Conventionally, neuronal development is regarded to follow a stereotypic sequence of neurogenesis, migration, and differentiation. We demonstrate that this notion is not a general principle of neuronal development by documenting the timing of mitosis in relation to multiple differentiation events for bipolar cells (BCs) in the zebrafish retina using in vivo imaging. We found that BC progenitors undergo terminal neurogenic divisions while in markedly disparate stages of neuronal differentiation. Remarkably, the differentiation state of individual BC progenitors at mitosis is not arbitrary but matches the differentiation state of post‐mitotic BCs in their surround. By experimentally shifting the relative timing of progenitor division and differentiation, we provide evidence that neurogenesis and differentiation can occur independently of each other. We propose that the uncoupling of neurogenesis and differentiation could provide neurogenic programs with flexibility, while allowing for synchronous neuronal development within a continuously expanding cell pool.
Investigating the timing of developmental hallmarks of a molecularly defined progenitor population in the retina reveals that mitotic divisions do not occur at a specific time in a cell's differentiation trajectory, but that neurogenesis and differentiation are uncoupled.
Timing of developmental hallmarks in a CNS progenitor population tracked in vivo.
The differentiation status of terminally dividing progenitors at mitosis varies greatly as cell cycle and differentiation are uncoupled.
Progenitor differentiation state is in lock‐step with post‐mitotic surrounding cells.
The nervous system is indisputably the most complex structure assembled during vertebrate ontogenesis. Consequently, the developmental processes underlying nervous system assembly must be precisely orchestrated. Neuronal development in the central and peripheral nervous systems (CNS, PNS) is widely accepted to require three major steps: (i) neurogenesis, the birth of neurons by progenitor cell mitosis, (ii) migration, the relocation of post‐mitotic neurons from their birthplace in proliferative zones to specific locations, and (iii) neuronal differentiation, the acquisition of molecular and morphological features that permit the integration of newly generated neurons into synaptic circuits. To date, the prevailing view is that these ontogenetic events are discrete steps along a stereotypic sequence, beginning with neurogenesis, followed by migration and concluding with neuronal differentiation. There is however, particularly in the PNS, evidence that some hallmarks of neuronal differentiation might already occur in progenitors (Rothman et al, 1980; Rohrer & Thoenen, 1987; DiCicco‐Bloom et al, 1990; Godinho et al, 2007; Attardo et al, 2008). These observations call into question the invariant developmental sequence of neurogenesis, migration, and differentiation, and raise the unresolved question: How stereotypic is the developmental program of a defined progenitor population in vivo?
Here, we examined the developmental fate of a molecularly defined CNS progenitor population (expressing visual homeobox gene 1, vsx1) that gives rise to the vast majority of a specific interneuron cell type (bipolar cells, BCs) in the zebrafish retina by terminal symmetric divisions (He et al, 2012; Weber et al, 2014). The swift development as well as the genetic and optical accessibility of zebrafish permitted us to follow the entire developmental program of vsx1+ progenitors, with single cell precision, in vivo. We used molecular, morphological, and cell biological markers of neuronal differentiation, in conjunction with chronic in vivo time‐lapse imaging, to determine the timing of mitosis in relation to a battery of developmental events. We discovered that for BCs, neurogenesis and multiple hallmarks of neuronal differentiation (such as somal positioning, neuronal marker expression, or neurite elaboration) are timed independently of each other. In other words, rather than dividing at a stereotypic point in their developmental trajectory, vsx1+ progenitors of BCs undergo terminal mitosis at markedly disparate stages of differentiation, suggesting that differentiation is not time‐locked to mitosis. However, the state of differentiation of a vsx1+ progenitor at mitosis is not arbitrary, but matches that of the post‐mitotic vsx1+ BCs in its vicinity.
