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The history of developmental and genetic analysis in the mouse has made it the model of choice for studying mammalian embryogenesis. Presently lacking is a simple technique for efficiently analyzing early mouse mutant phenotypes in utero. We demonstrate application of a real-time imaging method called ultrasound backscatter microscopy for visualizing mouse early embryonic neural tubes and hearts. This method was used to study live embryos in utero between 9.5 and 11.5 days of embryogenesis, with a spatial resolution close to 50 microns. Ultrasound backscatter microscope images of cultured embryos made it possible to visualize the heart chambers. This noninvasive imaging method was also used for analyzing a neural tube defect. The midhindbrain deletion associated with a null mutation of the Wnt-1 protooncogene was easily recognizable on ultrasound backscatter microscope images of 10.5- and 11.5-day embryos. Computer-generated volumetric renderings of the neural tube cavities were made from three-dimensional image data. This allowed a much clearer definition of the Wnt-1 mutant phenotype. These imaging techniques should be of considerable use in studying mouse development in utero.
We used the phosphatase substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6- chloro-4-[3H]-quinazolinone, with standard alkaline phosphatase-mediated immunohistochemical techniques, to visualize a number of antibodies that bind to adult zebrafish retinal tissue. This compound, known as the ELF (enzyme-labeled-fluorescence) phosphatase substrate, produces a precipitate that fluoresces at approximately 500-580 nm (bright yellow-green). We show that the precipitated product from the ELF phosphatase substrate has a number of characteristics that make it superior to fluorescein-labeled secondary reagents. The staining produced with the ELF substrate is much more photostable than that produced by fluorescein-labeled secondary reagents, thus allowing time to examine, focus, and photograph the ELF-labeled tissue under high magnification. Moreover, the ELF precipitate exhibits a Stokes shift of greater than 100 nm, a characteristic that has enabled us to overcome the problem of distinguishing signal from background in this autofluorescent tissue. In addition, we show that the ELF product's large Stokes shift makes the ELF substrate ideal for multicolor applications.
One of the first intercellular signalling events in the vertebrate embryo leads to mesoderm formation and axis determination. In the mouse, a gene encoding a new member of the TGF-beta superfamily, nodal, is disrupted in a mutant deficient in mesoderm formation (Zhou et al., 1993, Nature 361, 543). nodal mRNA is found in prestreak mouse embryos, consistent with a role in the development of the dorsal axis. To examine the biological activities of nodal, we have studied the action of this factor in eliciting axis determination in the zebrafish, Danio rerio. Injection of nodal mRNA into zebrafish embryos caused the formation of ectopic axes that included notochord and somites. Axis duplication was preceded by the generation of an apparent ectopic shield (organizer equivalent) in nodal-injected embryos, as indicated by the appearance of a region over-expressing gsc and lim1; isolation and expression in the shield of the lim1 gene is reported here. These results suggest a role for a nodal-like factor in pattern formation in zebrafish.
The ELF alkaline phosphate substrate can be used to fluorescently label a wide variety of biological targets. This substrate yields a bright, photostable yellow-green fluorescent precipitate at the site of enzymatic activity. ELF labeling can be as much as 40 times as bright and hundreds of times as photostable as labeling with conventional fluorophores and yields signals capable of very fine submicroscopic resolution. Signal development is also extremely rapid, making the signal amplification technology well suited for applications such as RNA in situ hybridization.
An antibody was used to detect antigens in zebrafish that appear to be homologous to the frog homeodomain-containing protein XlHbox 1. These antigens show a restricted expression in the anteroposterior axis and an anteroposterior gradient in the pectoral fin bud, consistent with the distribution of XlHbox 1 protein in frog and mouse embryos. In the somitic mesoderm, a sharp anterior limit of expression coincides exactly with the boundary between somites 4 and 5, and the protein level fades out posteriorly. A similar, graded expression of the antigen is seen within the series of Rohon-Beard sensory neurons of the CNS. We also immunostained the mutant spt-1 ('spadetail'), in which the trunk mesoderm is greatly depleted and disorganized in the region of XlHbox 1 expression. The defects stem from misdirected cell movements during gastrulation, but nervertheless, newly recruited cells that partially refill the trunk mesoderm express the antigen within the normal span of the anteroposterior axis. This finding suggests that the mutation does not delete positional information required for activation of the XlHbox 1 gene.