Rhonda Rogers

Targeted inactivation of the sodium-calcium exchanger (Ncx1) results in the lack of a heartbeat and abnormal myofibrillar organization

Contraction of cardiac muscle is triggered by an intracellular buildup of Ca2+ during excitation‐contraction (E‐C) coupling. The Na+/Ca2+ exchanger (Ncx1) is highly expressed in cardiomyocytes and is thought to serve a housekeeping function by maintaining a low intracellular Ca2+ concentration. However, its role in E‐C coupling is controversial. To determine the precise role of Na+/Ca2+ exchange in development of the mammalian heart, we used gene targeting to delete Ncx1. Heterozygous mice are normal and fertile, whereas Ncx1‐null embryos are growth‐retarded and survive to 11.0 days postcoitum but lack a spontaneously beating heart. Moreover, normal heart morphogenesis (specification, looping, and chamber formation) occurred relatively normally within Ncx1‐null embryos. In addition, Ncx1‐nulls displayed relatively normal transient Ca2+ signals when electrically stimulated. This suggests that the Ca2+ delivery mechanism was fundamentally intact, and that Ncx1‐null cardiomyocytes can regulate intracellular Ca2+ concentrations despite the absence of Ncx1. However, ultrastructural analysis revealed that Ncx1‐null cardiomyocytes have a complete lack of organized myofibrils and Z‐lines when compared with normal littermates. These data demonstrate that Ncx‐1 is a Ca2+‐ gene that is essential for normal cardiomyocyte development and function and may serve as an animal model for functionally related human congenital heart defects.

Periostin (an osteoblast-specific factor) is expressed within the embryonic mouse heart during valve formation.

Periostin was originally isolated as a osteoblast-specific factor that functions as a cell adhesion molecule for preosteoblasts and is thought to be involved in osteoblast recruitment, attachment and spreading. Additionally, periostin expression has previously been shown to be significantly increased by both transforming growth factor beta-1(TGFbeta1) and bone morphogenetic protein (BMP)-2. Likewise the endocardial cushions that form within embryonic heart tube (embryonic day (E)10-13) are formed by the recruitment, attachment and spreading of endocardial cells into the overlying extracellular matrix, in response to secreted growth factors of the TGFbeta and BMP families. In order to determine whether periostin is similarly involved in heart morphogenesis, in situ hybridization and reverse transcription-polymerase chain reaction were used to detect periostin mRNA expression in the developing mouse heart. We show for the first time that periostin mRNA is expressed in the developing mouse embryonic and fetal heart, and that it is localized to the endocardial cushions that ultimately divide the primitive heart tube into a four-chambered heart.

Decreased neural crest stem cell expansion is responsible for the conotruncal heart defects within the Splotch (Sp2H)/Pax3 mouse mutant

OBJECTIVE Several mouse models of cardiac neural crest cell (NCC)-associated conotruncal heart defects exist, but the specific cellular and molecular defects within cardiac NCC morphogenesis remain largely unknown. Our objective was to investigate the underlying mechanisms resulting in outflow tract defects and why insufficient cardiac NCC reach the heart of the Splotch (Sp(2H)) mouse mutant embryo. METHODS For this study we used in vitro cell culture techniques, in vivo mouse-chick chimeras, BrdU cell proliferation labeling, TUNEL labeling to visualize apoptosis and the molecular markers AP-2, Wnt-1 and Wnt-3a to characterize NCC morphogenesis in vivo. RESULTS Expression of the NCC marker AP-2 revealed an extensive reduction in migratory NCC, however the rates of cell proliferation and apoptosis were unaffected, and do not account for the Sp(2H) NCC-associated heart defects. Further expression analysis revealed that Wnt-1, but not Wnt-3a, is expressed at decreased levels within Sp(2H) and that the cardiac NCC fail to undergo normal NC stem cell proliferative expansion prior to migration while still in the neural folds. However, when placed into a wild-type matrix or a tissue culture environment, the Sp(2H) cardiac NCC could migrate normally. Additionally, this reduced population of Sp(2H) NC stem cells do migrate properly within the Sp(2H) environment, as observed by neurofilament expression and cardiac innervation. CONCLUSIONS Taken together, all these data indicate that the Sp(2H) defect is intrinsic to the NC stem cells themselves and that there is a decrease in the number of pre-migratory cardiac NCC that form. It appears that this decrease in NCC number is the primary defect that ultimately leads to a lack of a cardiac NCC-derived Sp(2H) outflow tract septum.

