Cell proliferation in the optic tectum decreases with visual system development
In the visual system of Xenopus laevis tadpoles retinal ganglion cells project axons to the contralateral optic tectum where they form synapses with tectal neurons (). Between stages 39 and 49, a period of 6-7 days, the visual system of Xenopus tadpoles develops rapidly to accommodate the behavioral needs of the animal. Retinal ganglion cells first innervate and transmit visual information to the optic tectum at stage 39 (Holt and Harris, 1983
) when the majority of cells in the tectum have radial glial morphology and neurons have very simple dendritic arbors (Wu et al., 1999
). An initial topographic retinotectal map is established by stage 45 (O'Rourke and Fraser, 1990
) and between stages 46 and 49 visual experience drives many aspects of visual circuit development pertaining to the detection and processing of visual inputs (Bestman and Cline, 2008
; Chiu et al., 2008
; Cline and Haas, 2008
; Engert et al., 2002
; Pratt and Aizenman, 2007
; Pratt et al., 2008
; Tao and Poo, 2005
) even as ventricular layer cells with radial glial morphology persist in the tectum (Tremblay et al., 2009
). Although it is well known that tectal ventricular layer cells proliferate throughout tadpole stages of development and generate neurons within the tectum (Peunova et al., 2001
; Straznicky and Gaze, 1972
), a potential relation between development of the functional visual circuit and cell proliferation has not been explored.
Figure 1 Developmental decrease in proliferative cells in the Xenopus tadpole optic tectum. A-C. The Xenopus optic tectum includes a functional visual circuit and proliferative ventricular layer cells. A. Phase contrast image of an albino Xenopus tadpole head. (more ...)
To test whether rates of cell proliferation in the optic tectum change over this period of visual system development, we exposed tadpoles at stage 46, 48 and 49 to 2 hr of XdU and either processed the brains immediately or allowed the animals to develop in normal rearing solution for another 22 hr before processing the brains as wholemounts for XdU immunodetection. We delivered XdU by exposing tadpoles to rearing solution containing 10 mM XdU for 2 hr. This method efficiently labels proliferative cells in the brain and allows greater control over XdU exposure time than standard injection methods (Peunova et al., 2001
). Brains were processed to detect XdU with antibodies and a complete confocal Z-series of images was collected through the midbrain of wholemount brains or cryostat sections. As previously reported, proliferating cells, identified by exposure to 3
H-thymidine (Straznicky and Gaze, 1972
) or BrdU-labeling (Peunova et al., 2001
) with a short survival time, line the ventricle (Figs and ). We counted XdU-labeled cells in the ventricular layer at the tectal midline using the dissector method and indexed cell counts to an estimated volume of 20,000 μm3
(see and methods). Stage 46 tadpoles have significantly more XdU-labeled cells (99.6 ± 2.4cells/20,000μm3
, n=8) than either stage 48 (36.1 ± 2.3 cells/20,000μm3
, n=8; p<0.05) or stage 49 (8.9 ± 0.8 cells/20,000μm3
, n=7; p<0.05) tadpoles (, Supplementary Movies S1
). The XdU-labeled cells continue to divide over the next 22 hr to approximately double the number of XdU-labeled cells (. Stage 46: 180.4±4.9 cells/20,000μm3
, n=8; Stage 48: 71.2±3.9 cells/20,000μm3
, n=8; Stage 49: 20.3±0.9 cells/20,000μm3
, n=7). These data suggest that proliferation gradually decreases between stages 46 and 49, a time interval during which the visual circuit matures.
Figure 3 Lineage and identity of proliferating neural progenitor cells in optic tectum. A-B. Stage 48 tadpoles were exposed to XdU for 2h in rearing solution and were either fixed immediately (A) or after 72 hrs of further development in the absence of XdU (B). (more ...)
