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The different cell types in the central nervous system develop from a common pool of progenitor cells. The nuclei of progenitors move between the apical and basal surfaces of the neuroepithelium in phase with their cell cycle, a process termed interkinetic nuclear migration (INM). In the retina of zebrafish mikre oko (mok) mutants, in which the motor protein Dynactin-1 is disrupted, interkinetic nuclei migrate more rapidly and more deeply to the basal side and more slowly to the apical side. We found that Notch signaling is predominantly activated on the apical side in both mutants and wildtype. Mutant progenitors are thus less exposed to Notch and exit the cell cycle prematurely. This leads to an overproduction of early-born retinal ganglion cells (RGCs) at the expense of later-born interneurons and glia. Our data indicate that the function of INM is to balance the exposure of progenitor nuclei to neurogenic vs. proliferative signals.
The enormous neuronal diversity present in the vertebrate central nervous system (CNS) arises from an apparently homogeneous progenitor population (Edlund and Jessell, 1999; Pearson and Doe, 2004). The mechanisms that generate the correct number and relative proportion of each cell type are incompletely understood. Which factors influence, at any given time, how many progenitors are undergoing a final neurogenic mitosis, producing two neurons, how many are dividing asymmetrically, producing one neuron and one progenitor, and how many are staying in a proliferative state, producing two new progenitors?
Neurons arise during development from a pseudostratified columnar epithelium, composed of mitotically active neuroepithelial cells. These cells have an elongated shape with cytoplasmic connections to both the apical (ventricular) and basal surfaces. Their nuclei occupy different levels within the epithelium depending on the phase of the cell cycle. Mitotic (M phase) nuclei are located in close proximity to the apical surface, while nuclei undergoing DNA synthesis (S phase) are displaced more basally. This characteristic movement of nuclei within CNS neuroepithelia was first predicted over 70 years ago based on histological observations (Sauer, 1935) and was termed interkinetic nuclear migration (INM) reviewed by (Baye and Link, 2008). Recently, the dynamics of INM have been shown to correlate with neurogenic cell divisions within the retina, such that neuroepithelia with more basal nuclear movements are biased to generate postmitotic daughters (Baye and Link, 2007). The precise developmental function of INM, however, has remained elusive.
The retina consists of six neuronal and one glial cell types, which differentiate in a stereotyped, yet overlapping, birth order (Cepko et al., 1996; Marquardt and Gruss, 2002; Poggi et al., 2005b). An extensive number of cell lineage studies have described that retinal progenitors are multipotent, giving rise to all the major neuronal and glial cell types (Holt et al., 1988; Turner and Cepko, 1987; Turner et al., 1990; Wetts and Fraser, 1988). Together, these observations have led to the proposal that retinal progenitors transit through a series of competence states in a fixed order, and during each one they are intrinsically able to generate one or a small subset of cell types (Livesey and Cepko, 2001).
In Drosophila, Notch signaling, through lateral inhibition, maintains neighboring cells in a multipotent, proliferative state, whereas downregulation of Notch is the prerequisite of neuronal differentiation (Chitnis, 1995). This mechanism appears to be largely conserved in the vertebrate retina (Jadhav et al., 2006; Nelson et al., 2006; Perron and Harris, 2000). Thus, as the progenitors produce the various cell types according to their competence state, Notch ensures at each step that a subset of the progenitors are retained for consecutive waves of neurogenesis. A missing piece in the puzzle is the mechanism that controls the dosage of Notch signaling, such that a fraction of progenitors exit the cell cycle and generate the appropriate number of each cell type.
Here we report a zebrafish mutant in which retinal progenitors exit the cell cycle prematurely and retinal neurogenesis is accelerated. The progenitor population is quickly depleted and gives rise to an imbalanced ratio of early cell fates versus late ones, reminiscent of Notch loss-of-function or a neurogenic gain-of-function. To our initial surprise, we discovered that it is caused by disruption of the motor protein Dynactin-1. We show that this mutation leads to defects in specific aspects of INM without affecting the intrinsic competence of the progenitors or their responsiveness to extrinsic cues. Furthermore, we demonstrate the existence of a Notch gradient within the neuroepithelium, with high levels in the apical domain. These results suggest that INM regulates the duration and level of exposure of progenitor nuclei to neurogenic signals.
