Dixdc1 is a DISC1-interacting protein
To determine if Dixdc1 interacts with DISC1 during embryonic development, we first performed biochemical experiments using embryonic day 14 (E14) brain tissue. We found that Dixdc1 and DISC1 co-immunoprecipitated at this embryonic time point, when neural progenitor proliferation is highly prevalent (), demonstrating that they form a complex in vivo. We then mapped the binding domains between Dixdc1 and DISC1 by creating and expressing full-length GFP-tagged Dixdc1 with FLAG-tagged DISC1 fragments in HEK293 cells. These experiments revealed that Dixdc1 binds most strongly to the C-terminus of DISC1 (fragment 4), and weakly to the middle region of DISC1 (fragment 3; ). Conversely, we mapped the region(s) of Dixdc1 which associate with DISC1. We generated and expressed FLAG-tagged Dixdc1 fragments with full-length GFP-DISC1 in HEK293 cells. We determined that DISC1 strongly binds to the N-terminal Dixdc1 region (fragment 2) that lies between the calpain homology and coiled-coil domains (). Taken together, these data demonstrate that Dixdc1 binds DISC1 in vivo during early brain development.
Dixdc1 is essential for neural progenitor proliferation
DISC1 is required for the regulation of neural progenitor proliferation and neuronal migration during embryonic cortical development (Kamiya et al., 2005
; Mao et al., 2009
) Given our data that Dixdc1 binds DISC1, we first examined the developmental expression profile of Dixdc1 to determine if it might have functions similar to DISC1. Western blot analysis of brain lysates from different ages revealed that the long and short isoforms of Dixdc1 (l-Dixdc1 and s-Dixdc1) are highly expressed as early as embryonic day 10 (E10) and persist throughout the neurogenic period (E11–17; Figure S1A
). However, following E18, the expression of s-Dixdc1, is downregulated to nearly undetectable levels, while l-Dixdc1 expression persists into adulthood. We then performed immunostaining on embryonic brains and found that Dixdc1 co-localizes with Nestin-positive radial glial cells, which give rise to neurons, in the E12 and E15 cortex (Figure S1B
). We further determined that Dixdc1 also co-localizes with the neuronal marker β-III tubulin (Tuj1) in the E15 and E17 cortex, demonstrating that Dixdc1 is expressed in both neural progenitor cells (radial glia) and postmitotic neurons (Figure S1C
). This staining pattern is in good agreement with a previous study that examined the expressed pattern of Dixdc1 using in situ
hybridization (Shiomi et al. 2003
). Together, the early expression pattern of Dixdc1 suggests that it may play a role in progenitor proliferation and neuronal differentiation.
To determine whether Dixdc1 regulates neural progenitor proliferation, we knocked down the expression of Dixdc1 using shRNA. We tested different shRNA constructs for their ability to reduce exogenous and endogenous Dixdc1 expression, and selected two that produced the most robust knockdown (Figure S2A–C
). We utilized in utero
electroporation to introduce Dixdc1 shRNA constructs together with a GFP-encoding construct into neural progenitor cells of the developing cortex in E13 mouse embryos, and analyzed brains at E16. We found that the knockdown of Dixdc1 resulted in a considerable change in cell distribution compared to scrambled shRNA controls. There was a significant loss of GFP-positive cells from the ventricular/subventricular zones (VZ/SVZ), a phenotype that is similar to that seen with DISC1 knockdown () (Mao et al., 2009
). This effect was also accompanied by a reduction of GFP-positive cells in the cortical plate (CP), suggesting an early migration defect. This resulted in Dixdc1 shRNA-treated brains having the majority of the GFP-positive cells accumulating in the intermediate zone (IZ) compared to control brains, which had an approximately equal percentage of GFP-positive cells in each of the three zones ().