Cells expressing MCM7 or Musashi decrease with development
We next tested whether MCM7 and Musashi1, which have been characterized as markers of proliferative cells in other experimental systems, change expression over the developmental period when XdU incorporation decreased. MCM7 is a part of minichromosome maintenance complex (MCM) of proteins and is expressed in cells with proliferative potential (Crevel et al., 2007
; Facoetti et al., 2006
; Khalili et al., 2003
). Proteins in the MCM complex are down-regulated when cells become quiescent, differentiated, or senescent (Facoetti et al., 2006
; Padmanabhan et al., 2004
). Antibodies to MCM7 are thought to label the population of cells that is not yet differentiated and is capable of proliferation (Blow and Dutta, 2005
). In the optic tectum, we find that MCM7-immunoreactive cells are present in the cell layers lining the ventricle () and partially overlap with the distribution of cells detected by incorporation of XdU following 2h exposure. MCM7 expression was down-regulated between stage 46 and 49 (), consistent with the decrease in XdU-incorporation over this time period.
Figure 2 Developmental decrease in MCM7 and Musashi1 immunoreactivity. Confocal images of 30 μm cryostat sections through the optic tectum of stages 46 (A, C) and 49 (B, D) tadpoles labeled with anti-MCM7 antibody (green; A1, B1) or anti-musashi1 antibody (more ...)
The Musashi proteins are highly conserved RNA binding proteins (Good et al., 1993
; Nakamura et al., 1994
; Sakakibara et al., 2001
), whose founding member, Musashi1, was originally discovered in Xenopus and named nrp1 (Richter et al., 1990
). In vertebrates, there are 2 genes, musashi1
, the latter of which is homologous to xrp1
in Xenopus. We focused on Musashi1 because it is expressed exclusively in the CNS and is enriched in CNS progenitor cells across phyla (Amato et al., 2005
; Good et al., 1993
; Kaneko et al., 2000
; Nakamura et al., 1994
; Sakakibara et al., 1996
; Sakakibara and Okano, 1997
) and because it is required for maintenance of neural stem cells (Okano et al., 2002
; Okano et al., 2005
; Sakakibara et al., 2001
; Sakakibara et al., 2002
), whereas musashi2/xrp1 is widely expressed throughout the body. Musashi1 protein expression is down-regulated in differentiated neurons (Kaneko et al., 2000
; Nakamura et al., 1994
; Sakakibara et al., 1996
; Sakakibara and Okano, 1997
). We find that musashi1-immunoreactive cells are present in the proliferative layer lining the ventricle of the optic tectum () and overlap with the distribution of MCM7-immunolabeled cells. Musashi1-immunoreactive cells decrease in number between stages 46 and 49 from 158.3±8.2 cells/20,0003
μm to 53±4.3 cells/20,0003
μm (p< 0.05, n=4, 4), consistent with the decrease in XdU- and MCM7- labeled cells. These data suggest that the pool of progenitor cells gradually decreases between stages 46 and 49.
Musashi-expressing cells are radial glial progenitor cells
To test whether the musashi1-immunoreactive cells are neural progenitors in the tadpole CNS, we exposed stage 48 tadpoles to XdU for 2 hr and fixed half of them immediately to analyze the distribution of XdU labeling and musashi1-immunoreactive cells. The remaining tadpoles survived for an additional 72 hours in the absence of XdU before fixation at stage 49. The majority of the cells labeled with a 2 hr exposure to XdU are located in the ventricular layer and label with antibodies to musashi1 (), indicating that musashi1-immunoreactive cells are progenitors. When XdU-labeled animals survived for an additional 72 hr (in the absence of further XdU exposure) the number of XdU-labeled cells increased (), indicating that the XdU-labeled neural progenitor cells continue to proliferate. Furthermore, after 72 hr, the XdU-labeled cells were distributed within the tectal cell body layer where mature tectal neurons are located ().
