The RSPO–LGR4/5–ZNRF3/RNF43 module controls liver zonation and size

LGR4/5 receptors and their cognate RSPO ligands potentiate Wnt/β-catenin signalling and promote proliferation and tissue homeostasis in epithelial stem cell compartments. In the liver, metabolic zonation requires a Wnt/β-catenin signalling gradient, but the instructive mechanism controlling its spatiotemporal regulation is not known. We have now identified the RSPO-LGR4/5-ZNRF3/RNF43 module as a master regulator of Wnt/β-catenin-mediated metabolic liver zonation. Liver-specific LGR4/5 loss of function (LOF) or RSPO blockade disrupted hepatic Wnt/β-catenin signalling and zonation. Conversely, pathway activation in ZNRF3/RNF43 LOF mice or with recombinant RSPO1 protein expanded the hepatic Wnt/β-catenin signalling gradient in a reversible and LGR4/5-dependent manner. Recombinant RSPO1 protein increased liver size and improved liver regeneration, whereas LGR4/5 LOF caused the opposite effects, resulting in hypoplastic livers. Furthermore, we show that LGR4(+) hepatocytes throughout the lobule contribute to liver homeostasis without zonal dominance. Taken together, our results indicate that the RSPO-LGR4/5-ZNRF3/RNF43 module controls metabolic liver zonation and is a hepatic growth/size rheostat during development, homeostasis and regeneration

Dual ISH-IHC

Interview with Kristie Wetzel Can you tell us about your work and why you developed a dual protocol? I work as an investigator in the developmental and molecular pathways lab at Novartis. Our group provides immunohistochemistry (IHC) and in situ hybridization (ISH) services not just to our own lab but also to other groups working in diverse disease areas. This means that we get many different sample types depending on the focus of the particular study; it might be tissue from human, mouse, rat... We developed a dual ISH-IHC protocol because fundamentally, we want to be able to see where the mRNA is accumulating. We want to identify which tissue and which cell type is producing it. That’s why RNAscope® ISH is really important in our work and that’s why we are using it. There are also certain scenarios where our targets don’t have very good antibodies, or using antibodies is difficult such as when profiling a secreted protein. What we get when we combine ISH and IHC is the ability to identify both the particular cell type by IHC and the mRNA profile by ISH. This information is of great interest to us. How was your dual ISH-IHC protocol set up? Were there any challenges? We tested a protocol where we first perfom the chromogenic RNAscope® assay (RNAscope®VS Red) followed by immunofluorescence staining. Our target was a lysozyme protein. Both of the reactions were automated on the Ventana Discovery system. To set this up, we first confirmed that the RNAscope® assay was working on our tissue samples and then we optimized our IHC protocols. We decided to do ISH followed by IHC because it was important to digest the tissue for our lysozyme antibody and the RNAscope® protocol has an antigen retrieval step followed by a protease treatment step. It took us a number of weeks and a number of iterations before we really got the protocol optimized but once we did, the resulting staining was really beautiful and provide a interesting data combination to see. Our major challenge was optimizing the IHC protocol as protein stability is affected by the RNAscope® pretreatments. It was important to make sure that the IHC was optimized independently of the RNAscope® ISH first before combining it in the dual reaction. Do you have any recommendations you’d like to share? I definitely recommend working out the individual protocols separately before combining them. You have to have a good signal individually first, but you also need to realize that even if they’re both working well individually, you’re still going to need to tweak and optimize once you combine them. The IHC protocol is definitely going to need optimizing due to the RNAscope® pretreatment - it’s also important to determine the optimal RNAscope® pretreatment protocol for each tissue type you have. Once this is established, it’s not going to change. Another thing I would recommend is following the RNAscope® protocol to the letter! It works very well if you do this. Figure 2. Dual ISH-IHC staining using chromogen labeled enzymes to identify mRNA expression in the stem cells, and protein expression in the Paneth cells in the mouse gut. Provided by Kristie Wetzel

