© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 1638-1648 doi:10.1242/dev.105874
RESEARCH ARTICLE
STEM CELLS AND REGENERATION
Cyp26 enzymes are required to balance the cardiac and vascular
lineages within the anterior lateral plate mesoderm
ABSTRACT
Normal heart development requires appropriate levels of retinoic acid
(RA) signaling. RA levels in embryos are dampened by Cyp26
enzymes, which metabolize RA into easily degraded derivatives.
Loss of Cyp26 function in humans is associated with numerous
developmental syndromes that include cardiovascular defects.
Although previous studies have shown that Cyp26-deficient vertebrate
models also have cardiovascular defects, the mechanisms underlying
these defects are not understood. Here, we found that in zebrafish, two
Cyp26 enzymes, Cyp26a1 and Cyp26c1, are expressed in the anterior
lateral plate mesoderm (ALPM) and predominantly overlap with vascular
progenitors (VPs). Although singular knockdown of Cyp26a1 or
Cyp26c1 does not overtly affect cardiovascular development, double
Cyp26a1 and Cyp26c1 (referred to here as Cyp26)-deficient embryos
have increased atrial cells and reduced cranial vasculature cells.
Examining the ALPM using lineage tracing indicated that in Cyp26deficient embryos the myocardial progenitor field contains excess atrial
progenitors and is shifted anteriorly into a region that normally solely
gives rise to VPs. Although Cyp26 expression partially overlaps with VPs
in the ALPM, we found that Cyp26 enzymes largely act cell nonautonomously to promote appropriate cardiovascular development. Our
results suggest that localized expression of Cyp26 enzymes cell nonautonomously defines the boundaries between the cardiac and VP fields
within the ALPM through regulating RA levels, which ensures a proper
balance of myocardial and endothelial lineages. Our study provides
novel insight into the earliest consequences of Cyp26 deficiency that
underlie cardiovascular malformations in vertebrate embryos.
KEY WORDS: Retinoic acid, Cyp26, Cardiovascular, Lateral plate
mesoderm, Zebrafish
INTRODUCTION
A balance of retinoic acid (RA) synthesis and degradation is
necessary for appropriate organogenesis (Otto et al., 2003; Uehara
et al., 2007; White et al., 2007; Niederreither and Dolle, 2008). In all
vertebrate embryos, a disruption of this balance, through excess
vitamin A (retinol) or RA, causes RA embryopathies (Lammer et al.,
1985; Pan and Baker, 2007), which have characteristic malformations
of the heart, thymus, central nervous system and craniofacial
structures. The necessity to limit endogenous embryonic RA levels
illustrates the importance of Cyp26 enzymes, which are members of
the p450 family of proteins that promote the degradation of RA (White
et al., 1997; Abu-Abed et al., 2001; Otto et al., 2003). In humans,
1
The Heart Institute, Molecular Cardiovascular Biology and Developmental Biology
Divisions, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229,
2
USA. Molecular and Developmental Biology Graduate Program, Cincinnati
Children’s Hospital Medical Center, Cincinnati, OH 45229, USA.
*Author for correspondence ( [email protected])
Received 8 November 2013; Accepted 11 February 2014
1638
Cyp26 deficiency has been implicated in several developmental
syndromes, most notably DiGeorge syndrome, Kippel Feil’s anomaly
and Antley Bixler syndrome (Fukami et al., 2010; Pennimpede et al.,
2010). Therefore, elucidating the requirements of Cyp26 enzymes
during vertebrate development will provide greater understanding of
the mechanisms underlying congenital defects found in human
syndromes.
Vertebrates have three conserved Cyp26 paralogs: Cyp26a1,
Cyp26b1 and Cyp26c1. Cyp26a1 is the earliest and most broadly
expressed during embryogenesis in all vertebrates, with expression in
the anterior and posterior of the embryos (Abu-Abed et al.,
2002; Kudoh et al., 2002; Dobbs-McAuliffe et al., 2004; Emoto
et al., 2005). In both mice and zebrafish, Cyp26a1-deficient embryos
have conserved hindbrain, limb and posterior trunk and tail defects
(Abu-Abed et al., 2001; Niederreither et al., 2002; Emoto et al., 2005).
All three Cyp26 enzymes are expressed in the hindbrain during early
development of vertebrates. Although Cyp26a1 deficiency alone
results in patterning defects that are not observed in Cyp26b1- and
Cyp26c1-deficient embryos, there is functional redundancy between
these enzymes during early embryogenesis. Double-deficient Cyp26a1
and Cyp26c1 and triple Cyp26a1-, Cyp26b1- and Cyp26c1-deficient
mice and zebrafish have progressively more severe hindbrain defects
than Cyp26a1 deficiency alone (Kudoh et al., 2002; Emoto et al., 2005;
Hernandez et al., 2007; Uehara et al., 2007, 2009). Specifically,
progressive loss of Cyp26 alleles causes an expansion of posterior
rhombomeres at the expense of more anterior rhombomeres, which is
consistent with the teratogenic effects of RA treatment. Furthermore,
the triple Cyp26-deficient mice die by E9.0 (Uehara et al., 2009),
suggesting there are additional early embryonic defects.
Although a primary focus of understanding the consequences of
Cyp26 deficiency has been neural development, as with RA treatment,
loss of Cyp26 enzymes affects numerous organs, including the heart.
During early development, the heart is particularly sensitive to excess
RA signaling, with surplus Vitamin A and RA being teratogenic
(Lammer et al., 1985; Osmond et al., 1991; Waxman and Yelon,
2009). Specifically, RA treatment causes similar overt malformations
of the atrial and ventricular chambers in all vertebrates examined.
Despite a long history examining the teratogenic consequences of
surplus RA, we still do not have a clear understanding of the
mechanisms underlying RA induced chamber malformations.
Furthermore, although previous studies in mice and zebrafish have
suggested that in Cyp26-deficient embryos there are gross cardiac
malformations, including looping defects (Abu-Abed et al., 2001;
Emoto et al., 2005; Ribes et al., 2007), the nature of these defects was
not analyzed in detail. Therefore, the consequences of increased
endogenous RA levels due to loss of Cyp26 enzymes in early cardiac
development are not currently understood.
In this paper, we sought to understand the earliest consequences
of Cyp26 deficiency on the cardiovascular field in vertebrate
embryos. Here, using zebrafish, we demonstrate that Cyp26a1 and
Cyp26c1 act redundantly to pattern the cardiovascular progenitor
DEVELOPMENT
Ariel B. Rydeen1,2 and Joshua S. Waxman1, *
fields within the anterior lateral plate mesoderm (ALPM).
Specifically, we found deficiency of both Cyp26a1 and Cyp26c1
causes shifts in the ALPM boundaries of the anterior vascular and
myocardial progenitor fields. The consequences of these shifts are
decreased endothelial cells and a specific expansion of atrial
cardiomyocytes, suggesting there is a fate transformation between
these two cardiovascular progenitor types in the nascent ALPM.
Furthermore, we show that the Cyp26 enzymes are required cell nonautonomously within the ALPM, indicating that Cyp26 enzymes are
required within a community of ALPM cells to restrict RA levels
required for proper patterning of the cardiovascular progenitor
fields. Therefore, our study provides novel insight into the earliest
requirements of Cyp26 enzymes in cardiovascular development,
suggesting that they are required to limit RA signaling that balances
the sizes of the atrial and VP fields.
RESULTS
Cyp26 enzyme expression in the ALPM
To understand the relationship of Cyp26 enzyme expression to
cardiovascular progenitors in the ALPM, we used two-color in situ
hybridization (ISH). We found that both cyp26a1 and cyp26c1 are
expressed in the ALPM at the 8-somite (s) stage (Fig. 1), consistent
with previous studies (Dobbs-McAuliffe et al., 2004; Zhao et al.,
2005; Hernandez et al., 2007). Although the expression patterns within
the ALPM differ slightly, the posterior limits of cyp26a1 and cyp26c1
expression only marginally overlap with the anterior limit of the
cardiac progenitor (CP) marker nkx2.5 (Fig. 1A-B0 ). By contrast, both
cyp26a1 and cyp26c1 predominantly overlap with the more anterior
early VP marker etv2 (Fig. 1C-D0 ). We did not examine cyp26b1
because previous studies demonstrated it is expressed solely in the
hindbrain at this stage (Zhao et al., 2005). Therefore, the expression
patterns of cyp26a1 and cyp26c1 within the ALPM suggest they are
closely associated with both types of cardiovascular progenitors.
