNAi screen to be performed in a whole vertebrate organism and reveals an important role for FatJ cadherin, acting via the Hippo signalling mediator Yap1, to control the 1 MRC Centre for Developmental and Biomedical Genetics, University of Sheffield, regionally restricted proliferation and differentiation of specific Sheffield, S10 2TN, UK. 2Department of Biomedical Science, University of Sheffield, Sheffield, S10 2TN, UK. 3Department of Molecular Biology and Biotechnology, interneuron classes. University of Sheffield, Sheffield, S10 2TN, UK.

*These authors contributed equally to this work MATERIALS AND METHODS † Present address: Division of Cell and Developmental Biology, College of Life Microarray analysis of genes expressed in the chicken neural tube Sciences, University of Dundee, Dundee, DD1 5EH, UK Total RNA extracted from stage 22 chicken spinal cords (100 embryos) ‡Authors for correspondence ([email protected]; [email protected]) was used to interrogate the Affymetrix whole chicken genome chip (ARK Genomics). Microarray data are available in the ArrayExpress database

Accepted 23 February 2011 (www.ebi.ac.uk/arrayexpress) under Accession Number E-TABM-701. DEVELOPMENT 1894 RESEARCH ARTICLE Development 138 (10)

Labelled cDNA targets were generated by reverse transcription of total RNA with modified dNTPs conjugated to aminoallyl. A secondary coupling reaction was then carried out to conjugate Cy3 dye to the cDNA. Dye-coupled cDNA was then used to probe genes on the microarray. This led to the identification of ~14,000 expressed genes, which were sorted according to identifiable protein domains using the ENSEMBL chicken genome assembly (version 2.1, May 2006 release). Forty of these genes contained cadherin domains.

RNAi vectors RNAi targeting vectors were designed as previously described (Das et al., 2006). However, the RNAi vectors used in this study were created using an updated RNAi vector (pRFPRNAiC) containing a MluI site instead of the AflII site in the microRNA expression cassette. Initially, two siRNA targets were generated for each cadherin and subsequently a third siRNA target was generated for each cadherin to ensure a maximal probability of the vectors producing a gene-specific knockdown. It has been suggested that employing at least two independent RNAi vectors for each target gene should significantly reduce the incidence of potential off-target effects (Echeverri et al., 2006). The target siRNA sequences for each cadherin and for Yap1 and Tead4 are listed in Table S1 in the supplementary material. All the RNAi plasmids described here are available as a community resource from our clone distributors (ARK-Genomics.org).

RNAi screening Two RNAi vectors for each gene were pooled and electroporated into stage 11-12 neural tubes. A third RNAi vector for each gene was subsequently electroporated into separate embryos. Electroporation procedure was as previously described (Das et al., 2006); however, the maximum DNA concentration in all electroporations was 0.5 g/l. After 48 hours of incubation, the embryos were analysed for defects caused by RNAi targeting of the particular cadherin. Primary phenotypic analysis was performed by analysing the location and morphology of RFP-positive cells. As a secondary screen, the expression patterns of Pax6, Pax7, Islet1, Lim1/Lim2, Nkx2.2 and Lmx1b (DSHB) were analysed by immunofluorescence. For all embryo sections studied, the electroporated side of the neural tube was compared with the contralateral unelectroporated side as a control. In addition, a luciferase RNAi vector (Das et al., 2006) was used as a negative control to ensure that expression of the RNAi cassette or of RFP was not responsible for the observed phenotype. The phenotypes detailed in this screen were observed in at least five different embryos targeted with the same RNAi vectors.

