Zebrafish as a model vertebrate for investigating chemical toxicity.

The pattern of blood flow in the developing heart has long been proposed to play a significant role in cardiac morphogenesis. In response to flow-induced forces, cultured cardiac endothelial cells rearrange their cytoskeletal structure and change their gene expression profiles. To link such in vitro data to the intact heart, we performed quantitative in vivo analyses of intracardiac flow forces in zebrafish embryos. Using in vivo imaging, here we show the presence of high-shear, vortical flow at two key stages in the developing heart, and predict flow-induced forces much greater than might have been expected for micro-scale structures at low Reynolds numbers. To test the relevance of these shear forces in vivo, flow was occluded at either the cardiac inflow or outflow tracts, resulting in hearts with an abnormal third chamber, diminished looping and impaired valve formation. The similarity of these defects to those observed in some congenital heart diseases argues for the importance of intracardiac haemodynamics as a key epigenetic factor in embryonic cardiogenesis.

Intracardiac fluid forces are an essential epigenetic factor for embryonic cardiogenesis

Costimulatory signals are required for activation of immune cells, but it is not known whether they contribute to other biological systems. The development and homeostasis of the skeletal system depend on the balance between bone formation and resorption. Receptor activator of NF-kappaB ligand (RANKL) regulates the differentiation of bone-resorbing cells, osteoclasts, in the presence of macrophage-colony stimulating factor (M-CSF). But it remains unclear how RANKL activates the calcium signals that lead to induction of nuclear factor of activated T cells c1, a key transcription factor for osteoclastogenesis. Here we show that mice lacking immunoreceptor tyrosine-based activation motif (ITAM)-harbouring adaptors, Fc receptor common gamma subunit (FcRgamma) and DNAX-activating protein (DAP)12, exhibit severe osteopetrosis owing to impaired osteoclast differentiation. In osteoclast precursor cells, FcRgamma and DAP12 associate with multiple immunoreceptors and activate calcium signalling through phospholipase Cgamma. Thus, ITAM-dependent costimulatory signals activated by multiple immunoreceptors are essential for the maintenance of bone homeostasis. These results reveal that RANKL and M-CSF are not sufficient to activate the signals required for osteoclastogenesis.

Costimulatory signals mediated by the ITAM motif cooperate with RANKL for bone homeostasis

Tube and lumen formation are essential steps in forming a functional vasculature. Despite their significance, our understanding of these processes remains limited, especially at the cellular and molecular levels. In this study, we analyze mechanisms of angioblast coalescence in the zebrafish embryonic midline and subsequent vascular tube formation. To facilitate these studies, we generated a transgenic line where EGFP expression is controlled by the zebrafish flk1 promoter. We find that angioblasts migrate as individual cells to form a vascular cord at the midline. This transient structure is stabilized by endothelial cell-cell junctions, and subsequently undergoes lumen formation to form a fully patent vessel. Downregulating the VEGF signaling pathway, while affecting the number of angioblasts, does not appear to affect their migratory behavior. Our studies also indicate that the endoderm, a tissue previously implicated in vascular development, provides a substratum for endothelial cell migration and is involved in regulating the timing of this process, but that it is not essential for the direction of migration. In addition, the endothelial cells in endodermless embryos form properly lumenized vessels, contrary to what has been previously reported in Xenopus and avian embryos. These studies provide the tools and a cellular framework for the investigation of mutations affecting vasculogenesis in zebrafish.

/pdf/cellular-and-molecular-analyses-of-vascular-tube-and-lumen-aijjtyws6q.pdf

Cellular and molecular analyses of vascular tube and lumen formation in zebrafish

Defects in cardiac function precede morphological abnormalities in fish embryos exposed to polycyclic aromatic hydrocarbons.

Mutations of the gene (TNNT2) encoding the thin-filament contractile protein cardiac troponin T are responsible for 15% of all cases of familial hypertrophic cardiomyopathy, the leading cause of sudden death in young athletes. Mutant proteins are thought to act through a dominant-negative mode that impairs function of heart muscle. TNNT2 mutations can also lead to dilated cardiomyopathy, a leading cause of heart failure. Despite the importance of cardiac troponin T in human disease, its loss-of-function phenotype has not been described. We show that the zebrafish silent heart (sih) mutation affects the gene tnnt2. We characterize two mutated alleles of sih that severely reduce tnnt2 expression: one affects mRNA splicing, and the other affects gene transcription. Tnnt2, together with alpha-tropomyosin (Tpma) and cardiac troponins C and I (Tnni3), forms a calcium-sensitive regulatory complex within sarcomeres. Unexpectedly, in addition to loss of Tnnt2 expression in sih mutant hearts, we observed a significant reduction in Tpma and Tnni3, and consequently, severe sarcomere defects. This interdependence of thin-filament protein expression led us to postulate that some mutations in tnnt2 may trigger misregulation of thin-filament protein expression, resulting in sarcomere loss and myocyte disarray, the life-threatening hallmarks of TNNT2 mutations in mice and humans.

Cardiac troponin T is essential in sarcomere assembly and cardiac contractility.

The chicken cardiac troponin T (cTnT) gene is representative of numerous cardiac and skeletal muscle-specific genes that contain muscle-CAT (MCAT) elements within their promoters. We examined the regulation of the chicken cTnT gene in vivo in zebrafish embryos, and in vitro in cardiomyocyte, myoblast, and fibroblast cultures. Defined regions of the cTnT promoter were linked to the green fluorescent protein (GFP) gene for in vivo analysis, and the luciferase gene for in vitro analysis. Injection of the cTnT promoter constructs into fertilized zebrafish eggs resulted in GFP expression in both heart and skeletal muscle cells reproducing the pattern of expression of the endogenous cTnT gene in the chicken embryo. Promoter deletion analysis revealed that the cis-regulatory regions responsible for cardiac and skeletal muscle-specific expression functioned in an equivalent manner in both in vitro and in vivo environments. In addition, we show that mutation of the poly-ADP ribose polymerase-I (PARP-I) binding site adjacent to the distal MCAT element in the chicken cTnT promoter produced a non-cell-specific promoter in vitro and in the zebrafish. Thus, the PARP-I transcriptional regulatory mechanism that governs muscle specificity of the chicken cTnT promoter is conserved across several chordate classes spanning at least 350 million years of evolution.

Anja Huq

Papers

Cardiac troponin T is essential in sarcomere assembly and cardiac contractility.

In vivo regulation of the chicken cardiac troponin T gene promoter in zebrafish embryos.