Stretching of the retinal pigment epithelium contributes to zebrafish optic cup morphogenesis

MorenoMármol etal. eLife 2021;10:e63396. DOI: https:// doi. org/ 10. 7554/ eLife. 63396 1 of 28 Stretching of the retinal pigment epithelium contributes to zebrafish optic cupmorphogenesis Tania MorenoMármol1,2, Mario LedesmaTerrón1, Noemi Tabanera1,2, Maria Jesús MartinBermejo1,2, Marcos J Cardozo1,2, Florencia Cavodeassi1,2†, Paola Bovolenta1,2* 1Centro de Biología Molecular Severo Ochoa, CSICUAM, c/ Nicolás Cabrera, 1, Campus de la Universidad Autónoma de Madrid, Madrid, Spain; 2CIBER de Enfermedades Raras (CIBERER), Madrid, Spain Abstract The vertebrate eye primordium consists of a pseudostratified neuroepithelium, the optic vesicle (OV), in which cells acquire neural retina or retinal pigment epithelium (RPE) fates. As these fates arise, the OV assumes a cup shape, influenced by mechanical forces generated within the neural retina. Whether the RPE passively adapts to retinal changes or actively contributes to OV morphogenesis remains unexplored. We generated a zebrafish Tg(E1-bhlhe40:GFP) line to track RPE morphogenesis and interrogate its participation in OV folding. We show that, in virtual absence of proliferation, RPE cells stretch and flatten, thereby matching the retinal curvature and promoting OV folding. Localized interference with the RPE cytoskeleton disrupts tissue stretching and OV folding. Thus, extreme RPE flattening and accelerated differentiation are efficient solutions adopted by fastdeveloping species to enable timely optic cup formation. This mechanism differs in amniotes, in which proliferation drives RPE expansion with a muchreduced need of cell flattening. Introduction The retinal pigment epithelium (RPE) is an essential component of the vertebrate eye, composed of a monolayer of pigmentenriched epithelial cells abutting the neural retina (NR) with a primary role in photoreception (Letelier etal., 2017). Despite the acquisition of specialized epithelial properties, RPE cells have a neural origin and share progenitors with the NR. These progenitors are organized in a pseudostratified neuroepithelium, known as optic vesicle (OV) or eye primordium. In amniotes, the OVs appear as balloonlike structures positioned at the sides of the anterior neural tube (MorenoMarmol etal., 2018). In zebrafish instead, these primordia are flat and form two bilayered structures with the outer and inner layers distally connected by a rim or hinge (Li etal., 2000). Under the influence of inductive signals (Gallardo and Bovolenta, 2018; Cardozo etal., 2020), the two layers activate different genetic programs that specify the cells of the inner layer and ventral outer layer as NR and those of the dorsal outer layer as RPE (Beccari etal., 2013; Buono and MartinezMorales, 2020; Buono etal., 2021). Whilst this specification occurs, the OV bends assuming a cuplike shape (MartinezMorales etal., 2017). The discovery of the ojoplano medaka fish mutant – affecting a transmembrane protein localized at the basal end feet of NR cells (MartinezMorales etal., 2009) – in which the OV remains unfolded, was instrumental to propose that basal constriction of NR progenitors is at the basis of OV bending (MartinezMorales etal., 2009). This basal constriction is mediated by the redistribution of the actomyosin cytoskeleton (MartinezMorales etal., 2009; NicolasPerez etal., 2016; Bryan etal., 2016), which also enables the apical relaxation of retinal cells (Sidhaye and Norden, 2017), RESEARCH ARTICLE *For correspondence: pbovolenta@ cbm. csic. es Present address: †St. George's, University of London, London, United Kingdom Competing interest: The authors declare that no competing interests exist. Funding: See page 24 Received: 23 September 2020 Preprinted: 24 September 2020 Accepted: 20 September 2021 Published: 21 September 2021 Reviewing Editor: Marianne E Bronner, California Institute of Technology, United States Copyright MorenoMármol etal. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. Research article Developmental Biology | Neuroscience MorenoMármol etal. eLife 2021;10:e63396. DOI: https:// doi. org/ 10. 7554/ eLife. 63396 2 of 28 enhanced by focal adhesions of the apical surface with the extracellular matrix molecules (ECM) such as laminin (Bryan etal., 2016). The importance of concomitant apical relaxation, especially of the cells positioned at the hinge, has also been supported in studies of mammalian retinal organoids (Eiraku etal., 2011; Okuda etal., 2018). Nevertheless and independently of their relative contribution, the acquisition of apical convexity and basal concavity in the NR epithelium are accepted drivers of the biomechanical forces that induce OV folding (Okuda etal., 2018). In zebrafish, this mechanism is reinforced by rim involution or epithelial flow, a process whereby progenitors at the hinge emit dynamic lamellipodia at the basal side and actively translocate from the ventral outer layer of the OV into the inner/retinal layer (Li etal., 2000; Sidhaye and Norden, 2017; Zheng etal., 2000; Heermann etal., 2015; Kwan etal., 2012; Picker etal., 2009). Periocular neural crest cells appear to facilitate this flow, in part by the deposition of the ECM (Bryan etal., 2020) to which the lamellipodia attach (Sidhaye and Norden, 2017; Heermann etal., 2015; Kwan etal., 2012). The result of this flow is an unbalanced cell number between the two layers, which should favour NR bending (Sidhaye and Norden, 2017; Heermann etal., 2015; Kwan etal., 2012). Whether this flow may also contribute to the concomitant cell shape modifications that the remaining outer layer cells undergo as they become specified into RPE, or conversely whether RPE specification favours the flow (Heermann etal., 2015), remain open questions. Indeed as the OV folds, the pseudostratified neuroepithelial cells of the OV dorsal outer layer progressively align their nuclei becoming a cuboidal monolayer in amniotes species (MorenoMarmol etal., 2018; MartinezMorales etal., 2004). In zebrafish, cuboidal cells further differentiate to a flat/ squamous epithelium (Zheng etal., 2000; Kwan etal., 2012) that spreads to cover the whole apical surface of the NR (Zheng etal., 2000; Cechmanek and McFarlane, 2017). In mice, failure of RPE specification, as observed after genetic inactivation of key specifier genes (i.e. Otx1/Otx2, Mitf, Yap/ Taz), enables RPE progenitors to acquire an NR fate (MartinezMorales etal., 2001; Bharti etal., 2006; Kim etal., 2016). The resulting optic cups (OCs) present evident folding defects (MartinezMorales etal., 2001), raising the possibility that specific RPE features are needed for OC formation. In line with this idea, a differential stiffness of the RPE vs. the NR layer has been proposed to drive the selforganization of mammalian organoids into an OC (Eiraku etal., 2011; Okuda etal., 2018; Nakano etal., 2012). Furthermore, generation of proper RPE cell numbers seems a requirement for eLife digest Rounded eyeballs help to optimize vision – but how do they acquire their distinctive shape? In animals with backbones, including humans, the eye begins to form early in development. A single layer of embryonic tissue called the optic vesicle reorganizes itself into a twolayered structure: a thin outer layer of cells, known as the retinal pigmented epithelium (RPE for short), and a thicker inner layer called the neural retina. If this process fails, the animal may be born blind or visually impaired. How this flat twolayered structure becomes round is still being investigated. In fish, studies have shown that the inner cell layer – the neural retina – generates mechanical forces that cause the developing tissue to curve inwards to form a cuplike shape. But it was unclear whether the outer layer of cells (the RPE) also contributed to this process. MorenoMarmol et al. were able to investigate this question by genetically modifying zebrafish to make all new RPE cells fluoresce. Following the early development of the zebrafish eye under a microscope revealed that RPE cells flattened themselves into long thin structures that stretched to cover the entire neural retina. This change was made possible by the cell’s internal skeleton reorganizing. In fact, preventing this reorganization stopped the RPE cells from flattening, and precluded the optic cup from acquiring its curved shape. The results thus confirmed a direct role for the RPE in generating curvature. The entire process did not require the RPE to produce new cells, allowing the curved shape to emerge in just a few hours. This is a major advantage for fastdeveloping species such as zebrafish. In species whose embryos develop more slowly, such as mice and humans, the RPE instead grows by producing additional cells – a process that takes many days. The development of the eye thus shows how various species use different evolutionary approaches to achieve a common goal. Research article Developmental Biology | Neuroscience MorenoMármol etal. eLife 2021;10:e63396. DOI: https:// doi. org/ 10. 