Artefacts in Volume Data Generated with High Resolution Episcopic Microscopy (HREM).

High resolution episcopic microscopy (HREM) produces digital volume data by physically sectioning histologically processed specimens, while capturing images of the subsequently exposed block faces. Our study aims to systematically define the spectrum of typical artefacts inherent to HREM data and to research their effect on the interpretation of the phenotype of wildtype and mutant mouse embryos. A total of 607 (198 wildtypes, 409 mutants) HREM data sets of mouse embryos harvested at embryonic day (E) 14.5 were systematically and comprehensively examined. The specimens had been processed according to essentially identical protocols. Each data set comprised 2000 to 4000 single digital images. Voxel dimensions were 3 × 3 × 3 µm3. Using 3D volume models and virtual resections, we identified a number of characteristic artefacts and grouped them according to their most likely causality. Furthermore, we highlight those that affect the interpretation of embryo data and provide examples for artefacts mimicking tissue defects and structural pathologies. Our results aid in optimizing specimen preparation and data generation, are vital for the correct interpretation of HREM data and allow distinguishing tissue defects and pathologies from harmless artificial alterations. In particular, they enable correct diagnosis of pathologies in mouse embryos serving as models for deciphering the mechanisms of developmental disorders.

FGF10 promotes regional foetal cardiomyocyte proliferation and adult cardiomyocyte cell-cycle re-entry.

AIMS: Cardiomyocyte proliferation gradually declines during embryogenesis resulting in severely limited regenerative capacities in the adult heart. Understanding the developmental processes controlling cardiomyocyte proliferation may thus identify new therapeutic targets to modulate the cell-cycle activity of cardiomyocytes in the adult heart. This study aims to determine the mechanism by which fibroblast growth factor 10 (FGF10) controls foetal cardiomyocyte proliferation and to test the hypothesis that FGF10 promotes the proliferative capacity of adult cardiomyocytes. METHODS AND RESULTS: Analysis of Fgf10(-/-) hearts and primary cardiomyocyte cultures reveals that altered ventricular morphology is associated with impaired proliferation of right but not left-ventricular myocytes. Decreased FOXO3 phosphorylation associated with up-regulated p27(kip) (1) levels was observed specifically in the right ventricle of Fgf10(-/-) hearts. In addition, cell-type-specific expression analysis revealed that Fgf10 and its receptor, Fgfr2b, are expressed in cardiomyocytes and not cardiac fibroblasts, consistent with a cell-type autonomous role of FGF10 in regulating regional specific myocyte proliferation in the foetal heart. Furthermore, we demonstrate that in vivo overexpression of Fgf10 in adult mice promotes cardiomyocyte but not cardiac fibroblast cell-cycle re-entry. CONCLUSION: FGF10 regulates regional cardiomyocyte proliferation in the foetal heart through a FOXO3/p27(kip1) pathway. In addition, FGF10 triggers cell-cycle re-entry of adult cardiomyocytes and is thus a potential target for cardiac repair.

Rapid high resolution three dimensional reconstruction of embryos with episcopic fluorescence image capture.

One of the overarching goals in developmental biology is the elucidation of mechanisms that elaborate form and function. To this end, an accurate morphological description of embryonic development is essential. However, visualizing dynamic changes in the three-dimensional (3D) structure of the developing embryo has been a "holy grail" in the field of developmental biology. The fundamental difficulties that have hindered all efforts in 3D reconstruction using two-dimensional (2D) image stacks revolve around the seemingly intractable problems of section registration and distortion. A remarkably simple solution has come about with the development of a new technique referred to as episcopic fluorescence image capture (EFIC). With EFIC imaging, tissue autofluorescence is used to image the block face prior to cutting each section. The 2D resolution obtained is close to that achieved by histology, and such 2D image stacks can be readily reconstructed in 3D. The 3D models generated provide fine structural details with resolution unmatched by 3D reconstructions obtained with any other imaging modalities. Given the perfect registration of EFIC image stacks, another important capability provided by EFIC is digital resectioning in any plane. This provides complete flexibility in the selection of optimal virtual sectioning planes for viewing different features in a specimen, and is invaluable for analyzing dynamic changes in tissue structure in the developing embryo. The capabilities provided by EFIC for rapid high resolution 3D reconstruction together with digital resectioning make this an unparalleled tool for characterizing morphogenetic events in the developing embryo. Although our review is focused on using EFIC for studying embryonic development, it is important to note that there is no intrinsic limitation on the size of the specimen that can be analyzed by EFIC imaging. Overall, EFIC should serve as an important imaging technique that will complement other 3D imaging modalities such as MRI and optical tomography. Given the feasibility of generating EFIC image stacks using cryoembedded or polyethylene glycol (PEG)-embedded specimens, there is the possibility that EFIC may be combined with 3D RNA or protein expression profiling. Together, such studies may help further elucidate the relationship between form and function.