Embryo fixation, immunohistochemistry and in situ hybridisation Fig. 1. A range of phenotypes are observed following loss of Embryo fixation, sectioning and immunohistochemistry were performed as cadherin function. Expression of red fluorescent protein (RFP) from previously described (Das et al., 2006). Antibodies used were against Pax6, RNAi vectors allows cell migration and morphology to be assessed. Pax7, Pax2, Evx1, Nkx2.2, Isl1, Lim1/Lim2, Lmx1b, N-cadherin, Nkx6.1 (A)RNAi-mediated knockdown of Fat2 cadherin causes an and En1 (DSHB); mouse anti--catenin (Sigma); rabbit anti-Dbx1 (Pierani accumulation of electroporated cells in the mantle zone (mz) of the et al., 1999); anti-Chx10 (S. Morton, Columbia University, NY, USA); neural tube. (B)A similar phenotype is observed after knockdown of rabbit anti-Pax6 (Covance); rabbit anti-Olig2 polyclonal and rabbit anti- Notch1 (Das et al., 2006), whereas cells targeted with luciferase RNAi RFP polyclonal (Chemicon); rabbit anti-phospho-histone H3 polyclonal (C) are located throughout the neural tube in both ventricular zone (vz) (Upstate); rabbit anti-GFP polyclonal (BD Bioscience); anti-Crb2 (P. and mantle zone. (D)Fat1 RNAi results in a reduction in size and cell Rashbass, University of Sheffield, UK); rabbit anti-pYap1 polyclonal (Cell number (32% fewer cells) in the electroporated side of the neural tube. Signaling Technology); and Alexa405-, Alexa488- and Alexa555- (G)Expression domain of Pax6 is reduced relative to the contralateral conjugated secondary antibodies were obtained from Molecular Probes side. (E,H)Loss of N-cadherin produces a decrease in cell number on (Invitrogen). Whole-mount in situ hybridisation was performed as the electroporated side, together with disruption of the apical surface previously described (Ohyama et al., 2005; Das et al., 2006) using of the neural tube, as marked by loss of -catenin expression (H, digoxigenin (DIG)-labelled (Roche) RNA probes. DIG-labelled antisense arrows). (F,I)Protocadherin 19 RNAi leads to a 50% increase in the riboprobes were generated from chicken EST plasmids ChEST187n19 number of cells on the electroporated side of the neural tube and an (FatJ); ChEST427d5 (Yap1) and ChEST580f24 (Tead4) [by linearization expansion of Pax6 expression compared with the contralateral side (I). with NotI and transcription with T3 RNA polymerase (Promega)]. After Some electroporated cells also migrate into the unelectroporated side sufficient staining development, embryos were cryosectioned at 30 m as of the neural tube (arrows in F), suggesting there may be some loss of described previously (Das et al., 2006). roof plate integrity. (J,K)RNAi-mediated knockdown of protocadherin 10 leads to a dorsal shift in expression of Pax6 (J) and Olig2 (K). BrdU incorporation studies (L,M)Knockdown of FatJ cadherin leads to an increased number of Neural tubes were electroporated as described above. After 48 hours Lim1+/Lim2+ cells. (N)This increase is spatially restricted to intermediate incubation at 38°C, 50 l of 0.01 M BrdU (Sigma) was injected under the regions of the neural tube (bottom panel, arrows); dorsal Lim1/Lim2-

vitelline membrane, the egg resealed and placed at 38°C. After 30 minutes expressing cells are unaffected by FatJ RNAi (N, top panel). DEVELOPMENT Patterned proliferation mediated by FatJ cadherin RESEARCH ARTICLE 1895 further incubation, embryos were harvested, fixed and cryosectioned as to the mantle zone (Fig. 1B, Fig. 2B) (Das et al., 2006), raising the previously described (Das et al., 2006) and immunohistochemistry was possibility that these cadherins may function in the same pathway. performed using 1/1000 mouse anti-BrdU monoclonal antibody (Sigma). We investigated this possibility by co-electroporating a Microscopy and image acquisition constitutively active form of Notch1 (Notch intracellular domain, Epifluorescence images were obtained using a Leica DM5000B upright NICD), shown previously to rescue the Notch RNAi phenotype microscope and confocal images were obtained using a Zeiss LSM510 (Kopan et al., 1996), with the Fat2 RNAi construct. Co- Meta. Section in situ images were obtained under Nomarski optics on a electroporation of NICD rescues the premature differentiation Leica DM-R microscope using a Leica digital camera. caused by Fat2 RNAi, and electroporated cells were now found throughout the neural tube in both the ventricular and mantle zones Co-electroporation of Notch intracellular domain (Fig. 2D). This suggests that Fat2 functions in the same pathway The pCS2-ICD plasmid expressing the intracellular domain of mNotch1 (NICD) was a kind gift from D. Henrique (IMM, University of Lisbon, as Notch1 to control neural progenitor differentiation. Portugal) and has been previously shown to constitutively activate Notch1 Knockdown of 14 cadherins caused a reduction in size of the signalling (Kopan et al., 1996). The plasmid was co-electroporated with electroporated side of the neural tube (Fig. 1D,E; see Fig. S1 in the the RNAi vectors [1 g/l NICD + 0.5 g/l of the relevant RNAi supplementary material), suggesting a decrease in cell number. No vector(s)] at stage 11-12 and the resulting phenotype was analysed at stage changes in dorsoventral (DV) patterning were detected; instead, the 25 as detailed above. domain of each homeodomain protein examined was small relative to the contralateral side (Fig. 1G; see Fig. S1 in the supplementary Western blotting Neural tubes were electroporated at stage 11-12 with FatJ RNAi or Luc material); this phenotype was not observed following introduction RNAi vectors and harvested after 72 hours incubation at 38°C. The of luciferase RNAi (see Fig. S1 in the supplementary material). intermediate region of the electroporated side of six or more neural tubes Knockdown of N-cadherin caused a similar defect, and additionally was dissected and lysed in Reporter Gene Lysis buffer (Roche). Each caused a loss of -catenin expression at the apical surface of the protein lysate (20 g) was separated by SDS-PAGE in a 10% neural tube (Fig. 1E,H) as observed previously for a mutation in polyacrylamide gel, transferred to a nitrocellulose membrane and western this gene in zebrafish (Lele et al., 2002). By contrast, knockdown blotting was performed using 1/1000 rabbit anti-pYap1 antibody (Cell of protocadherin 19 and cadherin 10 resulted in an increased Signalling Technology) in 10% BSA/1ϫTTBS followed by incubation number of cells in the neural tube and enlargement of the with 1/10,000 anti-rabbit HRP-conjugated secondary antibody and electroporated side. Concomitantly, there was a relative expansion detection by ECL. Each membrane was subsequently incubated with of the domain of expression of each homeodomain protein 1/5000 mouse anti-tubulin antibody (Sigma) and 1/5000 rabbit anti-RFP polyclonal (Chemicon) to evaluate protein amounts loaded and degree of examined (Fig. 1F,I; see Fig. S1 in the supplementary material). electroporation with RNAi vectors. Signal density was determined using A minor set of cadherins, when downregulated, caused very Quantity One software (Biorad). localised effects along the DV axis. RNAi-mediated knockdown of protocadherin 10 (Pcdh10) led to a change in DV patterning with