Dixdc1 regulates neural progenitor cell proliferation in vivo
To examine if the loss of GFP-positive cells from the VZ/SVZ was due to reduced neural progenitor proliferation, we injected BrdU into pregnant dams 24 hours prior to brain analysis at E16. Knockdown of Dixdc1 resulted in a significant decrease in BrdU incorporation into GFP-positive cells (), suggesting that Dixdc1 is required for neural progenitor proliferation. These results, together with the observed reduction in the number of GFP-positive cells in the VZ/SVZ, imply that the loss of Dixdc1 function leads to premature cell cycle exit and neuronal differentiation. To test this directly, we first performed an analysis of cell cycle exit by electroporating embryonic brains at E13, pulse labeling with BrdU at E15, and collecting brains at E16, followed by immunocytochemistry for GFP, BrdU and Ki67. Using this analysis, we found that Dixdc1 shRNA caused a significant increase in the number of cells exiting the cell cycle (; see Methods for calculation). To examine if this result directly leads to increased neuronal differentiation, we stained electroporated brains with a neuronal βIII-tubulin (Tuj1) antibody. Quantification of GFP-Tuj1 double-positive cells revealed that the knockdown of Dixdc1 led to a significant increase in the percentage of GFP-labeled cells that were also positive for Tuj1, demonstrating that the loss of Dixdc1 resulted in an increase in neuronal differentiation (). Since there is high expression of Tuj1 in the axons located in the SVZ, this can potentially lead to an overestimation of the percentage of GFP-Tuj1 double-positive cells. To circumvent this, we co-electroporated control or Dixdc1 shRNA plasmids together with an mCherry construct and an expression plasmid encoding GFP under the neuron-specific pNeuroD promoter (Yokota et al., 2007
). We quantified the percentage of mCherry-positive cells that were positive for pNeuroD-GFP, and found that the loss of Dixdc1 expression led to a significant increase in neuronal differentiation (), in accordance with our Tuj1 immunochemistry results.
Dixdc1 has also been shown to bind actin directly through its calpain homology domain (Wang et al., 2006
). It is therefore possible that the loss of Dixdc1 expression may produce progenitor proliferation defects by disrupting the apical-basal polarity of radial glial cells. To examine this possibility, we performed immunolabeling for Nestin, to label radial glia, and phalloidin staining for F-actin, which is highly concentrated in the end feet of radial glial cells at the apical surface. We found that the staining pattern of these markers was not disrupted after Dixdc1 knockdown (Figure S2E–G
). Taken together, these experiments demonstrate that the downregulation of Dixdc1 expression leads to reduced progenitor proliferation, which temporarily leads to premature cell cycle exit and increased neuronal differentiation.
Dixdc1 and DISC1 co-regulate Wnt-GSK3β/β-catenin signaling
Given our results that DISC1 and Dixdc1 bind one another in vivo
, and that DISC1 modifies the Wnt pathway in progenitor cells (Mao et al., 2009
), we hypothesized that Dixdc1 and DISC1 may functionally interact to modulate Wnt-mediated GSK3β/β-catenin signaling and thereby regulate neural progenitor proliferation. We first investigated the function of Dixdc1 in Wnt signaling, since Dixdc1 has been reported to stimulate TCF/LEF activity (Shiomi et al., 2003
). To measure Wnt-mediated GSK3β/β-catenin activity, we utilized a luciferase reporter construct containing eight copies of the TCF/LEF binding site (8XSuperTOPFLASH), since β-catenin stimulates transcription of genes containing TCF/LEF binding sites (Molenaar et al., 1996
; van de Wetering et al., 1997
). We knocked-down Dixdc1 expression, and determined that the Wnt3a-stimulated TCF/LEF-reporter activity was reduced by approximately 50% compared to shRNA controls (). We also found that overexpression of Dixdc1, compared to GFP alone, in cells that were stimulated with either control conditioned media (Control CM) or Wnt3a conditioned media (Wnt3a CM), resulted in a significant increase in TCF/LEF-reporter activity (). These findings are similar to our previously reported results for DISC1 (Mao et al., 2009
), and suggest that DISC1 and Dixdc1 may co-modulate the Wnt pathway in neural progenitor cells.
