Abstracts - Society for Developmental Biology

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Deafness and Renal disease), which can include craniofacial defects. Our zebrafish gata3 mutants display the range of
HDR symptoms, the severity of which is genetically modulated. Mutants in one inbred genetic background have nearly
wild-type phenotypes, while a second background has profound craniofacial defects. We used a microarray approach to
identify pathways that regulate this variability. Twenty-two zebrafish genes with clear human homologues were
differentially expressed across mutant, but not wild-type, embryos from the “mild” and “severe” backgrounds. We found
13 “protective”genes, those upregulated in mild mutants and/or downregulated in severe mutants and 9 “deleterious”
genes, upregulated in severe mutants and/or downregulated in mild mutants. We show that insulin receptor a (insra) is a
protective gene both necessary and sufficient for mild phenotypes and activator of Hsp90 ATPase homolog 1 (ahsa1) is a
deleterious gene, necessary and sufficient for severe phenotypes. These response genes tend to be broadly expressed
suggesting that they may be general regulators of craniofacial defects. Indeed, altering insra or ahsa1 levels changes the
phenotypic severityof both platelet-derived growth factor aand collagen type 11 a2 mutants. Collectively, our results
implicate a response gene network in regulating the phenotypic severity of craniofacial defects.
Program/Abstract # 99
The molecular mechanisms of SP8 activity during craniofacial development
Kasberg, Abi; Brunskill, Eric; Potter, Steve, Cincinnati Children's Hospital Research Foundation, Cincinnati, United
States
Craniofacial abnormalities such as cleft palate affect 1 in 700 live births. Despite recent advances, the pathways
responsible for causing craniofacial disorders remain largely unknown. We have shown that the mutation of the Sp8 gene,
which encodes a zinc finger transcription factor, resulted in a dramatic absence of most facial structures. Sp8 mutants
exhibited a failure of fusion along the midline, excencephaly, hyperterlorism, and cleft palate. In fact, careful skeletal
analysis showed a dramatic loss of many neural crest and paraxial mesoderm derived cranial bones. Analysis of SOX9
expression revealed that Sp8 mutant neural crest located adjacent to the forebrain fail to differentiate and instead remain in
a multipotent state. Immunofluorescent experiments showed high SP8 expression within the neuroepithelium including the
anterior neural ridge, as well as the epidermal ectoderm. Specific loss of Sp8 in the anterior neuroepithelium produced
mice with severe craniofacialmal formations most similar to the global Sp8 mutants, suggesting that anterior
neuroepithelial Sp8 expression is critical during craniofacial development. Despite studies that have identified the roles of
SP8 during limb outgrowth, the molecular targets of SP8 during craniofacial development remain unknown. Expression
analysis at E9.5 indicated that Gli2, Gli3, Fgf8 and Fgf17 are reduced in the mutant anterior neural ridge signaling center.
Gene expression analysis indicated a large reduction in the expression of Fgf17 in the olfactory pit of E10.5 mutants, but
no apparent change in Fgf8. Of interest, the results also showed increased Smoothened in E10.5 mutants. The gene
expression data coupled with the hypertelorism phenotype suggested an upregulation of SHH signaling in Sp8 mutants.
Remarkably, embryonic exposure to cyclopamine, a SHH inhibitor, resulted in significant rescue of the craniofacial
phenotype in Sp8 mutants. This suggests that elevated SHH signaling in the Sp8 mutants is a key effector of the mutant
phenotype. It is interesting to note that FGF signaling has previously been reported to be able to repress the SHH pathway.
Therefore, the data suggests that during craniofacial development SP8 might normally activate FGF signaling, which in
turn represses SHH signaling.
Program/Abstract # 100
Regulation of jaw development by LAR receptor protein tyrosine phosphatases
Stewart, Katherine, McGill University, Montreal, Canada
Malformations of the lower jaw are associated with many developmental syndromes, and may additionally result in
secondary defects of the oral cavity, including cleft palate. As such, understanding the normal patterning and specification
of the mandible, as well as the etiology of secondary defects, may provide important therapeutic opportunities to children
born within this spectrum of disorders. Recently we have demonstrated a requirement for the leukocyte antigen related
(LAR)- family of receptor protein tyrosine phosphatases (RPTPs) in craniofacial development. Embryos lacking both
RPTPσ and LAR exhibit micrognathia (small lower jaw), cleft palate, exencephaly (open neural tube) and open eyes at
birth. We have currently extended that to include abnormalities in cranial bones and soft tissues derived from the first
branchial arch, including defects in bones of the palate and mandible, as well as abnormal tongue shape. We have
determined that cleft palate occurs secondary to abnormal mandible formation as initial palatal shelf outgrowth appears
normal at E12.5, whereas misspecification of cartilage and bone in the anterior mandible is already apparent. However, the
initial stages of craniofacial development, including the population of the first branchial arch by cranial neural crest cells,
appear normal. Interestingly, concomitant loss of RPTPσ and LAR within the first branchial arch results in aberrant FGF
signaling activity, potentially altering the patterning and subsequent differentiation of the mandibular arch tissue.