Kessel, M.

Teichmann, U.

English

MPG.PuRe

Dev Genes Evol (2004) 214:96–98DOI 10.1007/s00427-003-0380-2E X P R E S S I ON NOTEUlrike Teichmann · Michael KesselHighly restricted BMP10 expression in the trabeculating myocardiumof the chick embryoReceived: 21 October 2003 / Accepted: 8 December 2003 / Published online: 14 January 2004 Springer-Verlag 2004Abstract Bone morphogenetic proteins (BMPs) controlthe development of diverse tissues during embryogenesis.Here, we report the expression of the BMP10 gene duringchick development. BMP10 transcription is confined tothe myocardium of atriae and ventricles in the formingheart from day 2 of development onwards (stage HH13).It correlates with the thickening of the innermost layer ofthe walls, i.e. the formation of myocardial ridges ortrabeculae.Keywords Heart · Bone morphogenetic protein ·Ventricle · Atrium · TGFbThe first period of heart morphogenesis in the chick is theformation of a straight tube from the lateral mesodermduring the first 30 h of embryogenesis. Subsequently, thetube loops towards the right side, extracardial cells arerecruited, and the morphogenesis of four primitivesubdivisions, the inflow tract, the primitive atrium, theprimitive ventricle and the outflow tract, is initiated(Larsen 1993). At the inflow side, part of the sinusvenosus becomes incorporated into the developing atriae,whereas the rest of the atriae becomes diminished to theauricles. Only the myocardium of the auricles begins tothicken and myocardial ridges, the trabeculae, appear onthe inner wall. At the outflow side of the heart theconotruncus, later the truncus arteriosus and the conuscordis, develops from an extracardial source of mesoder-mal cells, the secondary heartfield (Kelly et al. 2001;Mjaatvedt et al. 2001; Waldo et al. 2001). No trabecu-lation occurs in the outflow tract. The two ventricles arederivatives of the primary, paired heartfield. When theirmorphogenesis proceeds, the myocardium thickens andtrabeculae appear on the inner wall, including also theanterior portion of the muscular ventricular septum. Thetrabeculated myocardium is the essential, functionalcomponent of the contractile heart tube. Here, we reportthe cloning of a trabeculation specific gene, the chickenortholog of the previously isolated murine BMP-10 gene(Neuhaus et al. 1999).A chicken liver Lambda ZAP-cDNA library (Strata-gene) was screened under low stringency conditions witha 379-bp XbaI/PstI fragment from the 30end of the mouseBMP-9 ORF (Song et al. 1995). We obtained a cDNA of2,374 bp, which contains an open reading frame of1,149 bp followed by a 1,225-bp 30UTR with a polyA tail.A translation start site is missing as indicated by acomparison of the chick cDNA with the closely relatedmouse cDNAs and the consensus sequences (Kozak1984). The sequence is deposited in the EMBL databasewith the accession number AJ581667. The deduced aminoacid sequence shows the characteristics of a member ofthe BMP family. The N-terminal propeptide region isfollowed by the C-terminal mature region with conservedseven cysteines. Both regions are separated by a prote-olytic cleavage site (RIRR; Ozkaynak et al. 1992).Sequence comparisons reveal 77% amino acid identityto the murine BMP-10 protein, and 47% or 45% identityto the related chick dorsalin and mouse BMP-9 proteins,respectively. Therefore, we conclude to have cloned theorthologous chicken BMP10 gene.The embryonic expression was analyzed by whole-mount in situ hybridization in chick embryos. The firsttranscripts can be detected at HH13 (Hamburger andHamilton stage 13) in the ventrolateral wall of the hearttube (Fig. 1A, B). This area corresponds to the futureouter curvature, where the primitive ventricle will form.At HH15, the primitive myocardium has become multi-layered. BMP10 expression is detected in the innermyocardial layer (Fig. 1C, D), whereas the outer layeras well as the endocardium remain free of BMP10transcripts. In addition, BMP10 RNA is now found in thewall of the atrium. At HH18, BMP10 is expressed in theEdited by B. HerrmannU. Teichmann · M. Kessel ())Abteilung Molekulare Zellbiologie,Max-Planck-Institut fr Biophysikalische Chemie,Am Fassberg 11, 37077 Gttingen, Germanye-mail: mkessel1@gwdg.deTel.: +49-551-2011560Fax: +49-551-2011504trabeculating myocardial tissue of both the ventricle andthe atrium (Fig. 1E, F).Whole-mount in situ hybridization of isolated heartsfrom embryos ranging from HH21 to HH32 shows thatthe principle of this spatial pattern persists in allinvestigated stages (Fig. 2A–H). Expression in theventricles of the heart is confined to the innermosttrabeculae, whereas the compacting myocardial tissue onthe outer side is free of BMP10 RNA. The expression inthe ventricular trabeculae becomes very weak at stageHH32 (Fig. 2G), and is barely detectable on sections atday 7 of development. During the morphogenesis of theatriae, high BMP10 expression becomes restricted to theauricles, i.e. the trabeculated part of the atrial myocardi-um (HH32; Fig. 2H). BMP10 transcripts are not present inthe interventricular and interatrial septa and in themyocardium of