Download Larkins CE, Busey Long A, Caspary T. Dev Biol. 2012 Jul 1;367(1):15-24. Defective Nodal and Cerl2 expression in Arl13b(hnn) mutant node underlies its heterotaxia.

Developmental Biology 367 (2012) 15–24
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Developmental Biology
journal homepage: www.elsevier.com/locate/developmentalbiology
Defective Nodal and Cerl2 expression in the Arl13bhnn mutant node underlie
its heterotaxia
Christine E. Larkins a,b, Alyssa Bushey Long a, Tamara Caspary a,n
a
b
Department of Human Genetics, Emory University School of Medicine, 615 Michael Street, Suite 301, Atlanta, GA 30322, USA
Graduate Program in Biochemistry, Cell and Developmental Biology, Emory University, Atlanta, GA, USA
a r t i c l e i n f o
abstract
Article history:
Received 23 August 2011
Received in revised form
5 April 2012
Accepted 6 April 2012
Available online 24 April 2012
Specification of the left–right axis during embryonic development is critical for the morphogenesis of
asymmetric organs such as the heart, lungs, and stomach. The first known left–right asymmetry to
occur in the mouse embryo is a leftward fluid flow in the node that is created by rotating cilia on the
node surface. This flow is followed by asymmetric expression of Nodal and its inhibitor Cerl2 in the
node. Defects in cilia and/or fluid flow in the node lead to defective Nodal and Cerl2 expression and
therefore incorrect visceral organ situs. Here we show the cilia protein Arl13b is required for left right
axis specification as its absence results in heterotaxia. We find the defect originates in the node where
Cerl2 is not downregulated and asymmetric expression of Nodal is not maintained resulting in
symmetric expression of both genes. Subsequently, Nodal expression is delayed in the lateral plate
mesoderm (LPM). Symmetric Nodal and Cerl2 in the node could result from defects in either the
generation and/ or the detection of Nodal flow, which would account for the subsequent defects in the
LPM and organ positioning.
& 2012 Elsevier Inc. All rights reserved.
Keywords:
Nodal
Cerl2
Arl13b
Left–right axis
Introduction
The vertebrate body plan is established during embryonic
development through the specification of three axes: the anterior-posterior, dorsal-ventral, and left–right (LR). In vertebrates,
the LR axis is the final axis to be specified just before visceral
development, and its specification is necessary for asymmetric
development of organs, such as the heart, lungs, and stomach
(Peeters and Devriendt, 2006; Shiratori and Hamada, 2006). Work
over the last 15 years has provided tremendous advances in our
understanding of how the LR axis is established, but many steps
along the way, from the initial breaking of bilateral symmetry to
the development of asymmetric organs, remain elusive (Bisgrove
et al., 2003; Raya and Izpisua Belmonte, 2008; Speder et al., 2007;
Tabin, 2005; Vandenberg and Levin, 2009, 2010).
In mouse, the first known event required for establishment of
the LR axis is the leftward flow of extraembryonic fluid in the
node, which is created by the rotation of motile cilia on the node
surface (Nonaka et al., 2002; Nonaka et al., 1998; Okada et al.,
1999). At the molecular level, two proteins are initially expressed
symmetrically on the left and right side of the node: Nodal and its
inhibitor cerberus-like 2 (Cerl2) (Collignon et al., 1996; Lowe
n
Corresponding author. Fax: þ 404 727 3949.
E-mail address: [email protected] (T. Caspary).
0012-1606/$ - see front matter & 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ydbio.2012.04.011
et al., 1996). Once fluid flow is initiated, Nodal shows increased
expression at the left node periphery while Cerl2 is down
regulated on the left side of the node (Marques et al., 2004). As
Nodal feeds back to induce its own expression, Cerl2 repression on
the left side of the node would allow for the observed left-sided
increase in Nodal expression (Kawasumi et al., 2011; Norris et al.,
2002; Schweickert et al., 2010). While both Nodal and Cerl2 are
likely regulated in response to flow, the exact mechanisms of flow
detection and the cellular response to flow have been vigorously
debated. One hypothesis posits that the flow physically enriches
the left side of the node with a morphogen such as Sonic
Hedgehog (Shh) (Tanaka et al., 2005). Alternatively, another
hypothesis (the two cilia model) proposes that the flow generated
by motile cilia at the center of the node is detected via mechanosensory cilia at the node periphery (McGrath et al., 2003).
Although the debate is not settled, two recent observations are
consistent with the two cilia model: first, very little flow is
needed to break asymmetry within the node (Shinohara et al.,
2012); and second, the putative sensors in the mechanosensory
cilia at the node periphery were identified as Pkd1l1 and Pkd2
(Field et al., 2011; McGrath et al., 2003).
From the node, asymmetry is established throughout the
embryo. Nodal becomes expressed in the left lateral plate mesoderm (LPM) where it is required for correct asymmetric development of visceral organs. Evidence suggests that the Nodal ligand
itself diffuses from the node to the LPM to induce its own
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C.E. Larkins et al. / Developmental Biology 367 (2012) 15–24
expression there (Oki et al., 2007). Therefore, it is important that
the levels of active Nodal ligand be tightly regulated in the node
and LPM to achieve expression in only the left LPM.
