PMC

(A) Inhibition of Wnt signaling using XAV939 or heat shock–inducible, dominant negative Tcf3 (hsp70l:tcfΔC-EGFP) blocks Etv2-induced kdrl:GFP expression. However, activation of Wnt signaling using CHIR99021 or heat shock–inducible, constitutively active β-catenin (hsp70l:caβ-catenin-2A-TFP) also blocks Etv2-induced kdrl:GFP expression. (B) XAV939 dose dependently inhibits Etv2-induced kdrl:GFP expression. Kdrl:GFP expression was quantified by counting the number of GFP⁺ myotomes with a myotome being considered positive if >3 muscle fibers within a given myotome were GFP⁺. (C) CHIR99021 dose dependently inhibits Etv2-induced kdrl:GFP expression. (D) XAV939 (40 µM) inhibits Etv2-induced kdrl:GFP expression independent of heat shock time, while CHIR99021 (50 µM) inhibits at 22 and 24 h heat shocks, has no effect at 26 h heat shock, but enhances Etv2-induced kdrl:GFP expression at 28 h heat shock. For all data t test was used for statistical comparisons with (*) p<0.05 and (**) p<0.01.

(A) Extended time lapse imaging of the trunk of a kdrl:GFP/hsp70l:etv2 embryo showing ectopic GFP expressing cells change morphology from fiber-like (+12 h) to spindle-like (+20 h, +28 h) to form a network of thin cells (+48 h) and finally appear to form lumenized vessels (+72 h). The two panels shown at each time point are two magnifications of the same image. (B) Microangiography demonstrates the newly formed vessels are functional. Rhodamine dextran dye was injected into the circulation of a 4 dpf (+72 h post–heat shock) kdrl:GFP/hsp70l:etv2 embryo. Rhodamine labels within all of the newly formed vessels and no vascular leakage are observed.

(A) Blastula cell transplantation was performed from triple transgenic, mylpfa:mRFP/hsp70l:etv2/kdrl:GFP⁺, into wild-type embryos. Approximately 10 cells were transplanted per embryo. Transplanted embryos were raised until 22 hpf, at which point they were selected for embryos displaying mylpfa:mRFP expression in distinct regions absent in kdrl:GFP, region of interest (ROI) boxed in (B) corresponds to images in (C). These embryos were then either heat shocked or left as no heat shock controls. Embryos were then analyzed for mylpfa:mRFP/kdrl:GFP coexpression at 10 h post–heat shock and followed out to 42 h post–heat shock (C). A muscle cell labeled with the arrow undergoes transdifferentiation to form a lumenized vessel (C). (D) Quantification of transdifferentiation efficiency per muscle cell. Only clearly distinguishable muscle cells were counted. Thirty-eight chimeric embryos, 312 total cells, were observed in the heat-shocked condition, and 20 chimeric embryos, 143 total cells, were observed for the control non–heat shocked condition.

(A and B) Vascular genes are induced 8 h post-HS. (A) In situ hybridization (ISH) for kdrl demonstrates broad ectopic expression including in the trunk following HS, but normal vascular restricted expression in control embryos. The numbers in the higher magnification views represent the number of embryos exhibiting ectopic expression over the number observed. (B) Quantitiative RT-PCR (qPCR) for kdrl, kdr, tal1, fli1a, and erg 8 h post-HS versus non–heat shocked controls. (C) ISH for kdrl at 24 h post-HS demonstrates increase expression in the trunk (white arrows). The apparent age discrepancy between the control and the HS+24 h embryo is due to developmental delay caused by Etv2 overexpression. (D and E) Muscle genes are repressed by Etv2 overexpression. (D) ISH for myod1 demonstrating near complete loss of expression 4 h post-HS. The numbers in the higher magnification views represent the number of embryos exhibiting normal expression levels. (E) qPCR for muscle genes myod1, myog, myf6, mylpfa, and tnnt3a shows significantly decreased expression 4 h post–heat shock. qPCR was performed on three separate clutches. (**) p<0.01, t test versus non–heat shocked control.

(A) hsp70l:etv2/kdrl:GFP embryo at 27 h postfertilization (hpf) (high magnification view of trunk inset) and 48 hpf exhibit normal vascular-specific GFP expression and no Etv2-mCherry when not treated with heat shock (−HS+3 h and +24 h). Embryos heat shocked at 24 hpf exhibit normal vascular GFP expression and strong ubiquitous nuclear Etv2-mCherry expression at 27 hpf (+HS+3 h). By 24 h post–heat shock (48 hpf) robust ectopic GFP expression is present in the trunk. (B) Flow cytometric analysis of single cells isolated from three separate clutches of hsp70l:etv2/kdrl:GFP embryos treated with or without heat shock at 24 hpf and analyzed at 48 hpf. Ectopic Etv2 expression causes the percentage of GFP⁺ cells to increase from ∼2% to ∼8% of the total. T-test (*) p<0.05. (C) Response to Etv2 overexpression is developmentally restricted. Hsp70l:etv2/kdrl:GFP embryos heat shocked (HS) at 22, 24, 26, 28, or 30 hpf exhibit decreasing numbers of GFP⁺ cells and a shift from anterior trunk to tail. Heat shock after 30 hpf did not cause ectopic GFP⁺ cells. Dashed boxes highlight the Etv2 responsive cell populations.