Bipolar cell progenitor mitoses occur over an extended time‐period and relocate to non‐apical sites
In common with many parts of the developing vertebrate CNS, the retina begins as a pseudostratified neuroepithelium with spindle‐shaped progenitors that span its apico‐basal extent and undergo interkinetic nuclear migration, an oscillatory nuclear movement linked to specific cell cycle phases (Sauer, 1935; Baye & Link, 2008). At distinct but overlapping times, cells destined for different fates exit the cell cycle. Because mitotic divisions generally occur at the apical surface, newborn cells need to migrate varying distances to occupy their definitive locations within one of the emerging cellular laminae. Thus, while ganglion cells migrate furthest to occupy positions in the basal most part of the neuroepithelium, BCs have a shorter distance to relocate, and photoreceptors remain in situ at the apical surface. BCs, which are ultimately localized to the inner nuclear layer (INL) and confine their dendritic and axonal processes to the outer and inner plexiform layers (OPL, IPL), respectively, are generated over a protracted period, between 2 and 3 days post‐fertilization (dpf) in the zebrafish (He et al, 2012). Thus, early‐born cohorts of BCs are generated when the retinal neuroepithelium is not yet laminated, while later‐born cohorts are generated when the three cellular laminae are emerging.
To investigate the relationship between BC neurogenesis and differentiation, we examined the expression of vsx1, a transcription factor important for BC development (Passini et al, 1997; Chow et al, 2001; Vitorino et al, 2009; Shi et al, 2011), using a vsx1:GFP transgenic line (Kimura et al, 2008). In the zebrafish retina, vsx1 is expressed at low levels in the majority of committed, terminally dividing BC progenitors, up‐regulated during differentiation, and maintained at high levels in mature BCs (Vitorino et al, 2009). This developmental expression profile is faithfully reproduced in the bacterial artificial chromosome (BAC) vsx1:GFP transgenic line (Vitorino et al, 2009), so that we could follow nascent BCs from birth to maturity. Further, we took advantage of the zebrafish retina's gradient of development (Hu & Easter, 1999), to concurrently visualize immature (unlaminated) and more mature (laminated) parts of the retina and thus compare multiple states of BC differentiation in a single field of view.
At 2 dpf, we found low levels of GFP expression in the unlaminated part of the retina in which vsx1:GFP+ (henceforth referred to as vsx1+) cells span the entire apico‐basal axis. These vsx1+ cells represent both BC progenitors and post‐mitotic, undifferentiated BCs (Morgan et al, 2006; Randlett et al, 2013). By contrast, vsx1+ cells in the more mature, laminated part of the retina showed high levels of GFP expression and confined their processes to the IPL and OPL, suggestive of post‐mitotic, differentiated BCs (Fig 1A). To our surprise, immunostaining of vsx1:GFP retinas for phosphorylated histone H3 (pH3), a late G2/M‐phase marker (Hendzel et al, 1997), revealed vsx1+ pH3+ cells not only in the unlaminated retina but also in the INL of the laminated retina. This suggests that not only post‐mitotic BCs but also vsx1+ progenitors reside within the INL (Fig 1A–D). Notably, while vsx1+ progenitors in the unlaminated retina undergo mitosis at the apical surface, like “classical” progenitors in many parts of the CNS (Fig 1B), vsx1+ progenitors in the laminated retina undergo mitosis in the INL (Fig 1C), akin to previously described non‐apical progenitors (Godinho et al, 2007; Weber et al, 2014). Given the precocious expression of some neuronal characteristics in such non‐apical progenitors, we asked how similar vsx1+ progenitors were to the post‐mitotic vsx1+ BCs in their immediate surround.