RESULTS Cadherin genes influence neural tube development We identified cadherin domain-containing genes (cadherins) expressed in the embryonic chick neural tube by probing whole chicken genome microarrays with RNA isolated from stage (St) 22 neural tubes. The analysis identified 40 genes containing at least one cadherin domain expressed in the neural tube (see Table S1 in the supplementary material). To investigate the role of each cadherin in neural tube development, we generated three RNAi vectors for each gene (see Table S2 in the supplementary material), electroporated each into St 11-12 neural tubes and analysed embryos after 48 hours (St 25). As a primary phenotypic analysis, we examined the location and morphology of electroporated cells, visualised through red fluorescent protein (RFP) expression. As a secondary screen, expression patterns of the progenitor markers Pax6, Pax7 and Nkx2.2, and differentiation markers Islet1, Lim1/Lim2 and Lmx1b were analysed. Electroporated cells were compared with cells on the contralateral unelectroporated side and with cells electroporated with an RNAi control vector targeting luciferase. Electroporation of RNAi vectors targeting 18 cadherins caused no detectable Fig. 2. Rescue of Fat2 RNAi phenotype by NICD. (A)RNAi-mediated phenotype (see Table S1, Fig. S1 in the supplementary material). knockdown of Fat2 cadherin causes an accumulation of electroporated However, 22 others gave phenotypes that could be broadly cells in the mantle zone of the neural tube. (B,C)A similar phenotype is classified (see Table S1 in the supplementary material). observed after knockdown of Notch1 (Das et al., 2006) (B), whereas Knockdown of four cadherins (Fat2, brain cadherin, VE- cells targeted with luciferase RNAi (C) are located throughout the neural tube in both ventricular zone (vz) and mantle zone. (D,E)Co- cadherin and cadherin 18) resulted in accumulation of RFP+ cells electroporation of the Notch intracellular domain (NICD) rescues the in the mantle zone, a markedly different effect to that normally Fat2 knockdown (D), as well as the Notch1 knockdown phenotype (E). observed (Fig. 1A,C; see Fig. S1 in the supplementary material). The rescue is evident by the location of electroporated (RFP+) cells in This effect phenocopies knockdown of Notch1, where premature both the mantle zone and ventricular zone (compare A with D). This

