In collaboration with the Department of Neurology, AZM, Maastricht, an experimental chick model has been designed to simulate the development of a dermal sinus. Implantation of amniotic tissue in the closing neural tube results in a local persistent connection between the epidermis and the neural tube. In collaboration with The Universitair Medisch Centrum St. Radboud, Nijmegen, chick embryos have been treated with the methylation inhibitor adenosine dialdehyde (AdOx) which inhibits closure of the caudal neural tube. Gene expression analysis of these samples is currently being performed. In collaboration with Wageningen University, methods have been developed to determine chick blastoderm quality upon storage.
Our main focus is on the unraveling of the aetiology of SBA in the Axial defects mouse. The phenotype of this partially dominant model for lumbosacral neural tube defects highly resembles the human clinical picture. The elucidation of the Axd aetiology is thought to provide new prospects for genetic counselling and dietary intervention. The Axd research is divided into three major components: 1) identification of the Axd gene by positional cloning, 2) reduction of the penetrance of the Axd phenotype by dietary intervention and 3) analysis of the pathogenesis resulting in SBA in Axd.
Ad 1) a compelling Axd candidate gene has been identified by linkage analysis in affected embryos using a genome wide scan. This candidate gene is a close homologue of the previously identified Grhl3 gene which is mutated in the curly tail mouse, a model that has been extensively investigated by our research group. A molecular mechanism emerges in which the expression of two homologous transcription factors needs to be tightly regulated in order to allow proper closure of the caudal neural tube. Preliminary results indicate a genetic interaction between the curly tail and Axial defects models, corroborating the nature of the Axd gene.
Ad 2) a mouse embryo culture has been optimized in order to address the mechanism underlying the reported beneficial effect of methionine supplementation on the penetrance of the phenotype.
Ad 3) the axial level at which the neural tube closure defect first occurs has been accurately delineated by scanning electron microscopy analysis of 75 embryos derived from heterozygous crosses. Preliminary immunohistochemical results suggest that both apoptosis and proliferation rates are increased in mutant embryos.
In addition, mouse embryonic fibroblasts derived from wild type and mutant embryos were analysed for sensitivity to culturing in low methionine concentration, migration potential and proliferation rate. No differences could be detected. Global DNA methylation was determined using the methylation status of B1 SINE repetitive elements as read out. No difference could be detected between wild type and mutant embryonic genomic DNA methylation. Methionine supplementation by either intraperitoneal injection of pregnant dams or by food fortification did not reduce the penetrance of the Axd phenotype. Low breeding performance at the animal facility could have been related to this. The subsequent adjustment of the regular diet at the facility greatly improved general fertility but the supplementation experiments were not repeated as we now focus on in vitro supplementation which can be better controlled.
Selected publication
Matheson SF, Hu KQ, Brouns MR, Sordella R, VanderHeide JD, Settleman J.
Distinct but overlapping functions for the closely related p190 RhoGAPs in neural development.
Dev Neurosci. 2006;28(6):538-50.
Research group
Henny W.M. van Straaten, PhD, project leader
Madeleine R. Brouns, PhD
Technicians
Johan W.M. Hekking
Els Terwindt
Students
Carla Langejan, undergraduate student
Marjolein Caron, undergraduate student
Floortje Haest, undergraduate student
Toon Boselie, undergraduate student
Ivon van der Linden, graduate student UMC St. Radboud, Nijmegen
Inge Reijrink, graduate student, Wageningen University