Custom-designed nucleases afford a powerful reverse genetic tool for direct gene disruption and genome modification in vivo. Among various applications of the nucleases, homologous recombination (HR)-mediated genome editing is particularly useful for inserting heterologous DNA fragments, such as GFP, into a specific genomic locus in a sequence-specific fashion. However, precise HR-mediated genome editing is still technically challenging in zebrafish. Here, we establish a GFP reporter system for measuring the frequency of HR events in live zebrafish embryos. By co-injecting a TALE nuclease and GFP reporter targeting constructs with homology arms of different size, we defined the length of homology arms that increases the recombination efficiency. In addition, we found that the configuration of the targeting construct can be a crucial parameter in determining the efficiency of HR-mediated genome engineering. Implementing these modifications improved the efficiency of zebrafish knock-in generation, with over 10% of the injected F0 animals transmitting gene-targeting events through their germline. We generated two HR-mediated insertion alleles of sox2 and gfap loci that express either superfolder GFP (sfGFP) or tandem dimeric Tomato (tdTomato) in a spatiotemporal pattern that mirrors the endogenous loci. This efficient strategy provides new opportunities not only to monitor expression of endogenous genes and proteins and follow specific cell types in vivo, but it also paves the way for other sophisticated genetic manipulations of the zebrafish genome.

How astrocytes grow and integrate into neural circuits remains poorly defined. Zebrafish are well-suited for such investigations, but bona fide astrocytes have not been described in this system. Here, we characterize a zebrafish cell type that is remarkably similar to mammalian astrocytes that derive from radial glial cells and elaborate processes to establish their territories at early larval stages. Zebrafish astrocytes associate closely with synapses, tile with one another, and express markers including Glast and glutamine synthetase. Once integrated into circuits, they exhibit whole-cell and microdomain Ca 2+ transients, which are sensitive to norepinephrine. Finally, using a cell-specific CRISPR/Cas9 approach we demonstrate that fgfr3/4 are required for vertebrate astrocyte morphogenesis. This work provides the first visualization of astrocyte morphogenesis from stem cell to post-mitotic astrocyte in vivo , identifies a role for Fgf receptors in vertebrate astrocytes, and establishes zebrafish as a valuable new model system to study astrocyte biology in vivo .

Resent studies have identified Pygopus as a core component of the ␤-catenin/T-cell factor (TCF)/ lymphoid-enhancing factor 1 (LEF) transcriptional activation complex required for the expression of canonical Wg/Wnt target genes in Drosophila. However, the biochemical involvement of mammalian Pygopus proteins in ␤-catenin/TCF/LEF gene activation remains controversial. In this study, we perform a series of molecular/biochemical experiments to demonstrate that Pygo2 associates with histone-modifying enzymatic complexes, specifically the MLL2 histone methyltransferase (HMT) and STAGA histone acetyltransferase (HAT) complexes, to facilitate their interaction with ␤-catenin and to augment Wnt1-induced, TCF/LEF-dependent transcriptional activation in breast cancer cells. We identify a critical domain in Pygo2 encompassing the first 47 amino acids that mediates its HMT/HAT interaction. We further demonstrate the importance of this domain in Pygo2's ability to transcriptionally activate both artificial and endogenous Wnt target genes and to expand breast cancer stem-like cells in culture. This work now links mechanistically Pygo2's role in histone modification to its enhancement of the Wntdependent transcriptional program and cancer stem-like cell expansion.Epigenetic regulation underlies tissue development, homeostasis, and tumorigenesis and includes the modification of the chromatin in transcriptional activation or repression. The basic repeating unit of the chromatin is the nucleosome consisting of 146 bp of DNA wrapped around a histone octamer containing two copies of each of the histones H2A, H2B, H3, and H4. Methylation and acetylation of lysine (K) residues on histone H3 and H4 tails confer either activating or silencing effects on transcription. Dimethylation (me2) and trimethylation (me3) of H3K4 and acetylation (Ac) of H3K9/K14 are associated with transcriptional activation while H3K9 and H3K27 methylation is associated with transcriptional repression (49). Histone methylation is catalyzed by histone methyltransferases (HMTs) and reversed by histone demethylases, whereas the steady-state acetylation levels of histone proteins are achieved by the actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs) (11, 42).In Saccharomyces cerevisiae, a multisubunit complex containing the Drosophila trithorax-related protein Set1 has been shown to be responsible for mono-, di-, and trimethylation of histone H3K4 (7). In humans, multiple Set1-like HMT complexes with H3K4 HMT activities have been identified (12). Each of these complexes contains the SET domain-containing homologs of yeast Set1, including human Set1 (hSet1), MLL1 (mixed-lineage leukemia 1, also known as MLL, HRX, ALL1, or KMT2A), MLL2 (mixed-lineage leukemia 2, also known as HRX2 or KMT2B), MLL3 (mixed-lineage leukemia 3, also known as HALR or KMT2C), and MLL4 (mixed-lineage leukemia 4, also known as ALR or KMT2D) (2,16,22,30,37,46), which carry the enzymatic activity for the associated complexes. RbBP5, WDR5, and Ash2L, which are homologs ...

SummaryWnt-b-catenin signaling participates in the epithelial-mesenchymal transition (EMT) in a variety of cancers; however, its involvement in hepatocellular carcinoma (HCC) and downstream molecular events is largely undefined. HNF4a is the most prominent and specific factor maintaining the differentiation of hepatic lineage cells and a potential EMT regulator in HCC cells. However, the molecular mechanisms by which HNF4a maintains the differentiated liver epithelium and inhibits EMT have not been completely defined. In this study, we systematically explored the relationship between Wnt-b-catenin signaling and HNF4a in the EMT process of HCC cells. Our results indicated that HNF4a expression was negatively regulated during Wnt-b-catenin signaling-induced EMT through Snail and Slug in HCC cells. In contrast, HNF4a was found to directly associate with TCF4 to compete with b-catenin but facilitate transcription corepressor activities, thus inhibiting expression of EMT-related Wnt-b-catenin targets. Moreover, HNF4a may control the switch between the transcriptional and adhesion functions of b-catenin. Overexpression of HNF4a was found to completely compromise the Wnt-b-catenin-signaling-induced EMT phenotype. Finally, we determined the regulation pattern between Wnt-b-catenin signaling and HNF4a in rat tumor models. Our studies have identified a double-negative feedback mechanism controlling Wnt-b-catenin signaling and HNF4a expression in vitro and in vivo, which sheds new light on the regulation of EMT in HCC. The modulation of these molecular processes may be a method of inhibiting HCC invasion by blocking Wnt-b-catenin signaling or restoring HNF4a expression to prevent EMT.