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Dissection of keratin network formation, turnover and reorganization in living murine embryos.

Schwarz N, Windoffer R, Magin TM, Leube RE - Sci Rep (2015)

Bottom Line: Epithelial functions are fundamentally determined by cytoskeletal keratin network organization.However, our understanding of keratin network plasticity is only based on analyses of cultured cells overexpressing fluorescently tagged keratins.This mouse model will help to further dissect keratin network dynamics in its native tissue context during physiological and also pathological events.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cellular Anatomy, RWTH Aachen University, Aachen, Germany.

ABSTRACT
Epithelial functions are fundamentally determined by cytoskeletal keratin network organization. However, our understanding of keratin network plasticity is only based on analyses of cultured cells overexpressing fluorescently tagged keratins. In order to learn how keratin network organization is affected by various signals in functional epithelial tissues in vivo, we generated a knock-in mouse that produces fluorescence-tagged keratin 8. Homozygous keratin 8-YFP knock-in mice develop normally and show the expected expression of the fluorescent keratin network both in fixed and in vital tissues. In developing embryos, we observe for the first time de novo keratin network biogenesis in close proximity to desmosomal adhesion sites, keratin turnover in interphase cells and keratin rearrangements in dividing cells at subcellular resolution during formation of the first epithelial tissue. This mouse model will help to further dissect keratin network dynamics in its native tissue context during physiological and also pathological events.

No MeSH data available.


Related in: MedlinePlus

Trophectoderm-restricted expression of Krt8-YFP in a homozygous Krt8-YFP late blastocyst.(a, b) Brightfield image and corresponding fluorescence image showing Krt8-YFP exclusively in the outer trophectoderm layer, while the inner cell mass is completely negative (asterisk). (c) 3D-reconstruction of the fluorescence recording of 72 focal planes (1 μm steps) of the blastocyst shown in a. The relative fluorescence intensity is color coded. An animation of the reconstruction is presented in Supplementary Movie 4. Scale bar, 20 μm.
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f4: Trophectoderm-restricted expression of Krt8-YFP in a homozygous Krt8-YFP late blastocyst.(a, b) Brightfield image and corresponding fluorescence image showing Krt8-YFP exclusively in the outer trophectoderm layer, while the inner cell mass is completely negative (asterisk). (c) 3D-reconstruction of the fluorescence recording of 72 focal planes (1 μm steps) of the blastocyst shown in a. The relative fluorescence intensity is color coded. An animation of the reconstruction is presented in Supplementary Movie 4. Scale bar, 20 μm.

Mentions: To prove that the Krt8-YFP knock-in mice can be used to monitor keratin network formation and dynamics at high resolution in vivo, we decided to examine pre-implantation embryos. They are ideally suited for imaging due to their transparency, small size and the external location of their epithelial trophectodermal cells, which undergo rapid division during development. Imaging of live blastocysts revealed the expected trophectoderm-restricted keratin filament network (compare11 with Fig. 4). The 3D-animation in Supplementary Movie 4 further highlights the high degree of detail of the nascent cytoplasmic keratin network and prominent accumulations at cell-cell borders that can be achieved.


Dissection of keratin network formation, turnover and reorganization in living murine embryos.

Schwarz N, Windoffer R, Magin TM, Leube RE - Sci Rep (2015)

Trophectoderm-restricted expression of Krt8-YFP in a homozygous Krt8-YFP late blastocyst.(a, b) Brightfield image and corresponding fluorescence image showing Krt8-YFP exclusively in the outer trophectoderm layer, while the inner cell mass is completely negative (asterisk). (c) 3D-reconstruction of the fluorescence recording of 72 focal planes (1 μm steps) of the blastocyst shown in a. The relative fluorescence intensity is color coded. An animation of the reconstruction is presented in Supplementary Movie 4. Scale bar, 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4355630&req=5

f4: Trophectoderm-restricted expression of Krt8-YFP in a homozygous Krt8-YFP late blastocyst.(a, b) Brightfield image and corresponding fluorescence image showing Krt8-YFP exclusively in the outer trophectoderm layer, while the inner cell mass is completely negative (asterisk). (c) 3D-reconstruction of the fluorescence recording of 72 focal planes (1 μm steps) of the blastocyst shown in a. The relative fluorescence intensity is color coded. An animation of the reconstruction is presented in Supplementary Movie 4. Scale bar, 20 μm.
Mentions: To prove that the Krt8-YFP knock-in mice can be used to monitor keratin network formation and dynamics at high resolution in vivo, we decided to examine pre-implantation embryos. They are ideally suited for imaging due to their transparency, small size and the external location of their epithelial trophectodermal cells, which undergo rapid division during development. Imaging of live blastocysts revealed the expected trophectoderm-restricted keratin filament network (compare11 with Fig. 4). The 3D-animation in Supplementary Movie 4 further highlights the high degree of detail of the nascent cytoplasmic keratin network and prominent accumulations at cell-cell borders that can be achieved.

Bottom Line: Epithelial functions are fundamentally determined by cytoskeletal keratin network organization.However, our understanding of keratin network plasticity is only based on analyses of cultured cells overexpressing fluorescently tagged keratins.This mouse model will help to further dissect keratin network dynamics in its native tissue context during physiological and also pathological events.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cellular Anatomy, RWTH Aachen University, Aachen, Germany.

ABSTRACT
Epithelial functions are fundamentally determined by cytoskeletal keratin network organization. However, our understanding of keratin network plasticity is only based on analyses of cultured cells overexpressing fluorescently tagged keratins. In order to learn how keratin network organization is affected by various signals in functional epithelial tissues in vivo, we generated a knock-in mouse that produces fluorescence-tagged keratin 8. Homozygous keratin 8-YFP knock-in mice develop normally and show the expected expression of the fluorescent keratin network both in fixed and in vital tissues. In developing embryos, we observe for the first time de novo keratin network biogenesis in close proximity to desmosomal adhesion sites, keratin turnover in interphase cells and keratin rearrangements in dividing cells at subcellular resolution during formation of the first epithelial tissue. This mouse model will help to further dissect keratin network dynamics in its native tissue context during physiological and also pathological events.

No MeSH data available.


Related in: MedlinePlus