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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

Keratin network morphogenesis in Krt8-YFP embryos.(a, a′) Fluorescence and brightfield of 8-cell stage shows complete absence of Krt8-YFP. (b–b′′) Increasing diffuse fluorescence and appearance of fluorescent dots (b′′; arrowheads) is observed 7-8 hours after compaction (b′). Images are taken from Supplementary Movie 5. (c–c′) Fluorescence and brightfield of early Krt8-YFP blastocyst reveal dotted keratin fluorescence at cell borders and cytoplasmic keratin particles. (d–d′′) The fluorescence micrographs are taken from Supplementary Movie 6 and depict keratin dynamics from the early to the mid-blastula stage. Note the increasing fluorescent dots at the cell-cell borders and the appearance of cytoplasmic keratin particles and filaments. (e–e′) Fluorescence and brightfield microscopy of mid-blastocyst shows that the Krt8-YFP positive puncta become interconnected by filamentous structures. (f–f′′) Micrographs from Supplementary Movie 7 demonstrate the overall changes in distribution and arrangement of keratin particles in contrast to the stable arrangement of elongated keratin filaments and juxtamembraneous puncta. (g–g′) Krt8-YFP fluorescence and brightfield microscopy of a late blastocyst reveals an extensive cytoplasmic keratin network anchored to cell borders. (h–h′′) Images from Supplementary Movie 8 depict overall keratin network stability (arrowheads). All scale bars, 20 μm.
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f5: Keratin network morphogenesis in Krt8-YFP embryos.(a, a′) Fluorescence and brightfield of 8-cell stage shows complete absence of Krt8-YFP. (b–b′′) Increasing diffuse fluorescence and appearance of fluorescent dots (b′′; arrowheads) is observed 7-8 hours after compaction (b′). Images are taken from Supplementary Movie 5. (c–c′) Fluorescence and brightfield of early Krt8-YFP blastocyst reveal dotted keratin fluorescence at cell borders and cytoplasmic keratin particles. (d–d′′) The fluorescence micrographs are taken from Supplementary Movie 6 and depict keratin dynamics from the early to the mid-blastula stage. Note the increasing fluorescent dots at the cell-cell borders and the appearance of cytoplasmic keratin particles and filaments. (e–e′) Fluorescence and brightfield microscopy of mid-blastocyst shows that the Krt8-YFP positive puncta become interconnected by filamentous structures. (f–f′′) Micrographs from Supplementary Movie 7 demonstrate the overall changes in distribution and arrangement of keratin particles in contrast to the stable arrangement of elongated keratin filaments and juxtamembraneous puncta. (g–g′) Krt8-YFP fluorescence and brightfield microscopy of a late blastocyst reveals an extensive cytoplasmic keratin network anchored to cell borders. (h–h′′) Images from Supplementary Movie 8 depict overall keratin network stability (arrowheads). All scale bars, 20 μm.

Mentions: To document de novo keratin network formation, we imaged free-floating homozygous Krt8-YFP embryos. As expected, 8-cell stage embryos did not show any fluorescence signal until after they had compacted12 (Fig. 5a–a′, b, b′). Six to 8 hours after compaction, a diffuse cytoplasmic Krt8-YFP signal appeared and strongly fluorescent dots formed that accumulated at cell borders (Supplementary Movie 5; arrowheads in Fig. 5b′′). During the subsequent maturation of the compacted morula (Supplementary Movie 6; Fig. 5c–e′), the dotted signal at cell borders increased continuously. At the same time, fluorescent dots emerged throughout the cytoplasm and subsequently elongated into filamentous particles, giving rise to unstable reticular structures, predominantly in the cell periphery. Images recorded of mid-blastocysts at higher spatial and temporal resolution (Supplementary Movie 7; Fig. 5f–f′′) revealed that the cytoplasmic particles were highly flexible and moved around randomly. Occasionally, particles fused with each other into longer filaments that sometimes broke apart again. Gradually, a fully developed keratin filament network appeared (Fig. 5g–g′). The filaments within the network were stably integrated with only minor fluctuations (Supplementary Movie 8; arrowheads in Fig. 5h–h′′). The dotted cell border fluorescence persisted but became less prominent in comparison to the increasing cytoplasmic filament network. In addition, filamentous elements appeared that interconnected the dots. Immunofluorescence microscopy further revealed that the dots co-localized perfectly with the desmosomal marker desmoplakin (Fig. 6).


