The transcription factor Sox2 is both necessary and sufficient for the generation of sensory regions of the inner ear. It regulates expression of the Notch ligand Jag1 in prosensory progenitors, which signal to neighboring cells to up-regulate Sox2 and sustain prosensory identity. However, the expression pattern of Sox2 in the early inner ear is very broad, suggesting that Sox2-expressing progenitors form a wide variety of cell types in addition to generating the sensory regions of the ear. We used Sox2-CreER mice to follow the fates of Sox2-expressing cells at different stages in ear development. We find that Sox2-expressing cells in the early otocyst give rise to large numbers of non-sensory structures throughout the inner ear, and that Sox2 only becomes a truly prosensory marker at embryonic day (E)11.5. Our fate map reveals the organ of Corti derives from a central domain on the medial side of the otocyst and shows that a significant amount of the organ of Corti derives from a Sox2-negative population in this region.

Single/selective-plane illumination, or light-sheet, systems offer several advantages over other fluorescence microscopy methods for live, 3D microscopy. These systems are valuable for studying embryonic development in several animal systems, such as Drosophila, C. elegans and zebrafish. The geometry of the light path in this form of microscopy requires the sample to be accessible from multiple sides and fixed in place so that it can be rotated around a single axis. Popular methods for mounting include hanging the specimen from a pin or embedding it in 1-2% agarose. These methods can be particularly problematic for certain samples, such as postimplantation mouse embryos, that expand significantly in size and are very delicate and sensitive to mounting. To overcome the current limitations and to establish a robust strategy for long-term (24 h) time-lapse imaging of E6.5-8.5 mouse embryos with light-sheet microscopy, we developed and tested a method using hollow agarose cylinders designed to accommodate for embryonic growth, yet provide boundaries to minimize tissue drift and enable imaging in multiple orientations. Here, we report the first 24-h time-lapse sequences of post-implantation mouse embryo development with light-sheet microscopy. We demonstrate that light-sheet imaging can provide both quantitative data for tracking changes in morphogenesis and reveal new insights into mouse embryogenesis. Although we have used this approach for imaging mouse embryos, it can be extended to imaging other types of embryos as well as tissue explants.

Abstract:We present an analysis of imaging murine embryos at various embryonic developmental stages (embryonic day 9.5, 11.5, and 13.5) by optical coherence tomography (OCT) and optical projection tomography (OPT). We demonstrate that while OCT was capable of rapid highresolution live 3D imaging, its limited penetration depth prevented visualization of deeper structures, particularly in later stage embryos. In contrast, OPT was able to image the whole embryos, but could not be used in vivo because the embryos must be fixed and cleared. Moreover, the fixation process significantly altered the embryo morphology, which was quantified by the volume of the eye-globes before and after fixation. All of these factors should be weighed when determining which imaging modality one should use to achieve particular goals of a study. References and links1. J. Beckers, W. Wurst, and M. H. de Angelis, "Towards better mouse models: enhanced genotypes, systemic phenotyping and envirotype modelling," Nat. Rev. Genet. 10(6), 371-380 (2009 440-443 (2000). 4. K. Paigen and J. T. Eppig, "A mouse phenome project," Mamm. Genome 11(9), 715-717 (2000). Proc. Natl. Acad. Sci. U.S.A. 91(9), 3530-3533 (1994). 9. M. Dhenain, S. W. Ruffins, and R. E. Jacobs, "Three-dimensional digital mouse atlas using high-resolution MRI," Dev. Biol. 232(2), 458-470 (2001 95-105 (1999). 33. U. Morgner, W. Drexler, F. X. Kärtner, X. D. Li, C. Pitris, E. P. Ippen, and J. G. Fujimoto, "Spectroscopic optical coherence tomography," Opt. Lett. 25(2), 111-113 (2000). 34. S. Rugonyi, C. Shaut, A. Liu, K. Thornburg, and R. K. Wang, "Changes in wall motion and blood flow in the outflow tract of chick embryonic hearts observed with optical coherence tomography after outflow tract banding and vitelline-vein ligation," Phys.

Single/selective-plane illumination, or light-sheet, systems offer several advantages over other fluorescence microscopy methods for live, 3D microscopy. These systems are valuable for studying embryonic development in several animal systems, such as Drosophila, C. elegans and zebrafish. The geometry of the light path in this form of microscopy requires the sample to be accessible from multiple sides and fixed in place so that it can be rotated around a single axis. Popular methods for mounting include hanging the specimen from a pin or embedding it in 1-2% agarose. These methods can be particularly problematic for certain samples, such as postimplantation mouse embryos, that expand significantly in size and are very delicate and sensitive to mounting. To overcome the current limitations and to establish a robust strategy for long-term (24 h) time-lapse imaging of E6.5-8.5 mouse embryos with light-sheet microscopy, we developed and tested a method using hollow agarose cylinders designed to accommodate for embryonic growth, yet provide boundaries to minimize tissue drift and enable imaging in multiple orientations. Here, we report the first 24-h time-lapse sequences of post-implantation mouse embryo development with light-sheet microscopy. We demonstrate that light-sheet imaging can provide both quantitative data for tracking changes in morphogenesis and reveal new insights into mouse embryogenesis. Although we have used this approach for imaging mouse embryos, it can be extended to imaging other types of embryos as well as tissue explants.

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