In addition to Cerl2, Nodal is also inhibited by the TGF-beta
proteins Lefty1 and Lefty2, which are expressed at their highest
levels in the midline and LPM of the embryo, respectively, and are
targets of the Nodal ligand (Meno et al., 2001; Saijoh et al., 2000;
Yamamoto et al., 2003). Because Nodal can diffuse over long
distances to induce its own expression, Lefty1 acts as a barrier at
the midline preventing Nodal from inducing expression in the
right LPM (Meno et al., 1998; Meno et al., 2001). The expression of
Lefty2 in the left LPM limits the amount of time Nodal is expressed
there, and this expression pattern of both Lefty1 and Lefty2 creates
a situation in which the level of active Nodal ligand must reach a
concentration threshold in the LPM to overcome inhibition by the
Lefty proteins (Nakamura et al., 2006), thus adding another layer
to the tight regulation of active Nodal protein in the embryo.
Several other signaling events help mediate the left-sided
enrichment of Nodal in the node and its subsequent expression
in the left LPM. Wnt signaling, specifically Wnt3a, induces Notch
at the node, and Notch signaling is required for Nodal to be
enriched in the left node periphery (Krebs et al., 2003; Nakaya
et al., 2005; Raya et al., 2003). Shh signaling is needed to induce
expression of Gdf1 at the node periphery, which is required for
Nodal ligand diffusion to the left LPM (Tanaka et al., 2007; Zhang
et al., 2001). The genetic network of LR axis establishment
involves many players, some of which are still unknown, but
together they turn a localized leftward flow of extraembryonic
fluid into a robust LR asymmetry in the LPM that can guide
visceral organ development.
Here we describe the left–right axis defects found in the
Arl13b hennin (hnn) mouse mutant. Arl13bhnn mutants lack the cilia
protein Arl13b (ADP ribosylation factor-like 13b), a small regulatory GTPase, which leads to shortened cilia in the node (Caspary
et al., 2007). We show here that the Arl13bhnn mutants are
heterotaxic following an unusual pattern of Nodal expression: in
the LPM, Nodal initially shows delayed and randomized expression, with subsequent bilateral expression, while in the node
there is an inability to maintain asymmetric Nodal expression.
Interestingly, we found that although Nodal initially shows
expression in the node in a pattern that is similar to wild-type,
Cerl2 is not correctly down regulated on the left side of the node,
indicating that, in our mutants, the defect in Nodal expression
may be secondary to a defect in Cerl2 expression. Mouse mutants
that are unable to generate flow as well as mutants that cannot
detect flow also express Nodal and Cerl2 symmetrically within the
node raising the possibility that one or both of these processes is
disrupted in Arl13bhnn mutants.
Materials and methods
Mouse strains
The strains (Arl13bhnn, IFT172wim, BATgal, Patched-lacZ, and
NodallacZ) were genotyped as previously described and bred at
least 10 generations on the C3H background (Collignon et al.,
1996; Garcia-Garcia et al., 2005; Goodrich et al., 1997; Huangfu
et al., 2003; Maretto et al., 2003).
Phenotypic analysis
Timed matings of mice carrying the Arl13bhnn mutation were
performed, and embryos were harvested using somite number for
staging. In situ hybridization was as previously described, and
embryos were genotyped after analyzing expression patterns.
Antisense probes were generated from the following cDNA plasmids
(with sources): Nodal (Elizabeth Robertson), Gdf1 (Nancy Wall), Shh
and Foxa2 (Andy McMahon), NotchI (Janet Rossant), Lefty1 and Lefty2
(Hiroshi Hamada), Pitx2 (Axel Schweickart), Cerl2 (IMAGE clone ID
790229), and Dll1 (Radhika Atit).
Embryos homozygous for Arl13bhnn and carrying either the
BATgal, Patched-lacZ, or Nodal-lacZ reporter alleles were harvested
at E8.5 and fixed in 4% PFA with 0.2% glutaraldehyde for 15 min
(Goodrich et al., 1997; Maretto et al., 2003). Embryos were
washed (0.1 M phosphate buffer, 2 mM MgCl2, 0.01% sodium
deoxycholate, 0.02% NP-40) and then treated with 1 mg/mL
X-gal in 5 mM potassium ferricyanide and 5 mM potassium
ferrocyanide overnight at room temperature.
For organ analysis, embryos were harvested at E12.5. Organs
were removed from the body cavity to allow imaging of the lungs.
Images were taken using a Leica DM6000B upright fluorescence
microscope and processed using QCapture software.
