Author response: Retinal stem cells modulate proliferative parameters to coordinate post-embryonic morphogenesis in the eye of fish (2019)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics Abstract Combining clonal analysis with a computational agent based model, we investigate how tissue-specific stem cells for neural retina (NR) and retinal pigmented epithelium (RPE) of the teleost medaka (Oryzias latipes) coordinate their growth rates. NR cell division timing is less variable, consistent with an upstream role as growth inducer. RPE cells divide with greater variability, consistent with a downstream role responding to inductive signals. Strikingly, the arrangement of the retinal ciliary marginal zone niche results in a spatially biased random lineage loss, where stem- and progenitor cell domains emerge spontaneously. Further, our data indicate that NR cells orient division axes to regulate organ shape and retinal topology. We highlight an unappreciated mechanism for growth coordination, where one tissue integrates cues to synchronize growth of nearby tissues. This strategy may enable evolution to modulate cell proliferation parameters in one tissue to adapt whole-organ morphogenesis in a complex vertebrate organ. https://doi.org/10.7554/eLife.42646.001 eLife digest By the time babies reach adulthood, they have grown many times larger than they were at birth. This development is driven by an increase in the number and size of cells in the body. In particular, special types of cells, called stem cells, act as a reservoir for tissues: they divide to create new cells that will mature into various specialized structures. The retina is the light-sensitive part of the eye. It consists of the neural retina, a tissue that contains light-detecting cells, which is supported by the retinal pigment epithelium or RPE. In fish, the RPE and neural retina are replenished by distinct groups of stem cells that do not mix, despite the tissues being close together. Unlike humans, fish grow throughout adulthood, and their eyes must then keep pace with the body. This means that the different tissues in the retina must somehow coordinate to expand at the same rate: otherwise, the retina would get wrinkled and not work properly. Tsingos et al. therefore wanted to determine how stem cells in the neural retina and RPE co-operated to produce the right number of new cells at the right time. First, stem cells in the eyes of newly hatched fish were labelled with a visible marker so that their divisions could be tracked over time to build cell family trees. This showed that stem cells behaved differently in the neural retina and the RPE. Computer simulations of the growing retina explained this behavior: stem cells in the neural retina were telling the RPE stem cells when it was time to divide. Combining results from the simulations with data from the experiments revealed that a stem cell decided to keep up dividing partly because of its position in the tissue, and partly because of random chance. To be healthy, the body needs to fine-tune the number of cells it produces: creating too few cells may make it difficult to heal after injury, but making too many could lead to diseases such as cancer. Understanding how tissues normally agree to grow together could therefore open new avenues of treatment for these conditions. https://doi.org/10.7554/eLife.42646.002 Introduction To maintain proper proportions, growth must be regulated at the level of the whole body, the size of each organ, and the size of tissues within an organ (Roselló-Díez and Joyner, 2015). Some regulatory mechanisms are shared, while others are specific to each level or to particular organs (Lui and Baron, 2011; Roselló-Díez and Joyner, 2015). Systemic signals couple nutrition to growth to coordinate growth of all organs at the organismal level (Buchmann et al., 2014; Droujinine and Perrimon, 2016). In addition to extrinsic systemic factors, transplantation experiments showed that many organs, including the eye, grow autonomously according to intrinsic factors (Wallman and Winawer, 2004; Roselló-Díez and Joyner, 2015). Growth coordination mechanisms have been studied at the level of the whole organism and inter-organ communication (Buchmann et al., 2014; Droujinine and Perrimon, 2016), but feedback mechanisms between constituent tissues of an organ remain largely unexplored both experimentally and at a conceptual level (Buchmann et al., 2014). Teleost fish grow throughout their lives, increasing massively in size (Johns and Easter, 1977). The teleost medaka (Oryzias latipes) grows roughly ten-fold from hatching to sexual maturity within 2–3 months (Figure 1—figure supplement 1A). Unlike embryonic morphogenesis, during post-embryonic growth all organs