TeleFE: A New Tool for the Tele-Assessment of Executive Functions in Children

In recent decades, the utility of cognitive tele-assessment has increasingly been highlighted, both in adults and in children. The present study aimed to present TeleFE, a new tool for the tele-assessment of EF in children aged 6–13. TeleFE consists of a web platform including four tasks based on robust neuropsychological paradigms to evaluate inhibition, interference suppression, working memory, cognitive flexibility, and planning. It also includes questionnaires on EF for teachers and parents, to obtain information on the everyday functioning of the children. As TeleFE allows the assessment of EF both remotely and in-person, a comparison of the two modalities was conducted by administering TeleFE to 1288 Italian primary school children. A series of ANOVA was conducted, showing no significant effect of assessment modality (p > 0.05 for all the measures). In addition, significant differences by class emerged for all the measures (p < 0.001 for all the measures except p = 0.008 for planning). Finally, a significant sex effect emerged for inhibition (p < 0.001) and for the reaction times in both interference control (p = 0.013) and cognitive flexibility (p < 0.001), with boys showing a lower inhibition and faster reaction times. The implications of these results along with the indications for the choice of remote assessment are discussed

Distinct altered patterns of p27KIP1 gene expression in benign prostatic hyperplasia and prostatic carcinoma

Background: The p27(KIP1) gene, whose protein product is a negative regulator of the cell cycle, is a potential tumor suppressor gene; however, no tumor-specific mutations of this gene have been found in humans, This study was undertaken to identify and to assess potential alterations of p27(KIP1) gene expression in patients with benign prostatic hyperplasia (BPH) and patients with prostate cancer, Methods: We analyzed 130 prostate carcinomas from primary and metastatic sites, as well as prostate samples from normal subjects and from patients with BPH. Immunohistochemistry and in situ hybridization were used to determine the levels of expression and the microanatomical localization of p27 protein and messenger RNA (mRNA), respectively. Immunoblotting and immunodepletion assays mere performed on a subset of the prostate tumors. associations between alterations in p27(KIP1) expression and clinicopathologic variables were evaluated with a nonparametric test. The Kaplan-Meier method and the logrank test were used to compare disease-relapse-free survival. Prostate tissues of p27(KIP1) null (i.e., knock-out) and wild-type mice were also evaluated, Results: Normal human prostate tissue exhibited abundant amounts of p27 protein and high levels of p27(KIP1) mRNA in both epithelial cells and stromal cells. However, p27 protein and p27(KIP1) mRNA were almost undetectable in epithelial cells and stromal cells of BPH lesions, Furthermore, p27(KIP1) null mice developed enlarged (hyperplastic) prostate glands. In contrast to BPH, prostate carcinomas were found to contain abundant p27(KIP1) mRNA but either high or low to undetectable levels of p27 protein, Primary prostate carcinomas expressing lower levels of p27 protein appeared to be biologically more aggressive (two-sided P = .019 [Cox regression analysis]). Conclusions/Implications: On the basis of these results, we infer that loss of p27(KIP1) expression in the human prostate may be causally linked to BPH and that BPH is not a precursor to prostate cancer

α-Klotho regulates age-associated vascular mineralization and lifespan in zebrafish

α-Klotho is an anti-aging hormone regulating mineral homeostasis together with Fibroblast Growth Factor-23 (FGF23) in mammals. Cellular and molecular mechanisms through which these signals regulate age-related conditions, such as vascular mineralization remain intriguing. It is not known if aging mechanisms controlled by α-Klotho/FGF23 signaling are evolutionarily conserved. To this end, we generated knockouts carrying homozygous mutations in α-klotho and fgf23 in zebrafish. Both knockouts display an adult-onset decline in body condition and behavior, and an early-onset morbidity. Bulbus arteriosus, the outflow tract of zebrafish heart shows extensive mineralization in the mutants. RNA-seq analysis of kidney, heart and gills shows an ectopic activation of bone-remodeling pathway, extracellular matrix remodeling factors osteoclast differentiation and inflammation in the vasculature. Taken together, these findings indicate a conserved role of α-Klotho/FGF23 signaling in regulating vertebrate life span and identify molecular mechanisms underlying vascular mineralization