Cyp26 depletion produces a specific increase in atrial cells
To determine the role that Cyp26 enzymes play in early
cardiovascular development, we used previously characterized
morpholino oligonucleotides (MOs) to knockdown the Cyp26
enzymes (Hernandez et al., 2007; D’Aniello et al., 2013). We also
used the giraffe (gir) mutant, which contains a point mutation that
causes a truncation in Cyp26a1 (Emoto et al., 2005). Using the
characterized MOs and gir mutant, alone and in combination, we were
able to replicate previously published hindbrain phenotypes
(supplementary material Fig. S1A-F) (Hernandez et al., 2007).
Development (2014) 141, 1638-1648 doi:10.1242/dev.105874
Furthermore, at 36 hours post-fertilization (hpf), singular knockdown
of Cyp26a1 visibly recapitulates the gir mutant phenotype, while
Cyp26c1-deficient embryos appear overtly normal (supplementary
material Fig. S2A,C) (Hernandez et al., 2007; D’Aniello et al., 2013).
Double knockdown of cyp26a1 and cyp26c1 (referred to as Cyp26deficient embryos) or injection of cyp26c1 MO into gir mutant
embryos (referred to as gir+C) both caused more severe phenotypes,
with larger pericardial edema and smaller heads compared with
cyp26a1-deficient and gir embryos alone (supplementary material
Fig. S2A-F). We also found that Cyp26-deficient embryos had
increased GFP expression in the RA reporter line Tg(12XRARE-ef1a:
EGFP)sk72 (supplementary material Fig. S3A-D) (Waxman and
Yelon, 2011), suggesting there is increased RA signaling in Cyp26deficient embryos. Therefore, we were significantly depleting
Cyp26a1 and Cyp26c1 with these tools, and proceeded to analyze
cardiovascular development.
We first examined expression of the cardiac differentiation markers
myl7 (pan-cardiac), vmhc (ventricular) and amhc (atrial) in Cyp26deficient embryos using ISH and RT-qPCR at the 20-22 s stages.
Although myl7 will eventually be expressed in all cardiomyocytes, at
this stage it predominantly marks ventricular cardiomyocytes (Yelon
et al., 1999). Although myl7, vmhc and amhc were unchanged in
singular knockdowns, we found that Cyp26-deficient embryos had
expanded amhc expression (Fig. 2A-N). Similar increases in amhc
expression without changes in myl7 or vmhc were also found in gir+C
embryos (supplementary material Fig. S4A-L) and embryos treated
with ketoconazole (Keto), which is a pharmacological inhibitor of
cytochrome p450 enzymes (supplementary material Fig. S5A-G).
Next, we determined whether the increase in amhc expression
reflected an increase in atrial cardiomyocytes by depleting the Cyp26
enzymes from Tg(–5.1myl7:DsRed-NLS) embryos and counting the
numbers of cardiomyocytes at 36 hpf. Although there was no overt
difference in heart morphology at 36 hpf between control, singular
and double-deficient embryos (supplementary material Fig. S6A-F),
we found that Cyp26-deficient embryos had a significant increase in
atrial cardiomyocytes (Fig. 2O), whereas singular knockdown of
cyp26a1 or cyp26c1 did not affect cardiomyocyte number.
Interestingly, the specific increase in atrial cells in Cyp26-deficient
embryos is remarkably similar to the effects of modest increases in
RA signaling (Waxman et al., 2008; Waxman and Yelon, 2009;
D’Aniello et al., 2013). Therefore, our data demonstrate that depletion
of both Cyp26 enzymes is required to produce a specific increase in
atrial differentiation that is reminiscent of modest increases in RA
signaling resulting from RA treatment.
Fig. 1. Cyp26 expression partially overlaps with
cardiovascular progenitors. (A-B0 ) Whole-mount double
in situ hybridization of cyp26a1 and cyp26c1 expression (red)
relative to nkx2.5 (blue) in wild-type embryos at 8 s.
(C-D0 ) cyp26a1 and cyp26c1 expression relative to etv2 (blue)
in wild-type embryos at 8 s. A0 -D0 correspond to boxed areas in
A-D, respectively. All images are dorsal views with anterior
upwards. Black arrows in A0 -D0 indicate cells with overlapping
expression. White arrows in C0 and D0 indicate cells without
overlapping expression.
1639
DEVELOPMENT
RESEARCH ARTICLE
RESEARCH ARTICLE
Development (2014) 141, 1638-1648 doi:10.1242/dev.105874
Loss of Cyp26 enzymes leads to rostral shifts in the
progenitor fields of the ALPM
Because we found an increase in atrial differentiation in Cyp26deficient embryos, we wanted to determine how Cyp26 deficiency
affects the CP pools in the ALPM. To examine this, we used ISH and
RT-qPCR to examine CP markers at the 8 s stage. Unfortunately, at this
time point we could not examine a specific effect on atrial progenitors,
because there are currently no known atrial specific progenitor marker
genes. Instead, we examined nkx2.5, which is primarily expressed in
ventricular progenitors at 8 s, and hand2, which marks both atrial and
ventricular progenitors (Schoenebeck et al., 2007). We found that in
Cyp26-deficient embryos, both nkx2.5 and hand2 expression were
shifted anteriorly (Fig. 3A-D), although their expression levels were
relatively unchanged (Fig. 3M). These results suggest that loss of
Cyp26 enzymes leads to an anterior shift in the CPs within the ALPM.
We next examined gata4 because it is expressed more broadly in
both cardiac and VPs of the ALPM. Consistent with the anterior shift
of nkx2.5 and hand2 expression, we found that in Cyp26-deficient
embryos, gata4 expression was truncated, with the posterior boundary
shifted anteriorly in the ALPM, and significantly decreased compared
with wild-type siblings (Fig. 3E,F,M). Furthermore, gir+C-deficient
and Keto-treated embryos exhibited a similar shift of nkx2.5
expression within the anterior ALPM, although gata4 was
somewhat less affected in these embryos (supplementary material
Fig. S7A-H and Fig. S8A-D). The anterior shift in the CPs and
truncation of gata4 expression predicted that the VPs may also be
1640
truncated in Cyp26-deficient embryos. Consistent with this notion, we
found that in Cyp26-deficient, gir+C-deficient and Keto-treated
embryos the VP markers etv2 and tal1 were truncated within the
ALPM at 8 s (Fig. 3G-J,M; supplementary material Fig. S7C,D,G,H,
K,L,O,P and Fig. S8E-H), which correlated with decreased expression
in Cyp26-deficient embryos (Fig. 3M). As etv2 marks both vascular
and myeloid progenitors at this stage (Lee et al., 2008; Sumanas et al.,
2008), we wanted to determine whether one or both these progenitor
populations are affected in Cyp26-deficient embryos. We found that
the amount of spi1b-expressing anterior myeloid progenitors,
although shifted anteriorly, was not decreased in Cyp26-deficient
embryos (supplementary material Fig. S9), suggesting that loss of etv2
and tal1 in Cyp26-deficient embryos reflects a loss in VPs.
Next, we wanted to understand whether the borders of the adjacent
cardiac and VP fields remain distinct or were altered in Cyp26deficient embryos. To determine this, we performed double ISH
using probes for zsyellow and etv2 in Tg(nkx2.5:ZsYellow) embryos.
These experiments were carried out using Tg(nkx2.5:ZsYellow)
embryos because more robust two-color ISH was able to be achieved
with a zsyellow probe relative to nkx2.5. In wild-type embryos, the
early CP field lies directly posterior to the early VP field within the
ALPM (Fig. 4A,A0 ). Interestingly, although there was an anterior
shift in nkx2.5 expression in Cyp26 deficient embryos, nkx2.5 and
etv2 expression were no longer located adjacent to each other
(Fig. 4B,B0 ). Instead, there was a small but noticeable gap between
etv2 and nkx2.5 expression in Cyp26-deficient embryos (Fig. 4B,B0 ).