(A and B) Dose-dependent rescue of XAV939 inhibition of kdrl:GFP expression by the addition of Wnt activator CHIR99021. (A) Images of the trunk of 48 hpf embryos following treatment with the noted compounds and heat shock at 24 hpf. Note that 40 µM XAV939 alone almost completely inhibits ectopic GFP expression and the addition of 5–25 µM of CHIR99021 can rescue this inhibition with 15 µM being the most affective dose. CHIR99021 doses greater than 50 µM do not rescue XAV939 inhibition. (B) Quantification of the observations in (A). t test, (*) p<0.05 comparing Control (Con) to drug treated and (#) p<0.05 comparing XAV939 (40 µM) to XAV939 (40 µM) plus varying doses of CHIR99021. (C) Schematic of muscle cell transdifferentiation into endothelial cells. Muscle cells exhibiting permissive levels of canonical Wnt activity are responsive to a pulse of Etv2 expression, resulting in repression of muscle gene expression and initiation of vascular gene expression. Cells expressing vascular genes respond to VEGF signals and change morphology from muscle fiber to thin multibranched and finally to lumenized patent endothelium.

(A) Immunostained sections through the trunk of 48 hpf hsp70l:etv2/fli1a:EGFP embryos that were untreated (control) or heat shocked at 24 hpf (HS+24 h). Sections were stained for GFP and fast muscle myosin. Nuclei are stained with DAPI in the mergeDAPI panels. fli1a:EGFP is normally expressed in the intersomitic vessels (ISVs) and axial vessels (AVs) of control sections. However, following heat shock, many fast muscle myosin positive cells were also GFP positive (A). ROI is the region of interest highlighted by the dashed box in each panel. One section from 20 different embryos was observed for each treatment group with similar results within each group. (B) Confocal projection images of a kdrl:GFP ⁺ and mylpfa:mRFP ⁺ double positive muscle fiber (arrow) in a living embryo 12 h post–heat shock. (C) Time lapse imaging of the trunk (left column) and at the single cell level (right column) of a mylpfa:mRFP/hsp70l:etv2/kdrl:GFP triple transgenic embryo beginning at 8 h post–heat shock (t₀+8 h). Heat shock occurred at 24 hpf. A few Etv2-mCherry⁺ nuclei are present in the first panel (arrowhead). The normal GFP⁺ intersomitic vessels (ISVs) and axial vessels (AVs) are labeled. mylpfa:mRFP labels fast muscle fibers in red. In the trunk, GFP expression first appears in muscle fibers between t₀+8 h to t₀+10 h and progresses in an caudal to rostral wave. mRFP⁺ fibers induce GFP expression and then soon switch off mRFP expression. ISV sprouts appear to apoptose and regress (asterisks). At the single cell level, mRFP⁺ fibers become GFP⁺ and then change morphology, a single cell is highlighted by a dashed outline in the right column.

(A–D) Control kdrl:GFP embryos (A) or kdrl:GFP/hsp70l:etv2 (B–D) embryos following heat shock at 24 hpf and imaged at 24 h or 36 h post–heat shock. Control embryos exhibit normal vascular kdrl:GFP expression (A), while kdrl:GFP/hsp70l:etv2 embryos exhibit the ectopic GFP and morphological changes previously described (B–D). (E–H) VEGFAa morpholino (VEGF-MO) treated embryos lack intersomitic vessels (E) but still induce kdrl:GFP in muscle fibers (F). However, kdrl:GFP ⁺ muscle fibers do not undergo the normally observed morphological changes following heat shock–induced expression of Etv2 (G,H). (I–L) Overexpression of VEGFAa¹²¹ (VEGF OE) driven by the hsp70l promoter results in disorganization and expansion of the normal vasculature (I). Following heat shock–induced expression of Etv2, no significant change in the number of muscle fibers expressing kdrl:GFP is observed (J). However, the morphological changes observed are accelerated in the presence of elevated VEGF (K,L). (M–P) Treatment of embryos with SU5416, a Kdr inhibitor, similarly inhibits intersomitic vessel development in control embryos (M). However, drug treatment does not inhibit induction of kdrl:GFP following heat shock–induced Etv2 expression in muscle (N). The morphology and survival of these fibers is compromised when Kdr is inhibited (O,P). (Q–V) Removal of VEGF inhibitor SU5416 24 h following heat shock allows for survival and maturation of transdifferentiated cells. Kdrl:GFP/hsp07l:etv2 embryos were heat shocked at 22 hpf and then treated with DMSO carrier or SU5416 for 24 h at which point the drug was either maintained (S,T) or removed (U,V) and the embryos were allowed to develop until 72 hpf. (Q,R) DMSO controls exhibit a transdifferentiated vascular network similar to that in untreated controls. (S,T) Sustained SU5416 treatment largely abolishes the kdrl:GFP⁺ vascular network. (U,V) Removal of SU5416 24 h post–heat shock results in the development of a vascular network similar to controls (Q,R), suggesting VEGF signaling modulates the survival and maturation of muscle-derived vessels and not the initial induction. For all experiments at least 20 embryos were observed with similar results.