Studies in mammalian cortex indicate that radial glia are neural progenitors (Kriegstein and Alvarez-Buylla, 2009
), however the potential role of radial glial cells as neural progenitors in midbrain subcortical structures has not been established. We find that musashi1-immunoreactive cells in the tadpole optic tectum extend a radial process to the pia, indicating that they have radial glial morphology (). To further characterize the musashi1-immunoreactive cells, we labeled radial glial cells in stage 47 tadpoles by bulk electroporation of a CMV::eGFP expression plasmid into ventricular layer cells (Haas et al., 2002
). The day after electroporation, eGFP is expressed in cells with radial glial morphology lining the ventricle (Haas et al., 2002
; Tremblay et al., 2009
). The majority of eGFP-expressing radial glial cells in stage 47 optic tectum are musashi1-immunoreactive (). These results show that a 2 hr exposure to XdU in vivo labels musashi1-immunoreactive neural progenitors and that musashi1-immunoreactive cells are radial glia.
Visual deprivation increases cell proliferation in the optic tectum
The developmental decrease in progenitor cell proliferation correlates with the maturation of the functional visual system in Xenopus tadpoles and suggests that visual circuit function may negatively regulate progenitor cell activity. To test whether visual experience regulates cell proliferation in the tadpole optic tectum, animals were reared in a 12 hr light/12 dark cycle until stage 46 when rates of cell proliferation are still relatively high (). Tadpoles were separated into 3 groups: One group continued under the normal 12 hr light/12 dark cycle, called ‘ambient light’. The second group was provided with enhanced visual stimulation (from an array of LEDs flashing on and off at 1Hz (Sin et al., 2002
)) during the 12 hr light period of the light/dark cycle and the third group was deprived of visual stimulation (by keeping them in the dark, see diagram in ). All tadpoles were kept in the dark during the 12 hr dark period of light/dark cycle.
Figure 4 Visual deprivation increases cell proliferation in the optic tectum. A. Timeline of the experimental protocol. The 12 dark/12h light rearing conditions are shown as shaded and white bars. During the 12h ‘light’ period animals were either (more ...)
We determined the division history of proliferating cells in the optic tectum using an assay that is based on a double-label protocol in which animals were exposed to two deoxyuridine analogs, CldU and IdU, at an interval longer than the cell cycle to identify cells which remain proliferative (Encinas and Enikolopov, 2008
; Vega and Peterson, 2005
) as shown in . CldU and IdU can be identified by immunostaining in Xenopus optic tectum without significant cross-reactivity or labeling bias (Supplemental Figure S1
). We refer to CldU and IdU generically as XdU. We provided a 2-hour exposure to the first deoxyuridine analog X1
dU (either CldU or IdU; green in ) and allowed tadpoles to grow in the absence of X1
dU label for 24 hr, followed by a 2 hr exposure to the second deoxyuridine analog X2
dU (either CldU or IdU; red in ). We used a 24h interval in between the X1
dU and X2
dU exposures based on the observation that the number of XdU-labeled cells approximately doubled over 24 hr (). The optic tectum was then analyzed for the presence of X1
dU and X2
dU labeling in 30 μm cryostat sections. Three labeling combinations are predicted: 1. cells with only X1
dU (green in ) 2. cells with only X2
dU label (red in ) and 3. cells labeled with both XdU's (yellow in ). The number of cells labeled with X1
dU (green) relative to the total number of labeled cells is the fraction that were in S phase at the first XdU exposure, but were not in S phase at the time of exposure to X2
dU. This value gives an estimate of the cells that exit the cell cycle between the times of exposure to X1
dU and X2
dU. The number of cells labeled with X2
dU (red) relative to the total number of labeled cells is the fraction that was in S phase only at the second XdU exposure but not during exposure to X1
dU. This value would represent a potential recruitment of quiescent progenitors to proliferate. The number of cells labeled with both XdUs (yellow) relative to the total number of labeled cells is the fraction of cells that were in S phase during both XdU exposure periods and therefore provides an estimate of the population of progenitors that remained proliferative during the observation window. Although this protocol does not identify all cells that are proliferating or all cells that remain in the cell cycle over the 24 hr interval (Encinas and Enikolopov, 2008
; Vega and Peterson, 2005
), it allows us to compare cell proliferation and cell cycle parameters in tadpoles reared in a normal 12 hr light/12 dark cycle (referred to as ambient light) with animals provided with either enhanced visual stimulation or deprived of visual stimulation.
