Bone morphogentic proteins (BMPs) play an important role in cardiac development. Using an in vitro explant analysis, we show that BMPs are crucial for myocardium formation. As a first approach to identify which BMP may be involved in myocardium formation in intraand extracardiac mesenchyme in vivo, a survey of the expression patterns of BMP2, -4, -5, -6, and -7 mRNA is prepared by in situ hybridization in chicken embryonic hearts from HH5 to 44. During recruitment of mesodermal cells to the outflow tract myocardium (HH10 -23), BMP2, -4, -5, and -7 mRNA are expressed in the distal myocardial border and the flanking mesenchyme. After completion, BMP2 and -4 mRNA become restricted to the mesenchyme and BMP5 and -7 mRNA to the myocardium. At the venous pole, BMP2, -5, and -7 mRNA are expressed in the distal myocardial border of the caval vein, while BMP2, -5, -6, and -7 mRNA are expressed in the distal myocardium around the pulmonary vein. BMP4 mRNA is expressed in the adjacent mesenchyme at both sides. During muscularization of the atrioventricular cushions and the tricuspid valve, the cardiomyocytes that protrude into the mesenchyme express BMP2, -4, -5, and -7 mRNA, whereas BMP6 mRNA is expressed in the cushion mesenchyme. The myocardial protrusions formed in the mesenchymal proximal outlet septum express BMP4, -5, and -7 mRNA, while BMP2 and -6 mRNA are expressed in the mesenchyme. The spatiotemporal expression patterns of these BMPs in relation to myocardium formation at the distal ends and within the heart suggest a role for BMPs in myocardium formation. During delamination of the valves, BMP4 and -6 mRNA are expressed at the ventricular side of the forming mitral valve, BMP4 mRNA at the ventricular side of the forming tricuspid valve, and BMP2, -4, and -6 mRNA at the vascular side of the forming semilunar valves. © 2004 Wiley-Liss, Inc.Key words: bone morphogenetic protein; TGF␤ superfamily; cardiovascular development; myocardialization; chicken; myocardium; valve formationIn chicken, the primary linear heart tube forms by fusion of the paired heart-forming regions that develop anterolaterally in the splanchnic mesoderm. The primary heart tube comprises an outer myocardial layer and an inner endocardial layer that are separated by an extended extracellular matrix or cardiac jelly ). Recent advances suggest that in the anterior half of the early embryo, Wnt signaling is inhibited, on which Bone morphogentic protein (BMP) signaling promotes cardiogenesis in the anterior lateral mesoderm (Marvin et al., 2001;Tzahor and Lassar, 2001). The resulting heart-forming regions can be identified by the onset of expression of cardiac-enriched transcription factors, like Nkx2.5 and Gata4 (Schultheiss et al., 1995Andree et al., 1998;Schlange et al., 2000). Members of the BMP family are expressed in the endoderm and ectoderm overlaying the precardiac mesoderm. Ectopic application of BMP2 medial to the precardiac mesoderm results in a medial broaden-*Correspondence to: Maurice J.B. van

Availability of a standard human melanocyte cell line with unlimited growth potential and otherwise normal melanocytic properties will greatly facilitate research in melanocyte biology and in vitro studies on the etiology of pigmentary disorders and melanoma. Using a retroviral vector, E6 and E7 open reading frames of human papilloma virus type 16 (HPV 16) have been introduced into cultured normal human melanocytes. Cells selected by increased resistance to geneticin conveyed by the vector and expressing E6E7 mRNA have been cloned to ensure genetic homogeneity. Since their establishment as primary cells, cloned PIG1 cells have undergone more than twice the amount of population doublings of senescent parental cells. Moreover, in passage numbers when parental cells had become senescent, proliferation of clonal cells was retained at levels exceeding those of normal human melanocytes in third passage by 100%. Further characterization has revealed that the cells remain dependent on tetradecanoyl phorbol 13-acetate (TPA) for growth and do not proliferate in soft agar nor form tumors in nude mice. The antigenic profile of the cells was slightly altered as compared to parental cells, but was incomparable to that of M14 melanoma cells. Importantly, PIG1 cells contain more melanin pigment than parental cells.

Myosin heavy-chain (MHC) isoforms are major structural components of the contractile apparatus of the heart muscle. Their spatio-temporal patterns of expression have been used as a tool to dissect cardiac development and differentiation. Although extensively investigated, controversy still exists concerning the expression patterns of atrial (AMHC), ventricular (VMHC), and cardiac myosin heavy-chain (CMHC) during development in the heart. In this study, we describe that probe length, probe concentration, and staining time in the non-radioactive in situ hybridization procedure seriously influence the observed pattern of MHC expression and the subsequent interpretation, explaining the divergent opinions in the field. Using a variety of external and internal controls for the in situ hybridization procedure, we demonstrate that both AMHC and VMHC are expressed throughout the entire heart tube during early development. During subsequent development, VMHC becomes restricted to the ventricles, whereas AMHC remains expressed in the atria, and, at substantially lower levels, is detected in the ventricles. These results are discussed in the context of methodological constraints of demonstrating patterns of gene expression. This manuscript contains online supplemental material at http://www.jhc.org . Please visit this article online to view these materials.

In this communication we describe the expression pattern of BMP10 mRNA during cardiac development in chickens. BMP10 is considered an important factor in the regulation of cardiac growth and trabeculation in the murine embryo. We identified chicken Ests, which are similar to mouse and human BMP10 in the UMIST database. The cDNA clone that contained most sequences was obtained, verified by sequence analysis, and used to determine the spatiotemporal pattern of gene expression. BMP10 mRNA is initially expressed at HH10 in the myocardium of the arterial pole of the heart tube, anterior to the interventricular groove. Between HH14 and HH22, BMP10 mRNA becomes broadly expressed in the outflow tract, the distal part of the inflow tract, and the trabeculated part of the developing ventricles and atria. From HH31 onward, BMP10 mRNA expression decreases in the ventricular myocardium by first disappearing from the compact myocardium and then from the tips of the trabecules. At HH44, BMP10 mRNA is expressed only in the trabeculated myocardium of the atria and the endocardium of the ventricles. The observed expression pattern of BMP10 mRNA suggests that it may play a role in regulating the formation of the ventricular wall and trabecules.

The presence of mycobacterial antigens in leprosy skin lesions was studied by immunohistological methods using monoclonal antibodies (MAbs) to Mycobacterium leprae-specific phenolic glycolipid I (PGL-I) and to cross-reactive mycobacterial antigens of 36 kd, 65 kd, and lipoarabinomannan (LAM). The staining patterns with MAb to 36 kd and 65 kd were heterogeneous and were also seen in the lesions of other skin diseases. The in situ staining of PGL-I and LAM was seen only in leprosy. Both antigens were abundantly present in infiltrating macrophages in the lesions of untreated multibacillary (MB) patients, whereas only PGL-I was occasionally seen in scattered macrophages in untreated paucibacillary lesions. During treatment, clearance of PGL-I from granulomas in MB lesions occurred before that of LAM, although the former persisted in scattered macrophages in some treated patients. This persistence of PGL-I in the lesions paralleled high serum anti-PGL-I antibody titers but was not indicative for the presence of viable bacilli in the lesions. Interestingly, we also observed a differential expression pattern of PGL-I and LAM in the lesions of MB patients with reactions during the course of the disease as compared with those without reactions. In conclusion, the in situ expression pattern of PGL-I and LAM in MB patients may assist in early diagnosis of reactions versus relapse.