Dixdc1 and DISC1 co- regulate Wnt-GSK3β/β-catenin signaling
To further elucidate the concomitant roles of these proteins, we examined whether DISC1 and Dixdc1 functioned together to regulate Wnt-TCF/LEF signaling. We overexpressed Dixdc1 and DISC1 together, and observed that when cells were stimulated with either Control CM or Wnt3a CM, the expression of the two genes together additively activated TCF/LEF-reporter activity compared to either gene alone (). We then tested the relationship between Dixdc1 and DISC1 in Wnt signaling. First, we found that treatment with both Dixdc1 and DISC1 shRNA together resulted in significantly reduced TCF/LEF-reporter activity compared to knockdown of either gene alone, demonstrating the interaction between these genes strongly regulate Wnt signaling (). Interestingly, we also found that the decreased TCF/LEF-reporter activity due to Dixdc1 knockdown could be completely rescued by the overexpression of DISC1 (). Conversely, the DISC1 shRNA-mediated decrease in TCF/LEF-reporter activity could be rescued by overexpression of Dixdc1 (), suggesting that Dixdc1 and DISC1 can functionally compensate for each other's TCF/LEF activity when the reciprocal gene is overexpressed. Interestingly, we also found that overexpressing Dixdc1 Fragment 2, which inhibits interaction between DISC1 and Dixdc1 (Figure S7
), also significantly reduced TCF/LEF-reporter activity, demonstrating the interaction between DISC1and Dixdc1 is required for Wnt signaling (). Importantly, overexpression of a degradation-resistant β-catenin, which stabilizes its expression levels, also rescues the Dixdc1 shRNA-mediated decrease in reporter activity (), demonstrating that β-catenin signals downstream of Dixdc1, similar to DISC1 (Mao et al., 2009
). Finally, we determined that the Dixdc1 shRNA-mediated reduction in TCF/LEF-reporter activity could be completely rescued by application of GSK3β inhibitor (SB216763), suggesting that Dixdc1 regulates β-catenin through GSK3β, similar to DISC1 ().
Since these experiments were performed in vitro, we asked if Dixdc1 and DISC1 co-regulated Wnt-TCF/LEF signaling in vivo. Using in utero electroporation, we found that both DISC1 and Dixdc1 shRNA significantly reduced TCF/LEF-reporter activity in neural progenitor cells to the same degree (). Similar to the in vitro results, we found there was a further significant decrease in TCF/LEF-reporter activity when expression of both DISC1 and Dixdc1 were reduced in vivo. In addition, we found the DISC1 or Dixdc1 shRNA reduced TCF/LEF-reporter activity could be completely rescued by overexpression of the reciprocal gene (). Lastly, we performed gain of function experiments in vivo, and demonstrated that overexpression of either Dixdc1 or DISC1 alone significantly increases TCF/LEF-reporter activity, while overexpression of both genes together additively enhances TCF/LEF signaling (). Taken together, these results demonstrate both Dixdc1 and DISC1 are necessary for Wnt signaling, and functionally interact in an additive manner to regulate Wnt-GSK3β/β-catenin signaling in neural progenitor cells.
Dixdc1 and DISC1 functionally interact to modulate neural progenitor proliferation
Our results demonstrating that Dixdc1 and DISC1 are both required for Wnt-GSK3β/β-catenin signaling suggested that these factors functionally interact to regulate neural progenitor proliferation in vivo
. To test this, we knocked down expression of Dixdc1, DISC1 or both genes together in vivo
, and observed that the double gene knockdown led to significantly fewer GFP-positive cells remaining in the VZ/SVZ compared to knocking down expression of either gene alone (). In addition, we found the double gene knockdown also led to significantly reduced Brdu incorporation, increased cell cycle exit and increased neuronal differentiation compared to reducing expression of either gene alone (, Figure S3
). These data suggest that Dixdc1 is a major partner of DISC1 in the regulation of neural progenitor proliferation.
Dixdc1 and DISC1 functionally interact to regulate neural progenitor proliferation
Since our previous results demonstrated that loss of Dixdc1 or DISC1 leads to reduced Wnt signaling which can be restored by overexpression of the reciprocal gene, we hypothesized that overexpression of either gene would also rescue the other's loss-of-function cellular phenotype in the developing cortex. For these experiments, we first co-expressed full length DISC1 together with Dixdc1 shRNA in the developing mouse cortex. Remarkably, we found that DISC1 overexpression could completely rescue the Dixdc1 shRNA-mediated loss of GFP-positive cells from the VZ/SVZ (). Further analysis revealed that the reduction in BrdU incorporation, increase in cell cycle exit and increased neuronal differentiation due to Dixdc1 shRNA were also completely rescued (, Figure S3
). These experiments demonstrate that the overexpression of DISC1, which stimulates TCF/LEF activation, is sufficient to restore normal progenitor proliferation after Dixdc1 knockdown.
