© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 367-376 doi:10.1242/dev.099408
RESEARCH ARTICLE
Src64B phosphorylates Dumbfounded and regulates slit diaphragm
dynamics: Drosophila as a model to study nephropathies
ABSTRACT
Drosophila nephrocytes are functionally homologous to vertebrate
kidney podocytes. Both share the presence of slit diaphragms that
function as molecular filters during the process of blood and
haemolymph ultrafiltration. The protein components of the slit
diaphragm are likewise conserved between flies and humans, but the
mechanisms that regulate slit diaphragm dynamics in response to
injury or nutritional changes are still poorly characterised. Here, we
show that Dumbfounded/Neph1, a key diaphragm constituent, is a
target of the Src kinase Src64B. Loss of Src64B activity leads to a
reduction in the number of diaphragms, and this effect is in part
mediated by loss of Dumbfounded/Neph1 tyrosine phosphorylation.
The phosphorylation of Duf by Src64B, in turn, regulates Duf
association with the actin regulator Dock. We also find that
diaphragm damage induced by administration of the drug puromycin
aminonucleoside (PAN model) directly associates with Src64B
hyperactivation, suggesting that diaphragm stability is controlled by
Src-dependent phosphorylation of diaphragm components. Our
findings indicate that the balance between diaphragm damage and
repair is controlled by Src-dependent phosphorylation of diaphragm
components, and point to Src family kinases as novel targets for the
development of pharmacological therapies for the treatment of kidney
diseases that affect the function of the glomerular filtration barrier.
KEY WORDS: Dumbfounded, Src64B, Slit diaphragm
INTRODUCTION
Drosophila nephrocytes, the cells involved in removal of waste
products from the haemolymph, have a filtration diaphragm that
shares similarities at the ultrastructural, molecular and functional
levels with the vertebrate slit diaphragm (SD), the molecular filter
of the vertebrate kidney glomerular filtration barrier (PrietoSánchez, 2009; Weavers et al., 2009; Zhuang et al., 2009).
Nephrocytes are highly specialised cells whose surface is covered
by extensive membrane invaginations, the labyrinthine channels,
where most of their endocytic activity takes place. The entrance to
these channels is capped by a filtration diaphragm, 40 nm wide and
ultrastructurally similar to the SD, that restricts the passage of
macromolecules according to their size. The extracellular domains
of Dumbfounded (Duf, also known as Kirre) and Sticks and stones
(Sns) [the Drosophila orthologues of Neph1 (also known as Kirrel)
and nephrin, respectively], which are the main components of the
filtration diaphragm, contribute to the structure of the porous filter.
Their intracellular domains interact with other proteins of the SD
Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Nicolás Cabrera 1,
28049, Madrid, Spain.
*Present address: Instituto de Parasitología y Biomedicina López-Neyra, CSIC,
Avenida del Conocimiento s/n, 18100, Granada, Spain.
‡
Author for correspondence ([email protected])
Received 23 May 2013; Accepted 22 October 2013
protein complex in a way similar to that found in the vertebrate SD
(Benzing, 2004; Patrakka and Tryggvason, 2007). Thus, Duf
interacts with Polychateoid (Pyd), the orthologue of ZO-1, and Sns
interacts with Cindr, the orthologue of CD2AP (Weavers et al.,
2009).
In addition to its well-established structural role, the vertebrate
SD also functions as a signalling node that controls fundamental
aspects of podocyte biology such as transcriptional regulation,
cytoskeleton dynamics, cell survival and endocytosis (reviewed by
Aaltonen and Holthöfer, 2007; Benzing, 2004; Garg et al., 2007a;
Patrakka and Tryggvason, 2010). The importance of SD integrity for
kidney ultrafiltration is highlighted by the fact that congenital and
acquired glomerular diseases that affect the SD severely alter the
morphology of the podocytes, inducing the regression of the foot
processes, a phenomenon known as foot process effacement, that
results in massive proteinuria (Patrakka and Tryggvason, 2009).
However, the molecular mechanisms that lead to foot process
effacement and proteinuria are far from being understood (Kriz et
al., 2013). It is known that podocyte injury associates with changes
in the levels of nephrin and Neph1 phosphorylation. However,
published data are contradictory, as some authors claim that
podocyte injury results in an increase in nephrin and Neph1 tyrosine
phosphorylation, whereas others, using the same experimental
model of induced nephrosis, found a correlation between podocyte
damage and a decrease in phosphorylation (reviewed by Hattori et
al., 2011). Therefore, it is not clear whether increase or decrease of
nephrin and Neph1 phosphorylation is associated with injury and
whether injury is the cause or the consequence of changes in nephrin
and Neph1 phosphorylation. In Drosophila, mutations in duf and sns
also result in loss of filtration diaphragms and in radical changes in
nephrocyte architecture and function (Prieto-Sánchez, 2009;
Weavers et al., 2009; Zhuang et al., 2009). Taking into account all
the similarities previously described, we proposed that the
nephrocyte filtration diaphragm could be a suitable model with
which to study the formation, maintenance and repair of the
vertebrate SD (Weavers et al., 2009) (a view supported by Dow and
Romero, 2010; Simons and Huber, 2009; Zhang et al., 2013).
In this study, we investigate whether regulation by
phosphorylation of one of the main constituents of the diaphragm
occurs in Drosophila nephrocytes. We find that Src64B is
responsible for Duf tyrosine phosphorylation in nephrocytes and that
its activity regulates Duf association with the actin regulator Dock.
Using loss- and gain-of-function experiments, we show that
filtration diaphragm stability requires physiological levels of Src64B
activation. Thus, Src64B loss-of-function mutant nephrocytes
present less SD and altered morphology, including nephrocyte
agglutination and partial regression of labyrinthine channels,
whereas hyperactivation of Src64B leads to massive loss of SD.
Interestingly, diaphragm injury caused by both attenuation or
hyperactivation of Src64B can be repaired upon return to
physiological levels of Src64B activity. We also show that the
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Development
Antonio S. Tutor, Silvia Prieto-Sánchez* and Mar Ruiz-Gómez‡
RESEARCH ARTICLE
Development (2014) doi:10.1242/dev.099408
puromycin aminonucleoside (PAN) model of induced nephrosis,
which is extensively used in vertebrates, is applicable to Drosophila
and that the resulting injury to nephrocytes correlates with increased
activity of Src64B. Our results imply that hyperactivation of
Src64B, and hence hyperphosphorylation of SD constituents, is a
cause of diaphragm injury. In this manner, our findings suggest that
Src family kinases (SFK) should be considered as novel targets for
the development of new therapies in the treatment of congenital and
acquired glomerular diseases associated to dysfunction of the SD.