We identified a new mutation in the zebrafish mikre oko (mok) locus (Doerre and Malicki, 2001; Wehman et al., 2005). The moks309 mutant (formerly named bugs309) has smaller eyes and a protruding lens and is first recognizable at 4 days post fertilization (4 dpf; Figures 1A, B). As reported for other mok mutant alleles, photoreceptors are absent at 5 dpf (Figures 1C, D) (Doerre and Malicki, 2001; Tsujikawa et al., 2007). Analysis of embryonic stages revealed that photoreceptors are initially produced in normal numbers, but subsequently die by apoptosis between 2.5 and 3 dpf (Figures 1E, F; Supplemental Figure S1). In addition, we noticed a marked increase in the number of cells located in the ganglion cell layer (GCL) in moks309 at 5 dpf (290±11 cells/section, compared to 204±11 in wildtype; n=4 larvae each; one central section per larva analyzed; p<0.005; Fig. 1C, D). These extra cells appear to be differentiated retinal ganglion cells (RGCs), as they express the RGC-specific marker Zn5 (neurolin/DM-GRASP) and form an optic nerve.
To test the possibility that the excess production of RGCs might be related to the loss of photoreceptors, we crossed the moks309 mutant to the Tg(Atoh7:GFP) transgenic line, in which atoh7 regulatory sequence drives a green fluorescent protein (GFP) reporter in RGC precursors and early differentiated RGC. The atoh7 (ath5) gene encodes a basic helix-loop-helix transcription factor and acts as a proneural gene for RGC fate (Kay et al., 2001; Masai et al., 2000). We analyzed moks309 retinas at 48 hours post-fertilization (hpf), before the onset of photoreceptor differentiation and when mutant embryos are morphologically undistinguishable from wildtype siblings. Already at this early stage of retinogenesis, moks309 mutants show an increased number of RGCs as identified by their nuclear position in the innermost layer and the expression of GFP (Figures 1G, H, 108.6±3.8 cells/section in mutant retinas, compared to 82.4±3.1 in wildtype, n=5 larvae each, p<0.001). It is therefore unlikely that RGC overproduction is a response to a defect in photoreceptor survival.
RGCs are the first neurons born in the retina between 26 and 36 hpf. RGC precursors express atoh7 for a short time before their last mitosis (Poggi et al., 2005a) and downregulate it as they differentiate. RNA in situ hybridization revealed that initiation of atoh7 is unaffected in mok mutants. However, atoh7 expression is retained in the mutant progenitor population after 36 hpf, when RGC production normally subsides (Supplemental Figure S2) (Masai et al., 2000). Thus, the wave of neurogenesis producing RGCs is extended in the mok mutant.
In wildtype retina, RGCs are located exclusively in the GCL, as evidenced by their expression of the POU domain transcription factors Pou4f2 (Brn3b) and Pou4f3 (Brn3c) (Xiao et al., 2005). In mok mutants, however, some of the extra RGCs are found in the inner nuclear layer (INL) (Supplemental Figure S3; Figures 2B, G). In Tg(atoh7:GFP) fish, mature RGCs, as well as cells that were competent earlier to produce RGCs (but followed a different cell fate), remain fluorescently labeled for several days after the atoh7 promoter is turned off, due to perdurance of GFP (Masai et al., 2003). In wildtype retina, GFP-labeled cells accumulate predominantly in the GCL. In moks309 mutants, by contrast, cells in the enlarged GCL, as well as cells located outside the GCL (Figures 2A, F), are strongly GFP-labelled. This finding indicates that a greater number of progenitors in mok mutants become competent to produce RGCs.
