FatJ acts via the Hippo mediator Yap1 to restrict the size of neural progenitor cell pools.
ABSTRACT 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.
- SourceAvailable from: ncbi.nlm.nih.gov[Show abstract] [Hide abstract]
ABSTRACT: Metazoan cells are exposed to a multitude of signals, which they integrate to determine appropriate developmental or physiological responses. Although the Hippo pathway was only discovered recently, and our knowledge of Hippo signal transduction is far from complete, a wealth of interconnections amongst Hippo and other signaling pathways have already been identified. Hippo signaling is particularly important for growth control, and I describe how integration of Hippo and other pathways contributes to regulation of organ growth. Molecular links between Hippo signaling and other signal transduction pathways are summarized. Different types of mechanisms for signal integration are described, and examples of how the complex interconnections between pathways are used to guide developmental and physiological growth responses are discussed. Features of Hippo signaling appear to make it particularly well suited to signal integration, including its responsiveness to cell-cell contact and the mediation of its transcriptional output by transcriptional co-activator proteins that can interact with transcription factors of other pathways.Seminars in Cell and Developmental Biology 04/2012; 23(7):812-7. · 6.20 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Understanding the molecular nature of human cancer is essential to the development of effective and personalized therapies. Several different molecular signal transduction pathways drive tumorigenesis when deregulated and respond to different types of therapeutic interventions. The Hippo signaling pathway has been demonstrated to play a central role in the regulation of tissue and organ size during development. The deregulation of Hippo signaling leads to a concurrent combination of uncontrolled cellular proliferation and inhibition of apoptosis, two key hallmarks in cancer development. The molecular nature of this pathway was first uncovered in Drosophila melanogaster through genetic screens to identify regulators of cell growth and cell division. The pathway is strongly conserved in humans, rendering Drosophila a suitable and efficient model system to better understand the molecular nature of this pathway. In the present study, we review the current understanding of the molecular mechanism and clinical impact of the Hippo pathway. Current studies have demonstrated that a variety of deregulated molecules can alter Hippo signaling, leading to the constitutive activation of the transcriptional activator YAP or its paralog TAZ. Additionally, the Hippo pathway integrates inputs from a number of growth signaling pathways, positioning the Hippo pathway in a central role in the regulation of tissue size. Importantly, deregulated Hippo signaling is frequently observed in human cancers. YAP is commonly activated in a number of in vitro and in vivo models of tumorigenesis, as well as a number of human cancers. The common activation of YAP in many different tumor types provides an attractive target for potential therapeutic intervention.Clinical and translational medicine. 01/2014; 3:25.
- [Show abstract] [Hide abstract]
ABSTRACT: The large atypical cadherin Fat is a receptor for both Hippo and planar cell polarity (PCP) pathways. Here we investigate the molecular basis for signal transduction downstream of Fat by creating targeted alterations within a genomic construct that contains the entire fat locus, and by monitoring and manipulating the membrane localization of the Fat pathway component Dachs. We establish that the human Fat homolog FAT4 lacks the ability to transduce Hippo signaling in Drosophila, but can transduce Drosophila PCP signaling. Targeted deletion of conserved motifs identifies a four amino acid C-terminal motif that is essential for aspects of Fat-mediated PCP, and other internal motifs that contribute to Fat-Hippo signaling. Fat-Hippo signaling requires the Drosophila Casein kinase 1_ encoded by discs overgrown (Dco), and we characterize candidate Dco phosphorylation sites in the Fat intracellular domain (ICD), the mutation of which impairs Fat-Hippo signaling. Through characterization of Dachs localization and directed membrane targeting of Dachs, we show that localization of Dachs influences both the Hippo and PCP pathways. Our results identify a conservation of Fat-PCP signaling mechanisms, establish distinct functions for different regions of the Fat ICD, support the correlation of Fat ICD phosphorylation with Fat-Hippo signaling, and confirm the importance of Dachs membrane localization to downstream signaling pathways.Development 01/2013; · 6.60 Impact Factor
DEVELOPMENT AND STEM CELLSRESEARCH ARTICLE
Neural progenitor cells are patterned along the dorsoventral axis
of the neural tube, proliferate, then migrate laterally and
differentiate into defined neuronal classes (Kintner, 2002;
Bylund et al., 2003). The tight regulation of patterning,
proliferation and differentiation ensures an appropriate balance
of progenitor versus differentiated cells that is key to tissue
homeostasis. Signalling pathways mediating these events are
therefore crucial to the proper size, composition and functioning
of the nervous system (Dahmane et al., 2001). However, an
outstanding question is that of how appropriate progenitor and
differentiated cell numbers are exacted. Recent evidence has
implicated the Hippo pathway in such regulation, showing that
components of the Hippo signalling pathway govern neural
progenitor cell number (Cao et al., 2008).
Cadherin proteins are extracellular proteins that play important
roles in cell adhesion and cell signalling (Halbleib and Nelson,
2006). The Fat cadherins comprise a subfamily containing the
largest proteins in the superfamily (Tanoue and Takeichi, 2005).