7554/ eLife. 63396 3 of 28 correct OC folding in mice (Carpenter etal., 2015). However, studies addressing the specific contribution of the RPE to OV folding are currently lacking. Here, we report the generation of a Tg(E1-bhlhe40:GFP) zebrafish transgenic line with which we followed the beginning of RPE morphogenesis under both normal and interfered conditions. We show that, whereas in amniotes, including humans, the developing RPE undergo proliferation to increase its surface with a less evident cell flattening, zebrafish RPE cells rapidly cease proliferation and expand their surface by reducing their length along the apicobasal axis and extending in the mediolateral direction with a tissue autonomous process that depends on cytoskeletal reorganization. Localized interference with either the retinal or the RPE actomyosin and microtubule cytoskeleton shows that RPE flattening generates a mechanical force that actively contributes to OV folding, complementing the force generated by the basal constriction of the NR. This mechanism represents an efficient solution to match the increased apical surface of the NR layer in a fastdeveloping vertebrate species such as zebrafish. Results Generation of a specific reporter line to study zebrafish RPE development Detailed analysis of zebrafish RPE morphogenesis has been hampered by the lack of a suitable transgenic line, in which RPE cells could be followed from their initial commitment. The E40 (bhlhe40) gene, a basic helixloophelix family member, encodes a light and hypoxiainduced transcription factor (also known as Dec1, Stra13, Sharp2, or Bhlhb2) involved in cell proliferation and differentiation as well as in the control of circadian rhythms (Yamada and Miyamoto, 2005). In neurulating zebrafish embryos, its expression is limited to cells of the prospective RPE (Figure1A; Cechmanek and McFarlane, 2017; Yao etal., 2006), representing a potentially suitable tissue marker. We used predictive enhancer and promoter epigenetic marks at different zebrafish developmental stages (Bogdanovic etal., 2012) to scan the bhlhe40 locus for the presence of conserved and active regulatory regions. The promoter and four potential enhancers (E1–4; Figure1B) appeared to be active between 80% epiboly and 24 hpf, encompassing the early stages of zebrafish eye development (Bogdanovic etal., 2012). These enhancers were selected, amplified, and tested using the ZED vector (Bessa etal., 2009) as potential drivers of gene expression in the prospective RPE. The resulting F0 embryos were raised to adulthood and screened. Only the E1 enhancer drove specific and restricted GFP reporter expression into the prospective RPE. The corresponding fishes were further crossed to establish the stable transgenic line Tg(E1-bhlhe40:GFP) used in this study. Timelapse studies of the Tg(E1-bhlhe40:GFP) progeny confirmed that the transgenic line faithfully recapitulated the bhlhe40 mRNA expression profile detected with ISH (Figure1A and C). GFP reporter expression appeared in a discrete group of neuroepithelial cells in the dorsomedial region of the OV (16–17 hpf) and expanded both posteriorly and ventrally (Figure1C; Figure1—video 1 and Figure1—video 2), so that, by 24 hpf, GFPpositive cells appeared to wrap around the entire inner NR layer. 3D reconstructions of selected embryos further confirmed the fast (about 7 hr) expansion of the GFPpositive domain forming an outer shell for the eye (Figure1D). Apart from a faint and very transient signal in some early NR progenitors — likely due to the existence of negative regulatory elements not included in the construct — no GFP expression was observed in regions other than the RPE during this process. However, after the formation of the OC, reporter expression appeared also in the ciliary marginal zone (CMZ), the pineal gland, and few neural crest cells surrounding the eye (Figure1C; Figure1—videos 1–3). These additional domains of expression coincided with the reported bhlhe40 mRNA distribution (Yao etal., 2006) and represented no obstacle for using the transgenic line as a tool to follow the early phases of RPE generation. Indeed, very early activation represents an important advantage of the Tg(E1-bhlhe40:GFP) line over other presently available transgenic lines that allow visualizing the RPE (Zou etal., 2006; Miesfeld and Link, 2014). The suitability of the Tg(E1-bhlhe40:GFP) line for the identification of the very first RPE cells is supported by the onset of the reporter expression in the dorsomedial OV region, coinciding with previous fate map predictions (Zheng etal., 2000; Kwan etal., 2012). To further verify this notion, we took advantage of the characteristic of the fluorescent Kaede protein (Ando etal., 2002) that switches from green to red emission upon UV illumination. Embryos were injected with Kaede mRNA and neuroepithelial cells located at the most dorsomedial region of the OV were UV illuminated at Research article Developmental Biology | Neuroscience MorenoMármol etal. eLife 2021;10:e63396. DOI: https:// doi. org/ 10. 7554/ eLife. 63396 4 of 28 P E1 E2 E3 E4 H3K27ac dome 80% epiboly 24hpf 48hpf dome 80% epiboly 24hpf 48hpf H3K4me3 H3K36me3 24hpf B bhlhe40 24hpf18hpf 16hpf14hpf nt OV nt OC D V A P ol il ol/RPE il/NR A D 18hpf 24hpf 30hpf20hpf E RPE NR lens cmz E1-bhlhe40:GFP rx3:GAL4;UAS:RFP C ol il A P ML t=75 t=145 t=215 t=285 t=355 t=0 nc lens E1-bhlhe40:GFP A P ML A P ML Kaede PhC-Kaede Figure 1. The Tg(E1-bhlhe40:GFP) line is a suitable tool to study early retinal pigment epithelium (RPE) generation. (A) Frontal cryosections of 14–24 hpf wildtype (wt) embryos hybridized in toto with a bhlhe40specific probe. mRNA is first detected in the dorsal most region of the optic vesicle (OV) outer layer (arrowhead) and then expands ventrally. (B) UCSC Genome Browser view of H3K27ac (purple, potential active enhancers), H3K4me3 (green, potentially active promoters), and H3K36me3 (light blue, transcriptionally active regions) tracks obtained for four zebrafish developmental stages: dome, 80% epiboly, 24 hpf, 48 hpf related to the upstream bhlhe40 genomic locus (50kb). The black arrow at the bottom indicates bhlhe40 position and direction. The promoter (P) and the four selected enhancers (E1–4) are highlighted with a colourcoded box. (C) Time frames from in vivo timelapse recording of a Tg(E1-bhlhe40:GFP;rx3:GAL4;UAS;RFP) embryo between 14 and 24 hpf. Time is indicated in min. Note that the GFP reporter signal matches the bhlhe40 mRNA distribution in A. (D) 3D reconstruction of the prospective RPE from Tg(E1-bhlhe40:GFP) embryos at the stages indicated in the panel. (E) Dorsal view of a wt embryo injected with Kaede mRNA (green) at 12 hpf. A group of cells in the dorsal region of the outer layer was photoconverted (magenta, panel on the left) and the embryo visualized at 30 hpf (right panel). Magenta labelled cells cover the entire RPE region. Black and white dashed lines delineate the OV, neural tube, and virtual lumen in A, C. Abbreviations: A, anterior; cmz, ciliary margin zone; il, inner layer; l, lateral; m, medial; NR, NR; OC, OC; ol, outer layer; OV, optic vesicle; P, posterior; RPE, retinal pigment epithelium. Scale bars: 100µm (A–D); 50µm, E. The online version of this article includes the following video for figure 1: Figure 1—video 1. Dorsal view of the optic vesicle (OV) to optic cup (OC) transition visualized in a double Tg(E1-bhlhe40:GFP; rx3:GAL4;UAS:RFP Figure 1 continued on next page Research article Developmental Biology | Neuroscience MorenoMármol etal. eLife 2021;10:e63396. DOI: https:// doi. org/ 10. 