Marker expression in progenitors matches the surrounding post‐mitotic bipolar cells
GFP levels during mitotic division in vsx1+ pH3+ progenitors showed a striking (3.8‐fold) increase between the unlaminated and laminated regions of the developing retina (Fig 1E), which correlated well with the GFP levels in surrounding vsx1+ pH3− cells (Fig 1F; the surrounding vsx1+ cells in laminated regions are expected to be ~90% post‐mitotic BCs on average; for an estimate of this number see Appendix Supplementary Materials and Methods). Furthermore, comparison of vsx1:GFP levels in pH3+ progenitors and pH3− surrounding cells across the entire developmental gradient in single retinas revealed a linear increase along the gradient (Fig 1F, inset). Hence, with regard to vsx1 expression, progenitors in the laminated retina are more similar to their BC neighbors than to their early dividing peers and form a continuum with regard to vsx1 promoter activity in lock‐step with surrounding BC differentiation. Direct time‐lapse observation of vsx1:GFP levels confirmed a parallel increase of fluorescence levels in BC progenitors and surrounding post‐mitotic BCs in vivo (Fig EV1). Moreover, based on the decay of GFP in a vsx2:GFP BAC line (Vitorino et al, 2009), vsx2 down‐regulation was similarly linked to the progression of differentiation along the retinal gradient independent of mitotic status.
To further establish this similarity in molecular differentiation of vsx1+ progenitors and their post‐mitotic neighbors, we examined two additional molecular markers of BC differentiation: cone‐rod homeobox (Crx) and Ribeye a. Crx is a transcription factor expressed in mature photoreceptors and BCs (Liu et al, 2001; Shen & Raymond, 2004). In the unlaminated retina, antibody staining for Crx (combined with pH3) revealed little or no expression in vsx1+ progenitors or the vsx1+ cells in their vicinity, whereas in laminated regions, high levels of Crx expression were found in virtually all vsx1+ progenitors and surrounding post‐mitotic vsx1+ BCs (Fig 1G). The levels of Crx antibody staining in mitotic vsx1+ progenitors along the differentiation gradient strongly correlated with that of the vsx1+ cells in their immediate surround (Fig 1H). Moreover, time‐lapse imaging of a crx:mCFP transgenic line (Suzuki et al, 2013), in which Crx+ cells are faithfully labeled (Fig EV2A), regularly revealed non‐apical crx:mCFP+ mitotic divisions in the laminated retina (Fig EV2B and C). Finally, we analyzed the expression of Ribeye a, a structural protein of ribbon synapses in photoreceptors and BCs (Wan et al, 2005). As expected, using fluorescence in situ hybridization, we found ribeye a mRNA only in the laminated retina, where post‐mitotic cells predominate (Fig EV2D and E). Notably, we also observed ribeye a mRNA‐containing cells that were pH3+ (Fig EV2F). The fact that these cells were located in the INL suggests they are BC progenitors. Using a transgenic line designed to report ribeye a expression in BCs (ctbp2:mEGFP; Odermatt et al, 2012), we observed ctbp2:mEGFP+ cells dividing at non‐apical locations, giving rise to BCs (Fig EV2G). Hence, even with regard to a marker linked to synaptic structures unique to BCs in the inner retina, we find that BC progenitors co‐differentiate with post‐mitotic BCs in the surround. We next asked whether this similarity extended beyond molecular markers to cellular morphology and dynamics.