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

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

Keratin network morphogenesis in Krt8-YFP embryos.(a, a′) Fluorescence and brightfield of 8-cell stage shows complete absence of Krt8-YFP. (b–b′′) Increasing diffuse fluorescence and appearance of fluorescent dots (b′′; arrowheads) is observed 7-8 hours after compaction (b′). Images are taken from Supplementary Movie 5. (c–c′) Fluorescence and brightfield of early Krt8-YFP blastocyst reveal dotted keratin fluorescence at cell borders and cytoplasmic keratin particles. (d–d′′) The fluorescence micrographs are taken from Supplementary Movie 6 and depict keratin dynamics from the early to the mid-blastula stage. Note the increasing fluorescent dots at the cell-cell borders and the appearance of cytoplasmic keratin particles and filaments. (e–e′) Fluorescence and brightfield microscopy of mid-blastocyst shows that the Krt8-YFP positive puncta become interconnected by filamentous structures. (f–f′′) Micrographs from Supplementary Movie 7 demonstrate the overall changes in distribution and arrangement of keratin particles in contrast to the stable arrangement of elongated keratin filaments and juxtamembraneous puncta. (g–g′) Krt8-YFP fluorescence and brightfield microscopy of a late blastocyst reveals an extensive cytoplasmic keratin network anchored to cell borders. (h–h′′) Images from Supplementary Movie 8 depict overall keratin network stability (arrowheads). All scale bars, 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Keratin network morphogenesis in Krt8-YFP embryos.(a, a′) Fluorescence and brightfield of 8-cell stage shows complete absence of Krt8-YFP. (b–b′′) Increasing diffuse fluorescence and appearance of fluorescent dots (b′′; arrowheads) is observed 7-8 hours after compaction (b′). Images are taken from Supplementary Movie 5. (c–c′) Fluorescence and brightfield of early Krt8-YFP blastocyst reveal dotted keratin fluorescence at cell borders and cytoplasmic keratin particles. (d–d′′) The fluorescence micrographs are taken from Supplementary Movie 6 and depict keratin dynamics from the early to the mid-blastula stage. Note the increasing fluorescent dots at the cell-cell borders and the appearance of cytoplasmic keratin particles and filaments. (e–e′) Fluorescence and brightfield microscopy of mid-blastocyst shows that the Krt8-YFP positive puncta become interconnected by filamentous structures. (f–f′′) Micrographs from Supplementary Movie 7 demonstrate the overall changes in distribution and arrangement of keratin particles in contrast to the stable arrangement of elongated keratin filaments and juxtamembraneous puncta. (g–g′) Krt8-YFP fluorescence and brightfield microscopy of a late blastocyst reveals an extensive cytoplasmic keratin network anchored to cell borders. (h–h′′) Images from Supplementary Movie 8 depict overall keratin network stability (arrowheads). All scale bars, 20 μm.
Mentions: To document de novo keratin network formation, we imaged free-floating homozygous Krt8-YFP embryos. As expected, 8-cell stage embryos did not show any fluorescence signal until after they had compacted12 (Fig. 5a–a′, b, b′). Six to 8 hours after compaction, a diffuse cytoplasmic Krt8-YFP signal appeared and strongly fluorescent dots formed that accumulated at cell borders (Supplementary Movie 5; arrowheads in Fig. 5b′′). During the subsequent maturation of the compacted morula (Supplementary Movie 6; Fig. 5c–e′), the dotted signal at cell borders increased continuously. At the same time, fluorescent dots emerged throughout the cytoplasm and subsequently elongated into filamentous particles, giving rise to unstable reticular structures, predominantly in the cell periphery. Images recorded of mid-blastocysts at higher spatial and temporal resolution (Supplementary Movie 7; Fig. 5f–f′′) revealed that the cytoplasmic particles were highly flexible and moved around randomly. Occasionally, particles fused with each other into longer filaments that sometimes broke apart again. Gradually, a fully developed keratin filament network appeared (Fig. 5g–g′). The filaments within the network were stably integrated with only minor fluctuations (Supplementary Movie 8; arrowheads in Fig. 5h–h′′). The dotted cell border fluorescence persisted but became less prominent in comparison to the increasing cytoplasmic filament network. In addition, filamentous elements appeared that interconnected the dots. Immunofluorescence microscopy further revealed that the dots co-localized perfectly with the desmosomal marker desmoplakin (Fig. 6).

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