F. repair through paracrine control of liver cell function and regulation of appropriate collagen deposition. This article has an associated First Person interview with the first author of the paper. expression increased in the absence of hedgehog signaling, and VLK was reported to synergize with the hedgehog effector glioma-associated oncogene 3 (Gli3) to coordinate the kinetics of chondrocyte differentiation (Probst et al., 2013). Conversely, VLK negatively regulated hedgehog signaling by interacting with the extracellular domain of Smoothened, thereby leading to its lysosomal degradation (Kim et al., 2020). Furthermore, VLK-dependent phosphorylation of repulsive guidance molecule B was shown to modulate Wnt3a activity, which is crucial for the formation of neuronal circuitries (Harada et al., 2019). Despite its critical role in development and the identification of a broad range of extracellular substrates is expressed in hepatocytes and in non-parenchymal liver cells of adult mice. Histological analyses and immunofluorescence staining of liver tissue from mice at embryonic day 18 (E18), postnatal day 2 (P2), postnatal day 5 (P5) and at 6?weeks of age revealed predominant Retaspimycin VLK expression in a subset of clustered cells at E18 and P2. Their distribution and nuclear morphology, as well as staining of serial sections with antibodies against VLK and the hepatocyte marker albumin (ALB), suggest that most of the VLK-positive cells are hepatocytes (Fig.?1A,B; Fig.?S1A). In adult mice, VLK staining was most pronounced in cholangiocytes, the epithelial cells lining the bile ducts, as confirmed by co-localization with the Retaspimycin cholangiocyte markers cytokeratin 19 (CK19, also known as KRT19) (Fig.?1B) and osteopontin (OPN, also known as SPP1) (Fig.?S1B). The predominant expression of VLK in cholangiocytes of adult liver was confirmed with two additional VLK peptide antibodies (i.e. VLK 289 and VLK 404) (Fig.?S1C). Their specificity was demonstrated by immunofluorescence staining of human embryonic kidney 293 cells expressing simian vacuolating Retaspimycin virus 40 (SV40) large T antigen (HEK 293T cells) transfected with a VLK expression plasmid (Fig.?S1D). Open in a separate window Fig. 1. VLK is expressed in a stage-specific manner in hepatocytes and cholangiocytes of mouse liver. (A,B) Representative photomicrographs of mouse liver sections at the indicated developmental stages stained with Hematoxylin and Retaspimycin Eosin (H&E) (A) or analyzed by immunofluorescence for VLK (red) and CK19 (green) (B). Arrowheads in B indicate cell clusters of VLK-positive cells. Nuclei were counterstained with Hoechst 33342 (blue). PV, portal vein; BD, bile duct. mRNA in mouse liver at the indicated ages, visualized by RNA-Scope hybridization (red). (red) and mRNAs were used as positive Retaspimycin and negative controls, respectively. Nuclei were counterstained with Hoechst. expression relative to expression by qPCR. hybridization confirmed the increase in expression in cholangiocytes of adult mice compared with P1 mice, and the concomitant downregulation in hepatocytes during postnatal liver development, although mRNA was still detectable in this cell type in adult mice (Fig.?1C). The specificity of the probe was validated in HEK 293T cells overexpressing recombinant Itga11 VLK (Fig.?S1E). expression levels in whole liver increased in 6-week-old mice compared with postnatal mice as determined by quantitative real-time PCR (qPCR) (Fig.?1D). VLK modifies the mitogenic properties of the hepatocyte and cholangiocyte secretome Hepatocytes and cholangiocytes have a high secretory activity, therefore, we speculated that VLK is secreted from these cells and modifies their secretome, which may affect other liver cells in a paracrine manner. To test this possibility, we generated hepatocyte-specific knockout mice by breeding mice with floxed alleles (Probst et al., 2013) with mice expressing Cre recombinase under control of the albumin promoter (Alb-Cre mice) (Postic et al., 1999). The latter allow expression of transgenes in hepatocytes with an onset during late prenatal development, and thus during the time when VLK expression is high in this cell type (Postic et al., 1999; Weisend et al., 2009). The mice with hepatocyte-specific loss of VLK [Alb-VLK; designated knockout (KO) mice in this article] and respective control (Ctrl) mice with floxed alleles, but lacking Cre, were first used to establish an model to study autocrine and paracrine effects of the hepatocyte-derived secretome on fibroblast behavior. Consistent with the data, primary hepatocytes from adult Ctrl mice only weakly expressed VLK, which was only detectable at the RNA level (Fig.?2A). However, their reprogramming into expandable bipotent cells, which express the progenitor marker sex-determining region Y (SRY)-related high mobility group (HMG)-box gene 9 (SOX9) (Fig.?2A,B), strongly promoted.