Background: Several Cre reporter strains of mice have been described, in which a lacZ gene is turned on in cells expressing Cre recombinase, as well as their daughter cells, following Cremediated excision of a loxP-flanked transcriptional "stop" sequence. These mice are useful for cell lineage tracing experiments as well as for monitoring the expression of Cre transgenes. The green fluorescent protein (GFP) and variants such as EYFP and ECFP offer an advantage over lacZ as a reporter, in that they can be easily visualized without recourse to the vital substrates required to visualize β-gal in living tissue.

Lysophosphatidic acid (LPA) exerts a variety of biological responses through specific receptors: three subtypes of the EDG-family receptors, LPA 1 , LPA 2 , and LPA 3 (formerly known as EDG-2, EDG-4, and EDG-7, respectively), and LPA 4 /GPR23, structurally distinct from the EDG-family receptors, have so far been identified. In the present study, we characterized the action mechanisms of 3-(4-[4-([1-(2-chlorophenyl)ethoxy]carbonyl amino)-3-methyl-5-isoxazolyl] benzylsulfanyl) propanoic acid (Ki16425) on the EDG-family LPA receptors. Ki16425 inhibited several responses specific to LPA, depending on the cell types, without any appreciable effect on the responses to other related lipid receptor agonists, including sphingosine 1-phosphate. With the cells overexpressing LPA 1 , LPA 2 , or LPA 3 , we examined the selectivity and mode of inhibition by Ki16425 against the LPA-induced actions and compared them with those of dioctyl glycerol pyrophosphate (DGPP 8:0), a recently identified antagonist for LPA receptors. Ki16425 inhibited the LPA-induced response in the decreasing order of LPA 1 Ն LPA 3 Ͼ Ͼ LPA 2 , whereas DGPP 8:0 preferentially inhibited the LPA 3 -induced actions. Ki16425 inhibited LPA-induced guanosine 5Ј-O-(3-thio)triphosphate binding as well as LPA receptor binding to membrane fractions with a same pharmacological specificity as in intact cells. The difference in the inhibition profile of Ki16425 and DGPP 8:0 was exploited for the evaluation of receptor subtypes involved in responses to LPA in A431 cells. Finally, Ki16425 also inhibited LPA-induced longterm responses, including DNA synthesis and cell migration. In conclusion, Ki16425 selectively inhibits LPA receptor-mediated actions, especially through LPA 1 and LPA 3 ; therefore, it may be useful in evaluating the role of LPA and its receptor subtypes involved in biological actions.Lysophosphatidic acid (LPA) has been shown to elicit diverse biological actions, including Ca 2ϩ mobilization, change in cAMP accumulation, change in cell shape and motility in association with actin rearrangement, and proliferation in a variety of cell types (Moolenaar, 1999;Contos et al., 2000;Ye et al., 2002). Extracellular LPA has also been shown to be involved in certain diseases, such as atherosclerosis and cancer (Xu et al., 1995(Xu et al., , 2001Siess et al., 1999;Maschberger et al., 2000). LPA was first thought to be released from activated platelets; however, a major part of extracellular LPA has been shown to be produced from lysophosphatidylcholine by lysophospholipase D, which was previously called autotaxin (Sano et al., 2002;Tokumura et al., 2002;Umezu-Goto et al., 2002). The concentration of plasma LPA is about 100 nM, and its serum concentration can be as high as 5 M (Sano et al., 2002). LPA increases low-density lipoprotein during its oxidation, activates endothelial cells (Siess et al., This work was supported in part by a research grant grants-in-aid for scientific research from the Japan Society for the Promotion of Science and by research gr...

We demonstrate wavefront sensorless aberration correction in a two-photon excited fluorescence microscope. Using analysis of the image-formation process, we have developed an optimized correction scheme permitting image-quality improvement with minimal additional exposure of the sample. We show that, as a result, our correction process induces little photobleaching and significantly improves the quality of images of biological samples. In particular, increased visibility of small structures is demonstrated. Finally, we illustrate the use of this technique on various fresh and fixed biological tissues.

While GDNF signaling through the Ret receptor is critical for kidney development, its specific role in branching morphogenesis of the epithelial ureteric bud (UB) is unclear. Ret expression defines a population of UB "tip cells" distinct from cells of the tubular "trunks," but how these cells contribute to UB growth is unknown. We have used time-lapse mosaic analysis to investigate normal cell fates within the growing UB and the developmental potential of cells lacking Ret. We found that normal tip cells are bipotential, contributing to both tips and trunks. Cells lacking Ret are specifically excluded from the tips, although they contribute to the trunks, revealing that the tips form and expand by GDNF-driven cell proliferation. Surprisingly, the mutant cells assumed an asymmetric distribution in the UB trunks, suggesting a model of branching in which the epithelium of the tip and the adjacent trunk is remodeled to form new branches.

While it is clear that the normal branching morphogenesis of the ureteric bud (UB) is critical for development of the metanephric kidney, the specific patterns of branching and growth have heretofore only been inferred from static images. Here, we present a systematic time-lapse analysis of UB branching morphogenesis during the early development of the mouse kidney in organ culture. Metanephric primordia from Hoxb7/GFP transgenic embryos were cultured for 3-4 days, and GFP images of the UB taken every 30 min were assembled into movies. Analysis of these movies (available as )revealed that the UB is a highly plastic structure, which can branch in a variety of complex patterns, including terminal bifid, terminal trifid, and lateral branching. To examine kinetic parameters of branching and elongation, skeletal representations of the UB were used to measure the number of segments and branch points and the length of each segment as a function of time and of branch generation. These measurements provide a baseline for future studies on mutant kidneys with defects in renal development. To illustrate how these quantitative methods can be applied to the analysis of abnormal kidney development, we examined the effects of the MEK1 inhibitor PD98059 on renal organ cultures and confirmed a previous report that the drug has a specific inhibitory effect on UB branching as opposed to elongation.