|
Gene |
Clone |
Species |
Stages |
Anatomy |
myc.L
|
|
laevis
|
NF stage 10
to
NF stage 12.5
|
involuting marginal zone
|
myc.L
|
|
laevis
|
NF stage 14
|
neural crest
,
preplacodal ectoderm
|
myc.L
|
|
laevis
|
NF stage 22
|
branchial crest
,
eye
,
cranial neural crest
|
myc.L
|
|
laevis
|
NF stage 27
to
NF stage 28
|
brain
,
ventral blood island
,
pharyngeal arch
,
branchial arch
,
eye
,
[+]
|
myc.L
|
|
laevis
|
NF stage 35 and 36
|
brain
,
heart
,
alimentary system
,
eye
,
aortic arch
,
[+]
|
|
|
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). |
|
|
|
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. 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. |