The eye is our most important organ for sensing and recognizing our environment. In humans and other vertebrates, the eye forms from an outgrowth of the brain as the embryo develops. This outgrowth is called the optic vesicle and it is rapidly transformed into a cup-shaped structure known as the optic cup. Defects in this process prevent the optic cup from closing completely, which leads to a severe condition called Coloboma—one of the most frequent causes of blindness in children.

The optic cup has two distinct layers: the inside layer—known as the neuroretina—contains light sensitive cells and is surrounded by the other layer called the pigmented epithelium. It is thought that the neural retina is made from cells from the side of the optic vesicle that faces the lens, and the pigmented epithelium is formed by cells from the other side of the vesicle. This is a plausible explanation and is well accepted, but it cannot explain how the neuroretina can become five times larger as the cup forms.

Heermann et al. addressed this problem by using four-dimensional in vivo microscopy to follow individual cells as the optic cup forms in living zebrafish embryos. The experiments show that the neuroretina is made of cells from both sides of the optic vesicle. Cells from the back of the optic vesicle (furthest away from the lens) join the rest of the cells by moving around the outside rim of the cup.

Further experiments found that a signaling molecule called BMP—which is crucial to the normal development of the eye—controls the flow of cells around the developing optic cup. This factor needs to be carefully controlled during the development of the eye; when BMP activity was artificially increased, the flow of cells stopped, resulting in neuroretinal tissue developing in the wrong place (in the outer layer of the optic cup).

The experiments also reveal that the stem cells in the retina—which divide to produce new cells throughout the life of the zebrafish—originate from two distinct areas in the optic vesicle.

Heermann et al.'s findings challenge the textbook model of eye development by revealing that cells from both sides of the optic vesicle contribute to the neuroretina and that retinal stem cells originate from a specific place in the developing eye. A future challenge will be to understand how the movement of the cells into the neuroretina is coordinated to make a perfectly shaped eye.

The bi-layered optic vesicles of vertebrates are formed through a bilateral evagination of the late prosencephalon. In teleosts, this process is driven by a migration of single cells that undergo a subsequent intercalation into the epithelium of the expanding optic vesicle (Rembold et al., 2006, England et al., 2006, Sinn and Wittbrodt, 2013, Ivanovitch et al., 2013). The oval optic vesicle develops into a hemispheric bi-layered optic cup through a process that involves major morphological transformations. A long-held view of this process proposes that the lens-averted epithelium of the optic vesicle differentiates into the retinal pigmented epithelium (RPE), while the epithelium facing the lens gives rise to the neuroretina, which subsequently bends around the developing lens (Chow and Lang, 2001; Fuhrmann, 2010; Walls, 1942). This neuroepithelial bending is driven by a basal constriction of lens-facing retinal progenitor cells (RPC) (Martinez-Morales et al., 2009) (Bogdanović et al., 2012), which ultimately reduces the space occupied by an individual RPC at the basal surface. However, we observed that this is accompanied by a 4.7-fold increase in the overall basal optic cup surface area (Figure 1A–C). To identify the cellular origin of this massive increase, we performed in vivo time-lapse microscopy in zebrafish at the corresponding stages, starting at 16.5 hpf (Figure 1D–L, Video 1), in a transgenic line expressing a membrane-coupled GFP in retinal stem and progenitor cells (Rx2::GFPcaax).

Figure 1

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Strikingly, and in contrast to the former model (Chow and Lang, 2001; Fuhrmann, 2010; Walls, 1942), our analysis shows that almost the entire bi-layered optic vesicle gives rise to the neural retina (Figure 1D–I), with the marked exception of a small lens-averted patch (see below). The majority of the lens-averted epithelium (Figure 1D,G, between arrowheads) serves as a neuro-epithelial reservoir, which eventually is fully integrated into the lens-facing neuro-epithelium (Video 1). This occurs through a sheet-like flow of lens-averted cells into the forming optic cup (Figure 1E,H). This epithelial flow is independent of cell proliferation (Figure 2—figure supplement 1, Video 2) as demonstrated by aphidicolin treatment. The process is highly reminiscent of gastrulation movements and explains the marked increase of the lens-facing basal neuroretinal surface area. Notably, a small domain of the lens-averted epithelium exhibits a different morphology and behavior. As optic cup formation proceeds, this region flattens, enlarges, exhibits the morphological characteristics of RPE, and eventually ceases expressing RX2, a marker for retinal stem and early progenitor cells (Figure 1H, asterisks, Video 3, in between arrows).

