Characterization of primary cilia during the differentiation of retinal ganglion cells in the zebrafish
© Lepanto et al. 2016
Received: 23 December 2015
Accepted: 29 March 2016
Published: 6 April 2016
Retinal ganglion cell (RGC) differentiation in vivo is a highly stereotyped process, likely resulting from the interaction of cell type-specific transcription factors and tissue-derived signaling factors. The primary cilium, as a signaling hub in the cell, may have a role during this process but its presence and localization during RGC generation, and its contribution to the process of cell differentiation, have not been previously assessed in vivo.
In this work we analyzed the distribution of primary cilia in vivo using laser scanning confocal microscopy, as well as their main ultrastructural features by transmission electron microscopy, in the early stages of retinal histogenesis in the zebrafish, around the time of RGC generation and initial differentiation. In addition, we knocked-down ift88 and elipsa, two genes with an essential role in cilia generation and maintenance, a treatment that caused a general reduction in organelle size. The effect on retinal development and RGC differentiation was assessed by confocal microscopy of transgenic or immunolabeled embryos.
Our results show that retinal neuroepithelial cells have an apically-localized primary cilium usually protruding from the apical membrane. We also found a small proportion of sub-apical cilia, before and during the neurogenic period. This organelle was also present in an apical position in neuroblasts during apical process retraction and dendritogenesis, although between these stages cilia appeared highly dynamic regarding both presence and position. Disruption of cilia caused a decrease in the proliferation of retinal progenitors and a reduction of neural retina volume. In addition, retinal histogenesis was globally delayed albeit RGC layer formation was preferentially reduced with respect to the amacrine and photoreceptor cell layers.
These results indicate that primary cilia exhibit a highly dynamic behavior during early retinal differentiation, and that they are required for the proliferation and survival of retinal progenitors, as well as for neuronal generation, particularly of RGCs.
KeywordsRetina Cilia Retinal ganglion cell Neurogenesis
Developmental processes are carried out based on a complex interaction between information inherited from the parent cell and time/tissue-specific environmental cues. The vertebrate retina is one of the most organized tissues in the body. To achieve this unique organization, dividing neuroepithelial cells must give rise to differentiating neuroblasts in a highly controlled and orderly fashion. Even though retinal ganglion cells (RGCs) are the first neuroblasts to be born, how these cells arise and differentiate into the correct neuronal type, with its corresponding morphology and connections, is still not completely understood.
Both cell type-specific expression of transcription factors and tissue-derived positional and trophic factors are likely to interact to achieve a mature and fully functional retina. Several signals from the environment have been shown to influence cell position and differentiation. For example, Notch signaling in relation to interkinetic nuclear migration has been linked to cell cycle withdrawal, the first step in the differentiation process . The basal lamina of the neuroepithelium (the “inner limiting membrane”) has also been shown to play a critical role as RGC axon extension and orientation depends on the presence of Laminin . Another contributing signaling molecule is Sonic Hedgehog (Shh), which is needed for spreading the wave of RGC and amacrine cell differentiation across the retina, as well as later on for photoreceptor cell differentiation [3–7]. Importantly, during RGC differentiation in vivo, neuroepithelial polarity is transiently maintained: it has been shown that polarity determinants are apically-positioned during the initial stages of differentiation, while in vitro these determinants show an erratic behavior . Therefore, the tissue impinges constraints on the inherited differentiation program, guiding it to achieve the mature functional structure.
In recent years it has been shown that one of the main signaling hubs in cells, which has a critical role during development, is the primary cilium. Primary cilia are microtubule-based organelles that extend from a modified centriole, the basal body, protruding as an extension of the plasma membrane. Cilia are enriched in moieties required for sensing and transducing a number of signaling cascades that have been shown to rely on this particular cellular structure, including Wnt and Shh . These findings, coupled with the ubiquitous presence of primary cilia in different cell types, explain why defects in the formation, maintenance and function of this organelle result in a range of clinical manifestations that have been grouped under the term ciliopathies [10, 11]. Importantly, central nervous system associated phenotypes, including structural defects, mental retardation and retinal degeneration, are hallmark phenotypes of several ciliopathies .