DEVELOPMENT
Fig. 2. Cyp26-deficient embryos have increased atrial differentiation and cell number. (A-L) Cardiac differentiation marker expression in wild-type, cyp26a1deficient, cyp26c1-deficient and cyp26-deficient embryos. Singular knock down of cyp26a1 (D-F) or cyp26c1 (G-I) does not affect cardiac differentiation. Double
knockdown (J-L) shows an expansion of amhc expression. (M) Area measurements indicate that amhc expression is expanded in Cyp26-deficient embryos. For
myl7: WT, n=38; Cyp26a1-deficient, n=12; Cyp26c1-deficient, n=18; Cyp26-deficient, n=30. For vmhc: WT, n=40; Cyp26a1-deficient, n=19; Cyp26c1-deficient,
n=16; Cyp26-deficient, n=37. For amhc: WT, n=33; Cyp26a1-deficient, n=17; Cyp26c1-deficient, n=17; Cyp26-deficient, n=41. (N) RT-qPCR indicates that only
amhc expression is increased in Cyp26-deficient embryos. (O) Cardiomyocyte counts reveal a specific increase in atrial cells in Cyp26-deficient embryos at
36 hpf. WT, n=53; Cyp26a1-deficient, n=15; Cyp26c1-deficient, n=15; Cyp26 deficient, n=51. All images are dorsal views with anterior upwards. Significant
differences compared with controls are indicated (*P<0.05). Error bars indicate s.d.
RESEARCH ARTICLE
Development (2014) 141, 1638-1648 doi:10.1242/dev.105874
Fig. 3. ALPM progenitor markers are shifted anteriorly in
Cyp26-deficient embryos. In situ hybridization of (A,B)
nkx2.5, (C,D) hand2, (E,F) gata4, (G,H) etv2, (I,J) tal1 and
(K,L) dhrs3a in wild-type and Cyp26-deficient embryos.
(A-D) There is an anterior shift in nkx2.5 and hand2
expression in Cyp26-deficient embryos. Arrows in A and C
indicates distances between expression and posterior eye.
Arrows in B and D indicate border between expression and
posterior eye, which abut in Cyp26-deficient embryos.
(E-J) gata4, etv2 and tal1 expression is truncated in Cyp26deficient embryos compared with wild-type siblings.
(K,L) dhrs3a expression is expanded in Cyp26-deficient
embryos. Arrows indicate length of expression. The posterior
boundaries of dhrs3a expression did not overtly change in
control sibling and Cyp26-deficient embryos. (M) RT-qPCR of
cardiovascular progenitor genes. Cyp26-deficient embryos
have decreased gata4, etv2 and tal1 expression compared
with wild-type siblings. (N) Total ALPM length is the same in
wild-type and Cyp26-deficient embryos. For WT: n=22 (etv2),
n=10 (nkx2.5), n=21 (dhrs3a). For Cyp26-deficient: n=17
(etv2), n=12 (nkx2.5), n=20 (dhrs3a). All images are dorsal
views with anterior upwards. Significant differences compared
with controls are indicated (*P<0.05). Error bars indicate s.d.
(Waxman et al., 2008; Sorrell and Waxman, 2011), may be expanded.
To determine whether the progenitor field posterior to the CP field is
expanded in Cyp26-deficient embryos, we examined dhrs3a, which is
a RA-responsive gene that lies immediately posterior to the CPs
(Waxman et al., 2008; Feng et al., 2010). We found a rostral shift in the
anterior border and overall expansion of dhrs3a expression at 8 s in
Cyp26-deficient embryos (Fig. 3K,L). We then wanted to determine
whether the overall length of the ALPM was affected in Cyp26deficient embryos at 8 s. We found that the combined length of etv2,
nkx2.5 and dhrs3a expression is unchanged between wild-type and
Cyp26-deficient embryos (Fig. 3N). Altogether, these data indicate
that Cyp26 deficiency at this stage does not alter the size of the ALPM,
but instead produces a realignment of the progenitor field boundaries
within the ALPM.
Fig. 4. Loss of Cyp26 enzymes leads to differential shifts in
cardiac progenitor markers. (A-B0 ) Double in situ
hybridization of nkx2.5 (red) and etv2 (blue) shows a small gap
between expression domains in Cyp26-deficient embryos that
is not present in wild-type siblings. (C-D0 ) Double in situ
hybridization with hand2 (blue) and etv2 (red) shows no space
between expression domains in wild-type and Cyp26-deficient
embryos. A-D are dorsal views with anterior upwards. A0 -D0 are
dorsal views with anterior leftwards. A0 -D0 correspond to boxed
areas in A-D, respectively. Arrows indicate the borders
between expression domains.
1641
DEVELOPMENT
We found the space between nkx2.5 and etv2 intriguing, because at
this stage nkx2.5 is primarily a ventricular progenitor marker.
Therefore, we examined the boundaries of hand2 and etv2, as hand2
expression has been proposed to be a better marker of the entire CP
field at 8 s (Schoenebeck et al., 2007). In contrast to what was found
with nkx2.5, we found that the hand2 and etv2 expression domains
were directly adjacent in both wild-type and Cyp26-deficient
embryos. Therefore, our data suggest that loss of Cyp26 enzymes
leads to a differential shift in CP markers, and potentially an expanded
atrial specific progenitor population.
Previous studies of gir mutants have shown there is an anterior shift
in the forelimb (pectoral fin) bud field within the ALPM (Emoto et al.,
2005), suggesting the hypothesis that the progenitor field immediately
posterior to the cardiac field, which encompasses the forelimb field
Inhibition of RA restores ALPM marker expression within
Cyp26-deficient embryos
Although previous data suggest that the role of Cyp26 enzymes is to
specifically degrade RA (White et al., 1997), we wanted to exclude
the possibility that the phenotypes are due to off target effects of the
MOs. Therefore, we treated wild-type siblings and Cyp26-deficient
embryos with either DMSO (vehicle control) or 2.5 μM DEAB, a
pharmacological inhibitor of the major RA-synthesizing enzyme
Raldh2 (Russo et al., 1988). We found that treating Cyp26-deficient
embryos with 2.5 μM DEAB, a dose that produces phenotypes
consistent with moderate loss of RA signaling (Maves and Kimmel,
2005), was able to rescue the shifts in cardiac and VP markers in
the ALPM (Fig. 5A-I), suggesting that increased RA due to
Cyp26 depletion is the cause of the differential anterior shifts of
ALPM lineages.
Cyp26 enzymes are necessary to balance the vascular and
cardiac lineages
We wanted to examine more closely the cell fates of the cardiovascular
progenitors from the ALPM. In particular, we wanted to gain insight
into the source of the surplus atrial cells, as we have no definitive
markers for atrial progenitors. Therefore, we performed lineage-tracing
experiments using caged-fluorescein to determine the fates of
progenitors in the ALPM at 7-9 s, similar to work described
previously (Fig. 6A) (Schoenebeck et al., 2007; Waxman et al.,
2008). Consistent with previous fate maps (Schoenebeck et al., 2007),
our fate map showed that in wild-type embryos, all endothelial and
endocardial cells arose from progenitors in the ALPM that were anterior
to the tip of the notochord (Fig. 6B-D,G). Moreover, progenitor cells
labeled anterior to the midbrain-hindbrain boundary (MHB)
exclusively gave rise to cranial vasculature and endocardium
(Fig. 6B-D,G). Consistent with the truncated expression of VPs
markers in Cyp26-deficient embryos, we did not find endothelial- or
endocardial-fated cells posterior to the MHB (Fig. 6B-D,G), whereas
labeled progenitors anterior to the MHB that gave rise to the endothelial
cells were significantly less common (Fig. 6B-D,G). With respect to the
atrial and ventricular progenitors, in wild-type embryos the anterior
limit of the CPs was between the tip of the notochord and the MHB,
Development (2014) 141, 1638-1648 doi:10.1242/dev.105874
which again was consistent with previous fate maps (Fig. 6B,E,G)
(Serbedzija et al., 1998; Schoenebeck et al., 2007). However, in
Cyp26-deficient embryos we found that myocardial-fated cells
sometimes originated as anteriorly as adjacent to the posterior eye,
which was never found in the fate maps from wild-type embryos
(Fig. 6B,E). Moreover, we found that the myocardial progenitors
found in the anterior of Cyp26-deficient embryos gave rise to both
atrial and ventricle cells (Fig. 6B,E), suggesting there is an anterior
shift of both these CPs.