RESULTS
Dumbfounded is tyrosine phosphorylated by Src family
kinases both in Drosophila S2 cells and in nephrocytes
In vertebrates, the slit diaphragm components nephrin and Neph1
are transiently phosphorylated on tyrosine residues during
development and after podocyte injury (Harita et al., 2008;
Lahdenperä et al., 2003; Verma et al., 2006; Verma et al., 2003). We
therefore anticipated that Duf, the Drosophila orthologue of Neph1,
might also be a substrate for tyrosine phosphorylation. This was
indeed the case, as Duf was detected with anti-phosphotyrosine
368
antibodies in protein extracts of Drosophila S2 cells transiently
transfected with a GFP-tagged version of full-length Duf (DufGFP). Furthermore, tyrosine phosphorylation of Duf was increased
in S2 cells treated with the tyrosine phosphatase inhibitor sodium
orthovanadate (Fig. 1A). In addition, we found that endogenous Duf
was phosphorylated in tyrosine residues in third instar larval
nephrocytes (Fig. 1B). As both nephrin and Neph1 are
phosphorylated by Fyn (Harita et al., 2008; Verma et al., 2003), we
thought that a SFK member might be responsible for Duf tyrosine
phosphorylation. Indeed, when the S2 cells experiments were
repeated in the presence of the SFK-specific inhibitor PP2A,
tyrosine phosphorylation of Duf was eliminated (Fig. 1C). These
data suggest that Src family kinases are involved in Duf
phosphorylation.
Src64B is responsible of Duf phosphorylation in nephrocytes
To identify the tyrosine kinases responsible for endogenous Duf
phosphorylation, we analysed whether the two Drosophila Src
orthologues Src42A and Src64B, as well as the cytoplasmic
kinases Btk29A (Btk family kinase), Csk (C-terminal Src kinase)
Development
Fig. 1. Dumbfounded is phosphorylated by Src64B. (A) Immunoblot analysis with anti-phospho-tyrosine and anti-GFP of S2 cells transfected with Duf-GFP
with or without phosphatase inhibitor and immunoprecipitated with anti-GFP, showed that Duf is phosphorylated in tyrosine in S2 cells. The histogram refers to
three independent experiments. Data are mean±s.e.m. (B) Endogenous Duf precipitated from larval nephrocytes is tyrosine phosphorylated. (C) Incubation of
S2 cells with the SFK inhibitor PP2A abolished Duf tyrosine phosphorylation. (D,E) Cortical and medial views of representative images of control (D, n=4) and
Src64B attenuated (E, n=9) nephrocytes showing accumulation of Duf/Pyd complexes at the cell membrane in D,E and their mobilisation towards cell-cell
contacts (arrowheads) and intracellularly (arrows) in E. The asterisks indicate the cells depicted at higher magnification on the right (see also supplementary
material Fig. S1). (F) Attenuation of Src64B caused a substantial reduction in the amount of endogenous tyrosine phosphorylated Duf. (G) Co-transfection of
S2 cells with Duf-GFP and Src64B increased the levels of Duf tyrosine phosphorylation. (H) Effect on Duf tyrosine phosphorylation of co-transfections with DufGFP and the wild-type, the inactive (K312R) or the constitutively active (Y547F) variants of Src64B. (I) Wild-type Scr64B, but not Src64BK312R, associated
with Duf in S2 cells.
RESEARCH ARTICLE
Development (2014) doi:10.1242/dev.099408
and Shark (Src homology2, ankyrin repeat, tyrosine kinase) were
expressed in third instar larval nephrocytes. We found that Src42A,
Src64B and Btk29A were strongly expressed at this stage, whereas
Csk and shark were expressed at low or undetectable levels
(supplementary material Fig. S1). Next, we used UAS-RNAi lines
for these kinases to attenuate their expression in larval nephrocytes
(see supplementary material Fig. S1 for control of Src42A and
Scr64B attenuation). To identify the requirements for Src42A,
Src64B and Btk29A, we analysed the consequences of loss-offunction conditions for these genes on the distribution of both Duf
and Pyd in nephrocytes. We expected that interfering with Duf
phosphorylation could have an effect on the regulation and/or
stability of the Duf-Pyd complex (Prieto-Sánchez, 2009; Weavers
et al., 2009). Expression of UAS-RNAi-Src42A or UAS-RNAiBtk29A in nephrocytes (by using Pros-Gal4 that drives expression
in garland nephrocytes from stage 16 onwards) (Weavers et al.,
2009) did not significantly affect Duf or Pyd localisation
(supplementary material Fig. S1 and not shown). However,
attenuation of Src64B induced nephrocyte agglutination and
redistribution of both Duf and Pyd proteins. Thus, in contrast to
control nephrocytes (Fig. 1D), Src64B attenuated nephrocytes
maintained many cell contacts between them and although Duf and
Pyd were still present at the cell surface, both proteins
accumulated strongly at cell contact membranes and could be also
detected intracellularly (arrowheads and arrows in Fig. 1E).
Moreover, Duf from Src64B attenuated nephrocytes showed a
strong reduction in tyrosine phosphorylation relative to wild type
(Fig. 1F). To verify that Src64B phosphorylates Duf, S2 cells were
transiently co-transfected with tagged full-length Duf (Duf-GFP)
and either wild-type Src64B, a kinase-constitutively active
form (Y547F) or a kinase-dead variant (K312R) (Kussick
and Cooper, 1992). We observed an increase in tyrosine
phosphorylation of Duf in the presence of wild-type Src64B and
Src64BY547F, and its suppression in the presence of Src64BK312R
(Fig. 1G,H). Furthermore, under these conditions we could coimmunoprecipitate wild-type Src64B, but not Src64BK312R with
Duf (Fig. 1I), indicating that Src64B associates with Duf, and that
this interaction requires Src64B kinase activity. These observations
point to Src64B as the kinase determining the state of
phosphorylation of endogenous Duf.