Abnormal skeletogenesis occurs coincident with increased apoptosis in theSplotch (Sp2H) mutant: Putative roles forPax3 andPDGFR? in rib patterning

Although the importance of the transcription factor Pax3 has been identified in many embryological processes including neural crest cell migration, neural tube closure, and limb muscle formation, its role in proper formation of the ribs has not been well characterized. We have used the Splotch mouse which has a mutation in the Pax3 gene to determine what role Pax3 may play in rib morphogenesis. Homozygous Splotch embryos collected from days 13.5 to 16.5 of gestation displayed severe rib abnormalities including fusions at both the proximal and distal regions. Given its expression in the dermomyotome, we sought to determine when Pax3 expression in the Splotch mutant initially becomes abnormal and which rib segment progenitors may be affected. Prior to somite differentiation at 9.5 dpc (days post coitum), Pax3 is normally expressed in the somites; after differentiation, however, Pax3 expression is diminished in Splotch embryos. We also determined that significantly increased levels of apoptosis occur in the thoracic region by 11.0 dpc relative to wild‐type littermates. Because Patch mouse mutants which fail to express PDGF α‐receptor also have rib abnormalities, we sought to determine whether Pax3 may influence the expression of this receptor. By in situ hybridization, we determined that initially the expression of PDGFRα is normal in Splotch mutants at 10.0 dpc, but decreases by 12.5 dpc in the thoracic somite region, suggesting that Pax3 may act upstream of PDGFRα. Taken together, our results show that Pax3 expression is important for normal rib development and that increased apoptosis occurs in the thoracic muscles. We suggest that Pax3 may influence the expression of PDGFRα, and that the reduced and/or deficient thoracic muscles may contribute to the resulting rib abnormalities. Anat Rec 255:353–361, 1999.

Role of Sodium‐Calcium Exchanger (Ncx1) in Embryonic Heart Development

Abstract: Na+/Ca2+ exchanger (Ncx‐1) is highly expressed in cardiomyocytes, is thought to be required to maintain a low intracellular Ca2+ concentration, and may play a role in excitation‐contraction coupling. Significantly, targeted deletion of Ncx‐1 results in Ncx1‐null embryos that do not have a spontaneously beating heart and die in utero. Ultrastructural analysis revealed gross anomalies in the Ncx1‐null contractile apparatus, but physiologic analysis showed normal field‐stimulated Ca2+ transients, suggesting that Ncx‐1 function may not be critical for Ca2+ extrusion from the cytosol as previously thought. Using caffeine to empty the intracellular Ca2+ stores, we show that the sarcoplasmic reticulum is not fully functional within the 9.5‐dpc mouse heart, indicating that the sarcoplasmic reticulum is unlikely to account for the unexpected maintenance of intracellular Ca2+ homeostasis. Using the Ncx1‐lacZ reporter, our data indicate restricted expression patterns of Ncx1 and that Ncx1 is highly expressed within the conduction system, suggesting Ncx1 may be required for spontaneous pacemaking activity. To test this hypothesis, we used transgenic mice overexpressing one of the two known adult Ncx1 isoforms under the control of the cardiac‐specific a‐myosin heavy chain promoter to restore Ncx1 expression within the Ncx1‐null hearts. Results indicate that the transgenic re‐expression of one Ncx1 isoform was unable to rescue the lethal null mutant phenotype. Furthermore, our in situ results indicate that both known adult Ncx1 isoforms are coexpressed within the embryonic heart, suggesting that effective transgenic rescue may require the presence of both isoforms within the developing heart.

Segmental expression of aggrecan in the non-segmented perinotochordal sheath underlies normal segmentation of the vertebral column.

The embryonic vertebral column is derived from the unsegmented axial mesenchyme surrounding the notochord, and its development and differentiation are influenced by the notochord. The role of cartilage in determining the ultimate pattern of the segmental skeleton has been well documented, but a gene whose segmental expression corresponds to the pattern of the developing skeleton has yet to be identified. We show that chick aggrecan is initially expressed within the entire length of the notochord, and as development proceeds, aggrecan expression becomes restricted to the surrounding perinotochordal sheath in a segmental pattern, mirroring the differentiated somite pattern.