A non-cell-autonomous actin redistribution enables isotropic retinal growth
[['Acquiring the correct size and shape during development is a crucial prerequisite for tissue and organ function .', 'In diverse tissues , such as the Drosophila salivary gland [1] or the vertebrate retina [2] , shape characteristics are established early in development and need to be retained throughout growth .', 'This necessitates an isotropic rescaling of the initial tissue shape ( Fig 1A ) .', 'How such uniform , isotropic rescaling is achieved through cell and tissue level processes , however , is not yet well explored .', 'Nevertheless , some of the molecular factors governing cell and tissue geometry have been extensively studied .', 'Both microtubule reorganization and cortical actomyosin rearrangements can influence shapes of individual epithelial cells , as well as the shape of whole tissues [3–10] .', 'When studying these factors , most investigations , so far , explored anisotropic tissue shape changes occurring during development , while isotropic growth and shape maintenance are not as well understood .', 'In addition , previously , tissue growth and shape were primarily explored in two dimensions [5 , 9 , 11–13] .', 'Thus , a thorough evaluation of how tissue growth is coordinated by cell and tissue parameters in 3D is currently lacking .', 'One tissue that allows analyzing cell and tissue shape during growth is the curved vertebrate retinal neuroepithelium .', 'The retinal neuroepithelium establishes its shape early in development by formation of the optic cup [2 , 14] and grows continuously afterwards ( Fig 1A ) .', 'During this proliferative growth , the neuroepithelium is pseudostratified [15] , with cells attached to both the apical side and the basal lamina .', 'Nuclei are located along the apicobasal axis , except in mitosis , as all cells divide apically [16] .', 'Such pseudostratified epithelia ( PSE ) are evolutionarily conserved and serve as organ precursors in a wide range of species [15] .', 'In the retinal neuroepithelium , as well as in most other PSE , cells progressively increase their height as the tissue proliferates and grows [15 , 17–19] .', 'So far , however , it is not understood what drives this cell elongation over development and whether and how it is linked to the overall tissue growth and shape maintenance .', 'To investigate how growth and shape coordinate in the developing retinal neuroepithelium , we used zebrafish to analyze cell- and tissue-wide shape changes during proliferative growth .', 'We generated a quantitative tissue-wide 3D dataset , which allowed us to break down 3D growth into individual cellular components through balance equations .', 'Our analysis revealed that isotropic growth relies on apicobasal cell elongation .', 'This cell elongation is governed by cellular actin redistribution that occurs in a non-cell-autonomous manner .', 'Furthermore , cell elongation occurs in concert with continued proliferation and needs an unperturbed extracellular matrix ( ECM ) .', 'Our data are validated by a simplified theoretical model and balance equations that account for the main aspects of tissue changes .', 'If actin redistribution and thereby cell height increase is impaired , tissue shape maintenance does not take place , and the tissue gets distorted upon further growth .'], ['In order to understand how cell and tissue shape changes are linked during growth of the proliferating retinal PSE ( Fig 1A and 1B ) , we quantified cell and tissue geometry of the retinal neuroepithelium over development .', 'By performing tissue-wide 3D analysis ( Fig 1B and 1C and S1 Movie ) , we analyzed growth in 4–6 h intervals between 20 h post fertilization ( hpf ) , when morphogenesis of the optic cup is complete , and 48 hpf , a stage when neuronal differentiation is ongoing , ( Fig 1A and 1B , 10 embryos/time point ) .', 'For details of the analysis , we refer the reader to the Materials and methods part .', 'In brief , the whole volume of the tissue and its apical surface were manually outlined in 3 dimensions .', 'These outlines were used to obtain 3D tissue segmentations , using Imaris , and subsequently allowed for analysis of cell number , size , and shape in the entire retinal PSE ( Fig 1C ) .', 'While apoptosis was negligible between 20 hpf and 48 hpf ( S1 Fig ) [20] , the total cell number increased 8-fold , from about 2 , 200 to about 18 , 000 ( Fig 1D , S1 Fig ) .', 'Concurrently , single cell volumes decreased by almost two-thirds , from 440 to 140 μm3 ( Fig 1E ) .', 'Overall , the tissue volume increased exponentially by a factor of approximately 4 . 