Phagocytic myeloid cells provide the principle line of immune defence during early embryogenesis in lower vertebrates. They may also have important functions during normal embryo morphogenesis, not least through the phagocytic clearance of cell corpses arising from apoptosis. We have identified two cDNAs that provide sensitive molecular markers of embryonic leukocytes in the early Xenopus embryo. These encode a peroxidase (XPOX2) and a Ly-6/uPAR-related protein (XLURP-1). We show that myeloid progenitors can first be detected at an antero-ventral site in early tailbud stage embryos (a region previously termed the anterior ventral blood island) and transiently express the haematopoetic transcription factors SCL and AML. Phagocytes migrate from this site along consistent routes and proliferate, becoming widely distributed throughout the tadpole long before the circulatory system is established. This migration can be followed in living embryos using a 5 kb portion of the XLURP-1 promoter to drive expression of EGFP specifically in the myeloid cells. Interestingly, whilst much of this migration occurs by movement of individual cells between embryonic germ layers, the rostral-most myeloid cells apparently migrate in an anterior direction along the ventral midline within the mesodermal layer itself. The transient presence of such cells as a strip bisecting the cardiac mesoderm immediately prior to heart tube formation suggests that embryonic myeloid cells may play a role in early cardiac morphogenesis.

Background-Increased circulating levels of the cardiac polypeptide hormones atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) may be observed after orthotopic cardiac transplantation. Both the hypertrophic and inflammatory processes in the allograft may contribute to this increase, but no mechanistic explanation has been suggested for this observation. Methods and Results-Plasma immunoreactive ANF and BNP determinations were performed in 10 consecutive transplant patients. These were correlated with degree of rejection as reflected by histopathological findings at serial endomyocardial biopsies. Three patients had associated hemodynamic measurements and blood samples 24 hours before and after transplantation. All rejection episodes that received treatment were accompanied by a marked increase in BNP plasma levels to ϾϷ400 pg/mL. Steadily increasing BNP levels preceded overt rejection as assessed by histopathological criteria. The increase in plasma BNP was not always accompanied by an increase in ANF, which suggests the specific upregulation of BNP gene expression during acute rejection episodes. Treatment of the acute rejection episodes led to a substantial decrease of BNP plasma levels. Conclusions-The significant selective increase in plasma BNP levels found in the present study has not been previously described. This finding provides a new insight into the mechanism of allograft rejection and the modulation of natriuretic peptide synthesis and release. Furthermore, although preliminary, the data suggest that BNP plasma levels could form the basis for a new, noninvasive screening test to predict acute cardiac allograft rejection. Because treatment with the antilymphocyte monoclonal antibody OKT3 (murine monoclonal antibody to the CD3 antigen of the human T-cell) decreased BNP plasma levels, cytokine production by T-cells may mediate the selective increase in circulating BNP. (Circulation. 1999;100:287-291.)Key Words: transplantation Ⅲ natriuretic peptides Ⅲ atrial natriuretic factor Ⅲ myocarditis Ⅲ pathology U nder certain pathophysiological conditions, synthesis and release of the cardiac natriuretic peptides (NP) atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) are significantly augmented in both atrial and ventricular cardiocytes. Increased production of these hormones by the ventricle is a hallmark of cardiac hypertrophy, failure, 1,2 and inflammation. 3,4 The former increase reflects the reexpression of the cardiac fetal phenotype seen with chronic hemodynamic overload, but the basis for the increased production of NP in myocarditis has not yet been defined.We reported that after cardiac transplantation, ANF plasma concentrations remain elevated, reaching levels comparable to those found before transplantation despite the normalization of filling pressures and the renin-angiotensin-aldosterone system. 5 Elevated BNP plasma levels have also been found after cardiac transplantation. 6 -8 Because BNP is predominantly of ventricular origin, it would be expected that repla...

Adprhl1, a member of the ADP-ribosylhydrolase protein family, is expressed exclusively in the developing heart of all vertebrates. In the amphibian Xenopus laevis, distribution of its mRNA is biased towards actively growing chamber myocardium. Morpholino oligonucleotide-mediated knockdown of all Adprhl1 variants inhibits striated myofibril assembly and prevents outgrowth of the ventricle. The resulting ventricles retain normal electrical conduction and express markers of chamber muscle differentiation but are functionally inert. Using a cardiac-specific Gal4 binary expression system, we show that the abundance of Adprhl1 protein in tadpole hearts is tightly controlled through a negative regulatory mechanism targeting the 5′-coding sequence of Xenopus adprhl1. Over-expression of full length (40 kDa) Adprhl1 variants modified to escape such repression, also disrupts cardiac myofibrillogenesis. Disarrayed myofibrils persist that show extensive branching, with sarcomere division occurring at the actin-Z-disc boundary. Ultimately, Adprhl1-positive cells contain thin actin threads, connected to numerous circular branch points. Recombinant Adprhl1 can localize to stripes adjacent to the Z-disc, suggesting a direct role for Adprhl1 in modifying Z-disc and actin dynamics as heart chambers grow. Modelling the structure of Adprhl1 suggests this cardiac-specific protein is a pseudoenzyme, lacking key residues necessary for ADP-ribosylhydrolase catalytic activity.

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The mechanisms by which transcription factors, which are not themselves tissue restricted, establish cardiomyocyte-specific patterns of transcription in vivo are unknown. Nor do we understand how positional cues are integrated to provide regionally distinct domains of gene expression within the developing heart. We describe regulation of the Xenopus XMLC2 gene,which encodes a regulatory myosin light chain of the contractile apparatus in cardiac muscle. This gene is expressed from the onset of cardiac differentiation in the frog embryo and is expressed throughout all the myocardium, both before and after heart chamber formation. Using transgenesis in frog embryos, we have identified an 82 bp enhancer within the proximal promoter region of the gene that is necessary and sufficient for heart-specific expression of an XMLC2 transgene. This enhancer is composed of two GATA sites and a composite YY1/CArG-like site. We show that the low-affinity SRF site is essential for transgene expression and that cardiac-specific expression also requires the presence of at least one adjacent GATA site. The overlapping YY1 site within the enhancer appears to act primarily as a repressor of ectopic expression, although it may also have a positive role. Finally, we show that the frog MLC2 promoter drives pan myocardial expression of a transgene in mice, despite the more restricted patterns of expression of murine MLC2 genes. We speculate that a common regulatory mechanism may be responsible for pan-myocardial expression of XMLC2 in both the frog and mouse, modulation of which could have given rise to more restricted patterns of expression within the heart of higher vertebrates.

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Many details of cardiac chamber morphogenesis could be revealed if muscle fiber development could be visualized directly within the hearts of living vertebrate embryos. To achieve this end, we have used the active promoter of the MLC1v gene to drive expression of green fluorescent protein (GFP) in the developing tadpole heart. By using a line of Xenopus laevis frogs transgenic for the MLC1v-EGFP reporter, we have observed regionalized patterns of muscle formation within the ventricular chamber and maturation of the atrial chambers, from the onset of chamber formation through to the adult frog. In f1 generation MLC1v-EGFP animals, promoter activity is first detected within the looping heart tube and delineates the forming ventricular chamber and proximal outflow tract throughout their development.