Kersti K. Linask

N-Cadherin Is Required for the Differentiation and Initial Myofibrillogenesis of Chick Cardiomyocytes

To investigate initial stages of cardiac myofibrillogenesis, heart-forming mesoderm was excised from stage 6 chick embryos and explanted on fibronectin-coated coverglasses. The explants were fixed at various times and immunofluorescently stained with antibodies to N-cadherin, alpha-catenin, beta-catenin, sarcomeric myosin, pan and sarcomeric alpha-actinins, or rhodamine phalloidin. After 7 hours in culture the cells appeared epithelial. N-cadherin, alpha- and beta-catenin, pan alpha-actinin, and F-actin showed circumferential localization at cell borders. No cells in the explant were positive for sarcomeric alpha-actinin or sarcomeric myosin at this stage. Sarcomeric alpha-actinin and sarcomeric myosin were detected around 10 hours after plating. Sarcomeric alpha-actinin initially appeared as small beads along thin actin filaments. Mature Z-lines began to be organized at 20 hours, at the same time the cells started to contract. When the rat monoclonal antibody NCD-2, which inhibits N-cadherin function, was added to the culture at early time-points, cells lost cell-cell contacts, became spherical in shape, and contained tangled actin fibers. The expression of sarcomeric alpha-actinin and sarcomeric myosin was suppressed. These results indicate that 1) the precardiac mesoderm explant cells differentiate and form well-organized myofibrils in culture, 2) N-cadherin-mediated cell-cell interactions are necessary for early differentiation of cardiomyocytes and organization of myofibrils.

Differential expression of flectin in the extracellular matrix and left‐right asymmetry in mouse embryonic heart during looping stages

A novel extracellular matrix protein flectin (250 kD M(r)) shows specific left-right asymmetric expression before and throughout the looping process during heart development in avian embryos [Tsuda et al., 1996]. Flectin is a candidate molecule to provide directionality to the looping process in the avian model. In this study on mouse embryonic heart development, flectin is shown to be developmentally regulated and to be expressed in a specific asymmetric fashion, but in a different pattern from that observed in avian hearts. The molecules involved in development tend to be the same, but timing of expression, modulation, and asymmetry are different. In the mouse embryo, flectin is expressed symmetrically when the cardiogenic plate is formed. As looping progresses, flectin expression becomes asymmetric. There is right side predominance at the outflow tract and left side predominance at the ventricular portion of the tubular heart. The left side predominance of flectin develops in an anteroposterior direction, while right side predominance of the outflow tract remains relatively unchanged. These differential expression patterns of flectin decrease once the looping process is completed. After looping, flectin becomes restricted to the epicardium and subepicardial extracellular regions. In inv/inv mice, a known mouse model for human situs inversus, in which the directionality of heart looping is inverted, flectin expression pattern is mirror image of that of normal mouse embryos during looping stages. Our study indicates that, in the mouse, flectin shows a specific asymmetric expression pattern after initiation of heart looping and that this asymmetric expression pattern is related to the directionality of looping. The remodeling of the extracellular matrix (ECM) including specific flectin expression begins with the looping process. This morphogenetic change of the ECM coincides with the differentiation of each region of the tubular heart.

Sodium-calcium exchanger (NCX-1) and calcium modulation: NCX protein expression patterns and regulation of early heart development

Ouabain‐induced inhibition of early heart development indicated that Na/K‐ATPase plays an important role in maintaining normal ionic balances during differentiation of cardiomyocytes (Linask and Gui [1995] Dev Dyn 203:93–105). Inhibition of the sodium pump is generally accepted to affect the activity of the Na+‐Ca++ exchanger (NCX) to increase intracellular [Ca++]. These previous findings suggested that Ca++ signaling may be an important modulator during differentiation of cardiomyocytes. In order to identify a connection between heart development and NCX‐mediated Ca++ regulation, we determined the embryonic spatiotemporal protein expression pattern of NCX‐1 during early developmental stages. In both chick and mouse embryos, NCX‐1 (the cardiac NCX isoform) is asymmetrically expressed during gastrulation; in the right side of the Hensens node in the chick, in the right lateral mesoderm in the mouse. At slightly later stages, NCX‐1 is expressed in the heart fields at comparable stages of heart development, in the chick at stage 7 and in the mouse at embryonic day (ED) 7.5. By ED 8 in the mouse, the exchanger protein displays a rostrocaudal difference in cardiac expression and an outer curvature‐inner curvature ventricular difference. By ED 9.5, cardiac expression has increased from that seen at ED8 and NCX‐1 is distributed throughout the myocardium consistent with the possibility that it is important in regulating initial cardiac contractile function. Only a low level of expression is detected in inflow and outflow regions. To substantiate a role for the involvement of calcium‐mediated signaling, using pharmacologic approaches, ionomycin (a Ca++ ionophore) was shown to perturb cardiac cell differentiation in a manner similar to ouabain as assayed by cNkx2.5 and sarcomeric myosin heavy chain expression. In addition, we show that an inhibitor of NCX, KB‐R7943, can similarly and adversely affect early cardiac development at stage 4/5 and arrests cardiac cell contractility in 12‐somite embryos. Thus, based upon NCX‐1 protein expression patterns in the embryo, experimental Ca++ modulation, and inhibition of NCX activity by KB‐R7943, these results suggest an early and central role for calcium‐mediated signaling in cardiac cell differentiation and NCXs regulation of the initial heartbeats in the embryo.