Progenitor morphology and cell biology correspond to the surrounding post‐mitotic bipolar cells
To examine individual cells of the vsx1+ BC lineage, we generated a transgenic Gal4‐driver line (referred to as Q26) that was selected to label a sparse subset of vsx1+ cells. As Gal4 expression in Q26 is restricted to vsx1+ BCs and their progenitors in laminated parts of the retinal gradient (Appendix Fig S1), we almost exclusively observed non‐apically dividing Q26+ progenitors, which we followed by time‐lapse imaging as they terminally divided to produce BCs (Fig 2A). We examined the cleavage plane in dividing Q26+ progenitors (n = 65) and found divisions along the circumferential, apico‐basal, and centro‐peripheral axes with no preference for a particular orientation, in line with other reports of non‐apically dividing progenitors (Kimura et al, 2008; Weber et al, 2014). During mitosis, the vast majority of Q26+ progenitors had limited their processes to the plexiform layers (Fig 2A at 0′, Fig 2B), as is characteristic for BCs in the mouse and zebrafish retina (Morgan et al, 2006; Randlett et al, 2013) but not for apically dividing progenitors (Das et al, 2003; Miyata et al, 2004; Noctor et al, 2004). Prior to mitotic division however, the processes of non‐apically dividing progenitors extended beyond the OPL or IPL (see Fig 2A at −89′) and over time remodeled to become restricted to the synaptic layers. We asked whether this morphological remodeling occurred in a fixed time‐window relative to mitotic division and focused our analyses, for technical reasons (see Appendix Supplementary Materials and Methods) on the retraction of processes from the apical surface. We found that apical process retraction occurred over an extended period of time (18 min to > 9 h) prior to mitotic division (Fig 2C). In contrast, for apically dividing BC progenitors in the unlaminated retina, apical process retraction to the OPL was a post‐mitotic event (as observed in vsx1:GFP), but again occurred over an extended time span following mitotic division (a few min to > 8 h). Thus, a single differentiation step, the remodeling of the apical process, occurs both pre‐ and post‐mitotically, and over a time span of more than 17 h relative to mitosis. Notably however, our time‐lapse recordings in the Q26 line suggested that apical process remodeling is locally coordinated. When we identified progenitors that had just undergone apical process retraction to the OPL and asked whether post‐mitotic BCs in the immediate vicinity had also done the same (Fig 2D), we found that, on the population level, apical process remodeling occurred concurrently (Fig 2E). Moreover, once pruned, the apical and basal processes of pre‐mitotic progenitors could form lateral arbors. While these arbors regressed during mitosis, at earlier time points we could not distinguish them from the dendritic and axonal arbors of surrounding post‐mitotic BCs (brackets in Fig 3A and E; Fig 4D at −645′), suggesting that the morphological processes of BC differentiation proceed independent of progenitor mitosis. In accordance with observations from other systems and species (Miyata et al, 2001; Das et al, 2003; Saito et al, 2003; Kosodo et al, 2008) the basally directed process of dividing Q26+ progenitors exhibited either splitting or asymmetric inheritance by one daughter cell followed by new outgrowth by the other daughter (Appendix Fig S2). Remarkably, when new process outgrowth was observed, it exhibited directed targeting of the IPL without overshooting beyond it.
Next, we asked whether vsx1+ progenitors undergo interkinetic nuclear migration. We used an mRNA construct encoding a fusion protein between mOrange2 and proliferating cell nuclear antigen (PCNA; Fig 3A and Appendix Fig S3) that allows determination of progenitor cell cycle phase by distinct nuclear localization patterns (Leonhardt et al, 2000; Leung et al, 2011). Similar to classical progenitors, vsx1+ progenitors in the unlaminated region exhibited rapid apically directed interkinetic nuclear migration during G2 before mitosis at the apical surface (Fig 3B and Appendix Fig S3). This movement was absent in non‐apically dividing vsx1+ progenitors, making their nuclear movements largely indistinguishable from the post‐mitotic BCs in their immediate vicinity (Fig 3A and C). However, the non‐apically dividing vsx1+ progenitors always translocated their nuclei to the INL/OPL interface prior to mitosis. This movement can be explained by the location of the centrosome. By expressing fluorescently tagged centrin4, we found that BC centrosomes actively relocated from the apical surface to the INL/OPL interface (Fig 3D and E), resulting in a dendritic, rather than a somatic location at maturity (Fig EV3). Like apical process retraction, centrosome relocation occurred at the same time for progenitors and surrounding, post‐mitotic BCs (Figs 3D and EV3). Thus, nucleokinesis and centrosome relocation represent further differentiation steps for which vsx1+ progenitors are time‐locked to their post‐mitotic neighbors.