To test whether directly increasing TCF/LEF signaling rescues the Dixdc1 shRNA-mediated progenitor proliferation defects, we co-expressed Dixdc1 shRNA together with a degradation-resistant β-catenin construct. Importantly, we found that the Dixdc1 shRNA-mediated defects in cortical VZ/SVZ cell positioning, decrease in BrdU incorporation, increase in cell cycle exit and increased neuronal differentiation were all completely restored to control shRNA levels when β-catenin levels were stabilized (; Figure S4
). However, the loss of cells in the cortical plate after Dixdc1 knockdown was not rescued after stabilizing β-catenin levels, suggesting β-catenin signaling plays no role in neuronal migration. This demonstrates that β-catenin mediates TCF/LEF signaling and neural progenitor proliferation downstream of Dixdc1, similar to our previously reported results regarding DISC1 (Mao et al., 2009
Dixdc1 and DISC1 regulate progenitor proliferation, but not neuronal migration, via β-catenin signaling
We also examined whether overexpression of Dixdc1, which stimulates Wnt-TCF/LEF activity, could rescue the neural progenitor proliferation deficits induced by DISC1 shRNA. We co-electroporated embryonic brains with full-length Dixdc1 and DISC1 shRNA, and determined that the reduced neural progenitor proliferation, increased cell cycle exit and increased neuronal differentiation were completely rescued compared to the DISC1 shRNA conditions (, Figure S3
). Together, these studies demonstrate that the loss-of-function of one gene in vivo
leads to the cellular phenotypes observed due to reduced downstream Wnt-TCF/LEF signaling in neural progenitor cells. Furthermore, these deficits can be rescued by overexpression of the reciprocal gene. These data suggest that progenitor proliferation is tightly regulated downstream of the DISC1-Dixdc1 interaction by the levels of β-catenin, which modulate TCF/LEF signaling.
Dixdc1 regulates neuronal migration in the developing cortex
Initial studies of DISC1 reported that it regulates the radial migration of newborn neurons during the later, but not early, stages of cortical development (Kamiya et al., 2005
; Mao et al., 2009
). To analyze whether Dixdc1 regulates migration, we performed in utero
electroporation with Dixdc1 shRNA at a later time point (E15) and analyzed brains at E19, a period of time when neuronal migration is prevalent. We found that knocking down the expression of Dixdc1 resulted in a profound migration defect, where the majority of GFP-positive cells were arrested in the IZ compared to control conditions, where the majority of GFP-positive migrated to the upper cortical plate (Figure S5A
). Since knocking down expression of Dixdc1 led to progenitor proliferation defects (), we confirmed that the inhibition of migration was not due to disrupted neural differentiation by designing a Dixdc1 shRNA-encoding plasmid under the control of the pNeuroD promoter to restrict Dixdc1 knockdown specifically to neurons (Figure S2D
). Using this approach, we confirmed that the knockdown of Dixdc1 in neurons also inhibited neuronal migration (Figure S5A
). To determine if the inhibition of neuronal migration was a temporary effect, we electroporated E15 embryonic brains with Dixdc1 shRNA, and analyzed the brains at postnatal day 6 (P6). Interestingly, the downregulation of Dixdc1 expression caused a persistent neuronal migration deficit (Figure S5B
), where GFP-positive cells were still arrested in the IZ which, at this time point, is populated with white matter projections. The inhibited radial migration could also be due to a change in cell fate, where Dixdc1 shRNA-expressing cells may acquire the identity characteristic of neurons present in the deeper cortical layers. Therefore, we stained the cortex with different layer specific markers. First, we found that GFP-positive cells lacking Dixdc1 still expressed the mature neuronal marker NeuN, demonstrating that these cells are postmitotic neurons (Figure S5C
). Second, we observed no difference in the proportion of GFP-positive cells immunoreactive for the upper layer markers Cux1 and Tbr1, or the deeper layer marker Foxp2, compared to control conditions (Figure S5D–F
). Collectively these data strongly argue that Dixdc1 is required for neuronal migration during cortical development.
