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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IV.6.vii Subproject 7 - The Identification and Characterization of Transcriptional Networks in Early Cardiovascular Development:

This Subproject will use microarray techniques to generate data on vertebrates comparable to the Drosophila data in Subproject 6. It compares our knowledge of regulatory pathways in Xenopus with those in Drosophila. It also attempts to link the computational and genomic methods developed in Subprojects 1-6 with the generation of data required for the comprehensive models of organogenesis proposed in Project 3.

The cardiovascular system forms the vertebrate embryo's first functional organ. At present, however, the heart, while well studied, remains little understood. Much of what we know about early heart development was discovered through observation and embryonic manipulation.

Our goal is to identify the transcriptional pathways that control vertebrate cardiogenesis using a comprehensive set of gene expression experiments on the African Clawed Frog, Xenopus laevis. Computer modeling will generate hypothetical transcriptional networks, and follow-up experiments will refine this model.

At the current level of molecular detection, differentiation begins at around stage 27. At this time, the cells fated to contribute to the muscular layer of the heart, the myocardium, begin to express markers of terminal myocardial differentiation, such as cardiac troponin (TnIc), myosin heavy chain alpha (MHCα and myosin light chain 2 (MLC-2) (Drysdale et al., 1994; Logan and Mohun, 1993; O'Brien et al., 1993; Chambers et al., 1994).

Four families of transcription factors play critical early roles in the specification and differentiation of the heart: tinman-related genes (Tonissen et al.,1994; Evans et al., 1995; Newman et al., 2000; Newman and Krieg, 1998; Fu et al., 1998; Grow and Krieg, 1998): the GATA-4/5/6 subfamily (Laverriere et al., 1994; Kelley et al., 1993; Kuo et al., 1997; Molkentin et al., 1997; Gove et al., 1997), the SAP domain gene myocardin (Wang et al., 2001), and members of the MADS box transcription factor family (Chambers et al., 1992).

In Drosophila, the homeobox transcription factor tinman was found to be absolutely required for the formation of the dorsal vessel, the insect equivalent of the heart (Tonissen et al., 1994). The MADS box transcription factor, D-mef2, is required for the differentiation of all muscle cell types (Bour et al., 1995; Lilly et al., 1995). Multiple transcription factors regulate cardiogenesis. Tinman is required for D-mef-2 expression in the heart (Gajewski et al., 1997). Other experiments have shown that GATA-4, GATA-5 and Nkx2-5 can recognize regulatory elements in the genes encoding cardiac-specific proteins (Lee et al., 1998; Molkentin et al., 1994).