Primary cilia have been studied in the context of neuronal differentiation in different regions of the central nervous system. It has been shown for example that this organelle plays a role in progenitor cell proliferation in the cerebellum [13, 14], proliferation of progenitors and integration of neurons in the hippocampus [15, 16], and in the migration of neuroblasts in the mouse developing cortex [17, 18]. Early electron microscopy studies have shown that both neural tube and retinal neuroepithelial cells have an apically localized primary cilium, and that RGC neuroblasts of the mouse developing retina have a primary cilium that also displays a polarized localization, being positioned at the tip of the retracting apical process [19, 20]. Therefore, it is possible that the primary cilium is playing a role in RGC differentiation.
In this work we performed an in-depth characterization of the presence and localization of cilia during the differentiation of RGCs in the zebrafish retina combining electron and confocal microscopy with time-lapse video microscopy in live embryos. As an in vivo marker for cilia, we used a zebrafish transgenic line expressing EGFP fused to the carboxy-terminus of the small GTPase Arl13b (Arl13b-GFP; ). Arl13b, which belongs to the Arl/Arf family of GTPases involved in microtubule dynamics and membrane traffic, is specifically localized to the ciliary axoneme and is an essential protein for cilia maintenance in zebrafish and mice [22, 23]. In addition, we evaluated retinal development in conditions where cilia integrity was compromised. Our data show that RGCs primary cilia are highly dynamic organelles, changing in size and position during the differentiation process. The double knockdown of ift88 and elipsa, two genes important for cilia formation and maintenance, shows that this organelle plays a role both during progenitor cell proliferation and maintenance, as well as during neurogenesis. Thus, our data provide important information that will help in achieving a more complete understanding of the role of primary cilia in RGC differentiation.
Fish breeding and care
Zebrafish were maintained and bred in a stand-alone system (Tecniplast), with controlled temperature (28 °C), conductivity (500 μS/cm2) and pH (7.5), under live and pellet dietary regime. Embryos were raised at temperatures ranging from 28.5 to 32 °C and staged in hours post-fertilization (hpf) according to Kimmel and collaborators . We used wild-type (SAT; ) and different previously established transgenic lines in this work: Tg(actb2:Arl13b-GFP)hsc5 (Arl13b-GFP, kindly provided by B. Ciruna; ), Tg(atoh7:gap43-EGFP)cu1 (atoh7:gap-GFP; ), Tg(atoh7:gap43-RFP)cu2 (atoh7:gap-RFP; ), SoFa1 (atoh7:gap-RFP/ptf1a:cytGFP/crx:gap-CFP; ). In addition, we generated a double transgenic line crossing atoh7:gap-RFP and Arl13b-GFP. All the manipulations were carried out following the approved local regulations (CEUA-Institut Pasteur de Montevideo, and CNEA).
The morpholino oligomers (MOs) used in this study were obtained from Gene Tools (Philomath, USA) and included those previously used to target zebrafish elipsa and ift88 translational initiation: elipsa-ATG (GGCTACCGATTCGTTCATGGCATCA; ) and ift88-ATG (GCCTTATTAAACAGAAATACTCCCA; IFT88 MO3, ). We also used newly designed morpholinos to target the splicing of ift88 and elipsa mRNA: ift88-SP (AACAGCAGATGCAAAATGACTCACT) which targets the exon 3 - intron 3 boundary; elipsa-SP (CTGTTTTAATAACTCACCTCGCTGA) which targets the exon 1 - intron 1 boundary. All MOs were injected in the yolk of 1–4 cell-stage embryos, at a maximum volume of 4 nL. As control, we used matching doses of a standard MO (CCTCTTACCTCAGTTACAATTTATA) from Gene Tools (Philomath, USA). When considered necessary, we co-injected a double amount per embryo of the standard anti-p53 MO .
