Modeling Biological Networks

• IV.6.vii Subproject 7 – The Identification and Characterization of Transcriptional Networks in Early Cardiovascular Development
  • IV.6.vii.a Introduction
  • IV.6.vii.b Background and Significance
  • IV.6.vii.c Preliminary Result
    • IV.6.vii.c.1 Clones for in vitro RNA synthesis
    • IV.6.vii.c.2 Xenopis laevis embryonic heart microarray
  • IV.6.vii.d Specific Aims
  • IV.6.vii.e Research Plan
    • IV.6.vii.e.1 Aim 1
    • IV.6.vii.e.2 Aim 2
    • IV.6.vii.e.3 Aim 3
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We currently have clones for all of the relevant transcription factors described in this project. All of these genes have been inserted into proven vectors designed for in vitro mRNA synthesis, and confirmed to be functional. Additionally, we have a variety of useful markers of cardiovascular differentiation, including Cardiac Troponin I, MLC2, and XMsr.

To block the function of the entire family of tinman-related genes, two dominant inhibitory mutants were created through the substitution of a conserved Leucine residue with a Proline between helix 2 and helix 3 of the homeodomain in both XNkx2-3 and 2-5, and were named XNkx2-3LP and XNkx2-5LP respectively. Expression of either XNkx2-3LP or XNkx2-5LP mRNA in the presumptive heart regions results in the complete loss of cardiac differentiation (Grow and Krieg, 1998).

To study gene expression during cardiovascular development requires a microarray. We selected clones from a recently created Xenopus laevis embryonic heart library constructed in the lab of Dr. P. Krieg at the University of Arizona. 2,496 of these clones have been sequenced. Our pilot embryonic heart microarray, KEH1, includes 4,608 unsequenced clones from the library, the sequenced clones, 96 cloned genes of interest (Nkx2-5, GATA-4, GATA-5, etc.), internal housekeeping controls (ODC, β-actin, Ef1α, etc.), and external controls (Alien Spot Report from Stratagene). Test hybridizations with Xenopus laevis adult heart RNA have likewise confirmed the quality of this microarray (data not shown).

In October of 2002 we added an additional 8,448 unsequenced clones to the existing set and printed a KEH2 microarray with over 15,000 features. While the use of unsequenced clones will include the redundant representation of many genes, a study of the sequenced clones suggests that KEH2 should contain at least 12,000 different genes.

1. The generation of an in vivo temporal profile of heart field gene expression, based on expression profiling of GATA-4-injected animal caps from specification to early differentiation.
2. The creation of a hypothetical cardiogenic transcriptional network model from the expression profiles from the overexpression and suppression of genes known to be involved in cardiogenesis.
3. Testing and refining of the cardiogenic transcriptional network model through the further characterization of implicated genes, whole mount in situ hybridization, and morphological effect on heart development.