Early identification of mutant embryos prior to the manifestation of a defect facilitates the study of dysmorphogenesis. The In(l)lRk inversion was used as a cytogenetic marker to distinguish embryonic day 9 (D9) splotch (Sp) and splotch-delayed $(Sp sp{d})$ mouse mutants from heterozygous and wild-type littermates, and cellular aspects of abnormal neurulation and NCC migration were examined before inherent neural tube defects (NTDs) and deficiencies in neural crest cell (NCC) derivatives developed. In vitro analysis of NCC emigration from D9 neural tube explants revealed a delay in the release of NCCs from mutant neural tubes compared to controls, suggesting that the primary effect of the mutation was intrinsic to the neuroepithelium. Immunofluorescent localization of the neural cell adhesion molecule (N-CAM) antibody in situ demonstrated an increased intensity of antibody fluorescence in mutant tissue compared to controls, and further characterization by immunoblot analysis showed an altered embryonic N-CAM profile in both Sp and $Sp sp{d}$ mutants at D9 of gestation. The importance of N-CAMs in mediating cellular organization and communication has been well documented, supporting the idea that an alteration in this adhesion mechanism could result in the types of defects seen in splotch locus mouse mutants.

Cell adhesion molecules are known to play crucial roles in a variety of developmental processes. The neural cell adhesion molecule N-CAM is strongly implicated in neurulation and neural crest cell (NCC) migration and was thus studied in splotch (Sp) neural tube defect mutant embryos. At the 20 somite-stage of gestation day 9, Sp N-CAM was found to contain polysialic acid (PSA) side chains which are normally only present beginning at gestational day 11. Younger embryos at 12 and 14 somites also showed the presence of PSA on N-CAM, which was absent in controls. Enzymatic removal of PSA from N-CAM resulted in isoforms which migrated identically to PSA-free N-CAM isoforms in SDS-polyacrylamide gels. The post-translational modification of N-CAM appears to be the primary target of the Sp gene. In view of N-CAM's importance during development, an alteration at a critical stage is likely to result in the cascade of abnormalities seen in Sp mutants. / A new genotyping assay was also implemented for examination of N-CAM in Sp and other related wildtype strains.

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The spatial and temporal patterning of sequence specific transcription factors (SSTFs) contributes to cell type specification and organ formation during embryogenesis. Homeodomain transcription factors are evolutionally conserved among invertebrate and vertebrate animals. They are responsible for body segmentation and organogenesis. Lbx1 and Pitx2 both are homeodomain transcription factors contributing to SSTF pattern formation during multiple organ formations. We studied how homeodomain transcription factors regulate SSTF and non-SSTF genes in a population-specific manner using the Lbx1[superscript EGFP] and Pitx2[superscript LacZ] mouse models. We have studied the role of Lbx1 in dorsal horn interneuron specification and Pitx2 in forelimb muscle formation. The two top non-SSTF target genes, NPY and Chmp2b, of Lbx1 are studied for expression pattern and potential neuronal function in neural tube. The T box, Hox gene families and Pax genes were identified as Pitx2 target genes via microarray analysis and their expression pattern were analyzed in forelimb. The expression domains of signaling molecules were altered in absence of Pitx2, suggesting that Pitx2 played a general role in pattern formation in forelimb mesenchyme. / Graduation date: 2013

