Deletion of primary cilia in adult NSCs in the DG
Primary cilia are present in stem/progenitor cells and granule neurons in the adult hippocampal DG (Breunig et al., 2008
). To conditionally ablate primary cilia in NSCs postnatally, we used previously described transgenic mice expressing Cre recombinase under control of the mGFAP promoter (Garcia et al., 2004
). The Ift20
gene encodes an IFT protein essential for ciliogenesis (Follit et al., 2006
). To target Ift20
cells, we engineered mGFAP-Cre mice carrying floxed Ift20
) resulting in Ift20fl/fl
mutant) mice. Ift20
mutant mice were born at the expected Mendelian ratios and were indistinguishable from Ift20+/+
::mGFAP-Cre (wild-type) littermates. The mGFAP promoter drives Cre recombinase expression in the hippocampal DG and in the lateral ventricles (LVs), giving rise to recombined cells (A–D
). In this strain of mice, the mGFAP-Cre transgene in GFAP-expressing NSCs is not active during fetal development but is activated postnatally (Garcia et al., 2004
), and a few detectable Cre-positive cells start to appear in the DG and LVs around postnatal day 7 (data not shown), evidencing that normal development of these brain regions are not affected. Isolated neural stem/progenitor cells from Ift20
mutants lacked primary cilia, whereas primary cilia were readily detectable in wild-type cells (data not shown). To verify that primary cilia are absent in radial NSCs in the DG of Ift20
mutant mice in vivo
, we examined GFAP+
cells using littermates of Ift20
mutant mice crossed with mice that express a gene encoding a cyan fluorescent protein fused to a nuclear localization signal (CFPnuc) driven by the nestin enhancer (nestin-CFPnuc) (Encinas et al., 2006
). The use of CFPnuc and GFAP expression helped to visualize radial NSCs in the DG. Confocal analysis with adenylyl cyclase III (ACIII), a marker for primary cilia, verified the lack of primary cilia in radial NSCs within the subgranule layer of DG in Ift20
mutant mice crossed to nestin-CFPnuc mice (E–H
). In addition, by staining for the proliferative cell marker PCNA, we confirmed that radial NSCs and amplifying progenitors in cell cycle in Ift20
mutants do not have primary cilia (I
Figure 1. Primary cilia are ablated in GFAP-expressing NSCs from Ift20fl/fl::mGFAP-Cre (Ift20 mutant) mice (Ift20+/+::mGFAP-Cre; wild type). Double immunostaining in 8-week-old Ift20 mutant mice shows Cre expression (red) in GFAP+ cells (green; arrowheads) in the (more ...)
Normal morphology of brain structures in adult Ift20 mutant mice
We conducted several experiments to detect potential morphological abnormalities in the brain structures after postnatal deletion of Ift20 gene. At 8 weeks of age, the Ammon's horn of the hippocampus in Ift20 mutants exhibited normal morphology (A,B). Moreover, the DG of these mice showed normal organization and was the same size as that seen in wild-type mice (C,D). Both in wild-type and Ift20 mutant mice, staining using the neuronal marker NeuN revealed numerous nuclei of cells corresponding to mature neurons representing granule cells, without any evidence of altered density (E,F). In addition, the GFAP+ astrocytes in the DG, hilus, molecular layer, and the remainder of the hippocampus were present in Ift20 mutant mice in similar numbers and with similar morphologies as in wild-type mice (G,H).
Figure 2. Neuromorphological characterization of the DG in 8-week-old Ift20 mutant (right panels) and wild-type (left panels) mice. A, B, NeuN (red) immunofluorescence shows preserved layering in hippocampal subregions (CA1, CA2, CA3, and DG) in Ift20 mutant and (more ...)
The SGZ contains a heterogeneous population of stem/progenitor cells. One type is the radial NSCs (or type 1 cells), which are relatively quiescent, show morphologies characteristic of radial glia (Seri et al., 2001
), and express the precursor cell marker protein NESTIN and the astrocytic protein GFAP (Seri et al., 2004
). To determine whether the organization of the radial GFAP+
NSCs was defective in the mutant DG, we costained for GFAP and NESTIN and observed a similar pattern of radial processes in both genotypes (I
). This finding indicates that the lack of cilia in radial NSCs does not appreciably alter their morphology of radial astrocytes in the SGZ.
