Abstracts - Society for Developmental Biology

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dizygotic twin studies have shown that they can exhibit different severities of alcohol-induced developmental defects
suggesting that genetic variation influences FASD severity. The Raldh2 gene in the human population shows the existence
of variants that differ by an amino acid, which in turn might affect the enzymatic activity. Here, we use Xenopus embryos
to determine the genetic/enzymatic predisposition to FASD by analyzing the different Raldh2 isoforms in the presence of
ethanol in vivo. We use our retinoic acid signaling rescue assay and compare between the different human Raldh2 alleles
to determine which alleles modify the enzymatic activity such that it increases or decreases the risk for the embryo to
develop FASD as a result of ethanol exposure. The results of these studies will increase our knowledge on the effects of
ethanol on embryonic development, will demonstrate the feasibility of the approach and will establish the experimental
conditions to screen for genetic predisposition to FASD.
Program/Abstract # 296
Human BMP receptor mutations causing fibrodysplasia ossificans progressiva lead to ligand-independent receptor
activation in zebrafish embryos
Mucha, Bettina E, Division of Human Genetics and Molecular Biology, and Division of Biochemical Genetics,
Philadelphia, United States; Zinski, Joseph; Hashiguchi, Megumi (Department of Cell and Developmental Biology,
Philadelphia, PA, United States); Shore, Eileen M (Departments of Orthopedic Surgery and Genetics, and the Center for
Research in FOP and Related Disorders, Philadelphia, PA, United States); Mullins, Mary C (Department of Cell and
Developmental Biology, Philadelphia, PA, United States)
The human dominant disease fibrodysplasia ossificans progressiva (FOP) is caused by mutations in the Alk2 BMP
receptor. Most patients with classic FOP carry the mutation Arg206His, changing the tertiary structure of the intracellular
Alk2 GS domain and causing hyperactivation. Two mutations of Gly328 (Gly328Trp/Glu) cause a more severe FOP
phenotype, but it is unclear how these mutations cause disease. We used the zebrafish embryo to investigate the activity of
these codon 328 FOP mutations. In zebrafish, BMP signaling plays an important role in patterning embryonic dorsalventral
axial tissues. mRNA misexpression studies are therefore an excellent tool to study the effect of these mutations on
BMP signaling in vivo. Alk2 mRNA containing either Arg206His (cFOP), or a variant mutation (Gly328Trp, Gly328Glu)
was injected into wild type (WT) or BMP7 deficient embryos at the one-cell stage. Phenotypes were assessed at 24 hpf for
ventralization or dorsalization. Early and mid-gastrula embryos were analyzed for phospho-Smad1/5 expression and for
markers of ventral and dorsal tissues, respectively. Mutations in the G328 codon cause ventralization of WT embryos and
rescue BMP7 deficient embryos, but to a lesser extent than the cFOP mutation. Phospho-Smad1/5 expression is increased
in all injected embryos, but more profoundly in the cFOP group. In summary, although in silico modeling has not
demonstrated changes in the tertiary structure of the G328 mutant Alk2 receptors, evidence from this study suggests that
G328 mutants also confer increased receptor activation and ligand-independent signaling via Smad phosphorylation.
Future studies will investigate the mechanism through which alk2 G328 mutations cause hyperactivation.
Program/Abstract # 297
GATA4 A new biomarker for Rhabdomyosarcoma ?
Nemer, Georges; Haidar, Wiam; Saab, Raya, American University of Beirut, Beirut, Lebanon; Nemer, Mona (University
of Ottawa, Ottawa, Canada)
Background: We have previously shown that GATA4, a zinc finger cardiac-enriched transcription factor, is expressed in
subsets of undifferentiated skeletal muscle cells where it acts as a negative regulator of myogenesis. GATA4 is expressed
in the widely used c2c12 mouse myoblast cell line where a rapid decrease of the endogenous GATA4 binding activity
occurs after switching cells to a differentiation-promoting medium. These findings identify GATA4 as a novel regulator of
skeletal myogenesis and suggest that GATA4 and MyoD play pivotal and opposing roles during the premyogenicmyogenic
transition. Objective: We aim at establishing a role for GATA4 in rhabdomyosarcoma the skeletal-related form
of cancer, which is the most prevalent soft tissue sarcoma in children. The ultimate goal being to show that modulation of
GATA4 might open the way towards designing an efficient therapeutical regimen to treat the disease. Results: We have
shown that GATA4 is expressed in both embryonal (JR-1) and alveolar (Rh30 and Rh41) rhabdomyosarcoma cell lines. In
addition serum withdrawal did affect the expression profile of GATA4 in the cells but not as much as in the C2C12 model.
We characterized a new microRNA that could regulate GATA4 expression in both Rhabdomyosarcoma cells lines. Finally
we showed that GATA4 is differentially expressed in rhabdomyosarcoma biopsies collected from different patients.
Conclusion: The results showed for the first time an expression of GATA4 in Rhabdomyosarcoma and hypothetically
linked this expression to the inability of the cells to differentiate into skeletal muscle. This goes along with our previous
results on C2C12 whereby over expression of GATA4 blocks their differentiation into myotubes. We hope to establish
GATA4 as a novel biomarker for Rhabdomyosarcoma and link its expression profile and intensity to the severity of the
phenotype and response to treatment.