First published online May 9, 2008
doi: 10.1242/10.1242/dev.011296
Development 135, 1903-1911 (2008)
Published by The Company of Biologists 2008
A Myc-Slug (Snail2)/Twist regulatory circuit directs vascular development
Claudia O. Rodrigues1,2,
Steve T. Nerlick3,
Elsie L. White1,
John L. Cleveland1,4,* and
Mary Lou King3,*
1 Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN
38105, USA.
2 Department of Molecular and Cellular Pharmacology, University of Miami Miller
School of Medicine, Miami, FL, USA.
3 Department of Cell Biology, University of Miami Miller School of Medicine,
Miami, FL, USA.
4 Department of Cancer Biology, The Scripps Research Institute-Florida, Jupiter,
FL 33458, USA.
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Fig. 1. Xc-Myc expression and morpholino knockdown. (A) In
situ hybridization of Xc-Myc expression during development. (i)
Gastrula: expression in involuting mesoderm (M) that flanks the yolk plug.
(ii) Neurula: expression in anterior region and in the lateral edges of neural
crest (NC). (iii) Early tailbud: expression in the developing eye (E) and
migratory neural crest cells (MNC). (iv) Tailbud ( stages 27/28):
expression in eyes (E), brain (B), somites (S), pharyngeal arches (PA) and
ventral blood islands (VBI). (v) Stage 35/36: expression is predominant in
head and heart region (eyes, E; brain, B; heart, H; rostral lymph sac, RLS;
aortic arches, AA). (B) The Xc-Myc morpholino blocks translation of
Xc-Myc. In vitro translation of transcripts for wild-type (Xc-Myc) or a
morpholino-resistant mutant (Xc-Myc-Mut) in the absence (-) or presence (+) of
Xc-Myc morpholino (Mo) is shown. Control morpholinos were
non-specific (A) or a scrambled Xc-Myc (B) morpholino. (C) Knockdown of
Xc-Myc protein expression in vivo at different stages. Western blot of Xc-Myc
expression in embryos injected with Xc-Myc morpholino (Mo);
uninjected (uninj); loading control (input).
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Fig. 2. Knockdown of Xc-Myc provokes edema and compromises organogenesis.
(A) Embryos injected with Xc-Myc morpholino (Xc-Myc
Mo) at the one-cell stage developed edema in the head and heart region
(white arrows). By stage 45 (right panels) massive edema and
associated defects in organogenesis were obvious in Xc-Myc knockdown tadpoles
(Xc-Myc Mo). (B-D) Histological analysis (stage 37) of Xc-Myc
knockdown embryos revealed profound developmental defects: somites (S), spinal
cord (SC), notochord (N), abdominal cavity (ac), heart (H), pronephros (PN),
dorsal aorta (DA) and cardinal vein (CV). (C) The heart is shown. (D) The
region containing the dorsal aorta and cardinal vein.
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Fig. 3. Xc-Myc knockdown impairs endothelial cell development.
(A,B) Whole mount in situ hybridization (stage 37) for
endothelial lineage marker X-msr; (B) enlargement of the embryos
shown in A. X-msr staining is reduced and diffuse in Xc-Myc knockdown
embryos throughout, and the vitelline vein network (vvn), aortic arches (aa),
tunica vasculosa lentis (tvl), anterior cardinal vein (acv) and rostral lymph
sac (RLS) are reduced or missing. H, heart; pcv, posterior cardinal vein; isv,
intersegmental vessels. (C) Histology of posterior cardinal vein (pcv)
and dorsal aorta at stage 37. Uninj, uninjected control; Xc-Myc Mo,
Xc-Myc morpholino-injected embryos. The vessels found in Xc-Myc
morpholino-injected embryos have remarkably thin walls and empty lumens.
Analyses based on five or six embryos from two different experiments.
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Fig. 4. Edema provoked by Xc-Myc knockdown is associated with massive hemorrhage
and impaired expression of X-ERG, Scl and
Prox1. (A) The vascular networks of uninjected (Uninj) and
Xc-Myc morpholino (Mo)-injected embryos were visualized by staining
blood cells with benzidine. Hemorrhagic areas are present throughout body
proper of Mo-injected embryos. H, heart; aa, aortic arches; vvn, vitelline
vein network; pcv, posterior cardinal vein; da, dorsal aorta. (B) The
effects of Xc-Myc knockdown on the expression of genes involved in vascular
development was assessed by qRT-PCR in control (dark blue bars) and
Xc-Myc morpholino-injected (light blue bars) embryos at stage 28.