Drosophila Fat (dFat) was first identified as a putative tumour
suppressor gene involved in planar cell polarity and tissue size
regulation via the Hippo signalling pathway (Mahoney et al.,
1991; Matakatsu and Blair, 2004; Willecke et al., 2006). Of the
four vertebrate orthologues of dFat, Fat4 (also called FatJ) shows
the greatest homology to dFat (Matakatsu and Blair, 2006) and
is expressed in a variety of tissues with active PCP signalling
(Rock et al., 2005), including the kidney, where loss of Fat4
alters orientated cell divisions and leads to kidney dysfunction.
Fat4 additionally plays a role within the CNS: loss of Fat4
expression in the cerebellum reduces the apical membrane
compartment, suggesting a role in apical-basal polarity (Ishiuchi
et al., 2009). Furthermore, mouse Fat4–/–embryos display a
wider spinal cord than wild-type littermates, suggesting that
FatJ/Fat4 might play a role in spinal cord development (Saburi
et al., 2008).
We have previously developed a system for robust knockdown
of chicken genes (Das et al., 2006). In the present study, we employ
this system to knockdown the expression of all cadherin domain-
containing genes expressed within the chicken neural tube and
thereby analyse cadherin gene function during neural development.
Our unbiased approach defines a number of cadherin genes that
may play a role in cell patterning, proliferation and differentiation
within the developing spinal cord. Of these, we have studied one,
FatJ, in detail. FatJ is localised to progenitor cells within
intermediate regions along the dorsoventral axis of the neural tube.
Our studies reveal that FatJ acts via the Hippo pathway mediator
Yap1 to regulate the size of the dp4-vp1 progenitor cell pools and
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
regionally restricted proliferation and differentiation of specific
MATERIALS AND METHODS
Microarray analysis of genes expressed in the chicken neural tube
Total RNA extracted from stage 22 chicken spinal cords (100 embryos)
was used to interrogate the Affymetrix whole chicken genome chip (ARK
Genomics). Microarray data are available in the ArrayExpress database
(www.ebi.ac.uk/arrayexpress) under Accession Number E-TABM-701.
Development 138, 1893-1902 (2011) doi:10.1242/dev.064204
© 2011. Published by The Company of Biologists Ltd
1MRC Centre for Developmental and Biomedical Genetics, University of Sheffield,
Sheffield, S10 2TN, UK. 2Department of Biomedical Science, University of Sheffield,
Sheffield, S10 2TN, UK. 3Department of Molecular Biology and Biotechnology,
University of Sheffield, Sheffield, S10 2TN, UK.
*These authors contributed equally to this work
†Present address: Division of Cell and Developmental Biology, College of Life
Sciences, University of Dundee, Dundee, DD1 5EH, UK
‡Authors for correspondence (email@example.com;
Accepted 23 February 2011
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
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,‡
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 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).
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
Embryo fixation, sectioning and immunohistochemistry were performed as
previously described (Das et al., 2006). Antibodies used were against Pax6,
Pax7, Pax2, Evx1, Nkx2.2, Isl1, Lim1/Lim2, Lmx1b, N-cadherin, Nkx6.1
and En1 (DSHB); mouse anti--catenin (Sigma); rabbit anti-Dbx1 (Pierani
et al., 1999); anti-Chx10 (S. Morton, Columbia University, NY, USA);
rabbit anti-Pax6 (Covance); rabbit anti-Olig2 polyclonal and rabbit anti-
RFP polyclonal (Chemicon); rabbit anti-phospho-histone H3 polyclonal
(Upstate); rabbit anti-GFP polyclonal (BD Bioscience); anti-Crb2 (P.
Rashbass, University of Sheffield, UK); rabbit anti-pYap1 polyclonal (Cell
Signaling Technology); and Alexa405-, Alexa488- and Alexa555-
conjugated secondary antibodies were obtained from Molecular Probes
(Invitrogen). Whole-mount in situ hybridisation was performed as
previously described (Ohyama et al., 2005; Das et al., 2006) using
digoxigenin (DIG)-labelled (Roche) RNA probes. DIG-labelled antisense
riboprobes were generated from chicken EST plasmids ChEST187n19
(FatJ); ChEST427d5 (Yap1) and ChEST580f24 (Tead4) [by linearization
with NotI and transcription with T3 RNA polymerase (Promega)]. After
sufficient staining development, embryos were cryosectioned at 30 m as
described previously (Das et al., 2006).
BrdU incorporation studies
Neural tubes were electroporated as described above. After 48 hours
incubation at 38°C, 50 l of 0.01 M BrdU (Sigma) was injected under the
vitelline membrane, the egg resealed and placed at 38°C. After 30 minutes
Development 138 (10)
Fig. 1. A range of phenotypes are observed following loss of
cadherin function. Expression of red fluorescent protein (RFP) from
RNAi vectors allows cell migration and morphology to be assessed.
(A)RNAi-mediated knockdown of Fat2 cadherin causes an
accumulation of electroporated cells in the mantle zone (mz) of the
neural tube. (B)A similar phenotype is observed after knockdown of
Notch1 (Das et al., 2006), whereas cells targeted with luciferase RNAi
(C) are located throughout the neural tube in both ventricular zone (vz)
and mantle zone. (D)Fat1 RNAi results in a reduction in size and cell
number (32% fewer cells) in the electroporated side of the neural tube.
