Hypoxia stimulates tumor angiogenesis by inducing the expression of angiogenic molecules. The negative regulators of this process, however, are not well understood. Here, we report that hypoxia induced the expression of insulin‐like growth factor binding protein‐6 (IGFBP‐6), a tumor repressor, in human and rodent vascular endothelial cells (VECs) via a hypoxia‐inducible factor (HIF)‐mediated mechanism. Addition of human IGFBP‐6 to cultured human VECs inhibited angiogenesis in vitro. An IGFBP‐6 mutant with at least 10,000‐fold lower binding affinity for IGFs was an equally potent inhibitor of angiogenesis, suggesting that this action of IGFBP‐6 is IGF‐independent. The functional relationship between IGFBP‐6 and vascular endothelial growth factor (VEGF), a major hypoxia‐inducible angiogenic molecule, was examined. While VEGF alone increased angiogenesis in vitro, co‐incubation with IGFBP‐6 abolished VEGF‐stimulated angiogenesis. The in vivo role of IGFBP‐6 in angiogenesis was tested in flk1:GFP zebrafish embryos, which exhibit green fluorescence protein in developing vascular endothelium, permitting visualization of developing blood vessels. Injection of human IGFBP‐6 mRNA reduced the number of embryonic inter‐segmental blood vessels by ∼40%. This anti‐angiogenic activity is conserved in zebrafish because expression of zebrafish IGFBP‐6b had similar effects. To determine the anti‐angiogenic effect of IGFBP‐6 in a tumor model, human Rh30 rhabdomyosarcoma cells stably transfected with IGFBP‐6 were inoculated into athymic BALB/c nude mice. Vessel density was 52% lower in IGFBP‐6‐transfected xenografts than in vector control xenografts. These results suggest that the expression of IGFBP‐6 in VECs is up‐regulated by hypoxia and IGFBP‐6 inhibits angiogenesis in vitro and in vivo.

Neuronal survival and growth in the embryo is controlled partly by trophic factors. For most trophic factors (such as Insulin like growth factor-1, IGF1), the ability to regulate cell survival has been attributed to the PI3K/Akt kinase cascade. This study presents data illustrating the role of PI3K/Akt in attainment of normal brain size during zebrafish embryogenesis. Blocking PI3K with inhibitor LY294002 caused a significant reduction in brain size (in addition to global growth retardation) during zebrafish embryogenesis. This PI3 Kinase inhibition-induced brain size decrease was recovered by the overexpression of Myr-Akt, a constitutive form of Akt. Further analysis reveals that expressing exogenous Myr-Akt significantly augmented brain size. Whole mount in situ hybridization analysis of several marker genes showed that Myr-Akt overexpression did not alter brain patterning. Furthermore, the expression of Myr-Akt was found to protect neuronal cells from apoptosis induced by heat shock and UV light, suggesting that inhibition of neuronal cell death may be part of the underlying cause of the increased brain size. These data provide a foundation for addressing the role of PI3K/Akt in brain growth during zebrafish embryogenesis.

Craniofacial malformations are common congenital birth defects and usually caused by abnormal development of the cranial neural crest cells (CNCCs). Some nucleolar ribosome biogenesis factors are implicated in neural crest disorders also known as neurocristopathies. However, the underlying mechanisms linking ribosome biogenesis and NCC development remains to be elucidated. Here we report a novel zebrafish model with a CRISPR/Cas9 generated esf1 mutation, which exhibits severe NCC‐derived pharyngeal cartilage loss and defects in the eyes, brain and heart. The expression of several typical NCC markers, including sox10, dlx2a, nrp2b, crestin, vgll2a and sox9a, were reduced in the head of the esf1 mutants, which indicates that esf1 plays a role in the development of zebrafish NCCs. We demonstrate that, similar to the yeast, loss of esf1 in zebrafish leads to defects in 18S rRNA biogenesis and ribosome biogenesis. We also show that strong upregulation of p53 signaling as well as apoptosis, and poor proliferation in mutants. Inactivation of p53 rescues the early tissue defects and pharyngeal cartilage loss observed in esf1 mutants, indicating that increased cell death and pharyngeal cartilage defects observed in esf1 mutants are mediated via up‐regulated p53 signaling pathways. Based on transplantation analysis, we found that esf1 functions in NCC in a cell autonomous fashion. Together, our results suggest that esf1 is required for NCC development and pharyngeal cartilage formation. These studies provide a potential model for investigating the relationship between ribosome biogenesis defects and craniofacial neurocristopathies.

Hypoxia-inducible factor-1 (HIF-1) is a critical transcription factor that regulates the expression of genes involved in cellular adaptation to low oxygen levels. Aberrant regulation of the HIF-1 signaling pathway is linked to various human diseases. Previous studies have established that HIF-1α is rapidly degraded in a von Hippel-Lindau protein (pVHL)-dependent manner under normoxic conditions. In this study, we find that pVHL binding protein 1 (VBP1) is a negative regulator of HIF-1α but not HIF-2α using zebrafish as an in vivo model and in vitro cell culture models. Deletion of vbp1 in zebrafish caused Hif-1α accumulation and upregulation of Hif target genes. Moreover, vbp1 was involved in the induction of hematopoietic stem cells (HSCs) under hypoxic conditions. However, VBP1 interacted with and promoted the degradation of HIF-1α in a pVHL-independent manner. Mechanistically, we identify the ubiquitin ligase CHIP and HSP70 as new VBP1 binding partners and demonstrate that VBP1 negatively regulated CHIP and facilitated CHIP-mediated degradation of HIF-1α. In patients with clear cell renal cell carcinoma (ccRCC), lower VBP1 expression was associated with worse survival outcomes. In conclusion, our results link VBP1 with CHIP stability and provide insights into underlying molecular mechanisms of HIF-1α-driven pathological processes.