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Development
Fig. 2. Src64B regulates filtration
diaphragm stability. (A-B′′) Cortical and
medial views of wild-type (A,A′, n=8) and
Scr64BKO (B-B′′, n=12) nephrocytes stained
with anti-Duf, anti-Pyd and TOPRO, showing
the agglutinated phenotype (B′) and the
delocalisation of Duf/Pyd complexes from the
outer membrane (A,A′) towards cell-cell
contacts (white arrowhead in B′) and the
interior of the cell (arrow in B′). B′′ (at the
bottom of the figure) is a detail of the
nephrocyte marked with the empty arrowhead
in B′, showing internalisation of diaphragms.
(C-E) Transmission electron micrographs of
wild-type (C, n=5) and Src64BKO (D,E, n=6)
nephrocytes illustrating a reduction in the
density of diaphragms (arrows in D′, compare
with C′), their absence (arrowhead in D′) and
their internalisation (arrowheads in D′′) in
Src64BKO. (E,E′) Cell membranes rich in
electron-dense material are present
(arrowhead) between the agglutinated
nephrocytes. (F) Quantification of SD
visualized per μm in wild-type and Src64BKO
TE micrographs (n=3). Data are mean±s.e.m.
(G,H) Scanning electron micrographs of wildtype (G, n=11) and Src64BKO (H, n=12)
nephrocytes. (I) Immunoblots showing the
reduction in the level of tyrosine
phosphorylation of endogenous Duf in
Src64BKO nephrocytes. The asterisks in A-B′
show the cell enlarged in the insets; the bars
in C and D show the extension of the cortical
region; the rectangles in C-E show the
regions enlarged in C′,D′ (1), D′′ (2) and E′. N,
nucleus; e, cisterna of rER; v, vacuole. n
refers to number of larvae examined, each
containing ~20 nephrocytes.
RESEARCH ARTICLE
Development (2014) doi:10.1242/dev.099408
Src64B is required to maintain the integrity of the
nephrocyte filtration diaphragm
To further evaluate the requirement for Src64B-mediated
phosphorylation in nephrocytes, we analysed amorph Src64BKO
mutants (O’Reilly et al., 2006). The observed defects were more
severe than those induced by RNAi-mediated attenuation (Fig.
2A,B). The close apposition of mutant nephrocytes (Fig. 2B,E)
could be indicative of a fusion event. However, examination of these
cells revealed that they were separated by cell membranes
(arrowheads in Fig. 2B′,E′) that accumulated both Duf and Pyd (Fig.
2B′). In addition, the nephrocyte membranes exposed to
haemolymph showed reduced amounts of Duf and Pyd complexes
(arrowheads in insets, Fig. 2B,B′, compare with 2A,A′), which
suggested a lower density of filtration diaphragms. Furthermore, Duf
and Pyd were ectopically present inside the nephrocyte (arrows in
Fig. 2B′) and in some instances both proteins could be detected in
internal protrusions at the cortical region (arrowheads in Fig. 2B′′).
These phenotypes were further analysed at the ultrastructural level.
Transmission electron micrographs of Src64KO nephrocytes revealed
changes in their overall morphology (Fig. 2C-E). The most significant
were the shortening and reduction in numbers of labyrinthine channels
(bars in Fig. 2C,D, details in 2C′,D′′ and quantification in 2F) with the
consequent displacement of vacuoles and organelles towards the
370
periphery of the cell and nephrocyte agglutination (Fig. 2E). We also
observed regions practically devoid of diaphragms (arrowhead in Fig.
2D′) and more than one diaphragm closing the entrance of the
labyrinthine channels (arrowheads in Fig. 2D″). The formation of
ectopic diaphragms, which is never observed in wild-type
nephrocytes, correlates with the accumulation of Duf and Pyd at
internal protrusions from the membrane (Fig. 2B″) and could reflect
their internalisation. In addition, electron-dense material, reminiscent
of adherens junctions, was observed at the membrane in regions of
nephrocytes apposition (Fig. 2E′). Scanning electron microscopy of
Src64KO nephrocytes showed a surface smoother than in wild-type
cells (Fig. 2G,H), confirming a reduction of labyrinthine channels.
Finally, tyrosine phosphorylation of Duf in Src64BKO nephrocytes was
significantly reduced (Fig. 2I). Together, these results indicate that
Src64B is the SFK that phosphorylates Duf in nephrocytes and that
Src64B is required to maintain nephrocyte diaphragm stability,
regulating the localisation of the Duf-Pyd complex at the membrane
exposed to haemolymph.
Phosphorylation of Tyr810 is critical for Duf function in
nephrocytes
Duf contains five tyrosine residues susceptible to being
phosphorylated (see Materials and methods). When each of these
Development
Fig. 3. Phosphorylation of tyrosine 810 of Duf is crucial for its
function in nephrocytes. (A) Quantitative immunoblot analysis of
S2 cells transfected with GFP-tagged wild-type Duf or the variant
DufY810A showed a 30% reduction in the global levels of Duf
tyrosine phosphorylation in DufY810A. Data are mean±s.e.m.
(B-D) Overexpression of wild-type Duf (B, n=12), DufY810A (C,
n=15) or Duf-extra (D, n=7) induced no major effects (B) or
nephrocyte agglutination (C,D; arrowheads indicate apposing cell
membranes). (E,F) Loss of Duf causes absence of diaphragms and
an extreme agglutination phenotype (E, n=9). (E) Immunostaining of
larval nephrocytes. The middle panel shows a magnification of a
medial section of the nephrocytes indicated in the inset. The
remaining Pyd is located to cell-cell contact membranes, where it
colocalised with Fasciclin 3 (arrowheads) or appears as punctuated
intracellular staining. Bi-nucleated nephrocytes are present as in the
wild type (F, n=3). (F) Transmission electron micrograph showing
the absence of diaphragms (1) and the presence of cell membranes
(arrowheads in 2,3) between agglutinated nephrocytes. The right
panels show higher magnifications of the regions indicated by
rectangles in the left panel. (G-I) Both Duf wild-type (G, n=9) and
Duf Y810A (H, n=8) rescued the lack of filtration diaphragms
phenotype of Df(1)dufsps1 (cortical sections). However, DufY810A
failed to completely rescue nephrocyte agglutination (arrowheads in
H). Duf-ext did not rescue at all (I, n=5). Left panels show z-stack
projections of groups of nephrocytes. In B-D and G-I, right panels
illustrate higher magnifications of cortical and medial sections of the
nephrocyte indicated in left panels by asterisks. n refers to number
of larvae examined, each containing ~20 nephrocytes.