(G)Expression domain of Pax6 is reduced relative to the contralateral
side. (E,H)Loss of N-cadherin produces a decrease in cell number on
the electroporated side, together with disruption of the apical surface
of the neural tube, as marked by loss of -catenin expression (H,
arrows). (F,I)Protocadherin 19 RNAi leads to a 50% increase in the
number of cells on the electroporated side of the neural tube and an
expansion of Pax6 expression compared with the contralateral side (I).
Some electroporated cells also migrate into the unelectroporated side
of the neural tube (arrows in F), suggesting there may be some loss of
roof plate integrity. (J,K)RNAi-mediated knockdown of protocadherin
10 leads to a dorsal shift in expression of Pax6 (J) and Olig2 (K).
(L,M)Knockdown of FatJ cadherin leads to an increased number of
Lim1+/Lim2+cells. (N)This increase is spatially restricted to intermediate
regions of the neural tube (bottom panel, arrows); dorsal Lim1/Lim2-
expressing cells are unaffected by FatJ RNAi (N, top panel).
further incubation, embryos were harvested, fixed and cryosectioned as
previously described (Das et al., 2006) and immunohistochemistry was
performed using 1/1000 mouse anti-BrdU monoclonal antibody (Sigma).
Microscopy and image acquisition
Epifluorescence images were obtained using a Leica DM5000B upright
microscope and confocal images were obtained using a Zeiss LSM510
Meta. Section in situ images were obtained under Nomarski optics on a
Leica DM-R microscope using a Leica digital camera.
Co-electroporation of Notch intracellular domain
The pCS2-ICD plasmid expressing the intracellular domain of mNotch1
(NICD) was a kind gift from D. Henrique (IMM, University of Lisbon,
Portugal) and has been previously shown to constitutively activate Notch1
signalling (Kopan et al., 1996). The plasmid was co-electroporated with
the RNAi vectors [1 g/l NICD + 0.5 g/l of the relevant RNAi
vector(s)] at stage 11-12 and the resulting phenotype was analysed at stage
25 as detailed above.
Neural tubes were electroporated at stage 11-12 with FatJ RNAi or Luc
RNAi vectors and harvested after 72 hours incubation at 38°C. The
intermediate region of the electroporated side of six or more neural tubes
was dissected and lysed in Reporter Gene Lysis buffer (Roche). Each
protein lysate (20 g) was separated by SDS-PAGE in a 10%
polyacrylamide gel, transferred to a nitrocellulose membrane and western
blotting was performed using 1/1000 rabbit anti-pYap1 antibody (Cell
Signalling Technology) in 10% BSA/1?TTBS followed by incubation
with 1/10,000 anti-rabbit HRP-conjugated secondary antibody and
detection by ECL. Each membrane was subsequently incubated with
1/5000 mouse anti-tubulin antibody (Sigma) and 1/5000 rabbit anti-RFP
polyclonal (Chemicon) to evaluate protein amounts loaded and degree of
electroporation with RNAi vectors. Signal density was determined using
Quantity One software (Biorad).
Cadherin genes influence neural tube
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
phenotype (see Table S1, Fig. S1 in the supplementary material).
However, 22 others gave phenotypes that could be broadly
classified (see Table S1 in the supplementary material).
Knockdown of four cadherins (Fat2, brain cadherin, VE-
cadherin and cadherin 18) resulted in accumulation of RFP+ cells
in the mantle zone, a markedly different effect to that normally
observed (Fig. 1A,C; see Fig. S1 in the supplementary material).
This effect phenocopies knockdown of Notch1, where premature
differentiation of cells results in their migration from the ventricular
to the mantle zone (Fig. 1B, Fig. 2B) (Das et al., 2006), raising the
possibility that these cadherins may function in the same pathway.
We investigated this possibility by co-electroporating a
constitutively active form of Notch1 (Notch intracellular domain,
NICD), shown previously to rescue the Notch RNAi phenotype
(Kopan et al., 1996), with the Fat2 RNAi construct. Co-
electroporation of NICD rescues the premature differentiation
caused by Fat2 RNAi, and electroporated cells were now found
throughout the neural tube in both the ventricular and mantle zones
(Fig. 2D). This suggests that Fat2 functions in the same pathway
as Notch1 to control neural progenitor differentiation.
Knockdown of 14 cadherins caused a reduction in size of the
electroporated side of the neural tube (Fig. 1D,E; see Fig. S1 in the
supplementary material), suggesting a decrease in cell number. No
changes in dorsoventral (DV) patterning were detected; instead, the
domain of each homeodomain protein examined was small relative
to the contralateral side (Fig. 1G; see Fig. S1 in the supplementary
material); this phenotype was not observed following introduction
of luciferase RNAi (see Fig. S1 in the supplementary material).