7554/ eLife. 63396 5 of 28 the 15 hpf stage to ensure that no differentiation had yet occurred (Figure1E). Embryos were let develop until 30 hpf. Photoconverted cells were found throughout the thin outer layer of the OC (Figure1E), confirming that the entire RPE derives from the dorsomedial OV region. Neuroepithelial cell flattening drives RPE expansion at OV stages Tg(E1-bhlhe40:GFP) embryos were thereafter used to dissect the extensive changes in cell shape that are associated with the acquisition of RPE identity (Zheng etal., 2000; Cechmanek and McFarlane, 2017). At OV stage all retinal progenitors present a columnarlike morphology characteristic of embryonic neuroepithelia (Figure2A and A’). As soon as RPE progenitors begin to express the transgenic GFP reporter, their apicobasal length rapidly and progressively reduces (Figure2A–C’), so that the cells first assume a cuboidal shape (Figure2B and B’) and then become flat, forming a squamous epithelial monolayer overlaying the apical surface of the NR (Figure2C and C’). At 30 hpf, RPE cells presented a polygonal, frequently hexagonal, morphology (Figure2D and D’), with an apical surface area that, on average, became about eightfold larger than that observed in progenitor (PN) cells (Figure2F; RPE‾a: 354.8 ± 100.3 μm2 vs. PN‾a: 43.7 ± 7.8 μm2). In contrast, the abutting apical surface of NR cells slightly shrank as compared to that of PN cells (Figure2E, E’ and F; NR‾a: 22.5 ± 2.9 μm2 vs. PN‾a: 43.7 ± 7.8 μm2) while maintaining a constant apicobasal length. The latter observation agrees with previous reports showing that the conelike morphology of NR progenitors represents only a slight modification of the progenitor columnar shape (NicolasPerez etal., 2016; Sidhaye and Norden, 2017). To obtain a quantitative analysis of the dynamic changes that RPE tissue, as whole, underwent during OV folding, we performed a morphometric characterization of the images from Figure1—videos 1–3. To this end, the fluorescent information from the Tg(E1bhlhe40:GFP) reporter was discretized into seven different segments that were individually analysed along the recording time (Figure3— figure supplement 1; Materials and methods). The combined quantification of the different segments (Figure3A; Figure3—figure supplement 1) showed that, between stages 17 and 21 hpf, the overall thickness of the RPE tissue underwent, on average, a flattening of more than threefold (from a mean of about 24–8µm; Figure3B). Flattening occurred with a central to peripheral direction, so that RPE cells closer to the hinges were the last ones to flatten (Figure2C and C’). In parallel, the overall RPE surface underwent an approximately twofold expansion between 17 and 22 hpf (from approximately 1.1–2.2 × 103 μm2; Figure3C; Figure1—video 1 and Figure1—video 2), reflecting the large increase in the apical area observed in each individual cell at later stages (Figure2). In line with the idea that cell flattening is per se sufficient to account for whole tissue enlargement, the RPE volume only slightly changed between 17 and 20 hpf with a slope increase of 0.47 × 103 μm3/h (Figure3D). To provide further support to this idea, we analysed the RPE volume variation in comparison with the growth of the entire OC in two time windows: from 17 to 22 hpf (Figure1—videos 1–3) and from 24 to 37 hpf (Figure3—video 1), using GFP (RPE) and RFP (eye) reporter signals from the double Tg(E1-bhlhe40:GFP; rx3:GAL4;UAS;RFP) line or from the Tg(E1-bhlhe40:GFP) line injected with the pCS2:H2BRFP mRNA (Figure3E and F). Signal quantification showed that the eye underwent a marked and linear volume increase (slope: 5.54 × 104 μm3/hr from 17 to 22 hpf and 3.6 × 104 μm3/ hr from 24 to 37 hpf) as compared to that of the RPE (Figure3D and G). Between 20 and 22 hpf the reporter starts being expressed in the posterior and, to a lesser extent, in the anterior CMZ (GFPCMZ domain, Figure1—videos 1 and 2). Consistently with the onset of GFPCMZ expression, RPE reporter volume suddenly expanded between 20 and 22 hpf (slope: 1.25 × 104 μm3/hr; Figure3G) to then slow back between 24 and 37 hpf (slope: 1.2 × 103 μm3/hr; Figure3G). Confirming this association, only the embryo). https://elifesciences.org/articles/63396/figures#fig1video1 Figure 1—video 2. Dorsal view of the optic vesicle (OV) to optic cup (OC) transition visualized in a double Tg(E1-bhlhe40:GFP; rx3:GAL4;UAS:RFP embryo). https://elifesciences.org/articles/63396/figures#fig1video2 Figure 1—video 3. Lateral view of the optic cup (OC) folding visualized in a Tg(E1bhlhe40:GFP) embryo injected with H2BRFP mRNA (magenta) related to Figure1, frame rate 1/5min. https://elifesciences.org/articles/63396/figures#fig1video3 Figure 1 continued Research article Developmental Biology | Neuroscience MorenoMármol etal. eLife 2021;10:e63396. DOI: https:// doi. org/ 10. 7554/ eLife. 63396 6 of 28 Figure 2. The retinal pigment epithelium (RPE) converts from a pseudostratified to a squamous epithelium during optic vesicle (OV) folding by increasing individual cell surface. (A–C’) Confocal images of frontal cryosections of Tg(E1-bhlhe40:GFP) embryos immunostained for GFP (green) and β-catenin (white) and counterstained with Hoechst (blue). Note that the RPE rapidly decreases its thickness white straight line in (A–C) and cells change from columnar (14 hpf, arrow in A’) to cuboidal (16 hpf, arrow in B’) and then flat shape (22 hpf, arrow in C’). White dashed lines delineate eye contour and virtual lumen in A–C. (D–E’) Confocal images of the posterior RPE (D, D’) and neural retina (NR) (E, E’) regions of an eye cup dissected from 30 hpf Tg(E1-bhlhe40:GFP) embryos immunostained for GFP (green) and β-catenin (white) and counterstained with Hoechst (blue). Images in D’, E’ are high power views of the areas boxed in white box in D, E. Note the hexagonal morphology (yellow arrow in D’) of RPE cells (average area 354.8 ± 100.3 μm2) in contrast to the small and roundish crosssection of retinal progenitors (average area 22.5 ± 2.9 μm2; yellow arrow in E’). (F) The graph represents the average area of individual OV progenitors and NR and RPE cells (n = 15–19). The average area is calculated using cells from five different embryos. Data represent mean ± SD, ****p < 0.0001. ns, nonsignificant. Scale bar: 50µm. The online version of this article includes the following source code for figure 2: Source data 1. The source data 1 is the excell file that is already correcly linked. Research article Developmental Biology | Neuroscience MorenoMármol etal. eLife 2021;10:e63396. DOI: https:// doi. org/ 10. 7554/ eLife. 63396 7 of 28 AB 17 19 21 15 20 25 Thickness (um) hpf 4 Volume (um3) x104 2 3 17 19 21 hpf Surface (um2) x103 1.5 2 2.5 17 19 21 hpf CD hpf 17 19 21 26 30 34 3 7.5 Volume (um3) x104 100 200 rx3 volume H2B volume GFP volume EF G Figure 3. Retinal pigment epithelium (RPE) volume is conserved during initial tissue morphogenesis. (A) Image on the left represents the reconstruction of a single frame from Figure1—video 2 Tg(E1-bhlhe40:GFP; rx3:GAL4;UAS:RFP embryo) showing the optic vesicle/optic cup (OV/OC) in red and the RPE in green. The segments in which the RPE was discretized are depicted with black dashed lines. The image on the right shows the RPE reconstruction obtained after filtering. Double arrow points to RPE thickness. (B–D) The graphs show how the RPE thickness (B, calculated as volume/ surface), surface (C), and volume (D) change as a function of the developmental stage. (E) 3D reconstructions of raw (left) and processed (right) versions Figure 3 continued on next page Research article Developmental Biology | Neuroscience MorenoMármol etal. eLife 2021;10:e63396. DOI: https:// doi. org/ 10. 7554/ eLife. 