This project was the first study of the genetics and embryo-pathology of exencephaly in a partially inbred mouse stock, SELH/Bc. Exencephaly was found in 17% of SELH fetuses. Analysis of day 8-9 gestation embryos indicated that SELH embryos were collectively normal in general development, but delayed in neural tube closure relative to overall or general development compared to two normal strains of mice, ICR/Be and SWV/Bc. Exencephaly was observed to be caused by a failure of fusion of the cranial neural folds in the mesencephalon region in SELH. All SELH embryos appeared to be abnormal in their pattern of cranial neural tube closure. They fail to make initial contact at the prosencephalon/mesencephalon junction region of the cranial neural folds (the first fusion in the cranial neural folds in normal embryos). SELH embryos, fused their anterior neural folds via an alternate (possibly passive) mechanism compared to normal strains of mice (SWV/Bc, and ICR/Be), by fusing the folds in a "zipper-like" fashion from the rostral base of the prosencephalon. This closure of the neural tube in genetically liable embryos by an abnormal sequence of events suggests a new model for anterior neural tube closure failure. Liability to exencephaly appeared to be fixed in the SELH stock. Of the 53 SELH males tested, all produced exencephaly. SELH animals were found to be heterogeneous in the frequency of exencephaly they produced, indicating that there are still genes segregating in the stock which affect the ability of embryos to complete anterior neural tube closure. Exencephaly in SELH does not appear to be caused by an autosomal dominant, sex-linked dominant or recessive, or simple autosomal recessive single gene, although F2, BCl, and BC2 exencephaly frequencies (after an outcross to ICR/Be) suggest that only a small number of genes are involved. A marked excess of female exencephalics was observed in SELH, F2, BCl, and BC2 fetuses. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Neural tube defects (NTDs) are a world health issue, affecting approximately 1 in every 1000 live births. These congenital defects arise from the improper closure of the neural tube during development, resulting in significant, life-threatening malformations of the central nervous system. Although it has been observed that supplementing women of child-bearing age with folates greatly decreases the chances of having an NTD affected baby, unfortunately these defects still occur. It is accepted that these complex disorders arise from a combination of genetic, environmental, and dietary influences. One such dietary influence is the one-carbon metabolism metabolite, homocysteine. Homocysteine is a byproduct of methylation reactions in the cell that exists in an inverse homeostasis with folate. Homocysteine can also undergo a transformation that allows it to then react with exposed lysine or cysteine residues on proteins, in a process known as N-homocysteinylation or S-homocysteinylation respectively. High levels of homocysteine have been long correlated with many disease states, including NTDs. One potential mechanism by which homocysteine confers its negative effects is through protein N-homocysteinylation. Here, a novel and high-throughput assay for N-homocysteinylation determination is described. This assay is shown to be accurate with mass spectrometry then shown to be biologically relevant using known hyperhomocysteinemia mouse models. This assay was then applied to a cohort of neural tube closure staged mouse embryos with two different genetic mutations that have previously been shown to predispose mice to NTDs. The genotypes explored here are mutations to the LRP6 gene and the Folr1 gene, both of which have been described as folate-responsive NTD mouse models. It was seen that maternal diet and embryonic genotype had the largest influence on the developmental outcome of these embryos; however, the inverse relationship between folate and homocysteine seemed to be established at this early time point, emphasizing the importance of the balance in one-carbon metabolism. One of these genes, LRP6, was then explored in a human cohort of spina bifida cases. Four novel mutations to the LRP6 gene were found and compared to the mouse model used in the previous study. One of the mutations found in the human population was seen to mimic that of the LRP6 mouse model, therefore expanding the potential of this NTD model. / text

The central nervous system is formed from epiblast precursor cells through Neurulation. Neural induction can be studied in its main aspects in vitro. However, the process is poorly understood, especially in regard to when and how a cell becomes specified, and then committed, to be a neural cell. It is, on the other hand, well established that neural formation requires absence or, inhibition of the BMP signalling both in vivo and in vitro. ID1 is a direct target of BMP signalling with major influence on in vitro neural differentiation. A cDNA library screen, looking for transcription factors negatively regulated by ID1, reported TWIST1, along with only two other proteins. Twist1 expression is upregulated during in vitro neural differentiation. Furthermore, targeted deletion of Twist1 has dramatic consequences on anterior neural development. Twist1 knock-out mice fail to form the closed neural tube in the prospective brain, followed by exencephaly and, early embryonic death. In this thesis I investigate the influence on in vitro neural differentiation of a TWIST1 constitutively active form, insensitive to ID1 inhibition. I report that this transcriptionally active TWIST1 accelerates neural differentiation, in vitro and, biases it, towards dorsal phenotypes. I provide, for the first time, evidence for Twist1 expression in the neural tissue, observed weakly in a restricted domain, temporally and spatially, in the dorsal part of the neural tube. I propose a new model for TWIST1 influence at this level. I also investigate how TWIST1 actions depend on levels of expression and dimer choice. I found that, TWIST1 can exert its neural modulating actions only at low levels, as high levels divert a cell fate towards non-neural lineages.