To test the effectiveness of splice-blocking morpholinos we performed RT-PCR (primers sequences are available upon request). Total RNA was extracted from 30 morphant or wild-type embryos using TRIzol reagent (Invitrogen) and cDNA was prepared using the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen).
Blastula stage embryos were used for transplantation experiments. Cells from the donor embryos were transplanted into the animal pole of hosts, following standard procedures. After transplantation embryos were incubated in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4) plus 10 mM Hepes pH 7.4, 0.00005 % methylene blue and Penicillin/Streptomycin (Sigma) at 32 °C.
Embryos were grown in 0.003 % phenylthiourea (Sigma) from 10 hpf onwards to delay pigmentation, and fixed overnight at 4 °C, by immersion in 4 % paraformaldehyde in phosphate saline buffer (PBS; pH 7.4).
For whole-mount immunostaining all subsequent washes were performed in PBS containing 1 % Triton X-100. Further permeability was achieved by incubating the embryos in 0.25 % trypsin-EDTA for 10–15 min at 0 °C. Blocking was for 30 min in 1 % bovine serum albumin (BSA), 1 % Triton X-100 in PBS. The primary antibodies, diluted in the blocking solution, were used as follows: zn8 (ZIRC, Oregon), 1/100; anti-activated Caspase 3 (AbCam), 1/500; anti-histone H3 pSer10 (Santa Cruz), 1/300, anti-acetylated tubulin (Sigma), 1/750; anti-γ tubulin (Sigma), 1/500. The secondary antibodies used were: anti-rabbit IgG-TRITC (Life Technologies), 1/1000; anti-mouse IgG-Alexa 488 (Life Technologies), 1/1000; anti-mouse IgG1-Alexa 488 (Life Technologies), 1/1000; anti-mouse IgG2b-Alexa 568 (Life Technologies), 1/1000. When necessary, TRITC-conjugated phalloidin (Sigma) was mixed with the secondary antibody. Nuclei were fluorescently counterstained with methyl green . All antibody incubations were performed overnight at 4 °C. Embryos were mounted in 70 % glycerol in 20 mM Tris buffer (pH 8.0) and stored at 4 °C or −20 °C.
Five day-old embryos were fixed as described above, washed in PBS and cryoprotected in 30 % sucrose in PBS overnight at 4 °C. They were then embedded in OCT (Tissue-Tek) and quickly frozen in liquid N2. Transverse cryosections (10 μm) were made on a Reichert-Jung Cryocut E cryostat and adhered to gelatin subbed slides. Mounting was made using 70 % glycerol in 20 mM Tris buffer (pH 8.0).
Observation of whole embryos or cryosections was performed using a Leica TCS-SP5 (for all in vivo and some fixed material imaging) or a Zeiss LSM800 (for some fixed material imaging) laser confocal microscopes, with 63x 1.4 NA oil immersion or 20x 0.7 NA and 63x 1.3 NA glycerol:water (80:20) or water immersion objectives.
In vivo confocal microscopy
Around 30 hpf embryos were selected, anesthetized using 0.04 mg/mL MS222 (Sigma) and mounted in 0.8 % low melting-point agarose (Sigma) over n° 0 glass bottom dishes (MaTek). After agarose solidification and during overnight image acquisitions, embryos were kept in Ringer’s solution (116 mM NaCl, 2.9 mM KCl, 1.8 mM CaCl2, 5 mM HEPES pH 7.2) with 0.04 mg/mL MS222. Live acquisitions were made using a Leica TCS-SP5 laser confocal microscope with a 20x 0.7 NA objective and glycerol:water (80:20) immersion medium. Stacks around 60 μm-thick were acquired in bidirectional mode, at 1 μm spacing and 512 × 512 pixel resolution every 10 or 15 min (the acquisition time for each embryo was approximately 45 s).
