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Talin (tln) binds and activates integrins to couple extracellular matrix–bound integrins to the cytoskeleton; however, its role in heart development is not well characterized. We identified the defective gene and the resulting cardiovascular phenotypes in zebrafish tln1fl02k mutants. The ethylnitrosourea-induced fl02k mutant showed heart failure, brain hemorrhage, and diminished cardiac and vessel lumens at 52 h post fertilization. Positional cloning revealed a nonsense mutation of tln1 in this mutant. tln1, but neither tln2 nor -2a, was dominantly expressed in the heart and vessels. Unlike tln1 and -2 in the mouse heart, the unique tln1 expression in the heart enabled us, for the first time, to determine the critical roles of Tln1 in the maintenance of cardiac sarcomeric Z-disks and endothelial/endocardial cell integrity, partly through regulating F-actin networks in zebrafish. The similar expression profiles of tln1 and integrin β1b (itgb1b) and synergistic function of the 2 genes revealed that itgb1b is a potential partner for tln1 in the stabilization of cardiac Z-disks and vessel lumens. Taken together, the results of this work suggest that Tln1-mediated Itgβ1b plays a crucial role in maintaining cardiac sarcomeric Z-disks and endothelial/endocardial cell integrity in zebrafish and may also help to gain molecular insights into congenital heart diseases.—Wu, Q., Zhang, J., Koh, W., Yu, Q., Zhu, X., Amsterdam, A., Davis, G. E., Arnaout, M. A., Xiong, J.-W. Talin1 is required for cardiac Z-disk stabilization and endothelial integrity in zebrafish.

Although cardiac stem cells (CSCs) and tissue engineering are very promising for cardiac regenerative medicine, studies with model organisms for heart regeneration will provide alternative therapeutic targets and opportunities. Here, we present a review on heart regeneration, with a particular focus on the most recent work in mouse and zebrafish. We attempt to summarize the recent progresses and bottlenecks of CSCs and tissue engineering for heart regeneration; and emphasize what we have learned from mouse and zebrafish regenerative models on discovering crucial genetic and epigenetic factors for stimulating heart regeneration; and speculate the potential application of these regenerative factors for heart failure. A brief perspective highlights several important and promising research directions in this exciting field.

The Lycat homologue in zebrafish maps to the deletion interval of the cloche mutant in which hematopoietic and endothelial cell lineages are affected. However, its definitive relationship to cloche is inconclusive, partly due to inadequate expression data of Lycat from any organisms. We precisely examined the temporal and spatial expression patterns of Lycat in mouse using RNA in situ hybridization, immunostaining, and BAC transgenesis. Lycat is initially expressed in developing heart, lung, and somites, and later becomes progressively restricted to all vascular smooth muscle cells. In adult ovaries, Lycat turns on in oocytes during the transition from primary to secondary follicles. Expression of the Lycat/reporter transgene in the extraembryonic mesoderm, cardiogenic mesoderm, and primitive streak, but not extraembryonic endoderm at E7.5, suggests its potential roles in regulating cardiac, smooth muscle, hematopoietic and endothelial lineages. Promoter mapping assay by transient transgenesis identifies a novel cardiac-specific regulatory region in the Lycat locus. Developmental Dynamics 239:1827–1837, 2010. © 2010 Wiley-Liss, Inc.

Mouse embryonic stem cells are pluripotent cells that are derived from the inner cell mass of blastocysts. When induced to synchronously enter a program of differentiation in vitro, they form embryoid bodies that contain cells of the mesodermal, hematopoietic, endothelial, muscle, and neuronal lineages. Here, we used a panel of marker genes with early expression within the germ layers (oct-3, Brachyury T, Fgf-5, nodal, and GATA-4) or a variety of lineages (flk-1, Nkx-2.5, EKLF, and Msx3) to determine how progressive differentiation of embryoid bodies in culture correlated with early postimplantation development of mouse embryos. Using RNA in situ hybridization, we found that the temporal and spatial relationships existing between these marker genes in vivo were maintained also in vitro. Studying the onset of marker gene expression allowed us also to determine the time course of differentiation during the formation of embryoid bodies. Thus, stages equivalent to embryogenesis between implantation and the beginning of gastrulation (4.5-6.5 d.p.c.) occur within the first two days of embryoid body differentiation. Between days 3 and 5, embryoid bodies contain cell lineages found in embryos during gastrulation at 6.5 to 7.0 d.p.c., and after day 6 in culture, embryoid bodies are equivalent to early organogenesis-stage embryos (7.5 d.p.c.). In addition, we demonstrate that the panel of developmental markers can be applied in a screen for stage- or lineage-specific genes. Reporter gene expression from entrapment vector insertions can be co-localized with expression of specific markers within the same cell during embryoid body formation as well as during embryogenesis. Our results thus demonstrate the power of embryoid body formation as an in vitro model system to study early lineage determination and organogenesis in mammals, and indicate that they will prove to be useful tools for identifying developmental genes whose expression is restricted to particular lineages.

Mammalian development is orchestrated by a variety of cellular proteins with expression that is regulated precisely. Although some of the genes encoding these factors have been identified, largely by homology to those of simpler organisms, the majority of them presumably remain unknown. We report here on the results of a large-scale genetic screen that can potentially lead to the identification of many of these unidentified genes in mice. The method we developed takes advantage of the fact that many of the factors that regulate early development are expressed at highly specific stages of early embryogenesis. We therefore established a method for tagging candidate developmental genes by virtue of their expression in a stage-specific manner during formation of embryoid bodies without a bias for their expression in undifferentiated embryonic stem (ES) cells. Of 2,400 ES cell clones with random insertions of retroviral vectors carrying a human placental alkaline phosphatase reporter gene (AP), 41 clones exhibited stage-specific reporter gene expression during embryoid body formation. The majority of these insertions were in genes that are not expressed in undifferentiated ES cells. Eleven ES cell clones with characteristic patterns of AP reporter gene expression in vitro were chosen for further examination in vivo for AP expression in developing embryos. Ten ES cell clones exhibited AP expression between day 7.5 and day 10.5 of development. Clones that showed restricted reporter gene expression in vitro also exhibited similar temporally and spatially restricted AP expression in vivo. Sequence analysis of genomic DNA flanking several vector insertions and corresponding cDNAs suggested that several of the insertions identified a previously unidentified gene. Thus, screening for reporter gene expression during embryoid body formation provides an efficient means of enriching clones that contain vector insertions into potentially novel genes that are important for regulating different stages of early postimplantation development.