We counted XdU-labeled cells along the midline ventricular layer in sections through the optic tectum and found that tadpoles subjected to reduced visual experience had more XdU-labeled cells in the same volume (79 ± 7.6 cells/20,000μm3, n=5) compared to animals that were exposed to either ambient light (48.7 ± 5.0 cells/20,000μm3, n=5; p<0.05) or enhanced visual stimulation (44 ± 7.3 cells/20,000μm3, n=5; p<0.05; ). Similar indices of XdU-labeled cells were seen in the optic tecta of tadpoles exposed to either enhanced visual activity or ambient light. These data suggest that visual deprivation for 2 days increases cell proliferation in the optic tectum. They further suggest that the amount of visual activity provided by ambient light is sufficient to reduce cell proliferation in the tectum.
Next, we determined the fraction of X1dU-labeled cells that also incorporated X2dU, as an estimate of the cell population that continued to proliferate over the 24h period. Animals deprived of visual experience have a significantly larger fraction of X1dU and X2dU double-labeled cells (82.1 ± 1.9%) compared to tadpoles exposed to either ambient light (62.4 ± 1.6%, p<0.05) or enhanced visual stimulation (70.9 ± 3.5%; p<0.05; ). A smaller proportion of double-labeled cells in animals with visual experience indicates that relatively fewer X1dU-labeled cells were in S phase of the cell cycle at the time of exposure to the second label, X2dU. Furthermore, visual experience results in a higher fraction of cells labeled with only X1dU compared to visual deprivation () consistent the idea that visual experience changes the fate of tectal progenitors so they exit the cell cycle or become quiescent. Together, these data indicate that visual system activity decreases proliferative activity, while reduced visual experience maintains cells in a proliferative state by decreasing cell cycle exit and maintaining cells in the progenitor pool at the expense of differentiation.
Visual experience increases neuronal differentiation
The data presented above suggest that visual experience may change the fate of the progeny of tectal progenitor cells so they differentiate into neurons. To test whether visual experience promotes neuronal differentiation of newly generated cells, animals were reared in their normal 12h light/12h dark conditions until stage 47 and exposed to XdU for 2h in rearing solution. Animals were then either deprived of visual experience for 48h or exposed to enhanced visual stimulation as described in . We used N-β-tubulin antibodies to label differentiated neurons (Moody et al., 1996
). Visual experience increases the proportion of XdU-labeled cells that differentiated into N-β-tubulin-labeled neurons over the intervening 48h compared to that seen in visually-deprived animals (61.7 ± 1.5% vs 40.2 ± 1.1%, p<0.05, n=11,11 animals, respectively). These results indicate that visual experience changes the fate of progeny to exit the cell cycle and differentiate into neurons.
Figure 5 Visual experience increases neuronal differentiation. Stage 47 tadpoles were exposed to XdU for 2hr and allowed to develop in absence of XdU for 48 hrs while subjected to either enhanced visual stimulation or reduced visual stimulation as described in (more ...)
Visual deprivation expands the neural progenitor cell population by increasing Musashi1 expression
The reduced proliferation seen in animals with visual experience could result from two types of mechanisms in relation to neural progenitor cells. The number of neural progenitor cells could remain constant while the cell divisions occur less frequently or the size of neural progenitor pool could decrease with visual system activity. Either mechanism would result in a decreased fraction of X1dU and X2dU double-labeled cells and a reciprocal increase in the fraction of cells that stop dividing after X1dU exposure. To differentiate between these two possibilities we tested whether the musashi1-expressing neural progenitor cell population is affected by visual experience. Animals were reared under normal conditions to stage 47 when they were either deprived of visual experience or provided with visual experience, as shown in . The number of musashi1-expressing cells was significantly lower in tecta of animals with visual experience (267.2 ± 15.3 cells/20,000μm3, n=7) compared to tecta of visually-deprived animals (426.9 ± 32.8 cells/20,000μm3, n=7; p<0.05) (). These results are consistent with a model in which the decrease in cell proliferation seen with visual stimulation results from a reduction in number of musashi1-expressing neural progenitor cells, whereas visual deprivation increases the pool of musashi1-expressing neural progenitor cells.
