Due to the internal nature of mammalian development, much of the research performed is of a static nature and depends on interpolation between stages of development. This approach cannot explore the dynamic interactions that are essential for normal development. While roller culture overcomes the problem of inaccessibility of the embryo, the constant motion of the medium and embryos makes it impossible to observe and record development. We have developed a static mammalian culture system for imaging development of the mouse embryo. Using this technique, it is possible to sustain normal development for periods of 18-24 h. The success of the culture was evaluated based on the rate of embryo turning, heart rate, somite addition, and several gross morphological features. When this technique is combined with fluorescent markers, it is possible to follow the development of specific tissues or the movement of cells. To highlight some of the strengths of this approach, we present time-lapse movies of embryonic turning, somite addition, closure of the neural tube, and fluorescent imaging of blood circulation in the yolk sac and embryo.

Congenital megacolon develops in transgenic mice that overexpress the homeobox-containing gene, Hoxu-4. The current study was done to identify abnormalities of the terminal colon that might account for the phenotype. The terminal bowel of transgenic mice was compared with that of control and lethal spotted (ZslZs) mice, a strain in which megacolon also develops. The terminal colon of the transgenic mice contained fewer ganglia than that of controls, but was hypoganglionic, rather than aganglionic like that of ZslZs mice. The neurons present in the adult transgenic colon were significantly increased in size and a subset of very large neurons (>40 p. m in maximum diameter) were observed. Electron microscopic studies of young adult transgenic mice revealed that the ganglia and nerves of the myenteric plexus had the ultrastructure of extraenteric peripheral nerve rather than that of the enteric nervous system (ENS). The myenteric ganglia in the transgenic animals contained Schwann cells associated with a basal lamina that enveloped axons completely and individually, instead of glia. Although collagen is excluded from the ganglia and thin nerve fibers of the normal ENS, a collagen-containing endoneurium surrounded each of the axon-Schwann cell units of the abnormal nerve fibers of the transgenic colon. Some of the neurons of the transgenic mice were located in these nerve bundles rather than in ganglia. There were also smooth muscle abnormalities in the terminal bowel of the transgenic mice. Wide gaps were present in the longitudinal muscle of the transgenic mice; these gaps contained ganglia that were in contact with the adventitia. These longitudinal smooth muscle cells were more irregular than those of controls and they contained fewer puncta adherens; moreover, a larger proportion of the volume of the cytoplasm of transgenic smooth muscle cells was occupied by organelles. Finally, an extensive thickening and reduplication of the basal lamina surrounding the smooth muscle cells of the muscularis mucosa was observed in the transgenic colon and resembled 0 1993 WILEY-LISS, INC.that found in ZslZs mice. These data suggest that both smooth muscle and the innervation of the terminal bowel of neonatal Hoxu-4 transgenic mice are structurally abnormal. Although some of the abnormalities seen in Hoxu-4 transgenic mice are similar to those which arise in Zslls mice, the two conditions are not identical. In both animals, the data are consistent with the hypothesis that the defects arise as a result of a defective interaction between the precursors of enteric neurons and smooth muscle. o 1993 Wiley-Liss, Inc.

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The intron of the mouse Hoxa-4 gene acts as a strong homeotic response element in Drosophila melanogaster leg imaginal discs. This activity depends on homeodomain binding sites present within a 30 bp conserved element, HB1, in the intron. A similar arrangement of homeodomain binding sites is found in many other potential homeotic target genes. HB1 activity in Drosophila imaginal discs is activated by Antennapedia and more posterior homeotic genes, but is not activated by more anterior genes. Testing a reporter gene construct with mutated binding sites in mouse embryos shows that HB1 is also active in the expression domains of posterior Hox genes in the mouse neural tube.

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