Amira data and label files and 3D pdfs

These files contain a human embryonic section series that was published previously [1,2,3] so no informed consent was obtained for this investigation. The embryos were left over material from induced abortions on social indication performed at the Gynaecology Department of the Tartu University Hospital, Estonia. They had been collected with permission of the Medical Ethics committees of the University of Tartu, Estonia, and of the University of Amsterdam, the Netherlands. The investigation conformed to the principles outlined in the Declaration of Helsinki. The embryos were fixed in 4% paraformaldehyde after collection.<br>After exclusion of outward abnormalities, embryos had been staged on the basis of outward appearances before they were dehydrated, immersed in butanol, and embedded in paraffin [1]. The embryos are of Carnegie Stage (CS) 10, 11, 12, 13, 14, 15, 16, 18, 20 and 23. <br>The embryos had previously been sectioned at 7 (CS10, 11, 12, 13, 14, 15, 16, 18, 23) or 10 µm (CS20) thickness. Thereafter, sections containing parts of the heart had been immunofluorescently stained with Troponin I (1 : 250; MAB1691, Chemicon or 1:250; Hytest, 4T21/2 (only on CS11, 12 and 13)) or, on alternating sections, Troponin I, SERCA2a (1:250; ab2817, Abcam) [4] and MF20 (produced in house after [5]).<br>With a fluorescence microscope Leica DM6000, driven by ImagePro Plus 6.2 software (Media Cybernetics), each stained section had been photographed (Sizarov et al., 2010) and was imported in Amira 6.5.0 (Konrad-Zuse-Zentrum Berlin; FEI SAS, Thermo Fisher Scientific) (Fig. 1A). The x and y values of the voxel size were set to correspond with the actual tissue size on a section. The z value corresponded with section thickness. If a section was severely damaged, it was substituted with a copy of an adjacent section.<br>The AlignSlices module of Amira software allows for automatic alignment of the sections without deformation correction, while permitting manual adjustments of transformation and rotation. This was done for all hearts. Next, all recognisable cardiac structures were manually segmented on each individual section. The description of all label definitions can be found in the main manuscript. <br>The interactive 3D pdfs of the hearts were created using the Generate Surface tool in Amira with the Smoothing option set to None. Hereafter, the Surface (.surf) file was simplified in the Simplification editor to 1,000,000 faces with the options Preserve slice structure, Fast and Create level-of-detail switched on. Hereafter, the Amira surface views were imported in Fiji (ImageJ 1.53c) in order to generate Universal 3D (.u3d) files that could be imported as 3D Object in Adobe Acrobat Pro DC (version 2020.013.20064) after clustering of labels for the navigation panel in Deep Exploration (version 6.5 CSE, Corel DESIGNER Technical Suite X5). A custom made user interface based on javascripts is embedded in the files. The 3D pdfs can be viewed in recent versions of Adobe Reader on MS Windows or MacOS systems with javascript and playing of 3D content enabled. With the button rows on the left side of the pdf, individual or clusters of structures can be made visible (right button), transparent (middle button) or invisible (left button). By clicking and holding the left mouse button, the model will rotate when the mouse is moved. Clicking and holding the right mouse button will zoom the model when the mouse is moved. Clicking and holding both mouse buttons translates the model when the mouse is moved.<br><br><br>[1] A. Sizarov, J. Ya, B.A. De Boer, W.H. Lamers, V.M. Christoffels, A.F.M. Moorman, Formation of the building plan of the human heart: Morphogenesis, growth, and differentiation, Circulation. 123 (2011) 1125–1135. doi:10.1161/CIRCULATIONAHA.110.980607.<br>[2] A. Sizarov, H.D. Devalla, R.H. Anderson, R. Passier, V.M. Christoffels, A.F.M. Moorman, Molecular analysis of patterning of conduction tissues in the developing human heart, Circ. Arrhythmia Electrophysiol. 4 (2011) 532–542. doi:10.1161/CIRCEP.111.963421.<br>[3] A. Sizarov, W.H. Lamers, T.J. Mohun, N.A. Brown, R.H. Anderson, A.F.M. Moorman, Three-dimensional and molecular analysis of the arterial pole of the developing human heart, J. Anat. 220 (2012) 336–349. doi:10.1111/j.1469-7580.2012.01474.x.<br>[4] A. Sizarov, R.H. Anderson, V.M. Christoffels, A.F.M.M. Moorman, Three-dimensional and molecular analysis of the venous pole of the developing human heart, Circulation. 122 (2010) 798–807. doi:10.1161/CIRCULATIONAHA.110.953844.<br>[5] D. Bader, T. Masaki, D.A. Fischman, Immunochemical analysis of myosin heavy chain during avian myogenesis in vivo and in vitro, J. Cell Biol. 95 (1982) 763–770. doi:10.1083/jcb.95.3.763.<br><br><br>