Transmission electron microscopy
Embryos were fixed at 26, 35 and 48 hpf by immersion in fixative solution (4 % paraformaldehyde, 2.5 % glutaraldehyde in PBS, pH 7.2–7.4). Then the head was dissected and incubated overnight at 4 °C. The fixed material was washed in PBS, post-fixed in 1 % osmium tetroxide in distilled water for 1 h, and washed in distilled water. Samples were dehydrated through a graded (25, 50, 75, 95, 100 %) ethanol-water series, transferred to acetone for 2 × 20 min, and infiltrated with araldite resin through a series of steps (2:1 acetone:araldite for 30 min, 1:1 acetone:araldite for 30 min, 1:2 acetone:araldite for 30 min, 100 % araldite overnight at 4 °C). On the next day, the material was transferred into flat-embedding molds with freshly made araldite resin and oriented as desired. The embedded samples were cured at 60 °C for 48 h. Blocks were semi-thin sectioned at 500 nm using a RMC MT-X ultramicrotome, stained with boraxic methylene blue and examined in a light microscope. Once the area of interest was reached, ultrathin 70 nm sections were obtained and mounted on formvar-coated copper grids. Sections were stained for 2 h in 2 % aqueous uranyl acetate followed by staining in Reynold’s lead citrate for 10 min. Observation and acquisition was performed using a Jeol JEM 1010 transmission electron microscope operated at 100 kV, equipped with a Hamamatsu C4742-95 digital camera.
Images were analyzed using Fiji . Primary cilia length in Kupffer’s vesicle was measured manually in maximum intensity z-projections of the confocal stacks. Volume measurements (whole retina and zn8-positive region) were performed using intensity-based thresholding aided by manual selection. After that, the volume was obtained using 3D Roi Manager and 3D Objects Counter plugins [32, 33]. Cell counting (pH3 and activated Caspase 3-positive cells) was also done manually on the confocal stacks with the aid of the MTrackJ plugin . For the fluorescence profile analysis of the retina in SoFa transgenic embryos, confocal planes from a 10 μm-deep stack were projected using intensity average and then a 100 μm-long and 20 μm-wide line selection was drawn across the retina. The fluorescence intensity along this line was measured for each channel using the Intensity Toolset from Imperial College of London FILM facility (http://www.imperial.ac.uk/medicine/facility-for-imaging-by-light-microscopy/equipment/software---fiji/). In some cases, bleed-through from the GFP signal into the CFP and RFP channel was detected. In these cases we processed the images as follows: 1) we calculated a normalization factor for the GFP channel = maximum GFP intensity / (maximum RFP intensity due to bleed-through – (minus) average RFP intensity in RGC layer); 2) we divided the GFP channel over this ratio generating a new image that corresponded to the bleed-through signal, 3) we subtracted this new image from the RFP channel. The same procedure was implemented for the CFP channel when necessary. In order to quantify the integrated fluorescence intensity from each layer, the boundaries of the region below the fluorescence curve corresponding to that layer were set to the point corresponding to the 50 % of the peak intensity (see Fig. 11b). The percentage of the signal corresponding to each cell type was calculated as the ratio between the area under the plot corresponding to each cell type layer and the area under the plot corresponding to that channel as a whole.
In all cases, box plots represent the 25–75 % quartiles, the horizontal lines represent the median, and the short horizontal lines are the minimal and maximal values. All statistical analyses were performed as previously described [35, 36] using Past software . As a routine, the datasets were checked for normality using Shapiro-Wilk normality test and for homogeneity of variances using Levene’s test. In the case of normal and homogeneous data, we performed a Student’s t-test for media comparison. If homogeneity requirement was not met, we performed a rank transformation . In the case of non-normal data, we performed a Mann–Whitney test.
