Increased NCAM Expression and Vascular Development in Trisomy 16 Mouse Embryos: Relationship with Nuchal Translucency

0031-3998/05/5806-1222 PEDIATRIC RESEARCH Copyright © 2005 International Pediatric Research Foundation, Inc. Vol. 58, No. 6, 2005 Printed in U.S.A. Increased NCAM Expression and Vascular Development in Trisomy 16 Mouse Embryos: Relationship with Nuchal Translucency MIREILLE N. BEKKER, JENNY B. ARKESTEIJN, NYNKE M.S. VAN DEN AKKER, STANLEY HOFFMAN, SANDRA WEBB, JOHN M.G. VAN VUGT, AND ADRIANA C. GITTENBERGER-DE GROOT Department of Obstetrics and Gynecology [M.N.B., J.M.G.V.], VU University Medical Center, 1081 HV, Amsterdam, the Netherlands; Department of Anatomy and Embryology [M.N.B., J.B.A., N.M.S.A., A.C.G.G.], Leiden University Medical Center, 2300 RC, Leiden, the Netherlands; Department of Cell Biology [S.H.], Medical University of South Carolina, Charleston, SC 29425; Department of Basic Medical Sciences, Anatomy and Developmental Biology [S.W.], St. George Hospital Medical School, London, SW17 ORE, U.K. ABSTRACT Increased nuchal translucency in the human fetus is associated with chromosomal abnormalities, enlarged jugular lymphatic sacs, cardiac defects and changed flow through the ductus venosus. The developmental background of this nuchal edema in relation to the associated anomalies remains elusive. We studied the morphologic correlation between neurogenesis and vasculogenesis in neck, heart, and ductus venosus region of wild type and trisomy 16 mice embryos (E10- E18), using an antibody against Neural Cell Adhesion Molecule (NCAM). Trisomy 16 mice are a model for the above described human phenotype. From E12 trisomy 16 mice showed an altered arrangement of cranial nerves IX, X and XI, which are positioned between the carotid artery, jugular vein and enlarged lymphatic sac. The vagal nerve was significantly smaller, compared with wild type embryos. NCAM was over expressed in both neuronal and cardiovascular structures in trisomy 16 mice, being particularly prominent in the 4th and 6th pharyngeal arch arteries, and the Ultrasonographic measurement of nuchal translucency (NT) in the human fetus at gestational age between 10 to 14 wk is a common screening method to detect trisomy 21. Increased NT is associated with a spectrum of anomalies, including aneuploidy, isolated cardiac defects, and changed flow velocities through the ductus venosus (1,2). The developmental background of increased NT and associated findings is still insufficiently understood. Recently, a disturbed lymphangiogenesis has been suggested as a basis for the nuchal edema because of the concomitant abnormal enlargement and persistence of the jugular lymphatic sacs (JLS) in both human fetuses and mouse embryos with Received February 2, 2005; accepted April 29, 2005. Correspondence: Adriana C. Gittenberger-de Groot, Ph.D., Department of Anatomy and Embryology, Leiden University Medical Center, PO Box 9602, 2300RC Leiden, the Netherlands; email: acgittenlumc.nl DOI: 10.1203/01.pdr.0000187795.82497.31 ductus venosus. In the 4th and 6th pharyngeal arch arteries, NCAM over expression was located to the part of the vessel wall that is closely related to the vagal and recurrent nerve. In case of 4th pharyngeal arch artery abnormalities NCAM expression, on the other hand, was reduced. In conclusion, the interaction between neurogenesis and vasculogenesis is disturbed in the trisomy 16 mouse model, and might be a common denominator in the spectrum of anomalies associated with increased nuchal translucency. (Pediatr Res 58: 1222–1227, 2005) Abbreviations JLS, jugular lymphatic sac NCAM, Neural Cell Adhesion Molecule NT, nuchal translucency PAA, pharyngeal arch artery RUNX1, Runt-related gene-1 nuchal edema (3,4). This theory explains both the regional accumulation of the fluid in the neck region and the temporary character of the NT enlargement. Other suggested theories, like temporary cardiac failure (5,6) and the alteration of extracellular matrix components (7) might be part of the causal pathway, but fail to completely elucidate this phenomenon. A study (3) of trisomy 16 embryos showed that the endothelium of the enlarged JLS was abnormally thickened. Also, there was a very close correlation of nerves with the JLS and other vascular structures. A mutual influence of nerves and endothelium could play a role in the development of nuchal translucency, since vessels and nerves are known to share many developmental genes (8). The hypothesis for this study was that a disturbance in the interaction between neurogenesis and vasculogenesis is a common denominator in the spectrum of associated (lymphatic) 1222 NCAM EXPRESSION AND NUCHAL TRANSLUCENCY vascular and cardiovascular abnormalities in fetuses with increased NT. Innervation and vascular morphology in neck, heart, and ductus venosus region of wild type and trisomy 16 mouse embryos were investigated. Trisomy 16 mice embryos have nuchal edema, enlarged JLS and cardiac defects. Also, the trisomy 16 mouse is proposed as an animal model for human trisomy 21 as the mouse chromosome 16 contains the syntenic region for the human chromosome band 21q22 (9). In this model we studied the expression of Neural Cell Adhesion Molecule (NCAM) because it is expressed both in nerves and the cardiovascular system (10). NCAM is of special interest, as this protein is regulated by Runt-related gene-1 (RUNX1), which is described as triplicated gene in the trisomy 16 mouse and human trisomy 21 (11,12). METHODS Embryos. Wild type and trisomy 16 mouse embryos from gestational age day 10 (E10) to E18 d (Table 1) were collected (13). The study was approved by the Animal Care and Use Committee of St. George Hospital, London. The embryos were fixed in 4% paraformaldehyde at 4°C overnight. After dehydrating the embryos with xylene, they were embedded in paraffin and serially sectioned (5 ␮m). The sections were alternately mounted on 5 subsequent slides, so that 5 different staining procedures could be performed. Immunohistochemistry. Consecutive sections were stained with an NCAM antibody. Using an antibody against NCAM (provided by S. Hoffman, Charleston, S.C., U.S.A.) in an immunohistochemical staining all cells derived from neural tissue are stained. First, the slides were deparaffinated. Between all steps the slides were rinsed twice with phospate-buffered saline (PBS) and once with PBS/0.05%Tween unless indicated otherwise. Before staining, the slides were microwaveprocessed by heating them 3 times for 4 min to 99°C in a citric acid buffer (0.01 M in distilled water, pH ⫽ 6.0). Then the slides were rinsed twice with PBS and incubated for 15 min in a 0.3% H2O2 solution to block the endogenous peroxidase activity. Subsequently, the slides were incubated overnight with the specific antibody against NCAM. After that, the sections were incubated for 90 min with the second antibody, GAR-Ig. The third antibody used was R-PAP, with which the slides were incubated for 90 min. Afterward, the slides were rinsed twice with PBS and once with Tris/Maleate (pH 7.6). As chromogen, DA (...truncated)