63396 8 of 28 tissue segments very close to the posterior CMZ had a volume larger than that of the RPE at 17–20 hpf (Figure3—figure supplement 1), whereas the GFPpositive RPE domain located in the most central regions presented a volume undistinguishable from that detected at previous stages. In sum, a comparison of the dynamics slopes from the GFPRPE domain and OV regions suggests that the volume of the RPE grows at very low pace (0.47 × 103 μm3/hr) – despite the rather drastic morphological changes of its cells – whereas the whole OV expands at a pace ~25 times faster (1.25 × 104 μm3/ hr; Figure3D). Taken all together, this morphometric analysis indicates that the expansion of the RPE in zebrafish occurs by recruiting a limited number of cells that undergo profound cell shape changes: from a neuroepithelial to squamous morphology. RPE flattening is a tissue autonomous process required for proper OV folding Both external interactions and intracellular processes determine the shape of a cell and define its mechanical properties (Totaro etal., 2018). Thus, in principle, RPE flattening might occur as a ‘passive’ process, triggered by the forces that the NR and hinge cells exert on the RPE (MorenoMarmol etal., 2018; Heermann etal., 2015). Alternatively, it might depend on cell or tissue autonomous cytoskeletal rearrangements, involving, for example, myosin II activity, which controls the acquisition of a flat epithelial morphology in other contexts (Tee etal., 2011; Vishavkarma etal., 2014). Discriminating between these two possibilities has been technically difficult. Experiments directed to assess the mechanisms of OV folding have used whole embryo bathing in drugs such as blebbistatin (NicolasPerez etal., 2016; Sidhaye and Norden, 2017), a specific myosin II inhibitor (Rauscher etal., 2018). Such an approach hampers the assessment of the potential influence of NR over RPE morphogenesis (and vice versa) as well as the relative contribution of the two tissues to OV folding. We sought to overcome this limitation by spatially localized interference with the cytoskeletal organization of either the RPE or NR and by recording the tissue autonomous and nonautonomous consequences. Nevertheless, to begin with, we reproduced the whole embryo bathing approach used by others (NicolasPerez etal., 2016; Sidhaye and Norden, 2017), focusing on the yet unreported effect that blebbistatin had on the RPE. Tg(E1-bhlhe40:GFP) embryos were bathed either in blebbistatin or its diluent (DMSO) at 17 hpf (the onset of RPE specification; Figure4A–C) and then let develop up to 19.5 hpf, when embryos were analysed. DMSOtreated (control) embryos developed normally forming an OC surrounded by a squamous RPE (Figure4B). In blebbistatintreated embryos, NR cells did not undergo basal constriction and the OV remained unfolded (Figure4C), as previously described (NicolasPerez etal., 2016; Sidhaye and Norden, 2017). Notably, in almost all the embryos analysed (n = 44/49), RPE cells did not flatten but remained cuboidal in shape (Figure4C). A similar phenotype was observed after treatment with paranitroblebbistatin, a noncytotoxic and photostable version of blebbistatin (Figure4D). These observations support that lack of OV folding is associated with alterations in both the retina and RPE. To uncouple the two events, we turned to the photoactivable compound azidoblebbistatin (Ableb), which binds covalently to myosin II upon twophoton irradiation, thus permanently interfering with myosin II activity in a spatially restricted manner, as already proven (Kepiro etal., 2012; Kepiro etal., 2015). Tg(E1-bhlhe40:GFP) 17 hpf embryos were bathed in Ableb or in DMSO and irradiated in a of a frame from Figure1—videos 1 and 2. (F) 3D reconstructions of raw (left) and processed (right) versions of a frame from Figure3—video 1. (G) Quantification of RPE and eye volume based on Figure1—videos 1 and 2 (rx3 volume quantification) and Figure3—video 1 (H2B volume quantification) along developmental stages. The online version of this article includes the following video, source data, and figure supplement(s) for figure 3: Source data 1. Quantification of RPE thickness along time and space. Figure supplement 1. Retinal pigment epithelium (RPE) region selection from the GFPpositive domain. Figure supplement 1—source data 1. Quantification of RPE volume in space and time. Figure 3—video 1. Lateral view of optic cup (OC) growth visualized in a Tg(E1-bhlhe40:GFP) embryo injected with H2BRFP mRNA (magenta), related to Figure3, frame rate 1/5min. https://elifesciences.org/articles/63396/figures#fig3video1 Figure 3 continued Research article Developmental Biology | Neuroscience MorenoMármol etal. eLife 2021;10:e63396. DOI: https:// doi. org/ 10. 7554/ eLife. 63396 9 of 28 Figure 4. Retinal pigment epithelium (RPE) flattening is a myosindependent cell autonomous process required for proper optic vesicle (OV) folding. (A–J) Confocal images of dorsally viewed Tg(E1-bhlhe40:GFP) embryos before (17 hpf; A) and 2.5hr after incubation (19.5 hpf) with either DMSO (B, E, H), blebbistatin (C), paranitroblebbistatin (D), or azidoblebbistatin (Ableb) (F, G, I, J) with (G, J) or without irradiation (F, I) in the prospective RPE (F–G) or Figure 4 continued on next page Research article Developmental Biology | Neuroscience MorenoMármol etal. eLife 2021;10:e63396. DOI: https:// doi. org/ 10. 7554/ eLife. 63396 16 of 28 Amniots Slow development Zebrafish Fast development cell flattening reduction of proliferation cell proliferation slight reduction of A-B length B A Nocodazole Ablebb NR OV folding into OC Perturbed folding Ablebb RPE ?? Figure 8. Summary of speciesspecific modes of retinal pigment epithelium (RPE) differentiation and its contribution to optic vesicle (OV) folding. (A) The drawing on the top represent the dynamic of OV folding into an optic cup (OC). Green double arrow indicated RPE flattening, blue arrow rim involution whereas pink arrows indicate retinal basal constriction. Bottom row summarizes the alterations in OV folding observed after localized interference with RPE and neural retina (NR) cytoskeleton. (B) Schematic representation of the differential mechanisms by which the RPE in zebrafish Figure 8 continued on next page Research article Developmental Biology | Neuroscience MorenoMármol etal. eLife 2021;10:e63396. DOI: https:// doi. org/ 10. 7554/ eLife. 63396 17 of 28 of cell division, the RPE grows in a conventional proliferationbased mode that correlates with a less evident flattening of RPE cells (Figure7F). Discussion The cup shape of the vertebrate eye is thought to optimize vision (Goldsmith, 1990). This shape is acquired very early in development as the result of specification and morphogenetic events, during which the NR and the RPE arise. Studies in teleosts (zebrafish and medaka) together with mammalian organoid cultures have recently demonstrated a fundamental contribution of NR progenitors in driving the acquisition of this cup shape (MorenoMarmol etal., 2018; MartinezMorales etal., 2017). The role of the RPE progenitors in this process has instead not been properly clarified. In this study, we have filled this gap and analysed the folding of the zebrafish OV from the RPE perspective. This analysis has been possible thanks to the generation of a new RPE reporter line Tg(E1-bhlhe40:GFP), in which GFP expression appears in the domain fated to originate the RPE. Following the cells arising from this domain, we show that RPE surface expansion is an active and tissue autonomous process required for OV folding. This expansion largely occurs by extreme cell flattening with little contribution of cell proliferation, a mechanism that sets zebrafish RPE morphogenesis apart from that of other analysed vertebrate species, in which proliferation accounts for RPE growth. Our analysis together with a previous report (Cechmanek and McFarlane, 2017) shows that the onset bhlhe40 expression coincides spatially and temporally with that of zebrafish RPE specification. Thus, the Tg(E1-bhlhe40:GFP) line serves as an early tissuespecific marker that even precedes the appearance of previously accepted Otx or Mitf tissue specifiers, as confirmed in a parallel transcriptomic analysis (Buono etal., 2021). Bhlhe40 expression in the RPE is conserved at least in mouse and humans (Buono etal., 2021; CohenTayar etal., 2018; Hu etal., 2019), suggesting a possible relevant function in this tissue. However, its CRISP/Cas9 inactivation, alone or in conjunction with that of the related bhlhe41, mitfa, and mitfb, had no evident consequences on zebrafish RPE development, at least in our hands (data not shown). One possible