Confocal images of the periderm (A1) and basal epidermis (A2) showing localisation of E-cadherin (magenta) and aPKC (green) along various cell-heights at 48hpf in wild type embryos. Note 0 µm is the apical most section in both periderm as well as in the basal epidermis. Transverse sections of the epidermis covering the eye showing localisation of (B1) E-cadherin (magenta) and aPKC (yellow) and (B2) E-cadherin (magenta) and Lyn-GFP (green). Dotted arrows mark the cell boundary (B2) used for line intensity profiles for E-cadherin (B3) and Lyn-GFP (B4) in the Periderm (P) and for E-cadherin (B5) in Basal epidermis (BE). Quantification of E-cadherin levels along apicobasal axis at the normalised cell heights in the periderm (C1) and the basal epidermis (C2) at different developmental time points. Arrowheads in B1 and B2 point to maximum levels of E-cadherin at the interface of the two layers. Solid arrows in B1 point to aPKC localisation. Dotted lines mark the base of the epidermis. Scale bar is equivalent to 10 µm (A1, A2, B1, B2). AU = Arbitrary Units. Source file with fluorescence intensities for periderm and basal epidermis is available as Figure 1—source data 1 and 2 respectively.

Confocal Z-stack showing localisation of E-cadherin (magenta), Lyn-GFP (green) and aPKC (yellow) in the periderm (A). Confocal Z-stacks depicting the apical to basal localisation of Lgl2 (green) and E-cadherin (magenta) in the periderm (B) and basal epidermis (C) at 48hpf. Transverse section of the epidermis over the eye showing localisation of E-cadherin (magenta) and Lgl2 (green) (D). Dotted lines in (D) mark the base of the epidermis. 0 µm = apical; Scale bar represents 10 µm (A, B, C, D).

Confocal analysis of E-cadherin (magenta) immunolocalisation along the apicobasal axis (0 µm is apical) in the periderm (A) and basal epidermis (B) in wild-type embryos from 24hpf to 72hpf. Scale bar equals to 10 µm.

Immunostaining of β-catenin reveals that the adherens junctions are present along the apicobasal axis in periderm (A) and basal epidermis (B) at the mentioned time-points. 0 µm = apical; Scale bar represents 10 µm.

Immunolocalisation of E-cadherin along the apicobasal axis (0 µm is apical) in wild-type (WT) sibling and has/apkc mutant in the periderm (A1) and basal epidermis (B1) at 48hpf. Comparison of height of cells (A2, B2) and apical perimeter (A3, B3) in the periderm (A2, A3) and basal epidermis (B2, B3) of the two genetic conditions. Graphs showing polarised localisation of E-cadherin across normalised cell height in the periderm (A4) and basal epidermis (B4). A graph showing noise index in E-cadherin localisation in WT siblings versus has/apkc mutants (A5). Arrows in A4 pointing to the abnormal localisation of E-cadherin. Scale bars in A1, B1 are equivalent to 10 µm. AU = Arbitrary Units; WT = wild type, sib = sibling/s and has/apkc = has/apkc mutants. Asterisk indicates significant difference at p<0.05 and n.s. means non-significant difference observed by Mann Whitney U test. Source file with fluorescence intensities for periderm and basal epidermis is available as Figure 2—source data 1 and 2, respectively. Statistical analysis with p values for all the graphs of periderm and basal epidermis is available as Figure 2—source data 3 and 4, respectively.

Immunolocalisation of aPKC (green) and E-cadherin as well as p63 (basal epidermis marker; both magenta) in the periderm and basal epidermis at 24hpf (A1, A2) and 48hpf (B1, B2) showing absence of aPKC in the basal epidermis. Scale bar in A2 and B2 corresponds to 10 µm.

Transverse section through the dorsal head epidermis at 48hpf showing localisation of E-cadherin (magenta) and Lyn-GFP (green) along the apicobasal axis in WT siblings and has/apkc mutants (A). Dotted lines in (A) mark the base of the epidermis. Confocal micrographs showing localisation of E-cadherin (magenta) and Lyn-GFP (green) along the apicobasal axis (0 µm is apical) in the periderm in WT sibling and has/apkc mutant embryos at 48hpf (B). Scale bar represents 10 µm (A, B).