Characterization of primary cilia in early stages of retina differentiation
Likewise, our TEM analysis showed that in 35 hpf retinas most of the primary cilia are localized to the apical surface of the neuroepithelium, extending into the subretinal space (Fig. 1e-h). These cilia displayed different angles with respect to the neuroepithelial surface, albeit no correlation was evident between this angle and the region of the retina being examined. At the ultrastructural level, all these apical cilia had a 9 + 0 axoneme (Fig. 1i) and most (69/94) were immerse in a deep ciliary pocket (Fig. 1e), although in a few cases this structure was either incomplete (7/94; Fig. 1f) or absent (18/94; Fig. 1g-h). Some of the observed cilia projected into invaginations of retinal pigment epithelium (RPE) cells (Fig. 1h). Figure 1j presents a quantification of morphometric parameters of these apical cilia. Less frequently, we also observed basal bodies in close association with the plasma membrane but without a visible axoneme (Fig. 1k). In order to ascertain if these features were also present in a completely undifferentiated retinal neuroepithelium, we also analyzed apical primary cilia ultrastructure in 26 hpf embryos using TEM. Interestingly, we found that these cilia were morphologically similar to those from 35 hpf embryos, except for their length: cilia were significantly longer in 26 hpf retinas (Fig. 1l).
Cilia dynamics in early neuroblasts
A primary cilium usually re-appeared during the retraction of the apical process, localizing to its tip, and remaining visible until the end of the retraction (Fig. 3b; Movie in Additional file 2; Fig. 4, denoted as ■). The position along the neuroepithelium where these primary cilia became evident again was variable among different cells, but most frequently we first detected the cilium in the central region of the neuroepithelium (i.e.,: halfway in the retraction process; Fig. 3c). The time of appearance of the primary cilium relative to the initiation of apical process retraction was also highly variable, although in most cases it happened within the following 1.5 h (Fig. 3d). Given the possibility that resolution constraints of our time-lapse experiments could hinder the visualization of cilia at early stages of retraction, we also analyzed the process by TEM. Cilia, both with and without a ciliary pocket, were observed on retracting apical processes either in close proximity to the apical surface of the neuroepithelium or in more basal positions, always pointing towards the apical surface (Fig. 3e). Importantly, this analysis allowed us to confirm the presence of apically localized primary cilia in retracting processes and showed that, at least in some cells, a primary cilium is present during the early stages of apical retraction. A summary of the behavior of all cells analyzed by time-lapse microscopy is shown in Fig. 4.
Characterization of cilia in differentiating RGCs
If we consider only those cells that showed a primary cilium after completion of apical process retraction and/or during the initial steps of dendrite formation, the primary cilium remained in the apical side of the cell during the imaging period in half of them (Fig. 4, denoted as “A”). Surprisingly, in the other half, primary cilia transiently lost their apical localization, occupying apparently random positions around the cell body, and rapidly moving from one position to another (Fig. 5e; Movie in Additional file 5; Fig. 4, denoted as “NA”). In TEM studies of 35 hpf embryos, fully formed cilia were rarely found in basally localized neuroblasts without an apical process. However, short cilia were eventually visualized, which exhibited different localizations and orientations around the cell (Fig. 5f and g). Interestingly, our time-lapse experiments showed that in cells bearing a primary cilium at the onset of dendritogenesis, the cilium was invariably localized to the base of the protrusion that finally branched to form the dendrites. This was the case even when cilia moved around the cell body before eventually reaching that position (Fig. 5e, Movie in Additional file 5).
Cilia dysfunction leads to early retinal differentiation defects
We reasoned that the decrease in retina and ganglion cell layer volume, like the apparent delay in RGC differentiation, could be due either to reduced cell proliferation, to increased cell death, or a combination of both. To discriminate between these possibilities we performed whole-mount immunofluorescence against phosphorylated Histone H3 (pH3), present in mitotic cells from late prophase to the end of telophase, and for activated Caspase 3, indicative of early stages of apoptosis. We then quantified pH3 positive cells in the ventro-nasal region of the retina and the total number of activated Caspase 3-positive cells per eye, in embryos of different developmental stages. Regarding cell proliferation, while we did not observe differences at 24 hpf, our results showed a significant decrease in mitotic index in morphant embryos at 36 hpf (Fig. 10e). In the case of cell death, we observed an increase in apoptotic nuclei at 48 hpf while not at an earlier stage (36 hpf; Fig. 10f).