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Our data highlight that almost the entire optic vesicle contributes to the formation of the neural retina. This new perspective on optic cup formation raises the question of how the elongated oval optic vesicle is transformed into the hemispheric optic cup. We addressed this by 4D imaging of optic cup formation using a nuclear label (H2BGFP) (Figure 2A). We found, concomitant with lens formation, a prominent epithelial flow around the temporal perimeter of the forming optic cup. An involution of cells from the domain of the retinal pigmented epithelium (RPE) into the domain of the neuroretina had been proposed (Li et al., 2000). Such reorganization of the lens-averted and the lens-facing epithelia, affecting the temporal optic cup, has been subsequently described (Picker et al., 2009) and confirmed (Kwan et al., 2012). It was proposed that such ‘rim movements’ could occur around most of the optic vesicle circumference (Kwan et al., 2012).

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Our data confirm a flow around the temporal perimeter and additionally demonstrate epithelial flow around the nasal perimeter into the forming optic cup. We uncover that the direction of the epithelial flow primarily establishes two distinct neuroretinal domains (nasal and temporal) separated by the static dorsal and ventral poles of the forming eye (Figure 2D, Figure 3A). We use these poles as dorsal and ventral reference points throughout the manuscript. Importantly, the prominent rotation of the eye cup only occurs after the epithelial flow has ceased (24–36 hpf, Schmitt and Dowling, 1994).

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The prospective RPE remains in the lens-averted domain and expands in conjunction with the bi-furcated flow of the neuroretina from the lens-averted into the lens-facing domain (Figure 2A,B, Video 3). To further address the transformation of the elongated, oval optic vesicle into the hemispheric optic cup, we quantified cellular movements along the dorso-ventral axis. We found that the most prominent movements leading to the extension in the dorsal ventral axis occurred in the ventral domain (Figure 2C). A key step in the formation of the ventral neuroretina is the formation of the optic fissure at the ventral pole of the optic vesicle. Lens-averted epithelium flows through this fissure into the forming optic cup to constitute the ventral neuroepithelium (Figure 2D). Taken together, we present a model of optic cup formation, driven by gastrulation-like epithelial flow from the lens-averted into the lens-facing epithelium of the forming optic cup. The epithelium flows in two domains around the temporal and nasal rim, respectively and through the optic fissure of the forming optic cup. Overall, this has far-reaching implications for different aspects of eye development. One is the establishment of the retinal stem cell niche in the ciliary marginal zone (CMZ) (Centanin et al., 2011), the distal rim of the optic cup/retina.

To address whether the CMZ domain originates from a mixed population of progenitor cells that have been ‘set aside’, or from a predefined coherent domain, we analyzed the transition from optic vesicle to optic cup in 3D over time (4D) (Video 4). By tracking individual cells, we identified the origin of the distal retinal domain, the future CMZ, as two distinct domains (nasal and temporal) within the lens-averted epithelium at the optic vesicle stage (Figure 3A,B, Video 5). Based on tracking information, we noticed distinct phases during the flow from the lens-averted domain towards the CMZ (Figure 3D). Although cells show high motility in an early phase (Figure 3D, 1), the directed flow is established only in a later phase (Figure 3D, 2), in which cells ultimately flow to the rim of the forming optic cup (Figure 3D).

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As indicated above, the dorsal pole of the optic vesicle remains static (Figure 2D). Thus, the presumptive dorsal CMZ domain either originates from the lens-facing neuroretina or, alternatively, is established secondarily at a later time point, like the ventral CMZ in the region of the optic fissure. The identification of lens-averted domains as the source of the future nasal and temporal CMZ is consistent with the hypothesis of a distinct origin of retinal stem cells. Our data support a scenario in which the entire optic vesicle is initially composed of stem cells that at the lens-facing side respond to a signal to take a progenitor fate.

We propose a tight coupling of morphogenesis with cell determination by inductive signals derived from the surface ectoderm to explain the successive spreading of retinal differentiation from the center to the periphery (Sinn and Wittbrodt, 2013). Accordingly, lens-averted stem cells might retain their stem cell fate because they are exposed to that signal at the latest point in time. An alternative hypothesis is that stemness might require an active process at the interface to the RPE; it is also possible that both scenarios are involved. Both scenarios are consistent with the expression pattern of rx2, which is initially found in the entire optic vesicle and subsequently is confined to the CMZ. Strikingly, rx2-positive cells of the CMZ represent multipotent retinal stem cells (Reinhardt, Centanin et al., submitted).

