Three-dimensional reconstruction of gene expression patterns during cardiac development.
The study of the genetic regulation of embryonic development requires the three-dimensional (3D) mapping of gene expression at the microscopic level. Despite the recent burst in the number of methods focusing on 3D reconstruction of embryonic specimens, an adequate and accessible 3D reconstruction protocol for the visualization of patterns of gene expression is lacking. In this communication we describe a protocol that was developed for the 3D visualization of patterns of gene expression determined by in situ hybridization (ISH) on serial sections. The method still requires tissue sectioning, due to penetration limits of the specific staining agents into whole embryo preparations. With regard to expenditure of resources, i.e., hardware, software, and time, the protocol is relatively undemanding. Because the variation between specimens requires the visualization of multiple specimens per stage, it was decided to "do more, less well." The current protocol, therefore, results in reconstructions of sufficient, but not the highest, quality. The use of the protocol is demonstrated on a series of serially sectioned mouse hearts, ranging from embryonic day 8.5 to 14.5. The myocardium of the hearts was identified by ISH using a mixture of specific mRNA probes and reconstructed.
Soufan
Alexandre T
AT
Experimental and Molecular Cardiology Group, Academic Medical Center, 1105 AZ, Amsterdam, The Netherlands.
Ruijter
Jan M
JM
van den Hoff
Maurice J B
MJ
de Boer
Piet A J
PA
Hagoort
Jaco
J
Moorman
Antoon F M
AF
D000818:Animals; D002319:Cardiovascular System / Q000196:embryology / Q000254:growth & development*; D004195:Disease Models, Animal; D020869:Gene Expression Profiling / Q000379:methods*; D018507:Gene Expression Regulation, Developmental / Q000235:genetics*; D021621:Imaging, Three-Dimensional / Q000379:methods*; D051379:Mice