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Using a study on Xenopus embryos, the role of retinoic acid in the development of heart tube during cardiogenesis is examined.

Characterization of embryonic cardiac pacemaker and atrioventricular conduction physiology in Xenopus laevis using noninvasive imaging. Am J Physiol Heart Circ Physiol 286: H2035-H2041, 2004; 10.1152/ajpheart.00807.2003.--Congenital heart defects often include altered conduction as well as morphological changes. Model organisms, like the frog Xenopus laevis, offer practical advantages for the study of congenital heart disease. X. laevis embryos are easily obtained free living, and the developing heart is readily visualized. Functional and morphological evidence for a conduction system is available for adult frog hearts, but information on the normal properties of embryonic heart contraction is lacking, especially in intact animals. With the use of fine glass microelectrodes, we were able to obtain cardiac recordings and make standard electrophysiological measurements in 1-wk-old embryos (stage 46). In addition, a system using digital analysis of video images was adapted for measurement of the standard cardiac intervals and compared with invasive measurements. Video images were obtained of the heart in live, pharmacologically paralyzed, stage 46 X. laevis embryos. Normal values for the timing of the cardiac cycle were established. Intervals determined by video analysis (n = 53), including the atrial and ventricular cycle lengths (473 [+ or -] 10 ms and 464 [+ or -] 19 ms, respectively) and the atrioventricular interval (169 [+ or -] 5 ms) were not statistically different from those determined by intrathoracic cardiac recordings. We also present the data obtained from embryos treated with standard medications that affect the human conduction system. We conclude that the physiology of embryonic X. laevis cardiac conduction can be noninvasively studied by using digital video imaging. Additionally, we show the response of X. laevis embryonic hearts to chronotropic agents is similar but not identical to the response of the human heart. conduction system

The experimental manipulation of early embryologic events, resulting in the misexpression of the homeobox transcription factor pitx2, is associated with subsequent defects of laterality in a number of vertebrate systems. To clarify the role of one pitx2 isoform, pitx2c, in determining the left-right axis of amphibian embryos, we examined the heart and gut morphology of Xenopus laevis embryos after attenuating pitx2c mRNA levels using chemically modified antisense oligonucleotides. We demonstrate that the partial depletion of pitx2c mRNA in these embryos results in alteration of both cardiac morphology and intestinal coiling. The most common cardiac abnormality seen was a failure of rightward migration of the outflow tract, while the most common intestinal laterality phenotype seen was a full reversal in the direction of coiling, each present in 23% of embryos injected with the pitx2c antisense oligonucleotide. An abnormality in either the heart or gut further predisposed to a malformation in the other. In addition, a number of other cardiac anomalies were observed after pitx2c mRNA attenuation, including abnormalities of atrial septation, extracellular matrix restriction, relative atrial-ventricular chamber positioning, and restriction of ventricular development. Many of these findings correlate with cardiac defects previously reported in pitx2 null and hypomorphic mice, but can now be assigned specifically to attenuation of the pitx2c isoform in Xenopus. Keywords: Xenopus; Embryo; pitx2; Heart; Asymmetry; Antisense; Oligonucleotide

Expression of the Xenopus homolog of the mammalian transcription factor AP-2 [alpha] (XAP-2) is activated throughout the animal hemisphere shortly after the midblastula transition, and becomes restricted to prospective epidermis by the end of gastrulation, under the control of BMP signal modulation. Elevated expression in the future neural crest region begins at this time. Ectopic expression of XAP-2 can restore transcription of epidermal genes in neuralized ectoderm, both in ectodermal explants and in the intact embryo. Likewise, loss of XAP-2 function, accomplished by injection of antisense oligonucleotides or by overexpression of antimorphic XAP-2 derivatives, leads to loss of epidermal and gain of neural gene expression. These treatments also result in gastrulation failure. Thus, AP-2 is a critical regulator of ectodermal determination that is required for normal epidermal development and morphogenesis in the frog embryo. Key Words: bone morphogenetic protein; gastrulation; antisense oligonucleotide; epidermis.

Xenopus laevis provides a number of advantages to studies on cardiovascular development. The embryos are fairly large, are easy to obtain, and can develop at ambient temperature in simple buffer solutions. Although classic descriptions of heart development exist, the ability to use whole-mount immunohistochemical methods and confocal microscopy may enhance the ability to understand both normal and experimentally perturbed cardiovascular development. We have started to examine the early stages of cardiac development in Xenopus, seeking to identify antibodies and fixatives that allow easy examination of the developing heart. We have used monoclonal antibodies (mAbs) raised against bovine cardiac troponin T and chicken tropomyosin to visualize cardiac muscle, a goat antibody recognizing bovine type VI collagen to stain the lining of vessels, and the JB3 mAb raised against chicken fibrillin, which allows the visualization of a variety of cardiovascular tissues during early development. Results from embryonic stages 24-46 are presented.

An examination of two key parameters, which influence the efficiency of triplex-forming oligonucleotides (TFO)-induced recombination, binding affinity of the TFO for the target site and the distance between the target side and the mutation to be corrected is reported. The result indicate that TFO-induced recombination requires high-affinity binding but can affect sites hundreds of base pairs away from the position of triplex formation.

Heart valve formation is initiated by an epithelial-mesenchymal cell transformation (EMT) of endothelial cells in the atrioventricular (AV) canal. Mesenchymal cells formed from cardiac EMTs are the initial cellular components of the cardiac cushions and progenitors of valvular and septal fibroblasts. It has been shown that transforming growth factor [Beta] (TGF[Beta]) mediates EMT in the AV canal, and TGF[Beta]1 and 2 isoforms are expressed in the mouse heart while TGF[Beta] 2 and 3 are expressed in the avian heart. Depletion of TGF[Beta]3 in avian or TGF[Beta]2 in mouse leads to developmental defects of heart tissue. These observations raise questions as to whether multiple TGF[Beta] isoforms participate in valve formation. In this study, we examined the localization and function of TGF[Beta]2 and TGF[Beta]3 in the chick heart during EMT. TGF[Beta]2 was present in both endothelium and myocardium before and after EMT. TGF[Beta]2 antibody inhibited endothelial cell-cell separation. In contrast, TGF[Beta]3 was present only in the myocardium before EMT and was in the endothelium at the initiation of EMT. TGF[Beta]3 antibodies inhibited mesenchymal cell formation and migration into the underlying matrix. Both TGF[Beta]2 and 3 increased fibrillin 2 expression. However, only TGF[Beta]2 treatment increased cell surface [Beta]-1,4-galactosyltransferase expression. These data suggest that TGF[Beta]2 and TGF[Beta]3 are sequentially and separately involved in the process of EMT. TGF[Beta]2 mediates initial endothelial cell-cell separation while TGF[Beta]3 is required for the cell morphological change that enables the migration of cells into the underlying ECM. Key Words: cardiogenesis; valve development; cushion formation.