Knockdown of N-cadherin caused a similar defect, and additionally
caused a loss of -catenin expression at the apical surface of the
neural tube (Fig. 1E,H) as observed previously for a mutation in
this gene in zebrafish (Lele et al., 2002). By contrast, knockdown
of protocadherin 19 and cadherin 10 resulted in an increased
number of cells in the neural tube and enlargement of the
electroporated side. Concomitantly, there was a relative expansion
of the domain of expression of each homeodomain protein
examined (Fig. 1F,I; see Fig. S1 in the supplementary material).
A minor set of cadherins, when downregulated, caused very
localised effects along the DV axis. RNAi-mediated knockdown of
protocadherin 10 (Pcdh10) led to a change in DV patterning with
Patterned proliferation mediated by FatJ cadherin
Fig. 2. Rescue of Fat2 RNAi phenotype by NICD. (A)RNAi-mediated
knockdown of Fat2 cadherin causes an accumulation of electroporated
cells in the mantle zone of the neural tube. (B,C)A similar phenotype is
observed after knockdown of Notch1 (Das et al., 2006) (B), whereas
cells targeted with luciferase RNAi (C) are located throughout the
neural tube in both ventricular zone (vz) and mantle zone. (D,E)Co-
electroporation of the Notch intracellular domain (NICD) rescues the
Fat2 knockdown (D), as well as the Notch1 knockdown phenotype (E).
The rescue is evident by the location of electroporated (RFP+) cells in
both the mantle zone and ventricular zone (compare A with D). This
suggests that Fat2 is involved in Notch1 signalling.
a dorsal shift in the expression of both Pax6 (Fig. 1J) and Olig2
(Fig. 1K). Neither marker appeared to be expanded; instead, the
entire domain of each appeared dorsally shifted. The size of the
electroporated side of the neural tube was unchanged compared
with the contralateral side, suggesting that the effects observed
were likely to be due to a change in patterning.
Finally, FatJ RNAi appeared to produce a small, but robustly
reproducible, increase in the number of Lim1+/Lim2+cells. This
phenotype was apparent in only a subset of Lim1+/Lim2+cells,
notably those in medial regions along the dorsoventral axis (Fig.
1L,M, arrows). Lim1+/Lim2+cells in dorsal regions of the neural
tube appeared unaffected by electroporation of FatJ RNAi vectors,
whereas those in intermediate regions were expanded in number
(Fig. 1N). Together, this raises the possibility that FatJ knockdown
affects cell proliferation/differentiation, but that its effects are
localised to a regionally defined domain of the neural tube. We
therefore focused our attention on this cadherin, to confirm the
increase in Lim1+/Lim2+cells and clarify the mechanism that
governs Lim1+/Lim2+cell numbers.
We first confirmed that all three FatJ RNAi vectors, which
target different regions of FatJ mRNA (Fig. 3; see Table S2 in the
supplementary material), knock down FatJ expression and
confirmed that luciferase RNAi vector has no effect. To further
ensure that the effects of the FatJ RNAi vectors were not mediated
by an ‘off-target’ effect (see Echeverri et al., 2006), we compared
the ability of each FatJ RNAi vector to increase production of
Lim1+/Lim2+interneurons (Fig. 3G-I). Each vector caused at least
a 20% increase in the number of cells expressing Lim1+/Lim2+,
with the most effective vector (FatJ RNAi C) producing an
average of 32% more Lim1+/Lim2+cells (Fig. 3M). We conclude
that FatJ regulates the number of Lim1+/Lim2+neurons in the
Given that Lim1/Lim2 is expressed in a wide set of interneurons
(dl2, dl4, dl6, v0 and v1 subclasses), we next defined more
precisely which interneuron types were affected by FatJ RNAi, by
analysing embryos for Isl1, Pax2, Lmx1b, Evx1, En1 and Chx10
expression, which mark dI3, dI4+6, dI5, v0, v1 and v2
interneurons, respectively (Fig. 4). This analysis revealed that
interneurons in domains dI4, dI5, dI6, v0 and v1 were affected
(Fig. 4B-F), whereas those in domains dI3 and v2 were not (Fig.
4A,G). We conclude that FatJ knockdown results in the specific
expansion of differentiated dI4-v1 interneurons.
FatJ expression is spatially restricted
One possible explanation for the effect of FatJ RNAi in only a
subset of Lim1+/Lim2+cells is that FatJ itself is spatially
restricted to intermediate regions of the neural tube. To examine
this, we analysed FatJ mRNA expression over the period St10-
Development 138 (10)
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.
Patterned proliferation mediated by FatJ cadherin
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
St22 and found that its expression is restricted to intermediate
regions along the DV axis. Expression is limited to
ventricular/subventricular zone areas and appears to correlate
with dp4-vp1 progenitor regions, overlapping with expression
domains for progenitor markers Pax6, Pax7 and Dbx1, but not
Nkx6.1 (Fig. 5A-F, Fig. 3J-L). This pattern of expression is
reminiscent of that of Pax6, which is regulated by Shh and
Wnt/BMP signalling and the Gli activity gradient (Briscoe et al.,
2000; Timmer et al., 2002).
