E-cadherin expression pattern during zebrafish embryonic epidermis development

Epidermis development in zebrafish (Danio rerio)

In zebrafish, an immature epidermis establishes soon after the end of gastrulation, constituted by the surface layer, enveloping layer (EVL), and the inner epidermal basal layer (EBL)^3,4. EVL arises at mid-blastula stage (2.5 hours post fertilization; hpf) covering the whole embryo⁵. During epiboly EVL maintains tight joins to the yolk syncytial layer (YSL), and becomes the migration substrate for the underlying deep cells spreading during gastrulation⁶. At the tail bud stage (8–10 hpf), the epiblast forms the outer germ layer, the ectoderm, characterized as a pseudo-epithelial germ layer. From the non-neural ectoderm arises the EBL, which covers the whole embryonic surface underneath the EVL to form a two-layered epithelium by 10 hpf. At 24 hpf, the epidermis is a distinctive bilayer in which the basal layer actively produces collagen to form the basal membrane and the primary dermal stroma^7,8. It is after three weeks that the epidermis becomes further stratified and develops into the adult teleost four-layered epidermal structure: cuticle, surface, intermediate and basal stratums⁹. In adult zebrafish, the EVL cells are replaced by those derived from basal keratinocytes⁸. Thus, the epidermis of adult zebrafish, as in mice, derives from basal stem cells, further expanding the similarities of epidermal ontogeny across vertebrates¹⁰.

The importance of studying E-cadh in epidermis

E-cadh is member of a superfamily of cadherins, calcium-dependent cell-cell adhesion molecules forming junctions along the apicolateral membranes of adjacent cells^11–13. E-cadh plays a key role in determining cell polarity and differentiation, and thereby in the establishment and maintenance of metazoan tissue homeostasis^2,14. As epithelia are constituted by cell phenotypes with the maximum polarity and whose identity is primarily specified by E-cadh, it is key to know its expression in the epidermis establishment and maintenance¹⁵. Furthermore, due to the mechano-transduction activity coupled to the acto-myosin cytoskeleton remodeling, E-cadh is involved in processes such as cell division orientation in planar polarized epithelia¹⁵ and collective cell migration¹⁶. Relevant as well is that E-cadh has been characterized as a potent suppressor of invasion and metastasis in epithelia¹⁷, which are usually located in direct contact with mutagenic and/or carcinogenic agents responsible for 85–90 % of human cancers¹⁸.

In zebrafish, E-cadh transcripts and proteins are maternally deposited. Reduced levels of the maternal and zygotic protein have been proved to delay epiboly progression with lethal phenotypes⁵. E-cadh is required for blastomeres adhesion during the cleavage stage and later during gastrulation and epiboly^19–21. Indeed, as epiboly proceeds, EVL directs cell migration and the spreading of cells of the deep cells layer (DCL) in a process that requires dynamic cell contacts remodeling mediated by E-cadh⁶. Once the bi-layered epidermis is established, E-cadh role becomes more refined by keeping its integrity while actively remodeling cell-cell contacts within each layer. At tissue scale, this leads to cell rearrangements which establish regular geometric patterns, and loss of E-cadh results in altered epidermis topology^5,14,22. Strikingly, scarce knowledge exists regarding epidermal spatiotemporal expression of E-cadh after epiboly stages. By combining 3D-deconvolution and segmentation of AJs in epidermal cells we were able to obtain a quantitative profile of E-cadh expression during normal epidermis morphogenesis from embryonic-to-larval life of zebrafish.

Zebrafish husbandry

Zebrafish strain of T/AB genetic background was used as wild-type. Male and female adults of 8-months-old were obtained from the Institute of Molecular and Cellular Biology of Rosario (IBR-CONICET-UNR), Argentina, and maintained at 28°C on a 14-h light/10-h dark cycle. Adult fishes were kept in rectangular glass tanks of 12 liters at a density of (1–2 fishes/liter). In each tank, chlorine free water was constantly aerated and filtered (ATMAN hang On filter HF 0100), and renovated by 1/3 twice a week, water temperature was maintained with a heater (Atman 200W). Water pH was kept between 7.8–8.2, salinity was maintained between 350–600 TDS and nitrates were controlled using biological films included in the filtering system. Fishes were fed twice a day with dried flakes (TetraMin) and twice a week with freshly hatched artemia cysts.

After breeding, laid eggs were collected and maintained at 28°C. Then, embryos and larvae were staged according to Kimmel et al.²³. Around 20 to 25 embryos were collected at 2.5, 18, 24, 31, 48 and 72 hpf, then dechorionated and sedated with buffered tricaine methylsulfonate (MS-222, Sigma) prior to fixation. Approximately, 10–15 fixed embryos per stage were processed for immunofluorescence detection of E-cadh.