differentiation of cells results in their migration from the ventricular suggests that Fat2 is involved in Notch1 signalling. DEVELOPMENT 1896 RESEARCH ARTICLE Development 138 (10) a dorsal shift in the expression of both Pax6 (Fig. 1J) and Olig2 by an ‘off-target’ effect (see Echeverri et al., 2006), we compared (Fig. 1K). Neither marker appeared to be expanded; instead, the the ability of each FatJ RNAi vector to increase production of entire domain of each appeared dorsally shifted. The size of the Lim1+/Lim2+ interneurons (Fig. 3G-I). Each vector caused at least electroporated side of the neural tube was unchanged compared a 20% increase in the number of cells expressing Lim1+/Lim2+, with the contralateral side, suggesting that the effects observed with the most effective vector (FatJ RNAi C) producing an were likely to be due to a change in patterning. average of 32% more Lim1+/Lim2+ cells (Fig. 3M). We conclude Finally, FatJ RNAi appeared to produce a small, but robustly that FatJ regulates the number of Lim1+/Lim2+ neurons in the reproducible, increase in the number of Lim1+/Lim2+ cells. This neural tube. phenotype was apparent in only a subset of Lim1+/Lim2+ cells, Given that Lim1/Lim2 is expressed in a wide set of interneurons notably those in medial regions along the dorsoventral axis (Fig. (dl2, dl4, dl6, v0 and v1 subclasses), we next defined more 1L,M, arrows). Lim1+/Lim2+ cells in dorsal regions of the neural precisely which interneuron types were affected by FatJ RNAi, by tube appeared unaffected by electroporation of FatJ RNAi vectors, analysing embryos for Isl1, Pax2, Lmx1b, Evx1, En1 and Chx10 whereas those in intermediate regions were expanded in number expression, which mark dI3, dI4+6, dI5, v0, v1 and v2 (Fig. 1N). Together, this raises the possibility that FatJ knockdown interneurons, respectively (Fig. 4). This analysis revealed that affects cell proliferation/differentiation, but that its effects are interneurons in domains dI4, dI5, dI6, v0 and v1 were affected localised to a regionally defined domain of the neural tube. We (Fig. 4B-F), whereas those in domains dI3 and v2 were not (Fig. therefore focused our attention on this cadherin, to confirm the 4A,G). We conclude that FatJ knockdown results in the specific increase in Lim1+/Lim2+ cells and clarify the mechanism that expansion of differentiated dI4-v1 interneurons. governs Lim1+/Lim2+ cell numbers. We first confirmed that all three FatJ RNAi vectors, which FatJ expression is spatially restricted target different regions of FatJ mRNA (Fig. 3; see Table S2 in the One possible explanation for the effect of FatJ RNAi in only a supplementary material), knock down FatJ expression and subset of Lim1+/Lim2+ cells is that FatJ itself is spatially confirmed that luciferase RNAi vector has no effect. To further restricted to intermediate regions of the neural tube. To examine ensure that the effects of the FatJ RNAi vectors were not mediated this, we analysed FatJ mRNA expression over the period St10-

Fig. 3. Knock down of FatJ mRNA by RNAi vectors and FatJ expression pattern. (A-F)RNAi-mediated knockdown of FatJ was verified by in situ hybridisation. Electroporation with FatJ RNAi vectors A and B led to a marked reduction in FatJ expression, restricted to the electroporated side of the neural tube (A-C), whereas luciferase RNAi vectors had no effect on the expression of FatJ mRNA (D-F). (G-I)Similar levels of FatJ mRNA knockdown were produced by the other FatJ RNAi vectors (data not shown) and all three RNAi vectors produced a similar expansion of the Lim1/Lim2 domain when electroporated individually.Average percentage increase of Lim1+/Lim2+ cells shown, error bars are s.e.m. (J) At St 10, high levels of FatJ mRNA are detected in the ventral somites with lower levels of expression throughout the neural tube with the exception of ventral and dorsal-most regions. nt, neural tube; scl, sclerotome. (K,L)At St 19 and 22, FatJ is expressed at higher levels in the intermediate region of the neural tube. Expression is restricted to a diamond-shaped domain that appears to correspond to the dp4-vp1 progenitor domains. (M)Quantification of the number of Lim1+/Lim2+ cells in the neural tube of these embryos reveals all three vectors produced an increase of ~20-30% more Lim1+/Lim2+

cells compared with the unelectroporated side. Data are mean±s.e.m. DEVELOPMENT Patterned proliferation mediated by FatJ cadherin RESEARCH ARTICLE 1897

