Expression of hLAMP-1-Positive Particles During Early Heart Development in the Chick.

Heart development requires coordinated activity of various factors, the disturbance of which can lead to congenital heart defects. Heart lectin-associated matrix protein-1 (hLAMP-1) is a matrix protein expressed within Hensen's node at Hamburger-Hamilton (HH) stage 4, in the lateral mesoderm by HH stages 5-6 and enhanced within the left pre-cardiac field at HH stage 7. At HH stages 15-16, hLAMP-1 expression is observed in the atrioventricular canal and the outflow tract. Also, the role of hLAMP-1 in induction of mesenchyme formation in chick heart has been well documented. To further elucidate the role of this molecule in heart development, we examined its expression patterns during HH stages 8-14 in the chick. In this regard, we immunostained sections of the heart during HH stages 8-14 with antibodies specific to hLAMP-1. Our results showed prominent expression of hLAMP-1-positive particles in the extracellular matrix associated with the pre-cardiac mesoderm, the endoderm, ectoderm as well as neuroectoderm at HH stages 8-9. After formation of the linear heart tube at HH stage 10, the expression of hLAMP-1-stained particles disappears in those regions of original contact between the endoderm and heart forming fields due to rupture of the dorsal mesocardium while their expression becomes confined to the arterial and venous poles of the heart tube. This expression pattern is maintained until HH stage 14. This expression pattern suggests that hLAMP-1 may be involved in the formation of the endocardial tube.

Antisense approaches for elucidating ranavirus gene function in an infected fish cell line.

Viral virulence/immune evasion strategies and host anti-viral responses represent different sides of the continuing struggle between virus and host survival. To identify virus-encoding molecules whose function is to subvert or blunt host immune responses, we have adapted anti-sense approaches to knock down the expression of specific viral gene products. Our intention is to correlate knock down with loss of function and thus infer the role of a given viral gene. As a starting point in this process we have targeted several structural and catalytic genes using antisense morpholino oligonucleotides (asMO) and small, interfering RNAs (siRNA). In proof of concept experiments we show the feasibility of this approach and describe recent work targeting five frog virus 3 genes. Our results indicate that both 46K and 32R, two immediate-early viral proteins, are essential for replication in vitro, and confirm earlier findings that the major capsid protein, the largest subunit of the viral homolog of RNA polymerase II, and the viral DNA methyltransferase are also essential for replication in cell culture.

Retinoic acid administration is associated with changes in the extracellular matrix and cardiac mesenchyme within the endocardial cushion.

Retinoic acid has been associated with a number of cardiac defects, some of which seem to be related to changes in the endocardial cushions. Studies in mice and older chick embryos have suggested that these defects may be associated with a decrease in mesenchymal cell formation within the cushion. In a previous report we showed that retinoic acid lowered the number of mesenchymal cells in a culture bioassay of mesenchyme formation and that this response was due to retinoic acid modifying the production of particulate matrix from the myocardium. In this study, we have extended these observations to the embryo by implanting a retinoic acid coated bead into the embryo and examined the effect on cardiac mesenchyme formation and in the production of the particulate matrix. In all cases the addition of retinoic acid resulted in a decrease in the number of mesenchymal cells invading the endocardial cushions. In addition retinoic acid increased the production of hLAMP-1 and fibronectin but not transferrin, confirming our earlier report. Finally, we measured the volume of the cushion and calculated the cell density of both the inferior and superior cushions. The results suggest that the superior cushion is more sensitive to retinoic acid treatment than the inferior cushion. Collectively, these results support our earlier work that suggests that the mechanism of retinoic acid cardiac abnormalities involves a disruption in the production of particulate matrix from the myocardium and a subsequent decrease in cardiac mesenchyme cells that results in a malformed cardiac cushions.

Retinoic acid inhibition of cardiac mesenchyme formation in vitro correlates with changes in the secretion of particulate matrix from the myocardium.