To further confirm the possibility that RGC generation and/or differentiation is preferentially reduced after primary cilia impairment, we analyzed the extension of the inner plexiform layer (IPL) through whole-mount staining with TRITC-conjugated phalloidin (Fig. 11d). Embryos injected with control morpholino and fixed at 48 hpf presented a thin IPL layer only in the ventro-nasal region of the retina, while at 60 hpf the IPL was thicker and extended throughout the whole neural retina. Embryos injected with elipsa-SP/ift88-SP MOs and fixed at 48 hpf had a smaller patch of IPL restricted to the region adjacent to the optic nerve exit, where the first RGCs differentiate (Fig. 11d, arrowhead). At 60 hpf, the IPL of morphant embryos had extended throughout the retina, but appeared thinner and more disorganized than in control embryos (Fig. 11d, insets).
Dynamic apical cilia in the early neurogenic retinal neuroepithelium
Pioneering electron microscopy studies showed, many decades ago, the presence and apparent dynamics of primary cilia in the undifferentiated and differentiating neuroepithelium, both in the neural tube and the retina [19, 20]. In recent years, the finding that primary cilia act as cellular “antennae” has renewed the interest of developmental neurobiologists in understanding the possible roles of these organelles in neurogenesis and neuronal differentiation. Here, we sought to characterize the behavior and possible functions of primary cilia in these processes, starting by assessing their presence and localization in the embryonic zebrafish retina before and around the initial stages of neurogenesis. It must be noted that because of the morphological features of primary cilia, particularly their small size and the fact that there is only one per cell, added to their functional properties (cilia are constantly disassembled and reassembled along the cell cycle), it is simply not possible to assure that a cell that does not display a detectable cilium is actually a non-ciliated cell. We observed the presence of numerous primary cilia in the early retina, both before and shortly after the onset of neurogenesis, indicating that probably most retinal neuroepithelial cells are ciliated. These cilia mostly localized apically, and were pointing towards the sub-retinal space. In addition, most of them presented a deep ciliary pocket, a structure that has been documented in different cell types both in vitro and in vivo (reviewed in ). Some early studies suggested a link between the ciliary pocket and different stages of ciliation, as well as the possibility of different pathways in cilia formation. More recently however, it has been shown that the ciliary pocket is a site of active endocytosis [44, 45]. Interestingly, its presence, size and morphology were extremely variable among retinal neuroepithelial cells, which may therefore suggest differences in their cellular activity.
The accumulation of centrosomes at the apical side of different neuroepithelia has been extensively documented, and even used as a cell polarity marker [39, 46]. In the present work, we observed that many of these apical centrosomes are actually primary cilia basal bodies. However, we also found a few cases of apically localized centrosomes that were not nucleating an axoneme (although they usually appeared docked at the plasma membrane), as well as short primary cilia inside cytoplasmic vesicles. These possibly represented different stages of cilia formation , even though we cannot distinguish if these cells are cycling progenitors or neuroblasts. Our in vivo experiments showed that both situations are possible: while there is a cilia cycle related to the cell cycle (i.e.,: in progenitor cells), cilia in postmitotic neuroblasts may also be highly dynamic. We found that neuroepithelial cells committed to exit the cell cycle (evidenced by the expression of atoh7:gap-RFP), presented a primary cilium until a short time before mitosis. Accordingly, studies in chick neural tube have shown that primary cilia are lost during mid-G2 in order for the centrosome to engage in mitosis . Regarding postmitotic cells, our in vivo experiments interestingly showed that some of these cells had visible primary cilia while still attached to the apical border, although these organelles were transiently lost in many cells around the period of detachment (see below).