4 over 28 h , with a rate of about 0 . 055/h and a volume doubling time of 12 . 5 h ( Fig 1F ) .', 'In addition to decreasing their volume , cells changed their shape , becoming thinner ( Fig 2A ) and more apicobasally elongated ( Fig 2C ) .', 'The largest change in aspect ratio and thereby cell shape occurred between 36 hpf and 42 hpf ( Fig 2D and 2E ) , as did the largest increase in cell numbers ( Fig 1D ) , suggesting differences between an early ( before 36 hpf ) and later growth phase ( after 36 hpf ) .', 'We next examined the overall changes in tissue area ( Fig 2B ) and height ( Fig 2C ) over time .', 'Before 36 hpf , tissue height stayed fairly constant ( Fig 2C ) , and most of the volume growth was accounted for by area expansion ( Fig 2B and 2G ) .', 'After 36 hpf , however , a significant increase of tissue height was seen , which accounted for a larger fraction of tissue growth ( Fig 2G ) .', 'While single cells elongated and changed their aspect ratio ( Fig 2E and 2F ) , the tissue area ( Fig 2B ) and thickness ( Fig 2C ) roughly scaled with each other , such that the aspect ratio of the tissue remained constant ( Fig 2D and 2E ) .', 'This may seem in contradiction with the observation that area expansion occurs at constant height before 36 hpf .', 'However , during this phase , the area expansion is small enough ( Fig 2B ) for the tissue aspect ratio not to be significantly affected ( Fig 2E ) .', 'Overall , our measurements indicate that , while cell shape changes , height and area expansion of the retinal PSE occur concurrently to maintain a fixed tissue aspect ratio .', 'The fact that the retinal PSE appears to grow homogeneously suggested that mitotic events are distributed homogenously and without a preferred direction in the apical plane of the epithelium .', 'To test this notion , we analyzed the distribution of mitosis throughout the proliferative phase using a custom tool ( Fig 3A and Materials and methods ) that allowed the projection of the 3D coordinates of all apically dividing cells onto a 2D density heatmap ( Fig 3B ) .', 'This analysis confirmed that mitotic cells were distributed evenly across the apical surface at all developmental stages ( Fig 3B ) , making proliferative growth homogeneous .', 'Cell division angles in the plane of the epithelium were observed by live imaging and did not show a strong alignment along a preferred direction ( Fig 3C and S2 Movie ) .', 'Our mitotic density heatmaps ( Fig 3B ) in combination with the mitotic index analysis ( Fig 3D ) showed that mitotic activity peaked at approximately 36–42 hpf .', 'To understand whether this mitotic peak was due to an increased mitotic duration or due to overall cell cycle shortening , we analyzed cell cycle length by live imaging of a mosaically expressed nuclear marker , using light sheet microscopy to reduce phototoxicity ( S3 Movie ) [21] .', 'Manual cell tracking ( N = 250 , 20 embryos ) revealed that , as the length of mitosis remained constant ( S2 Fig ) , average cell cycle lengths shortened from 7 . 3 h at 30 hpf to 5 . 3 h at 42 hpf ( Fig 3E ) and became less variable , as seen by the reduced coefficient of variation ( Fig 3E ) .', 'Thus , the observed peak in proliferation between 36 hpf and 42 hpf resulted from an overall cell cycle shortening .', 'The cell cycle length analysis in the proliferative growth phase revealed that the rate of cell proliferation varied around an average value of approximately 0 . 11 cell divisions per cell per hour ( Fig 3E , S4 Fig ) .', 'Furthermore , our measurements showed that at 48 hpf , about 18 , 000 cells inhabit the retina ( Fig 1D ) .', 'However , if all cells were continuously dividing at an average rate of 0 . 11/h starting from 2 , 177 cells at 20 hpf , the number of cells at 48 hpf should reach about 47 , 000 .', 'To understand this discrepancy in cell number at 48 hpf , we analyzed the fraction of cells that exited the cell cycle .', 'We assessed the number of differentiating cells between 36 hpf ( the stage when neurogenesis starts [22] ) and 48 hpf ( S2 Fig ) using fluorescence-activated cell sorting ( FACS ) of the SoFa transgenic line , which labels all emerging retinal neurons [23] .', 'A model in which progenitors divide at a constant rate of 0 . 