The post‐mitotic differentiation status of late‐born bipolar cells is similar to the early‐born bipolar cell population in their vicinity
We assessed the rate of differentiation of the BC progeny resulting from non‐apically dividing vsx1+ progenitors in the laminated region of the retina. First, we monitored vsx1‐driven GFP expression levels in post‐mitotic BCs over the course of 10 h after their exit from the cell cycle and found a steady increase in fluorescence intensity (Fig 4A). Ten hours post‐division, GFP expression levels in BC sibling pairs were remarkably similar to each other (Fig 4A and B) and remained similar to the cells in their surround, which largely comprised of earlier‐born post‐mitotic BCs (Fig 4C).
Next, we used an established transgenic line, ctbp2:mCherry‐ctbp2 (Pelassa et al, 2014), to examine the emergence of ribbon synapses in BC axon terminals. We assessed the time interval between exit from the cell cycle and the appearance of ribeye a clusters (marker of ribbon synapses) as an indication of presynaptic differentiation in vsx1+ BCs (Fig 4D). For late‐born BCs derived from non‐apically dividing vsx1+ progenitors, the average time interval was 6.5 h. By contrast, for early‐born BCs this interval was longer than 13 h on average (Fig 4E). Thus, late‐born BCs acquire features of presynaptic differentiation with greater speed than their earlier‐born counter‐parts. Finally, we measured the lateral extent of the terminal axonal arbors of BCs derived from non‐apical vsx1+ progenitors 10 h after they exited the cell cycle, and found this to be remarkably similar to that of the earlier‐born post‐mitotic BCs in their vicinity (Fig 4F). Taken together, late‐born BCs continue to differentiate in lock‐step with earlier‐born BCs in their vicinity, including the elaboration of markers of axonal and synaptic differentiation, thus contributing to synchrony in local neuronal development.
Experimentally delaying cell division does not delay vsx1+ progenitor differentiation
Our experiments have established that along the developmental gradient, BC progenitors blend into the differentiation landscape that surrounds them with regard to their morphological, cell biological, and molecular characteristics. Moreover, the “head‐start” gained by pre‐mitotic differentiation in late‐dividing progenitors continues in their BC progeny, so that the lag in differentiation between late‐born and early‐born BCs is remarkably low. Two potential scenarios could explain these observations: (i) Multiple, “fixed” vsx1+ progenitors exist, each of which undergoes mitosis at a stereotypic time point in the cell's differentiation trajectory. (ii) Alternatively, neurogenesis and differentiation could be uncoupled from each other, so that mitotic divisions could occur at various points in any given vsx1+ cell's differentiation program. To distinguish between these two possibilities, we delayed BC progenitor divisions using hydroxyurea and aphidicolin (HUA). HUA treatment rapidly reduced the number of cells entering the G2/M‐phase (Appendix Fig S4), but a small number of progenitors continued to divide, albeit with a prominent delay. We could now ask whether during delayed progenitor mitosis, differentiation stalled (as implied by the existence of “fixed” progenitors) or whether it continued and remained in synchrony with surrounding post‐mitotic BC differentiation (as predicted by the “uncoupling” scenario; Fig 5A). We tracked 10 vsx1+ progenitors in HUA‐treated embryos from late S‐phase until mitosis. Knowing that progenitors, in control experiments, divided on average 142 ± 3.9 min after the onset of late S‐phase, we could determine when a HUA‐treated cell should have divided (“expected” mitosis) and measured the delay with which the division actually occurred (“observed” mitosis, delay range approximately 3.5–9 h; Figs 5B and EV4). Because we could predict with a high degree of accuracy (86.4%), whether progenitors, in control conditions, would undergo mitosis at the apical surface or in the INL well before the divisions occurred (see Appendix Supplementary Materials and Methods), we could ask whether the HUA‐induced delay of mitosis would shift divisions from apical to non‐apical locations (as an “uncoupling” scenario would imply). Nine of the 10 HUA‐treated cells fulfilled criteria that identified them to be destined to divide apically. However, all 10 cells instead underwent mitosis in the INL after process remodeling, suggesting that they had been shifted from an apical to a non‐apical phenotype simply by delaying mitosis (Fig 5C). Furthermore, vsx1:GFP expression levels in the 10 delayed progenitors increased from the point of expected mitosis to observed mitosis (Fig 5D). Notably, even hours after the delayed mitosis occurred, progenitors still matched the fluorescence levels of cells in their immediate surround (Fig 5E), suggesting uncoupling of cell division and differentiation. Together, these findings support the “uncoupling” scenario laid out above and hence argue for an independence of neurogenesis and differentiation programs during BC development.
