engleski

Morphology of spina bifida in splotch mouse mutants

Spina bifida is one of the most common congenital defects in humans, as it affects 0, 1% of newborns. Different types of this malformation, from the benign spina bifida occulta to the disabling myelomeningocela have common embryological base: defect of the neural tube closure during embryo development. Spina bifida affects lumbosacral region, i.e. region of junction between primary and secondary neurulation, which involve different morphogenetic mechanisms. In the same way development of the cranial part of the embryo differs from the caudal part. The cranial part is derived from three germ layers created during gastrulation (primary mechanism of embryo development), and the caudal from a mass of undifferentiated cells in the posterior end of the tail, named tail bud. In the cranial direction cells of the tail bud differentiate into neural tube, notochord and the tail gut, while paraaxial mesenchyme segments in the somites (secondary mechanism of embryo development) (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. The main goal of this work was to morphologically describe development of spina bifida in splotch mouse mutant. Splotch was first described in 1947, as a spontaneous mouse mutation of Pax3 gene affecting embryo development (2, 3). 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. Splotch homozygous embryos aged from 10.5 to 12.5 days were isolated. 11.5 and 12.5 days old embryos were recognized by visible open neural tube. As 10.5 days old homozygotes were not morphologically recognizable, PCR based genotypization of splotch embryos was developed. It included isolation of DNA by isopropanole - ethanole method, constructing of primers specific for mutated and normal Pax3 sequence and polymerase chain reaction. Embryos of all stages 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. 11.5 days old splotch embryos showed open neural tube in the middle part of the embryo tail (fig.1 and 2) which is in some cases combined with exencephaly 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. To find exact location of the onset of spina bifida, 10.5 days old splotch mouse embryos were isolated. It allowed us to find that spina bifida in splotch mutants originates in the caudal part of the neural tube. Described finding that spina bifida originates in the region of secondary neural tube, i.e tissue derived from the tail bud, may lead to explanation that spina bifida is a defect of transition zone between structures derived by primary and secondary body formation mechanisms. The observed malformations in mice give insight in the morphogenetic mechanisms and consequences of spina bifida in humans.

spina bifida; splotch; embryo; mouse

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