Nuclear Receptor Subfamily 2 Group F Member 1a (nr2f1a) Is Required for Vascular Development in Zebrafish

<div><p>Genetic regulators and signaling pathways are important for the formation of blood vessels. Transcription factors controlling vein identity, intersegmental vessels (ISV) growth and caudal vein plexus (CVP) formation in zebrafish are little understood as yet. Here, we show the importance of the nuclear receptor subfamily member 1A (nr2f1a) in zebrafish vascular development. Amino acid sequence alignment and phylogenetic analysis of nr2f1a is highly conserved among the vertebrates. Our in situ hybridization results showed <i>nr2f1a</i> mRNA is expressed in the lateral plate mesoderm at 18 somite stage and in vessels at 24–30 hpf, suggesting its roles in vasculization. Consistent with this morpholino-based knockdown of <i>nr2fla</i> impaired ISV growth and failed to develop fenestrated vascular structure in CVP, suggesting that <i>nr2f1a</i> has important roles in controlling ISV and CVP growth. Consequently, <i>nr2f1a</i> morphants showed pericardial edema and circulation defects. We further demonstrated reduced ISV cells and decreased CVP endothelial cells sprouting in <i>nr2f1a</i> morphants, indicating the growth impairment of ISV and CVP is due to a decrease of cell proliferation and migration, but not results from cell death in endothelial cells after morpholino knockdown. To test molecular mechanisms and signals that are associated with <i>nr2f1a</i>, we examined the expression of vascular markers. We found that a loss of <i>nr2f1a</i> results in a decreased expression of vein/ISV specific markers, <i>flt4, mrc1</i>, vascular markers <i>stabilin</i> and <i>ephrinb2.</i> This indicates the regulatory role of nr2f1a in controlling vascular development. We further showed that <i>nr2f1a</i> likely interact with <i>Notch</i> signaling by examining <i>nr2f1a</i> expression in <i>rbpsuh</i> morphants and DAPT-treatment embryos. Together, we show nr2f1a plays a critical role for vascular development in zebrafish.</p></div

Regulatory relationships between <i>nr2f1a</i> and Notch.

<p>(<b>A–B</b>) <i>nr2f1a</i> expression is downregulated in <i>rbpsuh</i> morphants (<b>B</b>) as compared to untreated control embryos (<b>A</b>). (<b>D–E</b>) <i>nr2f1a</i> expression is downregulated after treatment with DAPT (<b>E</b>) as compared to DMSO control embryos (<b>D</b>). (<b>C, F</b>) Quantification of the relative <i>nr2f1a</i> expression level by real-time qPCR assay shows a ∼50% reduced expression in <i>rbpsuh</i> morphants (<b>C</b>) and DAPT-treated embryos (<b>F</b>) compared to uninjected embryos and DMSO-treated controls. Scale bars represent 100 µm in A, B, D and E.</p

nr2f1a is conserved among vertebrates.

<p>(<b>A</b>) Comparison of the amino acid sequence of zebrafish nr2fIa to orthologs from other species by using ClustalW2 software. Identical amino acids are marked (*) below the sequence. The yellow bar represents a putative ligand binding domain and green box area indicates a zinc-finger DNA binding domain. Squared box indicates a conserved LLLRLP motif. (<b>B</b>) Schematic drawing shows that zebrafish nr2f1a protein contains 411 amino acids with the DNA binding domain (DBD) and ligand binding domain (LBD). (<b>C</b>) Cladograms provided from phylogenetic analysis of nr2f1a orthologs in different species using ClustalW2-phylogeny with the neighbor-joining method. The number (bootstrap values) at the nodes represent the possibility of groupings between 2 different nr2f members from different species. (<b>D</b>) Percent amino acid identity matrix among mouse (<i>Mus_musculus</i>) Nr2f2 and zebrafish (<i>Danio_rerio</i>) <i>nr2f1a, nr2f1b</i> and <i>nr2f2.</i></p