11/h then enabled us to account for the number of neurons and total number of cells .', 'In this model , all divisions give rise to progenitors prior to 35 hpf , while 35% of the divisions give rise to committed precursors/neurons [22 , 24] after 35 hpf ( Fig 3F and S1 Text ) .', 'Using both cell division and differentiation rates calculated from our data , one can account for the total number of cells at different developmental stages of the retinal PSE ( Fig 3F ) .', 'We next used tissue volume , cell volume , and cell number measurements to decompose the volume change in the tissue into', '( i ) changes of average cell volume and', '( ii ) changes in the number of cells through cell division .', 'The cumulative logarithmic rates of change of these quantities are shown in Fig 2H .', 'We verified that the sum of different contributions adds up to 0 within the experimental standard deviation .', 'We found that , because the rate of volume growth is about twice as low as the relative rate of cell number increase , the average cell volume is decreasing at roughly the same relative rate as the tissue volume is increasing ( Fig 2H ) .', 'Taken together , our data show that overall , the retinal PSE increases its volume homogeneously and without a strong preferred direction in the plane of the epithelium , and the cell division rate is about twice as large as the rate of volume growth , leading to a decrease in average cell volume before 35 hpf .', 'The combined decrease in cell volume and increase in cell number lead to increased cell density in the retinal PSE ( Fig 1A” , S2 Fig ) .', 'We wondered whether this increase in cell density could influence proliferative growth of the retina .', 'Such a hypothesis was posed in the 1970s , in which it was postulated that proliferative PSE growth can be constrained by a self-inflicted proliferative trap [17] .', 'It was suggested that this trap arises from the fact that all cell divisions occur exclusively at the apical surface , where all PSE cell nuclei migrate prior to division [15 , 17 , 25] .', 'When too many nuclei are packed under the apical surface , or too many cells divide simultaneously , the availability of the apical surface was hypothesized to become a spatial limitation to further proliferation ( Fig 3G , S3 Fig ) .', 'To this date , however , we are not aware of an experimental dataset that allowed to test this idea .', 'With our quantitative dataset at hand ( Figs 1 and 2 ) , we first analyzed the fraction of the apical surface occupied by rounded mitotic cells between 20 hpf and 48 hpf .', 'This analysis showed that mitotic cells never occupied more than 20% of the total apical surface , even at times of highest mitotic activity ( S3 Fig ) .', 'We then calculated a theoretical maximal number of proliferative cells in the conical volume beneath a single mitotic cell ( Fig 3H ) , given its cell cycle parameters , and compared this value to the number of cells stacked beneath a dividing cell at the apical surface in our measurements .', 'Again , the measured numbers were consistently far from the calculated theoretical maximum throughout development .', 'Together , these results show that no proliferative trap exists in the growing retinal PSE and that proliferative retinal growth is not constrained by the availability of the apical surface .', 'With this , we provide , to the best of our knowledge , the first measurements of PSE nuclear packing in relation to its proliferation through development and show that the suggested proliferate trap [17 , 19 , 25 , 26] is not a general phenomenon that applies to all pseudostratified tissues .', 'While proliferative growth was not constrained by the apical surface , the number of nuclei stacked in the tissue nevertheless increased during the proliferative phase ( Fig 4A and 4A’ ) .', 'We analyzed this increase in nuclear stacking throughout development by dividing the length of the apicobasal axis containing nuclear signal by the average nuclear length ( Fig 4A and 4A’ ) .', 'Between 36 hpf and 42 hpf , nuclear stacking increase was most prominent , with the average number of stacked nuclei rising from 2 . 8 to 5 . 