Results
Arl13bhnn mutants have heterotaxia
Midgestation Arl13bhnn embryos display a variety of phenotypes including randomized heart looping, an indication of LR axis
defects (Caspary et al., 2007; Garcia-Garcia et al., 2005). To
characterize the extent of the LR axis defects in Arl13bhnn mutants,
we examined the orientation of the heart, lungs, and stomach in
E12.5 embryos. Normally, the left lung has a single lobe, whereas
the right lung has four, so we determined the left versus right lung
by the presence of lobes. We found that in Arl13bhnn mutants, the
lungs most often showed left isomerism (44.4% of embryos) and
normal orientation at a high rate (38.9%), although the right lung
had smaller and fewer lobes (Fig. 1(A)–(F), Table 1, and data not
shown). The heart and stomach orientation were almost completely randomized and were not coordinated. For example, the
heart could be on the left while the stomach was on the right
(Fig. 1(A)–(F), Table 1) indicating that, in addition to the global
defect in specifying the LR axis, individual organs interpret the LR
axis distinctly, a defect called heterotaxia. This anomaly is seen in
several other mouse mutants with LR defects, as well as in human
laterality disorders (Chen et al., 1998; Lin et al., 1999; Lowe et al.,
2001; Meno et al., 1998; Peeters and Devriendt, 2006).
Nodal and Pitx2 are misexpressed in the LPM of Arl13bhnn mutants
Asymmetric organ development is guided by the asymmetric
expression of Nodal and its downstream target Pitx2 in the LPM,
both of which we assessed through in situ hybridization (Campione
et al., 1999; Lin et al., 1999; Liu et al., 2001; Ryan et al., 1998;
Shiratori et al., 2001). In all cases, we scored molecular expression
patterns prior to genotyping alleviating any ascertainment bias.
Wild-type embryos display Nodal expression in the left LPM starting
at the 2–3 somite stage, and this expression is extinguished by the
6–7 somite stage (Fig. 2(A)–(D)). In Arl13bhnn mutants, we saw an
initial delay in Nodal expression in the LPM until the 4–5 somite
stage. Nodal began to be expressed in the LPM at the 4–5 somite
stage, but still half of the embryos at this stage did not show Nodal
expression (Fig. 2(H)). Those embryos with expression at the 4–5
somite stage most often showed expression in the left LPM or
bilaterally (Fig. 2(H)). At the 6–7 somite stage, when most wild-type
embryos have no expression in the LPM (Fig. 2(A)–(D)), almost all
Arl13bhnn embryos had Nodal expression that was bilateral
(Fig. 2(E)–(H)). Therefore, in Arl13bhnn mutants, not only is there a
spatial defect in Nodal expression, but there is also a temporal
defect. This expression pattern was in marked contrast to mutants
C.E. Larkins et al. / Developmental Biology 367 (2012) 15–24
lacking cilia. Consistent with previous reports, we observed bilateral
Nodal expression in the LPM in IFT172wim mutants starting at early
somite stages (Huangfu et al., 2003). Because there is not a delay in
Nodal expression in mutants lacking cilia, our data indicate that
Nodal expression in the LPM is more severely affected in Arl13bhnn
embryos with abnormal cilia than in IFT172wim embryos lacking cilia
altogether.
In the LPM and splanchnopleure of E8.5 and E9.5 Arl13bhnn
embryos, we found that Pitx2 was most often expressed bilaterally or exclusively on the left side of the embryo (Fig. 3(A), (B)–
(D)). This pattern is consistent with the laterality in the lungs of
Arl13bhnn mutants, which most often showed left isomerism or
17
normal orientation (Table 1). Arl13bhnn embryos lacking Pitx2
expression were most often found at early somite stages, suggesting Pitx2 expression is also delayed in the absence of Arl13b.
The midline barrier in Arl13bhnn mutants is lost
Because we saw a large number of Arl13bhnn embryos with
bilateral Nodal expression, we wanted to examine a possible defect
in maintaining the midline barrier. In wild-type embryos, Nodal from
the LPM turns on expression of its inhibitor Lefty1 in the prospective
floor plate at the 2–3 somite stage, and Lefty1 expression is
extinguished by the 6–7 somite stage due to loss of Nodal expression
in the LPM (Meno et al., 1998; Yamamoto et al., 2003). Our previous
analysis of Arl13bhnn shows that by E9.5, mutants lack a floorplate in
the caudal embryo (Caspary et al., 2007); however, we did see
expression of the floor plate marker Foxa2, as well as Lefty1, in
mutant embryos at E8.5 (Fig. 4). Interestingly, Lefty1 was not
expressed in the midline at the 6–7 somite stage of our mutant
embryos, although Nodal expression persisted in the LPM (Fig. 4(A)–
(F), Fig. 2(G) and (H)). As there is no floorplate at E9.5 in our mutants,
these results could indicate a loss of floorplate integrity in Arl13bhnn
mutants. While examining Lefty1 expression, we also observed Lefty2
expression and found it follows the same pattern as Nodal in the LPM
(Fig. 4(A) and (B), (D) and (E), n¼13).
