Has2 expression in heart forming regions is independent of BMP signaling

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Abstract

Heart septation and valve malformations constitute the most common birth defects. These cardiac structures arise from the endocardial cushions through dynamic interactions between cells and the extracellular matrix (cardiac jelly). Targeted deletion of the hyaluronan synthase-2 (Has2) gene in mice results in an absence of cardiac jelly and endocardial cushions, a loss of vascular integrity, and embryonic death at E9.5. Despite the requirements for Has2 and its synthetic product hyaluronan (HA) in the developing cardiovascular system, little is known about the normal expression pattern of Has2 or the factors regulating Has2 gene transcription during development. Bmp signaling is an important regulator of cardiac myogenesis, and is also important for endocardial cushion formation. The current study defines the embryonic expression pattern of Has2 and explores the regulation of Has2 gene expression by Bmp signaling. In situ hybridization studies demonstrate dynamic Has2 expression patterns during myocardial cell development and cardiac tube formation, formation of the cardiac endocardial cushions, and cushion invasion by valve primordial cells. Despite overlapping regional expression of Bmp2 in the late gastrula anterior lateral endoderm and Has2 in the adjacent cardiogenic mesoderm, application of noggin-expressing CHO cells beneath the endoderm failed to perturb normal Has2 expression. Thus, in contrast to many genes expressed in the heart forming region, regulation of Has2 in the cardiogenic mesoderm is independent of Bmp signaling.

Section snippets

Expression of Has2 mRNA during early avian embryogenesis

Whole mount in situ hybridization was used to define Has2 expression in the developing chick embryo (Fig. 1). At HH St. 4 Has2 expression was observed diffusely in the anteriorly migrating hypoblast adjacent to and anterior to Hensen's node and in the primitive streak. By HH St. 6, Has2 mRNAs were localized in a peripheral ring extending bilaterally from the posterior third of the primitive streak anteriorly in two parallel stripes on either side of the streak and the neuroectoderm, and finally

Discussion

Has2-produced hyaluronan (HA) is a critical component of the extracellular milieu during vertebrate development; in particular, its requirements during heart morphogenesis are well-recognized (Baldwin et al., 1994, Bernanke and Markwald, 1979, Camenisch et al., 2000, Haddon and Lewis, 1991). Despite this, little is known regarding Has2 expression and regulation in the developing cardiovascular system. In this report, we define the expression of Has2 during early avian embryonic development and

Embryo collection

Fertilized white leghorn chicken eggs were incubated at 37 °C and embryos were collected between Hamburger Hamilton (HH) stages 4–24, corresponding to embryonic (E) days 0.5–4 (Hamburger & Hamilton, 1951). Embryos were either fixed overnight in 4% paraformaldehyde (PFA) for in situ hybridization or cultured ex vivo (described below). Timed FVBN mouse matings were established with the morning of vaginal mucous plug defined as E0.5. Embryos were removed from euthanized females at the indicated

Acknowledgements

We would like to thank the PANDAs for their dedication to improving the health of children. This work was supported by NHLBI P01 HL063926, R01 HL074070 and R01 HL 077493.

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