Retinoic acid has been associated with a variety of cardiac defects. A percentage of these defects are related to changes in the endocardial cushions. Studies in mice and older chick embryos have shown a decrease in mesenchymal cell formation attributable to retinoic acid and have suggested that retinoic acid was affecting the extracellular matrix. In this study we have tested the effect of retinoic acid on cardiac mesenchyme formation in vitro and then tested retinoic acid treated myocyte cultures for changes in the expression of hLAMP-1, fibronectin and transferrin members of the particulate matrix that is required for mesenchyme formation. Initial experiments tested the effect of retinoic acid on mesenchymal cell formation first in atrioventricular canal and outflow tract explant cultures and then in AV endothelial monolayer cultures using myocyte conditioned media or the particulate matrix fraction from retinoic acid treated myocyte cultures. In all cases, mesenchymal cell formation was suppressed while no suppression was observed when MyoCM was included with retinoic acid. Protein analysis showed that retinoic acid had a stimulatory effect on protein synthesis. ELISA assays revealed that retinoic acid treated myocyte cultures contained significantly more hLAMP-1 and fibronectin than either normal or DMSO controls. However, transferrin was not affected by retinoic acid treatment in these experiments. Our results suggest that retinoic acid affects the expression of the particulate matrix and that these changes may be responsible for the observed decrease in mesenchymal cell formation.

Role of vitamin A in the formation of congenital heart defects.

Retinoic acid, the biologically active form of vitamin A, is a critical player in normal development. The concentration of retinoic acid is highly regulated by the embryo to prevent either a deficit or an excess of this molecule, conditions that have been shown to produce cardiac defects that vary depending on the severity and the timing of the insult. The vast majority of these defects are associated with the valves or the membranous septa of the heart, suggesting a problem with the formation of the cardiac mesenchyme from both within and outside the heart. While the exact role of retinoic acid in cardiac development is not known, it is believed that retinoic acid influences development by up- or down-regulating cardiac specific genes. This review briefly discusses the role of cardiac mesenchyme and cardiac neural crest in septation of the heart. This is followed by a discussion of vitamin A metabolism and the cardiac defects associated with abnormal levels of retinoic acid. Finally, a mechanism is proposed concerning the ways abnormal levels of retinoic acid lead to similar cardiac defects by disrupting the production of the extracellular matrix.

Partial purification of HLAMP-1 provides direct evidence for the multicomponent nature of the particulate matrix associated with cardiac mesenchyme formation.

H-LAMP-1 is a 283 kDa protein that is involved in the transformation of endothelial cells into mesenchyme within the AV canal and proximal outflow tract of the heart. This protein is part of the particulate matrix that has been suggested to be composed of multicomponent complexes that have been termed cardiac adherons. However, to date no direct evidence has been provided that these proteins are complexed into an adheron-like particle. This report provides the first such evidence by showing that purification of hLAMP-1, under gentle conditions, results in the isolation of multiple bands of similar molecular weight within the fractions that contain anti-hLAMP-1 activity.

Retinoic acid directs cardiac laterality and the expression of early markers of precardiac asymmetry.

Formation of the left/right body axis is a critical early step in embryogenesis. The heart loop is one of the first clearly recognizable morphological asymmetries, and the molecular pathway which dictates this laterality is now beginning to be understood. We report here that the left and right precardiac fields of chick differ in their sensitivity to retinoic acid (RA); while RA applied to the right precardiac field at gastrulation randomizes heart looping, left side treatment induces situs inversus only at high RA concentrations. We identified two extracellular matrix proteins, the heart-specific lectin-associated matrix protein-1 (hLAMP1) and the fibrillin-related protein recognized by the antibody JB3, which are distributed asymmetrically within the precardiac fields at the head process stage. In normal embryos, JB3 expression is enhanced within the right precardiac field, and hLAMP-1 is enriched within the left. RA treatment predictably altered the expression of these proteins in a manner consistent with subsequent heart laterality: RA treatments which randomize heart loop direction also equalized or reversed the left/right JB3 and hLAMP-1 distribution prior to heart tube fusion. The existence of asymmetrically expressed extracellular matrix proteins within precardiac regions suggests that interactions between cardiocytes and their environment may contribute to heart laterality determination and looping.

Identification of a 283-kDa protein component of the particulate matrix associated with cardiac mesenchyme formation.

The elaboration of cardiac mesenchyme is controlled by a particulate form of extracellular matrix that is produced by the myocardium. This matrix consists of at least 7-10 major proteins or protein subunits. However, at present only a few of these proteins have been identified (i.e., ES130, fibronectin, transferrin). We report here the identification of a 283-kDa protein associated with this complex. This protein was identified using a new monoclonal antibody that is highly specific for this protein. The antibody does not react with human plasma fibronectin, chicken fibronectin or chicken cytotactin. In addition, this antibody inhibits the formation of mesenchyme when used in a bioassay of mesenchyme formation.