Fig. 4. Detailed analysis of increase in interneurons following FatJ knockdown. (A)Following loss of FatJ, there is no change in the number of cells expressing the marker Islet1 (Isl1), which marks the dI3 class of interneurons. (B-F)Loss of FatJ results in an increase in specific interneuron number, relative to the contralateral side: 9.52% more Pax2+ cells (B; dI4 and dI6 interneuron); 16.11% more Lmx1b+ cells (C; dI5 interneurons); 15.24% more Lim1+/Lim2+ cells (D; dI2, dI4, dI6, v0 and v1 interneuron classes; 20.00% more Evx1+ cells (E; marking v0 interneurons); 17.91% more En1+ cells (F; v1 interneurons). (G)Loss of FatJ does not increase the number of cells expressing Chx10 (v2 interneuron class). Therefore, the increase in interneuron number is observed from the dI4 to v1 interneurons. Average cell counts for each marker on both sides of the neural tube are shown graphically on the right of the figure (error bars show s.e.m.). Statistical significance of the cell counts was determined using a paired

Student’s t-test. DEVELOPMENT 1898 RESEARCH ARTICLE Development 138 (10)

St22 and found that its expression is restricted to intermediate FatJ knockdown alters progenitor cell number regions along the DV axis. Expression is limited to Two alternative possibilities could account for the enhanced ventricular/subventricular zone areas and appears to correlate differentiation of dI4-v1 interneurons after FatJ knockdown. First, with dp4-vp1 progenitor regions, overlapping with expression a decrease in FatJ activity could lead to acceleration in the domains for progenitor markers Pax6, Pax7 and Dbx1, but not differentiation of progenitor cells in the dp4-vp1 domain. In this Nkx6.1 (Fig. 5A-F, Fig. 3J-L). This pattern of expression is case, knockdown of FatJ should lead to a decrease in proliferating reminiscent of that of Pax6, which is regulated by Shh and progenitors within these domains. An alternate possibility is that Wnt/BMP signalling and the Gli activity gradient (Briscoe et al., FatJ governs the proliferating progenitor cells themselves, a 2000; Timmer et al., 2002). decrease in its activity leading to greater numbers of progenitor

Fig. 5. Loss of FatJ increases progenitor cell number and does not cause premature differentiation. (A-F)Comparison of FatJ expression (A) with the progenitor markers Pax6 (B), Pax7 (C), Dbx1 (D) and Nkx6.1 (E). A schematic of the defined progenitor and differentiated neuron domains is shown, for reference, in F. (G,H)Loss of FatJ expression does not alter the domain of the neural progenitor markers Dbx1 (G, green cells), Nkx6.1 (G, blue cells) or Pax6 (H). In each case, the domain of expression is similar on the electroporated side and the unelectroporated side of the neural tube. (I)BrdU incorporation is increased in cells targeted by FatJ RNAi, resulting in ~14% more BrdU-positive cells within the normal FatJ expression domain (boxed region). (J)There is a ~20% increase in mitotic cells marked by phospho-histone H3 (pH3) within the FatJ expression domain (boxed region). (K,L)Quantification of BrdU+ (K) and pH3+ (L) cells reveals statistically significant increases in number of mitotically active cells after FatJ RNAi. (M-O)Detailed quantification shows that FatJ knockdown leads to statistically significant increases in the number of Pax6+ (O) and Dbx1+ (N) cells, but does not change the number of Nkx6.1+ cells (M), which lie ventral to the domain of FatJ expression. Cell counts show average cell number±s.e.m.; statistical significance was determined using a paired Student’s t-test. (P)Loss of FatJ expression leads to an increased number of En1-expressing cells (left-hand panel, electroporated; right-hand panel, control); however, none of the En1+ cells co-express Pax6

(arrows). DEVELOPMENT Patterned proliferation mediated by FatJ cadherin RESEARCH ARTICLE 1899

Fig. 6. Expression of neural progenitor and apical proteins following FatJ knockdown. (A,B)Loss of FatJ expression does not alter the domain of the neural progenitor markers Pax7 (A) or Nkx2.2 (B). (C,D)In each case, the domain of expression is similar on the electroporated side and the unelectroporated side of the neural tube. Loss of FatJ does not affect the expression of Crumbs2 (C) or N-cadherin (D), as both proteins are markers of the apical cell surface; this suggests that the increase in progenitor cells is not due to disrupted apicobasal polarity. All sections are counterstained with DAPI to show nuclei.