must scale with the increasing body size while fully functioning. In the eye, continuous growth must be additionally balanced with continuous shape-keeping: Proper optics, and thus vision, requires a precise 3D shape. Highly visual shallow water fish such as medaka have near-perfect hemispherical eyes (Fernald, 1990; Nishiwaki et al., 1997; Beck et al., 2004). The growth rates of all eye tissues must perfectly match, otherwise the organ would deform, akin to a bimetallic strip. Thus, the eye of fish provides an excellent system to explore how anatomically and functionally distinct tissues coordinate to grow and maintain the shape of an organ in functional homeostasis (Johns and Easter, 1977; Centanin et al., 2014). The vertebrate eye consists of multiple concentric tissues, including the neural retina (NR) and the retinal pigmented epithelium (RPE) (Figure 1A; Table 1). In fish and amphibians, these tissues grow from a ring-shaped stem cell niche in the retinal periphery: the ciliary marginal zone (CMZ) (Johns, 1977; Harris and Perron, 1998; Amato et al., 2004). The CMZ can be subdivided into a peripheral stem- and a central progenitor cell domain; stem cells are believed to have the potential for indefinitely many cell divisions while progenitor cells divide only a handful of times (Raymond et al., 2006; Centanin et al., 2014; Wan et al., 2016; Shi et al., 2017). At the very periphery of the CMZ, about 5 rows of cells express the stem cell marker retina-specific homeobox gene 2 (Rx2) (Reinhardt et al., 2015; Wan et al., 2016; Tang et al., 2017). The CMZ is a bi-partite niche, with tissue-specific stem cells for NR and RPE (Shi et al., 2017). In medaka, stem cells for NR and RPE are strictly separate, as demonstrated by transplantations at blastula stage and genetic recombination after hatching (Centanin et al., 2011; Centanin et al., 2014). Thus, medaka NR and RPE are independently growing tissues with identical topology. Table 1 List of abbreviations used throughout the main text. https://doi.org/10.7554/eLife.42646.003 NRneural retinaRPEretinal pigment epitheliumCMZciliary marginal zoneRx2retina-specific homeobox gene 2ArCoSarched continuous stripesGFPgreen fluorescent proteinOca2oculo-cutaneous albinism 2IdU5-Iodo-2′-deoxyuridine Figure 1 with 2 supplements see all Download asset Open asset Clonal labelling enables analysis of growth patterns in NR and RPE. (A) Schematic anatomy of the fish eye. (B) Growth patterns of retinal cell population (concentric rings) and individual clones. (C) False color immunostained NR of 3 week old Rx2::ERT2Cre, GaudíRSG fish with ArCoS and concentric rings of IdU-labelled cells. Overnight IdU pulses were at 1 and 2.5 weeks of age. Leftover undissected autofluorescent tissue fragments cover the far right of the cup-shaped retina. (D) Proximal view of clones induced in the NR of Rx2::ERT2Cre, Gaudí2.1 fish. Maximum projection of confocal stack of GFP immunostaining in false colors. (E) Proximal view of unpigmented lineages induced in the RPE by mosaic bi-allelic knockout of Oca2 using CRISPR/Cas9. Focused projection of brightfield focal stack. Images in (D) and (E) have been rotated to place the optic nerve exit (pink asterisk) ventrally; the embryonic retina is circled with a pink dashed line. https://doi.org/10.7554/eLife.42646.004 As a population, CMZ cells appositionally add new cells in concentric rings as shown by label incorporation with thymidine analogues (Johns, 1977; Centanin et al., 2011). Individual stem cells labelled by genetic markers form clonal progeny in so-called Arched Continuous Stripes (ArCoS; Figure 1B) (Centanin et al., 2011; Centanin et al., 2014). Medaka NR stem cells produce the full complement of neuronal cells in apico-basal clonal columns (Figure 1—figure supplement 2A’–B) (Centanin et al., 2011; Centanin et al., 2014; Lust and Wittbrodt, 2018). These differentiated retinal cells grow little in size (Johns, 1977), retain their relative position over time (Johns, 1977; Centanin et al., 2011), and have negligible death rates (Johns and Easter, 1977; Stenkamp, 2007). Thus, the only parameter available to NR and RPE to coordinate their growth rates is the proliferation of the tissue-specific CMZ stem cells. Stem cells have long been defined by an unlimited self-renewal capacity (Watt and Hogan, 2000; Clevers and Watt, 2018). Two general strategies underlie long-term maintenance of stem cells: 1) a deterministic model where every single division produces a stem- and a progenitor daughter cell (‘invariant asymmetry’); and 2) a stochastic model where cells divide symmetrically, and the daughter cells have a probability to stay as stem cells or commit to a progenitor fate (‘neutral drift’) (Watt and Hogan, 2000; Clevers and Watt, 