The long term goal of this research is to elucidate a mechanistic model for the normal cell behaviors which occur in the lateral plate mesoderm prior to the formation of the limb bud and to specifically address the potential root causes of the limb defects exhibited in Holt-Oram Syndrome patients. Human genetic studies have linked Holt-Oram Syndrome to haplo-insufficiency in the T-box transcription factor Tbx5. This proposal will determine how gene dosage affects organogenesis at the cell behavior level, especially with regards to how directional cell migrations in the limb field are dependent upon the proper functions of tbx5 and its downstream effector genes. We will utilize state- of-the-art cell-labelin techniques combined with new technologies in computer-aided cell tracking analyses to characterize the cell movements of the entire lateral plate mesoderm-derived limb field prior to and during the formation of the limb bud. We will analyze these movements under wild-type conditions and under conditions in which the tbx5 is functionally knocked down to various levels, modeling the haplo-insufficiency effects observed in Holt-Oram Syndrome patients. In addition, these experiments will characterize the role of specific signaling molecules during pre-formation phases of limb bud development to investigate if they are being used as cell migration cues, and if so, whether experimental re-introduction of these signaling cues may be able to rescue the loss-of-limb phenotypes exhibited by Holt-Oram Syndrome patients.
This study seeks to establish a mechanistic model for the normal cell behaviors that contribute to the initial formation of the limb bud in order to better understand the root cause of birth defects of the arm or leg. The morphogenesis of the lateral plate mesoderm precursors of the limb bud will be analyzed under wild-type and altered levels of tbx5 gene function;defects in the TBX5 gene has been shown to lead to Holt-Oram Syndrome in human patients. We hypothesize that the debilitating upper arm defects exhibited by these patients may in part be due to the loss of signaling molecules used as directionality cues by specific limb bud precursors and that the experimental re-introduction of these signals may rescue the loss of limb phenotypes exhibited by Holt-Oram Syndrome patients.