FatJ knockdown alters progenitor cell number
Two alternative possibilities could account for the enhanced
differentiation of dI4-v1 interneurons after FatJ knockdown. First,
a decrease in FatJ activity could lead to acceleration in the
differentiation of progenitor cells in the dp4-vp1 domain. In this
case, knockdown of FatJ should lead to a decrease in proliferating
progenitors within these domains. An alternate possibility is that
FatJ governs the proliferating progenitor cells themselves, a
decrease in its activity leading to greater numbers of progenitor
Development 138 (10)
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
cells, and hence greater numbers of differentiated interneurons. In
this case, knockdown of FatJ should lead to an increase in
To distinguish these possibilities, and determine whether FatJ
expression is required for differentiation, or for the
establishment/maintenance of proliferating progenitors, we
quantified the number of progenitor cells in defined subdomains
along the DV axis of the neural tube after FatJ RNAi. Loss of FatJ
did not affect the general pattern of any of the markers examined
(Fig. 5G,H; Fig. 6A,B). Moreover, loss of FatJ expression did not
affect the number of progenitor cells in the vp2 domain (judged by
Nkx6.1 expression; Fig. 5G,M; Table 1), the vp3 domain (Nkx2.2
expression; Fig. 6B; Table 1) or the dp3 domain (low expression of
Pax6; Pax7 expression) (Fig. 5H, Fig. 6A, Table 1). Thus, progenitor
cells lying outside the FatJ expression domain were unaffected by its
reduction. By contrast, a small but consistent increase (10-20%,
Table 1) in the number of Dbx1+(Fig. 5G,N), Pax6 (strong positive)
(Fig. 5H,O) and Irx3+ cells (Table 1) was detected. This suggests
that reduction in FatJ expression leads to an increased number of
progenitors in the dp4-vp1 domains of the neural tube.
To begin to address the mechanism underlying the increase in
progenitor number, we analysed BrdU incorporation. A 14.65%
increase in the number of proliferating cells was detected in the
ventricular zone after FatJ RNAi (Fig. 5I,K; Table 2). Similarly, we
observed a 21.92% increase in the number of mitotic cells marked
by phospho-histone H3 (Fig. 5J,L; Table 2). These data suggest that
the increased number of differentiated interneurons can be explained
by an increased number of progenitors within the corresponding
progenitor pool. To further test this idea, we performed double
labelling analyses with Pax6 and En1. An increased number of En1+
cells was detected after FatJ knockdown, but no double-positive cells
were detected (Fig. 5P), indicating that En1+cells differentiated
properly and did not simply express En1 precociously.
FatJ is the closest orthologue of the Drosophila Fat (dFat)
gene, which plays a crucial role in planar cell polarity (Fanto et
al., 2003; Matakatsu and Blair, 2006) and interacts with Pals1 to
organise the apical membrane domain (Ishiuchi et al., 2009). To
determine whether the increase in progenitor cell number is
caused by defects in progenitor cell polarity, we examined
expression of the apical proteins N-cadherin, Crumbs2 and Par3.
Loss of FatJ did not appear to affect expression of any of these
markers (Fig. 6C,D; data not shown), suggesting that apical-
basal polarity is unaffected by loss of FatJ. Our data indicate that
FatJ limits the number of progenitor cells within its domain of
expression through a mechanism other than a disruption in
Patterned proliferation mediated by FatJ cadherin
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.
Table 1. Number of cells expressing progenitor markers after FatJ RNAi
Average number of cells
Marker Number of sectionsElectroporated Unelectroporated Change (%)s.e.m. (electroporated)s.e.m. (unelectroporated)
FatJ controls cell proliferation through the Hippo
Drosophila Fat controls tissue size by activating the Hippo
pathway, leading to phosphorylation of the transcriptional activator
Yorkie (Yki) (Willecke et al., 2006). Yki normally associates with
the transcription factor Scalloped (Sd) to promote cell proliferation
(Wu et al., 2008). Activation of the Hippo pathway sequesters Yki
in the cytoplasm via Yki phosphorylation or by direct binding of
Yki by the Hippo pathway components Expanded and Warts,
thereby preventing association of Yki with Sd and stopping cell
proliferation (Zhang et al., 2008; Oh et al., 2009). Intriguingly, the
Hippo pathway has recently been shown to control neural
progenitor cell number in the neural tube and expression of
dominant repressor versions of the vertebrate orthologues of Yki
and Sd (Yap1 and Tead1, respectively) can produce an increase in
the number of Lim1+/Lim2+cells, similar to the phenotype
observed in this study (Cao et al., 2008). This raises the possibility
that FatJ acts through the Hippo pathway to control the number of
cells within the dp4-vp1 domain. To test this, downstream
components of the Hippo pathway were knocked down
simultaneously with FatJ knockdown. RNAi mediated knockdown
of Yap1 was confirmed by in situ hybridisation: electroporation of
Yap1 RNAi constructs produced a significant loss of Yap1
expression within cells expressing the RNAi constructs (Fig. 7A),
whereas expression of luciferase RNAi vectors had no effect on
Yap1 expression (Fig. 7B). Notably, Yap1 expression is observed
solely in the ventricular/subventricular zones of the neural tube;
therefore, the FatJ expression domain comprises a subset of Yap1-
expressing cells. Knockdown of Yap1 (Fig. 7C,F) and Tead4 (the
closest vertebrate orthologue of Sd, not shown) alone had no
significant affect on the number of cells expressing Lim1/Lim2.
