SUMMARYThe epicardium is a mesothelial cell layer essential for vertebrate heart development and pertinent for cardiac repair post-injury in the adult. The epicardium initially forms from a dynamic precursor structure, the proepicardial organ, from which cells migrate onto the heart surface. During the initial stage of epicardial development crucial epicardial-derived cell lineages are thought to be determined. Here, we define an essential requirement for transcription factor Tcf21 during early stages of epicardial development in Xenopus, and show that depletion of Tcf21 results in a disruption in proepicardial cell specification and failure to form a mature epithelial epicardium. Using a mass spectrometry-based approach we defined Tcf21 interactions and established its association with proteins that function as transcriptional co-repressors. Furthermore, using an in vivo systems-based approach, we identified a panel of previously unreported proepicardial precursor genes that are persistently expressed in the epicardial layer upon Tcf21 depletion, thereby confirming a primary role for Tcf21 in the correct determination of the proepicardial lineage. Collectively, these studies lead us to propose that Tcf21 functions as a transcriptional repressor to regulate proepicardial cell specification and the correct formation of a mature epithelial epicardium. KEY WORDS: Tcf21, Proepicardial organ, Heart developmentTcf21 regulates the specification and maturation of proepicardial cells ZnCl 2 , 1 μM CaCl 2 , 0.5% Triton X-100 (v/v), 150 mM NaCl, 4 μg/ml DNase, 1/100 (v/v) Protease Inhibitor Cocktail and 1/100 Phosphatase Inhibitor Cocktail] using 5 ml lysis buffer/g cell powder. Lysates were homogenized, subjected to centrifugation, and supernatants incubated for 1 hour with 7 mg magnetic beads (M270 Epoxy Dynabeads, Invitrogen) conjugated with anti-EGFP antibodies (Cristea et al., 2005). Proteins were eluted by incubation for 10 minutes (70°C) in 30 µl 1× LDS sample buffer (Invitrogen) containing 1× Reducing Agent (Invitrogen), followed by shaking at room temperature for 10 minutes. Panna In-solution digestion, mass spectrometry analysis and data processingProtein IP eluates were prepared as described Greco et al., 2011;Tsai et al., 2012). Briefly, IP eluates were mixed with 8 M urea in aqueous 0.1 M Tris-HCl pH 8.0, applied to ultrafiltration Vivacon 500 units (Sartorius Stedim), and centrifuged at 14,000 g for 40 minutes at 20°C. Samples were washed, alkylated and digested with trypsin (Promega) overnight at 37°C. Resulting peptides were collected by centrifugation, acidified with trifluoroacetic acid, concentrated by vacuum centrifugation, and desalted using Empore C18 StageTips (Rappsilber et al., 2007;. Peptides were analyzed by nLC-MS/MS using a Dionex Ultimate 3000 RSLC system coupled online to an LTQ-Orbitrap Velos mass spectrometer (ThermoFisher Scientific) Tsai et al., 2012). Peptides were fragmented by collisioninduced dissociation (CID) and the MS/MS spectra were extracted by Proteome Discoverer (ThermoFishe...

The proper dissection of the molecular mechanisms governing the specification and differentiation of specific cell types requires isolation of pure cell populations from heterogeneous tissues and whole organisms. Here, we describe a method for purification of nuclei from defined cell or tissue types in vertebrate embryos using INTACT (isolation of nuclei tagged in specific cell types). This method, previously developed in plants, flies and worms, utilizes in vivo tagging of the nuclear envelope with biotin and the subsequent affinity purification of the labeled nuclei. In this study we successfully purified nuclei of cardiac and skeletal muscle from Xenopus using this strategy. We went on to demonstrate the utility of this approach by coupling the INTACT approach with liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomic methodologies to profile proteins expressed in the nuclei of developing hearts. From these studies we have identified the Xenopus orthologs of 12 human proteins encoded by genes, which when mutated in human lead to congenital heart disease. Thus, by combining these technologies we are able to identify tissue-specific proteins that are expressed and required for normal vertebrate organ development.

Organ growth occurs through the integration of external growth signals during the G1 phase of the cell cycle to initiate DNA replication. Although numerous growth factor signals have been shown to be required for the proliferation of cardiomyocytes, genetic studies have only identified a very limited number of transcription factors that act to regulate the entry of cardiomyocytes into S phase. Here, we report that the cardiac para-zinc-finger protein CASZ1 is expressed in murine cardiomyocytes. Genetic fate mapping with an inducible Casz1 allele demonstrates that CASZ1-expressing cells give rise to cardiomyocytes in the first and second heart fields. We show through the generation of a cardiac conditional null mutation that Casz1 is essential for the proliferation of cardiomyocytes in both heart fields and that loss of Casz1 leads to a decrease in cardiomyocyte cell number. We further report that the loss of Casz1 leads to a prolonged or arrested S phase, a decrease in DNA synthesis, an increase in phospho-RB and a concomitant decrease in the cardiac mitotic index. Taken together, these studies establish a role for CASZ1 in mammalian cardiomyocyte cell cycle progression in both the first and second heart fields.

Larval, or tadpole-stage Xenopus laevis frogs are a popular research model for developmental biology and disease studies. Existing euthanasia guidance documents offer recommendations for both eggs and adult stages, yet do not specifically address the larval stage. Data evaluating effective euthanasia methods for groups of X. laevis tadpoles would therefore be useful. The goal of the current study was to evaluate the efficacy of various immersion euthanasia procedures on tadpoles: tricaine methanesulfonate (MS222) at 6 g/L, eugenol at 800 μL/L and rapid chilling (2 to 4 °C). We also evaluated tadpoles at various developmental stages (NF stages 46, 47 and 49). Tadpoles (n = 70) were exposed to euthanasia solution for 15 min, and controls (n = 40) were placed in housing tank water for 15 min. All animals were then placed in recovery tanks containing housing tank water for 4 h to confirm irreversibility of each agent. Cessation of the heartbeat was assessed at the end of euthanasia solution exposure and at each hour thereafter. We found that immersion in a 6 g/L solution of MS222 resulted in 100% euthanasia of all larval stages tested. Conversely, eugenol produced variable euthanasia rates that were affected by both age group and batches of stock solutions. Rapid chilling was completely ineffective as a euthanasia method in our study. Based on our findings, we recommend MS222 as an effective and practical means of euthanizing large numbers of X. laevis tadpoles.

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