Our study is the first, to our knowledge, to address the relative timing of neurogenesis, migration, and differentiation for a molecularly defined CNS population in vivo and to elucidate the effects of such timing on progenitor characteristics. In contrast to the widely held view, we found that developing neurons did not adopt a stereotypic sequence of neurogenesis followed by migration and subsequent differentiation. Rather, to our surprise, the developmental trajectories that progenitors adopted were variable and accommodated remarkable flexibility. This resulted in vsx1+ BC progenitors with a wide variety of molecular, morphological, and cell biological characteristics. Importantly, rather than representing many distinct populations, the differentiation status of these progenitors formed a continuum in lock‐step with the differentiation of surrounding post‐mitotic BCs along the developmental gradient of the retina. Accordingly, cells dividing in the laminated parts of the retina were more BC‐like than their early dividing counter‐parts. Indeed, without the aid of time‐lapse imaging or cell cycle markers it would have been impossible to distinguish between pre‐mitotic progenitors and post‐mitotic cells. Our results support the conclusion that a stereotypical and fixed sequence of ontogenetic events is not essential during neuronal development. Thus, at least for terminally dividing progenitors, which generate a substantial part of the CNS neuronal population (Nakashima et al, 2015), mitosis does not have to occur before neuronal differentiation is initiated (or at any specific step thereafter), but rather can be flexibly intercalated between other developmental steps.
Our findings could explain two previous intriguing observations: First, that neuronal progenitors in different parts of the nervous system show precocious signs of differentiation prior to cell cycle exit (Rothman et al, 1980; Rohrer & Thoenen, 1987; DiCicco‐Bloom et al, 1990; Miyata et al, 2004; Godinho et al, 2007; Attardo et al, 2008; Prasov & Glaser, 2012); and second, that blocking mitosis does not halt neuronal differentiation in many parts of the Xenopus CNS including the retina, spinal cord, and brain stem (Harris & Hartenstein, 1991). Our results now offer a unifying explanation for these previous observations, suggesting that they might simply result from a fundamental uncoupling of cell cycle and neuronal differentiation during normal development. Indeed, the increasingly recognized prevalence of precocious progenitors in many parts of the nervous system of a range of species [e.g., basal progenitors in the neocortex (Haubensak et al, 2004; Miyata et al, 2004; Noctor et al, 2004); neural crest‐derived PNS progenitors (Rothman et al, 1980; Rohrer & Thoenen, 1987; DiCicco‐Bloom et al, 1990); retinal progenitors (Godinho et al, 2007; Prasov & Glaser, 2012)], implies that such uncoupling may be a general principle of neural development.
What would be the advantages of uncoupling neurogenesis and neuronal differentiation? By comparison with the orderly sequence of developmental events in the classical model, we suggest that the uncoupled model provides two advantages, namely speed and synchrony (Fig EV5). In the classical model, because differentiation can only be initiated following mitosis, the time required for the majority of cells in a defined population to differentiate is dictated by the delay in their birth dates. By contrast, in the uncoupled model, differentiation steps can already occur at the progenitor stage, permitting differentiation across the population to be faster. Furthermore, despite the extended time span over which mitotic divisions can occur, cells in a given population differentiate in relative synchrony. The uncoupling of mitosis and differentiation could thus be particularly pertinent for the assembly of essential neural circuits where swift maturation is paramount for survival (Nikolaou & Meyer, 2015).
