engleski

Development of the caudal part of the neural tube in the mouse mutants splotch and truncate

The morphogenesis of the caudal part of the embryo includes formation of a mass of undifferentiated cells in the posterior end of the tail, referred as 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). Development of the caudal part of the neural tube in truncate and splotch mouse mutants, where tail development is affected, was analyzed. 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. 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 morphology of the tail bud and irregularities of the caudal ends of the axial structures. Truncate homozygous embryos exhibited partially disturbed development of the notochord. In both 11.5 and 12.5 days old embryos, notochord was fragmented. In the parts of the tail, where notochord was missing, neural tube showed a variable morphology. Near the base of the tail neural tube morphology was comparable to the controls while in the distal regions it lacked floor plate and developing spinal ganglia were displaced in ventral direction. Occasionally, in the tip of the tail extensive cell death was present and secondary neurulation was disturbed. The observed malformations indicated that the normal sequence of tail development required coordinated interactions among tail axial structures. In splotch primary defect of the neural tube affected development of the tail bud and the neighboring structures, while opposite to that in truncate embryos discontinuities of the notochord changed the morphology of the neural tube.

mouse; neural tube; splotch; truncate

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