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Microarray Analysis of Genes Orchestrating Craniofacial Development
Leo R. Otake, MD, PhD and John A. Persing, MD.
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).
Synopsis of 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.
Cell Differentiation
The locus wnt5a was found to be down regulated 1.85 fold in KO vs wild type littermates. Wnt5a has been shown to be
required for cranial neural crest cell migration in zebra fish (Barallo-Gimeno et al., 2003); in mice the midline interfrontal
suture is neural crest derived in contrast to the sagittal suture which has neural crest cells interposed between the parietal
bones which are derived from mesoderm (Wang et al. 2005).
Locus Forkhead Box G1 (FoxG1) was down regulated 1.50 fold in KO littermates. This locus has been implicated in the
development of the telencephalon in mouse (Storm et al. 2006).
The locus endothelin receptor B which was upregulated 1.54 fold in KO mice had been implicated in congenital anomalies
associated with Hirschprung’s Disease; of note these anomalies include craniofacial, skeletal, and limb anomalies (Moore,
SW, 2006).
Extracellular Space
Expression levels of biglycan (bgn) and decorin (dcn), are decreased1.74 and 1.87 fold, respectively, in KO mice. As loss of
bgn and dcn have been shown to lead to osteogenic cell apoptosis as well as to decreased sequestration of tgf-1 in the ECM
(Bi et al., 2005), the loss of these two mediators of bone development in the KO mice would be consistent with the lack of
calvarial development which is observed.
Interestingly, a 1.67 fold decrease in the fibrillin locus was noted in KO mice; this locus was shown to be part of a genomic
deletion resulting in craniofacial abnormalities and mental delay in a human patient (Garcia-Minaur et al., 2005). In contrast,
overexpression of the transthyretin gene in mice was found to result in fronto-nasal hypoplasia (Noguchi et al., 2002); this
locus is upregulated 1.60 fold in the KO mouse and would be consistent with its hypoplastic phenotype.
Extracellular Matrix
The expression level of procollagen XIa1 was 2.55 fold decreased in the KO mouse. Interestingly, a defect in collagen XIa1
has been associated with human Marshall Syndrome, the presentation of which includes craniofacial abnormalities (Griffith
et al., 1998).
Receptor Activity
The expression of EphA4 was decreased 1.89 fold in KO mice. The expression of EphA4 has been shown to be associated
with patterning of cranial neural crest cells (Ishii et al, 2005). Given that in mice the midline interfrontal suture is neural
crest derived in contrast to the sagittal suture which has neural crest cells interposed between the parietal bones which are
derived from mesoderm (Wang et al. 2005), alterations in the expression of this gene would be consistent with the calvarial
defect noted in the KO mouse phenotype.
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
Future Investigations
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.
References
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