In the retina, all neuronal cell types and Müller glia cells arise from a common progenitor pool (Holt et al., 1988; Turner and Cepko, 1987; Wetts and Fraser, 1988). Therefore, we speculated that overproduction of RGCs could lead to depletion of the progenitor pool available for the genesis of later-born cells. To test this hypothesis, we investigated the presence of markers for other cell types. Müller glia are absent or strongly reduced in number in mutant retinas at 5 dpf, as revealed by immunohistochemical staining for glutamine synthetase (GS, Figures 2C, H). The same pattern was observed for bipolar cells, as shown by decreased PKC (Figures 2D, I). We confirmed this result by in situ hybridization for vsx1, which encodes a transcription factor marking bipolar cells at early stages of differentiation (Passini et al., 1997). vsx1 RNA is decreased in moks309 mutant retinas (Supplemental Figure S3). In contrast, no differences were observed in numbers of horizontal or amacrine cells, based on expression of GAD65/67, parvalbumin, and an amacrine-specific transgenic reporter (Kay et al., 2001) (Supplemental Figure S4). As reported above, photoreceptors are eliminated by apoptosis in the moks309 mutants, but their specification and number seem unaffected at earlier stages (Tsujikawa et al., 2007). Together these results show that the moks309 mutation causes a severe depletion of bipolar cells and Müller glia.
Factors that delay cell cycle exit in the retina give rise to an excess of later-born cell types, while prematurely forcing progenitor cells to become postmitotic increases the generation of RGCs (Ohnuma et al., 2002). Therefore, we suspected that the cell-fate switch observed in moks309 would be accompanied by abnormalities in the timing of cell cycle exit. To confirm this, we performed double-labeling experiments with two thymidine analogs, IdU and BrdU. We allowed IdU and BrdU incorporation for periods longer than a full cell cycle (12 hours), in order to label the total population of proliferative cells. We injected IdU into the developing embryos at 26 hpf, followed by BrdU at 38 hpf, and finally fixed the embryos at 50 hpf. Cells that are positively labeled for IdU, but not BrdU, had undergone the last S phase between 26 and 38 hpf, when RGCs are generated in wildtype. moks309 retinas show an approximate 25% increase in IdU-positive and BrdU-negative cells compared to wildtype retinas (Figure 2 I–P; 33.5±1.9 cells/section in mutant; 25.2±1.1 in wildtype, n>8 larvae each; p<0.005). This result was confirmed by similar double labelling experiments using BrdU, injected at 28 hpf, and the mitotically active cell marker PCNA (proliferating cell nuclear antigen). Embryos were fixed at 40 hpf and stained for PCNA (Supplemental Figure S5). Taken together, our data demonstrate that more cells leave the cell cycle during the first wave of neurogenesis in moks309, thus biasing the neurons to adopt the RGC fate.
To investigate the cell autonomy of the mutation, we analyzed mutant/wildtype chimeras following blastomere transplantations at the 1000-cell stage. If mok acted non-autonomously, then mutant clones in a wildtype environment should generate retinal cell types in normal proportions. Conversely, if mok functioned intrinsically in progenitor cells to promote neurogenesis, then mutant clones should produce an excess of RGCs and fewer INL cells regardless of their genetic environment. Clonal analysis showed that the latter possibility is correct. While wildtype clones in a wildtype retina give rise to neurons located in the GCL, INL, and ONL in a 3:5:2 ratio respectively, mutant clones produce these cells in a 5:3:2 ratio (Figures 3A, B; n = 1447 wildtype and 649 mutant cells counted in total). We conclude that mok acts cell-autonomously.
We further observed that wildtype clones in moks309 mutant retinas tend to be excluded from the GCL and produce fewer RGCs (2:6:2 ratio; n = 786 cells counted; Figure 3C) than when they develop in a wildtype environment (2:6:2 ratio for mutant cells in wildtype vs. 3:5:2 for wildtype cells in wildtype). Mutant clones in moks309 retinas show an increase of RGCs (4:5:1 ratio; n = 223 cells counted; Figure 3D) compared to mutant clones in wildtype. This result suggests that cell-fate switches of genotypically mutant progenitors are even more dramatically biased towards the RGC fate when they are in a wildtype environment than when they are surrounded by other mutant cells (5:3:2 vs. 4:5:1). This is probably due to intercellular feedback regulation within the developing retina. For instance, newborn RGCs limit the production of additional RGCs by secreting the GDF11, which suppresses expression of atoh7 in uncommitted progenitors (Kim et al., 2005). Consistent with this view, wildtype progenitors are more likely to generate RGCs when transplanted into an environment in which RGCs are absent, as in lakritz zebrafish mutants, which carry a null mutation in atoh7 (Poggi et al., 2005a). Our transplantation data suggest that such negative feedback attenuates the fate switch in moks309 retinas.
