Regulation of Vegfr3 Expression: CRISPR/Cas Genetic Screenings and New Cellular and Mouse Models

Abstract

Vegfr3 (Vascular Endothelial Growth Factor Receptor 3) is a member of the vascular endothelial tyrosine kinase transmembrane receptor family and a key driver of lymphatic vessel development and lymphangiogenesis. The expression of this receptor is tightly regulated both in the blood and lymphatic endothelium. However, despite its relevance, the molecular mechanisms that control Vegfr3 expression in different contexts of physiology and disease and the phenotypic consequences of its deregulation have not been sufficiently explored. Using a forward genetic approach combined with the power of the CRISPR/Cas gene editing system, we have performed genome wide genetic screenings searching for genes potentially involved in Vegfr3 transcription activation. First, we have generated two Vegfr3 reporter cell lines, one in ESCs (Vegfr3EGFPluc) and the other in imLECs (Vegfr3Kat) in which an EGFP-luciferase fusion protein or the TurboFP365 (Katushka) fluorescent protein, respectively, are expressed under the endogenous transcriptional control of Vegfr3. Using the genome-wide transcription activation CRISPR library (CRISPRa SAM-3 pooled library) and sorting of cells with EGFP or Katushka fluorescene above the one of the control cells, we have identified a new gene potentially related with Vegfr3 activation in Vegfr3EGFPluc reporter ESCs, Plpp2, and two different genes using the Vegfr3Kat reporter LECs: Wnt5a and ������3-Galnt1. The potential implication of each one of these genes in Vegfr3 transcription activation is discussed in this work, however, a more extensive analysis in vitro and in vivo is still required to elucidate the molecular mechanisms that lead to Vegfr3 activation by these genes and its physiological relevance. In parallel, we have generated a new reporter BAC-transgenic mouse that allows imaging of lymphatic vessels and lymphangiogenesis by monitoring Katushka expression and fluorescence. The far-red excitation and emission spectra of Katushka, with peaks at 588 and 635 nm, respectively, are important advantages for in vivo tissue detection of this reporter. We have verified that in the Tg.Vegfr3Kat reporter mouse, the expression of Katushka recapitulates Vegfr3 expression in vivo in all developmental stages and colocalizes with lymphatic markers but not with blood endothelial markers. Moreover, we show that this model can be used to trace and to quantify in vivo lymphangiogenesis associated to pathological processes such as inflammation in the adult. Finally, we have developed a unique mouse model that allows for the spatial and temporal upregulation of Vegfr3 expression in vivo. This mouse model combines the tamoxifen inducible Vegfr3CreERT2 and the Rosa26LSL-rtTA-EGFP knockin alleles, already established, with a tetO-Vegfr3 transgene targeted to the Col1A1 locus. In this model, administration of both tamoxifen and doxicycline leads to reversible overexpression of Vegfr3 driven by the Vegfr3CreERT2 allele, and therefore exclusively in those cells that physiologically express Vegfr3. The initial characterization of this model shows that Vegfr3 overexpression during postnatal dermal lymphatic development causes lymphatic hypoplasia by inducing excessive sprouting and vessel shortening that interferes with normal network formation. Mechanistically, we propose that excessive ERK activation mediated by Vegfr3 sustained overexpression, alters the balance between ERK and Notch signaling in LECs inhibiting proliferaton and inducing an hypersprouting phenotype that interferes with normal lymphangiogenesis. In summary, this PhD work provides new genetic tools to better understanding lymphangiogenesis in development and disease, and contributes to the discovery of new molecular mechanisms that control Vegfr3 expression and function