Metformin Inhibits Cyst Formation in a Zebrafish Model of Polycystin-2 Deficiency.

Autosomal dominant polycystic kidney disease (ADPKD) is a common kidney disease caused by mutations in PKD1 or PKD2. Metformin reduces cyst growth in mouse models of PKD1. However, metformin has not been studied in animal models of PKD2, and the cellular mechanism underlying its effectiveness is not entirely clear. This study investigated the effects of metformin on cyst formation in a zebrafish model of polycystin-2 deficiency resulting from morpholino knockdown of pkd2. We added metformin (2.5 to 20 mM) to the embryo media between 4 and 48 hours post fertilisation and observed pronephric cyst formation by using the wt1b promoter-driven GFP signal in Tg(wt1b:GFP) pkd2 morphants. Metformin inhibited pronephric cyst formation by 42-61% compared with the untreated controls. Metformin also reduced the number of proliferating cells in the pronephric ducts, the degree of dorsal body curvature, and the infiltration of leukocytes surrounding the pronephros. Moreover, metformin treatment increased the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and enhanced autophagy in the pronephros. Our data suggest that metformin reduces cyst formation through activation of the AMPK pathway and modulation of defective cellular events such as proliferation and autophagy. These results also imply that metformin could have therapeutic potential for ADPKD treatment.

Leptospiral outer membrane protein LipL32 induces inflammation and kidney injury in zebrafish larvae.

Leptospirosis is an often overlooked cause of acute kidney injury that can lead to multiple organ failure and even death. The principle protein that conserved in many pathogenic leptospires is the outer membrane protein LipL32. However, the role of LipL32 in the pathogenesis of renal injury in leptospirosis is not entirely clear. Here we studied the effects of LipL32 on the developing kidney in zebrafish larvae. Incubation of zebrafish larvae with Leptospira santarosai serovar Shermani induced acute tubular injury predominantly in the proximal pronephric ducts. Furthermore, microinjection of lipl32 mRNA or recombinant LipL32 protein into zebrafish larvae increased macrophage accumulation and disrupted the basolateral location of NA-K-ATPase in pronephric ducts. These changes led to substantial impairment of the pronephric kidney structure. We further demonstrated that morpholino knockdown of tlr2, but not tlr4, reduced the LipL32-induced leukocyte infiltration and kidney injury. These data demonstrate that LipL32 contributes to the renal pathology in leptospirosis and gives some clues to the potential virulence of LipL32. Our results support the use of zebrafish as a model organism for studying the disease mechanism of leptospiral infection. This model might permit the future exploration of the virulence and molecular pathways of different leptospiral outer membrane proteins.

The transcription factor hairy/E(spl)-related 2 induces proliferation of neural progenitors and regulates neurogenesis and gliogenesis.

The study of molecular regulation in neural development provides information to understand how diverse neural cells are generated. It also helps to establish therapeutic strategies for the treatment of neural degenerative disorders and brain tumors. The Hairy/E(spl) family members are potential targets of Notch signaling, which is fundamental to neural cell maintenance, cell fate decisions, and compartment boundary formation. In this study, we isolated a zebrafish homolog of Hairy/E(spl), her2, and showed that this gene is expressed in neural progenitor cells and in the developing nervous system. The expression of her2 required Notch activation, as revealed by a Notch-defective mutant and a chemical inhibitor, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). The endogenous expression of Her2 was altered by both overexpression and morpholino-knockdown approaches, and the results demonstrated that Her2 was both necessary and sufficient to promote the proliferation of neural progenitors by inhibiting the transcription of the cell cycle inhibitors cdkn1a, cdkn1ba, and cdkn1bb. Her2 knockdown caused premature neuronal differentiation, which indicates that Her2 is essential for inhibiting neuronal differentiation. At a later stage of neural development, Her2 could induce glial differentiation. The overexpression of Her2 constructs lacking the bHLH or WRPW domain phenocopied the effect of the morpholino knockdown, demonstrating the essential function of these two domains and further confirming the knockdown specificity. In conclusion, our data reveal that Her2 promotes progenitor proliferation and maintains progenitor characteristics by inhibiting neuronal differentiation. Together, these two mechanisms ensure the proper development of the neural progenitor cell pool.

The epigenetic factor Kmt2a/Mll1 regulates neural progenitor proliferation and neuronal and glial differentiation.

Multiple epigenetic factors play a critical role in cell proliferation and differentiation. However, their function in embryogenesis, especially in neural development, is currently unclear. The Trithorax group (TrxG) homolog KMT2A (MLL1) is an important epigenetic regulator during development and has an especially well-defined role in hematopoiesis. Translocation and aberrant expression of KMT2A is often observed in many tumors, indicating its proto-oncogenic character. Here, we show that Kmt2a was essential for neural development in zebrafish embryos. Disrupting the expression of Kmt2a using morpholino antisense oligonucleotides and a dominant-negative variant resulted in neurogenic phenotypes, including downregulated proliferation of neural progenitors, premature differentiation of neurons, and impaired gliogenesis. This study therefore revealed a novel function of Kmt2a in cell proliferation and differentiation, providing further insight into the function of TrxG proteins in neural development and brain tumors.

Dner inhibits neural progenitor proliferation and induces neuronal and glial differentiation in zebrafish.

Delta/notch-like epidermal growth factor (EGF)-related receptor (DNER) is a single-pass transmembrane protein found to be a novel ligand in the Notch signaling pathway. Its function was previously characterized in the developing cerebellum and inner ear hair cells. In this study, we isolated a zebrafish homolog of DNER and showed that this gene is expressed in the developing nervous system. Overexpression of dner or the intracellular domain of dner was sufficient to inhibit the proliferation of neural progenitors and induce neuronal and glial differentiation. In contrast, the knockdown of endogenous Dner expression using antisense morpholino oligonucleotides increased the proliferation of neural progenitors and maintained neural cells in a progenitor status through inhibition of neuronal and glial differentiation. Through analysis of the antagonistic effect on the Delta ligand and the role of the potential downstream mediator Deltex1, we showed that Dner acts in Notch-dependent and Notch-independent manner. This is the first study to demonstrate a role for Dner in neural progenitors and neuronal differentiation and provides new insights into mediation of neuronal development and differentiation by the Notch signaling pathway.