2018). One tenet of this model is neutral competition: Stem cells randomly displace one another, resulting in the ‘loss’ of lineages where all progeny commit to a progenitor fate until the entire niche is occupied by a single clone (Colom and Jones, 2016; Clevers and Watt, 2018). Strikingly, the medaka retina diverges from the neutral drift model. The CMZ maintains a polyclonal stem cell population for both the NR and the RPE, and in particular NR stem cells undergo asymmetric self-renewing divisions throughout the life of the animal (Centanin et al., 2011; Centanin et al., 2014). It remains unclear whether stem cell proliferation in the CMZ follows a purely deterministic model, or whether it follows a strategy in-between invariant asymmetry and neutral drift. In this work we combine in vivo and in silico clonal analysis in the NR and RPE of medaka to address how these tissues coordinate their growth rates. We find that RPE stem cells have highly variable cell division timing consistent with a downstream role in the control hierarchy, whereas NR stem cells display less variability consistent with an upstream role in inducing growth in nearby tissues. Our simulation predicts that the spatial segregation of stem and progenitor CMZ domains is an emergent property, as the topology of the retinal niche preconditions the retina to a spatially biased neutral drift. NR stem cells deviate from a purely random drift model by preferential division axis orientation and differential modulation of division parameters along the CMZ circumference. We propose that during post-embryonic growth of the teleost eye, the NR CMZ forms a hub for integrating external and internal stimuli that affect cell division parameters, which ultimately direct the growth and shape of the entire eye. Results Clonal analysis indicates NR and RPE follow different post-embryonic growth modes Retinal cells follow an exquisite spatiotemporal order (Figure 1B–C, Figure 1—figure supplement 1B). Thus, clones derived from stem cells are a frozen record of past cell divisions (Centanin et al., 2011; Centanin et al., 2014), offering a window of opportunity to study stem cell properties in the NR and RPE. We experimentally generated NR ArCoS by randomly labelling individual NR stem cells using the Rx2::ERT2Cre, Gaudí2.1 line in hatchling medaka, and analyzing the eyes in adult fish as previously described (Centanin et al., 2014; Reinhardt et al., 2015). The Rx2 promoter drives the inducible Cre recombinase in stem cells at the very periphery of the CMZ (Reinhardt et al., 2015). A recombined stem cell generates a stripe of GFP-positive progeny in an otherwise GFP-negative retina (Centanin et al., 2014). In proximal view, NR ArCoS emanated as rays from the central embryonic retina, the part of the eye that was already differentiated at the timepoint of Cre-mediated recombination (Figure 1D). We visualized RPE ArCoS by mosaic knockout of pigmentation using CRISPR/Cas9 targeted to the gene oculo-cutaneous albinism 2 (Oca2), which is required for melanosome maturation (Fukamachi et al., 2004; Lischik et al., 2019). RPE stem cells with a bi-allelic mutation in Oca2 generate unpigmented stripes, analogous to RPE ArCoS obtained by transplantation (Centanin et al., 2011). RPE ArCoS frequently branched, forming irregular stripes variable in size and shape (Figure 1E). These qualitative differences in clonal pattern suggested that despite their identical topology, the division behavior of NR and RPE stem cells differed. A 3D agent based model of retinal tissues Clonal data generates a distribution of outcomes that is challenging to analyse and easy to misinterpret (Klein et al., 2007). The curved retinal surface and spatial extent of the niche pose a further challenge. We overcome these challenges by comparing experimental clonal data with simulated clonal data from a 3D agent based cell-center overlapping spheres model built in the platform EPISIM (Sütterlin et al., 2013; Sütterlin et al., 2017; Sütterlin, 2019). This modelling technique represents cells as discrete objects (e.g. spheres) that physically interact through forces acting on the cell centers; the spheres are allowed to slightly overlap to simulate cell deformability and allow a tight cell packing (Sütterlin et al., 2013; Sütterlin et al., 2017). This level of abstraction is ideally suited to the tightly packed pseudocrystalline mosaic of retinal cells (Johns, 1981; Nishiwaki et al., 1997; Pérez Saturnino et al., 2018), and has been used previously to model clonal data in skin and gut epithelia (Osborne et al., 2010; Buske et al., 2011; Li et al., 2013). Our retinal tissue model consists of a layer of spheres (representing either NR or RPE cells) on a hemisphere (representing the rest of