RESEARCH ARTICLE
Development (2014) doi:10.1242/dev.099408
Fig. 4. Dock associates with Duf phosphorylated in Tyr810. (A) dock expression in wild-type larval nephrocytes (n=5). (B) Immunoblot analysis of S2 cells
co-transfected with either the wild-type or the Y810A Duf variant and Dock in the presence of wild-type Src 64B or the kinase-dead variant Src64 K312R as
indicated. Dock co-immunoprecipitates with wild-type Duf (lanes 1, 2) in a phosphorylation-dependent way: the amount of Dock immunoprecipitated in S2 cells
increases in the presence of ectopic Src64B (lane 2) in comparison with endogenous S2 Src64B (lane 1), whereas there is no interaction in the presence of
Duf Y810A (lane 3) or Src64B K312R (lane 4). (C) Attenuation of dock in nephrocytes induces occasional agglutination of nephrocytes (arrowhead) and the
delocalisation of Duf and Pyd towards the interior of the cell (n=12). Duf and Pyd granules are segregated (arrows) in the medial sections. The right panels
show higher magnifications of the nephrocyte depicted in the top-left panel by an asterisk. n refers to number of larvae examined, each containing ~20
nephrocytes.
Phosphorylation of Tyr810 is crucial for Duf interaction with
Dock
Next, we searched for candidate proteins that bind to Duf upon
tyrosine phosphorylation. As Tyr810 fulfils the consensus for an SH2
domain-binding motif, we investigated the SH2-containing adaptor
proteins Drk (Downstream of receptor kinase) and Dock
(Dreadlocks), the Drosophila orthologues of Grb2 and Nck, on the
basis of their ability to associate with Neph1 and nephrin,
respectively (Harita et al., 2008; Jones et al., 2006; Verma et al.,
2006). As dock and drk were expressed in larval nephrocytes (Fig.
4A and not shown), we examined whether their products associated
with Duf in a phosphorylation-dependent way. S2 cells were cotransfected with GFP-tagged Duf (wild-type or duf-Y810A) and
either V5-tagged Drk or V5-Dock, together with or without Src64B.
Dock (Fig. 4B), but not Drk (not shown), was coimmunoprecipitated with phosphorylated Duf. Strong interaction
was found only in the presence of exogenous Src64B kinase (lane
2). In its absence (lane 1) or in the presence of the kinase-dead
variant Src64BK312R (lane 4), weak or no interaction was detected,
respectively. Furthermore, replacing Tyr810 by Ala completely
abolished Duf-Dock interaction (lane 3). Hence, Tyr810 and its
phosphorylation are crucial for Duf-Dock association.
Drosophila Dock acts as an adaptor of the actin cytoskeleton (Rao
and Zipursky, 1998); thus, a role for Dock for nephrocyte cell
architecture could be anticipated. Owing to the embryonic lethality
of dock loss-of-function alleles, we resorted to RNAi-mediated
attenuation to analyse its role in nephrocytes. dock-attenuated
nephrocytes showed fewer Duf-Pyd complexes at the membrane.
Most interestingly, Duf/Pyd complexes redistributed towards the
interior of the cell (Fig. 4C), suggesting that Dock recruitment by
Duf mediates cytoskeleton rearrangements that are important for the
stability of the filtration diaphragm.
Hyperactivation of Src64B induces nephrocyte
agglutination and loss of filtration diaphragms
After establishing the importance of Duf tyrosine phosphorylation
for the stability of the diaphragm, we examined the effect of Src64B
hyperactivity. Overexpression of the constitutively active form of
Src64B (UAS-Src64BY547F) induced extreme nephrocyte
agglutination (Fig. 5A,B). This was accompanied by severe loss of
filtration diaphragms, visualised as a large decrease of Duf and Pyd
at the nephrocyte surface and their accumulation in the interior of
these cells (Fig. 5A). Moreover, Duf from these experimental
nephrocytes displayed increased tyrosine phosphorylation (Fig. 5C;
Fig. 7). The severity of the phenotype might indicate cell death.
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Development
tyrosines was mutated to alanine and transfected into S2 cells, only
Duf-Y638A, Duf-Y724A and Duf-Y810A showed reduced tyrosine
phosphorylation (40%, 20% and 30% relative to wildtype,
respectively), suggesting that these residues are phosphorylated in
S2 cells (Fig. 3A; data not shown). Significantly, Tyr810
(FSAIYGNPY) fulfils the consensus for a Src kinase substrate motif
(pY[A/G/S/T/E/D]) and also for a -SH2 domain binding motif
(pYXN).
Overexpression (Pros-Gal4) of Duf-Y638A, Duf-Y705A, DufY724A and Duf-Y814A caused no apparent effect, as is the case for
the wild-type Duf protein (Fig. 3B and data not shown). However,
overexpression of Duf-Y810A induced nephrocyte agglutination (Fig.
3C) similar to that generated by overexpression of a Duf protein
lacking its intracellular region (Duf-ext; Fig. 3D). As these transgenic
lines are inserted at the same chromosomal site, the differences in
phenotype cannot be attributed to different levels of overexpression,
and therefore the similarity between Duf-Y810A and Duf-ext
overexpression suggest that impeding Duf phosphorylation at Tyr810
affects duf function in a similar manner to the deletion of its
intracellular region. The contribution of Tyr810 phosphorylation to duf
function was verified in rescue experiments in a dufsps1 mutant
background. Lack of duf causes absence of filtration diaphragms and
agglutination of nephrocytes (Fig. 3E,F). This last phenotype was
verified by the presence of membranes separating apposing
nephrocytes visualised by Fasciclin 3 staining (Fig. 3E) and electron
microscopy (Fig. 3F), and consequently it does not correspond to
nephrocyte hyperfusion, as proposed by Helmstädter et al.
(Helmstädter et al., 2012). The expression of one copy of UAS-duf
(Sns-Gal4) fully rescued the dufsps1 phenotype (Fig. 3G), whereas
UAS-duf-ext was ineffectual (Fig. 3I) and UAS-duf-Y810A caused only
a partial rescue (Fig. 3H). Nephrocytes were less agglutinated than in
dufsps1, with Duf and Pyd accumulating at the apposed membranes as
in Scr64BKO mutants (Fig. 3H, compare with Fig. 2B; see also
supplementary material Fig. S2 for a phalloidin staining). These
results indicate that phosphorylation of Tyr810 is important for duf
function and is needed for the maintenance of nephrocyte architecture.