reason for the absence of an evident RPE phenotype is functional redundancy with other untested members of the large family of the BHLH transcription factors or that the gene has only later functions as reported (Abe etal., 2006). However, we favour the alternative possibility that zebrafish RPE specification does not occur stepwise as in other species (MartinezMorales etal., 2004; Fuhrmann etal., 2014) but ‘en bloc’ with an almost simultaneous activation of all differentiation genes. This would make the inactivation of one or two genes insufficient to perturb fate acquisition. Such a mechanism is expected to provide robustness to a process that takes place in just few hours and finds support in present and past findings (Buono etal., 2021; Cechmanek and McFarlane, 2017). Indeed, we and others Cechmanek and McFarlane, 2017 have shown that, by the time the OV starts to bend, the large majority of RPE cells have already left the cell cycle and have acquired a differentiated squamous morphology by undergoing a marked surface enlargement in the mediolateral direction and a reduction of the apicobasal axis. The net result is an overall modest volume increase. Furthermore, blocking cell division as the OC forms does not interfere with RPE expansion (Cechmanek and McFarlane, 2017), strongly supporting a primary role of cell stretching in RPE expansion. Consistently, transcriptomic analysis shows that during this same lag of time, RPE cells repress genes characteristic of 16 hpf OV progenitors, such as vsx1, and acquire the expression of RPEspecific genes. These include blocks of transcription factors, such as known RPE specifiers (i.e. otx, mitf) and regulators of epidermal specification (i.e. tfap family members, known regulator of keratin gene expression; Leask etal., 1991) as well as several cytoskeletal components, most prominently a large number of keratins and other desmosomal components found in squamous epithelia (Buono etal., 2021). Thus, in just few hours (from 16 to 18 hpf) RPE cells acquire the molecular machinery required for their conversion from a neuroepithelial to a squamous and likely highly coupled epithelium. Our study shows that this conversion relays on a tissue autonomous cytoskeletal reorganization without the influence of the morphogenetic events occurring in the nearby NR. Indeed, local interference with actomyosin or microtubule dynamics is sufficient to retain RPE cells into a cuboidal (upper row) and in amniotes (lower row) expands its surface during OV folding morphogenesis. In zebrafish, the RPE enlarges its surface by cell stretching; in amniotes, including in humans, the RPE instead expands by cell proliferation with a less pronounced need of cell flattening. Figure 8 continued Research article Developmental Biology | Neuroscience MorenoMármol etal. eLife 2021;10:e63396. DOI: https:// doi. org/ 10. 7554/ eLife. 63396 18 of 28 or neuroepithelial configuration, respectively, without affecting their specification. In contrast, localized interference with NR bending has no effect on RPE flattening. Notably, our studies also suggest that the RPE acts in a ‘syncytiallike’ manner, as mosaic interference with microtubule polymerization seems to impact in the shape of the adjacent cells if not on the entire tissue. This is perhaps not surprising given that mature RPE cells have been reported to be chemically coupled (Pearson etal., 2004; Bao etal., 2019). Furthermore, the presumptive RPE of the chick (unpublished observations) and zebrafish (Buono etal., 2021) expresses high levels of connexion proteins (i.e. Gap43), which are responsible for the ‘syncytiallike’ behaviour observed in brain astrocytes (Buskila etal., 2019). This together with the additional observation that st18 RPE cells express many desmosomal proteins (Buono etal., 2021) indicate that the tissue becomes tightly connected very soon, perhaps behaving as a community (Gurdon, 1988). The extreme flattening of the zebrafish RPE cells makes the resolution of their cytoskeletal components difficult with in vivo confocal microscopy, hampering the complete understanding of how the actomyosin cytoskeleton promotes the acquisition of a squamous configuration. In other contexts, a flat morphology is associated with the presence of actomyosin stress fibres that compress the nucleus (Tee etal., 2011; Vishavkarma etal., 2014). Myosin II is essential for this compressive role and its inhibition with blebbistatin causes the loss of the flat morphology (Tee etal., 2011; Vishavkarma etal., 2014), as we have observed in blebbistatinand Ablebtreated embryos. It is thus possible that a similar nuclear compression may occur in the RPE cells as they flatten, although we were unable to detect stress fibres around the nucleus, likely due to plasma membrane proximity. Remodelling of the microtubular cytoskeleton seems to aid further RPE cell flattening. Microtubules change their orientation during RPE morphogenesis, from being aligned along the apicobasal axis of the cells at the onset of RPE morphogenesis, to becoming aligned with the planar axis in squamous RPE cells. A similar process has been described during the morphogenesis of the Drosophila amnioserosa (Pope and Harris, 2008), in which cells also change from a columnar to a squamous morphology. In these cells, actin accumulation at the apical edge seems to provide resistance to the elongation of microtubules, which thus bend, leading to a 90° rotation of all subcellular components. This rotation is accompanied by a myosindependent remodelling of the adherens junctions (Pope and Harris, 2008), a process that may also take place during RPE flattening. Although additional studies are needed to clarify the precise dynamics of the cytoskeletal reorganization underlying RPE differentiation, our study demonstrates that cytoskeletal dynamics occurs in a tissue autonomous manner. In contrast to other studies (NicolasPerez etal., 2016; Sidhaye and Norden, 2017), we have used a photoactivable version of blebbistatin that has allowed us to determine the individual contribution of the NR and RPE to OV folding. As a drawback, this approach allows to activate the drug only in relatively small patches of tissue. It was thus rather remarkable to observe that failure of RPE flattening in small regions was sufficient to decrease OV folding. This suggests that RPE stretching represents an additional and relevant mechanical force that, together with retinal basal constriction and rim involution, contributes to zebrafish eye morphogenesis (Figure8A). This flattening and stretching together with a substantial expression of keratins (Buono etal., 2021) may confer a particular mechanical strength to the zebrafish RPE, which, in turn, may constrain the NR at the same time favouring rim involution (Heermann etal., 2015). The latter possibility is supported by the observation that inner layer cells seem to accumulate at the hinge in the absence of RPE flattening. Alternatively, this accumulation may simply reflect that rim cell involution depends on intrinsic microtubule polymerization, although previous studies have discarded this possibility (Sidhaye and Norden, 2017). These marked morphogenetic rearrangements can thus be seen as an efficient solution adopted in fastdeveloping species to make eye morphogenesis feasible in a period that does not allow for proliferationbased tissue growth. The perhaps obvious question is whether similar morphogenetic rearrangements are needed in other vertebrates to form the remarkably conserved cup shape of the eye. So far, rim involution has been reported only in teleost species where it may represent a fast mode of increasing the surface of the inner layer of the OV, thus favouring its bending (Sidhaye and Norden, 2017; Heermann etal., 2015; Kwan etal., 2012). This idea is well in agreement with previous data showing that between 16 and 27 hpf the number of cells in the outer layer of the OV decreases from about 587 to 432, whereas that of the inner layer increases in a way that cannot be explained solely by proliferation (Zheng etal., 2000). In other species, this cell displacement may not be needed as the layer can Research article Developmental Biology | Neuroscience MorenoMármol etal. eLife 2021;10:e63396. DOI: https:// doi. org/ 10. 