5 nuclei , respectively ( Fig 4A , gray bars ) .', 'The length of the apicobasal nuclear zone ( Fig 4A ) , however , differed from the tissue thickness assessed in our initial growth analysis ( Fig 2C ) .', 'One explanation for this discrepancy could be that nuclei do not occupy the entire length of the tissue .', 'Inspecting the tissue in more detail , we indeed noted that , prior to the significant increase in nuclear stacking , the basal zone of the retinal PSE was devoid of nuclei ( Fig 4A’ ) .', 'This resulted in a shorter nuclear zone compared to the entire tissue thickness ( Fig 4A’ ) .', 'This nuclear exclusion zone was diminished by 42 hpf , the time at which the major increase in nuclear stacking is observed .', 'From this stage on , nuclei occupied the entire apicobasal tissue axis ( Fig 4A and 4A’ ) .', 'We next asked how nuclei might be restrained from occupying the basal region before 42 hpf .', 'It was previously reported that a basolateral actin accumulation is present in cells of the early zebrafish optic cup [2] .', 'We therefore asked whether this actin accumulation persisted at later stages and whether it might be linked to the basal nuclear exclusion zone ( Fig 4B ) .', 'To this end , we analyzed the distribution of actin signal intensity along the apicobasal axis of the cells , in relation to nuclear distribution ( Fig 4C–4E ) .', 'Before 40 hpf , the basolateral actin accumulation was consistently observed ( Fig 4B–4D ) .', 'More specifically , at 30 hpf , the basolateral actin accumulation took up 26% of the total cell height , or an average 12 μm of the apicobasal axis ( Fig 4C ) , both on the single cell ( Fig 4B” ) and tissue-wide level ( Fig 4B , 4B’ and 4B”’ ) .', 'However , at 36 hpf , the accumulation started to disappear ( Fig 4D ) , spanning an average 20% of the cell axis ( 9 μm ) .', 'At 42 hpf , the basolateral actin pool was drastically reduced ( Fig 4B , 4B”’ and 4E ) , occupying only the most basal 8% ( 5 μm ) of the cells’ apicobasal axis .', 'At all stages , this tissue-wide basolateral actin accumulation was in precise negative correlation with the nuclear exclusion zone ( Fig 4C–4E ) .', 'To test whether this actin accumulation was indeed responsible for the exclusion of nuclei from basal positions , we abolished the basolateral actin at 30 hpf using the Rho-kinase ( ROCK ) inhibitor , Rockout [2 , 27] .', 'This treatment led to a disappearance of both the actin accumulation ( Fig 4F ) and the basal nuclear exclusion zone , as nuclei were consequently occupying the entire apicobasal axis ( Fig 4F ) .', 'Thus , a basolateral actin accumulation creates a basal nuclear exclusion zone , and only once it disappears nuclei occupy basal positions in the tissue .', 'The redistribution of actin was not only linked to an increase in nuclear stacking but also coincided with the cell height increase observed after 36 hpf ( Figs 2C and 5A ) .', 'As contraction and tension of the actin cytoskeleton can actively affect cell shape in many different developmental contexts [28–30] , we asked whether actin redistribution was involved in cell elongation .', 'To test this notion , we analyzed actin redistribution in cells by comparing the ratios of the lateral actin signal intensity to basal- and apical-most actin signal intensity throughout the proliferative phase ( Fig 5B , S6 Fig ) .', 'We found that , as development progressed , actin was depleted from the lateral cell cortex and concentrated at the basal and apical cell attachments , relative to its lateral distribution ( Fig 5B , S6 Fig ) .', 'If actin distribution was indeed involved in controlling tissue height , one simple explanation could be that the changing cell aspect ratio is dictated by a balance of forces between the line tensions in the apical and basal actin epithelial belt and the surface tension exerted by the actin cortex along the lateral cell interfaces ( S4 Fig and S1 Text ) .', 'The actin redistribution observed between 30 hpf and 42 hpf would then drive cell height elongation by favoring a contraction of the apical and basal perimeter at the cost of an expansion of the lateral surface area .', 'We incorporated this possibility in our simplified description of tissue growth by considering a mechanical balance equation that gives the cell shape as a function of the lateral surface tension and the apicobasal line tensions ( S4 Fig , S1 Text ) .', 'We also proposed an overall description of tissue growth with a constant rate of volume growth and a constant rate of progenitor cell divisions .', 'We further assumed that a transition occurs at approximately 35–36 hpf , at which progenitors start to differentiate , and actin redistributes from the lateral surfaces to apical and basal epithelial belts ( Fig 5C and 5D ) .', 'Taking into account the balance equations discussed earlier ( S1 Text ) , this simplified description accounted for