the organ that is not explicitly modelled; Figure 2A). The RPE is a monolayer, thus each model cell corresponds to one RPE cell. In the NR, CMZ stem cells form a monolayer, and their differentiated progeny arrange in multiple neuronal layers (Johns, 1977; Raymond et al., 2006). We observed that clonal progeny of CMZ stem cells retained close proximity with little spread tangential to the retinal surface, forming clonally related ‘columns’ (Figure 1—figure supplement 2A’-B) (Centanin et al., 2011; Centanin et al., 2014; Lust and Wittbrodt, 2018). We took advantage of this fact to abstract each differentiated clonal column as a single cell in the simulation. Figure 2 with 3 supplements see all Download asset Open asset Feedback between proliferation and organ growth affects the simulated clonal pattern. (A) Initial condition and properties of the agent based model of the growing fish retina. Virtual embryonic retina in light green. CMZ cells are assigned unique colors for virtual clonal analysis. (B’) Simulated IdU pulse-chase experiment. First pulse: 200–220 hr, second pulse: 400–420 hr. Screenshot from 435 hr. Virtual cells incorporate IdU when they divide and half of the signal is passed on to each daughter cell. (B’’) Cell age (hours elapsed since last cell division) forms a gradient with the oldest cells in the virtual embryonic retina. (C’) In the inducer growth mode, the modelled tissue signals upstream to drive growth of other tissues in the organ. (C’’) Representative screenshot of inducer growth mode. (C’’’) Sample of 10 clones from (C’’). Colors: ratio of full perimeter by bounding rectangle perimeter, a metric for shape complexity. (C’’’’) Cell division intervals plotted against the mean average overlap. (D’) In the responder growth mode, control of the growth of the modelled tissue is downstream of an external signal. (D’’) Representative screenshot of the responder growth mode. (D’’’) Sample of 10 clones from (D’’) evaluated by the same shape metric as in (C’’’). (D’’’’) Cell division intervals plotted against the mean average overlap. Note the higher range of values for cells over the threshold overlap of 0.2. https://doi.org/10.7554/eLife.42646.007 In vivo, the spatial extent of the CMZ stem cell domain is believed to be defined by cues such as nearby blood vessels (Wan et al., 2016; Tang et al., 2017). Therefore, we defined the virtual stem cell domain with a fixed size of 25 µm, that is 5 rows of cells, reflecting the endogenous scale of the Rx2-expressing CMZ domain (Reinhardt et al., 2015; Wan et al., 2016; Tang et al., 2017). In vivo, NR stem cells divide predominantly asymmetrically, but also undergo symmetric divisions (Centanin et al., 2014). The rates of asymmetric and symmetric divisions are unknown; likewise, it is unknown whether these rates are deterministically defined or an emergent property of an underlying stochastic system. Since stochastic cell divisions successfully describe the proliferation of committed retinal progenitor cells in larval zebrafish (Wan et al., 2016), we used a simple stochastic mechanism for our initial model. Virtual stem cells commit to divide with a fixed probability pdivision=126h−1 and intervals between subsequent cell divisions must fulfill a minimum cell cycle length tcellCycle=24h. These values lie within a biologically plausible range estimated from experimentally measured growth rates and a parameter scan of the simulation (Appendix 1 section 3.3). All divisions are symmetric, resulting in two stem cells; cells differentiate and stop cycling when they exit the virtual CMZ after being pushed out by cellular crowding. To prevent physically implausible cell crowding, cell-center based models include a density-dependent inhibition of cell division (Pathmanathan et al., 2009; Osborne et al., 2017; Sütterlin et al., 2017). In our model, inhibition occurs in cells whose average overlap with all neighbors exceeds a fraction of the cell’s diameter given by the model parameter δol_threshold (Figure 2—figure supplement 1; Appendix 1, section 2.4). Based on in vivo observations (Lyall, 1957; Johns, 1977; Ohki and Aoki, 1985), the growing virtual eye gradually moves cells apart as it expands, thus decreasing cell density (Figure 2—figure supplement 2; Appendix 1 section 2.2). Continuous proliferation in the CMZ counteracts this decrease in vivo (Johns, 1977; Johns and Easter, 1977); likewise, the ever-increasing virtual cell population optimally fills the hemisphere at all times (Video 1; Video 2). Our model distills the complexity of the system and replicates the exquisite spatiotemporal growth order observed in vivo (Figure 2B’, B’’). Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. 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