7554/ eLife. 63396 19 of 28 grow by cell division. In a similar way, we have shown here that in slower developing species, RPE cells maintain a higher proliferation rate that contributes substantially to the increase of RPE surface while undergoing less marked changes in cell shape (Figure8B). This correlation is visible in medaka, despite its relative evolutionary proximity to zebrafish (FurutaniSeiki and Wittbrodt, 2004), and is maximal in human embryos. Indeed, in humans, the RPE layer is composed of cells with a neuroepithelial appearance and a high proliferation rate, despite the expression of OTX2, considered a tissue specifier. Thus, in mammals, full commitment of the OV outer layer to an RPE identity may occur over a prolonged period of time and not ‘en bloc’ as in zebrafish, as suggested by comparing RNAseq data of RPE cells from human CS13–16 embryos (Hu etal., 2019) with those from equivalent stages in zebrafish (Buono etal., 2021). Human RPE cells from CS13 to CS16 embryos are still enriched in the expression of proliferation associated genes (Hu etal., 2019) but not of those typical of squamous epithelia as in zebrafish (Buono etal., 2021). A slow acquisition of RPE identity may also explain why, in mice, inactivation of genes such as Otx2, Mitf, or Yap causes the RPE layer to adopt NR characteristic (MartinezMorales etal., 2001; Kim etal., 2016; Nguyen and Arnheiter, 2000), whereas this feature that has never been reported after equivalent manipulations in zebrafish (Lane and Lister, 2012; Miesfeld etal., 2015), or why FGF8 can push the amniote but not the zebrafish RPE layer to acquire an NR identity (MartinezMorales etal., 2005). As a reflection of this slower differentiation in amniotes, RPE cells can largely retain their neuroepithelial morphology and adopt a final cuboidal – but not squamous – appearance at a slower and speciesspecific pace. We thus propose that RPE cell stretching vs. cell addition are different solutions adopted by species with different rates of development to reach a common goal: an appropriate equilibrium between the surface of the RPE and that of the NR. Indeed, the present study together with previous observations (Carpenter etal., 2015) and in silico models (Okuda etal., 2018; Eiraku etal., 2012) support that this equilibrium is a prerequisite for proper OV folding. Materials and methods Continued on next page Key resources table Reagent type (species) or resource Designation Source or reference Identifiers Additional information Gene (Danio rerio)bhlhe40 ENSEMBL ENSDARG0000004060 Ref. 28 Strain, strain background (Oryzias latipes) Wildtype, adult cab strain CBMSO fish room NBRP Medaka (https:// shigen. nig. ac. jp/ medaka/) Strain, strain background (Mus musculus)Wildtype BALB/c CBMSO animal facility https://www. jax. org/ jaxmiceandservices Strain, strain background (Danio rerio) Adult wildtype AB/tupl strain CBMSO fish room ZIRC (https:// zebrafish. org/ home/ guide. ph) Genetic reagent (Danio rerio)Tg(E1bhlhe40:GFP) Transgenic line generated in this study Details in Materials and methods, ‘Generation of the Tg(E1bhlhe40:GFP) line’ section Genetic reagent (Danio rerio) Tg(rx3:Gal4VP16;UAS:GFP) PMID:22819672 ZFIN Cat# ZDBGENO12110583, RRID:ZFIN_ZDB-GENO-121105-83 Ref. 48 Biological sample (Homo sapiens) Paraffin sections of human embryonic eye primordia Human Dev. Biology Resource (http://www. hdbr. org/) Recombinant DNA reagent ZED vector PMID:19653328 Ref. 31 Recombinant DNA reagent Bidirectional UAS:GFP PMID:19363289 Ref. 45 Recombinant DNA reagent pQTEVSTMN1 Addgene# 31326 RRID:Addgene_31326 Research article Developmental Biology | Neuroscience MorenoMármol etal. eLife 2021;10:e63396. DOI: https:// doi. org/ 10. 7554/ eLife. 63396 20 of 28 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent UAS: STMN1 Construct generated in this study Details in Materials and methods, ‘Gal4UASmediated expression’ Recombinant DNA reagent pCS2Kaede PMID:17406330 Ref. 34 Recombinant DNA reagent pCS2H2bmRFP Addgene# 53745 RRID:Addgene_53745 Recombinant DNA reagent pCS2EB3GFP PMID:12684451 Ref. 43 Antibody AntiBrdU (mouse) BectonDickinson IF(1:200), Antibody AntiGFP (chicken polyclonal) Abcam Cat# ab13970, RRID:AB_300798 IF(1:2000) Antibody Anti-βcatenin (mouse monoclonal) BD Transduction Laboratories Cat# 610153, RRID:AB_397554 IF(1:400) Antibody AntiZO1 (rabbit monoclonal) Invitrogen IF(1:400) Antibody Antilaminin (rabbit polyclonal) Sigma Cat# L9393, RRID:AB_477163 IF(1:200) Antibody Antiotx2 (rabbit polyclonal) Abcam Cat# ab76748, RRID:AB_1524130 IF(1:1000) Antibody AntiKi67 (rabbit polyclonal) Abcam Cat# ab15580, RRID:AB_443209 IF(1:500) Commercial assay or kit GatewayTM LR ClonaseTM Enzyme Mix Invitrogen 11791019 Commercial assay or kit pCR8/GW/TOPO TA Cloning Kit Invitrogen K250020 Commercial assay or kit mMessage mMachine SP6 transcription kit Invitrogen AM1340 Commercial assay or kit NucleoSpin RNA Cleanup kit Macherey Nagel 740948.50 Chemical compound, drug Blebbistatin Calbiochem BlebbistatinCAS674289555Calbioche, 100 µM Chemical compound, drug Paranitroblebbistatin Optopharma DRN111 20 µM Chemical compound, drug Azidoblebbistatin Optopharma DRA081 5 µM Chemical compound, drug Nocodazole Sigma M1404 10 ng/µl Chemical compound, drug BrdU Roche B23151 5 mg/ml Software, algorithm SPSS CSIC bioinformatic resources RRID:SCR_002865 IBM (https://www. ibm. com/ uken/ products/ spssstatistics) Software, algorithm MATLAB CSIC bioinformatic resources RRID:SCR_001622 MathWorks (https://www. mathworks. com/ products/ getmatlab. htm) Other DAPI stain Invitrogen D1306 Continued Animals Adult zebrafish (Danio rerio) were maintained under standard conditions at 28°C on 14/10 hr light/ dark cycles. AB/Tübingen strain was used to generate the transgenic lines and as control wild type. Embryos and larvae were kept in E3 medium (5mM NaCl, 0.17mM KCl, 0.33mM CaCl2, 0.33mM Research article Developmental Biology | Neuroscience MorenoMármol etal. eLife 2021;10:e63396. DOI: https:// doi. org/ 10. 7554/ eLife. 63396 21 of 28 MgSO4) supplemented with Methylene Blue (Sigma) at 28°C and staged according to somite number and morphology (Kimmel etal., 1995). The Tg(E1-bhlhe40:GFP) and Tg(rx3:Gal4;UAS:RFP) (Weiss etal., 2012) lines were maintained in the same conditions and crossed to generate the Tg(E1-bhlhe40:GFP;rx3:GAL4;UAS;RFP) line. Wildtype medaka fish (Oryzias latipes) of the cab strain were maintained at 28°C on a 14/10hr light/dark cycle. Embryos were staged as described (Iwamatsu, 2004). Fertilized chick embryos (Santa Isabel Farm, Cordoba, Spain) were incubated at 38°C in a humidified rotating incubator until the desired stage. Embryos were inspected for normal development and staged according to Hamburger and Hamilton, 1992. Wildtype BALB/c mice were in pathogenfree conditions at the CBMSO animal facilities, following current national and European guidelines (Directive 2010/63/EU). The day of the appearance of the vaginal plug was considered as embryonic day (E)0.5. All experimental procedures were approved by the CBMSO and Comunidad Autónoma de Madrid ethical committees. Human tissue Paraffin sections of human embryonic eye primordia were provided by the Joint MRC/Wellcome Trust (grant# MR/R006237/1) Human Developmental Biology Resource (http:// hdbr. org). Sections corresponded to samples CS13, -14, -15, and -16. CS staging allowed to determine the age of embryo as days post ovulation based on morphological landmarks (O’Rahilly and Müller, 2010). Generation of the Tg(E1-bhlhe40:GFP) line Predictive enhancer and promoter epigenetic marks (Bogdanovic etal., 2012) were used to identify different potential regulatory elements of the bhlhe40 gene (Figure1B). Each region was amplified by PCR with specific primers (Supplementary file 1) and cloned using the pCR8/GW/TOPO TA Cloning Kit (Invitrogen). Plasmids were checked for enhancer insertion and the Gateway LR Clonase Enzyme Mix (Invitrogen) was used for recombination with the ZED vector (Bessa etal., 2009). The resulting constructs were injected together with Tol2 mRNA to generate the corresponding transgenic embryos, which were screened using a transgenesis efficiency marker present in the ZED vector (cardiac actin