Nodal asymmetry is initiated but not maintained in the node
of Arl13bhnn mutants
Fig. 1. Organ laterality in Arl13bhnn mutants shows heterotaxia, while Arl13bhnn;
NodallacZ/ þ shows right isomerism. (A)–(C) Wild-type E12.5 embryos. (D)–(F)
E12.5 Arl13bhnn mutant embryos. (G)–(I) E12.5 Arl13bhnn; NodallacZ/ þ embryos.
(B) A ventral view of the internal organs of the wild-type embryo shows normal
placement of the heart with its apex towards the left. (C) A dorsal view of the
organs shows the left–right asymmetry of the lungs, where there is a single left
lobe and 4 right lobes. The stomach can be seen on the left side of the embryo. (E)
A ventral view shows the heart apex toward the left side of the Arl13bhnn mutant
embryo. (F) A dorsal view of the same embryo in (D) and (E) shows the stomach on
the right, as well as a single lobe on the left and right lung. (H) A ventral view of
the Arl13bhnn; NodallacZ/ þ mutant shows the heart apex toward the left. (I) A dorsal
view shows there are small lobes on both the left and right lung, with the stomach
in the center of the embryo. Inset is a ventral view of the lungs, with arrowheads
pointing to the lobes. rv, right ventricle; lv, left ventricle; st, stomach; l, left;
r, right.
Nodal and Cerl2 are initially expressed symmetrically in the crown
cells of the node and are then asymmetrically expressed as flow
commences at early somite stages (Nonaka et al., 1998; Okada et al.,
1999). The expression of Cerl2, an inhibitor of Nodal, is reduced on
the left while being maintained on the right, while Nodal expression
is increased on the left (Marques et al., 2004; Schweickert et al.,
2010). This pattern of Cerl2 expression results in an increase in active
Nodal protein on the left side of the node, which leads to increased
Nodal expression in the left LPM (Kawasumi et al., 2011; Oki et al.,
2009). We examined Nodal and Cerl2 expression in the node, and
surprisingly, we saw that almost half of Arl13bhnn embryos had
increased Nodal expression on the left side of the node at presomite
stages, which was nearly identical to wild-type (Fig. 5(A) and (B)).
However, that asymmetry was gradually lost in Arl13bhnn mutant
embryos at later somite stages (Fig. 5(A) and (B)). Consistent with
Cerl2 inhibiting Nodal, we saw that Cerl2 expression was not down
regulated on the left side of the Arl13bhnn node to the same extent as
wild-type from the presomite to 5 somite stage (Fig. 5(D) and (E)),
indicating that the gradual loss in Nodal asymmetry could be due to
an inability to down regulate Cerl2.
Left biased Nodal asymmetry is not initiated in the node of IFT172wim
mutants
We examined mutants completely lacking cilia, the IFT172wim
mutants, to see if they had similar defects in Nodal enrichment in
Table 1
Organ laterality in wild-type, Arl13bhnn, and Arl13bhnn; NodallacZ mutants. The orientation of the heart, lungs, and stomach were determined for wild-type and mutant
embryos. n is the number of embryos examined and the percentage of total embryos examined is shown.
Heart
Wild-type
Arl13bhnn/hnn
Arl13bhnn/hnn; NodallacZ/ þ
Lungs
Stomach
Left (%)
Right (%)
n¼
Normal (%)
Reversed (%)
Left isom (%)
Right isom (%)
n¼
Left (%)
Right (%)
Center (%)
n¼
100
57.9
62.5
0
42.1
37.5
6
19
8
100
38.9
0
0
6
0
0
44.4
0
0
11.1
100
6
18
5
100
52.4
0
0
42.9
12.5
0
4.8
87.5
6
21
8
18
C.E. Larkins et al. / Developmental Biology 367 (2012) 15–24
Fig. 2. Nodal expression is disrupted in Arl13bhnn mutants. (A)–(C), (E)–(G), (I)–(K), (M)–(O) in situ hybridization for Nodal. (A)–(C) Wild-type embryos showing Nodal
expression in the left LPM starting at the 2–3 somite stage (A) and being extinguished at the 6–7 somite stage (C). (E)–(G) Arl13bhnn mutant embryos showing no LPM
expression at the 2–3 somite stage (E), right-sided expression at the 4–5 somite stage (F), and bilateral expression at the 6–7 somite stage (G). (I)–(K) IFT172wim mutants
showing bilateral Nodal expression starting at the 2–3 somite stage. (M)–(O) Arl13bhnn; IFT172wim mutants have bilateral expression of Nodal starting at the 2–3 somite
stage (M). (D), (H), (L), (P) Graphs quantifying the number of embryos, with each Nodal expression pattern broken down by somite stage.
the node (Huangfu et al., 2003). If Arl13bhnn mutants completely
lacked fluid flow in the node, we might expect them to show a
similar pattern of Nodal expression to mutants lacking cilia. In
IFT172wim mutants, almost half of the embryos showed asymmetric Nodal expression in the node, but increased expression
was seen more often on the right side of the node than in either
wild-type or Arl13bhnn mutant embryos (Fig. 5(C)). Our finding
that Arl13bhnn mutants showed Nodal expression in the node in a
pattern that is distinct from mutants with no cilia suggests that
the Nodal expression defect in the node of Arl13bhnn mutants is
not due to a complete loss of fluid flow.