We demonstrated that cell motility and thus tissue fluidity are a prerequisite for neuroretinal flow. These characteristics are likely maintained through signaling, raising the question of which system might be involved. A likely candidate might be BMP, which has been linked to mobility in other tissues during development. In heart jogging, for example, BMP has an ‘antimotogenic’ effect (Veerkamp et al., 2013). BMP signaling is important for various aspects of vertebrate eye development such as the enhancement of RPE and the inhibition of optic cup/neuroretina development (Fuhrmann et al., 2000; Hyer et al., 2003; Müller et al., 2007; Steinfeld et al., 2013), the formation of the dorso-ventral axis (Behesti et al., 2006; Holly et al., 2014; Koshiba-Takeuchi et al., 2000; Sasagawa et al., 2002), and the induction of the optic fissure (Morcillo et al., 2006). Specific regions of the eye also seem to depend on the modulation of BMP signaling by the expression of a BMP antagonist (Sakuta et al., 2001, French et al., 2009).

We analyzed BMP signaling activity by assays based on the phosphorylation of the Smads 1/5/8 and the activation of a BMP signaling reporter (Laux et al., 2011). BMP signaling is mainly elevated in the temporal domain and to a lesser degree in the nasal domain of the optic vesicle (16.5hpf, Figure 4A,B,D,E). At 21.5 hpf BMP signaling is confined to the dorsal pole of the optic cup (Figure 4C,F). The transcriptional BMP sensor is activated with a delay and shows a more confined area of activity (compare Figure 4A–C to Figure 4D–F).

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To address the means by which BMP activity is restricted, we analyzed the activity of prominent BMP antagonists follistatin a (fsta) (Thompson et al., 2005), and bambi (bambia) (French et al., 2009). Fsta was expressed in two domains, a nasal and a temporal domain (Figure 4G–I and Figure 5A), whereas bambi was only expressed in the temporal domain of the optic vesicle (Figure 4J,K) and the dorsal domain of the optic cup (Figure 4L). The regions of fsta expression correspond to the domains showing neuroretinal flow during optic cup formation.

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To address the importance of localized BMP signaling in wild-type embryos, we expressed BMP4 in the entire eye using an Rx2 proximal cis regulatory element (Figure 5B), which overrides the localized BMP antagonist in the optic vesicle and optic cup.

In BMP reporter fish (Laux et al., 2011), we addressed BMP signaling activity under control and experimental conditions. At the optic cup stage, moderate BMP signaling activity was observed in the dorsal retina of control fish (Figures 4F and 5C). The pan-ocular expression of BMP4 resulted in a strong response of the reporter, indicating pan-ocular BMP4 signaling (Figure 5D).

Strikingly resembling the BMP dependent ‘antimotogenic’ effect (Veerkamp et al., 2013), pan-ocular BMP expression arrested epithelial flow during optic cup formation. Time-lapse in vivo microscopy revealed that cells in the lens-averted part of the future neuroretina remained in the prospective RPE domain and did not contribute to the optic cup (Video 6 and 7). This ultimately resulted in an apparently ectopic domain of neuroretina that arose from a morphogenetic failure, rather than from a trans-differentiation of RPE (Figure 6D, Fig. 6—figure supplement 1,2,3, Videos 8,9,10). The severity of the phenotype correlated well with levels of fsta expression in the optic vesicle and was most prominent in the temporal domain of the optic vesicle. These findings highlight the importance of the modulation of BMP signaling for epithelial fluidity during the transformation from optic vesicle to optic cup. We propose that the repression of BMP signaling is crucial to mobilize the lens-averted retinal epithelium, causing it to flow and eventually constitute the neural retina to a large extent.

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We further investigated the implications of impaired epithelial flow for subsequent steps of eye development (e.g., fate of the optic fissure). After initiation of neuroretinal differentiation in control embryos, the undifferentiated domains are restricted to the un-fused optic fissure margins and the forming CMZ. Both can be visualized by the expression of Rx2 (Figure 6A,B). The impairment of neuroretinal flow, however, resulted in a mis-organization of the optic fissure. Here, the undifferentiated Rx2-expressing domain was found at the ultimate tip of the lens-averted neuroretinal domain, which failed to flow into the optic cup and persisted in the prospective RPE (Figure 6C). As a result, the temporal optic fissure margin, in particular, failed to extend into the optic fissure (Figure 6D–E). This also holds true, but to a lesser extent, to the nasal optic fissure margin (Figure 6D). As a result, the two fissure margins cannot converge resulting in a persisting optic fissure, a coloboma. Macroscopically, the pan-ocular expression of BMP4 results in phenotypes including a ‘Plattauge’ (flat-eye) (Figure 6G), in which the ventral part of the eye is strongly affected and a milder phenotype (Figure 6F), in which the ventral retina develops, but with a persisting optic fissure.