To gain insight into the molecular basis of the cell-fate regulation defect, we positionally cloned the mok gene. The moks309 phenotype is perfectly linked to a nonsense point mutation in Dynactin-1 (Dnct1, p150Glued), introducing a stop codon at amino acid 867 and a complete deletion of the C-terminal third of this protein. This region contains important protein-protein interaction domains responsible for the binding of Dnct1 to other dynactin subunits like Arp1 and p25 (Schroer, 2004). In Drosophila, the glued allele that carries a similar truncation in the dnct1 orthologue, is not incorporated in the dynactin complex (McGrail et al., 1995). Western-blot of whole embryo extracts showed that Dnct1 protein is weakly detected in both mutants and wildtype immediately after fertilization and for the first three days of development, indicating that it is maternally supplied (Figure 4B). In moks309 mutants, Dnct1 protein is no longer detectable at 4 dpf (Figure 4A). Consistent with these data, the expression of dnct1 RNA is ubiquitous and weak at early stages (data not shown) and is enriched at 3 dpf in most of the larval CNS, including the GCL and part of the INL (Figures 4C, D). To demonstrate that dnct1 is the gene affected in the moks309 mutation, we designed a splicing morpholino oligonucleotide (MO) to disrupt dnct1 function in wildtype embryos (Draper et al., 2001). MO-injected embryos phenocopied moks309 mutants, including thickening of the GCL and absence of differentiated photoreceptors (Supplemental Figure S6). In conclusion, we predict that moks309 is a null or a strong hypomorph of dnct1.
Dynactin mediates the interaction of the dynein motor with many, if not all, of its cargoes and allows the motor to traverse the microtubule lattice over long distances by increasing its processivity (King and Schroer, 2000). Loss of function analysis during Drosophila eye development revealed that dynactin is required for correct nuclear migration and maintenance of nuclear position within postmitotic photoreceptors (Fan and Ready, 1997; Whited et al., 2004). A similar nuclear positioning defect is observed in zebrafish photoreceptor cells carrying a different mok mutation (Tsujikawa et al., 2007). We therefore speculated that, in moks309 mutants, the INM of neuroepithelial cells could be altered and that this could in turn be responsible for the effects on cell cycle exit. To test this hypothesis, we analyzed the positions of neuroepithelial nuclei during late G2/M phase using phosphorylated histone H3 (PH3) as a marker. PH3-positive nuclei in wildtype are located close to the apical (ventricular) surface of the neural retina at all time points analyzed (24, 36 and 48 hpf; Figure 4E, and data not shown, 47/47 cells in two retinas at 48 hpf). In contrast, about 40% of PH3-positive nuclei are positioned ectopically, towards the basal side, in moks309 mutants (Figure 4F; 25/63 cells in three retinas at 48 hpf).
Time-lapse analysis of single-cell INM revealed a significant increase in the maximal-basal position and a faster-than-normal apical-to-basal movement of the nuclei in mutant cells (Supplemental Movie 1, Supplemental Movie 2; Figures 4G, H; Supplemental Table 1). Similar results were obtained in dnct1-MO injected embryos. The basal-to-apical migration velocity was also reduced in the absence of dnct1 function, although the effect in this direction was less pronounced. The net result is that interkinetic nuclei in mok mutants migrate faster to the basal surface and further basally, take longer to return, and often enter mitosis before they have reached the apical domain. In the wildtype retina, the depth of INM is an approximate predictor of a neurogenic cell division upon return to the apical side (Baye and Link, 2007a). We therefore hypothesized that this net change in INM promotes production of neurons.
An alternative hypothesis posits that cell polarity may be disrupted in moks309 mutants. Previous work indeed established that intrinsic cell polarity is essential for the relationship between nuclear position and neurogenesis (Baye and Link, 2007a). However, analysis of a series of apical and basal markers (laminin, ZO-1, aPKCζ) showed a correct localization in the mutant retinas. We also investigated the distribution of a GFP-tagged Par3 protein (Pard3), which partially overlaps with the adherens junction-associated actin bundles (Wei et al., 2004). Apical localization of Pard3-GFP was indistinguishable from wildtype (Supplemental Figure S7). These findings are consistent with a recent study showing that polarity of photoreceptors is intact in mok mutants (Tsujikawa et al., 2007). Cumulatively, these observations do not support the interpretation that apical-basal polarity defects underlie the displaced mitosis and neurogenic phenotypes in mok retina.