A subset of SBA lectin-binding proteins isolated from myocardial-conditioned media transforms cardiac endothelium into mesenchyme.

It has been shown that the inductively active proteins for cardiac mesenchyme formation are localized to a particulate form of extracellular matrix that resembles adheron-like complexes. These complexes are extractable from the embryonic heart using EDTA and can be visualized with the lectin SBA (Glycine max). In addition, the growth medium obtained from embryonic myocardial cell cultures has also been shown to support mesenchyme formation. However, except for the identification of EDTA extract and conditioned media, all previous experiments analyzing this system have relied on negative type results (i.e. the loss of biological activity) to show a relationship between the particulate matrix and the transformation process. We report here that SBA affinity chromatography can be used to partially purify a subset of proteins from myocardial conditioned medium which elicits the transformation of endothelial cells into mesenchyme. In addition, a polyclonal antibody made against this subset of proteins is specific for the in situ particulate matrix and recognizes several proteins in conditioned medium and EDTA extracts. This antibody is also specific for matrix particulates in other areas of the embryo that undergo an epithelial/mesenchymal interaction. These results provide the most direct evidence to date that conditioned medium is equivalent to the hypothesized inductively active particulate matrix. In addition, the data provides evidence that conditioned medium can be used to identify the functional role of the components of the particulate matrix in mesenchyme formation.

Multiple glycoproteins localize to a particulate form of extracellular matrix in regions of the embryonic heart where endothelial cells transform into mesenchyme.

Cells derived from an epithelial-mesenchymal transformation within the atrioventricular canal and outflow tract are involved in the partitioning of the early embryonic heart into a four-chambered organ. This transformation process has been shown to proceed from an inductive interaction between the myocardium and competent, target endothelial cells within these regions of the heart. Interestingly, immunohistochemistry revealed the presence of fibronectin-positive particulates within the matrix of mesenchyme-forming regions (Mjaatvedt et al., 1987). This particulate matrix is extractable by EDTA and can elicit the epithelial-mesenchymal transformation in culture (Mjaatvedt and Markwald, 1989). Analysis of EDTA extracts of embryonic heart tissue revealed the presence of fibronectin and about 40 unidentified proteins, 6 of which appeared to be enriched in the biologically active 100,000g pellet fraction (Mjaatvedt and Markwald, 1989). Based on these and other data we have proposed that the particulate matrix is composed of a multicomponent complex of fibronectin and one or more of the low-molecular-weight proteins in this pellet. The purpose of the present study was to begin a biochemical characterization of the nonfibronectin proteins thought to be present in the matrix particulates. Given that many matrix constituents are glycoproteins, lectins were used to initially characterize the particulate constituents. Of the lectins tested, soybean agglutinin (SBA) was found to be specific only for matrix particulates. Histochemical analyses showed that SBA and antibodies against fibronectin colocalized regionally and temporally to the same matrix particulates in embryonic heart tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase: immunochemical characterization of the lung enzyme from pregnant rabbits.

A polyclonal antibody was produced in guinea pig against the lung NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) purified from pregnant rabbits. Western blot analysis demonstrated that the protein identified by this antibody in the 105,000g supernatant fraction of lung tissue from pregnant rabbits had a molecular mass of 30 kDa and comigrated with the purified PGDH. The specific activity of the lung PGDH in pregnant rabbits (25- to 28-day gestations) was 36.7 nmol NADH formed/min/mg protein compared to 0.3 nmol NADH formed/min/mg protein in nonpregnant rabbits. Although the PGDH activity in the lung cytosol of nonpregnant rabbits was inhibited by the anti-lung PGDH antibody, the 30-kDa protein was not detected by Western blot analysis. An examination of this 30-kDa protein during the gestational period indicated that the protein was present after 10 days and the amount of the protein increased from Day 10 to Day 28. This increase in the immunochemically reactive protein correlated with the marked increase in PGDH specific activity between 10 and 28 days. An immunochemically reactive protein also was observed in the ovary of 25- to 28-day pregnant rabbits and the specific activity of the ovary PGDH was 19.3 nmol NADH formed/min/mg protein. Only trace levels of the PGDH activity were detected in the ovaries of nonpregnant rabbits. A 30-kDa protein was not detected by the anti-rabbit lung PGDH in brain, kidney, bladder, uterus, liver, and heart tissue of pregnant or nonpregnant rabbits. When rabbit or human placental cytosol was examined with the anti-rabbit lung PGDH only faint 30-kDa bands were observed by Western blot analysis. A monoclonal antibody prepared against human placental PGDH did not recognize the 30-kDa band in the pregnant rabbit lung. Localization studies indicated a marked increase in immunochemical staining in pulmonary epithelial cells of pregnant rabbits as compared to nonpregnant rabbits. Lung epithelial cells but not endothelial cells were identified as containing the PGDH.

