Differentiated keratinocytes extend cytoneme-like cellular protrusions

(A) Airineme-like protrusion (arrowhead), and (B) cytoneme-like protrusions (arrowheads) labelled in krt4:palmEGFP injected keratinocytes. (C) Keratinocytes expressing krt4 extend cytoneme-like protrusions most frequently during metamorphic stages [6.5-7.5 SSL; N=457 cells, 19 larvae total]. A cytoneme-like protrusion (arrowhead) labeled for (D) F-actin, revealed by LifeAct-mRuby but did not colocalize (E) with tubulin, revealed by Tuba-mCherry. (F) Keratinocyte cytonemes extend faster than they retract (N=125 cells, 4 larvae total), with an average length (G) of 18.21µm (N=190 cells, 4 larvae total). Scale bars: 20µm (A, B, D, E). Error bars indicate mean ± SEM.

Keratinocyte cytonemes establish physical contact with underlying undifferentiated keratinocytes

(A-A”) Postembryonic zebrafish epidermis of Tg(krt4:lyn-EGFP;krtt1c19e:lyn-tdTomato) comprises three distinct layers. (A) Expression of lyn-EGFP in krt4+ cells in the periderm layer. (A’-A”) Expression of lyn-tdTomato (pseudo-colored magenta) in krtt1c19e+ cells in (A’) the intermediate layer and (A”) the basal layer. Note that the green circular labelling in (A’, arrowheads) represents mucous-secreting goblet cells. (Chang and Hwang, 2011) (B) Undifferentiated keratinocytes (KC) extend significantly fewer cytoneme-like protrusions (P<0.0001, N=49, 3 larvae; N=91 cells, 6 larvae total). (C) Cytonemes from fully differentiated keratinocytes make physical contact with undifferentiated keratinocytes in the intermediate layer (red dotted circles). Note that dotted lines indicate the layers in the cross-sectional views. Statistical significances were assessed by Student t test. Scale bars: 20µm (A-A”, C). Error bars indicate mean ± SEM.

Cytoneme-mediated signaling regulates keratinocyte differentiation and proliferation

(A) Expression of cdc42DN effectively inhibits cytoneme extension (F2, 231 = 25.38, P<0.0001, N=234 cells, 10 larvae total). (B) Periderm layer of Tg(krt4:TetGBDTRE-v2a-Cdc42DN;krt4:lyn-EGFP;krtt1c19e:lyn-tdTomato) without drug treatment. (B’) Periderm layer of Tg(krt4:lyn-EGFP;krtt1c19e:lyn-tdTomato) with drug treatment. (B’’) Periderm layer of Tg(krt4:TetGBDTRE-v2a-cdc42DN;krt4:lyn-EGFP;krtt1c19e:lyn-tdTomato) with drug treatment exhibits disorganization and regions lacking krt4+ keratinocytes (arrowhead). Cytoneme inhibition increases krtt1c19e expression in the periderm (C) (F2, 387 = 226.7, P<0.0001, N=39 larvae total) and leads to an increased number of keratinocytes within the periderm (D) (F2, 18 = 27.36, P<0.0001, N=21 larvae total). (E) The cell death rate in the periderm is not affected by cytoneme inhibition, tested with acridine orange incorporation (F2, 17 = 3.192, P=0.0666, N=20 larvae total). Statistical significances were assessed by One-way ANOVA followed by Tukey’s HSD post hoc test. Scale bars: 20µm (B-B”). Error bars indicate mean ± SEM.

Cytonemes activate Notch in undifferentiated keratinocytes

(A) Postembryonic zebrafish epidermis of Tg(krt4:lyn-EGFP;krtt1c19e:lyn-tdTomato;TP1:H2BmCherry) shows Notch responsiveness in undifferentiated keratinocytes within the intermediate layer but not in the periderm or basal layer. (B) Inhibition of cytonemes reduces Notch responsiveness in undifferentiated keratinocytes. (F2, 33 = 48.27, P < 0.0001, N=36 larvae total). (C) Expression of DeltaC-mCherry fusion protein along the cytoneme (yellow arrowheads). (D-D”) Notch inhibition by expressing SuHDN in undifferentiated keratinocytes results in the disorganization of periderm (D”). (C) Properly organized periderm layer of Tg(krtt1c19e:tetGBDTRE-v2a-SuHDN;krt4:lyn-EGFP;krtt1c19e:lyn-tdTomato) without drug treatment and (C’) Tg(krt4:lyn-EGFP;krtt1c19e:lyn-tdTomato) with drug treatment. (E-G) Notch inhibition (E) increases krtt1c19e signal in the periderm (F2, 397 = 89.47, P < 0.0001, N=40 larvae total), (F) reduces Notch responsiveness in undifferentiated keratinocytes (F2, 32 = 13.18, P < 0.0001, N=35 larvae total), and leads to (G) an increased number of keratinocytes in the periderm (F2, 19 = 6.620, P = 0.0066, N=22 larvae total). Statistical significances were assessed by One-way ANOVA followed by Tukey’s HSD post hoc test. Scale bars, 20µm (A, C, D). Error bars indicate mean ± SEM.

Disruption of epidermal maintenance by Interleukin-17 receptor overexpression

Overexpression of il17rd or il17ra1a in krt4+ keratinocytes results in (A) a significant reduction in cytoneme extension (F2, 325 = 38.48, P < 0.0001, N=328 cells, 10 larvae total), and (B) decreased Notch responsiveness (F2, 34 = 23.16, P < 0.0001, N=37 larvae total). (C-C’) The periderm is disrupted in (C’) Tg(krt4:nVenus-v2a-il17rd;krt4:lyn-EGFP;krtt1c19e:lyn-tdTomato) compared to the (C) properly arranged periderm of Tg(krt4:lyn-EGFP;krtt1c19e:lyn-tdTomato), resulting in (D) an increased krtt1c19e signal in the periderm (D) (F2, 287 = 109.7, P < 0.0001, N=29 larvae total) and (E) an increased number of keratinocytes in the periderm (F2, 15 = 27.79, P < 0.0001, N=18 larvae total). Statistical significances were assessed by One-way ANOVA followed by Tukey’s HSD post hoc test. Scale bars: 20µm (C). Error bars indicate mean ± SEM.

Interleukin-17 regulates keratinocyte cytonemes

(A) Significant reduction in cytoneme extension in response to overexpressed il17a ligands in the epidermis (P<0.0001, N=3 larvae per group). (B) Cytoneme extension frequency increased significantly in il17a+/- mutants as compared to their wild-type siblings (P=0.0009, N=4 larvae for wild-type, N=3 for mutants). (C) FACS sorted krt4+ peridermal cells show significantly reduced Cdc42 expression (P= 0.001, N=5 independent experiments). (D) il17rd-overexpressing krt4+ keratinocytes exhibit disorganization of microridges (arrowheads). (E) Simultaneous overexpression of cdc42DN and il17rd or il17ra1a in krt4+ keratinocytes further reduces cytoneme extension (il17rd: P < 0.0001, N=164 cells, 7 larvae total, il17ra1a: P < 0.0001, N=115 cells, 6 larvae total). Statistical significances were assessed by Student t test. Scale bars: 20µm (C, G). Error bars indicate mean ± SEM.