Notch activation is known to delay neurogenesis (Furukawa et al., 2000; Gaiano et al., 2000; Morrison et al., 2000; Scheer et al., 2001). We observed that notch1a RNA is enriched in the apical domain of the neuroepithelium in zebrafish (Fig. 5A), extending earlier reports in chick (Murciano et al., 2002). In addition, the Notch receptor ligands DeltaB and DeltaC are mostly found in the basal half of the developing neuroretina (Figures 5B, C). Reflecting the localization of its transcript, DeltaC protein is restricted to the basal half of the developing neuroretina both in wildtype and mutant embryos (Figures 5E, F). Thus, Notch receptor and Delta ligands appear to be preferentially expressed on opposite sides of the neuroepithelium.
We asked if the Notch localization gradient leads to an apical-to-basal gradient in Notch signaling. Upon binding of ligand, Notch receptor undergoes a proteolytic cleavage. The cytosolic fragment (Notch intracellular domain, NICD) translocates to the nucleus where it modulates the transcription of target genes. A specific anti-NICD antibody has been used in mouse to detect the pattern of Notch activation in the developing brain (Tokunaga et al., 2004). Using this antibody in mouse retina sections, we detected Notch activation only in nuclei located at the apical surface, consistent with data in zebrafish (Figure 5J). her4 is one target gene of the Notch/Delta pathway that is upregulated by NICD and is involved in neurogenesis (Takke et al., 1999). Using the Tg(her4.1:dRFP) transgenic line, in which a short-lived form of red fluorescent protein (dRFP) is expressed under the control of the her4 promoter (Yeo et al., 2007), we monitored her4 expression in vivo by time-lapse analysis (Figure 5D; Supplemental Movie 3; Supplemental Figure S8). In 13/15 cells moving from basal to apical dRFP fluorescence increased over the two-hour observation period, while in 11/15 cells moving apical to basal the fluorescence decreased. This suggests that Notch signaling is activated as the nuclei move into the apical compartment and downregulated in nuclei that move in the opposite direction.
The apical distribution of Notch, together with the Dynactin-driven, basally directed migration of interkinetic nuclei, offers a mechanism by which the rate of neurogenesis could be regulated. Consistent with our model, the microtubule minus-ends, marked by centrioles, are located at the apical surface (Figures 5G, H, K) and microtubules are primarily oriented parallel to the AB axis (Figure 5I). Using anti-Dnct1 antibodies, we observed cytoplasmic punctate staining with enrichment at the apical surface both in zebrafish wildtype and mouse retinal neuroepithelia, but not in moks309 mutants (Figure 5G, H, K). BBS4 protein localization is dependent on Dynein motor function and disrupted in moks309 mutants (Tsujikawa et al., 2007) (Figure 5L; Supplemental Figure S9). Thus, Dnct1 likely exerts its influence on neurogenesis by controlling INM and cell body movement within this graded signaling environment. Nuclei moving further basally appear to down-regulate Notch, while cells that remain closer to the ventricular surface during INM retain high Notch activity levels.
An alternative way by which microtuble-associated motors could affect Notch/Delta signaling in the neuroepithelial cells is by altering the distribution and therefore function of the endocytic compartment. In fact, several studies have linked endosomal sorting and endocytosis to the regulation of Notch signaling (Le Borgne, 2006; Nichols et al., 2007). In particular, both Delta activity and Notch proteolytic activation require endocytic internalization and recycling. The small GTPase Rab11 regulates formation of recycling endosomes, though which Delta passes, and unequal distribution of this protein in daughter cells modulates asymmetric cell divisions in Drosophila sensory organ formation (Emery et al., 2005). Numb is another endocytic protein that regulates Notch activity through direct binding and by targeting the receptor for endocytosis (Berdnik et al., 2002; Guo et al., 1996; McGill and McGlade, 2003). We therefore investigated whether loss of Dnct1 activity could alter the distribution of GFP-tagged versions of both Numb and Rab11. We did not detect any alteration of these endocytic components in dnct1-MO-injected embryos compared to control embryos (Supplemental Figure S10). This result indicates that Dnct1 influences Notch signaling primarily by regulating INM in progenitor cells.