The loss of pen/lgl2 function has no effect on E-cadherin polarity. Immunolocalisation of E-cadherin at various cell heights (0 µm is apical) along the apicobasal axis in wild-type sibling (WT sib) and pen/lgl2 mutant in the periderm (A1) and basal epidermis (B1) at 72hpf. Comparison between WT siblings and pen/lgl2 mutants in the periderm (A2–A4) and the basal epidermis (B2–B4) for height of cells (A2, B2) and apical perimeter (A3, B3). Graphs showing polarised distribution of E-cadherin across normalised cell height in the periderm (A4) and basal epidermis (B4) in WT siblings and pen/lgl2 mutants. Scale bar corresponds to 10 µm in A1 and B1. AU = Arbitrary Units; WT = wild type and sib = sibling/s. Asterisk indicates significant difference (p<0.05) and n.s. means non-significant difference by Mann Whitney U statistical test. Source file with fluorescence intensities for periderm and basal epidermis is available as Figure 3—source data 1 and 2, respectively. Statistical analysis with p values for all the graphs of periderm and basal epidermis is available as Figure 3—source data 3 and 4, respectively.

Confocal images of E-cadherin (magenta) localisation in GFP clones (green) carrying cadherin-1 morpholino (cdh-1 MO) and control morpholino (Ctrl MO) in the periderm (A) and basal epidermis (D). (A1–A3) shows peridermal clone and its effect on E-cadherin localisation in the basal epidermal cells (A4–A6). (D4–D6) show a basal epidermis clone and its effect on E-cadherin localisation in the periderm (D1–D3). Graphs showing effect on E-cadherin levels across normalised cell height in the periderm (B, E) and basal epidermis (C, F) across different boundaries upon knockdown of e-cadherin in a clonal manner. The boundaries Clone-Clone (C–C), Clone-Non-clone (N–C), and Non clone-Non clone (N–N) are within the same layer with respect to the clones whereas boundaries showing Complete Overlap (CO), Partial Overlap (PO), and No Overlap (NO) are in the cells of the juxtaposed layer. Dotted line in (A1, A2) represents the position of the peridermal clone and the basal epidermal region below the clone (A4, A5). Similarly, dotted lines in (D4, D5) mark the clones in the basal epidermis and the peridermal region above the clone (D1, D2). Arrowheads point to the loss/reduction in E-cadherin staining at the cell membranes. Scale bar represents 10 µm in (A6, D6). AU = Arbitrary Units. Source file with fluorescence intensities for peridermal clone and E-cadherin analysis at the boundaries in the periderm (B) and basal epidermis (C) is available as Figure 4—source data 1 and 2, respectively. Fluorescence intensities for basal epidermal clones and analysis at the boundaries in periderm (E) and basal epidermis (F) is available as Figure 4—source data 3 and 4, respectively. Statistical analysis consisting of intensity comparisons is available as Figure 4—source data 5.

Morpholino tagged with Carboxyfluorescein (A2, B2, A5, and B5) and mCherry (A1, B1, A4 and B4) showing co-segregation in peridermal clones (A1–A3) but no localisation in basal epidermis (A4–A6). Similarly, clones in basal epidermis showing co-localisation with mCherry (B4–B6) but no localisation in periderm (B1–B3). Arrows point to cells showing co-segregation of labelled morpholino and mCherry. Dotted line in (A1, A2, A3) represents the position of the peridermal clone and the basal epidermal region below the clone (A4, A5, A6). Similarly, dotted lines in (B4, B5, and B6) mark the clones in the basal epidermis and the peridermal region above the clone (B1, B2, and B3). Asterisk marks fluorescence bleed through. Scale bar represents 10 µm (A6, B6). Std MO- Fluor = 3’- Carboxyfluorescein Standard Morpholino. Schematic (C1, C2) showing different types of boundaries defined for quantifications. The two epidermal layers, periderm (blue) and basal epidermis (magenta), overlaid on top of each other having either peridermal clone (green in C1) or basal epidermal clone (green in C2). Arrowheads demonstrate different types of boundaries namely, clone-clone (CC), Non-clone-Clone (NC), and Non clone-Non clone (NN) for analysing layer autonomous effect and complete overlap (CO), partial overlap (PO), and no overlap (NO) (C1, C2) for analysis of layer non autonomous effect.

Brightfield image of zebrafish embryos injected with Cdh-1 MO (A), Cdh-1 MO with mCherry tagged e-cad mRNA (B) and just e-cad-mCherry mRNA (C). Insets show a high zoom image of one of the embryos showing its stage; cdh-1 MO embryos are stuck at epiboly (A’), but the cdh-1MO + e-cad mRNA (B’) and e-cad mRNA (C’) embryos cross the epiboly stages. The numbers of embryos from various genotypes at the 70% epiboly stages are quantified in the table (D). Arrow shows the initiation of tail bud formation in (B’, C’).

