Modulation of Eomes Activity Alters the Size of the Developing Heart: Implications for In Utero Cardiac Gene Therapy

Abstract

Congenital heart disease is the most prevalent cause of infant morbidity and mortality in developed countries. The mechanisms responsible for many specific types of congenital cardiac malformations are strongly associated with gene abnormalities. However, at this time no strategies for gene therapy of the various congenital heart malformations have been investigated. In the present studies we focus on Eomesodermin (Eomes), a T-box transcription factor expressed in developing vertebrate mesoderm. Although Eomes is required for early mesodermal patterning and differentiation, the role of Eomes in cardiac development is unknown. In the present studies we demonstrate that Eomes is expressed in the developing heart, with a pronounced myocardial distribution in the Xenopus ventricle during late cardiac development. Using either a conditional dominant-interfering approach (GR-Eomes--engrailed) or an Eomes-activating approach (GR-Eomes-VP16) we demonstrate that manipulating Eomes activity during late cardiac development can either suppress ventricular development (GR-Eomes-enR) or increase ventricular myocardial size (GR-Eomes-VP16). Thus, a potential gene therapy approach for treating both congenital ventricular hypoplasia (e.g., the hypoplastic left heart syndrome) and hypertrophic cardiomyopathy is hypothetically implicit from the present results.

Tbr2 Is Essential for Hippocampal Lineage Progression from Neural Stem Cells to Intermediate Progenitors and Neurons

“…The following plasmids were separately injected into the ventricles of individual embryos: ires-GFP control, Tbr2 -ires-GFP, Tbr2 -VP16 (Ryan et al, 2004), and Tbr2 -engrailed (enR)(Ryan et al, 2004). Embryos were electroporated with paddle electrodes across the cerebrum, targeting the developing hippocampal field.…”

Wnt2 is a direct downstream target of GATA6 during early cardiogenesis

“…Of the relatively small number of up-regulated genes identified in our screen, some are known to be involved in the regulation of heart development, such as BMP4, Runx1, Eomes and Msx2, (Abdelwahid et al, 2001;Gattenlohner et al, 2003;Ryan et al, 2004). In addition, many of the upregulated genes are known to be expressed within the developing heart, although their functions have yet to be elucidated, such as Wnt2, Nr0b1 and Gas6 (Monkley et al, 1996;Bae et al, 1996;Rees et al, 1999).…”

Eomesodermin Requires Transforming Growth Factor-β/Activin Signaling and Binds Smad2 to Activate Mesodermal Genes

“…These two overlapping PCR products, containing the P445H mutation in the overlapping region, were then used with P399 plus P405 in a third "splice" PCR to obtain the full-length P445H sequence (1461 bp). The 1461-bp fragment was restriction digested and cloned 5Ј AflII-3Ј NotI in pKmRN3P (10). The resulting cDNA plasmid, DNxS2P445H/pKmRN3P, was linearized using SfiI and synthetic capped mRNA was synthesized as previously described (9) using a Megascript T3 kit (Ambion).…”

“…The GR PCR product was digested overnight with SmaI, and ligated into vector plasmid pBluescript RN3P (9), creating the plasmid p3ЈHGR (10). p3ЈHGR contains a Kozak sequence at its 5Ј end (ACAGCC-ACCATG, including initiator methionine; underlined in 5ЈSR-GR), and a "PG 4 P hinge" region at its 3Ј end, in-frame with the GR cDNA (9, 10) (underlined in 3ЈSP-GR).…”