promoter:RFP). Positive larvae were grown to adulthood (F0) and then individually outcrossed with wildtype partners to identify founders. Founders were analysed using confocal microscopy. One of the lines corresponding to the enhancer E1 was finally selected and used for subsequent studies. Gal4-UAS-mediated expression The UAS:STMN1 construct was generated from the bidirectional UAS:GFP vector, which allows simultaneous and comparable production of GFP and the gene product of interest under the same regulatory sequences (Paquet etal., 2009; Distel etal., 2010). The gene was amplified by PCR using specific primers (Supplementary file 1) flanked by StuI restriction sites and the Expand High Fidelity PCR System, using the pQTEVSTMN1 (Addgene# 31326) construct as a mould. The PCR product was digested with StuI (Takara) and cloned into the pCS2 vector and thereafter isolated together with the polyA sequence of the vector by digestion with HindIII and SacII (Takara) and subcloned into the UAS:GFP plasmid. The generated plasmid (30pg) was injected into the Tg(rx3:Gal4;UAS:RFP) (Weiss etal., 2012) line, together with Tol2 mRNA (50pg) to increase efficiency. Embryos micro-injection and drug treatments Embryos at one cell stage were injected using a Narishige microinjector and glass needles prepared by horizontally pulling standard capillaries (filament, 1.0mm, World Precision Instruments) with aP97 Flaming/Brown Micropipette Puller (Sutter Instrument Company). A total of 30 pg for DNA and between 50 and 100pg for mRNA in 1nl volume were injected in the embryos in the cell or the yolk, respectively. Drug treatments were performed on manually dechorionated embryos at the desired developmental stage in E3 medium. The following compounds were used: blebbistatin (100μM for 2.5 hr; Calbiochem); paranitroblebbistatin (20 μM; Optopharma), Ableb (5 μM for 15 min before photoactivation; Optopharma), and nocodazole (10ng/μl for 2.5hr; Sigma). Research article Developmental Biology | Neuroscience MorenoMármol etal. eLife 2021;10:e63396. DOI: https:// doi. org/ 10. 7554/ eLife. 63396 22 of 28 In vitro transcription The pCS2:Kaede, pCS2:EB3GFP, and pCS2:H2BRFP constructs were linearized and transcribed using the mMessage mMachine SP6 transcription kit (Invitrogen), following manufacturer’s instructions. After transcription mRNAs were purified using the NucleoSpin RNA Cleanup kit (Machery Nagel). In situ hybridization (ISH) otx1 (previously known as otx1b) and mitfa probes were gifts from Prof. Steve Wilson (UCL, London, UK). The bhlhe40 probe was generated by PCR from 24 hpf cDNA with specific primers Supplementary file 1 using the Expand High Fidelity PCR System. Reverse primers included the T3 promoter sequence to in vitro transcribe the PCR product. In vitro transcription was performed using T3 RNA polymerase and DIG RNA labelling Mix (Roche) following manufacturer’s instructions. Transcription products were precipitated with LiCl 0.4M and 3 volumes of ethanol 100% overnight at –20°C. Samples were centrifuged at 4°C and 12,000g for 30min, washed with ethanol 70%, and resuspended in 15µl of RNAsefree water and 15µl UltraPure Formamide (Panreac). ISH were performed as described (Cardozo etal., 2014). BrdU incorporation assays BrdU (Roche) was resuspended in DMSO (Sigma) to generate stocks of 50mg/ml that were kept at –20°C. For Tg(E1-bhlhe40:GFP) zebraand wildtype medaka fish groups of 15 embryos of stages comprised between 16 ss and 48 hpf were dechorionated and placed in BrdU solution (5mg/ml in E3 medium) for 30min on ice and then washed with fresh E3 medium. Embryos were let recover at 28°C for 10min before fixation in paraformaldehyde (PFA) 4% overnight at 4°C. For analysis in chick, BrdU (50mg/egg) was added to each embryo 30min before fixation. For analysis in mouse, pregnant dams were injected intraperitoneally with BrdU (50μg/g), sacrificed 1hr later and fixed. Chick and mouse embryos were immersion fixed in 4% PFA in 0.1M phosphate buffer, pH 7 at 4°C for 4hr and then washed in PBS and cryoprotected in 15% and 30%saccharose in 0.1M phosphate buffer . All embryos were cryosectioned and the sections hydrated with PBS 1X during 5min and incubated in HCl during 40min at 37°C. After HCl treatment, sections were rinsed with PBS 1X 10 times, and then processed for immunofluorescence as described below. The percentage of RPE proliferating progenitors was determined as the proportion of BrdUpositive cells over the total number of GFP (for E1-bhlhe40:GFP) or Otx2/Hoechst (medaka fish, chick, mouse embryos) positive cells in the RPE layer in each section. A minimum of three embryos and sections per embryo were counted (both eyes). Immunofluorescence Zebrafish embryos at the corresponding stage for each experiment were fixed with 4% (wt/vol) PFA (Merck) in 0.1M phosphate buffer overnight at 4°C. Wholemount immunofluorescence was performed as described (Cardozo etal., 2014). Alternatively, embryos were incubated in 15% sucrose – PBS overnight at 4°C, embedded in 7.5% gelatine (Sigma) 15% sucrose (Merck), frozen in isopentane (PanReac) between –30°C and –40°C and kept at –80°C. Cryosectioning was performed with a cryostat (Leica CM 1950) at 20µm thickness and dried overnight at room temperature. Chick and mouse embryos were collected, fixed 4% PFA, equilibrated in sucrose, and cryosectioned as above. Paraffin sections of human embryonic tissue were deparaffinized, washed in PBS, processed for antigen retrieval (10mM citrate buffer, pH6, for 5min at 110°C in a boiling chamber, Biocaremedical), and subsequently processed together with all other samples for immunofluorescence. Immunostaining was performed as described (Cardozo etal., 2014) using the following primary antibodies: mouse antiBrdU (1:200; BectonDickinson); chick antiGFP (1:2000; Abcam); mouse anti-βcatenin (1:400, BD Transduction Laboratories); mouse antiZO1 (1:400, Invitrogen); rabbit antilaminin (1:200, Sigma); rabbit antiOtx2 antibodies (1:1000; Abcam); rabbit antiKi67 (1:500, Abcam). The used secondary antibodies were conjugated with Alexa488, Alexa594, or Alexa647 (1:500; Thermo Fisher). Sections were counterstained with Hoechst (Invitrogen), mounted in Mowiol, and analysed by conventional and confocal microscopy. Kaede photoconversion Wildtype embryos were injected with Kaede mRNA. Embryos at 15 hpf with homogeneous green fluorescence were selected, mounted, and visualized under the Nikon AR1+ Confocal Microscope using a 20×/0.75 PlanApochromat objective. A region of interest (ROI) was drawn in the outer layer, corresponding to the putative position of the RPE progenitors, at a specific zposition and irradiated Research article Developmental Biology | Neuroscience MorenoMármol etal. eLife 2021;10:e63396. DOI: https:// doi. org/ 10. 7554/ eLife. 63396 23 of 28 with the 405nm laser at 21% of power for 10 loops to switch Kaede emission from green to red fluorescence. Due to confocality, photoconversion occasionally extended further than the selected plane, so that the tissues present above or below (i.e. ectoderm) also underwent photoconversion. After photoconversion embryos were let develop up to approximately 30 hpf stage, fixed and analysed by confocal microscopy for red fluorescence distribution. Ableb photoactivation Ableb (Kepiro etal., 2012) was photoactivated with a Zeiss LSM 780 Upright multiphoton FLIM system with a W PlanApochromat 20×/1.0 DIC M27 75mm WD 1.8mm dipping objective. For each eye a specific ROI was drawn including RPE cells identified by GFP fluorescence. Ableb was activated in the ROIs using 860nm wavelength and 20mW laser power (this corresponds to 9–14 µW/µm2 inside the ROI). Confocal imaging Embryos were mounted with the appropriate orientation in 1.5% low melting point agarose (Conda) diluted in E3 medium (for in vivo recording) or PBS (for fixed samples). Images were acquired either with a Nikon A1R + High Definition Resonant Scanning Confocal Microscope connected to an Inverted Eclipse TiE Microscope (20×/0.75 PlanApochromat, 40×/1.3 oil PlanFluor and 60×/1.4 oil PlanApocromat objectives) or with a Zeiss LSM710 Confocal Laser Scanning Microscope connected to a Vertical AxioImager M2 Microscope (40×/1.3 oil PlanApochromat, W NAchroplan 20×/0.5, W PlanApochromat 40×/1.0 DIC VISIR). 