Other brain regions that undergo considerable postnatal neurogenesis—including the cerebellar GCL and the olfactory bulb (OB) (data not shown)—showed normal size, morphology, and density of mature neurons in 8-week-old Ift20 mutant mice, suggesting that neurons in these regions had already formed before the conditional deletion of Ift20 affects primary cilia formation in the mutant mice.
Decreased adult hippocampal cell proliferation in Ift20 mutant mice
Ift20 mutant mice show gradual decrease in SGZ proliferation with a significant reduction already at 4 weeks of age (data not shown). To evaluate the proliferation of stem/progenitor cells in the SGZ of 8-week-old Ift20 mutant mice, we injected mice with the cell cycle marker BrdU. A 2 h pulse of BrdU (100 mg kg−1 body weight) resulted in many labeled cells in the SGZ of wild-type mice (A). By contrast, the number of BrdU-labeled cells in the DG of Ift20 mutants was 54.5% of the number in wild-type mice (5.51 ± 0.8 vs 10.11 ± 1.27 × 103 cells/mm3; p = 0.006) (A–C). The number of cells in the SGZ positive for the proliferative cell marker PCNA was similarly reduced (39.2% of wild type; 3.96 ± 0.54 vs 10.1 ± 0.81 × 103 cells/mm3; p < 0.000001) (D–F). These data indicate that proliferation is reduced approximately twofold in the SGZ of Ift20 mutant mice. To assess whether reduction in proliferation was maintained in Ift20 mutants at older stages, we analyzed 12-month-old mice. The number of Ki67-labeled cells in the DG was similarly reduced (61.5% of wild type; 0.51 ± 0.08 vs 0.84 ± 0.13 × 103 cells/mm3; p = 0.035) at this stage.
Figure 3. Ift20 mutant mice exhibit reduced neural precursor cell proliferation in the SGZ of the DG. A, B, BrdU (black) staining after a 2 h BrdU pulse (100 mg kg−1 body weight) showed less proliferation in the SGZ of the DG in 8-week-old Ift20 mutants (more ...)
A reduced number of proliferating cells in the DG of Ift20 mutants should decrease the number of neuroblasts. In agreement with this prediction, immunostaining with the neuroblast marker DCX revealed a reduced number of neuroblasts in the DG of 8-week-old (57.5% of wild type; 30.32 ± 4.48 vs 52.73 ± 4.33 × 103 cells/mm3; p < 0.001) (G–I) and 12-month-old (62.1% of wild type; 4.42 ± 0.29 vs 7.12 ± 0.59 × 103 cells/mm3; p < 0.0001) Ift20 mutant mice.
Newborn cells that are not recruited into functional circuits in the adult DG are eliminated by apoptotic cell death (Biebl et al., 2000
). To analyze whether the reduction in proliferating/DCX+
cells is due to apoptosis in the SGZ, we performed immunostaining for TUNEL+
cells. We observed similar numbers of apoptotic cells in the SGZ of Ift20
mutants and wild-type mice (J–L
Despite the fact that ablation of primary cilia in neural precursor cells of the adult SGZ significantly reduced neurogenesis, 8-week-old Ift20 mutant mice did not showed changes in proliferation in the subventricular zone (SVZ) of the LVs, as evidenced by BrdU incorporation and PCNA staining analyses (A–F). This observation was confirmed by DCX staining, which revealed that the density of neuroblasts in the SVZ was similar in Ift20 mutant and wild-type mice (data not shown). Further analysis at older stages (12 months of age) revealed a modest reduction in number of BrdU-labeled cells (83.7% of wild type; 24.47 ± 1.49 vs 29.24 ± 1.65 × 103 cells/mm3; p = 0.037) in the SVZ of Ift20 mutant mice.