Adults and embryos were handled according to the ARRIVE guidelines and to the national guidelines from the Advisory Committee on Ethics of the Facultad de Bioquímica y Ciencias Biológicas de la Universidad Nacional del Litoral, Santa Fe, Argentina (Res. 229 and 388/2006).

Embryos processing for immunodetection of E-cadh

All embryos were fixed in toto in Carnoy solution at room temperature (RT) for at least 2 h and processed according to Izaguirre et al.²⁴. Briefly, they were washed in PBS and permeated in 1% Triton X-100/PBS pH 7.4 for 1 h. Then, washed in PBS pH 7.4 and incubated in normal goat serum (catalogue number: S-1000 Vector Laboratories, Burlingame, CA) for 45 min, followed by overnight incubation with primary antibody anti E-cadh at 4°C, three washes in PBS, and incubation with secondary goat anti-mouse IgG-FITC antibody at RT in darkness for 2 h. Finally, they were rinsed in PBS and mounted in 50% Glycerol-PBS for microscopy imaging. Embryos directly incubated with secondary antibody and normal goat serum, were used as negative controls.

Antibodies. The 36/E-cadh monoclonal antibody recognizes the cytoplasmic domain of human E-cadh, regardless of phosphorylation status (clone 36 mouse IgG2a, catalogue number: 610181 Transduction Laboratories). It was diluted 1:150 and revealed with secondary goat anti-mouse IgG-FITC antibody (Sigma, catalogue number: F8771, St. Louis, MO) used at 1:100 dilution.

Microscope settings and image acquisition

The spatial distribution of E-cadh in zebrafish epidermis was analyzed by fluorescence microscopy followed by image deconvolution and cell segmentation in 3D. The trunk was selected for the ease of orientation and image acquisition within the studied periods. Images were acquired with an inverted wide field sectioning microscope Olympus IX83 coupled to a digital camera CMOS-ORCA-Flash 2.8 (Hamamatsu), and commanded by Olympus Cell Sens software v. 1.13. Raw images were processed using FIJI v. 3.0. Sampling in xy was 0.182 µm with z-step every 0.33 µm. The epidermis was completely scanned along the trunk region. Lamp power was set at 12 %, and exposure time was experimentally determined and fixed in 370 ms, in order to avoid pixel intensity saturation and to minimize photobleaching.

Deconvolution, intensity based segmentation of AJs and fluorescence intensity measurements

Deconvolution was applied to restore fluorescence, which improved contrast and z-resolution, enabling better definition of E-cadh in AJs for subsequent application of the 3D-segmentation tool. Quantification of E-cadh fluorescence intensity was carried throughout the epidermis bilayer (~ 6 μm) in calibrated 3D-ROIs set at 2500 µm² × 0.33 µm × 20 slices (16500 µm³). First, deconvolution was performed on individual 3D-ROI by applying Richardson-Lucy algorithm²⁵ running under the open source Deconvolution Lab 2 v 2.0.0, with a theoretical point spread function²⁶. The Trainable Weka Segmentation Plugin v. 3.1.0, a classification tool based on machine learning in FIJI²⁷ was applied on each deconvolved 3D-ROI so as to create a template that would automatically find the cell boundaries by providing trainable examples of membranes and cytosol (set as background). Each segmented 3D stack was further converted into 8-bit binary 3D-mask and multiplied by the corresponding deconvolved 3D-ROI to obtain the final “Result of Classification”. On each classified image E-cadh fluorescence was quantified as the sum of pixel intensities per 3D-ROI and expressed as raw integrated density (RawIntDen). This measurement was performed on at least six 3D-ROIs per embryo to cover the trunk region, in five embryos per developmental stage. The pipeline for the image processing, theoretical psf and classifier model files are available as Supplementary File 1 as well as an example output.

Fluorescence intensity measurements in individual EVL cells

On each classified image, 3D-ROIs of fixed volume (10 μm² × 3 μm deep) were selected along cell-cell contacts in EVL cells and fluorescence intensity was expressed as RawIntDen/cell-cell contact. To assess the fluorescence intensity in individual cells of the EVL, 3D-ROIs were manually outlined along cell perimeters to include the full membrane width and thickness and expressed as RawIntDen/cell area.