Low Temperature Mitigates Cardia Bifida in Zebrafish Embryos

<div><p>The coordinated migration of bilateral cardiomyocytes and the formation of the cardiac cone are essential for heart tube formation. We investigated gene regulatory mechanisms involved in myocardial migration, and regulation of the timing of cardiac cone formation in zebrafish embryos. Through screening of zebrafish treated with ethylnitrosourea, we isolated a mutant with a hypomorphic allele of <i>mil</i> (<i>s1pr2</i>)/<i>edg5</i>, called <i>s1pr2^as10</i> (<i>as10</i>). Mutant embryos with this allele expressed less <i>mil</i>/<i>edg5</i> mRNA and exhibited cardia bifida prior to 28 hours post-fertilization. Although the bilateral hearts of the mutants gradually fused together, the resulting formation of two atria and one tightly-packed ventricle failed to support normal blood circulation. Interestingly, cardia bifida of <i>s1pr2^as10</i> embryos could be rescued and normal circulation could be restored by incubating the embryos at low temperature (22.5°C). Rescue was also observed in <i>gata5</i> and <i>bon</i> cardia bifida morphants raised at 22.5°C. The use of DNA microarrays, digital gene expression analyses, loss-of-function, as well as mRNA and protein rescue experiments, revealed that low temperature mitigates cardia bifida by regulating the expression of genes encoding components of the extracellular matrix (<i>fibronectin 1</i>, <i>tenascin-c</i>, <i>tenascin-w</i>). Furthermore, the addition of N-acetyl cysteine (NAC), a reactive oxygen species (ROS) scavenger, significantly decreased the effect of low temperature on mitigating cardia bifida in <i>s1pr2^as10</i> embryos. Our study reveals that temperature coordinates the development of the heart tube and somitogenesis, and that extracellular matrix genes (<i>fibronectin 1</i>, <i>tenascin-c</i> and <i>tenascin-w</i>) are involved.</p></div

Loss of <i>nr2f1a</i> results in circulation defects and pericardial edema.

<p>(<b>A–E</b>) <i>nr2f1a</i>^e2i2 MO was injected into transgenic <i>Tg (fli:eGFP^y1; gata1:dsRed)</i> embryos with GFP-labeled endothelial cells (<b>A, C</b>) and dsRed-labeled blood cells (<b>B, D, E</b>). Loss of <i>nr2f1a</i> showed a mis-pattern at ISV and at the caudal vein plexus (CVP) and results in circulation defects in severe (<b>D</b>, no circulation and blood stock) or mild (<b>E</b>, blood stock at CVP and shorter circulation) at 52 hpf compared to wild-type fish (<b>B</b>). (<b>F</b>) Circulation defects at the ISV/DLAV (∼68% in morphants) and slow to lose axial circulation of the aorta/vein in the trunk region (∼58% in morphants) are quantitated in wt (n = 37) and nr2f1aMO (n = 42) at 48–52 hpf. (<b>G, H</b>) Representative edema fish and quantitative results from three independent experiments showed 65% of <i>nr2f1a</i> morphants (n = 69) with mild to severe pericardial edema compared to wt (n = 30). The scale bar in A–E represent 200 µm and in G is 500 µm.</p

Knockdown of nr2f1a can be rescued by overexpression of <i>nr2f1a</i>.

<p>In uninjected control embryos, intersegmental vessels (isv) have reached the DLAV at the dorsal region from 24–30 hpf (<b>A,</b><i>white arrowheads</i>) and caudal vein plexus (cvp) were formed honeycomb-like structures at the tail around 48 hpf (<b>E,</b><i>white arrows</i>). At the same stage, ISVs are stalled at mid-somite (<b>B,</b><i>hollow arrowheads</i>) and less/no honeycomb structure is formed at CVP (<b>F</b> with <i>no arrows</i>) in nr2f1a^e2i2 morphants. Overexpression of nr2f1a driven by fli promoter had no obvious defect in vasculature (<b>C, G</b>), but rescued the defect of ISV stalling (<i>solid arrowhead</i>) (<b>D</b>) and restored the honeycomb structure at CVP (<b>H</b>). (<b>I</b>) Quantification of percentage of completed ISV at 26 hpf shows a ∼40% increase in rescued embryos compare to <i>nr2f1a</i> morphants. Wild-type embryos had 62±3.1% complete ISV; <i>nr2f1aMO</i> had 18±8.0% complete ISV; <i>fli:nr2f1a</i> overexpression embryos had 63±2.6% complete ISV and rescued embryos had 57±2.7% complete ISV. (<b>J</b>) Quantification of loop formation at CVP showed a 2-fold increased in rescued embryos compared to <i>nr2f1a</i> morphants at 48 hpf. Wild-type embryos (19.6±0.9); <i>nr2f1aMO</i> (4.2±1.5); <i>fli:nr2f1a</i> overexpression embryos (22.6±1.3) and rescued embryos (10.6±1.6). (*** refers to p<0.0001, ** refers to p<0.001 and * refers to p<0.05 by an unpaired student’s t-test. Data are represented as means ± SEM). Scale bars are 100 µm for A–H.</p

