Platelet-Derived Growth Factor Receptor β Is Critical for Zebrafish Intersegmental Vessel Formation

Background: Platelet-derived growth factor receptor β (PDGFRβ) is a tyrosine kinase receptor known to affect vascular development. The zebrafish is an excellent model to study specific regulators of vascular development, yet the role of PDGF signaling has not been determined in early zebrafish embryos. Furthermore, vascular mural cells, in which PDGFRβ functions cell autonomously in other systems, have not been identified in zebrafish embryos younger than 72 hours post fertilization. Methodology/Principal Findings: In order to investigate the role of PDGFRβ in zebrafish vascular development, we cloned the highly conserved zebrafish homolog of PDGFRβ. We found that pdgfrβ is expressed in the hypochord, a developmental structure that is immediately dorsal to the dorsal aorta and potentially regulates blood vessel development in the zebrafish. Using a PDGFR tyrosine kinase inhibitor, a morpholino oligonucleotide specific to PDGFRβ, and a dominant negative PDGFRβ transgenic line, we found that PDGFRβ is necessary for angiogenesis of the intersegmental vessels. Significance/Conclusion: Our data provide the first evidence that PDGFRβ signaling is required for zebrafish angiogenesis. We propose a novel mechanism for zebrafish PDGFRβ signaling that regulates vascular angiogenesis in the absence of mural cells

Genome-Wide Association Study in East Asians Identifies Novel Susceptibility Loci for Breast Cancer

Genetic factors play an important role in the etiology of both sporadic and familial breast cancer. We aimed to discover novel genetic susceptibility loci for breast cancer. We conducted a four-stage genome-wide association study (GWAS) in 19,091 cases and 20,606 controls of East-Asian descent including Chinese, Korean, and Japanese women. After analyzing 690,947 SNPs in 2,918 cases and 2,324 controls, we evaluated 5,365 SNPs for replication in 3,972 cases and 3,852 controls. Ninety-four SNPs were further evaluated in 5,203 cases and 5,138 controls, and finally the top 22 SNPs were investigated in up to 17,423 additional subjects (7,489 cases and 9,934 controls). SNP rs9485372, near the TGF-β activated kinase (TAB2) gene in chromosome 6q25.1, showed a consistent association with breast cancer risk across all four stages, with a P-value of 3.8×10−12 in the combined analysis of all samples. Adjusted odds ratios (95% confidence intervals) were 0.89 (0.85–0.94) and 0.80 (0.75–0.86) for the A/G and A/A genotypes, respectively, compared with the genotype G/G. SNP rs9383951 (P = 1.9×10−6 from the combined analysis of all samples), located in intron 5 of the ESR1 gene, and SNP rs7107217 (P = 4.6×10−7), located at 11q24.3, also showed a consistent association in each of the four stages. This study provides strong evidence for a novel breast cancer susceptibility locus represented by rs9485372, near the TAB2 gene (6q25.1), and identifies two possible susceptibility loci located in the ESR1 gene and 11q24.3, respectively

A poly(ethylene glycol) three-dimensional bone marrow hydrogel

Three-dimensional (3D) hydrogels made from synthetic polymers have emerged as in vitro cell culture platforms capable of representing the extracellular geometry, modulus, and water content of tissues in a tunable fashion. Hydrogels made from these otherwise non-bioactive polymers can be decorated with short peptides derived from proteins naturally found in tissues to support cell viability and direct phenotype. We identified two key limitations that limit the ability of this class of materials to recapitulate real tissue. First, these environments typically display between 1 and 3 bioactive peptides, which vastly underrepresents the diversity of proteins found in the extracellular matrix (ECM) of real tissues. Second, peptides chosen are ubiquitous in ECM and not derived from proteins found in specific tissues, per se. To overcome this critical limitation in hydrogel design and functionality, we developed an approach to incorporate the complex and specific protein signature of bone marrow into a poly (ethylene glycol) (PEG) hydrogel. This bone marrow hydrogel mimics the elasticity of marrow and has 20 bone marrow-specific and cell-instructive peptides. We propose this tissue-centric approach as the next generation of 3D hydrogel design for applications in tissue engineering and beyond

Characterization of <i>pdgf</i>-<i>b</i> in the developing zebrafish embryo.

