Raymond T.H. Lee*, P.V. Asharani* and Tom J. Carney#
Institute of Molecular and Cell Biology (IMCB)
* Equal contribution
# Author for correspondence
Published in PLoS One on 6 January 2014.
Please see http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0084858
The epidermis of terrestrial vertebrates is a stratified epithelium and forms an essential protective barrier. It is continually renewed, with dead corneocytes shed from the surface and replaced from a basal keratinocyte stem cell population. Whilst mouse is the prime model system used for epidermal studies, there is increasing employment of the zebrafish to analyse epidermis development and homeostasis, however the architecture and ontogeny of the epidermis in this system are incompletely described. In particular, it is unclear if adult zebrafish epidermis is derived entirely from the basal epidermal stem cell layer, as in the mouse, or if the most superficial keratinocyte layer is a remnant of the embryonic periderm. Furthermore, a relative paucity of cellular markers and genetic reagents to label and manipulate the basal epidermal stem cell compartment has hampered research. Here we show that the type I keratin, krtt1c19e, is a suitable marker of the basal epidermal layer and identify a krtt1c19e promoter fragment able to drive strong and specific expression in this cell type. Use of this promoter to express an inducible Cre recombinase allowed permanent labelling of basal cells during embryogenesis, and demonstrated that these cells do indeed generate keratinocytes of all strata in the adult epidermis. Further deployment of the Cre-Lox system highlighted the transient nature of the embryonic periderm. We thus show that the epidermis of adult zebrafish, as in the mouse, derives from basal stem cells, further expanding the similarities of epidermal ontogeny across vertebrates. Future use of this promoter will assist genetic analysis of basal keratinocyte biology in zebrafish.
Figure Legend: Embryonic EVL is lost and is replaced by cells from the basal epidermis during metamorphosis
A-D’’: Confocal images of lateral views (A, C) and transverse cryosections (B-B’, D-D’’) of krt4:CreERt2; ubi:swtch (A-B’) and krtt1c19e:CreERt2; ubi:swtch (C-D’’) at 5dpf following 4-OHT mediated Cre conversion, and which have been immunofluorescently labelled with antibodies against mCherry (red; A-C, D’-D’’), ZO-1 (green; A, C), ΔNp63 (white; B’, D, D’’) and eGFP (green; D’’) and counterstained with DAPI (blue; B-B’, D-D’’). Upon treatment of 4-OHT, krt4:CreERt2 drove recombination, and thus mCherry expression, in ZO-1-positive/ ΔNp63-negative EVL cells (A-B’), whilst krtt1c19e:CreERt2 induced recombination in ΔNp63-positive/ZO-1-negative basal keratinocytes (C-D’’). E-F’’’: Time course of floxed krt4:CreERt2; ubi:swtch (E-E’’’) and krtt1c19e:CreERt2; ubi:swtch (F-F’’’) showing the same region of mCherry positive cells on the flank of representative individuals. Fluorescent images were taken at 12dpf (E, F), 22dpf (E’, F’) 32dpf (E’’, F’’) and 42dpf (E’’’, F’’’), and show that EVL cells are gradually lost, whilst clones of basal keratinocytes expand and stratify. n=24 per genotype. G-H’’: Transverse cryosections of the trunk (G-G’’) and fin (H-H’’) epidermis of 42hpf floxed krtt1c19e:CreERt2; ubi:switch transgenics, immunostained with antibodies against ΔNp63 (white; G, G’’, H, H’’), mCherry (red; G’-G’’, H’-H’’) and eGFP (green, G’’). Nuclei of all cells in the epidermis are marked by DAPI staining (blue; G-H’’). In contrast to 7dpf (C-D’’), mCherry is now found in both suprabasal and the most superficial ΔNp63-negative cell layers (examples of the latter highlighted by open arrowheads). Occasional superficial cells, not derived from floxed basal cells, can be seen (green cell highlighted by closed arrowhead G-G’’).
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