Spatiotemporal expression of <i>nr2f1a</i> during development.

<p>(<b>A</b>) <i>nr2f1a</i> expression can be observed at the 18S stage in the lateral plate mesoderm (lpm), the telencephalon (t), diencephalon (d) and hindbrain (h). (<b>A’</b>) Dorsal view of embryos show that <i>nr2f1a</i> is expressed at lpm and the axial vessels (av). (<b>B, B’</b>) At 24 hpf, <i>nr2f1a</i> is expressed in the telencephalon (t), diencephalon (d), hindbrain (h), as well as in vessels (v), and caudal vein plexus (CVP) of the trunk. <b>B’</b> is an enlargement of <b>B</b>. (<b>C, D</b>) Cross sections of embryos from B’ show that nr2f1a is expressed in dorsal aorta (da), posterior cardinal vein (pcv), and caudal vein plexus (CVP). (<b>E, E’</b>) At 30 hpf, <i>nr2f1a</i> expression continues in the head, vessels (v), intersegmental vessels (ISV) and caudal vein plexus (CVP) of the trunk. <b>E’</b> is an enlargement of <b>E</b>. Scale bars in all figures represent 100 µm.</p

<i>nr2f1a</i> is required for the growth of ISV cells.

<p>(<b>A–D</b>) TUNEL labeling and AO staining was used to detect apoptotic cells in wild type, and nr2f1a^e2i2 morphants. Although some apoptotic cells were observed on the skin, cell death in vascular regions was not elevated compared to wild type controls. (<b>E–H</b>) The number of cells forming each ISV counted in wild type control <i>Tg(kdrl:mCherry; fli1a:negfp)^y7</i> (<b>E</b>) and <i>nr2f1a</i> morphant embryos (<b>F</b>) at 32 hpf. (<b>G</b>) Proportional distribution of ISVs containing 1–5 cells and (<b>H</b>) average ISV cells per ISV counted in both wild type and <i>nr2f1a</i> morphant. (*** refers to p<0.0001 by an unpaired student’s t-test).</p

Morpholino-knockdown efficiency of <i>nr2f1a</i> in zebrafish embryos.

<p>(<b>A</b>) Scheme shows nr2f1a morpholino targeting the pre-mRNA structure of nr2f1a and suggested mis-splicing fragment which can be detected by a nr2f1a_MO_f and nr2f1a_MO_r primer set. (<b>B</b>) cDNA from uninjected controls or <i>nr2f1a</i> morphants (injected with 4 ng morpholino) underwent PCR with primers for the housekeeping control gene (<i>GAPDH</i>), or for the <i>nr2f1a</i> gene. Morphants injected with nr2f1a^e2i2, <i>GAPDH</i> levels are unchanged while the amount of wild-type <i>nr2f1a</i> product (683 bp) got diminished and a new lower molecular weight band appeared (120 bp), representing mis-splicing caused by the morpholino.</p

Reactive oxygen species (ROS) mediate mitigation of cardia bifida in zebrafish embryos incubated at low temperature.

<p>(A) <i>s1pr2^as10</i> mutant embryos raised at 22.5°C were treated with 50 or 150 µM N-acetyl cysteine (NAC) at the tailbud stage, and were then incubated at 22.5°C. Different degrees of myocardial migration defects were observed at the 26 ss. Class I (single heart tube), Class II (cardiomyocytes in close proximity but not in contact), and Class III (two separate hearts). (B) A proposed model showing how low temperature mitigates cardia bifida in zebrafish embryos. Statistical significance was determined using Student’s <i>t</i>-test. * indicates <i>p</i><0.05.</p