RESEARCH ARTICLE
Development (2014) doi:10.1242/dev.099408
Fig. 6. Administration of puromycin aminonucleoside induces
nephrocyte injury that correlates with Src64B hyperactivation.
(A,A′) Administration of puromycin aminonucleoside to wild-type larvae
induced nephrocyte injury, as revealed by mild agglutination (A) and
internalisation of Duf/Pyd complexes from the outer membrane (arrowheads
in A′) (n=11). The panels in A′ show details of the nephrocyte indicated by an
arrow in A. (B,C) Immunostainings of wild-type (B, n=5) and PAN-treated (C,
n=7) nephrocytes with anti-pSrc64B indicated that PAN administration
induced Src64B hyperactivation. n refers to number of larvae examined,
each containing ~20 nephrocytes.
However, when UAS-Src64BY547F was co-expressed with UASApoliner, a vital reporter of apoptosis (Bardet et al., 2008), no
signals of nephrocyte apoptosis were evident (supplementary
material Fig. S3). We further studied the effect of Src64B
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Nephrocyte injury correlates with Src64B hyperactivation
and its recovery requires physiological activity of Src64B
Puromycin aminonucleoside-induced nephrosis (PAN) in rodents is a
well-established model of renal injury that causes characteristic
morphological changes, which are associated with rearrangement of
the cytoskeleton, and proteinuria (Caulfield et al., 1976; Luimula et
al., 2002). These changes have been correlated with alterations in the
Development
Fig. 5. Hyperactivation of Src64B induces loss of filtration diaphragms
and nephrocyte agglutination. (A,A′) Overexpression of constitutively active
Src64B (Y547F) in nephrocytes causes the loss of filtration diaphragms. The
amount of Duf/Pyd complexes is reduced and they are delocalised from the
outer membrane (n=13, see also supplementary material Fig. S2).
(B) Scanning electron micrographs of nephrocytes overexpressing
Src64BY547F, showing agglutination of nephrocytes and their smooth cell
surface (compare with Fig. 2F, n=7). (C) Overexpression of Src64BY547F
promoted Duf tyrosine phosphorylation. To obtain similar inputs of Duf in both
lanes, the amount of protein extract used in the overexpression of
Src64BY547F was augmented threefold. (D-F) Transmission electron
micrographs of wild-type (F, n=5) and Src64BY547F-overexpressing (D-D′′′,
n=4) nephrocytes illustrating the lack of filtration diaphragms and the
thickening of the basement membrane (D′′′, compare with F), the presence of
aggregates containing internalised bits of membrane (arrow in D′′) and the
agglutination phenotype (D,D′). Membranes containing adherens junctions
between agglutinated nephrocytes are present (arrow in D′). (E) Duf and Pyd
localised to electron-dense regions in apposing membranes between
agglutinated nephrocytes, as illustrated by cryo-immunogold electron
microscopy. Rectangles 1-3 in D indicate the regions enlarged in D′-D′′′,
respectively. Arrow in D′′′ indicates electron dense material. n refers to number
of larvae examined, each containing ~20 nephrocytes.
hyperactivity on nephrocyte architecture by transmission electron
microscopy (Fig. 5D,E). Sections at the nuclei level revealed
dramatic changes in nephrocyte organisation: the cells lacked a
cortical region (Fig. 5D), due to the absence of labyrinthine channels
and filtration diaphragms (Fig. 5D′′′); the organelles appeared close
to the membrane (Fig. 5D′′′); the basement membrane was thicker
with occasional inclusions (Fig. 5D′′′ bar and arrow, compare with
5F); and the cytoplasm contained intracellular structures composed
of membrane fragments (arrow in Fig. 5D′′). Furthermore, there
were membranes separating adjacent nephrocytes with regions rich
in adherens junctions (arrow in Fig. 5D′), which accumulate Duf and
Pyd (Fig. 5E). In summary, either reduced (as in Src64BKO mutants)
or increased (overexpression of activated Src64B) Src64B activity
in nephrocytes causes destabilisation of the filtration diaphragm
complex and changes nephrocyte architecture, including loss of
labyrinthine channels and agglutination of adjacent nephrocytes.
These changes are concomitant with changes in the levels of Duf
tyrosine phosphorylation and redistribution of Duf-Pyd complexes
from the nephrocyte surface towards the interior of the cell and
regions of cell-cell contact.
RESEARCH ARTICLE
Development (2014) doi:10.1242/dev.099408
levels of phosphorylation of nephrin and Neph1, although there is
disagreement over whether injury is associated with an increase (Garg
et al., 2007b; Harita et al., 2008) or decrease (Jones et al., 2006) of
phosphorylation. To examine the relationship between nephrocyte
injury and levels of Src64B activation, we reproduced the PAN model
of nephrosis in flies. We found that nephrocytes dissected from third
instar larvae cultured in food supplemented with puromycin
aminonucleoside showed a mild degree of agglutination (Fig. 6A,C,
compare with Fig. 2A) and Duf/Pyd complexes delocalised from the
membrane towards more internal regions (arrowheads in Fig. 6A′),
most likely owing to internalisation. Unequivocally, these structural
changes were accompanied by Src64B hyperactivity, revealed by antipY434 Src64B staining, specific for activated Src64B (Fig. 6C,
compare with 6B). Next, we used the TARGET system (McGuire et
al., 2003), which permits spatiotemporal control of gene expression,
to induce conditional damage to nephrocytes by Src64B
hyperactivation. Tubulin-Gal80ts; Pros-Gal4::UAS-Src64BY547F
individuals were raised at permissive temperature until the onset of
third instar larval stage (T0 in Fig. 7B) and then shifted to the
restrictive temperature for 10 hours, allowing expression of UASSrc64BY547F that caused nephrocyte damage that was characteristic of
hyperphosphorylation (T1 in Fig. 7B). Recovery of diaphragm
integrity was substantial 24 hours later (T2) and complete after
another 24-hour period (T3). Importantly, hyperactivation of Src64B
increased Duf phosphorylation, coincident with a reduction in total
amount of Duf (Fig. 7C), and recovery of diaphragm stability was
associated with restored normal Src64B activity and a return to
physiological levels of Duf and its phosphorylation status. These
results prompted us to investigate whether nephrocyte damage
induced by Src64B attenuation was also reversible by using the
TARGET system again to conditionally express UAS-Src64B-RNAi.