Other signaling pathways are grossly intact in Arl13bhnn mutants
at e8.5
Several signaling pathways are active in the node and affect
Nodal expression in the node and LPM. First, a Wnt3a null mutant
shows a similar delay in Nodal expression followed by bilateral
expression of Nodal in the LPM. Wnt3a is required for Notch
signaling at the node, which in turn is needed for Nodal
expression (Nakaya et al., 2005). There has also been a link
between cilia and Wnt signaling making it a candidate for
disruption in our mutants (Corbit et al., 2007; Germino, 2005;
Simons et al., 2005; Lancaster et al., 2011). To examine canonical
Wnt signaling activity in our mutant embryos, we incorporated
the BATgal reporter allele, which expresses the lacZ gene in
response to Wnt signaling activity (Maretto et al., 2003). We
saw no difference in b-galactosidase activity between wild-type
and mutant embryos (Fig. 6(A) and (B)). Likewise, we saw no
change in the expression of Dll1, a Wnt3a target, or of Notch1 and
its target, Lfng (Fig. 6(C)–(H)).
Another signaling pathway, Shh, induces expression of Gdf1 at
the node periphery, which is required for Nodal diffusion to the
LPM (Tanaka et al., 2007; Zhang et al., 2001). Shh signaling is also
known to be required for Nodal expression in the LPM (Tsiairis and
McMahon, 2009), and we know there are Shh signaling defects in
Arl13bhnn mutants at later stages (Caspary et al., 2007). At E8.5, we
saw no change in Shh expression, and using Patched-lacZ to
monitor Shh signaling activity, there was no difference between
wild-type and mutant embryos (Fig. 6(I)–(L)). Gdf1 expression was
C.E. Larkins et al. / Developmental Biology 367 (2012) 15–24
19
the b-galactosidase assay as well as in situ hybridizations for Nodal
(Fig. 7(D)–(F)). In the node, in situ hybridization showed that Nodal
expression was weakened such that any asymmetry could not be
recorded (Fig. 7(F)). Because Nodal acts to enhance its own expression in the node and induces its expression in the LPM, the simplest
interpretation of this genetic interaction is that the levels of active
Nodal are reduced in the Arl13bhnn mutant, although we cannot say
whether this effect is direct or indirect. A reduction in active Nodal in
the LPM could be the direct consequence of reduced active Nodal in
the node.
We confirmed the loss of Nodal expression in Arl13bhnn; NodallacZ/ þ
by examining Pitx2, and saw that it likewise was not expressed in the
LPM (Fig. 3(C) and (D)). These molecular patterns were reflected by
the organ laterality in the Arl13bhnn; NodallacZ/ þ mutants, which
showed right isomerism in the lungs, with the lobes being smaller
than wild-type, as well as a midline stomach (Fig. 1(G)–(I)).
To determine whether there was a defect in Nodal expression at
earlier stages in Arl13bhnn mutants, we also examined the Arl13bhnn;
NodallacZ/ þ at E7.5 and saw that its expression using b-galactosidase
detection was indistinguishable from wild-type (Fig. 7(A) and (D)).
Similarly, at E12.5 in Arl13bhnn; NodallacZ/ þ mutants, the only Nodal
signaling-dependent defect we saw was in the visceral LR axis, and
not in mesoderm or endoderm derivatives, which are specified by
Nodal in the pre-gastrulation embryo.
LPM Nodal expression requires cilia in Arl13bhnn mutants
Fig. 3. Pitx2 expression is disrupted in Arl13bhnn mutants. (A)–(C) Pitx2 in situ
hybridization in E8.5 embryos. (A) Wild-type with expression in the left LPM. (B)
Arl13bhnn embryo with bilateral Pitx2 expression. (C) Arl13bhnn; NodallacZ/ þ embryo
with no expression in the LPM. (D) Graph quantifies the number of embryos with
each expression pattern of Pitx2 in the LPM.
also normal (Fig. 6(M) and (N)). This is surprising since Gdf1 and
Ptch1-lacZ are Shh responsive genes and we previously showed Shh
signaling is abnormal in the absence of Arl13b, however, our
previous work shows that Shh signaling is not completely abolished in the Arl13bhnn mutants and we predict that at e8.5 there is
enough Shh signaling to allow for Ptch1 and Gdf1 expression. Thus,
while we cannot rule out subtle quantitative differences within the
pathways, in terms of the molecular events known to be critical in
establishing the LR axis, Wnt, Shh and Notch activities appear
grossly intact in the absence of Arl13b.