Figure 4. Proliferation in the SVZ of the LVs is similar in Ift20 mutant (right panels) and wild-type mice (left panels). A, B, BrdU staining in 8-week-old mice after a 2 h BrdU pulse (100 mg kg−1 body weight) showed no evident genotypic differences in (more ...)
Primary cilia regulate proliferation of hippocampal amplifying progenitors
To analyze the numbers of radial NSCs and amplifying progenitors in the SGZ of Ift20 mutants we used 8-week-old Ift20+/+:: mGFAP-Cre::nestin-CFPnuc mice. Staining for GFAP, an intermediate filament protein organized in the processes traversing granule layer in radial NSCs (type 1 cells), and for CFPnuc, which is localized to nuclei of cells expressing NESTIN (type 1 and type 2a cells) (A,B), allowed us to distinguish between radial NSCs (CFPnuc+ cells with radial GFAP+ processes) and amplifying progenitors (CFPnuc+ cells without GFAP+ radial processes). Total numbers of radial NSCs were similar in both genotypes (C), suggesting that the lack of primary cilia does not affect self-renewal and/or survival on this cell population. In contrast, the numbers of amplifying progenitors were significantly reduced in Ift20 mutants (79.8% of wild type; 6.74 ± 0.53 vs 8.45 ± 0.26 × 103 cells/mm3; p < 0.01) (C), indicating that primary cilia ablation specifically affected this cell population.
Figure 5. Number of adult hippocampal amplifying progenitors in cell cycle is reduced in Ift20fl/fl::mGFAP-Cre::nestin-CFPnuc (Ift20 mutant) mice. A, B, Confocal GFAP (red) and CFPnuc (green) double staining in DG of 8-week-old Ift20fl/fl::mGFAP-Cre::nestin-CFPnuc (more ...)
To determine how lack of primary cilia affects proliferation in radial NSCs and amplifying progenitors, we used staining for PCNA in combination with staining for GFAP and CFPnuc (D,E). Analysis of radial NSCs showed no difference between genotypes within both quiescent (PCNA− CFPnuc+ radial GFAP+) and proliferating (PCNA+ CFPnuc+ radial GFAP+) subpopulations (F). Quiescent amplifying progenitors (PCNA− CFPnuc+) showed no differences between genotype; however, the numbers of proliferating amplifying progenitors (PCNA+ CFPnuc+) were significantly reduced in Ift20 mutants (61.1% of wild type; 2.13 ± 0.24 vs 3.49 ± 0.29 × 103 cells/mm3; p < 0.01). These results indicate that the loss of primary cilia in the DG specifically reduced the numbers of proliferating, but not quiescent, amplifying progenitors without affecting the numbers of either proliferating or quiescent radial NSCs.
In the SVZ, analysis of stem/progenitor cells stained for CFPnuc in 8-week-old mice did not reveal major differences between mutant and wild-type mice (data not shown). These findings suggest that the lack of primary cilia in neural precursors does not affect the overall cell numbers in this neurogenic niche.
Reduced neurogenesis in the DG of Ift20 mutant mice
It remains unknown whether loss of cilia in adult NSCs would alter adult neurogenesis. To analyze the proportion of neurons that mature and integrate into the DG, we injected BrdU into 8-week-old mice for 5 d (twice daily, 12 h apart, at 50 mg kg−1
body weight) and examined the BrdU/NeuN-labeled cells 4 weeks after the last injection (A
). The number of BrdU+
cells in Ift20
mutant mice was 36.6% of that seen in wild-type mice (1267.9 ± 123.2 vs 3465.3 ± 413.2 cells/mm3
< 0.001) (B
). Independently, we also analyzed the cumulative numbers of postnatal newborn neurons integrated in the DG of Ift20
mutants. Crossing with mice harboring a reporter designated Z/EG (lacZ/Lox-Stop/EGFP) (Novak et al., 2000
), in which postnatal Cre recombination activates expression of an EGFP reporter, allowed lineage tracing of stem/progenitor progeny. Double staining for NeuN and EGFP in 8-week-old Ift20fl/fl
mutant) mice and Ift20+/+:
: mGFAP-Cre::Z/EG (wild-type) mice showed decrease in numbers of that EGFP+
double-positive cells in the DG of Ift20
mutant mice (31.9% of wild type; 147.8 ± 26.9 vs 462.5 ± 35.3 × 103
< 0.001). These data indicate that the cumulative (8 week) decrease in adult neurogenesis (approximately threefold) is similar to the neurogenesis rate measured over 5 d of BrdU incorporation, suggesting that the reduction in generation of new neurons in Ift20
mutants is constant over time.