We also found primary cilia that, albeit being localized to the apical region of neuroepithelial cells, emerged from a basal position with respect to adherens junctions. These cilia may be translocated or re-formed at the basolateral surface and the cilia that were observed inside cytoplasmic vesicles may correspond to intermediate stages of any of these processes. This observation is highly reminiscent of that reported in the mouse cortex where cells committed to delamination in the embryonic telencephalon present basolaterally localized primary cilia, the proportion of which increases at the onset of neurogenesis . In the zebrafish retina, we found the proportion of basolateral primary cilia to be the same at 26 and 35 hpf (stages before and after neurogenesis initiation, respectively), whereas ciliary length showed a decrease at the latter stage. Thus, cilia shortening might present a stronger correlation with neurogenesis in the zebrafish retina than basolateral cilia localization.
Cilia dynamics in differentiating RGCs
The appearance of the primary cilium during apical process retraction was highly variable both in time and position across the neuroepithelium, as was the period it remained visible (see Fig. 4). However, in all cells, the cilium remained apical throughout retraction, in accordance to previous work that reported an apically localized centrosome in RGCs all through retraction  and at the initial stages of dendrite formation . Consistently, apically-localized primary cilia in differentiating RGCs had also been reported in early electron microscopy studies of the mouse retina . Surprisingly, however, we also observed a highly dynamic primary cilium regarding localization around the cell body surface, from the end of retraction to just before the initiation of dendrite formation. These movements are fast (can be observed in 10 min intervals), and might be related to the necessary cellular rearrangements that occur during the period between axon and dendrite formation. Interestingly, these movements ceased at the onset of dendritogenesis, with the primary cilium localizing to the base of the growing dendrites. Similar movements of the centrosome of differentiating RGCs, although in a more prolonged period of time, were observed when knocking-down Laminin α1, an essential signal for neuronal orientation in the zebrafish retina . In tangentially migrating cortical interneurons, highly dynamic primary cilia that change in length and position during migration have been shown to be involved in sensing environmental Shh, and possibly other signaling molecules [17, 18]. Cortical neuroblasts form primary cilia postnatally, at a stage when their migration has ended . Likewise, dentate granule cells born in adult mice, form primary cilia after reaching their final positions in the hippocampus, around the time of dendrite formation and synaptic connection establishment . It has been shown that in these systems the primary cilium, which localizes to the base of the apical dendrite, is necessary for dendrite refinement and synapse formation [16, 52].
RGCs are preferentially affected by cilia dysfunction during retinal development
The particular localization and dynamics of cilia in differentiating RGCs made us wonder what roles the organelle might have in the generation or differentiation of these neurons. As a first approach to evaluate the physiological role of cilia in the developing retina, we opted for a knockdown strategy using morpholino oligomers. IFT88 and Elipsa are two ciliary proteins that have been shown to directly interact and to be essential for intraflagellar transport in the zebrafish, whose mutants and knockdowns have been reported to give clear and reproducible “ciliary phenotypes” [27, 28]. In our hands, the best results were obtained with a combination of MOs against these two genes, as we reasoned that through this approach we were going to maximize the chances of observing a ciliary phenotype with relatively low doses of individual MOs, avoiding cilia-independent alterations. In addition, we used splice-blocking MOs to avoid affecting early developmental processes. This combination of MOs effectively reduced cilia number and length in different organs and tissues, and gave a clearer ciliary phenotype than the individual MOs used at higher doses.