To further test the hypothesis that INM is crucially involved in regulating cell cycle exit and cell-fate determination, we examined the moks309 phenotype in different genetic backgrounds. First we crossed moks309 with lakritz (lakth241)(Kay et al., 2001). The lakth241 mutation disrupts Atoh7. In its absence, neuronal progenitors stay longer in a proliferative state and give rise to an excessive number of later-born neurons including bipolar cells (Kay et al., 2001). We hypothesized that if the primary reason for the cell-fate changes observed in moks309 is premature neurogenesis, then keeping retinal progenitor cells proliferative for a longer time, as in lakth241, would rescue late cell fates. Indeed, moks309; lakth241 double mutants have an increased number of bipolar cells compared to moks309 mutants (Figures 6C and 6D). Therefore, inhibition of early neurogenesis can rescue late cell fates in moks309 mutants.
Second, we overexpressed an activated form of the Notch receptor (NICD) in a temporally controlled manner using a heat shock promoter coupled to the Gal4/UAS system. If reduced exposure of retinal neuroepithelial cells to Notch causes premature cell cycle exit in moks309, then uniformly activating Notch should overcome the effect caused by altered INM in moks309 mutants. As previously reported, NICD overexpression between 27 hpf and 42 hpf forces central retina cells to adopt a Müller glia fate (Scheer et al., 2001). We found that moks309 retinas respond similarly to NICD overexpression, as indicated by staining for the glial marker GS, which was otherwise essentially absent in moks309 (Figures 6E–6H). This result shows that increased Notch signaling promotes gliogenesis both in wildtype and moks309 mutants. Taken together, these data demonstrate that mutant progenitor cells retain their competence to generate later-born neuronal types and glia.
INM requires factors that anchor the nucleus to motor proteins. Good candidates for this role are the KASH-domain protein family members. These proteins are localized to the outer nuclear envelope and mediate the interaction between the nucleus and the cytoskeleton acting in nuclear positioning in a variety of eukaryotic cells from fungi to mammals (Starr and Fischer, 2005). We therefore hypothesized that knockdown of KASH proteins would disrupt the coupling of the nucleus to the Dynein motor, perturbing INM and affecting neurogenesis similar to mok.
To test this hypothesis, we targeted the KASH-containing protein Syne2a. Interfering with Syne2a function at late stages of retina development results in photoreceptor nuclear displacement (Tsujikawa et al., 2007). First we globally reduced the levels of syne2a using a splice blocking MO. While high doses of syne2a-MO resulted in grossly malformed embryos (79/85 larvae), low doses had milder effects resulting in larvae with only slightly smaller eyes with normal lamination by 5 dpf (67/98 larvae). We detected nevertheless a dramatic reduction in the number of GS-positive Müller glia cells (37.7±1.2 cells/section, in control retinas, 21.2±1.2 cells/section in syne2a-MO injected retinas; n=4 in each group, p<0.001, Figure 7A, B), similar to the phenotype observed in moks309 retinas.
To separate the global effects of syne2a knockdown on embryonic development from specific effects in the developing retina, we overexpressed a dominant-negative, GFP-tagged Syne2a KASH domain in a mosaic fashion, under control of a heat-shock promoter (Tsujikawa et al., 2007). Plasmid DNA was injected at the 2–4 cell stage, and heat shock was applied at 23 hpf. KASH-expressing clones preferentially generated RGC neurons at the expense of INL and ONL cells (Figure 7C–E). These results are not the consequence of reduced INL or ONL cell survival, because similar overexpression experiments after 48 hpf did not cause immediate cell loss (Tsujikawa et al., 2007). These results reinforce the notion that interfering with INM, either through the motor protein complex or the nuclear anchor, perturbs cell cycle exit and neurogenesis.