E-cadherin regulates localisation of aPKC and Lgl2 in the epidermis. Confocal images showing GFP marked clones (A1, B1, C1) marking cadherin-1 morpholino (cdh-1 MO) or control MO (Ctrl MO) and immunolocalisation of E-cadherin (A2, B2, C2), aPKC (A3, A4) and Lgl2 (B3, B4, C3, C4). Arrowhead marks the loss of E-cadherin in morphant cells (A2, B2, C2). Different z-stacks depicting apical (A3) or basal domains (A4) of peridermal cells showing aPKC immunostaining. Arrow shows enrichment of aPKC in the basal domain of the cdh-1 morphant cells (A4). Intensity profiles for aPKC in the basal domain in non-clone (NC) cells (A5) and clone (C) cells (A6) were measured along the dotted arrows (in A4). Lgl staining in the periderm (B3, C4) or basal epidermis (B4, C3). Arrows show the high levels of Lgl in the morphant or juxtaposed cells (B3, B4, C3, and C4) upon e-cadherin knockdown. Graphs showing levels of Lgl across normalised cell height in the periderm (B5, C6) and basal epidermis (B6, C5) across different boundaries mentioned. Dotted line indicates the position of the clone in the periderm (B1–B3) or basal epidermis (C1–C3) and corresponding region in basal epidermis (B4) or periderm (C4), respectively. Scale bar represents 10 µm in (A4, B4, C4). AU = Arbitrary Units. Source file with fluorescence intensities for peridermal clone and boundary analysis in periderm (B5) and basal epidermis (B6) is available as Figure 5—source data 1 and 2, respectively. Fluorescence intensities for basal epidermis clone and analysis of boundaries in basal epidermis (C5) and periderm (C6) is available as Figure 5—source data 3 and 4, respectively. Statistical analysis of intensity comparisons along with p values is available as Figure 5—source data 5.

Immunostainings of embryos harbouring Ctrl MO (A) or cdh-1 MO (B) clones marked by GFP (A1, B1) showing E-cadherin (A2, B2) and β-catenin (A3, B3) localisation. Arrow heads (B2) and arrows (B3) mark the loss of E-cadherin and concurrent reduction in β-catenin localisation in morphant cells. Dotted lines demarcate the clones. Scale bar represents 10 µm (A3, B3).

Immunostaining of embryos having Ctrl MO (A) and cdh1 MO (B) clones marked by GFP (A1, B1) and showing e-cadherin knockdown (A2, B2), aPKC localisation in the basal epidermis (A3, B3) and the apical domain (A4, B4) as well as basal domain of the peridermal cells (A5, B5) lying above the basal epidermal clone. Note that there is no effect on aPKC localisation in the periderm when e-cadherin is knocked down in the basal epidermis. Dotted lines mark the basal epidermal clone and the peridermal region above the clone. Arrowheads (in B2) mark the loss of E-cadherin in the morphant clone. Scale bar equals to 10 µm (A5, B5).

Confocal micrographs of embryos with Ctrl MO (A) and cdh1 MO (B) clones labelled with GFP (A1, B1) and showing localisation of E-cadherin (A2, B2) and ZO-1 (A3, B3). Note that there is no effect of loss of e-cadherin function on ZO-1 localisation. Dotted lines represent the position of the peridermal clones. Arrowheads mark the reduction in E-cadherin localisation in morphant cells (B2). Scale bar is equivalent to 10 µm (A3, B3).

Confocal images showing expression and localisation of GFP tagged E-cadherin-FL and ΔEC1-EC2-Ecad (green) (A1, B1, C1) and localisation of E-cadherin (magenta) (A2, A4, B2, B4, C2), Lgl2 (A3, A5, B3, B5) and aPKC (C3, C4). Lgl staining in the periderm (A3, B5) or basal epidermis (B3, A5) is shown as heatmap with calibration bar provided for reference (A6, B6). Arrow shows high levels of Lgl in the ΔEC1-EC2-Ecad expressing cells or juxtaposed cells (A3, A5, B3, and B5). Dotted lines indicate the position of the clone in the periderm (A1–A3 and C1-C4) or basal epidermis (B1–B3) and corresponding region in basal epidermis (A4–A5) or periderm (B4–B5), respectively. Scale bar represents 10 µm in (A5, B5, C4).