To determine whether there is a relative increase in atrial
progenitors in Cyp26-deficient embryos, we next compared the
frequency that we observed atrial and ventricular progenitors to the
total number of times we labeled CPs from our lineage tracing.
Although the frequency of ventricular progenitors between
wild-type and Cyp26-deficient embryos was relatively similar,
95% (19/20) and 79% (22/28), respectively (P>0.2), we found a
strong trend towards an increase in the overall frequency of labeled
atrial progenitors, 30% (6/20) in wild type versus 60% (17/28) in
Cyp26-deficient embryos (P<0.1) (Fig. 6I). Furthermore, when we
examined the frequency of atrial cell contribution in the anterior
region of the ALPM that gave rise to ectopic myocardial cells
in Cyp26-deficient embryos (bracket in Fig. 6H), we found a
significant increase in the frequency of labeled cells that gave rise
to atrial cells, 69% (9/13) compared with 30% in wild-type
siblings (P<0.05) (Fig. 6H,I). Therefore, our lineage tracing data
suggest that in Cyp26-deficient embryos there is a shift in the
boundary between endothelial and CPs, and, importantly, that
there is an increase in the number of atrial progenitors within
the ALPM.
Loss of Cyp26 enzymes leads to disrupted cranial
vasculature development
Because we observed a reduction in the VP expression and reduced
frequency of cranial vasculature from our fate map, we asked
whether there were abnormalities in vascular morphology and/or
cell number at later stages in Cyp26-depleted embryos. To answer
this question, we used Tg(kdrl:nEGFP) embryos, which facilitated
analysis of cranial endothelial structures and cell counting. For the
Fig. 5. Inhibition of RA restores the sizes of the cardiovascular progenitor fields in Cyp26-deficient embryos. (A-D) nkx2.5 expression in wild-type, DEABtreated, Cyp26-deficient and Cyp26-deficient+DEAB-treated embryos. (C) Cyp26-deficient embryos have an anterior shift in nkx2.5 expression, which is rescued
in (D) DEAB-treated Cyp26-deficient embryos and comparable with wild-type and DEAB-treated embryos (A,B). Arrows in A-D indicate distances between
posterior eye and the anterior border of nkx2.5 expression. (E-H) etv2 expression in wild-type, DEAB-treated, Cyp26-deficient and Cyp26-deficient+DEABtreated embryos. (G) Cyp26-deficient embryos show a posterior truncation in etv2 expression, which is rescued in DEAB-treated Cyp26-deficient embryos (H).
(I) Measurements of length of etv2 expression and distance from the anterior tip of the embryo to anterior border of nkx2.5 expression. For etv2 measurements:
WT, n=27; DEAB-treated, n=30; Cyp26-deficient, n=17; Cyp26-deficient + DEAB-treated embryos, n=22. For nkx2.5 measurements: WT, n=27; DEAB-treated,
n=26; Cyp26-deficient, n=18; Cyp26-deficient + DEAB-treated embryos, n=22. All images are dorsal views with anterior upwards. Significant differences
compared with controls are indicated (*P<0.05). Error bars indicate s.d.
1642
DEVELOPMENT
RESEARCH ARTICLE
RESEARCH ARTICLE
Development (2014) 141, 1638-1648 doi:10.1242/dev.105874
purposes of this study, we considered anything anterior to the
first somite to be cranial vasculature. We found that the region
anterior to the first somite was considerably shortened in Cyp26deficient embryos compared with wild-type siblings (Fig. 7A,B).
Moreover, we found that the primordial midbrain channel and the
anterior and middle cerebral veins had significantly disrupted
morphology (Fig. 7A,B). When we counted the cranial endothelial
cells, we found that Cyp26-deficient embryos had a ∼40%
reduction in anterior endothelial and endocardial cells compared
with wild-type siblings (Fig. 7C-E). Although cyp26a1-deficient
and gir mutants have a modest truncation of etv2 and tal1
expression within the ALPM (supplementary material Fig. S7I,J,
M,N and Fig. S10E-H), this patterning defect does not lead to a
significant decrease in endothelial cell number at 28 hpf
(supplementary material Fig. S11A-C). Therefore, our data
suggest that the reduction of VPs in Cyp26-deficient embryos
leads to morphological abnormalities and decreased cranial
endothelial cell number.
Cyp26 enzymes act non-autonomously on the cardiovascular
progenitors
We next wanted to understand the cellular mechanism by which
Cyp26 enzymes act on the cranial endothelial progenitors, i.e. if the
Cyp26 enzymes are required within the VP cells or within
the surrounding environment to moderate RA levels. A clear
mechanism was not suggested by the expression patterns of the
Cyp26 enzymes and the VP markers because although there is
overlap between cyp26a1, cyp26c1 and etv2 expression, numerous
cells did not have overlapping expression (Fig. 1C-D0 ). To test where
the Cyp26 enzymes are required, we performed cell transplantation
and mosaic analysis using donor cells obtained from Tg(kdrl:
mCherry) embryos, which facilitated the observation of donorderived endothelial cells (Fig. 8A). We then scored the frequency of
contribution to the cranial and trunk vasculature at 48 hpf. When we
transplanted Cyp26-deficient donor cells into wild-type hosts, we
found that Cyp26-deficient donor cells contributed to the cranial
endothelial and endocardial cells at a slightly reduced, but not
statistically significant (P>0.2), frequency than that achieved when
wild-type donors were placed into wild-type hosts (Fig. 8B),
suggesting that Cyp26 enzymes are not required within the
individual VPs to promote proper specification and that the
surrounding wild-type cells can protect individual cells that have
reduced Cyp26 expression. Conversely, when we placed wild-type
cells into Cyp26-deficient embryos, we found there was a dramatic
decrease in the frequency of cranial endothelial and endocardial
contribution compared with when wild-type cells were transplanted
into wild-type hosts (Fig. 8B), suggesting that loss of Cyp26 enzymes
can cause inhibition of endothelial cell specification in individual
cells, presumably owing to excess RA in the local environment. In
both sets of experiments, we found that trunk endothelial cells
1643
DEVELOPMENT
Fig. 6. Lineage tracing of the ALPM of Cyp26-deficient embryos suggests a fate transformation between atrial and endothelial progenitors.
(A) Schematic of fate-mapping procedure. (B) Composite fate map of wild-type and Cyp26-deficient embryos, depicting the fates of uncaged cells.
(C-E) Individual maps to showing contribution to (C) endothelial cells, (D) endocardium and (E) myocardium. Line in C-E indicates MHB. (F,G) Percentages of
contribution anterior to the notochord and MHB in wild-type and Cyp26-deficient embryos. (H) Fate map of wild-type and Cyp26-deficient embryos depicting
chamber contribution of myocardial fated cells. Brackets indicate anterior cardiac region. (I) Percentages of total contribution to the chambers in wild-type and
Cyp26-deficient embryos. ey, eye; mhb, midbrain-hindbrain boundary; nc, notochord.
RESEARCH ARTICLE
Development (2014) 141, 1638-1648 doi:10.1242/dev.105874
Independent chamber specification defects in Cyp26
deficient embryos
contributed at the same frequency (Fig. 8B). Taken together, this set
of experiments suggests that loss of Cyp26 enzymes can have cell
non-autonomous consequences on the RA levels within ALPM,
which must be tempered to promote proper cranial endothelial and
endocardial cell specification.
DISCUSSION
In the present study, we examined the earliest requirements for
Cyp26 enzymes in cardiovascular development. We find that
Cyp26a1 and Cyp26c1 are redundantly required to limit the size of
the atrium and promote proper cranial endothelial and endocardial
development. Cyp26-deficient embryos have a rostral shift and
expansion of the CP field into the region of the ALPM that would
normally give rise to cranial endothelial and endocardial
progenitors, suggesting the increased RA in Cyp26-deficient
embryos promotes specification of atrial progenitors at the
expense of the endothelial progenitors. Importantly, our data
contrast with a previous model suggesting that RA promotes atrial
cell specification at the expense of ventricular cell specification.
