Basal keratinocytes contribute to all strata of the adult zebrafish epidermis - PubMed (original) (raw)

Basal keratinocytes contribute to all strata of the adult zebrafish epidermis

Raymond T H Lee et al. PLoS One. 2014.

Abstract

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Expression of krtt1c19e in basal keratinocytes.

In situ hybridisation of krtt1c19e at 24 hpf (A–E’), 48 hpf (F–H’) and 5 dpf (I), imaged laterally (A–D, F–G, I) or after cryosectioning (E–E’, H–H’). Overviews of embryos are shown at 24 hpf (A), 48 hpf (F) and 5 dpf (I), showing broad skin expression. Epidermal cells were visualised by counterstaining with DAPI (blue – E’, H’) or by Nomarski optics (B–C, F inset, E’, H’), and basal cell nuclei were immunolabelled using an antibody against ΔNp63 (red D–E’, H–H’). Strong krtt1c19e epidermal expression can be seen in the basal keratinocytes with the borders of overlying EVL cells intersecting basal cells (B - arrowheads). A gap in the krtt1c19e in situ signal is seen in the epidermis corresponding to the location of the migrating lateral line primordial (C). krtt1c19e expressing keratinocytes have ΔNp63 immunoreactive nuclei (D–E’, H–H’) and are seen below EVL cells in cryosections (E’, H’). A higher magnification of the tail region of the 48 hpf embryo is shown inset (F), with expression excluded from the fin epithelium, whilst a single layer of keratinocytes can be seen over the eye as part of the cornea (G).

Figure 2

Figure 2. Isolation and transient activity of a krtt1c19e promoter.

Map of the genomic context of the krtt1c19e gene in the type I keratin cluster on chromosome 19 (A) with a schematic of the intron/exon structure of the krtt1c19e gene and the upstream cki gene (B). The entire sequence upstream of krtt1c19e to the end of the cki was isolated and cloned upstream of egfp (C). Injection of this krtt1c19e:egfp construct into embryos yielded limited epidermal expression at 24 hpf but widespread eGFP expression by 48 hpf (C). E–L: Micrographs of eGFP positive epidermal cells in both the basal layer (E–H) and EVL (I–L), demonstrated by co-immunofluorescent labelling with antibodies against eGFP (green – E–L), ΔNp63 (red – E–G, L), E-cadherin (red - H) and ZO-1 (purple – I-K). At 24 hpf (E, I), 48 hpf (F, J) and 72 hpf (G–H, K–L) both ΔNp63 and E-cadherin positive basal cells are eGFP positive as are ZO-1 positive EVL cells. eGFP positive EVL cells can be seen above ΔNp63 positive nuclei (arrowheads - L).

Figure 3

Figure 3. Characterization of krtt1c19e :egfp transgenic larvae.

A–D’: Confocal images of eGFP expression in germline krtt1c19e:egfp transgenic larvae at 48 hpf (A, C–C’) and 7 dpf (B, D–D’). A–B: Lateral overviews of transgenic larvae indicating the krtt1c19e promoter drives eGFP expression in the epidermis of zebrafish larvae at all locations except the fins (extent of medial fins outlined by white line). C–D’: Immunofluorescent labelling of transgenic larvae cryosections demonstrates co-expression of eGFP (green; C–D’) and ΔNp63 (red; C–D’) in basal keratinocytes. Counterstaining with DAPI (blue; C–D’) and Pan-cadherin (white; C’, D’) highlights the eGFP negative region overlying EVL (demarcated by dashed lines; C’, D’). E–F’’’: Confocal images of the epidermis of germline krtt1c19e:lyn-tdtomato; krt4:lyn-egfp double transgenic larvae at 72 hpf (E–E’’) and 7 dpf (F–F’’’) immunofluorescently stained for eGFP (green; E’–E’’, F’, F’’’), tdTomato (red; E, E’’, F, F’’’), ΔNp63 (white; E, E’’) and ZO-1 (white; F’’–F’’’). The expression of membrane bound tdTomato delineates the ΔNp63 positive basal keratinocytes from the eGFP expressing ZO-1 positive EVL cells.

Figure 4

Figure 4. Characterization of krtt1c19e:egfp transgenic adult zebrafish.

A–C: Lateral micrographs of krtt1c19e:egfp transgenic adult zebrafish, showing eGFP expression weakly in the trunk region but strongly in the fins (A). Expression is also seen associated with neuromasts of the lateral line system in the body (A, B) and head (C). D–G’’’: Immunostaining of cryosections from the trunk region (D–E’’) or fin (F–G’’’) of transgenic adults demonstrates expression is in basal and suprabasal epidermal cells, with no expression visible in the superficial epidermal stratum (arrowheads D–D’’). eGFP is shown in green (D, D’’, E, E’’, F, F’’’, G, G’’’) and co-localises with ΔNp63 (red; E’–E’’, F’, F’’’, G’, G’’’) in basal and suprabasal keratinocytes, and is excluded from the most superficial keratinocytes (e.g. arrowheads F’’’, G’’’) visualised by DAPI (blue; D–G’’’), Phalloidin staining (white, D’–D’’) or Pan-cadherin staining (white; F’’–F’’’, G’’–G’’’).

Figure 5

Figure 5. Transplantation of basal epidermal cells demonstrates contribution to the EVL at late stages.

A: Schematic of transplantation strategy to test if the EVL of post metamorphosis larvae derives from the basal layer. Deep cells from krtt1c19e:lyn-tdtomato; krt4:lyn-egfp double transgenic embryos were transplanted into wild-type embryos after segregation of the EVL lineage at 4.3 hpf. Labelling of different epidermal cells was checked at 2 timepoints. B–G: Confocal images of the epidermis of representative recipient embryo at 7 dpf (B–D) and 26 dpf (E–G), immunostained for ΔNp63 (white; B,D–E, G), tdTomato (red; B,D–E, G) and eGFP (green; C–D, F–G). Transplanted deep cells only generate clones of basal cells expressing lyn-tdTomato at 7 dpf (B–D), but do not contribute to the EVL. The superficial layer is eGFP positive at 26 dpf indicating it is descended from the basal cells (E–G). n = 5 transplants were followed.

Figure 6

Figure 6. 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 5 dpf 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 12 dpf (E, F), 22 dpf (E’, F’) 32 dpf (E’’, F’’) and 42 dpf (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 42 hpf 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 7 dpf (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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This work was supported by the Biomedical Research Council of A*STAR (Agency for Science, Technology and Research), Singapore. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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