Figure 6. Adult hippocampal neurogenesis is reduced in Ift20 mutant mice. A, A 5 d BrdU injection protocol (twice daily, 12 h apart, at 50 mg kg−1 body weight) was used in 8-week-old Ift20fl/fl::mGFAP-Cre (Ift20 mutant) and Ift20+/+::mGFAP-Cre (wild-type) (more ...)
Colocalization of EGFP and NeuN markers suggested that neurons derived from radial NSCs that had undergone primary cilia ablation were present in the GCL of Ift20 mutants (data not shown). In addition, a stronger reduction in the numbers of granule cells (approximately threefold) compared with DCX+ cells (approximately twofold) hints at a potential role for primary cilia in the survival of DCX+ neuroblasts.
The presence of BrdU+ NeuN− cells in the SGZ suggested that some non-neuronal cells could retain BrdU for 4 weeks. We analyze the percentage of CFPnuc+ (NESTIN+) stem/progenitor cells that retain BrdU+ after 4 weeks in Ift20fl/fl:: mGFAP-Cre::nestin-CFPnuc (Ift20 mutant) mice. Compared with wild-type mice, Ift20 mutants show approximately twofold decrease in numbers of stem/progenitor cells that retain BrdU+ after 4 weeks (45.8% of wild-type; 1.89 ± 0.57 vs 4.13 ± 0.78% of BrdU+ cells among CFPnuc+ cells; p < 0.001) (G–J,L). This decrease is similar to that seen in global proliferation studies, suggesting that the survival of stem/progenitor cells that had previously divided is similar in Ift20 mutants and wild-type mice.
Analysis of BrdU+
cells in the OB 4 weeks after the last BrdU injection did not show differences between genotypes (G–K
). Furthermore, EGFP+
cells in the OB of 8-week-old Ift20fl/fl
::mGFAP-Cre::Z/EG mice derived from recombined GFAP+
NSCs in the LVs did not show any major defect in neurogenesis (data not shown). These results indicate that adult neurogenesis in the SVZ-OB is not significantly affected in Ift20
mutant mice, consistent with the previous finding that primary cilia in NSCs are largely dispensable for the development of the SVZ (Han et al., 2008
Ift20 mutant mice show altered spatial novelty recognition
One of the most striking challenges in the field of adult hippocampal neurogenesis is to elucidate a causal relationship between neurogenesis and learning and memory. To evaluate spatial recognition in Ift20 mutant mice, we used two different novel location–object recognition tests, a spatial pattern recognition test and a location novelty recognition test. Briefly, in the spatial recognition test, mice were habituated to three different objects in an open field. After familiarization trials, they were tested in a novelty recognition test in which one object was moved to a novel location in the arena. We found no genotypic difference in habituation to test objects across three familiarization trials: mice of both genotypes made fewer contacts with the objects over time (A). In contrast, when one of the objects was moved to a novel location, there were striking effects on reexploration of the displaced object, in which a renewed interest (increased contacts) in the object was observed in wild-type (p < 0.05) but not in Ift20 mutant mice. Post hoc analysis revealed significant differences between genotypes in reexploring the displaced object (p < 0.01).
Figure 7. Ift20 mutant mice exhibit altered spatial novelty recognition. A, In a spatial pattern recognition test (mice group 1), habituation to three plastic objects (farmer boy, horse, and cow) across three familiarization trials (T1, T2, and T3) was similar (more ...)