cells, and hence greater numbers of differentiated interneurons. In ventricular zone after FatJ RNAi (Fig. 5I,K; Table 2). Similarly, we this case, knockdown of FatJ should lead to an increase in observed a 21.92% increase in the number of mitotic cells marked proliferating progenitors. by phospho-histone H3 (Fig. 5J,L; Table 2). These data suggest that To distinguish these possibilities, and determine whether FatJ the increased number of differentiated interneurons can be explained expression is required for differentiation, or for the by an increased number of progenitors within the corresponding establishment/maintenance of proliferating progenitors, we progenitor pool. To further test this idea, we performed double quantified the number of progenitor cells in defined subdomains labelling analyses with Pax6 and En1. An increased number of En1+ along the DV axis of the neural tube after FatJ RNAi. Loss of FatJ cells was detected after FatJ knockdown, but no double-positive cells did not affect the general pattern of any of the markers examined were detected (Fig. 5P), indicating that En1+ cells differentiated (Fig. 5G,H; Fig. 6A,B). Moreover, loss of FatJ expression did not properly and did not simply express En1 precociously. affect the number of progenitor cells in the vp2 domain (judged by FatJ is the closest orthologue of the Drosophila Fat (dFat) Nkx6.1 expression; Fig. 5G,M; Table 1), the vp3 domain (Nkx2.2 gene, which plays a crucial role in planar cell polarity (Fanto et expression; Fig. 6B; Table 1) or the dp3 domain (low expression of al., 2003; Matakatsu and Blair, 2006) and interacts with Pals1 to Pax6; Pax7 expression) (Fig. 5H, Fig. 6A, Table 1). Thus, progenitor organise the apical membrane domain (Ishiuchi et al., 2009). To cells lying outside the FatJ expression domain were unaffected by its determine whether the increase in progenitor cell number is reduction. By contrast, a small but consistent increase (10-20%, caused by defects in progenitor cell polarity, we examined Table 1) in the number of Dbx1+ (Fig. 5G,N), Pax6 (strong positive) expression of the apical proteins N-cadherin, Crumbs2 and Par3. (Fig. 5H,O) and Irx3+ cells (Table 1) was detected. This suggests Loss of FatJ did not appear to affect expression of any of these that reduction in FatJ expression leads to an increased number of markers (Fig. 6C,D; data not shown), suggesting that apical- progenitors in the dp4-vp1 domains of the neural tube. basal polarity is unaffected by loss of FatJ. Our data indicate that To begin to address the mechanism underlying the increase in FatJ limits the number of progenitor cells within its domain of progenitor number, we analysed BrdU incorporation. A 14.65% expression through a mechanism other than a disruption in increase in the number of proliferating cells was detected in the apicobasal polarity.

Table 1. Number of cells expressing progenitor markers after FatJ RNAi Average number of cells Marker Number of sections Electroporated Unelectroporated Change (%) s.e.m. (electroporated) s.e.m. (unelectroporated) Pax7 5 127.60 128.00 –0.31 14.10 15.16 Irx3 5 163.80 147.20 11.28 18.73 20.71 Dbx1 6 29.50 25.17 17.22 2.95 2.61 Pax6 12 175.25 147.33 18.95 18.17 15.56 Nkx6.1 3 79.33 78.67 0.84 5.81 5.78 Nkx2.2 3 26.67 27.67 –3.61 2.60 3.38 DEVELOPMENT 1900 RESEARCH ARTICLE Development 138 (10)

Table 2. Number of mitotic cells after FatJ RNAi Average number of cells Marker Number of sections Electroporated Unelectroporated Change (%) s.e.m. (electroporated) s.e.m. (unelectroporated) BrdU 5 39.25 33.50 14.65 2.72 0.93 pH3 17 8.59 6.71 21.92 0.74 0.64

