Inhibition of Wnt activity induces heart formation from posterior mesoderm.

In the chick, heart mesoderm is induced by signals from the anterior endoderm. Although BMP-2 is expressed in the anterior endoderm, BMP activity is necessary but not sufficient for heart formation. Previous work from our lab has suggested that one or more additional factors from anterior endoderm are required. Crescent is a Frizzled-related protein that inhibits Wnt-8c and is expressed in anterior endoderm during gastrulation. At the same stages, expression of Wnt-3a and Wnt-8c is restricted to the primitive streak and posterior lateral plate, and is absent from the anterior region where crescent is expressed. Posterior lateral plate mesoderm normally forms blood, but coculture of this tissue with anterior endoderm or infection with RCAS-crescent induces formation of beating heart muscle and represses formation of blood. Dkk-1, a Wnt inhibitor of a different protein family, similarly induces heart-specific gene expression in posterior lateral plate mesoderm. Furthermore, we have found that ectopic Wnt signals can repress heart formation from anterior mesoderm in vitro and in vivo and that forced expression of either Wnt-3a or Wnt-8c can promote development of primitive erythrocytes from the precardiac region. We conclude that inhibition of Wnt signaling promotes heart formation in the anterior lateral mesoderm, whereas active Wnt signaling in the posterior lateral mesoderm promotes blood development. We propose a model in which two orthogonal gradients, one of Wnt activity along the anterior-posterior axis and the other of BMP signals along the dorsal-ventral axis, intersect in the heart-forming region to induce cardiogenesis in a region of high BMP and low Wnt activity.

Cerberus regulates left-right asymmetry of the embryonic head and heart.

BACKGROUND: Most of the molecules known to regulate left-right asymmetry in vertebrate embryos are expressed on the left side of the future trunk region of the embryo. Members of the protein family comprising Cerberus and the putative tumour suppressor Dan have not before been implicated in left-right asymmetry. In Xenopus, these proteins have been shown to antagonise members of the transforming growth factor beta (TGF-beta) and Wnt families of signalling proteins. RESULTS: Chick Cerberus (cCer) was found to be expressed in the left head mesenchyme and in the left flank of the embryo. Expression on the left side of the head was controlled by Sonic hedgehog (Shh) acting through the TGF-beta family member Nodal; in the flank, cCer was also regulated by Shh, but independently of Nodal. Surprisingly, although no known targets of Cerberus are expressed asymmetrically on the right side of the embryo at these stages, misexpression of cCer on this side of the embryo led to upregulation of the transcription factor Pitx2 and reversal of the direction of heart and head turning, apparently as independent events. Consistent with the possibility that cCer may be acting on bilaterally expressed TGF-beta family members such as the bone morphogenetic proteins (BMPs), this result was mimicked by right-sided misexpression of the BMP antagonist, Noggin. CONCLUSIONS: Our findings suggest that cCer maintains a delicate balance of different TGF-beta family members involved in laterality decisions, and reveal the existence of partially overlapping molecular pathways regulating left-right asymmetry in the head and trunk of the embryo.

A rod end deletion in the intermediate filament protein nestin alters its subcellular localization in neuroepithelial cells of transgenic mice.

Neuroepithelial and radial glial cells span between the ventricular and the pial surfaces of the neural tube and express two intermediate filaments (IFs), nestin and vimentin, which form a filamentous network throughout the length of the cells. In this report we study the polymerization characteristics of nestin and examine how mutations affect the assembly and localization of the nestin protein in cultured cells and in the developing CNS of transgenic mice. A wild-type rat nestin gene transfected into the IF-free SW13 cell line failed to assemble into a filamentous network but was incorporated into the existing IF network of a subclone expressing vimentin, demonstrating that nestin requires vimentin for proper assembly. In transgenic mice, rat nestin formed a network indistinguishable from that formed by endogenous nestin and vimentin, but a mutant form lacking five amino acids at the carboxy terminus of the rod domain was largely restricted to the pial endfeet. Since nestin mRNA is localized to the pial endfoot region we propose that both transgenes are translated there, but that the wild-type protein is preferentially incorporated into the IF network. These observations provide evidence for hierarchical assembly and a complex organization of the IF network along the ventricular-pial axis in the early CNS.

Percutaneous absorption of nicardipine and ketorolac in rhesus monkeys.

Vehicle effects on the percutaneous absorption of nicardipine base, nicardipine hydrochloride, ketorolac acid, and ketorolac tromethamine were determined using the rhesus monkey as an in vivo model for human skin penetration. Vehicles investigated included blends of propylene glycol, trimethylene glycol, ethanol, Azone, Tween 20, water, and long-chain fatty acids. Formulations were prepared such that the compound dose, application area, and percentage saturation of the compound in the vehicle were held constant. Variations in absorption of the compounds were therefore attributable to vehicle effects. Each formulation was applied to three monkeys for a period of 24 hr using 10 Hill Top Chambers. Plasma samples were taken at appropriate intervals for 36 to 48 hr. The results indicated that trimethylene glycol and Tween 20 did not enhance absorption of the test compounds despite claims by other investigators. Azone and ethanol provided moderate enhancement of both the rate and the extent of absorption, while long-chain fatty acids in combination with propylene glycol significantly enhanced penetration. In general, higher fluxes were observed with the more lipophilic compounds nicardipine base and ketorolac acid as compared to the hydrochloride and tromethamine salts.

Structure of the retinal chromophore in the hR578 form of halorhodopsin.

Halorhodopsin is a retinal-containing pigment that is thought to function as a light-driven chloride ion pump in the cell membrane of Halobacterium halobium. To address the role of the retinal chromophore in chloride ion transport, resonance Raman spectra have been obtained of the hR578 form of chromatographically purified halorhodopsin (hR). The close similarity of the frequencies and intensities of the hR578 Raman bands with those of light-adapted bacteriorhodopsin (bR568) shows that the chromophore in hR578 has an all-trans configuration and that the protein environment around the chromophore in these two pigments is very similar. In addition, hR578 exhibits a Raman line at 1633 cm-1 which is assigned as the stretching vibration of a protonated Schiff base linkage to the protein based on its shift to 1627 cm-1 in D2O. The reduced frequency of the Schiff base stretching vibration compared with bR568 (1640 cm-1) is shown to result from a reduction of its coupling with the NH in-plane rock. This may be due to a reduction in hydrogen-bonding between the Schiff base proton and an electronegative counterion in halorhodopsin.