Angiogenesis is an essential requirement for embryonic development and adult homeostasis. Its deregulation is a key feature of numerous pathologies and many studies have shown that members of the transforming growth factor beta (TGF-β) family of proteins play important roles in angiogenesis during development and disease. Betaglycan (BG), also known as TGF-β receptor type III, is a TGF-β coreceptor essential for mice embryonic development but its role in angiogenesis has not been described. We have cloned the cDNA encoding zebrafish BG, a TGF-β-binding membrane proteoglycan that showed a dynamic expression pattern in zebrafish embryos, including the notochord and cells adjacent to developing vessels. Injection of antisense morpholinos decreased BG protein levels and morphant embryos exhibited impaired angiogenesis that was rescued by coinjection with rat BG mRNA. In vivo time-lapse microscopy revealed that BG deficiency differentially affected arterial and venous angiogenesis: morphants showed impaired pathfinding of intersegmental vessels migrating from dorsal aorta, while endothelial cells originating from the caudal vein displayed sprouting and migration defects. Our results reveal a new role for BG during embryonic angiogenesis in zebrafish, which has not been described in mammals and pose interesting questions about the molecular machinery regulating angiogenesis in different vertebrates. genesis 53:583-603, 2015. © 2015 Wiley Periodicals, Inc.

Background The Transforming Growth Factor β (TGFβ) family is a group of related proteins that signal through a type I and type II receptors. Betaglycan, also known as the type III receptor (Tgfbr3), is a coreceptor for various ligands of the TGFβ family that participates in heart, liver and kidney development as revealed by the tgfbr3‐null mouse, as well as in angiogenesis as revealed by Tgfbr3 downregulation in morphant zebrafish. Results Here, we present CRISPR/Cas9‐derived zebrafish Tgfbr3‐null mutants, which exhibited unaltered embryonic angiogenesis and developed into fertile adults. One reproducible phenotype displayed by these Tgfbr3‐null mutants is delayed chordacentra mineralization, which nonetheless does not result in vertebral abnormalities in the adult fishes. We also report that the canonical TGFβ signaling pathway is needed for proper chordacentra mineralization and that Tgfbr3 absence decreases this signal in the notochordal cells responsible for this process. Conclusion Betaglycan's “ligand presentation” function contributes to the optimal TGFβ signaling required for zebrafish chordacentra mineralization.

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish embryos has been implemented. This technique takes advantage of the selectivity and sensitivity conferred by affinity labeling of a given receptor by its ligand with the specificity of the immunoprecipitation. We have used AFLIP to detect the type III TGF-β receptor (TGFBR3), also know as betaglycan, during early zebrafish development. AFLIP was instrumental in validating the efficacy of a TGFBR3 morphant zebrafish phenotype. In the first step, embryo protein extracts are prepared and used to generate I-TGF-β2-TGFBR3 complexes that are purified by immunoprecipitation. Later, these complexes are covalently cross-linked and revealed using SDS-PAGE separation and autoradiography detection. This technique requires the availability of a labeled ligand for, and a specific antibody against, the receptor to be detected, and shall be easily adapted to identify any growth factor or cytokine receptor that meets these requirements.

Neural progenitor cells (NPCs) of the subventricular zone proliferate in response to ischemic stroke in the adult mouse brain. Newly generated cells have been considered to influence recovery following a stroke. However, the mechanism underlying such protection is a matter of active study since it has been thought that proliferating NPCs mediate their protective effects by secreting soluble factors that promote recovery rather than neuronal replacement in the ischemic penumbra. We tested the hypothesis that this mechanism is mediated by the secretion of multimolecular complexes in extracellular vesicles (EVs). We found that the molecular influence of oxygen and glucose-deprived (OGD) NPCs-derived EVs is very limited in improving overt neurological alterations caused by stroke compared to our recently reported astrocyte-derived EVs. However, when we inhibited the ischemia-triggered proliferation of NPCs with the chronic administration of the DNA synthesis inhibitor Ara-C, the effect of NPC-derived EVs became evident, suggesting that the endogenous protection exerted by the proliferation of NPC is mainly carried out through a mechanism that involves the intercellular communication mediated by EVs. We analyzed the proteomic content of NPC-derived EVs cargo with label-free relative abundance mass spectrometry and identified several molecular mediators of neuronal recovery within these vesicles. Our findings indicate that NPC-derived EVs are protective against the ischemic cascade activated by stroke and, thus, hold significant therapeutic potential.

Using intracellular-controlled photochemistry to track dynamic organelle processes is gaining attention due to its broad applications. However, most of the employed molecular probes usually require toxic photosensitizers and complex bioanalytical protocols. Here, the synthesis and performance of two new subcellular probes (MitoT1 and MitoT2) are described. The probes undergo photooxidation in the damaged tissue of zebrafish, a model system for tissue regeneration studies. Using high-resolution confocal microscopy and fluorescence spectroscopy, we combine the mentioned photoinduced interconversion at the homeostatic membrane viscosity to track singlet oxygen activity selectively. The continuous and real-time biosensing method reported here provides a new approach for simultaneously detecting endogenous singlet oxygen and viscosity status.