We found that this was the case (supplementary material Fig. S4).
Therefore, our data demonstrate that fine-tuning of Src64B dependent
phosphorylation of Duf is paramount for diaphragm stability.
373
Development
Fig. 7. Hyperactivation of Src64B induces reversible nephrocyte damage that correlates with hyperphosphorylation of the filtration diaphragm
component Duf. (A) Experimental procedure used to manipulate Src64BY547F expression temporally in Tubulin-GAL80ts; Pros-GAL4::UAS-Src64BY547F
individuals. Data presented refer to three independent experiments. At 17°C, Gal80 prevents GAL4-dependent activation of UAS-Src64BY547F. Individuals were
raised 17°C until the onset of third instar larval stage (T0). Gal80 was inactivated by shifting to 29°C for 10 hours. Nephrocytes were dissected 1 hour after
returning to 17°C (T1) and every 24 hours thereafter (T2-T4). (B) Immunostaining of nephrocytes with anti-Duf and anti-Pyd antibodies. T0 nephrocytes were
undistinguishable from the wild type (n=6). T1 nephrocytes presented strong agglutination and loss of filtration diaphragms (n=9, see also supplementary
material Fig. S2). T2 nephrocytes showed a clear recovery, manifested by mild agglutination and partial re-localisation of Duf/Pyd complexes at the outer
membrane, n=14. Recovery was almost complete at T3 (n=10) and totally achieved at T4 (n=7). Left panels show z-stack projections of representative strings
of nephrocytes; right panels show cortical and medial sections of the nephrocyte, indicated by an asterisk in the left panels. Arrowheads indicate the outer
membrane. (C) Profile of Duf tyrosine phosphorylation and Src64B activation at different experimental times. Western blot and immunoblot analyses showed
that overexpression of Src64BY547F resulted in a strong hyperactivation of Src64B and in Duf hyperphosphorylation (see graph), and in a dramatic reduction
in the levels of Duf in nephrocytes. Return to physiological activity of Src64B resulted in recovery of Duf levels and in the state of phosphorylation. n refers to
number of larvae examined, each containing ~20 nephrocytes.
DISCUSSION
The dissection of the molecular mechanisms involved in the
regulation of SD stability in mammals has been hindered by access
to podocytes in vivo and by the requirement of podocyte functional
integrity for their viability. Thus, it is still unknown how SD
assembles during development and after injury, and which are the
major players that regulate the dynamic behaviour of SD. Both
issues are fundamental to understanding the molecular basis for
most diseases that lead to end-state renal failure (Patrakka and
Tryggvason, 2009). Given the high degree of similarity between
the podocyte slit diaphragm and the nephrocyte filtration
diaphragm at the molecular, ultrastructural and functional levels
(Prieto-Sánchez, 2009; Weavers et al., 2009; Zhuang et al., 2009),
we decided to use the fly nephrocyte to study in the whole
organism which aspects of podocyte development and behaviour
affect SD integrity under physiological and pathological
conditions. In this work, we show that the stability of the filtration
diaphragm is regulated by the activity of a tyrosine kinase of the
Src family, Src64B, which at least phosphorylates the main SD
constituent, Duf, and controls its association to the actin regulator
Dock.
Src64B phosphorylates Duf and regulates the structural
integrity of the nephrocyte filtration diaphragm
Many of the vertebrate slit diaphragm proteins are tyrosine
phosphorylated in normal glomeruli (Zhang et al., 2010).
Furthermore, tyrosine phosphorylation of nephrin and Neph1 by the
SFK Fyn is crucial for the stability of this protein complex and
therefore for glomerular filtration function (reviewed by Hattori et
al., 2011). Thus, upon phosphorylation, the cytoplasmic regions of
nephrin and Neph1 recruit the intracellular adaptors Nck and Grb2,
among others, that in turn regulate actin cytoskeleton reorganisation
(Harita et al., 2008; Jones et al., 2006; Tryggvason et al., 2006;
Verma et al., 2003). We found that the post-translational regulation
by phosphorylation of Duf, the orthologue of Neph1, is conserved
and that Src64B, a member of the non-receptor Src family tyrosine
kinases, is responsible for Duf tyrosine phosphorylation in
nephrocytes. Furthermore, Src64B function is necessary for the
structural integrity of the filtration diaphragm and for normal
nephrocyte morphology. In Src64B loss-of-function or knockdown
conditions, there is a reduction in the density of filtration
diaphragms at the nephrocyte cell membrane. Presumably, this is
due in part to their internalisation, as suggested by the accumulation
of both Duf and Pyd at protrusions extending inwards from the
membrane and the presence in these locations of structures dense to
electrons reminiscent of filtration diaphragms. In addition, we
observe delocalisation of Duf/Pyd complexes to cell contact
membranes that, at the ultrastructural level, are rich in adherens
junctions. The presence of adherens junctions in apposed
membranes is never found in wild-type mature nephrocytes but is
characteristic of embryonic nephrocytes prior to the formation of
filtration diaphragms (Prieto-Sánchez, 2009). All these alterations
are concomitant with changes in nephrocyte architecture, revealed
by their smoother surface and their agglutination. As the loss of
filtration diaphragms result in regression of labyrinthine channels,
and this is always associated with nephrocyte agglutination, we
interpret these morphological changes as being due to reallocation
of Duf/Pyd complexes from SD to adherens junctions. We suggest
that these morphological alterations are the nephrocyte equivalent
of podocyte foot process effacement, a feature common to all
proteinuric diseases and believed to be initiated by changes in the
actin cytoskeleton.
374
Development (2014) doi:10.1242/dev.099408
Scr64B-mediated phosphorylation of Duf regulates its
coupling to the cytoskeleton
Although Src64B might be involved in phosphorylation of other
components of the filtration diaphragm, including the fly nephrin
orthologue Sns, our data suggest that Duf is a main target of
Src64B-mediated phosphorylation and participates in the
reorganisation of the actin cytoskeleton. We find that, upon
phosphorylation of residue Tyr810, Duf can associate with the
adaptor protein Dock, the Drosophila orthologue of Nck, and that
phosphorylation of this residue is important for Duf function, thus
reproducing in nephrocytes the connection between SD and actin
cytoskeleton existing in podocytes. Our data also show that
attenuation of dock leads to dissociation of the Duf/Pyd complex,
although further studies will be necessary to reveal the molecular
basis of these observations. These results indicate that, through the
regulation of Duf-Dock interactions, Src64B controls the coupling
of SD protein complex to the actin cytoskeleton, and point to the
modulation of SFKs activity as a promising therapeutic target for the
treatment of proteinuric renal diseases.
