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Development
2008 Jun 01;13511:1903-11. doi: 10.1242/dev.011296.
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A Myc-Slug (Snail2)/Twist regulatory circuit directs vascular development.
Rodrigues CO
,
Nerlick ST
,
White EL
,
Cleveland JL
,
King ML
.
Abstract Myc-deficient mice fail to develop normal vascular networks and Myc-deficient embryonic stem cells fail to provoke a tumor angiogenic response when injected into immune compromised mice. However, the molecular underpinnings of these defects are poorly understood. To assess whether Myc indeed contributes to embryonic vasculogenesis we evaluated Myc function in Xenopus laevis embryogenesis. Here, we report that Xc-Myc is required for the normal assembly of endothelial cells into patent vessels during both angiogenesis and lymphangiogenesis. Accordingly, the specific knockdown of Xc-Myc provokes massive embryonic edema and hemorrhage. Conversely, Xc-Myc overexpression triggers the formation of ectopic vascular beds in embryos. Myc is required for normal expression of Slug/Snail2 and Twist, and either XSlug/Snail2 or XTwist could compensate for defects manifest by Xc-Myc knockdown. Importantly, knockdown of Xc-Myc, XSlug/Snail2 or XTwist within the lateral plate mesoderm, but not the neural crest, provoked embryonic edema and hemorrhage. Collectively, these findings support a model in which Myc, Twist and Slug/Snail2 function in a regulatory circuit within lateral plate mesoderm that directs normal vessel formation in both the vascular and lymphatic systems.
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 ventralblood 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).
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.
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 orβ -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.
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.
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.
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).
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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