the main aspects of the geometrical changes seen for the tissue between 20 hpf and 48 hpf ( Fig 5E–5G ) .', 'To experimentally test whether a link between actin redistribution , cell height increase , and uniform tissue growth existed , we used a histone-deacetylase 1 ( hdac1 ) zebrafish mutant that undergoes continued proliferation and does not enter differentiation programs , because of the loss of Hdac1-dependent Wnt and Notch inhibition [31 , 32] .', 'It was previously reported the hdac1−/− retinal PSE shows a perturbed tissue shape [31 , 32] ( S5 Fig ) .', 'Our live imaging confirmed that hdac1−/− mutants developed folds in the retinal PSE instead of the smooth tissue surface seen in controls ( S5 Fig , S4 Movie ) .', 'This shape disruption starts at stages at which differentiation would have begun in the wild-type retina ( S5 Fig , S4 Movie ) .', 'In previous studies , the emergence of this shape distortion was not explicitly explored , but it was reported that hdac1−/− mutants show disturbed actin organization [32] .', 'We thus set out to test whether a lack of timely actin redistribution could be linked to the tissue shape disruption seen in this mutant .', 'We imaged actin localization and cell height changes of control versus hdac1−/− neuroepithelia ( Fig 6A and S5 Movie ) .', 'This revealed that in hdac1−/− embryos , actin accumulation along lateral interfaces lasted substantially longer than in controls ( 70 hpf versus 40 hpf , Fig 6A–6C ) .', 'This was confirmed by the analysis of basal-to-lateral actin signal ratios , which , unlike the control , remained unchanged throughout development in the hdac1−/− embryos ( Fig 6D ) .', 'With the basolateral actin accumulation maintained in hdac1−/− tissue , cells did not elongate past 55 μm ( Fig 6E ) , a height value that is reached in controls at 30 hpf .', 'As a consequence of hdac1−/− cells not changing their shape as control cells ( Fig 6F ) , the hdac1−/− retinal PSE did not maintain its aspect ratio upon continued proliferation ( Fig 6F’ and 6F” , S5 Movie ) , leading to tissue shape changes rather than cell shape changes .', 'This indeed indicated a link between retinal PSE shape changes and actin rearrangements .', 'In accordance with this finding , our simplified theory predicted that the lack of actin redistribution leads to a decrease in tissue aspect ratio , as seen in the hdac1−/− mutant ( Fig 6G ) .', 'While retinal PSE shape was disturbed in the hdac1−/− neuroepithelium , the tissue nevertheless retained its pseudostratified characteristics , as shown by intact apical junctions and basal lamina ( S5 Fig ) and its mitoses occurring apically ( S5 Fig ) .', 'A similar sequence of tissue shape changes was seen when using an Hdac1 morpholino ( S5 Fig ) [31] or the Hdac1 inhibitor Trichostatin-A ( S5 Fig ) .', 'In all conditions , the end result of cell shape maintenance was the appearance of apical and basal epithelial folds ( S5 Fig ) [31 , 32] .', 'Taken together , these data suggest that actin redistribution from lateral cellular interfaces to the apical and basal epithelial belts drives cell and thereby tissue elongation .', 'When this reorganization does not occur , cells do not increase their height , leading to disturbed tissue shape .', 'We asked whether the depletion of actin from the lateral cellular interfaces is sufficient to drive cell elongation .', 'To test this , we used Rockout as we showed that this depletes the basolateral actin accumulation prematurely ( Fig 6H ) .', 'As shown before ( Fig 4F ) , this treatment led to nuclei redistributed along the complete apicobasal axis ( Fig 6H ) .', 'However , even after the actin accumulation was diminished and nuclei also inhabited basal positions , no increase in tissue height was observed ( Fig 6H , S6 Movie ) .', 'This counterintuitive result led us to further evaluate the effect of Rockout on overall tissue development .', 'We noted that in the Rockout-treated embryos , cell division rate was reduced compared to controls ( between 37 and 40 hpf , only 3 mitotic events were observed in the middle retinal section [S6 Movie] , compared to 78 mitotic events in wild type in the same retinal region [S5 Movie] ) .', 'This effect of Rockout on cell proliferation is most likely due to the effect the drug has on cytokinesis by inhibiting ROCK-mediated myosin light chain phosphorylation [33 , 34] .', 'This consequently affects the tissue growth rate .', 'Thus , actin disappearance from lateral cellular interfaces