Drosophila nephrocytes as a model of induced nephrosis
The advance in the knowledge of the molecular basis of human
diseases and their treatment constantly demands the development of
reliable and genetically amenable preclinical animal models, valid for
genetic screenings and drug discovery. The success of such preclinical
models relies on the rigorous assessment of the reproducibility of the
human disease under study in the animal model. We show here that
this is the case for Drosophila melanogaster and the study of some
nephropathies. The PAN model, which induces foot process
effacement and proteinuria, has become a mainstay in the study of
podocyte dysfunction (Pippin et al., 2009). Thus, the early response
of podocytes to PAN is to change the levels and distribution of SD
proteins such as nephrin and podocin, followed by flattening of foot
processes and proteinuria (Guan et al., 2004; Luimula et al., 2002).
We reasoned that if the analogy between podocyte and nephrocyte
filtration diaphragms was genuine and the earliest targets of PAN were
SD components, then the nephrocyte diaphragm should react to PAN
treatment in a similar way. Interestingly, this is what we observed.
Drug treatment induced delocalisation of Duf and Pyd from the outer
membrane and a mild degree of nephrocyte agglutination. Moreover,
this treatment has allowed us to firmly establish a correlation between
induced nephrocyte damage, Src64B hyperactivity and an increase in
the phosphorylation of SD components, in agreement with some
vertebrate data (Garg et al., 2007b; Harita et al., 2008). These results
confirm the similarity between both cell types and validate the use of
Drosophila to study SD behaviour during development and in
pathological conditions.
Src64B hyperactivity causes reversible nephrocyte damage
PAN treatment did not allow us to solve the controversial subject of
the causality between diaphragm injury and changes in
phosphorylation of SD components, as continuous exposure to the
drug could mask possible attempts of diaphragm repair. Therefore,
we used the advantages of Drosophila to manipulate gene function
spatiotemporally to induce the expression of the constitutively active
form of Src64B for a short temporal window and check whether it
provoked nephrocyte damage. This allowed us to establish a causal
relationship between Src64B hyperactivity and injury, and returning
to permissive temperature permitted analysis of diaphragm recovery
in the absence of further induced damage. In this way we could
establish that Src64B hyperactivity causes an increase in Duf
phosphorylation and a dramatic decrease in the total amount of this
Development
RESEARCH ARTICLE
RESEARCH ARTICLE
Development (2014) doi:10.1242/dev.099408
adherens junctions. This situation illustrates the dynamics of the
molecular interactions at play during diaphragm assembly, as it is the
opposite to what happens during development when Sns/Duf/Pyd
complexes mobilise from cell contact membranes to form filtration
diaphragms (Prieto-Sánchez, 2009). We would like to propose that
injury to nephrocytes by means of drug treatment or stress results in
hyperactivation of Src64B that increases Duf phosphorylation,
causing destabilisation of SD and internalisation for degradation of
their components (Fig. 8C). Whether the internalisation of Duf is a
result of more molecules of Duf being phosphorylated or
phosphorylation of Duf on other tyrosine residues will be the subject
of further investigation.
MATERIALS AND METHODS
Fly stocks
protein. These changes coincide with delocalisation of Duf and its
binding partner Pyd from the membrane towards the interior of the
cell and an extreme agglutination phenotype. Return to
physiological Src64B activity results in recovery of the nephrocytes,
coinciding with recuperation of Duf protein levels and degree of
phosphorylation. It is noteworthy that in no case did we find a
significant increase in Duf phosphorylation above the levels
observed before the damage, suggesting that recovery does not
require an increase in Duf phosphorylation relative to the
physiological levels present in mature nephrocytes.
In summary, our results indicate that, in Drosophila, the stability of
the filtration diaphragm depends on the activity of the SFK Src64B,
which phosphorylates Duf. Phosphorylation of Duf creates a new
docking site for its association with Dock, which might be
instrumental in organizing actin cytoskeleton and nephrocyte
architecture (Fig. 8A). Lack of Src64B induces a reduction of
filtration diaphragms and mobilisation of Duf/Pyd complexes towards
cell contact membranes, causing nephrocyte agglutination (Fig. 8B).
The fact that there are diaphragms in the absence of Src64B activity
indicate that Src64B is not involved in promoting the association of
the main diaphragm components (Sns, Duf, Pyd) but in regulating the
link between the diaphragm protein complex and the cytoskeleton,
which could be necessary to stabilise the complex at the outer
membrane, sealing the labyrinthine channels. We suggest that when
the association between SD and the cytoskeleton is weak or does not
take place, there is an internalisation of the SD components and their
redistribution to cell contact membranes, where they contribute to
Plasmid constructs
Online prediction servers NetPhos 2.0 (Blom et al., 1999), DISPHOS 1.3
(Iakoucheva et al., 2004) and PhosphoMotif Finder (Amanchy et al., 2007)
were used to identify Duf tyrosines residues susceptible to phosphorylation.
Duf mutant variants Y638A, Y705A, Y724A, Y810A and Y814A were
generated by PCR using as DNA template pBS-DufGFP-wt with the
QuikChange Site-Directed Mutagenesis Kit (Stratagene). PCR products
were sequenced and subcloned into pAC5.1 for cell culture experiments and
pUAST-attB vectors for generation of transgenic lines. EST clones LD42588
and LD12029 were used to clone V5-tagged versions of Dock and Drk,
respectively, in pAC5.1 vector. Src64B wt, Src64B K312R and Src 64B
Y547F were gifts from J. A. Cooper (FHCRC, WA, USA).
Cell culture and transfections
Drosophila Schneider (S2) cells were grown at 25°C in Insect-X press
(BioWhittaker, MD, USA) containing 7% fetal bovine serum. Cells were
transfected with 2 μg of the DNAs required using the Nucleofector Amaxa
procedure, as indicated in the manufacturer’s protocol (BioWhittaker). After
transfection, cells were plated in six-well plates (2×106 cells per well) and
incubated at 25°C for 48 hours. In specific cases, cells were treated with 10
mM orthovanadate (Sigma) or 5 mM PP2A inhibitor (Sigma) for 30 minutes
before harvesting the cells.