It was previously shown that exposing the developing eye to high levels of ectopically applied BMP can cause dorsalization, concomitant with a loss of ventral cell identities (Behesti et al., 2006). This is likely the cause for coloboma (Behesti et al., 2006; Koshiba-Takeuchi et al., 2000, Sasagawa et al., 2002). Our data based on stable BMP4 expression (rx2::BMP4) in the entire optic vesicle, however, conclusively show that early BMP4 exposure arrests neuroepithelial flow, resulting in a morphologically affected ventral retina. The ventral expression of vax2 in optic cups of rx2::BMP4 embryos indicates the maintenance of ventral retinal fates and argues against early transdifferentiation/dorsalization induced by BMP (Figure 6—figure supplement 4). Remarkably, the remaining lens-averted domain of those embryos, which was ectopically localized and was not integrated into the optic cup, eventually differentiated into neuroretina (Figure 6—figure supplement 1), as indicated by the expression of vsx1 (Kimura et al., 2008; Shi et al., 2011; Vitorino et al., 2009) and vsx2 (formerly Chx10) (Vitorino et al., 2009). Notably, a localization of neuroretina within the RPE domain might be mistaken for an RPE to neuroretina trans-differentiation, as proposed for other phenotypes (Araki et al., 2002; Azuma et al., 2005; Sakaguchi et al., 1997, Bankhead et al., 2015).

Even in amniotes, the histological analyses of consecutive stages of optic cup development are best interpreted as epithelial flow that also enlarges the retinal surface. This can even be appreciated during in vitro optic cup formation using mammalian embryonic stem cells (Eiraku et al., 2011).

Taken together, our data clearly show that during optic vesicle to optic cup transformation, the lens-averted part of the optic vesicle is largely integrated into the lens-facing optic cup by flowing around the distal rim of the optic cup including the forming optic fissure. Our data have far-reaching implications on the generation of the retinal stem cell niche of teleosts, as the last cells flowing into the optic cup will eventually constitute the CMZ. We identify a part of the lens-averted epithelium as the primary source of the RPE. The arrest of neuroepithelial flow by the ‘antimotogenic’ effect of BMP (Veerkamp et al., 2013) results in coloboma and thus highlights the importance of the flow through the fissure for the establishment of the ventral optic cup.

It is unlikely that the bending of the neuroretina provides the motor for the epithelial flow; in the opo mutant no ectopic neuroretina can be found, indicating that the flow persists, even in the absence of optic cup bending (Bogdanović et al., 2012). Consequently, forces established outside the neuroretina are likely to drive the flow. One tissue potentially involved is the mono-layered-forming RPE. We speculate that this tissue contributes to the flow by changing its shape from a columnar to a flat epithelium, massively enlarging its surface (Figure 1J–K, Video 3). This remains an interesting point, in particular given that epithelial flow is maintained even if cell proliferation is inhibited in both neuroretina and RPE.

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Author details

1. Stephan Heermann

   1. Centre for Organismal Studies Heidelberg, Ruprecht Karls Universität, Heidelberg, Germany
   2. Department of Molecular Embryology, Institute of Anatomy and Cell Biology, University Freiburg, Freiburg, Germany

   Present address

   Anatomie und Zellbiologie, Heidelberg University, Heidelberg

   Contribution

   SH, Final approval of the version to be published, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   stephan.heermann@cos.uni-heidelberg.de

   Competing interests

   The authors declare that no competing interests exist.

2. Lucas Schütz

   Centre for Organismal Studies Heidelberg, Ruprecht Karls Universität, Heidelberg, Germany

   Contribution

   LS, Final approval of the version to be published, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

3. Steffen Lemke

   Centre for Organismal Studies Heidelberg, Ruprecht Karls Universität, Heidelberg, Germany

   Contribution

   SL, Final approval of the version to be published, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

4. Kerstin Krieglstein

   Department of Molecular Embryology, Institute of Anatomy and Cell Biology, University Freiburg, Freiburg, Germany

   Contribution

   KK, Final approval of the version to be published, Conception and design, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

5. Joachim Wittbrodt

   Centre for Organismal Studies Heidelberg, Ruprecht Karls Universität, Heidelberg, Germany

   Contribution

   JW, Final approval of the version to be published, Conception and design, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   For correspondence

   jochen.wittbrodt@cos.uni-heidelberg.de

   Competing interests

   The authors declare that no competing interests exist.

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