Visualizing expression patterns of Shh and Foxf1 genes in the foregut and lung buds by optical projection tomography

Abstract

Congenital malformations of the foregut are common in humans. The respiratory and digestive tubes are both derived by division of the foregut primordium. Sonic hedgehog (Shh) and Fork head box F1 (Foxf1) genes encode regulatory molecules that play a pivotal role in gut and lung morphogenesis and are therefore important candidate genes to be examined in models of foregut developmental disruption. Optical projection tomography (OPT) is a new, rapid and non-invasive technique for three-dimensional (3D) imaging of small biological tissue specimens that allows visualization of the tissue distribution of RNA in developing organs while also recording morphology. To explore the application of OPT in this context, we visualized Shh and Foxf1 gene expression patterns in the mouse foregut and lung buds at several stages of development. Time-mated CBA/Ca mice were harvested on embryonic days 9-12. The embryos were stained following whole mount in situ hybridization with labelled RNA probes to detect Shh and Foxf1 transcripts at each stage. The embryos were scanned by OPT to obtain 3D representations of gene expression domains in the context of the changing morphology of the embryo. OPT analysis of Shh and Foxf1 expression in the foregut and lung buds revealed extra details of the patterns not previously reported, particularly in the case of Foxf1 where gene expression was revealed in a changing pattern in the mesenchyme around the developing lung. Shh expression was also revealed in the epithelium of the lung bud itself. Both genes were detected in complementary patterns in the developing bronchi as late as E12, showing successful penetration of molecular probes and imaging at later stages. OPT is a valuable tool for revealing gene expression in an anatomical context even in internal tissues like the foregut and lung buds across stages of development, at least until E12. This provides the possibility of visualizing altered gene expression in an in vivo context in genetic or teratogenic models of congenital malformations.

Adriamycin-Induced Models of VACTERL Association

“…Ptc expression was unchanged between treated embryos and controls [Ioannides et al, 2003]. With optical projection tomography (OPT), it was possible to visualise Shh and Foxf1 expression patterns in the foregut in detail [Sato et al 2008]. Hajduk et al [2011b] confirmed the diffuse expression pattern of Shh in the AMM foregut and additionally found an abnormal site of Shh expression in the dorsal foregut which correlated with the vicinity and thickness of notochord branches ( fig.…”

Genes into geometry: imaging for mouse development in 3D

“…of critical developmental genes [39,[47][48][49][50]. On the other hand, local mapping of gene expression relative to morphological change, during craniofacial [31] or lung [26] development for instance, provide insight into the possible roles of gene expression in mediating development.…”

Three-Dimensional (3D) Visualisation of the Cardiovascular System of Mouse Embryos and Fetus

“…This sets an upper specimen size limit. Mouse embryos, especially early embryos are small enough to become 3D visualised with OPT [73,[78][79][80]. Late mouse embryos and mouse fetus must be parted or dissected prior to staining and scanning (e.g.…”