Figure 6 Musashi1 is required for visual experience-dependent regulation of cell proliferation. A-C. Visual deprivation for 48h increases the number of Musashi1 (Msi1) immunoreactive neural progenitor cells compared to animals with enhanced visual experience. (more ...)
The experiments described above indicate that musashi1 expression correlates with the proliferative activity of tectal neural progenitor cells. To test whether musashi1 is required for the increase in cell proliferation in visually-deprived animals, shown in , we knocked down nrp1B, the Xenopus homolog of musashi1 using morpholino antisense oligonucleotides to nrp1B. To test the efficacy of knockdown, morpholinos against nrp1B or control scrambled morpholinos were electroporated into the right tectal lobe. After 48 hours, we compared the intensity of musashi1 immunoreactivity in ventricular layer cells of the right and left optic tecta. Morpholinos against nrp1B reduced the ratio of musashi1 immunoreactivity in the electroporated right tectum to the unelectroporated left tectum to 57.2 ± 0.03% (p<0.05), whereas control morpholinos did not show any significant difference in musashi1 immunoreactivity between right and left tecta after unilateral electroporation (Supplementary Data Figure 2
). We then electroporated stage 46 tadpoles in both tectal lobes with nrp1B morpholinos or control morpholinos and subjected animals to reduced visual stimulation for 60 hrs followed by 2hr exposure to XdU. Morpholinos against nrp1B significantly reduced the number of XdU-labeled cells (36.93 ± 1.94 cells/20,000μm3
, n= 14; ;) compared to control morpholinos (52.8 ± 1.7 cells, n= 16; p<0.05; ). Furthermore, coelectroporation of plasmid containing mouse musashi1 cDNA with morpholinos directed against Xenopus nrp1B mRNA completely rescued the morpholino-induced reduction in proliferation (62.5 ± 4.0 cells/20,000μm3
, n= 11; p<0.05; ). These data indicate that musashi1 protein levels are negatively regulated by visual activity in the optic tectum and that musashi1 is necessary for the increased cell proliferation seen in visually-deprived animals. Together with data presented in , the data show that progenitors increase musashi expression, increase proliferative activity and expand the progenitor pool in the absence of visual input but that visual experience triggers two changes in the system: 1. a decrease in musashi expression and a decrease in proliferative activity in radial glial cells and 2. an increase in the rate at which newly generated progeny differentiate into neurons.
To test whether musashi1 is sufficient to increase cell proliferation in the CNS we returned to stage 49 tadpoles, where musashi1 expression is relatively low in ventricular layer cells (). Electroporation of ventricular layer cells in stage 49 tadpoles with a dual promoter plasmid co-expressing mouse musashi1 and eGFP or eGFP alone labeled radial glial cells (). In vivo time-lapse images of eGFP+ cells co-expressing mouse musashi1 show a greater increase in eGFP-expressing cells over 6 days than seen in control animals, suggesting that exogenous expression of musashi1 increases proliferation of radial glia. Furthermore, we tested whether musashi1 expression in stage 49 tadpoles increases XdU incorporation. Stage 49 tadpoles were electroporated with a dual promoter plasmid co-expressing either eGFP alone or mouse musashi1 and eGFP and after 60 hr, animals were exposed to XdU for 2 hr immediately before sacrifice. Mouse musashi1 expression doubled the number of XdU-labeled cells compared to expression of eGFP alone (Musashi1: 21.3±1.3 cells/20,000μm3, n=17, eGFP: 10.9±0.9 cells/20,000μm3, n=18; p< 0.05).
Figure 7 Musashi1 expression is sufficient to increase cell proliferation. A.B. In vivo images of eGFP-expressing cells collected over 6 days after electroporation of stage 49 tadpoles with a dual promoter plasmid expressing eGFP alone (A) or eGFP and mouse Musashi1 (more ...)