However, knockdown of Yap1 or Tead4 together with FatJ (Fig.
7D,F) rescued the increase in Lim1+/Lim2+cells detected after
knockdown of FatJ alone (Fig. 7E,F). To further confirm this
rescue, dominant negative (DN) Yap1 or Tead4 constructs [detailed
in Cao et al. (Cao et al., 2008)] were co-electroporated with the
FatJ RNAi vector. In both cases, these were able to rescue the
increase in Lim1+/Lim2+cells observed after knockdown of FatJ
alone (Fig. 7G).
To further investigate regulation of the Hippo pathway by FatJ,
we compared the levels of phosphorylated Yap1 (pYap1) after FatJ
or Luciferase RNAi. Western blotting revealed decreased levels of
pYap1 following FatJ RNAi when compared with Luc RNAi
control samples (Fig. 7H). Similarly, decreased pYap1 levels were
observed within the FatJ expression domain on neural tube sections
after FatJ RNAi but not after Luciferase RNAi electroporation (Fig.
7I,J). Notably, the decrease in pYap1 was most pronounced at the
lateral edge of the ventricular zone (arrows in Fig. 7I) adjacent to
the mantle zone.
These data suggest that loss of FatJ signalling causes a decrease
in phosphorylation of the Hippo mediator Yap1. The upstream
Hippo pathway kinases Mst1/2 and Lats1/2 have been shown to
regulate neuronal progenitor proliferation by inhibiting Yap1
activity through Yap1 phosphorylation (Cao et al., 2008); however,
we were unable to detect any reduction in the levels of pMst1/2
after FatJ RNAi. Therefore, we are unable to distinguish whether
FatJ operates through the established Hippo pathway, or via a
parallel pathway that links FatJ and pYap1. Nonetheless, taken
together, our data show that loss of downstream Hippo components
can rescue the defect caused by loss of FatJ and implicates FatJ-
control of Hippo pathway mediators as a mechanism for limiting
the number of interneurons differentiating in the dp4-vp1 region of
the neural tube.
Our analyses provide proof-of-principle for large-scale RNAi
screening in the chick neural tube and provide insight into those
cadherins whose RNAi-mediated knockdown leads to early defects
in size, proliferation, cell differentiation and patterning of gene
expression. The large size of cadherin genes can preclude analysis
of their roles through gain-of-function approaches, highlighting
further the importance of the RNAi approach. Our studies
complement and support studies that document a role for cadherin
genes in early neural tube development, for example, showing
that N-cadherin is required to maintain the integrity of the
neuroepithelium (Lele et al., 2002). In addition, our studies
highlight the temporal effects of cadherins in neural tube
development: our screen did not reveal early roles for cadherins
previously shown to affect later development of the spinal cord,
including late migration of neural crest cells (Coles et al., 2007),
sorting of cell pools (Price et al., 2002) and synapse formation (Bao
et al., 2007).
Our detailed analysis of the FatJ knockdown phenotype provides
insight into a novel early role for cadherin function within the CNS,
revealing a mechanism to govern the balance of regionally
restricted progenitor pool cells and cognate interneuron numbers.
Local interneuron circuits play a major role in coordinating the
sensory-motor circuits that characterise the spinal cord. The
number of interneurons of a particular class is likely to be crucial
in achieving appropriate function of selective sensory-motor
circuits, and our studies provide insight into the manner in which
appropriate numbers of Lim1+/Lim2+interneurons are generated.
Our data show that FatJ restricts the size of progenitor pools in
which it is expressed. Loss-of-function of FatJ leads to a consistent
increase in progenitor cell number. These appear to differentiate
along their normal route, as evidenced by a consistent increase in
appropriate differentiated interneuron subtypes. Mouse Fat4–/–
embryos display a wider spinal cord than do wild-type littermates,
suggesting that the role of FatJ/Fat4 in neural progenitor expansion
might be conserved across vertebrate species (Saburi et al., 2008).
Intriguingly, our data reveal that FatJ restricts progenitor cell
numbers via mediators of the recently discovered Hippo signalling
pathway. Originally identified in Drosophila, activation of the
Hippo pathway by dFat prevents the transcriptional activation of
genes such as CyclinE and Diap1 that promote cell proliferation
and prevent apoptosis (Wu et al., 2003), and in this manner,
prevents excessive tissue growth. The Hippo pathway is conserved
in vertebrates with one or more orthologues of all components in
the pathway. Several of these orthologues can rescue the
corresponding Drosophila mutant phenotype (Wu et al., 2003; Lai
Development 138 (10)
Table 2. Number of mitotic cells after FatJ RNAi
Average number of cells
MarkerNumber of sectionsElectroporatedUnelectroporatedChange (%)s.e.m. (electroporated)s.e.m. (unelectroporated)
et al., 2005; Wei et al., 2007), suggesting a conserved role in
growth control. Mutation of many of these genes has been
implicated in human cancers (McClatchey and Giovannini, 2005;
Harvey and Tapon, 2007; Yokoyama et al., 2008), Fat4/FatJ has
recently been implicated as a breast tumour suppressor gene (Qi et
al., 2009), and Yap1 expression and nuclear localisation is
upregulated in Shh-associated medulloblastomas (Fernandez et al.,
2009). Recent studies show that the Hippo pathway controls the
number of neuronal progenitors in the neural tube by influencing
cell proliferation and apoptosis (Cao et al., 2008). Our studies now
reveal that regulation of the Hippo pathway mediator Yap1 in the
vertebrate neural tube occurs through FatJ, the closest vertebrate
homologue to dFat. Together, our data suggest that FatJ mediates
a robust mechanism for the acquisition of appropriate numbers of
cells in progenitor pools, and hence appropriate numbers of distinct
interneuron subtypes, along the DV axis of the neural tube.