the atrioventricular canal, where theendocardial cushions and the atrioventricular valves willform. Here, no trabeculated structures will develop. Theoutflow tract, also consisting of non-trabeculated myo-cardial tissue, is free of BMP10 transcripts.In conclusion, for the chicken BMP10 gene wedescribe an expression pattern which is much morerestricted than that found for other genes of the largeBMP family. The pattern is highly conserved betweenchick and mouse embryos (Neuhaus et al. 1999). Theextreme restriction to the inner myocardial layer impli-cates a strong correlation with the process of trabecula-tion. Trabeculation as well as BMP10 expression occuronly in myocardium derived from the primary, pairedheartfield. The areas of BMP10 expression in the ventriclecorrespond to the region where the heart chambers“balloon” out from the bended heart tube according tothe ballooning model discussed by Christoffels et al.(2000).Fig. 1A–F Expression of BMP10 during early stages of chickembryonic development. A, C, E Whole-mount in situ hybridiza-tion at HH13, HH15 and HH18 shows expression of BMP10exclusively in the embryonic heart (v ventricle, a atrium). In A, theshape of the heart is indicated by a dotted line. In E, the outflowtract is indicated by a dashed line. The level of the sections shownin B, D, and F is indicated. B, D, F Transversal sectionsdemonstrate that the staining is confined to the inner myocardiallayer only. The outer myocardial layer (om, black arrowhead) andthe endocard (en, open arrowhead) are free of BMP10 transcripts.Chick embryos were staged according to Hamburger and Hamilton(1951). The cloned BMP10 cDNA fragment was used to generateantisense RNA. The expression was analyzed by whole-mount insitu hybridization essentially as described by Wilkinson (1992) butadapted as follows. Tissues were permeabilized 315 min in abuffer containing 0.1% SDS, 1% NP40, 0.5% Desoxycholat,150 mM NaCl, 1 mM EDTA, 50 mM Tris pH 8.0 before theprehybridization. The pre-incubation steps before the hybridizationand the antibody binding were extended to 8 h. The hybridizationand the first two washing steps were performed at 70C. Theembryos were washed for 3 days in maleic acid buffers and 2 daysin NTMT to remove all traces of antibody. Paraffin sections wereobtained from embryos stained after whole-mount in situ hybrid-ization. Bar =100 mm97Acknowledgements We thank D. Treichel, L. Wittler, D. Spieler,J. Mnner and H. Neuhaus for discussion, W. Behrens for technicaland R. Altschffel for photographical assistance. The mouse BMP-9 cDNA used for screening was kindly provided by T. Celeste,Genetics Institute. This work was supported by grant SFB 271/A3of the Deutsche Forschungsgemeinschaft.ReferencesChristoffels VM, Habets PE, Franco D, Campione M, de Jong F,Lamers WH, Bao ZZ, Palmer S, Biben C, Harvey RP,Moorman AF (2000) Chamber formation and morphogenesisin the developing mammalian heart. Dev Biol 223:266–278Hamburger V, Hamilton HL (1951) A series of normal stages in thedevelopment of the chick embryo. J Morphol 88:49–92Kelly RG, Brown NA, Buckingham ME (2001) The arterial pole ofthe mouse heart forms from FGF10-expressing cells inpharyngeal mesoderm. Dev Cell 1:435–440Kozak M (1984) Compilation and analysis of sequences upstreamfrom the translational start site in eukaryotic mRNAs. NucleicAcids Res 12:857–872Larsen WJ (1993) Human embryology. Churchill Livingstone, NewYorkMjaatvedt CH, Nakaoka T, Moreno-Rodriguez R, Norris RA, KernMJ, Eisenberg CA, Turner D, Markwald RR (2001) Theoutflow tract of the heart is recruited from a novel heart-forming field. Dev Biol 238:97–109Neuhaus H, Rosen V, Thies RS (1999) Heart specific expression ofmouse BMP-10, a novel member of the TGF-beta superfamily.Mech Dev 80:181–184Ozkaynak E, Schnegelsberg PN, Jin DF, Clifford GM, Warren FD,Drier EA, Oppermann H (1992) Osteogenic protein-2. A newmember of the transforming growth factor-beta superfamilyexpressed early in embryogenesis. J Biol Chem 267:25220–25227Song JJ, Celeste AJ, Kong FM, Jirtle RL, Rosen V, Thies RS(1995) Bone morphogenetic protein-9 binds to liver cells andstimulates proliferation. Endocrinology 136:4293–4297Waldo KL, Kumiski DH, Wallis KT, Stadt HA, Hutson MR, PlattDH, Kirby ML (2001) Conotruncal myocardium arises from asecondary heart field. Development 128:3179–3188Wilkinson DG (1992) In situ hybridisation; a practical approach.Oxford University Press, LondonFig. 2A–H Expression ofBMP10 in the hearts during latestages of chick embryonic de-velopment. A, D, G Whole-mount in situ hybridization ofisolated hearts at HH21, HH27and HH32 show that BMP10RNA is present in the ventricles(v) and in the atria (a). Theoutflow tract (indicated bydashed line, oft) is free ofBMP10 transcript. The level ofthe sections is indicated. B, C,E, F, H Sections demonstratethat the staining becomes con-fined to the atrial wall and tothe innermost trabeculae (ar-rowheads) of the ventricles.Bar =100 mm98

Highly restricted BMP10 expression in the trabeculating myocardium of the chick embryo

Abstract

Similar works

Full text

Available Versions

MPG.PuRe