The initial spatial pattern recognition test relies on the ability of the animals to detect a change in the spatial patterning of objects relative to each other. In the location novelty recognition test, a more complex object exploration task, mice are tested for their ability to detect the displacement of a single object moved 45° in an arc around a circular field. Success requires the mice to use distal spatial cues to discriminate similar contexts. We found no genotypic difference in habituation to test the object across four familiarization trials (B). When the object (Erlenmeyer flask) was moved to a novel location, a significant renewed interest (increased contacts) in the object was observed in wild-type (p < 0.01) but not in Ift20 mutant mice. Post hoc analysis revealed significant differences between genotypes in reexploring the displaced object (p < 0.05). These results indicate that subtle changes in spatial context are not detected by the mutant mice and suggest decreased efficiency or impairment in spatial novelty recognition.
Ift20 mutant mice show delay in spatial learning
As a further more complex test of spatial learning, we used the Barnes maze, which consists of an elevated maze with 20 holes (A). An escape chamber is located in one of the holes, and mice learn to locate the chamber over 12 d, in four blocks of three trials. Using repeated-measures ANOVA of the numbers of errors to find the target hole, we found the interaction term between blocks and genotype to be significant (p < 0.05) (B), in that mutant mice performed poorly in the first blocks. Overall, Ift20 mutant mice made more errors in locating an escape chamber and ultimately escaping. Closer analysis showed that the difference was significant in blocks 2 and 3 (p < 0.05). A day later (following the 12 trials), when we performed a probe test in which the escape chamber was removed and time spent in the target quadrant and hole search strategies was assessed, the two groups of mice performed similarly (C,D). Analysis undertaken 2 weeks after the last trial (retention trial) showed that long-term memory was not affected (E). In addition, mice exposed to a reversal trial (in which the escape chamber was moved to a new location) did not show delayed learning, illustrating that the working memory strategies adopted by the mice to locate a new escape chamber location were preserved. A comparison of strategies used by wild-type versus mutant mice also revealed significant differences (F). Wild-type mice progressively increased the use of a spatial strategy across the first three blocks, whereas mutant mice were slower to shift to a spatial strategy and instead relied on random and/or serial strategies for longer periods. However, the spatial strategy used in Ift20 mutant mice in the last block of trials and in the retention trial was similar to that used by wild-type mice. Thus, Ift20 mutant mice could learn a spatial contingency but required more trials to do so, suggesting that they have delayed rather than permanently impaired spatial learning.
Figure 8. Ift20 mutant mice exhibit delayed spatial learning but preserved long-term memory. A, Schematic representation of Barnes maze test. B, In the Barnes maze, Ift20 mutants showed delayed spatial learning in finding the escape chamber (averaged into 4 blocks (more ...)
Additional behavioral tasks were performed to test for locomotor dysfunction, visual disabilities, lack of motivation, anxiety-related and depression-like behaviors (data not shown) with no differences between genotype.
Ift20 mutant mice show enhanced cued fear responses
Contextual and cued fear learning are induced by pairing neutral conditioned stimuli (tone plus light) with an aversive unconditioned stimulus (electric shock) in a specific context, and the extent of “freezing” behavior is used as an index of effective retrieval of the consolidated fear memory. Following pairing of a shock with a tone plus light combination in a specific context (conditioning), mice were tested in the same context without shock or tone plus light combination (context test) (A). Ift20 mutants showed slightly reduced patterns of freezing behavior than wild-type mice across the context test (nonsignificant) (B). In addition, mice were tested in a different context with the introduction of the tone plus light combination (cued test). Interestingly, Ift20 mutants showed higher levels of freezing during the exposure to the tone plus light combination (p < 0.05 and p < 0.01) (C), but not before, indicating that Ift20 mutants responded more robustly to the cue than wild-type mice.
Figure 9. Ift20 mutant mice exhibit enhanced fear responses to cued but not contextual fear conditioning. A, A 4 d fear conditioning protocol was used to evaluate contextual and cued fear conditioning. B, The freezing response to the same context than conditioning (more ...)
The greater levels in freezing behavior of Ift20 mutants after the introduction of the neutral stimuli (tone plus light) in a different context suggests that Ift20 mutant mice are biased toward cue-based strategies.