FatJ controls cell proliferation through the Hippo after FatJ RNAi. Therefore, we are unable to distinguish whether mediator Yap1 FatJ operates through the established Hippo pathway, or via a Drosophila Fat controls tissue size by activating the Hippo parallel pathway that links FatJ and pYap1. Nonetheless, taken pathway, leading to phosphorylation of the transcriptional activator together, our data show that loss of downstream Hippo components Yorkie (Yki) (Willecke et al., 2006). Yki normally associates with can rescue the defect caused by loss of FatJ and implicates FatJ- the transcription factor Scalloped (Sd) to promote cell proliferation control of Hippo pathway mediators as a mechanism for limiting (Wu et al., 2008). Activation of the Hippo pathway sequesters Yki the number of interneurons differentiating in the dp4-vp1 region of in the cytoplasm via Yki phosphorylation or by direct binding of the neural tube. Yki by the Hippo pathway components Expanded and Warts, thereby preventing association of Yki with Sd and stopping cell DISCUSSION proliferation (Zhang et al., 2008; Oh et al., 2009). Intriguingly, the Our analyses provide proof-of-principle for large-scale RNAi Hippo pathway has recently been shown to control neural screening in the chick neural tube and provide insight into those progenitor cell number in the neural tube and expression of cadherins whose RNAi-mediated knockdown leads to early defects dominant repressor versions of the vertebrate orthologues of Yki in size, proliferation, cell differentiation and patterning of gene and Sd (Yap1 and Tead1, respectively) can produce an increase in expression. The large size of cadherin genes can preclude analysis the number of Lim1+/Lim2+ cells, similar to the phenotype of their roles through gain-of-function approaches, highlighting observed in this study (Cao et al., 2008). This raises the possibility further the importance of the RNAi approach. Our studies that FatJ acts through the Hippo pathway to control the number of complement and support studies that document a role for cadherin cells within the dp4-vp1 domain. To test this, downstream genes in early neural tube development, for example, showing components of the Hippo pathway were knocked down that N-cadherin is required to maintain the integrity of the simultaneously with FatJ knockdown. RNAi mediated knockdown neuroepithelium (Lele et al., 2002). In addition, our studies of Yap1 was confirmed by in situ hybridisation: electroporation of highlight the temporal effects of cadherins in neural tube Yap1 RNAi constructs produced a significant loss of Yap1 development: our screen did not reveal early roles for cadherins expression within cells expressing the RNAi constructs (Fig. 7A), previously shown to affect later development of the spinal cord, whereas expression of luciferase RNAi vectors had no effect on including late migration of neural crest cells (Coles et al., 2007), Yap1 expression (Fig. 7B). Notably, Yap1 expression is observed sorting of cell pools (Price et al., 2002) and synapse formation (Bao solely in the ventricular/subventricular zones of the neural tube; et al., 2007). therefore, the FatJ expression domain comprises a subset of Yap1- Our detailed analysis of the FatJ knockdown phenotype provides expressing cells. Knockdown of Yap1 (Fig. 7C,F) and Tead4 (the insight into a novel early role for cadherin function within the CNS, closest vertebrate orthologue of Sd, not shown) alone had no revealing a mechanism to govern the balance of regionally significant affect on the number of cells expressing Lim1/Lim2. restricted progenitor pool cells and cognate interneuron numbers. However, knockdown of Yap1 or Tead4 together with FatJ (Fig. Local interneuron circuits play a major role in coordinating the 7D,F) rescued the increase in Lim1+/Lim2+ cells detected after sensory-motor circuits that characterise the spinal cord. The knockdown of FatJ alone (Fig. 7E,F). To further confirm this number of interneurons of a particular class is likely to be crucial rescue, dominant negative (DN) Yap1 or Tead4 constructs [detailed in achieving appropriate function of selective sensory-motor in Cao et al. (Cao et al., 2008)] were co-electroporated with the circuits, and our studies provide insight into the manner in which FatJ RNAi vector. In both cases, these were able to rescue the appropriate numbers of Lim1+/Lim2+ interneurons are generated. increase in Lim1+/Lim2+ cells observed after knockdown of FatJ Our data show that FatJ restricts the size of progenitor pools in alone (Fig. 7G). which it is expressed. Loss-of-function of FatJ leads to a consistent To further investigate regulation of the Hippo pathway by FatJ, increase in progenitor cell number. These appear to differentiate we compared the levels of phosphorylated Yap1 (pYap1) after FatJ along their normal route, as evidenced by a consistent increase in or Luciferase RNAi. Western blotting revealed decreased levels of appropriate differentiated interneuron subtypes. Mouse Fat4–/– pYap1 following FatJ RNAi when compared with Luc RNAi embryos display a wider spinal cord than do wild-type littermates, control samples (Fig. 7H). Similarly, decreased pYap1 levels were suggesting that the role of FatJ/Fat4 in neural progenitor expansion observed within the FatJ expression domain on neural tube sections might be conserved across vertebrate species (Saburi et al., 2008). after FatJ RNAi but not after Luciferase RNAi electroporation (Fig. Intriguingly, our data reveal that FatJ restricts progenitor cell 7I,J). Notably, the decrease in pYap1 was most pronounced at the numbers via mediators of the recently discovered Hippo signalling lateral edge of the ventricular zone (arrows in Fig. 7I) adjacent to pathway. Originally identified in Drosophila, activation of the the mantle zone. Hippo pathway by dFat prevents the transcriptional activation of These data suggest that loss of FatJ signalling causes a decrease genes such as CyclinE and Diap1 that promote cell proliferation in phosphorylation of the Hippo mediator Yap1. The upstream and prevent apoptosis (Wu et al., 2003), and in this manner, Hippo pathway kinases Mst1/2 and Lats1/2 have been shown to prevents excessive tissue growth. The Hippo pathway is conserved regulate neuronal progenitor proliferation by inhibiting Yap1 in vertebrates with one or more orthologues of all components in activity through Yap1 phosphorylation (Cao et al., 2008); however, the pathway. Several of these orthologues can rescue the

