Organogensis

VI.1 COORDINATORS
VI.2 PARTICIPANTS
VI.3 SUMMARY
VI.4 INTRODUCTION
VI.5 SPECIFIC AIMS
VI.6 BACKGROUND AND SIGNIFICANCE
VI.7 THEORETICAL FRAMEWORK
VI.8 PRELIMINARY RESULTS
VI.9 RESEARCH DESIGN AND METHODS

• VI.9.iv Cardiovascular Development
  • VI.9.iv.a Overview
  • VI.9.iv.b Preliminary Results
  • VI.9.iv.c Hypothesis
  • VI.9.iv.d Methodology
    • VI.9.iv.d.1 Assess the role of BMP-10 in enhancing embryonic cardiomyocyte proliferation in vitro
    • VI.9.iv.d.2 Overexpress BMP-10 in mouse myocardium to define the role of BMP-10 in cardiac growth and trabeculation
  • VI.9.iv.e Modeling of Cardiac Trabeculation
• [ Complete VI.9 Outline ]

VI.10 RELATIONSHIP TO CYTOSKELETON (PROJECT 2) AND BIOLOGICAL NETWORKS (PROJECT 1)
VI.11 TIMELINE

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VI.9.iv.d Methodology:
VI.9.iv.d.1 Assess the role of BMP-10 in enhancing embryonic cardiomyocyte proliferation in vitro:

Our first goal is to test whether BMP-10 has promotes cardiomyocyte growth. Previous studies have demonstrated that cardiac trabeculation is closely associated with ventricular growth at midgestation: 1) Mutant mice with a hypoplastic ventricular wall (decrease in cardiac growth) always show a significant reduction of ventricular trabeculae (Maclellan and Schneider, 2000; Rossant, 1996). 2) Increase of cardiac growth by conotruncal banding or unilateral vitelline artery banding increases the number and thickness of ventricular trabeculae (Keller, 1995).

Growth regulation of cardiomyocytes and ventricular trabeculation-compaction in the developing ventricular myocardium, must depend on one or more key signaling molecules to regulate the spatio-temporal expression of growth factors. Such molecules must show unique expression patterns during ventricular trabeculation-compaction and must have a growth regulatory function. Using cDNA differential display, we found that BMP-10 is up-regulated in FKBP12-deficient hearts. It expresses only in ventricular trabeculae and changes in time. Because BMP-10 is up-regulated in FKBP12-deficient hearts, and also because BMP-10 expression is up-regulated during ventricular trabeculation and down-regulated during compaction, we hypothesize that BMP-10 is a growth promoting factor. Since BMP-10 protein is not available currently, we are unable to supplement BMP-10 in culture media to directly assess its effect on the proliferation of cardiomyocytes. Instead, we will use two in vitro culture systems, a neonatal cardiomyocyte growth assay using CMV-BMP-10 stably transfected NIH3T3 cells and an in vitro cardiogenic assay using embryonic stem cells, to test the ability of BMP-10 to promote cardiomyocyte proliferation.

VI.9.iv.d.2 Overexpress BMP-10 in mouse myocardium to define the role of BMP-10 in cardiac growth and trabeculation.

BMP-10 expression in ventricles is prolonged and up-regulated in the FKBP12-deficient mice. To determine if forced overexpression of BMP-10 in myocardium induces abnormal ventricular structure, we will use the atrial natriuretic factor (ANF) promoter to drive BMP-10 expression. We will analyze the resulting phenotype histologically to compare with FKBP12 mutants.

The FKBP12 null mutation may lead to the abnormal expression of BMP-10 because FKBP12 affects BMP signaling. FKBP12-deficient fibroblast cells appear to have enhanced TGF-β signaling and a defect in cell cycle progression (Aghdasi et al., 2001). The expression pattern of BMP-10, indicates that altered BMP-10 expression plays a key function in transforming functional defects into a developmental defect in FKBP12-deficient mutants (i.e., the prolonged BMP-10 expression is responsible for the abnormal trabeculation and growth in FKBP12 mutant heart).

We will test this hypothesis by generating BMP-10-knockout and BMP-10/FKBP12 compound mutant mice and assess the resulting cardiac phenotypes. Our second strategy is to overexpress BMP-10 in the myocardium using a transgenic approach and determine if BMP-10 overexpression induces overgrown ventricular trabeculae. The idea is to mimic the situation in FKBP12 deficient mutants (i.e., BMP-10 expression is prolonged and up-regulated). Overexpressor transgenic mice are commonly generated by introducing a minigene, which contains a spatially and/or temporally specific promoter and a coding sequence of interest, into the mouse genome. We will use the human ANF promoter which restricts expression to both atria and ventricles in mouse embryonic heart (Field, 1993).

We will follow standard procedures to create the ANF-BMP-10 transgenic mice. A minigene, including the mouse BMP-10 cDNA and a mouse protamine (mp1) sequence. The mp1 sequence provides an intron as well as a 3' polyadenylation sequence. BMP-10 is under the transcriptional control of the cardiac-restricted ANF promoter. The resulting ANF-BMP-10 vector will comprise the ANF promoter, the mouse BMP-10 cDNA coding region with the transcription initiation site, a flag-tag placed at 3' of BMP-10 fragment, and the mp1-3'-untranslated region with a polyadenylation site. The flag-tag will allow us to use immunohistochemistry or Western blot to diagnosis the BMP-10 expression in transgenic heart and cells isolated from transgenic mice. Pronuclear injection and embryo transfer will be performed in Indiana University mouse transgenic core laboratory directed by Dr. L. Field.

VI.9.iv.e Modeling of Cardiac Trabeculation:

While the Shou laboratory has been involved primarily in experimental approaches, we have begun discussions with other Consortium members on the modeling of cardiac trabeculation using the CPM. Our identification of the spatiotemporal expression of growth and differentiation factors such as BMP-10 under conditions of normal cardiogenesis and impaired cardiogenesis in the FKBP12 mutant mouse, in conjunction with earlier studies on expression of BMP-2/BMP-4 (Nakajima et al., 2000), BMP-6 (Solloway et al., 1998), and BMP-5 and BMP-7 (Solloway and Robertson, 1999) provide a basis for modeling the relevant morphogen fields, which in the case of myocardial development regulate cell proliferation. Moreover, as with limb morphogenesis (see Section VI.9.ii, above), the extracellular matrix protein fibronectin increases at the onset of trabeculation (Icardo and Manasek, 1983), providing a potential haptotactic component that the CPM is ideally suited to handle. Finally, an existing biomechanical model of cardiac trabeculation (Taber and Zahalak, 2001) lacks specific reference to growth-regulating morphogen and haptotactic fields, although it provides a plausible account of mechanical aspects. We plan to devise a more comprehensive model taking both aspects into account using the CPM.