Dixdc1 and DISC1 do not regulate neuronal migration through GSK3β/β-catenin-mediated signaling
Our data suggest that Dixdc1 and DISC1 interact to regulate neural progenitor proliferation via the modulation of Wnt-GSK3β/β-catenin signaling. Since both Dixdc1 and DISC1 loss-of-function result in inhibited neuronal migration, we hypothesized that they also regulate migration together via Wnt signaling. To assess this, we electroporated E15 embryos with either Dixdc1 or DISC1 shRNA, and asked whether stimulation of TCF/LEF signaling by overexpression of the reciprocal gene would rescue the migration deficit. We found that, in both experimental conditions, overexpression of the reciprocal gene did not restore neuronal migration (). We then directly examined whether increasing β-catenin levels would rescue the migration deficits specifically in neurons after DISC1 or Dixdc1 knockdown. We co-electroporated Dixdc1 or DISC1 shRNA together with degradation-resistant β-catenin expressed under the neuronal pNeuroD promoter to restrict its expression to neurons. Using this assay, we did not observe a rescue of radial migration after Dixdc1 or DISC1 downregulation (). Together, these experiments suggest that Dixdc1 and DISC1 regulate migration via a pathway independent of Wnt-GSK3β/β-catenin signaling, which is in contrast to their regulation of progenitor proliferation via the Wnt-pathway.
Dixdc1 interacts with the DISC1-binding partner Ndel1
We next examined whether an alternative pathway mediates DISC1/Dixdc1-dependent radial migration. Since DISC1 has been previously reported to bind Ndel1 to regulate neuronal migration (Kamiya et al., 2005
), we hypothesized that Dixdc1 might also bind Ndel1 in vivo
, and regulate neuronal migration via a tripartite Dixdc1/DISC1/Ndel1 complex. In a tandem-affinity purification screen using HA and Flag epitope-tagged Ndel1 as bait, we discovered that Dixdc1 is indeed a Ndel1 binding partner (). We confirmed this interaction in HEK293 cells, finding that overexpressed GFP-tagged Dixdc1 interacted with endogenous Ndel1 (). Furthermore, we determined that Dixdc1 and Ndel1 could endogenously co-immunoprecipitate one another in E17 brain lysate together with Lis1, a known Ndel1 interacting partner that has important roles in radial migration (). To test if DISC1 was also part of the Dixdc1/Ndel1 complex, we performed co-immunoprecipitation experiments in brain tissue at E18. We determined that Dixdc1 can be co-immunoprecipitated in a complex containing DISC1 and Ndel1 (), suggesting that this tripartite complex exists at later embryonic time points. We then mapped the Ndel1-binding site(s) on Dixdc1 using Flag-tagged Dixdc1 fragments expressed together with GFP-tagged Ndel1 in HEK293 cells. Surprisingly, we found that Ndel1 binds to the same Dixdc1 N-terminal region as DISC1, the Dixdc1 fragment 2 (). This suggests that Dixdc1 may facilitate the interaction between DISC1 and Ndel1.
The DISC1/Dixdc1 complex interacts with Ndel1 in a developmental manner and is regulated by Cdk5-mediated phosphorylation of Dixdc1
Cdk5 phosphorylates Dixdc1 to regulate binding to Ndel1
Given that both DISC1 and Ndel1 bind to the same N-terminal region of Dixdc1, the interaction of this complex could be regulated by the modification of Dixdc1 in this region. We hypothesized that Dixdc1 may be phosphorylated, and therefore screened different kinases to determine the potential phosphorylation of the N-terminal region. We discovered that Dixdc1 is phosphorylated by cyclin-dependent kinase 5 (Cdk5) at serine 250, which falls within the region of Dixdc1 to which both DISC1 and Ndel1 bind (). Cdk5 has been previously demonstrated by our lab and others to be important for neuronal migration via its regulation of cortical layering through phosphorylation of a number of substrates (Niethammer et al., 2000
; Sasaki et al., 2000
; Tanaka et al., 2004
; Xie et al., 2006
; Xie et al., 2003
). Based on our discovery that the serine 250 residue of Dixdc1 is phosphorylated by Cdk5, we hypothesized that this phosphorylation event regulates the formation of the Dixdc1/DISC1/Ndel1 complex. To examine this, we asked whether inhibiting phosphorylation at residue 250 on Dixdc1, by changing serine to alanine (Ser250Ala), altered the binding of Dixdc1 to either DISC1 or Ndel1. We overexpressed flag-tagged Dixdc1 or a Dixdc1 Ser250Ala mutant together with GFP-tagged DISC1 or GFP-tagged Ndel1. Interestingly, we found significantly reduced binding of Dixdc1 Ser250Ala to Ndel1, but not DISC1, compared to wild-type Dixdc1 (). This suggests Cdk5-phosphorylation of Dixdc1 does not affect its binding to DISC1 and therefore does not affect its function in Wnt signaling or neural progenitor proliferation. To confirm this, we overexpressed Dixdc1 Ser250Ala and observed that it significantly potentiated Wnt-TCF/LEF reporter activity and increased neural progenitor proliferation similarly to WT-Dixdc1 (Figure S6A,B
). Furthermore, we found that knocking down expression of Ndel1 has no effect on Wnt signaling (Figure S6C
), suggesting Ndel1 itself is not in the canonical Wnt pathway. Taken together, these data demonstrate that the binding between Dixdc1 and Ndel1 is regulated by Cdk5-mediated phosphorylation of Dixdc1 at serine 250, which may regulate neuronal migration, but not neurogenesis or Wnt signaling.
