Comparative proteomic analysis reveals alterations in development and photosynthesis-related proteins in diploid and triploid rice

Six pairs of primers were designed for gene-specific transcript amplification. (DOC 25 kb

Transdifferentiation of Fast Skeletal Muscle Into Functional Endothelium in Vivo by Transcription Factor Etv2

<div><p>Etsrp/Etv2 (Etv2) is an evolutionarily conserved master regulator of vascular development in vertebrates. Etv2 deficiency prevents the proper specification of the endothelial cell lineage, while its overexpression causes expansion of the endothelial cell lineage in the early embryo or in embryonic stem cells. We hypothesized that Etv2 alone is capable of transdifferentiating later somatic cells into endothelial cells. Using heat shock inducible Etv2 transgenic zebrafish, we demonstrate that Etv2 expression alone is sufficient to transdifferentiate fast skeletal muscle cells into functional blood vessels. Following heat treatment, fast skeletal muscle cells turn on vascular genes and repress muscle genes. Time-lapse imaging clearly shows that muscle cells turn on vascular gene expression, undergo dramatic morphological changes, and integrate into the existing vascular network. Lineage tracing and immunostaining confirm that fast skeletal muscle cells are the source of these newly generated vessels. Microangiography and observed blood flow demonstrated that this new vasculature is capable of supporting circulation. Using pharmacological, transgenic, and morpholino approaches, we further establish that the canonical Wnt pathway is important for induction of the transdifferentiation process, whereas the VEGF pathway provides a maturation signal for the endothelial fate. Additionally, overexpression of Etv2 in mammalian myoblast cells, but not in other cell types examined, induced expression of vascular genes. We have demonstrated in zebrafish that expression of Etv2 alone is sufficient to transdifferentiate fast skeletal muscle into functional endothelial cells in vivo. Given the evolutionarily conserved function of this transcription factor and the responsiveness of mammalian myoblasts to Etv2, it is likely that mammalian muscle cells will respond similarly.</p></div

Fast skeletal muscle expresses ectopic endothelial genes following Etv2 overexpression.

<p>(A) Immunostained sections through the trunk of 48 hpf <i>hsp70l:etv2/fli1a:EGFP</i> 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. <i>fli1a:EGFP</i> 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 <i>kdrl:GFP</i>⁺ and <i>mylpfa:mRFP</i>⁺ 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 <i>mylpfa:mRFP/hsp70l:etv2/kdrl:GFP</i> 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. <i>mylpfa:mRFP</i> 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.</p

Ubiquitous Etv2 expression induces ectopic vascular gene expression in the trunk of 48 h zebrafish embryos.

<p>(A) <i>hsp70l:etv2/kdrl:GFP</i> 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 <i>hsp70l:etv2/kdrl:GFP</i> 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. <i>T-test</i> (*) <i>p</i><0.05. (C) Response to Etv2 overexpression is developmentally restricted. <i>Hsp70l:etv2/kdrl:GFP</i> 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.</p

A tightly controlled level of Wnt signaling is necessary for muscle to transdifferentiate into endothelial cells.

<p>(A and B) Dose-dependent rescue of XAV939 inhibition of <i>kdrl:GFP</i> 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). <i>t</i> test, (*) <i>p</i><0.05 comparing Control (Con) to drug treated and (#) <i>p</i><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.</p

Etv2 overexpression induces vascular gene expression and represses muscle gene expression.

<p>(A and B) Vascular genes are induced 8 h post-HS. (A) In situ hybridization (ISH) for <i>kdrl</i> 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 <i>kdrl</i>, <i>kdr</i>, <i>tal1</i>, <i>fli1a</i>, and <i>erg</i> 8 h post-HS versus non–heat shocked controls. (C) ISH for <i>kdrl</i> 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 <i>myod1</i> 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 <i>myod1</i>, <i>myog</i>, <i>myf6</i>, <i>mylpfa</i>, and <i>tnnt3a</i> shows significantly decreased expression 4 h post–heat shock. qPCR was performed on three separate clutches. (**) <i>p</i><0.01, <i>t</i> test versus non–heat shocked control.</p

Fast skeletal muscle converts to functional endothelial cells following Etv2 expression.

<p>(A) Extended time lapse imaging of the trunk of a <i>kdrl:GFP/hsp70l:etv2</i> 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) <i>kdrl:GFP/hsp70l:etv2</i> embryo. Rhodamine labels within all of the newly formed vessels and no vascular leakage are observed.</p

VEGF signaling is dispensable for induction but necessary for maturation of Etv2 induced vasculature.

<p>(A–D) Control <i>kdrl:GFP</i> embryos (A) or <i>kdrl:GFP/hsp70l:etv2</i> (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 <i>kdrl:GFP</i> expression (A), while <i>kdrl:GFP/hsp70l:etv2</i> 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 <i>kdrl:GFP</i> in muscle fibers (F). However, <i>kdrl:GFP</i>⁺ 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 <i>hsp70l</i> 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 <i>kdrl:GFP</i> 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 <i>kdrl:GFP</i> 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. <i>Kdrl:GFP/hsp07l:etv2</i> 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 <i>kdrl:GFP⁺</i> 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.</p