Arl13b and Nodal genetically interact
Our analysis shows that Arl13bhnn mutants have an inability to
maintain higher levels of Nodal expression in the left side of the node
and also have delayed Nodal expression in the LPM. Because Nodal is
in a positive feedback loop to enhance its expression in the node and
travels from the node to induce its expression in the LPM (Nakamura
et al., 2006; Norris et al., 2002; Oki et al., 2007), the expression
pattern we see in our mutants could be caused by a reduction in
active Nodal protein in Arl13bhnn mutants. To test this, we genetically
reduced Nodal in our Arl13bhnn mutants using the null NodallacZ allele
(Collignon et al., 1996). If Nodal is reduced in the Arl13bhnn mutant,
then a further reduction in Nodal by introducing the null allele
should cause a more severe Nodal expression phenotype. Indeed, the
Arl13bhnn; NodallacZ/ þ embryos were more severe, as they lacked
Nodal expression in the LPM at all somite stages according to both
In mutants that lack cilia and therefore have no flow, Nodal
asymmetry in the node is randomized and Nodal in the LPM is
bilateral. This predicts that removing cilia in combination with
Arl13b should rescue Nodal expression in the LPM. To this end, we
crossed Arl13bhnn mutants to IFT172wim and made double homozygous mutants that lack Arl13b as well as cilia. The double
mutant embryos showed bilateral expression of Nodal starting at
the 2–3 somite stage, reminiscent of IFT172wim mutants (Fig. 2(I)–
(P)) indicating that without cilia, Arl13b mutants can express
Nodal at the correct time.
Discussion
Here we characterized the left–right patterning defects in mice
lacking the small regulatory GTPase, Arl13b. We found that
Arl13bhnn embryos are heterotaxic due to misexpression of several
genes known to be critical in L–R patterning. In the node we
showed that Nodal and Cerl2 are expressed symmetrically in the
absence of Arl13b. As Arl13b is highly enriched in cilia and its loss
leads to short cilia in the node, it is easy to imagine that the
symmetric gene expression arises from abnormal nodal flow.
However, we cannot rule out the possibility that Arl13bhnn may
disrupt the ability for flow to be detected and this inability leads
to the symmetric expression of Nodal and Cerl2 in the node.
Organ laterality and the dose of Pitx2 in Arl13bhnn mutants
The heterotaxia we observed in Arl13bhnn mutants corresponded to the abnormal expression of the molecules that guide
organ laterality. This phenotype could yield important clues to
help us understand congenital defects in humans. In several
different LR axis-defective mutants and human syndromes, the
visceral organs may have different lateralities, even though a
single global LR axis decision has been specified. The cause of this
has been perplexing, but the suggestion is that individual organs
may require different doses of Pitx2, in terms of either the amount
of Pitx2 the developing organ is presented with or the length
of time the developing organ sees Pitx2, or both (Liu et al., 2001).
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C.E. Larkins et al. / Developmental Biology 367 (2012) 15–24
Fig. 4. Midline patterning is normal is Arl13bhnn mutants. (A)–(F) Lefty1 and Lefty2 in situ hybridization in wild-type (A) and (B) and Arl13bhnn (D) and (E). Graphs in (C) and
(F) quantify the number of embryos with Lefty1 expression in the midline. (G)–(J) Foxa2 in situ hybridization in wild-type (G) and (H) and Arl13bhnn (I) and (J).
The complex pattern of Nodal and Pitx2 expression in Arl13bhnn
mutants supports this idea. Because the lungs follow almost
exactly the Pitx2 expression pattern we saw in Arl13bhnn mutants,
they may only require a small dose of Pitx2 for LR specification to
occur. However, the heart and stomach obviously require more
precise amounts of Pitx2, as they developed with a random
orientation in Arl13bhnn mutants and chose a left or right side
independently of one another.
Patients with mutations in Arl13b display Joubert syndrome,
which is characterized by brain malformations (Cantagrel et al.,
2008). In the context of our analysis it is interesting to note that
laterality defects in Joubert patients are rare. Since Arl13b is a
GTPase and likely has multiple effectors, it will be interesting to
see whether any effectors are specific to LR specification. This
would also predict that distinct alleles of Arl13b may be identified
in non-Joubert patients who only exhibit laterality defects.
Defective Nodal expression in the LPM
In the Arl13bhnn LPM, we have shown that Nodal expression is
delayed and randomized followed by late bilateral expression.
This is most likely an indirect consequence of the other molecular
C.E. Larkins et al. / Developmental Biology 367 (2012) 15–24
21
Fig. 5. Arl13bhnn embryos show Nodal and Cerl2 expression defects in the node. (A)–(E) in situ hybridization for Nodal and Cerl2. The graphs show the expression patterns
broken down by somite stage. (A) wild-type embryo showing left-sided asymmetry of Nodal. (B) Arl13bhnn mutant embryo with symmetric Nodal expression in the node.
(C) IFT172wim embryo showing right-sided enrichment of Nodal. (D) wild-type embryo showing Cerl2 has higher levels of expression on the right side of the node. (E)
Arl13bhnn mutant showing slightly higher levels of Cerl2 expression in the left side of the node.