Materials and Methods
All experiments were performed according to regulations as approved by the local regulatory bodies. Zebrafish were maintained, mated, and raised as described in Mullins et al (1994). Embryos were kept in 0.3× Danieau's solution at 28.5°C and staged as previously described (Kimmel et al, 1995). Fish were either in an AB wild‐type or roy orbison (Ren et al, 2002) background. The transgenic lines used are listed in Appendix Table S1. We generated Tg(vsx1:Gal4)q26 (Q26) and Tg(14xUAS:memTagRFP‐T) by Tol2 mediated insertion (Kawakami, 2004). For details of constructs used to make transgenic fish and for transient injections see Appendix Supplementary Materials and Methods.
mRNA synthesis and injection
Plasmids were linearized (PCNA: NotI, centrin4: ApaI). Capped mRNA was produced using the Ambion mMESSAGE mMACHINE kit (Applied Biosystems) according to the manufacturer's instructions. mRNA was injected at 100 ng/μl into one‐ or two‐cell stage embryos.
Immunohistochemistry and in situ hybridization
Immunostaining to detect pH3, Crx, and GFP was performed using adaptations of previously published protocols either on cryosections (Williams et al, 2010) or on whole‐mount embryos (Hunter et al, 2011). In situ hybridization to visualize ribeye a mRNA was performed on whole‐mount embryos using a digoxigenin‐labeled riboprobe and Fast Red TR/naphthol AS‐MX (Sigma) to detect alkaline phosphatase activity. For a detailed description see Appendix Supplementary Materials and Methods.
In vivo imaging
Embryos were prepared for imaging as described previously (Godinho, 2011; Engerer et al, 2016). Between 10 and 18 h post‐fertilization (hpf), embryos were transferred to 0.3× Danieau's solution containing 0.003% 1‐phenyl‐2‐thiourea (PTU, Sigma) to inhibit melanin formation. At 2.25 days post‐fertilization (dpf), manually dechorionated embryos were anesthetized using 0.02% tricaine (PharmaQ) in medium containing PTU and embedded laying on their side in low‐melting agarose (0.7–0.8%, Sigma).
Fish were imaged starting at 2 dpf on an Olympus FV1000 confocal/2‐photon and an Olympus FVMPE‐RS 2‐photon microscope using water‐immersion objectives (Olympus 20×/NA 0.95, Olympus 25×/NA 1.05, Zeiss 40×/NA 1.0, Nikon 25×/NA 1.1, and Nikon 40×/NA 0.8) or a silicon‐immersion objective (Olympus 30×/NA 1.05). Embryos were maintained at 28.5°C during all in vivo recordings. At each time point z‐stacks were acquired of the peripheral retina, encompassing its entire circumference.
Images were viewed and processed using open‐source ImageJ/Fiji software (http://fiji.sc). The StackReg function was used for drift compensation in xy. Image panels were assembled in Photoshop CS5 (Adobe) and combined into figures using Illustrator CS5 (Adobe). The “Gaussian blur” function was used to filter noise for clarity. Gamma was not adjusted.
Hydroxyurea (Sigma) and aphidicolin (BioViotica) were used at a final concentration of 20 mM and 150 μM, respectively, in 0.3× Danieau's containing 1.0–1.7% DMSO. Embryos were injected with a p53 morpholino (0.5–1 mM, Gene Tools) at the one‐ or two‐cell stage to ameliorate HUA‐induced apoptosis (Girdler et al, 2013). At 2 dpf embryos were mounted in agarose as described for in vivo imaging above, but leaving the tail fin un‐embedded for better drug access. Retinas were imaged for one time point prior to HUA administration. The 0.3× Danieau's medium was replaced with HUA‐containing medium, and time‐lapse recording was immediately resumed. Recordings were generally limited to < 16 h after HUA addition as high levels of cell death were observed thereafter.