Cell morphology and area measurements

Cell morphology and cell area were assessed in EVL and EBL cells from previously selected 3D-ROIs. Round, 4-, 5-, 6-, 7- and 8-sided cells were counted using the “polygon selection tool” in the individual layers. Mean area was expressed in the image calibrated units. Cell packing index was scored for the EVL and expressed as mean of number of cells/ ROI area (2500 μm²). Area of penta- and hexagonal cells from EBL and EVL were compared for all stages (Supplementary File 2).

Statistical analysis

Five animals in the specified stages were obtained from three to five independent experiments. Differences in E-cadh levels between developmental stages were analyzed using a Linear Mixed Model (LMM). The assumptions of the model were checked graphically (linearity, homoscedasticity, normality of residuals and independence). The non-normality of the data was tested using the Shapiro-Wilk test. The variable “stage” was considered as fixed effects (24, 31, 48 and 72 hpf). The random effects of the model are the number of embryos per stage (5) and number of 3D-ROIs per embryo (at least 6). This number of ROIs per embryo was estimated in order to cover >90% of the embryo trunk for the selected stages.

Differences in mean cellular area and percentages for the observed polygon types were analyzed using LMM containing the same fixed and random effects but adding the variable “morphology”. The same statistical analysis was performed on the data set for the analysis of cell density and expressed as packing index (mean of number of cells / ROI area).

The Tukey’s test was used for post-hoc pair-wise comparison when an effect or an interaction was found significant. Significant differences are denoted with *p < 0.05, **p < 0.01. Data were analyzed with RStudio software’s version 1.1.453 and plotted with the BoxPlotR application or InfoStat software version 2018.

E-cadh expression during embryonic epidermis morphogenesis

E-cadh expression pattern was determined in wild type zebrafish during epidermis development from 2.5 (blastula period) to 72 hpf. E-cadh protein was clearly detected in embryos at the blastula stage on epiblast cells (EVL) (Figure 1a). By 18 hpf, during primary organogenesis both epidermis layers are already established. At this stage, E-cadh was observed in AJs in EVL cells and weakly detected in EBL cells (Figure 1b–c). At later stages E-cadh labeling was observed as well as cytoplasmic dots, presumably in endocytic vesicles (Figure 1d,g).

Figure 1. E-cadh immunofluorescence detection in zebrafish embryo epidermis.

a) at 2.5 hours post fertilization (hpf) b) at 18 hpf; c) Zoom of selection in b) showing a single puncta adherens in contacting EVL cells (arrow) and weak E-cadh detection in the EBL (arrowhead); d,g) at 48 hpf, showing cytoplasmic dot labeling in EVL cells ; e–h) 24 to 72 hpf embryos, displaying E-cadh distribution in trunk, clearly visible in underlying EBL cells from 24 hpf. Images are contrast enhanced maximum intensity projections of 20 optical slices, z-step: 0.33 µm, scale bar: 50 µm. Objective: UPLFLN 40X 1.3 NA oil.

In embryos at 24 hpf, E-cadh labeling was observed in vertices (puncta adherens) as well as in micro-clusters along lateral cell-cell contacts in the EVL (Figure 1e). At this stage the underlying EBL was barely visible, detected as a faint E-cadh immunolabeling. From 31 hpf onwards, fluorescence in the underlying EBL cells was clearly detected (Figure 1 f–h).

During the transition from embryo to larval stages the growing detection of E-cadh along cell-cell contacts parallels noticeable changes in cell size and morphology in the epidermis bilayer (Figure 1, e–h), which were further analyzed.

E-cadh levels were compared in the epidermis layers between stages 24, 31, 48 and 72 hpf, a period during which intense morphogenetic events lead to hatching and significant physiological changes occur for the resulting larvae epidermis to adapt to the aquatic environment. By implementing a 3D-Segmentation algorithm based on machine learning we were able to generate a mask to extract fluorescence intensity values along cell-cell contacts on 3D-ROIs covering the trunk epidermis bilayer and at each developmental stage (Figure 2a–d). With this approach we measured a significant increase of E-cadh levels between 31 and 48 hpf, consistent with the visible detection of E-cadh in the EBL and the visible increase in cell density in the EVL, which was subsequently quantitated.

Figure 2. Expression patterns of E-cadherin (E-cadh) during epidermis development from stage 24-to-72 hours post fertilization (hpf).

a) 24 hpf and b) 31 hpf, with incipient E-cadh labeling of the underlying epidermis basal layer (EBL) cells; c) 48 hpf; d) 72 hpf, with stronger detection of E-cadh in the EBL (arrow heads). Panels are representative 3D-ROIs classified images shown as sum of intensities in projections of 20-optical section stacks covering the epidermis thickness (6.6 μm); e) Box-plot of means of RawIntDen/ROI per embryo. Objective, 40X, NA 1.3 oil. Statistical significances, ** p<0.01, * p<0.05.