Additionally, our data suggest that the effects of Cyp26 deficiency
on endothelial specification within the ALPM are cell nonautonomous, providing the first evidence that, at least within the
mesoderm, Cyp26 enzyme-expressing cells can act as a sink that
affects RA levels within neighboring tissues. Thus, we have
uncovered a previously unappreciated mechanism by which Cyp26
enzymes are required to limit RA levels within the ALPM, which is
necessary for proper cardiovascular development through balancing
the sizes of the atrial and endothelial progenitor fields.
1644
Atrial progenitors are specified at the expense of VPs in
Cyp26-deficient embryos
Within the anterior of the embryo, previous studies have focused on
the consequences of Cyp26 deficiency to hindbrain patterning in mice
and zebrafish. Consistent with increases in RA, Cyp26-deficient
embryos have posteriorized hindbrains, which are manifested by
rostral shifts in the posterior rhombomeres at the expense of the
more anterior rhombomeres (Niederreither et al., 2002; Hernandez
et al., 2007; Uehara et al., 2007). Although these models have
cardiovascular malformations, the consequences, if any, of Cyp26
deficiency on the adjacent ALPM were not previously examined. We
find that the cyp26a1 and cyp26c1 expression partially overlaps with
the VPs and that Cyp26 deficiency results in differential effects on the
ALPM progenitor fields. Specifically, both our ISH analysis and
lineage tracing in Cyp26-deficient embryos support the observation
that the anterior endothelial progenitor field is truncated, the CP
(middle) field is rostrally shifted and modestly enlarged, whereas the
most posterior field of the ALPM, which in wild-type embryos
harbors forelimb progenitors (Emoto et al., 2005; Waxman et al.,
DEVELOPMENT
Fig. 7. Cyp26-deficient embryos have decreased anterior endothelial
cell number. (A,B) Optical sections of uninjected sibling and Cyp26-deficient
Tg(kdrl:nEGFP) embryos show that Cyp26-deficient embryos have
mispatterned cranial vasculature. (C,D) Representative immunostained
Tg(kdrl:nEGFP) embryos for counting the cranial endothelial and endocardial
cells (green). Somites and myocardial cells are indicated in red.
(E) Endothelial and endocardial cell counts at 28 hpf. WT, n=10; Cyp26deficient, n=11. All images are lateral views with anterior towards the right.
Arrowheads in A and B indicate primordial midbrain channel and the anterior
and middle cerebral veins. Arrows in A-D indicate the somite boundary.
Significant differences compared with controls are indicated (*P<0.05). Error
bars indicate s.d.
In all vertebrates, treatment of embryos with RA causes atrial
and ventricular malformations (Osmond et al., 1991; Stainier and
Fishman, 1992; Waxman and Yelon, 2009). Because of this and the
established functions of RA signaling in anterior-posterior patterning,
previous studies had proposed that excess RA signaling leads to
increased atrial specification at the expense of ventricular specification
(Yutzey et al., 1994; Hochgreb et al., 2003). In contrast to the previous
hypothesis, our complementary analysis of ISH, lineage tracing and
cardiomyocyte counting indicate that Cyp26 loss results in increased
atrial progenitor specification and atrial cardiomyocytes; this is
independent of effects on ventricular progenitor specification and cell
number, which were unchanged. Importantly, the independent effects
on chamber specification in Cyp26-deficient embryos corroborate
recent studies examining increases and decreases of RA signaling in
vertebrates using other methods (Waxman et al., 2008; Waxman and
Yelon, 2009; D’Aniello et al., 2013). Specifically, loss of Cyp26
results in chamber defects that are equivalent to treatment with
moderate RA concentrations (∼0.1 μM), although treatment
with higher concentrations of RA is able to eliminate both atrial and
ventricular progenitors (Waxman and Yelon, 2009). We propose that a
modest increase in RA in Cyp26-deficient embryos is not due to
inefficiency of the MO depletion, because we do not obtain
indications of more pronounced increases in RA using Keto
treatments and gir mutants injected with cyp26c1 MOs when
examining the heart and the hindbrain. Moreover, using these tools,
we recapitulate the effects on the hindbrain seen in previous studies
(Hernandez et al., 2007), which also have maximal phenotypes
resembling treatment with moderate concentrations of RA. Therefore,
we favor the hypothesis that a modest increase in RA signaling, due
to Cyp26 deficiency, is the maximal effect achievable from
endogenously produced RA. However, as numerous feedback
mechanisms of RA exist to maintain appropriate embryonic RA
levels (Niederreither et al., 1997; Dobbs-McAuliffe et al., 2004;
Emoto et al., 2005; Sandell et al., 2007, 2012; White et al., 2007;
D’Aniello et al., 2013), it is also feasible that diminished raldh2
expression, which occurs in Cyp26-deficient embryos (Emoto et al.,
2005), contributes to the attenuation of RA levels. Thus, our data
support a model where loss of Cyp26 enzymes results in endogenous
increases in RA that promote atrial cell specification independently of
effects on ventricular cell specification.
RESEARCH ARTICLE
Development (2014) 141, 1638-1648 doi:10.1242/dev.105874
Fig. 8. Cyp26 enzymes act non-autonomously to promote cranial endothelial specification. (A) Transplant protocol. Tg(kdrl:mCherry) embryos were
injected with a blue lineage tracer. Donor cells are then transplanted into the marginal zone of host embryos and scored for endothelial contribution at 48 hpf.
(B,C) Representative Tg(kdrl:mCherry) donor cells that incorporated into the cranial vasculature and trunk vasculature. (D) Transplant results indicating that
wild-type donor cells incorporate significantly less frequently into cranial vasculature and endocardium in Cyp26-deficient host embryos than in wild-type host
embryos. Numbers in parentheses indicate the number of embryos that had contributed to the designated tissue relative to the total embryos. Images are lateral
views with anterior rightwards. Significant differences compared with controls are indicated (*P<0.05).
lack endothelial cells, have an anterior expansion of hand2
expression within the ALPM that leads to surplus atrial
cardiomyocytes potentially at the expense of the lost endothelial
cells (Schoenebeck et al., 2007). Although the overt phenotypes
with respect to the ALPM and surplus atrial cell production are
remarkably similar in Cyp26-deficient and cloche embryos, there
are no other phenotypic indications that RA signaling and cloche are
functioning in the same genetic pathway, suggesting these similar
phenotypes are achieved by two independent mechanisms.
Therefore, our findings suggest that Cyp26 enzymes promote
vascular specification at the expense of atrial specification by
properly determining the boundaries between the atrial and
endothelial progenitor fields within the ALPM, providing a
significant advance in our understanding of the mechanisms
underlying cardiovascular defects in Cyp26-deficient embryos.
Cyp26 enzymes promote vascular specification
The effects of inappropriate RA signaling on endothelial development
have been studied in other contexts. The most detailed studies have
been of Raldh2-deficient mice, i.e. RA signaling-deficient mice,
which have increased endothelial cell proliferation (Lai et al., 2003;
Bohnsack and Hirschi, 2004). Conversely, Cyp26-deficient mice,
which are hypersensitive to RA, have cranial vascular defects when
mothers are fed a vitamin A-rich diet (Ribes et al., 2007).
Furthermore, Por knockout mice, which lack the oxidoreductase
required for all cytochrome p450 activity and have increased
embryonic RA, also show severe vascular defects and are early
embryonic lethal (Otto et al., 2003). Additionally, chick embryos
treated with a Cyp26 inhibitor during early somitogenesis have
pharyngeal arch artery defects (Roberts et al., 2006), whereas
zebrafish gir mutants have been shown to have minor defects in the
common cardinal veins (Emoto et al., 2005). However, the previous
1645
DEVELOPMENT
2008), is expanded. Interestingly, the total length of the ALPM at this
stage is unchanged, suggesting that the Cyp26 enzymes are necessary
to limit RA signaling that refines the boundaries of these progenitor
fields within a pre-specified ALPM territory.
Because previous studies have shown that the cardiac and VP
fields within the ALPM lay directly adjacent to each other and share
a common progenitor, we were particularly interested in how Cyp26
deficiency affected the borders of these fields. Unexpectedly, we
found that, in addition to the general rostral shift of the CPs, the
Cyp26 deficiency caused differential rostral shifts in the atrial and
ventricular progenitor fields. In wild-type embryos, both the CP
markers nkx2.5 (ventricular progenitors) and hand2 ( pan-CPs) lie
directly posterior and adjacent to the more anterior VP marker etv2.
