Mechanisms of cardiovascular pleiotropy and buffering in Cited2 deficiency. (360G-Wellcome-083228_Z_07_Z)

Aims: Our aim is to understand the developmental and molecular mechanisms of pleiotropy and buffering in Cited2 deficiency Key Goals: a) We will use conditional knockouts to identify the developmental mechanism of cardiac pleiotropy in Cited2 deficiency. Specifically, we will identify the spatial and temporal locus of Cited2 action in cardiovascular patterning and cell fate; and develop new tools to visualise conditionally deleted cells using EYFP for cell fate analysis. b) Use expression profiling to determine the molecular mechanism of pleiotropy and buffering in Cited2 deficiency. We will use a 2 x 4 x 3 fully-crossed factorial design, where the factors are Cited2 genotype (Cited2+/+ or Cited2 / ), strain (129S6, B6, 129S6XB6-F1, or B6X129S6-F1), and time (E7.5 , E8.5 or E9.5) to identify overdominant mechanisms that could explain heterosis, identify Cited2 target genes, and determine if variable target gene expression could explain heterosis-independent phenotypic variation. c) Analyse CITED2-protein interactions & upstream pathways to understand how they may buffer Cited2 deficiency. This will use proteomic techniques to identify CITED2-interacting proteins from ES cells, the effect of CHD-associated mutations on these interactions, investigate CITED2-T166 phosphorylation in vivo, and develop knockin alleles of T166N and FLAG-tagged CITED2 for in vivo studies.