Initial Retinoid Requirement for Early Avian Development Coincides With Retinoid Receptor Coexpression in the Precardiac Fields and Induction of Normal Cardiovascular Development

Vitamin A requirement for early embryonic development is clearly evident in the gross cardiovascular and central nervous system abnormalities and an early death of the vitamin A‐deficient quail embryo. This retinoid knockout model system was used to examine the biological activity of various natural retinoids in early cardiovascular development. We demonstrate that all‐trans‐, 9‐cis‐, 4‐oxo‐, and didehydroretinoic acids, and didehydroretinol and all‐trans‐retinol induce and maintain normal cardiovascular development as well as induce expression of the retinoic acid receptor β2 in the vitamin A‐deficient quail embryo. The expression of RARβ2 is at the same level and at the same sites where it is expressed in the normal embryo. Retinoids provided to the vitamin A‐deficient embryo up to the 5‐somite stage of development, but not later, completely rescue embryonic development, suggesting the 5‐somite stage as a critical retinoid‐sensitive time point during early avian embryogenesis. Retinoid receptors RARα, RARγ, and RXRα are expressed in both the precardiac endoderm and mesoderm in the normal and the vitamin A‐deficient quail embryo, while the expression of RXRγ is restricted to precardiac endoderm. Vitamin A deficiency downregulates the expression of RARα and RARβ. Our studies provide strong evidence for a narrow retinoid‐requiring developmental window during early embryogenesis, in which the presence of bioactive retinoids and their receptors is essential for a subsequent normal embryonic development. Dev. Dyn. 1998;213:188–198.

Effects of antisense misexpression of CFC on downstream flectin protein expression during heart looping

Dextral looping of the heart is regulated on multiple levels. In humans, mutations of the genes CFC and Pitx2/RIEG result in laterality‐associated cardiac anomalies. In animal models, a common read‐out after the misexpression of laterality genes is heart looping direction. Missing in these studies is how laterality genes impact on downstream morphogenetic processes to coordinate heart looping. Previously, we showed that Pitx2 indirectly regulates flectin protein by regulating the timing of flectin expression in one heart field versus the other (Linask et al. [ 2002 ] Dev. Biol. 246:407–417). To address this question further we used a reported loss‐of‐function approach to interfere with chick CFC expression (Schlange et al. [ 2001 ] Dev. Biol. 234:376–389) and assaying for flectin expression during looping. Antisense CFC treatment results in abnormal heart looping or no looping. Our results show that regardless of the sidedness of downstream Pitx2 expression, it is the sidedness of predominant flectin protein expression in the extracellular matrix of the dorsal mesocardial folds and splanchnic mesoderm apposed to the foregut wall that is associated directly with looping direction. Thus, Pitx2 can be experimentally uncoupled from heart looping. The flectin asymmetry continues to be maintained in the secondary heart field during looping. Developmental Dynamics 228:217–230, 2003.

Application of Plastic Embedding for Sectioning Whole-Mount Immunostained Early Vertebrate Embryos

Development of tissues and organs relies on the constant interplay of intracellular events and molecules in the microenvironment of the cells. Dynamics of patterning of molecules in a spatiotemporal manner become important in understanding any developing system. Immunohistochemistry has become an important technique for the study of specific proteins in the early embryo. Ability to localize molecules in the embryo with the greatest resolution possible provides much critical information regarding intracellular and extracellular localization of specific molecules and changes in their patterning over time. For example, an apical or basal cellular localization of a molecule may be critical in relation to its function. Thus, the definition and correlation of the spatiotemporal patterns of protein expression in the developing embryo with events of morphogenesis may give useful information on the possible roles of these molecules. Such analyses will provide a more precise basis for further experimentation. To obtain the highest resolution possible in antigen localization, a pre-embedding immunohistochemical technique followed by whole-mount plastic embedding and sectioning at 1 μm has been modified to make it useful for analyzing events of early chick and mouse morphogenesis in specific regions of the embryo. The description of the pre-embedding immunohistochemistry method and embedding procedure provided is an adaptation of the technique of Franklin and Martin (1) and others (2,3). This technique has been used with good results for analyses of whole chick embryos up to 48 h of development and for whole mouse embryos up to 9.5 d of gestation and isolated organs (i.e., heart) up to day 15 of gestation. For presentation purpose, this chapter is divided into two parts, dealing with chick embryos and mouse embryos, respectively.

Calcium Channel Blockade in Embryonic Cardiac Progenitor Cells Disrupts Normal Cardiac Cell Differentiation

We suggest that characterization of processes involved in differentiation of the pluripotential cardiac precursor cells in their embryonic environment will permit identifying pathways important for induction of diverse stem cells toward the cardiac phenotype. Phenotypic characteristics of cardiac cells are their contractile and electrical properties. The objective of the present study was to define whether calcium (Ca(++)) has a regulatory role in the pluripotential precursor cell population during commitment into cardiomyocytes. We used the chick embryo model because of ease of staging the embryos and visibility of heart development. Using the Ca(++) indicator Fluo-3/acetoxymethyl and confocal microscopy, we demonstrated the existence of higher free Ca(++) levels in the cardiogenic precursor cells than in neighboring cell populations outside of the heart fields. Subsequently, gastrulation stage 4/5 chick embryos were set up in modified New cultures in the medium containing either the L-type Ca channel blocker, diltiazem, or the N-type Ca channel inhibitor, ω-conotoxin. The embryos were incubated for 22-24 h during which time the control embryos developed, beating looping hearts. At the end of incubation, exposure to the L-type channel blockade with diltiazem resulted in an inhibition of cardiomyogenesis in the most posterior, uncommitted, part of the heart fields. N-type channel blockade with ω-conotoxin was less intense. Cells in the most anterior cardiogenic regions that were already committed at time of exposure continued to differentiate. Thus, regulation and maintenance of normal cytosolic Ca levels are necessary for the early steps of cardiomyocyte specification and commitment leading to differentiation.