<p><i>In situ</i> hybridization of embryos at 24, 48, and 72 hpf (A) using an anti-sense probe against <i>pdgf-b</i>. <i>pdgf-b</i> was expressed at all time points in the head and tail vasculature (arrowheads indicate <i>pdgf-b</i> staining in ISVs, red brackets indicate the dorsal aorta, and black brackets indicate the posterior cardinal vein in A). Sagittal (B) and transverse (C) JB-4 sections of 72 hpf embryos analyzed with whole mount <i>in situ</i> hybridization using a probe against <i>pdgf-b. pdgf-b</i> expression was localized near the dorsal aorta, posterior cardinal vein and ISVs (arrow in C indicates <i>pdgf-b</i> staining in dorsal aorta and posterior cardinal vein while the arrowhead indicates staining in the ISV). Neural tube (nt), notochord (nc), dorsal aorta (a), posterior cardinal vein (v).</p

PDGFR inhibition blocked ISV formation and extension.

<p><i>kdrl:GFP</i> embryos were treated at the shield stage (6 hpf) with DMSO alone, 0.1 µM PDGFR tyrosine kinase inhibitor V (inhV), or 0.25 µM inhV. Inhibition of PDGFRs resulted in a decrease in the number of angiogenic sprouts (asterisks in A) in the tail at 24 hpf (B) and a decrease in the number of complete ISVs (arrowheads in A) at 48 hpf (A and C) and 72 hpf (A and D). The morphology for all treatments at 24 hpf was normal (E). Embryos treated with 0.25 µM inhV began to show edema (arrowhead in F) and pooling of blood (arrow in F) at 48 hpf. Angiography at 72 hpf showed a decrease in blood circulation through the ISVs in embryos treated with inhV (G). *** indicates p<0.001. All data represent the mean +/− standard error.</p

Sequence and phylogenetic analysis of PDGFRβ.

<p>Alignment of the zebrafish PDGFRβ tyrosine kinase domain with known PDGFRβ proteins of human, mouse, and <i>Fugu</i> (A). Phylogenetic analysis of PDGFRβ and PDGF-B proteins (B). Arrows in A indicate the beginning and end of the tyrosine kinase domains of PDGFRβ.</p

PDGFRβ2 signaling was mediated by PI3 kinase.

<p><i>kdrl:GFP</i> embryos were injected at the one-cell stage with control morpholino oligonucleotide (MO), <i>pdgfrβ2</i> MO, or <i>pdgfrβ2</i> MO plus <i>pdgfrβ2</i> mRNA, and analyzed for angiogenic sprouting at 24 hpf in the presence of DMSO control or 100 nM of the PI3 kinase inhibitor Wortmannin. *** indicates p<0.001. All data represent the mean +/− standard error.</p

A dominant-negative PDGFRβ blocked ISV angiogenesis in zebrafish embryos.

<p>A heat shock-inducible dominant negative form of PDGFRβ was created by substituting the intracellular kinase domains of PDGFRβ with YFP (A, dnPDGFRβ-YFP). Heat shock at 10 hpf led to dnPDGFRβ-YFP expression within 7 hours after heat shock with no effect on overall morphology (B). Heat shock at 20 hpf and analysis at 48 hpf indicated an increase in ISV defects in heat shocked embryos positive for dnPDGFRβ-YFP versus heat shocked embryos negative for dnPDGFRβ-YFP (C and E, n = 23). Embryos heat shocked at 20 hpf that were positive for dnPDGFRβ-YFP at 48 hpf showed no overall morphology defects as compared to embryos heat shocked at 20 hpf negative for dnPDGFRβ-YFP (D). Arrowheads indicate complete ISVs. Asterisks indicate angiogenic sprouts. ** indicates p<0.01. All data represent the mean +/− standard error.</p