Specific Aims The objective of this project is to identify candidate interacting genes which are temporally differentially expressed during craniofacial development using the mouse animal model. The Affymetrix GeneChip Mouse Genome 430 2.0 Array has been utilized in this investigation. As the molecular underpinnings of craniofacial development are not well understood at the present time, modalities such as molecular diagnostics and gene therapy lie at early stages of development. A greater understanding of the genetic network involved in craniofacial development would be an asset towards elucidating the molecular mechanisms underlying craniofacial development in general as well as craniosynostosis in particular.
Significance Although genetic lesions associated with various congenital craniofacial syndromes have been identified, the interacting genetic networks/pathways that result in the syndromic craniosynostoses have not been clearly elucidated. The candidate human genes and mutations which have been associated with syndromes have been reported, including the homeobox protein MSX2 with Boston-type craniosyostosis, the family of fibroblast growth factor receptors (FGFR) type 1 (FGFR1) with Pfeifer type I, FGFR2 with Apert, Crouzon, and Pfeifer types I/II/III, FGFR3 with Apert/Acanthosis Nigricans and the transcription factor TWIST with Saethre-Chotzen.
A comprehensive examination allows for evaluation of the complex interplay of pathways which lead to the clinical phenotype. A compelling question lies in the observation that although Apert and Crouzon Syndromes are both associated with mutations in FGFR2, the clinical presentation on physical examination and temporal progression of craniosynostoses, as well as, mental functional outcome with Apert patients being more severely affected, differ so greatly; this suggests that alternative or differential dysregulation of similar pathways occurs downstream of the identified genetic lesion.
Experimental Design and Methods Dr. Hongbo Chi and Dr. Richard Flavell of the Department of Immunobiology, Yale School of Medicine; have developed a targeted, single locus gene knockout of the mouse MAP kinase kinase kinase 4 (MEKK4) gene. Mice which are homozygous for the gene knockout are viable to birth, but die shortly thereafter. These mice demonstrate severe derangements in craniofacial development, notably, the cranial vault fails to develop with attendant exencephaly and spina bifida. Additionally, the cortex fails to develop normally in the knockout mice. The development of such a striking craniofacial defect in the setting of a targeted, single gene knockout presents a unique opportunity to examine, using microarray technology, specific genetic pathways which have been disrupted.
Heads from E18 homozygous MEKK4 knockout and homozygous normal fetuses from the same mating were collected. Total cellular RNA was extracted and matched pairs of homozygous knockout and normal samples were applied to the Affymetrix mouse 430 v 2.0 total genomic microarray and quantitated using GeneSpring GX 7.3 (Agilent Technologies).
Initial Results Results from 2 pairs of Mekk4 wild-type and gene knockout E18 fetuses (total of 4 animals) are shown in table form (Table1). Microarray data were normalized and fold-changes in expression were determined and segregated into functional groups. Representative genes are presented if an alteration of expression in the given locus resulted in a reported phenotype in the literature relevant to craniofacial development.
Table 1: Functional Groupings of Genes with Altered Expression Levels
Cell Differentiation Wnt5a -1.85 Forkhead BoxG1 -1.50 Endothelin Recept B +1.54
Extracellular Space Biglycan -1.74 Dermatopontin -1.87 Decorin -1.55 Fibrillin -1.67 Endothelin Recept-B +1.54 Transthyretun +1.60
Extracellular Matrix Procollagen XIa1 -2.55 Fibrillin2 -2.34
Receptor Activity Eph Receptor A4 -1.89
Subsequent to the described examination of the gene expression profiles in the heads of the MEKK4 knockout mouse at the E18 stage of development, we propose to extend our analyses to compare expression profiles of other mutant mice with craniofacial defects. Starting with the initial state of absent calvarial vault development in the MEKK4 mouse, our objective is to gain a greater understanding of the repertoire of genes needed to orchestrate calvarial development in both a normal and abnormal synostotic state.