Phosphorylation of Dixdc1 and its interaction with DISC1/Ndel1 is essential for radial migration
Our data suggest that both DISC1 and Ndel1 bind to the same N-terminal region of Dixdc1, and that the phosphorylation of Dixdc1 at serine 250 is particularly important for its interaction with Ndel1. Based on these results, we first asked whether this phosphorylation site is essential for neuronal migration by inhibiting the phosphorylation of Dixdc1 at serine 250. To achieve this, we used a 3' UTR Dixdc1 shRNA construct that does not target the coding region, and compared the ability of full length WT-Dixdc1 versus the Dixdc1 Ser250Ala mutant to rescue migration. We observed that, while WT-Dixdc1 was able to significantly restore migration, the Dixdc1 Ser250Ala mutant was not able to rescue migration (). These results demonstrate that the Cdk5-mediated phosphorylation of Dixdc1 at serine 250, which regulates binding to the DISC1-interacting protein Ndel1, is essential for radial migration.
The phosphorylation of Dixdc1 and its interaction with the DISC1/Ndel1 complex is essential for radial migration
We also asked whether the interaction between Dixdc1 and DISC/Ndel1 is required for neuronal migration. We overexpressed HA-tagged Dixdc1 fragment 2 (181–370aa), which inhibited the interaction between Flag-tagged Dixdc1 and GFP-tagged Ndel1 or GFP-tagged DISC1 (Figure S7
). We then overexpressed the different peptide fragments of Dixdc1 together with GFP in vivo
and observed that the overexpression of Dixdc1 fragment 2 was sufficient to completely inhibit radial migration, producing a phenotype very similar to that seen with the application of Dixdc1 or DISC1 shRNA (). Interestingly, the adjacent Dixdc1 fragment 1, which includes the actin binding domain, also produced a neuronal migration phenotype, suggesting that Dixdc1 may also regulate actin dynamics. However, Dixdc1 fragments 3 and 4 did not disrupt migration, demonstrating that the C-terminus of Dixdc1 is not involved in migration. Taken together, these data strongly suggest that the interaction between Dixdc1 and DISC1/Ndel1 in vivo
, which is regulated by Cdk5-mediated phosphorylation of Dixdc1, is essential for the radial migration of neurons during cortical development.
Finally, to determine how Cdk5-mediated phosphorylation of Dixdc1 regulates migration, we analyzed the morphology of GFP-positive cells in vivo. Interestingly, we found that GFP-positive cells either lacking Dixdc1 or expressing Dixdc1 Ser250Ala failed to adopt the bipolar morphology that control shRNA or WT-Dixdc1-expressing cells acquired. Instead, these cells were arrested in the intermediate zone and displayed multiple thin and dystrophic processes (). To further determine if this morphological observation was due to a disrupted cytoskeletal network, we cultured GFP-positive cells from electroporated brains. Here we found that GFP-positive cells lacking Dixdc1 or expressing Dixdc1 Ser250Ala had overall diminished F-actin and α-tubulin staining in the neurites (). Taken together, this data suggests Dixdc1, and its phosphorylation by Cdk5, mediates neuronal migration by regulating cellular morphology through modulation of the actin and microtubule cytoskeleton.
Phosphorylation of Dixdc1 regulates the morphology and structural integrity of neurons