For details, see Appendix Supplementary Materials and Methods.
Mean values and standard error of the mean (SEM) were calculated using Microsoft Excel. We used the Mann–Whitney U‐test to compare datasets with GraphPad Prism 5. Data are presented as mean ± SEM unless indicated otherwise. P‐values < 0.05 are denoted with “*”, P < 0.01 with “**”, and P < 0.001 with “***”.
PE, PRW, TM, and LG conceived of the project and designed experiments. TM and LG supervised the project. PE performed all in vivo imaging experiments, with some help from NO. PE and PC performed in situ hybridization. PE, SCS, TY, NO, and BO generated new constructs and transgenic lines for this research. PE, PRW, TM, and LG wrote the paper with input from all authors.
Conflict of interest
The authors declare that they have no conflict of interest.
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We thank Kristina Wullimann for expert fish husbandry and Yvonne Hufnagel, Alexandra Graupner, and Monika Schetterer for expert technical and administrative support. We are especially grateful to Rachel Wong (U. Washington, Seattle), who generously supported this project in multiple ways, thanks to her, and Magdalena Götz and Tim Czopka for critical reading of an earlier version of this manuscript. We are grateful to M. Nonet (Washington University in St. Louis) for the pCold Heart Tol2 vector, M. Meyer (King's College London) for the 5xUAS:TagRFP‐T vector, T. Nicolson (Oregon Health & Science University and Vollum Institute) for the ribeye a cDNA template, R. Köster (Technische Universität Braunschweig) for pSK5xUAS:Centrin2‐YFP, and P. Raymond (University of Michigan) for the anti‐Crx antibody. The mOrange2‐PCNA‐19‐SV40NLS‐4 plasmid was a gift from Michael Davidson (Addgene plasmid # 57971). We thank S. Higashijima (National Institutes of Natural Sciences, Okazaki Institute for Integrative Bioscience) for the Tg(vsx1:GFP)nns5 and Tg(vsx2:GFP)nns1 BAC transgenic lines which were generated with the support of the National Bioresource Project of Japan. We also thank R. Wong for providing the following transgenic lines: Tg(pax6‐DF4:gap43‐CFP)q01, Tg(vsx1:MCerulean)q19, Tg(14xUAS:MYFP), and Tg(crx:MA‐CFP)q20, and L. Lagnado (University of Sussex, UK) for Tg(−1.8ctbp2a:mCherry‐ctbp2a)lmb7. The Tg(−1.8ctbp2:gap43‐EGFP)lmb1 line was made in the laboratory of L. Lagnado at MRC‐LMB, Cambridge. This project was made possible by funding to L.G. and T.M. from the Deutsche Forschungsgemeinschaft (DFG) through Collaborative Research Center 870 “Assembly and Function of Neuronal Circuits”, project A11. T.M. is further supported by the Center for Integrated Protein Science Munich (CIPSM, EXC 114), the European Research Council under the European Union's Seventh Framework Program (FP/2007‐2013; ERC Grant Agreement no. 616791), the Munich Center for Systems Neurology (SyNergy; EXC 1010), the DFG Priority Program 1710 (Mi694/4‐1), the DFG research grants Mi694/7‐1, 8‐1, and the German‐Israeli Foundation (I‐1200‐237.1/2012). T.M. is also associated with the German Center for Neurodegenerative Diseases (DZNE Munich). P.R.W. was supported by the Human Frontier Science Program and the Wings for Life Foundation. N.O. was supported by the Amgen Scholar program. S.C.S. and T.Y. were supported by a grant awarded to R. Wong (NIH EY14358). P.E. was supported by the DFG Research Training Group 1373 and the Graduate School of the Technische Universität München (TUM‐GS).
FundingCenter for Integrated Protein Science Munich EXC 114
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