We show here that a mutation in Dnct1, a microtubule-motor associated protein, perturbs INM in a selective fashion and results in an overproduction of early-born neurons in the zebrafish retina. The progenitor nuclei move more quickly and more deeply in the basal direction and more slowly apically. We further established that anti-neurogenic Notch signals are enriched on the apical side of the neuroepithelium in both mutants and wildtype. Combined with previous observations that progenitors whose nuclei migrate deep are more likely to produce postmitotic neuronal daughters following their return to the apical side (Baye and Link, 2007a), our studies suggest a mechanism by which INM co-operates with an apical-basal Notch gradient to select progenitors for cell cycle exit and apportion cell fates (Fig. 7F).
The phenotype of the mok mutant is reminiscent of manipulations of cell fate determinants, such as overexpression of bHLH proneural genes. We therefore asked if the mok mutation altered the intrinsic competence of progenitors. The bHLH transcription factor Atoh7 functions as a proneural factor to set neurogenic competency of early retinal cell types and is essential for RGC fate. Disruption of atoh7 eliminates RGCs and increases the number of progenitors that remain in the cell cycle (Brown et al., 2001; Kay et al., 2001; Wang et al., 2001). As a consequence of this enlarged progenitor pool, bipolar cells and glia are increased in number in the zebrafish lak (atoh7) mutant. Conversely, overexpression of atoh7 results in an excess of RGCs, resembling the mok phenotype (Hatakeyama and Kageyama, 2004; Vetter and Brown, 2001; Yan et al., 2005). In lak; mok double mutants, glia and bipolar neurons, which are severely reduced in mok single mutants, develop similarly to lak single mutants, demonstrating that mok mutant progenitors retain their potential to generate later-born neurons. Photoreceptors, which develop normally in lak single mutants, however, are absent in the double mutants, as they are in mok single mutants, due to a related but independent function of Dnct1 in photoreceptor nuclear positioning (Tsujikawa et al., 2007). In conclusion, Dnct1 does not affect the neurogenic competence of retinal progenitor cells.
Inhibition of Notch (Austin et al., 1995) or disruption of GDF11 (Kim et al., 2005) mimic some aspects of the mok phenotype. Notch prevents progenitors from leaving the cell cycle and from differentiating prematurely. Its activation during the time progenitors are competent to produce RGCs leads to a depletion of this cell type. GDF11, on the other hand, is secreted by differentiated RGCs to negatively regulate the number of new RGCs produced by suppressing atoh7. As a consequence, progenitors are more likely to generate RGCs when transplanted into an environment in which RGCs are absent, as in lak mutants (Poggi et al., 2005a). We made a complementary observation by transplanting wildtype cells into mok mutants. These clones tend to form fewer RGCs, suggesting that the negative feedback signals are intact in the mutant environment. Moreover, we show that constitutive activation of Notch in mok mutants blocks RGC production and leads to an overproduction of glia, as it does in wildtype. Together, these results demonstrate that mok mutant cells are still able to produce, and respond to, extrinsic regulators of cell fate.
A Notch gradient along the apical-basal axis of the neuroepithelium is likely to play a key role in neurogenesis. Notch mRNA is increased on the apical side, whereas Delta mRNA and protein are enriched basally. This results in a gradient of Notch transcriptional activity, as demonstrated by the higher concentration of Notch ICD in the apical domains and the increased her4 expression in cells whose nuclei move into the high-Notch environment. We have found no evidence that Notch signaling, neuroepithelial polarity, or the Notch gradient itself, are altered in mok mutants. Distribution of the endocytic pathway components Rab11 and Numb, which in other contexts regulate Notch, is unaltered. Moreover, an independent manipulation of the Dynein/Dynactin-dependent component of INM (disruption of the nuclear anchor protein Syne2a) perturbs cell fate decisions similarly to the mok mutation.
Between mitoses, a progenitor nucleus moves twice through a Notch spatial gradient. If the nucleus stays close to the apical side it will encounter high Notch levels throughout the cell cycle, and both of its daughters are likely to remain proliferative. On the other hand, if the nucleus is translocated more basally, Notch activity is reduced, predisposing the progenitor to produce one or two daughter neurons during its subsequent mitosis. It follows that, in mok mutants, the balance of neurogenic vs. proliferative divisions is shifted by decreasing exposure to Notch across the progenitor population. Given that both INM and Notch signaling compartments are ubiquitous features of CNS neuroepithelia (Frade, 2002) as well as non-neuronal epithelia (Bort et al., 2006), it seems likely that this mechanism is widely employed during embryonic development, growth, and regeneration.