was necessary but not sufficient to trigger cell elongation .', 'Instead , our experiments suggested that cell proliferation was another important factor to increase cell height in a growing PSE tissue .', 'Indeed , the increase in cell height observed after 36 hpf coincided with increased proliferation due to shortened cell cycles ( Fig 3D and 3E ) .', 'To directly test whether proliferation was involved in cell elongation , we blocked proliferation by hydroxyurea/aphidicolin treatment at 30 hpf .', 'In this condition , even after the lateral actin accumulation disappeared , the tissue height did not increase to the same amount as in controls ( Fig 6I , S7 Fig ) .', 'Using the same approach , we explored how proliferation inhibition influences tissue shape in the hdac1−/− mutant .', 'Here , actin did not get redistributed from basolateral positions , and the tissue did not increase its height , as seen before ( Fig 6A ) .', 'Because of the lack of ongoing proliferation , however , no tissue buckling or shape disturbance was observed ( S7 Fig ) .', 'Together , this set of experiments strongly argues that both the disappearance of actin from lateral cell interfaces and continued proliferation are required for the observed cell and tissue elongation .', 'Therefore , these two factors together ensure tissue shape scaling during retinal PSE growth .', 'Live imaging of F-actin revealed that the disappearance of actin from the lateral interfaces occurred simultaneously in all progenitor cells throughout the retinal PSE ( S7 Movie ) .', 'This observation could be explained 2-fold:', '( i ) a cell-intrinsic event is coordinated in all cells , or', '( ii ) cells react to an extrinsic , tissue-wide signal .', 'To differentiate between these two possibilities , we transplanted cells from control embryos into hdac1−/− embryos , in which the basolateral actin bias persisted ( Fig 7A and 7A’ ) .', 'Here , transplanted control cells preserved their actin distribution ( Fig 7A’ , 7C and 7C’ ) and did not increase their height , even at stages at which they would elongate in their native environment ( Fig 7A’ ) .', 'Conversely , when hdac1 morpholino was injected into 64-cell stage control embryos to achieve a mosaic distribution of morphant cells , these morphant cells lost the lateral actin accumulation similarly to neighboring control cells ( Fig 7B ) .', 'In this context , nuclei of hdac1 morphant cells occupied basal positions ( Fig 7B and 7D ) , and cells increased their height alongside their neighbors ( Fig 7B ) .', 'This confirmed that actin redistribution is a non-cell-autonomous event that coincides with cell height increase .', 'It has been previously shown that the ECM underlying growing tissues can actively influence their size and shape during organ development [35 , 36] .', 'All retinal PSE cells are basally attached to the ECM of the basal lamina , which contains collagen and laminin as main components ( S5 Fig ) [37] .', 'As forces mediated by cell–ECM attachments can affect actin polymerization and bundling in diverse epithelial cells [38 , 39] , we tested whether cell–ECM interactions were involved in the tissue-wide non-cell-autonomous actin reorganization in the retinal PSE .', 'We injected collagenase , an ECM-degrading metalloprotease , between the lens and the retinal neuroepithelium of hdac1+/− and hdac1−/− fish at 30 hpf and 42 hpf ( Fig 7E ) .', 'In the hdac1+/− control , as well as in the hdac1−/− tissues , collagenase indeed abolished the actin accumulation on lateral interfaces and led to the removal of the nuclear exclusion zone ( Fig 7E” and 7E”’ ) , which was not seen in PBS-injected controls ( Fig 7E’ ) .', 'This suggested an active role of the ECM in actin organization .', 'Thus , as the ECM is necessary for the distribution of lateral actin , it might also be driving its redistribution and , ultimately , the isotropic growth of the retinal PSE .', 'Together , these data show a strong relationship between actin redistribution and cell height increase , indicating that these parameters are mechanistically linked .', 'Cell-level actin redistribution occurs independently of cellular identity but is most likely dependent on a change in the ECM to allow the maintenance of tissue shape during growth .'], ['In this study , we used a combination of quantitative 3D tissue-wide analysis and long-term 3D live imaging to investigate tissue growth and shape .', 'We demonstrated how such a large-scale , quantitative approach