Morphometric analysis of the forebrain anomalies in the delayed Splotch mutant embryo

The early development of delayed Splotch mouse embryos was examined histologically using scanning electron microscopy and morphometric techniques. Embryos obtained from matings of mice heterozygous for the delayed Splotch gene exhibited a high incidence of lumbosacral (25%) or cephalic (7%) neural tube defects. The lumbosacral neural tube defects extended from the posterior neuropore region to the tip of the tailbud; cephalic neural tube closure defects were found in the hindbrain and midbrain regions. The frontal region of affected embryos was abnormal in that it was reduced in size, particularly in the developing midface. Histologically, the forebrain region of affected embryos appeared reduced, and the luminal surface of the neuroepithelium was often irregular and infolded. To quantify these alterations and to determine their contribution to the final form of the region, size and areal measurements were recorded and served as input for principal component and cluster analytic techniques. In affected embryos, significant reductions were found in lumen size, in neuroepithelial area but not thickness, and in overall area of forebrain but not hindbrain. Principal component analysis of data from unaffected embryos produced two factors, one containing hindbrain variables and the second forebrain variables; for the affected embryos, three factors were extracted. The first loaded on variables that measured the thickness and area of the neuroepithelium, the second on forebrain variables, and the third on hindbrain variables. Factor scores were then generated from a pooled analysis of normal and affected cases and were analyzed using cluster analysis. Three clusters were identified: one contained eight affected embryos with cephalic neural tube defects; another contained nine affected embryos with lumbosacral neural tube defects and five normal embryos; and the final cluster contained ten unaffected embryos. These results suggest a major role of the delayed Splotch gene on the neuroepithelium itself and support the suggested role of cerebrospinal fluid pressure in normal forebrain histogenesis.