Retinal sections were stained using standard protocols (Kay et al., 2001). E12 CD1 mice embryos (gift from Florence Lee) were fixed in 4% PFA and processed for cryosectioning according to standard protocols. The full list of primary and secondary antibody is given in Supplemental Information. Staining for gamma-tubulin required the treatment of the samples for 5 min in acetone at −20°C prior to blocking. Fixation and staining for alpha-˜tubulin was performed as described (Wehman et al., 2007). Apoptosis was detected by whole-mount TUNEL assay using the ApoTag kit (Chemicon). Whole-mount in situ hybridization was performed according to standard protocols and a list of antisense probes is reported in Supplemetal Information.
Cell proliferation was assayed by BrdU/IdU incorporation as previously described (Burns and Kuan, 2005; Kay et al., 2001) with the following modification. Embryos were injected with 10 mM BrdU or IdU solutions into the yolk and grown until fixation.
Chimeric embryos were generated using standard methods (Ho and Kane, 1990). Donor embryos were Tg(h2afv:GFP)kca6 to easily identify transplanted clones. Both donor embryos and chimeras were allowed to develop until 5 dpf to identify mutants. Chimeric larvae were then fixed and processed for immunohistochemistry.
All injections were performed at the 1-cell stage, unless specified otherwise. dnct1-MO were injected at a concentration of 20 mM. Sequence: 5’-ctgagggacggccggtctgtggagg. syne2a-MO were injected at 500 mM (high dose) or 50mM (low dose). The sequence has been described previously (Tsujikawa et al., 2007). Expression of Syne2a-KASH domain Pard3-GFP, EGFP-Numb, and EGFP-Rab11 was carried out as described (Geldmacher-Voss et al., 2003; Muto et al., 2006; Reugels et al., 2006; Tsujikawa et al., 2007). The pard3-EGFP contruct was injected at 2–4 cell stages to achieve sparse expression. EGFP-Numb and EGFP-rab11 where subcloned in the Tol2-based vector and injected with transposase RNA to achieve uniform expression (Kawakami, 2004).
To label nuclei of retinal progenitor cells, plasmid DNA encoding the Histone H2B-GFP fusion protein was microinjected into 1–4 cell stage embryos to label nuclei in a mosaic fashion throughout the embryo (Koster and Fraser, 2001; Meng et al., 1999). Embryos were derived from incrosses of heterozygous moks309 pairs. All embryos were grown in 0.003% 1-phenyl-2-thiourea (PTU) to block pigmentation. At 22 hpf, small pieces of tail tissue were used to PCR genotype either wildtype or mutant embryos prior to imaging. Following imaging, all embryos were allowed to develop and scored for their phenotypes in order to validate genotyping results.
At 26 hpf, labeled embryos were anesthetized with 0.05% Tricane in 0.003% PTU and embedded in 1.0% low-temperature melting agarose. Embryos were placed in a glass bottom culture dish and oriented so that the eye was facing up. GFP-labeled nuclei and dRFP labelled cells were imaged on a Nikon C1 confocal microscope. Transmitted light images were also collected during the time-lapse to enable accurate measurement of apical and basal surfaces during nuclear movements. Optical z-sections were collected at 2 µm steps every 12 minutes for 30–48 hours. These parameters were sufficient to capture M-phase for each cell while reducing photo-bleaching during the extended time course. Temperature was maintained throughout all experiments at 28.5°C using a stage incubator. Nuclear migration velocities were measured as described (Baye and Link, 2007b). For details, see Supplemental Information.
We thank D.Y. Stainier, S. Guo, J. Malicki, J. Lewis, I. Masai, P.A. Raymond, B. D. Perkins, A.B. Chitnis, N. Katsanis, K.T. Vaughan, R.B. Vallee, H.A. Ingraham for fish strains, antibodies, in situ probes and mouse embryos. We thank Jeremy Reiter for critically reading the manuscript. F.D.B. was supported by a Human Frontier Science Program long-term fellowship. A.M.W. was supported by an American Association of University Women dissertation fellowship. This work was supported by NIH grants EY013855 (HB), EY012406 (HB), EY01467 (BAL) and the March of Dimes Foundation (HB).
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