Transgenic Expression of A Venous Malformation Related Mutation, TIE2-R849W, Significantly Induces Multiple Malformations of Zebrafish

Abstract

A TIE2 mutation causing arginine-to-tryptophan substitution at residue 849 (TIE2-R849W) is commonly identified in heredofamilial venous malformation. However, there is no in vivo model to confirm the pathogenic role of TIE2-R849W. Humanized TIE2-R849W plasmid was constructed via PCR-mediated site-directed mutagenesis. After transcription and micro-injection, TIE2-R849W significantly induces multiple malformations in zebrafish: caudal vein plexus (CVP) defect, eye abnormalities, forebrain formation perturbations, and mandibular malformation. Histologically, these phenotypes accompany aphakia, confused retina plexiform layer, abnormal mandibular cartilage, ectopic myelencephalon proliferation and aberrant location of neurogliocytes. According to qRT-PCR, except for high expression of egfl7, the other CVP-related genes cd146, nr2f1a, and s1pr1 are not significantly different from control. TIE2-R849W also induced upregulation of the wnt signaling pathway. Gene array in vitro shows that under the effect of TIE2-R849W, consistent with high expression of pik3 and foxo1, high levels of egfl7, wnt9a, lrp5 and dkk1 were partly confirmed. This in vivo model directly identifies the venous-related pathogenic role of TIE2-R849W. Under up-regulation of TIE2-R849W, egfl7 could be considered a potential reason for venous defects. Moreover, the wnt pathway may perform an important role as a key trigger for head multi-malformations.

Monitoring antiangiogenesis of bevacizumab in zebrafish

“…Adobe Photoshop 7.0 software (Adobe, San Jose, CA, USA) was used to adjust the level, brightness, contrast, hue, and saturation of a series of images to optimize the visual representation. 23 …”

Cerebral cavernous malformations do not fall in the spectrum of PIK3CA-related overgrowth

“…Interestingly, multiple malformations such as head malformations, which fall into the phenotypic spectrum of human PROS, have been observed in zebrafish injected with the most common GOF TIE2 mutant ( R849W ). Vascular defects in caudal vein plexus formation in these zebrafish mirrors human VeMs,121 demonstrating similar vascular phenotypes in humans and zebrafish through VeMs-causative mutant forms of TIE2 . Since tie2 is located upstream of the PI3K/AKT/mTOR pathway, which plays a crucial role in the pathogenesis of VeMs, zebrafish were then injected with a constitutively active form of human myrAkt1, resulting in observations of wide-spread malformations, including skin hyperplasia/hypertrophy, also within the phenotype spectrum of human PROS 122.…”

“…Finally, the disappearance of DLAV formation was also observed in zebrafish injected with GOF MAP3K3 mutation (I441M) mRNA at ~48 hpf,4 suggesting that MAP3K3 is downstream of VEGFR in the PI3K/AKT/mTOR signalling cascade and plays an inhibitory role in embryonic artery development. Phenotypic differences in angiogenic events in zebrafish indicated that CCM signalling complex-mediated signalling does not intersect with the enhancement of RTKs (VEGFR, TIE2)/PI3K/AKT/mTOR signalling cascades 4 121 122…”

Application of zebrafish in the study of craniomaxillofacial developmental anomalies

“…Wnt plays a critical role in zebrafish craniofacial patterning, as well as tooth and bone development. Abnormal expression of related molecules in the signaling pathways may lead to craniofacial malformations, including significant eye defects and mandibular deformities (Du, Ma, Zhang, Zheng, & Wang, 2018; Nakamura et al, 2007; van de Water et al, 2001). Inhibition of wisp3 (WNT‐1 inducible signaling pathway protein 3) protein expression affects the size and shape of the pharyngeal cartilage of zebrafish (Nakamura et al, 2007).…”

“…Wnt plays a critical role in zebrafish craniofacial patterning, as well as tooth and bone development. Abnormal expression of related molecules in the signaling pathways may lead to craniofacial malformations, including significant eye defects and mandibular deformities (Du, Ma, Zhang, Zheng, & Wang, 2018;Nakamura et al, 2007;van de Water et al, 2001).…”