Previous work by others aimed at characterizing the in vivo functional role of genes involved in ciliogenesis in the zebrafish, such as elipsa, ift88, ift57 or ift172 [27, 28, 53, 54], showed, by analyzing single-mutants for these genes, that the major phenotype in the retina was the cell-autonomous progressive loss of photoreceptor cells, evident from 3 dpf onwards. Here, we also observed very little or no early retinal phenotype after the individual injection of MOs to ift88 and elipsa. Double-morphants, however, showed a significant and sustained reduction in retinal volume from early stages of differentiation, which correlated with both a decrease in the number of mitosis and an increase in cell death throughout the retina. The experiments on SoFa1 embryos also showed decreased numbers of all retinal cell types. This effect of cilia impairment on cell proliferation/survival was cell-autonomous, as can be concluded from the blastomere transplantation experiments. Interestingly these experiments indicated that MO-treated retinal cells had an extremely reduced capacity to compete against their neighboring cells, as: 1) very scarce transplanted morphant atoh7:gap-RFP cells could be detected in wild-type host embryos, and 2) wild-type transplanted atoh7:gap-GFP cells tended to appear earlier than, and in several cases overcame, the host atoh7:gap-RFP cells. Taken together, these results suggest that primary cilia are functional at the progenitor cell level, regulating mitosis and possibly cell cycle exit.
Interestingly, while we observed a 38 % reduction in total retina volume, the volume of the RGC layer transiently decreased up to 95 %. This effect was more evident at 48 hpf than at later stages, suggesting a partial recovery of neuronal differentiation as development advanced. In addition, the 48 hpf morphant RGC layer appeared to be in earlier developmental stages when compared to controls and the inner plexiform layer was only visible in morphant embryos at the anterior-ventral part of the retina, where cell differentiation begins , while it was complete in controls. Consistent with this supposition, we observed that transplanted wild-type atoh7:gap-GFP cells tended to differentiate much earlier than the surrounding atoh7:gap-RFP cells in morphant hosts. In vivo experiments in the SoFa1 fish line further indicated that the delay in neuronal generation was more prominent in RGCs than in other cell types. A proportional increase in the photoreceptor layer was also noticed, indicating that cell fate decisions were affected. A causal relationship between cell cycle regulation and neuronal cell fate choice has been reported in different regions of the central nervous system . Therefore, it could be possible that an altered cell cycle progression upon cilia disruption could account for the observed reduction in RGC number with respect to photoreceptors. Indeed, the manipulation of the timing of cell cycle exit in Xenopus retinal progenitors affected the generation of early neuronal cell types (as RGCs) at the expense of late-generated neurons (as bipolar cells) .
We have shown here that relatively short primary cilia are present in neural progenitors and early neuroblasts of the neural retina in the zebrafish. The most remarkable features of these cilia are that they tend to remain localized to the apical region of the cells, and that they become extremely dynamic particularly during neuroblast polarity transitions, such as apical detachment and between axon and dendrite formation. Finally, our cilia disruption experiments, knocking-down elipsa and ift88, underscore a cell-autonomous role for cilia at cell proliferation and survival, as well as in neuronal cell-type specification.
Ethics approval and consent to participate
All manipulations described including zebrafish have been approved by the local Animal Ethics Committee at Institut Pasteur de Montevideo (CEUA, reg. n° 010 / 2013) and by the Uruguayan National Animal Ethics Committee (CNEA, reg. n° 002/11).
Consent for publication
retinal ganglion cell
retinal pigment epithelium
transmission electron microscopy
We are grateful to all colleagues who generously shared diverse reagents and fish lines: Kristen Kwan provided several Tol2 kit plasmids, Bill Harris provided the atoh7:gap-RFP and -GFP, and the SoFa1 zebrafish lines, Brian Ciruna provided the Arl13b-GFP zebrafish line and plasmid. We also thank the technical assistance of Ana Paula Arévalo, Casandra Carrillo and Martina Crispo for fish care and maintenance; Marcela Díaz and Tabaré De Los Campos, for support on confocal microscopy and image processing. Finally, we thank Magdalena Cárdenas for advice and help with zebrafish embryo techniques.
The present work was funded by ANII (FCE_1_2011_1_5888 to FRZ), Institut Pasteur de Montevideo (Transversal Grant 2010 to JLB and FRZ), and PEDECIBA. PL had a SNB-ANII Doctoral Fellowship (POS_NAC_2012_1_8518). Funding sources had no role in the design of the studies.
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