TheCspg2Gene, Disrupted in thehdfMutant, Is Required for Right Cardiac Chamber and Endocardial Cushion Formation

Abstract

The heart defect (hdf) mouse is a recessive lethal that arose from a transgene insertional mutation on chromosome 13. Embryos homozygous for the transgene die in utero by embryonic day 10.5 postcoitus and exhibit specific defects along the anterior-posterior cardiac axis. The future right ventricle and conus/truncus of the single heart tube fail to form and the endocardial cushions in the atrioventricular and conus/truncus regions are absent. Because the hdf mouse mutation provided the opportunity to identify a gene required for endocardial cushion formation and for specification or maintenance of the anterior most segments of the heart, we initiated studies to further characterize the phenotype, clone the insertion site, and identify the gene disrupted. Chromosome mapping studies first identified the gene, Cspg2 (versican), as a candidate hdf gene. In addition, an antibody recognizing a glycosaminoglycan epitope on versican was found to be positive by immunohistochemistry in the extracellular matrix of normal wild-type embryonic hearts, but absent in homozygous hearts. Expression analysis of the Cspg2 gene showed that the 6/8, 6/9, and 7/9 Cspg2 exon boundaries were present in mRNA of normal wild-type embryonic hearts but absent in the homozygous mutant embryos. DNA sequence flanking the transgene was used to isolate from a normal mouse library overlapping genomic DNA segments that span the transgene insertion site. The contiguous genomic DNA segment was found to contain exon 7 of the Cspg2 in a position 3' to the transgene insertion site. These four separate lines of evidence support the hypothesis that Cspg2 is the gene disrupted by the transgene insertion in the hdf mouse line. The findings of this study and our previous studies of the hdf insertional mutant mouse have shown that normal expression of the Cspg2 gene is required for the successful development of the endocardial cushion swellings and the embryonic heart segments that give rise to the right ventricle and conus/truncus in the outlet of the looped heart.

Versican Deficiency Significantly Reduces Lung Inflammatory Response Induced by Polyinosine-Polycytidylic Acid Stimulation

“…The development of a conditionally versican-deficient mouse strain has been pivotal to further study the role of versican in inflammation in vivo. Total gene disruption preventing versican production (35) or production of mutant versican missing exon 3 (39) causes heart development defects that result in embryonic or neonatal lethality in homozygous animals. Recently, a mouse strain with floxed versican exon 2 (Vcan e2 fl/fl ) was generated by Watanabe and colleagues who used it to demonstrate the importance of versican in joint development and TGF-␤-dependent chondrocyte differentiation (40) and, more recently, and in cancer progression (41).…”

“…Interfering with versican accumulation in this ECM also inhibits leukocyte adhesion in vitro, suggesting that versican and HA may form an immunomodulatory complex in response to viral lung infection (32)(33)(34). However, specific roles for versican in the regulation of pulmonary inflammatory responses are not yet well defined due to lack of versican knock-out animals, which are embryonically lethal due to defective cardiac development (35). In this study, we examine the formation of HA-and versican-enriched ECM in lungs of conditionally versican-deficient mice, developed recently in our laboratory, in response to poly(I:C) as a surrogate for viral infection.…”

Versican: a novel modulator of hepatic fibrosis

“…36,41 It is essential in development, wherein versican null mice die during embryogenesis. 42 Conditional knockout of versican has demonstrated its importance both during chondrogenesis and in binding the profibrotic cytokine TGF-β. 43 Versican is a major component of the early fibroproliferative matrix in lung fibrosis where its deposition is associated with fibroblast migration and proliferation, preceding collagen deposition.…”

A Hyaluronan Binding Link Protein Gene Family Whose Members Are Physically Linked Adjacent to Chrondroitin Sulfate Proteoglycan Core Protein Genes

“…Engineered and spontaneous loss-of-function mutations in the versican (2) and aggrecan genes (3), result in mid-gestational and early postnatal lethalities, respectively. This illustrates the fundamental importance of these two CSPGs and the HA-CSPG aggregates to normal development and tissue structure and function.…”