3D reconstructions 3D videos (i.e. Figure1—videos 1–3) were generated from full stacks using the 3D project option in Fiji (Schindelin etal., 2012). RPE surface renderings were generated using Imaris (Bitplane), with a value of 6 in Surface Area Detail and 7 in Background Subtraction. Morphometric analysis Unless otherwise specified, morphometric analysis of cells and tissues was performed using Matlab (The Mathworks, Natick, MA) using the XYZ coordinates of the processed images or Fiji (Schindelin etal., 2012). This analysis was performed using previously processed fluorescent images from videos of Tg(E1-bhlhe40:GFP; rx3:GAL4;UAS:RFP) or Tg(E1-bhlhe40:GFP) and H2BRFPinjected embryos (Figure1—video 2 and Figure3—video 1), from which the signal corresponding to the RPE or the whole OV/OC were isolated semimanually with the help of Fiji macros and tools designed to select 3D structures. The RPEspecific GFP signal was processed with a median filter. In the case of Figure3—video 1, the background ramp for the GFP signal was neutralized in each frame via subtraction of a copy of itself after a greyscale morphological operation (Hassanpour etal., 2015; Arce, 2005). For all videos, the median intensity was thereafter established as the cutoff value for differentiating background and signal (i.e. pixel with an intensity lower than the cutoff were set to zero) for all images that were in both videos. The signal derived from H2B was localized in cell nuclei, and therefore it was postprocessed with a greyscale closing operation to fill empty spaces between nuclei. Morphometric analysis was performed in the resulting processed images. All values were calculated in microns by scaling the x, y, z coordinates according to the following: (0.62 μm × 0.62μm × 1.37μm) for Figure1—video 2 and (0.62 μm × 0.62μm × 1.07μm) for Figure3—video 1. Volumes (μm3) were calculated as the number of voxels with a value higher than 0. RPE surface (μm2) was calculated applying a secondorder linear adjustment on the plane YZ corresponding to the plane of the OV/OC hinges with the fit function available in Matlab (The Mathworks, Natick, MA). RPE thickness (μm) was determined as the result of volume (μm3)/surface (μm2). Unfortunately, semimanual RPE image extraction was not perfect, when GFP signal associated to CMZ development arises. To account for this problem, the GFP signal for each frame was divided into seven equivalent blocks using the x, y coordinates from the zprojection of each frame. In this case, RPE volume and surface were calculated independently in each one of the regions up to 20 hpf, when the most anterior block (now corresponding to the arising CMZ) was discarded from the analysis. For the subsequent frames the two anterior most blocks were discarded (Figure3—figure supplement 1). The total OV/OC volume (μm3) was determined using the red fluorescence from the Tg(rx3:GAL4;UAS:RFP) embryos at Research article Developmental Biology | Neuroscience MorenoMármol etal. eLife 2021;10:e63396. DOI: https:// doi. org/ 10. 7554/ eLife. 63396 24 of 28 17–22hpf. H2B expression was used to determine the volume of the OC (H2B volume in Figure3) as follows for each frame of Figure3—video 1: the maximum, Gaussian blur and minimum filters were applied to the image; subsequently, the convex hull (Hamburger and Hamilton, 1992) was calculated for the image to obtain the geometrical shape that covers all pixels with an intensity higher than 0, including the lens; finally, only the regions present in the image and the convex hull are used to define the H2B volume. Individual cell area was determined in cells located at a medial position of the OV for each cell type (progenitor, RPE, and NR); cell contour was drawn using the segmented line tool in Fiji (Schindelin etal., 2012). Apicobasal (AB) length (µm) of individual cells was estimated by manually tracing a line from the basal to the apical membrane in the zposition in which the nucleus had its larger surface using the straightline tool in Fiji (Schindelin etal., 2012). To account for possible developmental asynchrony when eyes from the same embryo were differentially treated (irradiated vs. nonirradiated), the AB length of the irradiated eye was normalized with that of the nonirradiated eye. Values above 1 indicated less RPE cell flattening in experimental eyes. The invagination angle was determined as previously described (Sidhaye and Norden, 2017) using manual drawing with the Fiji angle tool (Schindelin etal., 2012). The vertex of the angle was placed approximately in the centre of the basal surface of the NR and the vectors were drawn up to the edges of the CMZ. Angles were measured in the zpositions in which the irradiated RPE was maximally affected and compared to equivalent positions of control nonirradiated eyes. Values were normalized with those of the contralateral nontreated eye, to account for possible asynchronies. Statistical analysis All statistical analysis was performed with IBM SPSS Statistics version 20.0. The method used is indicated in each case together with the sample size. Acknowledgements We wish to thank Drs JR MartinezMorales and E Marti for critical reading of the manuscript; the confocal microscopy service of the CBMSO and CNIC (Centro Nacional de Investigaciones Cardiovasculares) for help with image acquisition and analysis and the fish facilities for caring of the zebrafish lines. This work was supported by grants from the Spanish AEI (BFU201455918P to FC; BFU201675412R with FEDER support, RED2018102553T and PID2019104186RB100 to PB), BBVA Foundation (N[16]_BBM_BAS_0078 to FC) and Fundación Ramon Areces2016 (to PB). TMM and ML were supported by FPU (FPU14/02867) and FPI (BES2015–073253) predoctoral contracts from the Spanish AEI, respectively. We also acknowledge a CBM Institutional grant from the Fundación Ramon Areces. Additional information Funding Funder Grant reference number Author Agencia Estatal de Investigación PID2019-104186RB-100 Paola Bovolenta Ministerio de Economía, Industria y Competitividad, Gobierno de España RED2018-102553-T Paola Bovolenta Ministerio de Economía, Industria y Competitividad, Gobierno de España BFU2016-75412-R Paola Bovolenta Ministerio de Economía, Industria y Competitividad, Gobierno de España BFU2014-55918-P Florencia Cavodeassi BBVA Foundation N[16]_BBM_BAS_0078 Florencia Cavodeassi Fundación Ramon Areces-2016 Paola Bovolenta Research article Developmental Biology | Neuroscience MorenoMármol etal. eLife 2021;10:e63396. DOI: https:// doi. org/ 10. 7554/ eLife. 63396 25 of 28 Funder Grant reference number Author The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Author contributions Tania MorenoMármol, Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing – original draft; Mario LedesmaTerrón, Formal analysis, Investigation, Methodology; Noemi Tabanera, Maria Jesús MartinBermejo, Investigation, Methodology; Marcos J Cardozo, Investigation, Visualization; Florencia Cavodeassi, Conceptualization, Funding acquisition, Supervision, Writing – original draft; Paola Bovolenta, Conceptualization, Funding acquisition, Supervision, Writing - review and editing Author ORCIDs Florencia Cavodeassi http:// orcid. org/ 0000000346096258 Paola Bovolenta http:// orcid. org/ 000000021870751X Ethics Complying with EU regulations. Decision letter and Author response Decision letter https:// doi. org/ 10. 7554/ 63396. sa1 Author response https:// doi. org/ 10. 7554/ 63396. sa2 Additional files Supplementary files • Transparent reporting form • Supplementary file 1. List of primers used in this study. Data availability All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for all the graphs shown in the study. References Abe T, Ishikawa T, Masuda T, Mizusawa K, Tsukamoto T, Mitani H. 2006. Molecular analysis of Dec1 and Dec2 in the peripheral circadian clock of zebrafish photosensitive cells. Biochemical and Biophysical Research Communications 351: 1072–1077. DOI: https:// doi. org/ 10. 1016/ j. bbrc. 2006. 10. 172, PMID: 17097613 Ando R, Hama H, YamamotoHino M, Mizuno H, Miyawaki A. 2002. An optical marker based on the UVinduced greentored photoconversion of a fluorescent protein. PNAS 99: 12651–12656. 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