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

OPEN SUBJECT AREAS: INTERMEDIATE FILAMENTS CELLULAR IMAGING Received 2 November 2014 Accepted 10 February 2015 Published 11 March 2015 Correspondence and requests for materials should be addressed to R.E.L. (rleube@ Dissection of keratin network formation, turnover and reorganization in living murine embryos Nicole Schwarz1, Reinhard Windoffer1, Thomas M. Magin2 & Rudolf E. Leube1 1 Institute of Molecular and Cellular Anatomy, RWTH Aachen University, Aachen, Germany, 2Translational Center for Regenerative Medicine and Institute of Biology, University of Leipzig, Leipzig, Germany. 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. ukaachen.de) K eratins are among the most abundant proteins in epithelial tissues providing resistance to environmental stress. Their essential contribution to epithelial function is attested to by a broad range of keratin-related human diseases with barrier defects, inflammation, hyperproliferation and dedifferentiation and by a growing number of murine knockouts with pronounced epithelial dysfunction1–3. Keratin function is considered to be dependent on a high degree of compositional and structural plasticity of the keratin cytoskeleton. The more than 50 keratin polypeptides assemble as obligatory type I/type II heterodimers, which are expressed in a contextspecific manner4,5. While assembly of keratin heterodimers into 10 nm filaments occurs spontaneously without proteinaceous co-factors or nucleoside triphosphates in vitro, in vivo assembly of keratins into desmosomeanchored networks appears to be much more complex. So far, our knowledge of keratin network plasticity has been restricted to analyses of cultured cells that overexpress fluorescence-tagged keratins6,7. It has been proposed that the observed dynamic processes are part of a spatially defined multistep turnover cycle2,7. How keratin networks become organized and react to various signals in functional epithelial tissues is, however, completely unknown. To address these questions, we wanted to generate a knock-in mouse producing fluorescence-tagged keratins, which mimic the properties of endogenous keratins as closely as possible. We selected enhanced yellow fluorescent protein (YFP) as a tag because of its superior brightness and stability8 in combination with low in vivo toxicity9. As a suitable target we chose the keratin 8 gene (Krt8) because it is expressed in all simple epithelia, in certain compartments of all multilayered epithelia, and in most carcinomas4,5. Furthermore, along with keratin 7, it is the first intermediate filament protein to be synthesized during embryogenesis10–15. Results Establishment of healthy homozygous Krt8-YFP knock-in mice. The YFP-encoding gene was inserted in frame at the end of the protein-coding sequence in exon 9 of the Krt8 gene by homologous recombination in embryonic stem cells (ESCs) as detailed in Fig. 1a. The insertion should not adversely affect transcription of the Krt8-coding sequence since all of the native gene regions are maintained in the recombined gene locus. Correct integration of the targeting construct into the Krt8 gene was verified by PCR and Southern blotting in selected ESC clones (Fig. 1b). Fluorescence microscopy was then performed to examine the expression of keratin 8-YFP (Krt8-YFP) in fixed differentiating ESCs. The comparison of the images in Fig. 1c and Fig. 1d shows that the fluorescent Krt8YFP filament network in recombinant ESCs is similar to that observed by anti-keratin 8 staining in differentiating SCIENTIFIC REPORTS | 5 : 9007 | DOI: 10.1038/srep09007 1 www.nature.com/scientificreports wild-type ESCs. Fluorescence microscopy of vital embryoid bodies derived from Krt8-YFP knock-in ESCs revealed a restricted expression of Krt8-YFP in the outer cell layer (Fig. 1e,e9), which is known to be keratin 8-positive16 and corresponds to the primitive endoderm17. Time-lapse fluorescence microscopy could be performed for extended periods on embryonic bodies. The example presented in Fig. 1e9–e999 and corresponding Supplementary Movie 1 depicts the substantial rearrangements of the keratin network during the cell cycle and in the highly motile outgrowing cells. Correctly targeted ESCs were subsequently used to generate Krt8YFP chimeric mice. The recombinant Krt8-YFPneo allele (Fig. 1a) was transmitted through the germline to transgenic offspring. These animals were subsequently crossed with the FLPu deleter strain18. The neomycin resistance cassette was successfully removed by flpmediated recombination as determined by PCR in the resulting offspring. Homozygous and heterozygous Krt8-YFP knock-in mice were born to heterozygous parents close to the expected Mendelian ratio (wildtype: 31%; heterozygous knock-in: 45%; homozygous knock-in: 24%; n 5 42). All knock-in mice developed normally, demonstrating that the Krt8-YFP fusion protein functionally substitutes for wild-type keratin 8, which, depending on the genetic background, has been shown to be essential for embryogenesis and intestinal function19,20. Matings of homozygous mice resulted in 5.6 pups/litter (n 5 139), which is in the expected range for the genetic background of the mouse strain (C57BL/6 and 129/Ola; http://www. informatics.jax.org/). The sex distribution of the offspring was also within the normal range (45% males). No obvious defects of keratin 8 expressing tissues were detected in hematoxylin-eosin-stained sections of homozygous Krt8-YFP knock-in mice (Supplementary Figure 1). Orthotopic localization of Krt8-YFP in fixed and vital tissues of adult mice. According to previous reports on keratin 8 expression4,5 Krt8-YFP is expected to be produced in simple and complex epithelia. Cytoskeletal extracts were therefore prepared from lung, liver, kidney, and intestine of adult knock-in mice to detect Krt8-YFP by immunoblotting. Fig. 2a shows that Krt8-YFP fusion proteins with the expected molecular mass of approximately 82 kDa are present in these tissues of homozygous kno (...truncated)