Here, we document a collection of ∼7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstrate reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates.DOI: http://dx.doi.org/10.7554/eLife.05338.001

Rationale: We previously identified somatic activating mutations in the KRAS gene in the endothelium of the majority of human sporadic brain arteriovenous malformations (bAVMs); a disorder characterized by direct connections between arteries and veins. However, whether this genetic abnormality alone is sufficient for lesion formation, as well as how active KRAS signaling contributes to AVM formation, remains unknown. Objective: To establish the first in vivo models of somatic KRAS gain of function in the endothelium in both mice and zebrafish in order to directly observe the phenotypic consequences of constitutive KRAS activity at a cellular level in vivo, and to test potential therapeutic interventions for AVMs. Methods and Results: Using both postnatal and adult mice, as well as embryonic zebrafish, we demonstrate that endothelial-specific gain of function mutations in Kras (G12D or G12V) are sufficient to induce bAVMs. Active KRAS signaling leads to altered endothelial cell morphogenesis and increased cell size, ectopic sprouting, expanded vessel lumen diameter, and direct connections between arteries and veins. Furthermore, we show that these lesions are not associated with altered endothelial growth dynamics or a lack of proper arteriovenous identity, but instead appear to feature exuberant angiogenic signaling. Finally, we demonstrate that KRAS-dependent AVMs in zebrafish are refractory to inhibition of the downstream effector PI3K, but instead require active MEK signaling. Conclusions: We demonstrate that active KRAS expression in the endothelium is sufficient for bAVM formation, even in the setting of uninjured adult vasculature. Furthermore, the finding that KRAS-dependent lesions are reversible in zebrafish suggests that MEK inhibition may represent a promising therapeutic treatment for AVM patients.

The transcriptional pathways activated downstream of vascular endothelial growth factor (VEGF) signaling during angiogenesis remain incompletely characterized. By assessing the signals responsible for induction of the Notch ligand delta-like 4 (DLL4) in endothelial cells, we find that activation of the MAPK/ERK pathway mirrors the rapid and dynamic induction of DLL4 transcription and that this pathway is required for DLL4 expression. Furthermore, VEGF/ERK signaling induces phosphorylation and activation of the ETS transcription factor ERG, a prerequisite for DLL4 induction. Transcription of DLL4 coincides with dynamic ERG-dependent recruitment of the transcriptional co-activator p300. Genome-wide gene expression profiling identified a network of VEGF-responsive and ERG-dependent genes, and ERG chromatin immunoprecipitation (ChIP)-seq revealed the presence of conserved ERG-bound putative enhancer elements near these target genes. Functional experiments performed in vitro and in vivo confirm that this network of genes requires ERK, ERG and p300 activity. Finally, genome-editing and transgenic approaches demonstrate that a highly conserved ERG-bound enhancer located upstream of HLX (which encodes a transcription factor implicated in sprouting angiogenesis) is required for its VEGFmediated induction. Collectively, these findings elucidate a novel transcriptional pathway contributing to VEGF-dependent angiogenesis.

Background Glioblastoma is the most common and aggressive type of primary brain tumor, as most patients succumb to the disease less than two years after diagnosis. Critically, studies demonstrate that glioma recruits surrounding blood vessels, while some work suggests that tumor stem cells themselves directly differentiate into endothelial cells, yet the molecular and cellular dynamics of the endothelium in glioma are poorly characterized. The goal of this study was to establish molecular and morphological benchmarks for tumor associated vessels (TAVs) and tumor derived endothelial cells (TDECs) during GBM progression. Methods Using In-Utero Electroporation and CRISPR/Cas9 genome engineering to generate a native, immunocompetent mouse model of glioma, we characterized vascular-tumor dynamics in three dimensions during tumor progression. We employed bulk and single-cell RNA-Sequencing to elucidate the relationship between TAV and TDECs. We confirmed our findings in a patient derived orthotopic xenograft (PDOX) model. Results Using a mouse model of glioma, we identified progressive alteration of vessel function and morphogenesis over time. We also showed in our mouse model that TDECs are a rare subpopulation that contributes to vessels within the tumor, albeit to a limited degree. Furthermore, transcriptional profiling demonstrates that both TAVs and TDECs are molecularly distinct, and both populations feature extensive molecular heterogeneity. Finally, the distinct molecular signatures of these heterogenous populations are also present in human glioma. Conclusions Our findings show extensive endothelial heterogeneity within the tumor and tumor microenvironment, and provide insights into the diverse cellular and molecular mechanisms that drive glioma vascularization and angiogenesis during tumorigenesis.