engleski

Morphological analysis of mouse mutants with disturbed development of the caudal part of the embryo

The morphogenesis of the caudal part of the embryo includes formation of a mass of undifferentiated cells in the posterior end of the tail, named the tail bud. In the cranial direction the tail bud is continuous to the neural tube, notochord and the tail gut, while paraaxial mesenchyme segments in the somites (1). As the mammalian embryo is difficult to access within the uterus for experimental manipulation, mouse mutants are invaluable tool for investigation of developmental mechanisms including those that take place in the caudal part of the mouse embryo. Development of the caudal part of the truncate and splotch mouse mutant, where tail development is affected, was analyzed. Splotch was first described in 1947, as a spontaneous mouse mutation of Pax3 gene affecting embryo development (2). Splotch homozygotes die during embryo development showing disorders of neural tube (spina bifida, exencephaly), abnormalities of neural crest cells (absence of sympathetic and spinal ganglia, Schwann cells, melanocytes and cells which migrate to the heart) and defects in somite structure. Corresponding mutation in humans is Waardenburg syndrome: defects of head development (deafness), hand musculature and localized absence of pigmentation. Truncate is a mouse strain with disorders in embryo development caused by spontaneously arisen mutation. Heterozygotes exhibit normal phenotype, while homozygotes have truncated tail, due to the partially absent notochord (3) Splotch and truncate homozygous embryos aged 11.5 and 12.5 days were isolated. They were fixed in a mixture of 1% paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer and postfixed in 1% osmium tetroxide. One group of embryos was embedded in Durcopan (Fluka) and serial semithin sections, perpendicular to the longitudinal tail axis, were obtained, stained with toluidine blue and examined by light microscopy. Second group of embryos after fixation and postfixation were dehydrated in ascending mixtures of ethanol and amyl acetate to 100% amyl acetate, dried with liquid CO2, sputter coated with gold and observed in the scanning electron microscope. Splotch 11.5 days old embryos showed open neural tube in the most cranial third of the embryo tail. However, development of other axial structures was not influenced by abnormal neural tube, as all axial structures were normal and continuous with the tail bud. In contrast, splotch 12.5 days old embryos showed open neural tube accompanied with disturbed development of other axial structures and disturbed morphology of the tail bud. It is suggested that a longer presence of open neural tube influenced development of all structures caudal from the defect. Truncate homozygous embryos exhibited partially disturbed development of the notochord. In both 11.5 and 12.5 days old embryos, notochord was not continuous with the tail bud, but with the ventral neuroepithelium. Also, discontinuous or, even doubled notochord was found. Lack of the notochord in the caudal part of the tail was accompanied with disturbed development of the neural tube and the somites. Neural tube was smaller in diameter, it lacked lumen and massive cell dead in neuroepithelium was found. In parts of the tail without notochord, somites were connected in the midline of the tail. The observed malformations indicate that the normal sequence of tail development requires the coordinated interactions among the tail axial structures, which can be related to pathogenesis of spina bifida in humans.

mouse; embryo; tail

nije evidentirano