We thank Gareth Howell and members of the ENSEMBL team at EBI for help
with analysis of the microarray data, and Kyoji Ohyama for help with
dissection of chick neural tubes and discussions. This work was supported by a
Patterned proliferation mediated by FatJ cadherin
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.
BBSRC grant to S.W. and M.P., and by Wellcome and MRC grants to M.P.
Microscopy was performed using the Light Microscopy Facility, University of
Sheffield, which is supported by Wellcome Trust grant number GR077544AIA.
Deposited in PMC for release after 6 months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material for this article is available at
Bao, H., Berlanga, M. L., Xue, M., Hapip, S. M., Daniels, R. W., Mendenhall,
J. M., Alcantara, A. A. and Zhang, B. (2007). The atypical cadherin flamingo
regulates synaptogenesis and helps prevent axonal and synaptic degeneration in
Drosophila. Mol. Cell. Neurosci. 34, 662-678.
Briscoe, J., Pierani, A., Jessell, T. M. and Ericson, J. (2000). A homeodomain
protein code specifies progenitor cell identity and neuronal fate in the ventral
neural tube. Cell 101, 435-445.
Bylund, M., Andersson, E., Novitch, B. G. and Muhr, J. (2003). Vertebrate
neurogenesis is counteracted by Sox1-3 activity. Nat. Neurosci. 6, 1162-1168.
Cao, X., Pfaff, S. L. and Gage, F. H. (2008). YAP regulates neural progenitor cell
number via the TEA domain transcription factor. Genes Dev. 22, 3320-3334.
Coles, E. G., Taneyhill, L. A. and Bronner-Fraser, M. (2007). A critical role for
Cadherin6B in regulating avian neural crest emigration. Dev. Biol. 312, 533-544.
Dahmane, N., Sanchez, P., Gitton, Y., Palma, V., Sun, T., Beyna, M., Weiner,
H. and Ruiz i Altaba, A. (2001). The Sonic Hedgehog-Gli pathway regulates
dorsal brain growth and tumorigenesis. Development 128, 5201-5212.
Das, R. M., Van Hateren, N. J., Howell, G. R., Farrell, E. R., Bangs, F. K.,
Porteous, V. C., Manning, E. M., McGrew, M. J., Ohyama, K., Sacco, M. A.
et al. (2006). A robust system for RNA interference in the chicken using a
modified microRNA operon. Dev. Biol. 294, 554-563.
Echeverri, C. J., Beachy, P. A., Baum, B., Boutros, M., Buchholz, F., Chanda, S.
K., Downward, J., Ellenberg, J., Fraser, A. G., Hacohen, N. et al. (2006).
Minimizing the risk of reporting false positives in large-scale RNAi screens. Nat.
Methods 3, 777-779.
Fanto, M., Clayton, L., Meredith, J., Hardiman, K., Charroux, B., Kerridge, S.
and McNeill, H. (2003). The tumor-suppressor and cell adhesion molecule Fat
controls planar polarity via physical interactions with Atrophin, a transcriptional
co-repressor. Development 130, 763-774.
Fernandez, L. A., Northcott, P. A., Dalton, J., Fraga, C., Ellison, D., Angers, S.,
Taylor, M. D. and Kenney, A. M. (2009). YAP1 is amplified and up-regulated in
hedgehog-associated medulloblastomas and mediates Sonic hedgehog-driven
neural precursor proliferation. Genes Dev. 23, 2729-2741.
Halbleib, J. M. and Nelson, W. J. (2006). Cadherins in development: cell
adhesion, sorting, and tissue morphogenesis. Genes Dev. 20, 3199-3214.
Harvey, K. and Tapon, N. (2007). The Salvador-Warts-Hippo pathway-an
emerging tumour-suppressor network. Nat. Rev. Cancer 7, 182-191.
Ishiuchi, T., Misaki, K., Yonemura, S., Takeichi, M. and Tanoue, T. (2009).
Mammalian Fat and Dachsous cadherins regulate apical membrane organization
in the embryonic cerebral cortex. J. Cell Biol. 185, 959-967.
Kintner, C. (2002). Neurogenesis in embryos and in adult neural stem cells. J.
Neurosci. 22, 639-643.