However, in Cyp26-deficient embryos, the nkx2.5 and etv2 borders
no longer abut, leaving a noticeable gap between the ventricular and
endothelial progenitor expression domains. Conversely, hand2,
which marks both atrial and ventricular progenitors, is still located
directly adjacent to the etv2 expression in Cyp26-deficient embryos,
suggesting that the hand2+/nkx2.5− expression domain adjacent to
the etv2 expression contains surplus atrial progenitors, which are at
the expense of the endothelial progenitors. Although our fate maps
of Cyp26-deficient embryos were not able to discern such a small
region of surplus atrial specification, they do complement the ISH
analysis and provide evidence that there is an increased frequency of
atrial progenitor specification, particularly in the more anterior zone
of the atrial progenitor field.
When considering our results, it is interesting to compare them
with recent observations from Schoenebeck et al. (Schoenebeck
et al., 2007), who studied cardiac and hematovascular patterning of
the ALPM in cloche mutants and etv2/tal1-deficient embryos.
Strikingly similar to what we have observed in Cyp26-deficient
embryos, cloche mutants and etv2/tal1-deficient embryos, which
studies of Cyp26- and Por-deficient embryos did not examine the
underlying mechanisms of the vascular defects.
We find that Cyp26-deficient embryos have aberrant cranial
vascular morphology and reduced cranial endothelial and endocardial
cell number, which is due to a truncation of the anterior endothelial
field within the ALPM. Because previous studies found that
modulation of RA signaling could affect endothelial proliferation,
we also examined proliferation. However, we did not find changes in
progenitor proliferation (supplementary material Fig. S12) nor did
we find increased apoptosis (supplementary material Fig. S13).
Therefore, we currently favor the model that increased RA levels in
Cyp26-deficient embryos primarily inhibits endothelial and
endocardial progenitor specification. Moreover, we also did not find
a difference in proliferation of differentiated endothelial cells in Cyp26
deficient embryos compared with wild-type siblings at later stages
(supplementary material Fig. S12), suggesting that the cranial vascular
defects in Cyp26-deficient embryos are not due to reduced endothelial
cell proliferation after they have differentiated. Altogether, our results
support that Cyp26 enzymes are required to promote proper
specification of the most anterior endothelial progenitors of the
ALPM, which give rise to the cranial vasculature and endocardium.
Cell non-autonomous requirements of Cyp26 enzymes on RA
signaling gradient
In addressing the cellular requirements for Cyp26 enzymes in
endothelial progenitor specification, we found that Cyp26 enzymes
are required cell non-autonomously to promote proper endothelial
specification, despite some overlap in expression with the endothelial
cell progenitors. These data suggest that Cyp26 enzymes are not
necessarily required within a single cell to control an endothelial
progenitor fate decision. Instead, a community of Cyp26-expressing
cells is the primary factor within the anterior embryo that creates an
environment for proper cardiovascular field establishment and
subsequent fate decisions. Additionally, as Cyp26 expression has
minimal overlap with the anterior CPs, this also suggests the
requirements of Cyp26 enzymes on CPs are indirect. Although it has
been suggested that Cyp26 enzymes may have cell non-autonomous
effects on RA that pattern the hindbrain in mice (Uehara et al., 2009),
this hypothesis was not tested experimentally. Instead, recent studies
in zebrafish have proposed that local RA degradation controls the RA
signaling gradient within the embryo, in effect arguing for a cell
autonomous role of Cyp26 enzymes that creates the RA gradient
(Hernandez et al., 2007). However, these studies focused primarily on
hindbrain patterning and did not formally rule out cell nonautonomous effects on RA levels due to Cyp26 enzymes. It is also
possible that Cyp26 enzymes can cause non-autonomous effects in
both the hindbrain and ALPM. However, it would be interesting if the
cellular mechanisms creating RA gradients within different tissues
differed, because, unlike the hindbrain, the cells of the ALPM are
mesenchymal in nature. Therefore, our transplantation experiments
are the first to suggest that loss of Cyp26 enzymes, at least in the
mesoderm, can non-autonomously affect the local RA levels that
affect the patterning of neighboring tissues.
Conclusions
In conclusion, excess embryonic RA is teratogenic in all vertebrates,
highlighting the importance of Cyp26 enzymes in limiting RA during
embryogenesis. To illustrate this, Cyp26-deficient vertebrate embryos
had poorly characterized cardiovascular defects, necessitating a better
understanding of the nature of these defects. Our studies provide novel
insight into the earliest consequences and mechanisms of Cyp26
deficiency that underlie atrial and cranial vascular malformations in
1646
Development (2014) 141, 1638-1648 doi:10.1242/dev.105874
vertebrate embryos, which will help us to understand the etiology of
developmental syndromes with elevated RA signaling in humans.
MATERIALS AND METHODS
Zebrafish husbandry and transgenic and mutant lines
Adult zebrafish (Danio rerio) were raised and maintained under standard
laboratory conditions (Westerfield, 2000). The transgenic lines used were:
Tg(–5.1myl7:DsRed-NLS) (Mably et al., 2003), Tg(−6.5kdrl:mCherry)ci5
(Proulx et al., 2010), Tg(12xRARE-ef1a:EGFP)sk72 (Waxman and Yelon,
2011), Tg(kdrl:nlsEGFP) (Blum et al., 2008), TgBAC(etv2:EGFP) (Proulx
et al., 2010) and TgBAC(−36nkx2.5:ZsYellow) (Zhou et al., 2011). The
giraffe (gir)/cyp26a1 mutant line was used (Emoto et al., 2005).
Morpholino oligonucleotide (MO) injections
Zebrafish embryos were injected with MOs at the one-cell stage. MO
sequences for cyp26a1 and cyp26c1 have been published previously
(Hernandez et al., 2007; D’Aniello et al., 2013). Singular knockdown of
cyp26a1 used a cocktail of 4 ng cyp26a1 MO1 and 2 ng cyp26a1 MO2.
cyp26c1 singular knockdown used 6 ng cyp26c1 MO. Double knockdown
of cyp26a1 and cyp26c1 was achieved using a cocktail of 2 ng cyp26a1
MO1, 1 ng cyp26a1 MO2 and 6 ng cyp26c1, which produced phenotypes
equivalent to cyp26a1/gir mutants injected with cyp26c1 MO. To
counteract non-specific MO-induced cell death, 3 ng p53 MO was used in
all injections (Robu et al., 2007).
In situ hybridization
Whole mount in situ hybridization (ISH) was performed using a previously
established protocol (Oxtoby and Jowett, 1993). All probes have been
previously reported: myl7 (formerly cmlc2; ZDB-GENE-991019), vmhc
(ZDB-GENE-991123-5), amhc (ZDB-GENE-031112-1), nkx2.5 (ZDBGENE-980526), hand2 (000511-1), dhrs3a (ZDB-GENE-040801-217),
eng2a (ZDB-GENE-980526-167), egr2b (formerly krox20; ZDB-GENE980526-283), etv2 (ZDB-GENE-050622-14), tal1 (ZDB-GENE-980526501), gata4 (ZDB-GENE-980526-476), hoxb4a (ZDB-GENE-990415-105),
spi1b (previously pu.1, ZDB-GENE-980526-164) and ZsYellow (accession
number: Q9U6Y4). Double ISHs were carried out essentially as described
previously (Prince et al., 1998). Both INT-BCIP (Roche) and Fast Red (Sigma)
were used. Embryos were de-yolked then flatmounted for imaging using a Zeiss
M2BioV12 stereomicroscope. For ISH experiments analyzing gir embryos, the
embryos were genotyped as described previously (Emoto et al., 2005).
Area and length measurements
Area measurements of myl7, vmhc, amhc and spi1b were carried out using
ImageJ as previously described (Waxman et al., 2008). Length of etv2,
nkx2.5, dhrs3a and spi1b expression was measured from lateral and dorsal
views with ImageJ. All statistical analysis was carried out using Student’s
t-test with P<0.05 considered to be statistically significant.
Immunohistochemistry and cell counting
Immunohistochemistry, cardiac cell counting and statistical analysis were
performed essentially as previously described (Waxman et al., 2008).