Fig. 6. Wnt and Shh signaling is intact in Arl13bhnn mutants. (A) and (B) whole mount b-galactosidase assay shows no difference in Wnt signaling between wild-type
(A) and Arl13bhnn mutant (B) embryos. (C)–(H) Whole mount in situ hybridization for Notch1 (C) and (D), Dll1 (E) and (F), and Lfng (G) and (H) shows that Notch signaling is
intact in Arl13bhnn mutants compared to wild-type. (I) and (J) Whole mount in situ hybridization for Shh is identical between wild-type and Arl13bhnn mutants. (K) and (L)
Patched-lacZ shows no change in Hedgehog response between wild-type and Arl13bhnn mutant embryos. (M) and (N) the Shh signaling target Gdf1 is expressed normally in
Arl13bhnn mutants.
defects we observed. The late bilateral expression of Nodal in the
LPM may result from the late loss of midline integrity in our
mutants. The Nodal inhibitor Lefty1 is normally expressed in the
midline in response to Nodal from the left LPM and prevents
Nodal from diffusing to the right LPM to induce expression there
(Meno et al., 1998; Sakuma et al., 2002; Yamamoto et al., 2003).
22
C.E. Larkins et al. / Developmental Biology 367 (2012) 15–24
Fig. 7. Arl13b interacts genetically with Nodal. (A) and (B) wild-type whole mount
b-galactosidase assay in embryos carrying the nodal-lacZ allele at e7.5 (A) and the
6–7 somite stage (B). (D) and (E) whole mount b-galactosidase assay in Arl13bhnn
mutant embryos carrying the nodal-lacZ allele at e7.5 (D) and the 6–7 somite stage
(E). (C) and (F) Whole mount in situ hybridization confirms the lack of Nodal
expression in Arl13bhnn mutant embryos with the Nodal-lacZ allele (F), while
expression is intact in wild-type embryos (C).
We found that Lefty1 was initially expressed in the midline, but
was lost at late somite stages. This loss of expression occurred
even though Nodal was still expressed in the LPM, which would
likely allow for Nodal to diffuse to the opposing LPM to induce its
expression there, thus causing bilateral LPM Nodal expression
only at late somite stages.
Another interpretation for the late bilateral expression of
Nodal in our mutants is that the inability to down regulate Cerl2
and the loss of asymmetric Nodal expression in the node leads to
equal levels of active Nodal protein on both sides of the node at
later somite stages causing activation of Nodal expression in both
the left and right LPM. This inability to down regulate Cerl2 could
also be the cause for delayed expression of Nodal in the LPM, as
there would be less active Nodal protein in the node to induce its
expression in the LPM. The lack of Nodal in the LPM of Arl13bhnn;
NodallacZ/ þ embryos supports the model that there is less active
Nodal protein in the LPM to activate its expression there. One
possibility is that Arl13b functions in Nodal signaling from the
node to the LPM, however we cannot determine this definitively
as the decreased Nodal expression in the node could also cause
abnormal expression of Nodal in the LPM.
The fact that mutants lacking cilia can effectively signal from
the node to the LPM while Arl13bhnn mutants do not is reminiscent of other L–R patterning mouse mutants possessing cilia. In
the node, Pkd2 and Pkd1l1 mutants display normal cilia but in the
LPM they lack a response to the Nodal signaling cascade (Field
et al., 2011). Inv mutants have cilia in the node that generate slow
abnormal flow towards the left, however they display delayed
Nodal expression in the right LPM (Lowe et al., 1996; Okada et al.,
1999). For inv mutants the delay may be due to altered levels of
Nodal signaling from the node as Cerl2 and Lefty1 expression
there is abnormal. Such a mechanism may underlie the Arl13bhnn
phenotype although it is unclear whether the direct comparison
can be made. Analysis of the Pkd1l1, Pkd2 and inv mutants
revealed that asymmetric gene expression is highly sensitive to
genetic background and the inv work was performed on a FVB
strain (Field et al., 2011; Lowe et al., 1996; Meno et al., 1996). We
work in the C3H strain background where the Pkd alleles were
also analyzed so can make direct phenotypic comparisons to
those. Pkd2 and Pkd1l1 mutants have normal cilia within the node
yet the Nodal signaling cascade is not activated in the LPM of
either mutant whereas the cascade is activated late in the absence
of Arl13b. The simplest model to reconcile these phenotypes
would posit that in the Pkd mutants, there is not enough active
Nodal being produced in the node to allow for Nodal induction in
the LPM, whereas in Arl13bhnn mutants, there is just enough Nodal
in the node to allow for activation of Nodal expression, albeit
delayed, in the LPM.
The fact that removing cilia in the context of Arl13b mutants
can rescue the activation of the Nodal signaling cascade suggests
that cilia remove positive and negative regulators important for
activation of Nodal. For example, we know that removal of cilia
causes loss of Lefty1 expression in the midline (Nakamura et al.,
2006), so removal of cilia in the Arl13bhnn mutants would likely
remove the Lefty1 inhibitor allowing for higher levels of active
Nodal in the LPM (Nakamura et al., 2006). It would be interesting
to delete Lefty1 in the Arl13bhnn mutants to determine if we see
the same phenotype that we found from removing cilia.
