Microvillar and ciliary defects in zebrafish lacking an actin-binding bioactive peptide amidating enzyme.

The assembly of membranous extensions such as microvilli and cilia in polarized cells is a tightly regulated, yet poorly understood, process. Peptidylglycine α-amidating monooxygenase (PAM), a membrane enzyme essential for the synthesis of amidated bioactive peptides, was recently identified in motile and non-motile (primary) cilia and has an essential role in ciliogenesis in Chlamydomonas, Schmidtea and mouse. In mammalian cells, changes in PAM levels alter secretion and organization of the actin cytoskeleton. Here we show that lack of Pam in zebrafish recapitulates the lethal edematous phenotype observed in Pam -/- mice and reveals additional defects. The pam -/- zebrafish embryos display an initial striking loss of microvilli and subsequently impaired ciliogenesis in the pronephros. In multiciliated mouse tracheal epithelial cells, vesicular PAM staining colocalizes with apical actin, below the microvilli. In PAM-deficient Chlamydomonas, the actin cytoskeleton is dramatically reorganized, and expression of an actin paralogue is upregulated. Biochemical assays reveal that the cytosolic PAM C-terminal domain interacts directly with filamentous actin but does not alter the rate of actin polymerization or disassembly. Our results point to a critical role for PAM in organizing the actin cytoskeleton during development, which could in turn impact both microvillus formation and ciliogenesis.

Characterization of trace metal content in the developing zebrafish embryo.

Trace metals are essential for health but toxic when present in excess. The maintenance of trace metals at physiologic levels reflects both import and export by cells and absorption and excretion by organs. The mechanism by which this maintenance is achieved in vertebrate organisms is incompletely understood. To explore this, we chose zebrafish as our model organism, as they are amenable to both pharmacologic and genetic manipulation and comprise an ideal system for genetic screens and toxicological studies. To characterize trace metal content in developing zebrafish, we measured levels of three trace elements, copper, zinc, and manganese, from the oocyte stage to 30 days post-fertilization using inductively coupled plasma mass spectrometry. Our results indicate that metal levels are stable until zebrafish can acquire metals from the environment and imply that the early embryo relies on maternal contribution of metals to the oocyte. We also measured metal levels in bodies and yolks of embryos reared in presence and absence of the copper chelator neocuproine. All three metals exhibited different relative abundances between yolks and bodies of embryos. While neocuproine treatment led to an expected phenotype of copper deficiency, total copper levels were unaffected, indicating that measurement of total metal levels does not equate with measurement of biologically active metal levels. Overall, our data not only can be used in the design and execution of genetic, physiologic, and toxicologic studies but also has implications for the understanding of vertebrate metal homeostasis.

The chick embryo as an expanding experimental model for cancer and cardiovascular research.

A long and productive history in biomedical research defines the chick as a model for human biology. Fundamental discoveries, including the description of directional circulation propelled by the heart and the link between oncogenes and the formation of cancer, indicate its utility in cardiac biology and cancer. Despite the more recent arrival of several vertebrate and invertebrate animal models during the last century, the chick embryo remains a commonly used model for vertebrate biology and provides a tractable biological template. With new molecular and genetic tools applied to the avian genome, the chick embryo is accelerating the discovery of normal development and elusive disease processes. Moreover, progress in imaging and chick culture technologies is advancing real-time visualization of dynamic biological events, such as tissue morphogenesis, angiogenesis, and cancer metastasis. A rich background of information, coupled with new technologies and relative ease of maintenance, suggest an expanding utility for the chick embryo in cardiac biology and cancer research.

Comprehensive timeline of mesodermal development in the quail small intestine.

BACKGROUND: To generate the mature intestine, splanchnic mesoderm diversifies into six different tissue layers each with multiple cell types through concurrent and complex morphogenetic events. Hindering the progress of research in the field is the lack of a detailed description of the fundamental morphological changes that constitute development of the intestinal mesoderm. RESULTS: We used immunofluorescence and morphometric analyses of wild-type and Tg(tie1:H2B-eYFP) quail embryos to establish a comprehensive timeline of mesodermal development in the avian intestine. The following landmark features were analyzed from appearance of the intestinal primordium through generation of the definitive structure: radial compartment formation, basement membrane dynamics, mesothelial differentiation, mesenchymal expansion and growth patterns, smooth muscle differentiation, and maturation of the vasculature. In this way, structural relationships between mesodermal components were identified over time. CONCLUSIONS: This integrated analysis presents a roadmap for investigators and clinicians to evaluate diverse experimental data obtained at individual stages of intestinal development within the longitudinal context of intestinal morphogenesis.

Identification of a novel developmental mechanism in the generation of mesothelia.

Mesothelium is the surface layer of all coelomic organs and is crucial for the generation of their vasculature. Still, our understanding of the genesis of this essential cell type is restricted to the heart where a localized exogenous population of cells, the proepicardium, migrates to and envelops the myocardium supplying mesothelial, vascular and stromal cell lineages. Currently it is not known whether this pattern of development is specific to the heart or applies broadly to other coelomic organs. Using two independent long-term lineage-tracing studies, we demonstrate that mesothelial progenitors of the intestine are intrinsic to the gut tube anlage. Furthermore, a novel chick-quail chimera model of gut morphogenesis reveals these mesothelial progenitors are broadly distributed throughout the gut primordium and are not derived from a localized and exogenous proepicardium-like source of cells. These data demonstrate an intrinsic origin of mesothelial cells to a coelomic organ and provide a novel mechanism for the generation of mesothelial cells.

Application of small organic molecules reveals cooperative TGFß and BMP regulation of mesothelial cell behaviors.

Epicardial development is a process during which epithelial sheet movement, single cell migration, and differentiation are coordinated to generate coronary arteries. Signaling cascades regulate the concurrent and complex nature of these three events. Through simple and highly reproducible assays, we identified small organic molecules that impact signaling pathways regulating these epicardial behaviors. Subsequent biochemical analyses confirmed the specificity of these reagents and revealed novel targets for the widely used dorsomorphin (DM) and LDN-193189 molecules. Using these newly characterized reagents, we show the broad regulation of epicardial cell differentiation, sheet movement, and single cell migration by Transforming Growth Factor ß (TGFß). With the DM analogue DMH1, a highly specific Bone Morphogenetic Protein (BMP) inhibitor, we demonstrate the cooperative yet exclusive role for BMP signaling in regulation of sheet migration. The action of DMH1 reveals that small organic molecules (SOM) can intervene on a single epicardial behavior while leaving other concurrent behaviors intact. All SOM data were confirmed by reciprocal experiments using growth factor addition and/or application of established non-SOM inhibitors. These compounds can be applied to cell lines or native proepicardial tissue. Taken together, these data establish the efficacy of chemical intervention for analysis of epicardial behaviors and provide novel reagents for analysis of epicardial development and repair.