can be used to decompose growth of tissues into geometric cell and tissue-wide parameters to extract fundamental rules of growth during development .', 'Our analysis on growth and shape showed that the retina growth is uniform and isotropic .', 'We find that this uniform 3D growth is enabled by the redistribution of the actin cytoskeleton in concert with continued proliferation , allowing for timely apicobasal cell elongation ensuring tissue shape maintenance ( summarized in Fig 8 ) .', 'To understand the links between growth and shape in a developing PSE in 3D , we generated a quantitative dataset of the retinal neuroepithelium growth phase .', 'This , together with a set of balance equations , allowed us to create an unprecedented characterization of growth .', 'We reveal that geometric changes at the single-cell level are essential to maintain tissue shape .', 'Single-cell shape changes were most prominent after 36 hpf , a stage at which tissue height increased .', 'In concurrence with this increase in tissue height , nuclei enter a previously unoccupied nuclear exclusion zone , leading increased nuclear stacking .', 'We find that the loss of this nuclear exclusion zone , as well as cell height increase , is linked to a cellular redistribution of actin .', 'When actin is maintained at lateral cell interfaces , cell elongation does not take place , and the scaling of tissue shape is consequently disturbed .', 'These findings , together with a simplified theoretical description , led us to propose a model in which the lateral actin accumulation generates a mechanical force acting against apicobasal cell elongation .', 'We postulate that the redistribution of actin leads to a change of distribution of forces generated in the cells , involved in driving apicobasal cell elongation .', 'Because a similar redistribution of tension has been suggested to drive cell elongation in the developing Drosophila wing disc PSE [40] , and due to the fact that all PSE generally thicken during growth , we propose that such a mechanism might be evolutionarily conserved in other developing PSE tissues of intermediate length .', 'How exactly height increase results from the observed actin redistribution will need to be further investigated .', 'Basolateral actin accumulation depends on an intact underlying ECM , as we found that disturbing ECM composition results in actin redistribution .', 'This , together with the fact that actin redistribution occurs non-cell autonomously , supports the idea that ECM rearrangements or cell–ECM attachments act as upstream modulators of actin cellular organization in the retinal PSE .', 'In line with this assumption , distinct ECM components have been shown to play an instructive role in regulated proliferation in the vertebrate central nervous system ( CNS ) as well as retinal development [41 , 42] .', 'Upstream drivers for the ECM-mediated actin redistribution here could be , for example , the Wnt or the Notch signaling pathway , as Hdac1 acts as inhibitor of Wnt and Notch , and both molecules have been implicated in modulating cell–ECM force transmission [43–45] .', 'Overall , our finding showcases an example of a developmental change in which the ECM can actively influence tissue size and shape during growth , as also seen , for example , in the Drosophila wing disc PSE [35] or the developing mouse salivary gland [36] .', 'Our results also demonstrate that tissue growth needs to be tightly coordinated to cell shape changes in time and space for successful tissue development .', 'We show that cell height increase occurs in concert with the neighboring tissue independently of how the cell would behave in its native environment .', 'In contrast , retinal neuronal differentiation is most likely a cell-autonomous program , as we observed that after washing out the hdac1 inhibitor Trichostatin-A , cells in the already folded retinal tissue regained the potential to differentiate and generate neuronal layers , in spite of the tissue’s perturbed shape ( S5 Fig ) .', 'Thus , it is likely that differentiation is uncoupled from tissue shape .', 'This lends further weight to the conclusion that precise coordination of cell-intrinsic and tissue-wide parameters is essential to generate the fully functional , differentiated tissue of correct size and shape .', 'We found that in the retinal PSE , apical surface availability does not pose a constraint on growth during the proliferative phase , as was suggested for other PSE [15 , 17 , 25] .', 'While it is possible that the retinal PSE is an exception due to its curved shape , a careful analysis of factors contributing to tissue growth in other PSE , similar to our study here , would allow testing of this old hypothesis [17] in other contexts .', 'As retinal cells need to change their geometry while the tissue grows , our study exemplifies that tissue shape maintenance during growth is not a default state but requires active preservation [46] .', 'We found that shape scaling of the retina depends on altering active forces in the cell that control the cell shape .', 'Such a dynamic force change is also seen in other contexts such as germ band elongation in Drosophila , in which actin redistributes along the apical plane of the epithelium [4] .', 'In the example presented here , however , we propose that cellular tension redistribution acts in 3D , along the lateral cell surfaces , as well as along apical and basal cell perimeters , to ensure scaling of shape during growth , rather than guiding changes in tissue shape .', 'Overall , we here elaborate on the important question of how organs coordinate growth with scaling of shape during development .', 'We believe that our quantitative dataset can consti
['Introduction', 'Results', 'Discussion', 'Materials and methods']
['Tissue shape is often established early in development and needs to be scaled isotropically during growth .', 'However , the cellular contributors and ways by which cells interact tissue-wide to enable coordinated isotropic tissue scaling are not yet understood .', 'Here , we follow cell and tissue shape changes in the zebrafish retinal neuroepithelium , which forms a cup with a smooth surface early in development and maintains this architecture as it grows .', 'By combining 3D analysis and theory , we show how a global increase in cell height can maintain tissue shape during growth .', 'Timely cell height increase occurs concurrently with a non-cell-autonomous actin redistribution .', 'Blocking actin redistribution and cell height increase perturbs isotropic scaling and leads to disturbed , folded tissue shape .', 'Taken together , our data show how global changes in cell shape enable isotropic growth of the developing retinal neuroepithelium , a concept that could also apply to other systems .']
['As the shape of a tissue or organ is usually important for its function , it is critical to understand both how tissues form with the correct size and how their shape is maintained during development .', 'However , so far , most studies have focused on how the shape of tissues changes during morphogenesis , while little is known about how tissues keep their shape during growth .', 'Many tissues , including the human nose and retina , establish their shape early in development and need to maintain this shape as the tissue grows .', 'To understand how shape is maintained during growth , cellular behaviors underlying isotropic growth need to be linked to overall tissue parameters .', 'To achieve this , 3D studies of entire tissues , with cellular resolution , are needed .', 'This study took advantage of the exceptional imaging possibilities of the developing zebrafish retina to accomplish this goal .', 'It identified cell elongation as the key cellular shape change that allows the retinal tissue to maintain its shape during growth .', 'This 3D analysis during development , together with a theoretical description , shows that spindle-like retinal cells elongate further thanks to a timely non-cell-autonomous redistribution of the cytoskeletal element actin .', 'Overall , this study provides a fascinatingly simple example of how isotropic growth of a tissue is achieved and , at the same time , showcases the severe consequences on tissue geometry that arise if cellular shape changes do not occur at the correct developmental time .']
['medicine and health sciences', 'fish', 'cell cycle and cell division', 'cell processes', 'ocular anatomy', 'vertebrates', 'geometry', 'animals', 'aspect ratio', 'animal models', 'osteichthyes', 'developmental biology', 'model organisms', 'mathematics', 'experimental organism systems', 'embryos', 'research and analysis methods', 'contractile proteins', 'embryology', 'actins', 'fertilization', 'proteins', 'biological tissue', 'biochemistry', 'cytoskeletal proteins', 'zebrafish', 'retina', 'eukaryota', 'cell biology', 'anatomy', 'epithelium', 'biology and life sciences', 'ocular system', 'physical sciences', 'organisms']