It is also possible that Arl13b has roles in the LPM independent
of its function in the node. A precise understanding would require
us to spatially delete Arl13b in the LPM which has proven
technically challenging as Arl13b protein requires 42 h to turn
over (our unpublished data). Regardless, the idea that Arl13b
affects the balance of positive and negative inputs to Nodal
signaling parallels its function in Shh signaling where we showed
it controls activation but not repression of Shh signaling (Caspary
et al., 2007).
Defective Nodal expression in the node
The primary LR axis specification defect in Arl13bhnn mutants is
the symmetric expression of Nodal and Cerl2 in the node as this can
explain all subsequent observations. Both Cerl2 and Nodal are
predicted to be downstream targets of fluid flow (Hirokawa et al.,
2006; Schweickert et al., 2010), and our mutants have stunted cilia
that, if motile, would likely be unable to generate the same quality of
leftward flow that would be found in wild-type embryos. It was
recently shown that very little fluid flow is required in the node to
generate asymmetric expression of Cerl2, and that in the absence of
flow, Cerl2 is symmetric (Shinohara et al., 2012). We saw that almost
50% of Arl13bhnn mutant embryos had higher levels of Nodal expression in the left side of the node at presomite stages, a pattern that
was nearly identical to wild-type. However, this pattern gradually
shifted to symmetric expression between the 2 to 5 somite stage.
This may indicate that our mutants generate a fluid flow that is too
weak to allow for the signaling events required for down regulation
of Cerl2, but this weak flow is sufficient for initial Nodal enrichment.
Consistent with this, we found that the inhibitor of Nodal, Cerl2, was
not down regulated normally in the left side of the node, and most
often showed equal levels of expression in both sides of the node. An
inability to down regulate Cerl2 could lead to less active Nodal
protein in the node, preventing the Nodal feedback loop necessary for
increasing Nodal expression there (Norris et al., 2002). To confirm
C.E. Larkins et al. / Developmental Biology 367 (2012) 15–24
that reduced levels of Nodal expression are due to the defect in Cerl2
expression, it will be necessary to generate Cerl2/ ; Arl13bhnn
double mutants and observe Nodal expression.
The symmetric Nodal and Cerl2 we observed in the Arl13bhnn
node could also be explained if Arl13b is required for detection of
flow in the node. The two cilia hypothesis invokes a Pkd2dependent mechanism for flow detection. Mutations in Pkd2 or
its binding partner, Pkd1l1 have normal cilia but LR defects
stemming from symmetric expression of both Nodal and Cerl2 in
the 3 somite node. As this mimics the Arl13bhnn phenotype, it
raises the possibility that Arl13bhnn mutants may also be unable to
detect flow. Arl13b is a member of the Arf family whose members
have well-established roles in protein transport including the
transport of Pkd2 to the cilium by Arf4 (Ward et al., 2011).
Furthermore, both Arl13b and Pkd mutants possess cilia yet
Nodal is delayed or absent in the LPM. This has always been
difficult to reconcile with the phenotype of mutants lacking cilia
which express Nodal in the LPM, albeit bilaterally. One model to
explain this would posit that cilia are required for both positive
and negative regulators that induce Nodal in the LPM and that
removing cilia ablates both, resulting in bilateral Nodal expression
by the 2–3 somite stage. In contrast, in Pkd or Arl13b mutants
where cilia are present (albeit distinctly) perhaps the balance of
the positive and negative regulation is distorted resulting in
distinct spatial and temporal patterns of Nodal expression in the
LPM. The bilateral Nodal we saw in the Arl13bhnn IFT172wim double
mutants is consistent with this interpretation.
Taken together our analysis shows that Arl13b controls left–
right patterning initially by disrupting the normal asymmetric
expression of Nodal and Cerl2 in the node. It remains possible that
this is due to distinct roles of Arl13b in both the generation and
detection of flow. In support of this, mutants lacking cilia do not
display presomitic node asymmetry of Nodal as Arl13b mutants
do. Mutants that cannot detect flow cannot activate the Nodal
signaling cascade in the LPM as Arl13b mutants do (Field et al.,
2011). We have recently been able to bypass the Arl13b dependent ciliogenesis defects in a cell culture model indicating that
Arl13b functions via distinct effectors (Mariani and Caspary,
personal communication). As we identify specific Arl13b effectors,
it will be interesting to determine if they distinguish roles of
Arl13b in generating and detecting flow in the node.
Acknowledgments
We thank Miao Sun, Nicole Umberger, and Karolina Piotrowska-Nitsche for helpful comments on the manuscript. This
work was funded by a Predoctoral Fellowship from the Greater
Southeast Region American Heart Association (C.E.L.), a Basil
O’Conner Starter Scholar Award from the March of Dimes (T.C.),
as well as a Hitchings Elion Career Development Award from the
Burroughs Wellcome Fund (T.C.).
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