we were unable to detect any reduction in the levels of pMst1/2 corresponding Drosophila mutant phenotype (Wu et al., 2003; Lai DEVELOPMENT Patterned proliferation mediated by FatJ cadherin RESEARCH ARTICLE 1901

Fig. 7. The FatJ knockdown phenotype can be rescued by knockdown of Yap1 and Tead4. (A)Knockdown of Yap1 by RNAi was verified using in situ hybridisation. A significant knockdown of Yap1 mRNA, normally expressed widely in the ventricular zone with the exception of the cells immediately dorsal to the floor plate, was observed following electroporation of Yap1 RNAi vectors (arrow). (B)Luciferase RNAi does not affect Yap1 expression. (C)RNAi-mediated knockdown of Yap1 has no significant effect on the number of Lim1+/Lim2+ cells in the neural tube. (D,E)Simultaneous knockdown of FatJ and Yap1 rescues the phenotype observed with FatJ knockdown (shown for reference in E). No difference is detected in the number of Lim1+/Lim2+ cells when compared with the unelectroporated control side. (F)Quantitative analysis showing knockdown of either Yap1 or Tead4 is able to rescue the increase in Lim1+/Lim2+ cells caused by loss of FatJ expression. FatJ RNAi leads to 16.77% more Lim1+/Lim2+ cells, whereas loss of Yap1 or Tead4 at the same time as loss of FatJ rescues the phenotype. Error bars are s.e.m., *P<0.05 (paired Student’s t-test). (G)Electroporation of dominant negative (DN) Yap1 or Tead4 constructs together with FatJ RNAi vectors results in normal numbers of Lim1+/Lim2+ interneurons, therefore rescuing the increase in Lim1+/Lim2+ cells observed with FatJ RNAi alone. Data are mean±s.e.m. (H)Western blot analysis of pYap1 levels 72 hours after electroporation reveals a decrease in pYap1 levels after FatJ RNAi when compared with the Luc RNAi sample. pYap1 and tubulin levels were analysed using Quantity-one software after collection using charge coupled device (CCD) camera detection of chemiluminescent signals and pYap1 values were normalised to tubulin levels. (I)pYap1 levels are reduced within the FatJ expression domain 72 hours after FatJ RNAi, particularly at the lateral edge of the ventricular zone adjacent to the mantle zone (arrows). The lateral boundary of the ventricular zone is indicated by a broken line. (J)pYap1 expression is unchanged 72 hours after electroporation with Luc RNAi vectors. et al., 2005; Wei et al., 2007), suggesting a conserved role in reveal that regulation of the Hippo pathway mediator Yap1 in the growth control. Mutation of many of these genes has been vertebrate neural tube occurs through FatJ, the closest vertebrate implicated in human cancers (McClatchey and Giovannini, 2005; homologue to dFat. Together, our data suggest that FatJ mediates Harvey and Tapon, 2007; Yokoyama et al., 2008), Fat4/FatJ has a robust mechanism for the acquisition of appropriate numbers of recently been implicated as a breast tumour suppressor gene (Qi et cells in progenitor pools, and hence appropriate numbers of distinct al., 2009), and Yap1 expression and nuclear localisation is interneuron subtypes, along the DV axis of the neural tube. upregulated in Shh-associated medulloblastomas (Fernandez et al., Acknowledgements 2009). Recent studies show that the Hippo pathway controls the We thank Gareth Howell and members of the ENSEMBL team at EBI for help number of neuronal progenitors in the neural tube by influencing with analysis of the microarray data, and Kyoji Ohyama for help with

cell proliferation and apoptosis (Cao et al., 2008). Our studies now dissection of chick neural tubes and discussions. This work was supported by a DEVELOPMENT 1902 RESEARCH ARTICLE Development 138 (10)

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