Vascular cell counts were done using Tg(kdrl:nEGFP) embryos that were
immunostained using MF20 (Stainier and Fishman, 1992) and chicken antiGFP (Invitrogen) primary antibodies and IgG2b-Tritc (Southern Biotech)
and anti-chicken (Southern Biotech) secondary antibodies, respectively. To
analyze the cranial vascular structures embryos, we imaged embryos using a
Zeiss Apotome. For endothelial cell counting, embryos were flattened in a
lateral orientation and imaged using a Zeiss M2BioV12 fluorescent
stereomicroscope. To analyze heart morphology at 36 hpf, MF20
(ventricle) and S46 (atrium) were used (Stainier and Fishman, 1992). To
assay apoptosis, activated caspase 3 (casp3) antibody (BD Bioscience) was
used. Cell proliferation in the VPs was assayed using a pHH3 antibody
(Millipore) to detect proliferating cells and the chicken anti-GFP antibody to
detect etv2-expressing progenitors in TgBAC(etv2:EGFP) embryos.
Embryos were then imaged at 8 s using a Nikon A1 confocal microscope
and at 24 hpf using a Zeiss Apotome.
DEVELOPMENT
RESEARCH ARTICLE
Real-time quantitative PCR (RT-qPCR)
cDNA was prepared from whole embryos as previously described
(D’Aniello et al., 2013). RT-qPCR using SYBR green PCR master mix
(Applied Biosystems) was performed under standard PCR conditions in
Bio-Rad CFX PCR machine. Expression levels of myl7, vmhc, amhc,
nkx2.5, gata4, hand2, etv2 and tal1 were standardized to β-actin. Data were
analyzed using 2−ΔΔCT Livak Method and Student’s t-test. Primer sequences
can be found in supplementary material Table S1.
Drug treatments
Embryos were treated with 25 μM ketoconazole (Sigma) beginning at
the two-cell stage. Embryos were treated with 2.5 μM DEAB
(4-diethylaminobenzaldehyde; Sigma) beginning at 50% epiboly.
Lineage tracing
Lineage tracing for fate-mapping experiments was performed essentially as
previously described (Schoenebeck et al., 2007; Waxman et al., 2008). Two
or three cells in the ALPM were uncaged between 7 s and 9 s using an Andor
Micropoint laser, and then imaged with a Zeiss AxioImager microscope.
Embryos were grown to 48 hpf then fixed, stained and scored for
contribution to the vascular and cardiac cells. Statistical analysis was
performed using a two-tailed Z-test where P<0.05 is considered significant.
Cell transplantation experiments
To assess cellular autonomy, Tg(kdrl:mCherry) donor embryos were
injected at the one-cell stage with Cascade blue-dextran (Invitrogen). At
the sphere stage, ∼20 cells were transplanted in the margin of wild-type
(control) and Cyp26-deficient host embryos. Host embryos were then grown
to 48 hpf and scored for contribution to cranial vasculature, endocardium
and trunk vasculature. Reciprocal experiments to test a cell-autonomous role
for the Cyp26 enzymes were performed similarly, except Cyp26-deficient
donor cells and wild-type hosts were used. Statistical analysis was
performed using a two-tailed Z-test where P<0.05 is considered significant.
Acknowledgements
The authors are grateful to E. D’Aniello and S. Sumanas for discussions and reading
of the manuscript, and to S. Sumanas for TgBAC(etv2:EGFP) embryos.
Competing interests
The authors declare no competing financial interests.
Author contributions
A.B.R. performed all experiments. A.B.R. and J.S.W. designed the experiments,
performed the data analysis and wrote the manuscript.
Funding
This work was supported by the National Institutes of Health (NIH) [R00 HL901126
and R01 HL112893-A1 to J.S.W.]. Deposited in PMC for release after 12 months.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.105874/-/DC1
References
Abu-Abed, S., Dollé , P., Metzger, D., Beckett, B., Chambon, P. and Petkovich, M.
(2001). The retinoic acid-metabolizing enzyme, CYP26A1, is essential for normal
hindbrain patterning, vertebral identity, and development of posterior structures.
Genes Dev. 15, 226-240.
Abu-Abed, S., MacLean, G., Fraulob, V., Chambon, P., Petkovich, M. and Dollé , P.
(2002). Differential expression of the retinoic acid-metabolizing enzymes CYP26A1
and CYP26B1 during murine organogenesis. Mech. Dev. 110, 173-177.
Blum, Y., Belting, H.-G., Ellertsdottir, E., Herwig, L., Lü ders, F. and Affolter, M.
(2008). Complex cell rearrangements during intersegmental vessel sprouting and
vessel fusion in the zebrafish embryo. Dev. Biol. 316, 312-322.
Bohnsack, B. L. and Hirschi, K. K. (2004). Red light, green light: signals that
control endothelial cell proliferation during embryonic vascular development.
Cell Cycle 3, 1506-1511.
D’Aniello, E., Rydeen, A. B., Anderson, J. L., Mandal, A. and Waxman, J. S.
(2013). Depletion of retinoic acid receptors initiates a novel positive feedback
mechanism that promotes teratogenic increases in retinoic acid. PLoS Genet. 9,
e1003689.
Development (2014) 141, 1638-1648 doi:10.1242/dev.105874
Dobbs-McAuliffe, B., Zhao, Q. and Linney, E. (2004). Feedback mechanisms
regulate retinoic acid production and degradation in the zebrafish embryo. Mech.
Dev. 121, 339-350.
Emoto, Y., Wada, H., Okamoto, H., Kudo, A. and Imai, Y. (2005). Retinoic acidmetabolizing enzyme Cyp26a1 is essential for determining territories of hindbrain
and spinal cord in zebrafish. Dev. Biol. 278, 415-427.
Feng, L., Hernandez, R. E., Waxman, J. S., Yelon, D. and Moens, C. B. (2010).
Dhrs3a regulates retinoic acid biosynthesis through a feedback inhibition
mechanism. Dev. Biol. 338, 1-14.
Fukami, M., Nagai, T., Mochizuki, H., Muroya, K., Yamada, G., Takitani, K. and
Ogata, T. (2010). Anorectal and urinary anomalies and aberrant retinoic acid
metabolism in cytochrome P450 oxidoreductase deficiency. Mol. Genet. Metab.
100, 269-273.
Hernandez, R. E., Putzke, A. P., Myers, J. P., Margaretha, L. and Moens, C. B.
(2007). Cyp26 enzymes generate the retinoic acid response pattern necessary for
hindbrain development. Development 134, 177-187.
Hochgreb, T., Linhares, V. L., Menezes, D. C., Sampaio, A. C., Yan, C. Y. I.,
Cardoso, W. V., Rosenthal, N. and Xavier-Neto, J. (2003). A caudorostral wave
of RALDH2 conveys anteroposterior information to the cardiac field. Development
130, 5363-5374.
Kudoh, T., Wilson, S. W. and Dawid, I. B. (2002). Distinct roles for Fgf, Wnt
and retinoic acid in posteriorizing the neural ectoderm. Development 129,
4335-4346.
Lai, L., Bohnsack, B. L., Niederreither, K. and Hirschi, K. K. (2003). Retinoic acid
regulates endothelial cell proliferation during vasculogenesis. Development 130,
6465-6474.
Lammer, E. J., Chen, D. T., Hoar, R. M., Agnish, N. D., Benke, P. J., Braun, J. T.,
Curry, C. J., Fernhoff, P. M., Grix, A. W., Jr, Lott, I. T. et al. (1985). Retinoic acid
embryopathy. N. Engl. J. Med. 313, 837-841.
Lee, D., Park, C., Lee, H., Lugus, J. J., Kim, S. H., Arentson, E., Chung, Y. S.,
Gomez, G., Kyba, M., Lin, S. et al. (2008). ER71 acts downstream of BMP,
Notch, and Wnt signaling in blood and vessel progenitor specification. Cell Stem
Cell 2, 497-507.
Mably, J. D., Mohideen, M.-A. P. K., Burns, C. G., Chen, J.-N. and Fishman, M. C.
(2003). heart of glass regulates the concentric growth of the heart in zebrafish.
Curr. Biol. 13, 2138-2147.
Maves, L. and Kimmel, C. B. (2005). Dynamic and sequential patterning of the
zebrafish posterior hindbrain by retinoic acid. Dev. Biol. 285, 593-605.