Kopan, R., Schroeter, E. H., Weintraub, H. and Nye, J. S. (1996). Signal
transduction by activated mNotch: importance of proteolytic processing and its
regulation by the extracellular domain. Proc. Natl. Acad. Sci. USA 93, 1683-1688.
Lai, Z. C., Wei, X., Shimizu, T., Ramos, E., Rohrbaugh, M., Nikolaidis, N., Ho,
L. L. and Li, Y. (2005). Control of cell proliferation and apoptosis by mob as
tumor suppressor, mats. Cell 120, 675-685.
Lele, Z., Folchert, A., Concha, M., Rauch, G. J., Geisler, R., Rosa, F., Wilson, S.
W., Hammerschmidt, M. and Bally-Cuif, L. (2002). parachute/n-cadherin is
required for morphogenesis and maintained integrity of the zebrafish neural
tube. Development 129, 3281-3294.
Mahoney, P. A., Weber, U., Onofrechuk, P., Biessmann, H., Bryant, P. J. and
Goodman, C. S. (1991). The fat tumor suppressor gene in Drosophila encodes a
novel member of the cadherin gene superfamily. Cell 67, 853-868.
Matakatsu, H. and Blair, S. S. (2004). Interactions between Fat and Dachsous
and the regulation of planar cell polarity in the Drosophila wing. Development
Matakatsu, H. and Blair, S. S. (2006). Separating the adhesive and signaling
functions of the Fat and Dachsous protocadherins. Development 133, 2315-
McClatchey, A. I. and Giovannini, M. (2005). Membrane organization and
tumorigenesis-the NF2 tumor suppressor, Merlin. Genes Dev. 19, 2265-2277.
Oh, H., Reddy, B. V. and Irvine, K. D. (2009). Phosphorylation-independent
repression of Yorkie in Fat-Hippo signaling. Dev. Biol. 335, 188-197.
Ohyama, K., Ellis, P., Kimura, S. and Placzek, M. (2005). Directed differentiation
of neural cells to hypothalamic dopaminergic neurons. Development 132, 5185-
Pierani, A., Brenner-Morton, S., Chiang, C. and Jessell, T. M. (1999). A sonic
hedgehog-independent, retinoid-activated pathway of neurogenesis in the
ventral spinal cord. Cell 97, 903-915.
Price, S. R., De Marco Garcia, N. V., Ranscht, B. and Jessell, T. M. (2002).
Regulation of motor neuron pool sorting by differential expression of type II
cadherins. Cell 109, 205-216.
Qi, C., Zhu, Y. T., Hu, L. and Zhu, Y. J. (2009). Identification of Fat4 as a
candidate tumor suppressor gene in breast cancers. Int. J. Cancer 124, 793-
Rock, R., Schrauth, S. and Gessler, M. (2005). Expression of mouse dchs1, fjx1,
and fat-j suggests conservation of the planar cell polarity pathway identified in
Drosophila. Dev. Dyn. 234, 747-755.
Saburi, S., Hester, I., Fischer, E., Pontoglio, M., Eremina, V., Gessler, M.,
Quaggin, S. E., Harrison, R., Mount, R. and McNeill, H. (2008). Loss of Fat4
disrupts PCP signaling and oriented cell division and leads to cystic kidney
disease. Nat. Genet. 40, 1010-1015.
Tanoue, T. and Takeichi, M. (2005). New insights into Fat cadherins. J. Cell Sci.
Timmer, J. R., Wang, C. and Niswander, L. (2002). BMP signaling patterns the
dorsal and intermediate neural tube via regulation of homeobox and helix-loop-
helix transcription factors. Development 129, 2459-2472.
Wei, X., Shimizu, T. and Lai, Z. C. (2007). Mob as tumor suppressor is
activated by Hippo kinase for growth inhibition in Drosophila. EMBO J. 26,
Willecke, M., Hamaratoglu, F., Kango-Singh, M., Udan, R., Chen, C. L.,
Tao, C., Zhang, X. and Halder, G. (2006). The fat cadherin acts through the
hippo tumor-suppressor pathway to regulate tissue size. Curr. Biol. 16, 2090-
Wu, S., Huang, J., Dong, J. and Pan, D. (2003). hippo encodes a Ste-20 family
protein kinase that restricts cell proliferation and promotes apoptosis in
conjunction with salvador and warts. Cell 114, 445-456.
Wu, S., Liu, Y., Zheng, Y., Dong, J. and Pan, D. (2008). The TEAD/TEF family
protein Scalloped mediates transcriptional output of the Hippo growth-
regulatory pathway. Dev. Cell 14, 388-398.
Yokoyama, T., Osada, H., Murakami, H., Tatematsu, Y., Taniguchi, T., Kondo,
Y., Yatabe, Y., Hasegawa, Y., Shimokata, K., Horio, Y. et al. (2008). YAP1 is
involved in mesothelioma development and negatively regulated by Merlin
through phosphorylation. Carcinogenesis 29, 2139-2146.
Zhang, L., Ren, F., Zhang, Q., Chen, Y., Wang, B. and Jiang, J. (2008). The
TEAD/TEF family of transcription factor Scalloped mediates Hippo signaling in
organ size control. Dev. Cell 14, 377-387.
Development 138 (10)