Expression was normalized to Gapdh.
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Fig. 5. Edema provoked by Xc-Myc knockdown is intrinsic and specific and Xc-Myc
overexpression induces ectopic vascular beds. (A) The percentage of
Xc-Myc morpholino-injected embryos that developed edema (light blue
bars) or appeared normal (dark blue bars) is shown. Co-injection of
Xc-Myc-Mut rescued the edema phenotype. (B) Representative
images of stage 45 control (i) or those injected with Xc-Myc
morpholino (ii), with both Xc-Myc-Mut mRNA and Xc-Myc
morpholino (iii) [note rescue of phenotype and hypervascularization (black
arrows)], or with Xc-Myc RNA (iv), showing hypervascularization and
ectopic vascular beds (black arrows). (C) The percentage of
Xc-Myc morpholino-injected embryos with hemorrhage. Co-injection of
Xc-Myc-Mut mRNA rescued the phenotype. Injection of either wild-type
Xc-Myc or mutant Xc-Myc-Mut mRNA induced
hypervascularization (red bars).
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Fig. 6. Slug or Twist rescue the vascular defects provoked by Xc-Myc
knockdown. (A) The percentage of embryos that appeared normal
(dark-blue bars) or showed edema associated with hemorrhage (light-blue bars)
after injection of Xc-Myc morpholino alone or mixed with
Xc-Myc-Mut, wild-type Xc-Myc, Slug, Twist or
β-galactosidase mRNAs. (B) Representative images of the
results in A. showing the rescue of the Xc-Myc knockdown phenotype by Slug or
Twist. (C) One blastomere of a two-cell stage embryo was injected with
Slug morpholino. In situ hybridization at the neural tube stage using
a probe specific for Xc-Myc showed that knockdown of Slug indeed
affects Xc-Myc expression in the region corresponding to the neural
crest (i-iii). On the injected side (black arrowheads) an accumulation of
Xc-Myc-expressing cells is evident, while on the uninjected side cells
expressing Xc-Myc have started to migrate (red arrow). A deviation in the body
axis is also evident (ii,iii). Despite affecting Xc-Myc expression
during neurulation, knockdown of Slug in two-cell embryos is not
sufficient to cause the edema and hemorrhagic phenotype (iv,v), as observed
upon Xc-Myc knockdown using Xc-Myc morpholino.
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Fig. 7. Xc-Myc or XSlug/XTwist targeted knockdown in lateral plate mesoderm but
not the neural crest lineage, provokes edema and hemorrhage. (A)
Map showing the blastomeres targeted for knockdown of Xc-Myc or
XSlug/XTwist in predominantly neural crest (V1.2) or lateral plate
precursors (D2.1 or V2.1). Correct targeting of the morpholinos is shown in
the next three images taken at neurula and tailbud when neural crest cells
start migrating. NC, neural crest; LPM, lateral plate mesoderm; Neph,
nephrotome. (B,C) Embryos were injected with a mixture of either
Xc-Myc morpholino (B) or XSlug/XTwist morpholino (C), and were
dextran fluorescently labeled with rhodamine. At stage 43, embryos were
stained with benzidine to reveal the location of blood. Knockdown of
Xc-myc or XSlug/XTwist within the lateral plate mesoderm
(3rd and 4th column), but not the neural crest (2nd column), provoked edema
(arrows) and hemorrhagic (arrowheads) phenotypes. Levels of blood in the V2.1
injected embryos (4th column) are significantly lower (dashed arrow).
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Fig. 8. A Myc-Slug/Twist regulatory circuit directs vasculogenesis and
lymphangiogenesis. Xc-Myc is required for the normal expression of the
XSlug and XTwist transcription factors, either of which are
sufficient to rescue the defects in vascular and lymphatic development
provoked by knockdown of Xc-Myc. Knockdown of XSlug also disrupts normal
patterns of Xc-Myc expression, indicating a positive-feedback loop.
Knockdown of both XSlug and XTwist in lateral plate
mesoderm, but not neural crest, results in the vascular defects observed after
knockdown of Xc-Myc, indicating both genes operate in the same pathway and
downstream of Myc. Either ectopic Slug or Twist rescue defects in endogenous
XSlug or XTwist expression induced by Xc-Myc knockdown,
indicating that they also regulate each the expression of one another.
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© The Company of Biologists Ltd 2008