Co-immunoprecipitations and western blots
Transfected S2 cells or larval nephrocytes were washed and lysed in
modified ice-cold RIPA lysis buffer [50 mM Tris (pH 7.4), 150 mM NaCl,
1 mM EDTA, 1% NP-40, 0.25% DOC] supplemented with phosphatase
inhibitor (1 mM NaVO4 and 25 mM NaF) plus proteases inhibitors for 6090 minutes at 4°C. After centrifugation (15,000 g, 15 minutes), lysate
aliquots were taken to assess protein overexpression and the rest of cellular
extracts were immunoprecipitated with the desired antibodies by overnight
incubation at 4°C. After incubation with protein A or G agarose and
extensive washing with lysis buffer, lysates and immunoprecipitates were
resolved by SDS-PAGE, and the gel was transferred to nitrocellulose
membranes to be probed with specific antibodies. Densitometry analysis was
carried out using Bio-Rad Quantity One software. The following antibodies
were used: anti-GFP (1:1000, Roche), anti-phosphotyrosine (1:1000,
Millipore), anti-Duf (1:100), anti-Src64B (1:500, A. O’Reilly) or anti-V5
(1:5000, Invitrogen).
375
Development
Fig. 8. Effect of Src64B activity on the stability of the nephrocyte slit
diaphragm. (A) In wild-type larval nephrocytes, Src64B phosphorylates Duf
at Tyr 810, creating a novel docking site for its association with Dock, a
regulator of actin cytoskeleton. This interaction stabilises the slit diaphragm
complex at the outer membrane, capping the entrance to the labyrinthine
channels. (B) In Src64B flies, failure to establish a stable link between the slit
diaphragm complex and the cytoskeleton entails the mobilisation of their
constituents towards sites of cell contact, inducing nephrocyte agglutination.
(C) Src64B hyperactivation, induced by overexpression of activated Src64B
or as result of external agents that result in nephrocyte injury, leads to an
increase in the levels of Duf phosphorylation, its mobilisation towards cell
contact membranes and its internalisation for degradation.
Flies were reared at 25°C, except when specifically indicated. The following
Drosophila strains were used: Oregon R, Df(1)Duf sps1 (deficiency made
using the P elements P[XP]d8289 and pBAC[RB]e03354 that eliminates 52 kb
of DNA, including Duf coding region) (Prieto-Sánchez, 2009), Src64BKO,
UAS-Src64B Y547F (A. O’Reilly, Fox Chase Cancer Center, USA), UASapoliner (J. P. Vincent, MRC, UK), UAS-Src64B RNAi (DGRC, Japan),
UAS-Src64 K312R (L. G. Fradkin, LUMC, The Netherlands), UAS-Src42A
RNAi, UAS-Btk29 RNAi, UAS-Csk RNAi and UAS-Nck RNAi (VDRC,
Austria), Apterous-GAL4; UAS-GFP, prospero-GAL4 (C. Doe, HHMI,
USA), tubulin-GAL80ts; prospero-GAL4 and sns-GC-GAL4 (S. Abmayr,
SIMR, USA).
RNA in situ hybridisation and immunohistochemistry
Larval nephrocytes were processed for in situ hybridisation and
immunohistochemistry as indicated by Weavers et al. (Weavers et al., 2009).
The following primary antibodies were used: anti-Duf (1:100), anti-Pyd
(1:100), anti-Fas3 (1:50, DSHB, IA, USA), anti-pSrc64B (1:500; A.
O’Reilly) and anti-Src42A (1:1000; S. Hayashi, RIKEN Center, Japan). A
15-minute counterstain with TO-PRO-3 (1:1000, Invitrogen) was used to
visualise nuclei. Confocal images were acquired using a Zeiss LSM 510
Meta or Zeiss LSM 710 microscope and processed using Adobe Photoshop
CS and ImageJ software.
Electron microscopy
Samples for transmission and scanning electron microscopy were processed
using standard techniques modified as described previously (Weavers et al.,
2009). For immunoelectron microscopy, dissected third instar larval
nephrocytes were fixed in 4% formaldehyde + 0.05% glutaraldehyde,
embedded in gelatin, cryosectioned and incubated with anti-Duf (1:5) and
anti-Pyd (1:5); 5 nm and 10 nm gold-conjugated secondary antibodies were
used. Samples for transmission electron microscopy were observed in a
Jem1010 (JEOL) instrument working at 80 kV.
Puromycin aminonucleoside treatment
Adult Oregon R flies were collected within the first 24 hours of their
emergence and fed with standard fly food supplemented with 400 μg/ml
puromycin aminonucleoside (Sigma). Wandering third instar larvae from
their progeny were dissected for nephrocyte analysis.
Acknowledgements
We are grateful to H. Skaer, B. Denholm, J. F. de Celis, S. Campuzano, J. Modolell
and other members of the lab for helpful comments and advice. We thank J. P.
Vincent, A. O’Reilly, L. G. Fradkin, C. Doe, S. Abmayr, S. Hayashi and J. A.
Cooper for sharing antibodies, reagents and flies. We also thank the Bloomington,
NIG-FLY and VDRC stock centres; and the BDGP and DSHB for providing fly
stocks, cDNA clones and antibodies. We thank S. Velázquez for technical
assistance; E. Caminero and M. Casado from the CONSOLIDER transgenic and
stock platforms; M. Guerra from the Electron Microscopy service of the CBMSO;
and E. Salvador from SEM Services of the SIDI-UAM for their technical support.
Competing interests
The authors declare no competing financial interests.
Author contributions
M.R.-G. and A.S.T. conceived and designed the experiments. A.S.T. and S.P.-S.
performed the experiments. A.S.T., S.P.-S. and M.R.-G. analysed the data.
M.R.-G. wrote the paper.
Funding
This work was supported by the Ministerio de Ciencia e Innovacion MICINN
[BFU2010-14884] to M.R.-G. and by the Spanish Ministry of Education and
Science (MEC) [CSD-2007-00008]. An institutional grant from Fundación Ramón
Areces to the CBMSO is also acknowledged.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.099408/-/DC1
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