Niederreither, K. and Dollé , P. (2008). Retinoic acid in development: towards an
integrated view. Nat. Rev. Genet. 9, 541-553.
Niederreither, K., McCaffery, P., Drä ger, U. C., Chambon, P. and Dollé , P. (1997).
Restricted expression and retinoic acid-induced downregulation of the
retinaldehyde dehydrogenase type 2 (RALDH-2) gene during mouse
development. Mech. Dev. 62, 67-78.
Niederreither, K., Abu-Abed, S., Schuhbaur, B., Petkovich, M., Chambon, P.
and Dolle, P. (2002). Genetic evidence that oxidative derivatives of retinoic acid
are not involved in retinoid signaling during mouse development. Nat. Genet. 31,
84-88.
Osmond, M. K., Butler, A. J., Voon, F. C. and Bellairs, R. (1991). The effects of
retinoic acid on heart formation in the early chick embryo. Development 113,
1405-1417.
Otto, D. M. E., Henderson, C. J., Carrie, D., Davey, M., Gundersen, T. E.,
Blomhoff, R., Adams, R. H., Tickle, C. and Wolf, C. R. (2003). Identification
of novel roles of the cytochrome p450 system in early embryogenesis: effects
on vasculogenesis and retinoic Acid homeostasis. Mol. Cell. Biol. 23,
6103-6116.
Oxtoby, E. and Jowett, T. (1993). Cloning of the zebrafish krox-20 gene (krx-20)
and its expression during hindbrain development. Nucleic Acids Res. 21,
1087-1095.
Pan, J. and Baker, K. M. (2007). Retinoic acid and the heart. Vitamin A 75, 257-283.
Pennimpede, T., Cameron, D. A., MacLean, G. A., Li, H., Abu-Abed, S. and
Petkovich, M. (2010). The role of CYP26 enzymes in defining appropriate retinoic
acid exposure during embryogenesis. Birth Defects Res. A Clin. Mol. Teratol. 88,
883-894.
Prince, V. E., Moens, C. B., Kimmel, C. B. and Ho, R. K. (1998). Zebrafish hox
genes: expression in the hindbrain region of wild-type and mutants of the
segmentation gene, valentino. Development 125, 393-406.
Proulx, K., Lu, A. and Sumanas, S. (2010). Cranial vasculature in zebrafish forms
by angioblast cluster-derived angiogenesis. Dev. Biol. 348, 34-46.
Ribes, V., Fraulob, V., Petkovich, M. and Dollé , P. (2007). The oxidizing enzyme
CYP26a1 tightly regulates the availability of retinoic acid in the gastrulating mouse
embryo to ensure proper head development and vasculogenesis. Dev. Dyn. 236,
644-653.
Roberts, C., Ivins, S., Cook, A. C., Baldini, A. and Scambler, P. J. (2006). Cyp26
genes a1, b1 and c1 are down-regulated in Tbx1 null mice and inhibition of Cyp26
enzyme function produces a phenocopy of DiGeorge Syndrome in the chick.
Hum. Mol. Genet. 15, 3394-3410.
Robu, M. E., Larson, J. D., Nasevicius, A., Beiraghi, S., Brenner, C., Farber,
S. A. and Ekker, S. C. (2007). p53 activation by knockdown technologies. PLoS
Genet. 3, e78.
1647
DEVELOPMENT
RESEARCH ARTICLE
RESEARCH ARTICLE
correct Nodal expression during early embryonic patterning. Genes Dev. 23,
1689-1698.
Waxman, J. S. and Yelon, D. (2009). Increased Hox activity mimics the teratogenic
effects of excess retinoic acid signaling. Dev. Dyn. 238, 1207-1213.
Waxman, J. S. and Yelon, D. (2011). Zebrafish retinoic acid receptors function as
context-dependent transcriptional activators. Dev. Biol. 352, 128-140.
Waxman, J. S., Keegan, B. R., Roberts, R. W., Poss, K. D. and Yelon, D. (2008).
Hoxb5b acts downstream of retinoic acid signaling in the forelimb field to restrict
heart field potential in zebrafish. Dev. Cell 15, 923-934.
Westerfield, M. (2000). The Zebrafish Book. A Guide for the Laboratory Use of
Zebrafish (Danio rerio). Eugene, OR: University of Oregon Press.
White, J. A., Beckett-Jones, B., Guo, Y.-D., Dilworth, F. J., Bonasoro, J., Jones,
G. and Petkovich, M. (1997). cDNA cloning of human retinoic acid-metabolizing
enzyme (hP450RAI) identifies a novel family of cytochromes P450. J. Biol. Chem.
272, 18538-18541.
White, R. J., Nie, Q., Lander, A. D. and Schilling, T. F. (2007). Complex regulation
of cyp26a1 creates a robust retinoic acid gradient in the zebrafish embryo. PLoS
Biol. 5, e304.
Yelon, D., Horne, S. A. and Stainier, D. Y. R. (1999). Restricted expression of
cardiac myosin genes reveals regulated aspects of heart tube assembly in
zebrafish. Dev. Biol. 214, 23-37.
Yutzey, K. E., Rhee, J. T. and Bader, D. (1994). Expression of the atrial-specific
myosin heavy chain AMHC1 and the establishment of anteroposterior polarity in
the developing chicken heart. Development 120, 871-883.
Zhao, Q., Dobbs-McAuliffe, B. and Linney, E. (2005). Expression of cyp26b1
during zebrafish early development. Gene Expr. Patterns 5, 363-369.
Zhou, Y., Cashman, T. J., Nevis, K. R., Obregon, P., Carney, S. A., Liu, Y., Gu, A.,
Mosimann, C., Sondalle, S., Peterson, R. E. et al. (2011). Latent TGF-beta
binding protein 3 identifies a second heart field in zebrafish. Nature 474, 645-648.
DEVELOPMENT
Russo, J. E., Hauquitz, D. and Hilton, J. (1988). Inhibition of mouse cytosolic
aldehyde dehydrogenase by 4-(Diethylamino)benzaldehyde. Biochem. Pharmacol.
37, 1639-1642.
Sandell, L. L., Sanderson, B. W., Moiseyev, G., Johnson, T., Mushegian, A.,
Young, K., Rey, J.-P., Ma, J.-x., Staehling-Hampton, K. and Trainor, P. A.
(2007). RDH10 is essential for synthesis of embryonic retinoic acid and is
required for limb, craniofacial, and organ development. Genes Dev. 21,
1113-1124.
Sandell, L. L., Lynn, M. L., Inman, K. E., McDowell, W. and Trainor, P. A. (2012).
RDH10 oxidation of vitamin A is a critical control step in synthesis of retinoic acid
during mouse embryogenesis. PLoS ONE 7, e30698.
Schoenebeck, J. J., Keegan, B. R. and Yelon, D. (2007). Vessel and blood
specification override cardiac potential in anterior mesoderm. Dev. Cell 13,
254-267.
Serbedzija, G. N., Chen, J. N. and Fishman, M. C. (1998). Regulation in the heart
field of zebrafish. Development 125, 1095-1101.
Sorrell, M. R. J. and Waxman, J. S. (2011). Restraint of Fgf8 signaling by retinoic
acid signaling is required for proper heart and forelimb formation. Dev. Biol. 358,
44-55.
Stainier, D. Y. R. and Fishman, M. C. (1992). Patterning the zebrafish heart tube:
acquisition of anteroposterior polarity. Dev. Biol. 153, 91-101.
Sumanas, S., Gomez, G., Zhao, Y., Park, C., Choi, K. and Lin, S. (2008). Interplay
among Etsrp/ER71, Scl, and Alk8 signaling controls endothelial and myeloid cell
formation. Blood 111, 4500-4510.
Uehara, M., Yashiro, K., Mamiya, S., Nishino, J., Chambon, P., Dolle, P. and
Sakai, Y. (2007). CYP26A1 and CYP26C1 cooperatively regulate anteriorposterior patterning of the developing brain and the production of migratory cranial
neural crest cells in the mouse. Dev. Biol. 302, 399-411.
Uehara, M., Yashiro, K., Takaoka, K., Yamamoto, M. and Hamada, H. (2009).
Removal of maternal retinoic acid by embryonic CYP26 is required for
Development (2014) 141, 1638-1648 doi:10.1242/dev.105874
1648