Cell geometry changes during embryo to larval transition

Changes in cell shape, area and density were quantitated in an attempt to correlate the observed increments in E-cadh expression with cell morphology changes characteristics of developing epithelia²⁸. Similar to the other epithelia, the morphogenetic processes leading to epidermis topology development involve cell morphology changes with a predominance of hexagonal geometry in the outermost layer^16,24,28. Therefore, the distribution of the cell polygons classes was analyzed in the EVL layer. While round-cells, 4- and 8-side cell polygons were only detected in 24, 31 and 48 hpf stages and represented no more than 7, 15 and 2 % respectively, pentagonal and hexagonal shaped cells predominated in all stages (Figure 3a). Hexagonal cells constituted approximately 34 % of EVL total cells in stage 24 hpf and 45–50% at 31, 48 and 72 hpf with a two-fold increase above pentagonal cells from 31 hpf. Hexagonal cells represented a ~ 50 % of the total cells analyzed consistent with previous reports for other species¹⁴.

Figure 3. Polygon class distribution in the EVL layer from 24 to 72 hpf.

a) Bar graph displaying mean percentages of x-sided polygons of embryos at 24 hpf (n = 81 cells), 31 hpf (n = 147 cells), 48 hpf (n = 133 cells) and 72 hpf (n = 102 cells). Hexagonal and pentagonal cells are the main cell morphologies observed in the EVL. b) Mean area of cell types on EVL at 24 hpf (EVL n= 73), 31 hpf (EVL n= 147), 48 hpf (EVL n= 133) and 72 hpf (EVL n= 102). Significant differences were found in the mean cell area for penta and hexagonal cells between stages (***p < 0.001). Data obtained from 30 3D-ROIs in five animals for each stage. The bars represent mean values ± standard deviations (SD).

For all polygon types assessed in the EVL the mean cell areas decreased within the same polygon type and significantly for pentagonal and hexagonal cells from 24 to 72 hpf (Figure 3b). In the EBL, and despite fewer cells were accessible for area measurements, this tendency was not evident (Supplementary Figure 2). Therefore, the visible increment in cell density in the EVL was characterized by an establishment of hexagonal cell morphology.

We hypothesized then, that the significant increment of E-cadh levels in the epidermis bilayer between 31 and 48 hpf could reflect a contribution of the appearance of more cell-cell contacts per area, the addition of more protein to cell-cell contacts and the emergent detection of the protein in the underlying EBL.

Then, to elucidate whether the increase could be due to the appearance of more cell-cell contacts per EVL area, cell density was estimated globally and for hexagonal cells, and expressed as cell packing indexes (Figure 4a, b). Together, these results showed a significant increment in cell density in the EVL that was characterized by the establishment of hexagonal cell morphology from 24 to 72 hpf. However, there was no significant change in cell density between 31 and 48 hpf, parallel to the increment in E-cadh.

Figure 4. E-cadh expression profile in individual cells of the enveloping layer (EVL).

a) Plot of means of cell packing index calculated as the average number of cells/µm²; b) Plot of means of hexagonal cell packing index calculated as the average number of hexagonal cells/µm²); c) Box-plot of means of RawIntDen/cell-cell contact volume; d) Box-plot of means of RawIntDen/average cell area. Statistical significances, ** p<0.01, ***p<0.001.

The separation of the EVL as a sub-stack in individual 3DROIs is not completely free from the fluorescence contribution of the underlying EBL layer. Therefore, to evaluate the contribution of individual EVL cells to the observed differences in global fluorescence intensity, this was quantified in individual cell-cell contacts of fixed volume (3D-ROIs, 10 μm² × 3 μm deep) in the EVL or in individual cells. With this approach overlapping contacts with the underlying EBL cells were excluded from the measurements. Mean RawIntDen values along cell-cell contacts revealed that more E-cadh may populate individual contacts during embryonic epidermis morphogenesis and contribute to the total fluorescence increase, although differences were not significant between developmental stages (Figure 4c). Then, fluorescence intensity was quantified in individual cells of the EVL manually outlined from the original 3D-ROIs, and expressed as RawIntDen/cell area (Figure 4d). As expected, the expression of E-cadh in the EVL followed a similar pattern as the one obtained for the global bilayer analysis. However, differences between 31 and 48 hpf were not significant. Together this data suggested that the main contribution to the significant increase in E-cadh in the bilayer is due to the growing detection of the protein in the underlying EBL observed at 48 hpf.