Comparative expression profiling of wild type Drosophila Malpighian tubules and von Hippel-Lindau haploinsufficient mutant

The von-Hippel Lindau (VHL) disease is a hereditary genetic disorder that predisposes to the onset of several highly vascularized benign and malignant tumors, developing with elevate frequency in the central nervous system and kidneys. The most-aggressive VHL tumor is ccRCC, the clear-cell renal cell carcinoma, affecting the kidney. VHL disease etiology can be attributed to the inheritance of a VHL loss-of-function allele, typically a deletion (Gnarra et al., 1994; Herman et al., 1994); this facilitates the somatic inactivation of the other allele (through amorphic mutations or gene silencing through promoter methylation), leading to the onset of the tumorous phenotype (Latif et al., 1993). This reveals the haploinsufficient behavior of the VHL gene. The high vascularization of VHL tumors can be explained considering that human VHL protein is the substrate-binding subunit of an E3 ubiquitin ligase (Lonergan et al., 1998; Iwai et al., 1999; Kamura et al., 1999) involved in the poly-ubiquitination of HIF-1α transcription factor. This post-translational modification leads HIF-1α to proteosomal degradation (Maxwell et al., 1999). Loss of VHL function causes the stabilization of HIF-1α, triggering cellular response and adaptation to hypoxic conditions (expression of genes involved in glycolysis, angiogenesis and erythropoiesis) (Bader and Hsu, 2012). While this represents the canonical function of VHL, other HIF-1α-independent function of VHL have been identified, thanks to the contribution of model organisms (Hsu, 2012). Indeed, VHL gene function is conserved and also Drosophila has a VHL homolog, the dVHL gene (Adryan et al., 2000; Aso et al., 2000). dVHL is involved in the development of Drosophila vascular system (Adryan et al., 2000; Hsouna et al., 2010) and in morphogenesis of follicular epithelium of the egg chamber (Duchi et al., 2010). Interestingly, some VHL functions are mediated by Awd, an endocytic mediator whose human orthologs are NME1/2 metastasis suppressors (Rosengard et al., 1989). Awd is broadly required during Drosophila development since it is involved in epithelial morphogenesis (Nallamothu et al., 2008; Woolworth et al., 2009; Ignesti et al., 2014) and required for maintaining genomic stability (Romani et al., 2017). Moreover, Awd is also present into the extracellular fluids of Drosophila larvae (Romani et al., 2016, 2018). In Drosophila, two pairs of monolayered epithelial Malpighian tubules, each composed of 100-150 cells, absolve to osmoregulation and excretion functions (Denholm and Skaer, 2009). Transcriptomic analysis of Malpighian tubules revealed that among genes that are here enriched there are homologs of human genes implicated into renal pathologies (Wang et al., 2004). This justifies the use of Drosophila Malpighian tubules as model system to gain insights into pathophysiology of human kidneys (Dow and Romero, 2010; Miller et al., 2013). The dVHL1.1 allele is a loss of function mutation of the dVHL locus (Duchi et al., 2010; Hsouna et al., 2010). dVHL1.1/+ flies mimic the genetic condition of VHL patients. We carried out a genome-wide gene expression profiling of whole Malpighian tubules dissected from Drosophila females both heterozygous for the dVHL1.1 mutation and with two wild type copies of the dVHL gene. The comparison of differentially expressed genes in the two genetic backgrounds potentially allows to identify genes that are sensible to dVHL functional copy number. Quality control assessments of the data were performed and results obtained from the differential expression analysis were confirmed by qRT-PCR. With this approach we aimed to provide a well-controlled dataset for a better understanding of the VHL disease. Indeed, even if further molecular and functional characterization are needed, human homologs of the differentially expressed genes, if existing, could have a role in the somatic inactivation of the wild type copy of VHL and/or into the very first phase of cancer onset.