Initial expression of type I procollagen in chick cardiac mesenchyme is dependent upon myocardial stimulation.

Formation of the atrioventricular (AV) mesenchyme is a critical step in early heart development. Endothelial cells are activated and transformed into a mesenchymal population that invades the cell-free myocardial basement membrane. This process can be duplicated in collagen gel culture, where it has been established that myocardium or its secretory products activate the endothelium. The purpose of the present study was to determine when these activated endothelial and/or mesenchymal cells start producing type I collagen in situ. These results were compared to those obtained from a culture model of mesenchyme formation. The production of type I collagen was monitored using a monoclonal antibody (M38) that recognizes the carboxy-terminal propeptide of human type I procollagen. The initial expression of the latter within activated AV endothelial and mesenchymal cells in ovo was 48 hr following activation. Prior to this time, only the myocardium was reactive with M38. AV explants of early hearts on collagen gels revealed staining of activated endothelial and mesenchymal cells with M38 after 48 hr in coculture with myocardial tissue. Explants that were prevented from activating (myocardium removed) never expressed the M38 antigen. Similarly, AV endothelial monolayers grown in the presence of myocardial conditioned medium activated and expressed type I collagen after 48 hr in culture, whereas those grown in standard medium did not. These results establish the initial expression of type I collagen within activated AV endothelium and mesenchyme. In addition, the data suggest that the expression of type I collagen within the AV mesenchyme may be dependent on extrinsic influences that induce the AV endothelium to transform into mesenchyme.

Surface coat material associated with the developing otic placode/vesicle in the chick.

Surface coat material (SCM) has been illustrated in association with the apical surfaces of numerous epithelia during morphogenesis. This study investigates the development of a SCM associated with the invaginating otic placode/vesicle in the chick. Glycoconjugate containing SCM was retained by the inclusion of cetylpyridinium chloride (CPC) in the fixative, histochemically visualized by using ruthenium red (RR) staining, and viewed by scanning (SEM) or transmission (TEM) electron microscopy. Initial characterization of the glycoconjugates present in this material was elucidated by using lectins conjugated to fluorescein isothiocyanate. Lectins utilized included concanavalin A (Con A), wheat germ agglutinin (WGA), and soybean agglutinin (SBA). Invagination of the otic placode was apparent as early as stage 12. By stage 15 the vesicle was beginning to separate from the surface ectoderm as evidenced by its aperture, which was altered in shape and reduced in size. All embryos fixed with glutaraldehyde containing either CPC or RR were shown to possess SCM associated with the surface ectoderm, particularly in the area of the otic placode/vesicle. Additional embryos were processed by cryofixation without prior aldehyde fixation; these also exhibited SCM. All lectins labelled the epithelium of the otic placode/vesicle. However, their binding patterns were not identical. The binding of Con A and WGA remained constant over the stages studied, while SBA increased as the otic vesicle developed. The data clearly indicate that otic placode morphogenesis is accompanied by the synthesis of SCM rich in glycoconjugates.

The effects of zinc deficiency on developing photoreceptors in the rat retina: a scanning microscopic study.

A study was undertaken to observe the effects of dietary zinc deficiency on the topographical morphology of developing photoreceptor (rod) cells in the sensory retina of the rat. Observations were made by means of scanning electron microscopy (SEM). Pups were utilized from three groups including (1) zinc deficient (2) pair-fed controls and (3) ad libitum controls. Animals ranged in age from birth to twenty days. Femur zinc levels were measured by flame atomic absorption spectrophotometry. A statistically significant increase in the number of photoreceptor cells continued through the 6th day, however, no morphological differences were noted among cells during the first 13 days. By day 14, outer segments appeared more elongated in ad libitum control tissues. No differences in outer segment length were apparent by day 20.