Cortical projection neurons maintain their identities in dissociated cultures
Projection neurons sharing particular pathways and targets can be identified in vivo
by their expression of particular transcription factors. Satb2 is expressed in the majority of callosal projection neurons, while Ctip2 or Tbr1 are expressed in neurons sending axons to subcortical targets (Arlotta et al., 2005
; Chen et al., 2005
; Alcamo et al., 2008
; Britanova et al., 2008
; Chen et al., 2008a
). To assess whether cortical neurons maintain their identities in culture we immunolabeled for Satb2 and Ctip2 at the time of plating and after 2, 4 or 6 days in culture. The percentage of neurons immunolabeled for Satb2 (Satb2pos
) steadily increases from 26% immediately after plating to a maximum of 62% at four days in vitro
(DIV) after which it rapidly diminishes (). Satb2pos
neurons show little to no labeling for either Ctip2 or Tbr1, which are expressed in a much lower proportion of cortical neurons (20% and 12% at 4div, respectively: ). The gradual increase in Satb2 expression in postmitotic neurons, its exclusion from neurons labeled for Ctip2 or Tbr1, and its subsequent loss closely resemble in vivo
observations (Alcamo et al., 2008
; Britanova et al., 2008
). The interval defining stable Satb2 expression, between 2 and 4 DIV, was used for all subsequent experiments.
Cortical neurons maintain their identity in dissociated cultures
All axons projecting corticofugally originate in deep cortical layers 5 and 6, whereas neurons having callosal projections reside mainly in superficial layers 2/3 and in layer 5a. We asked whether transcription factor expression in cultured neurons also reflects appropriate laminar destiny. In order to visualize lamina-specific cortical neurons in culture, ex-utero electroporation (EUE, see methods) was used to deliver yellow fluorescent protein (YFP) into progenitors of the ventricular zone at embryonic day (E)15.5, when layer 5 neurons are born in the rat somatosensory cortex, or E17.5, when layer 2/3 neurons are born (Bayer and Altman, 1990
; Koester and O'Leary, 1994
); cortices were dissociated immediately thereafter, cultured for 3 days, and then immunolabeled for transcription factors. Quantitative analysis indicates that half of the E15-born neurons express Satb2 () and many neurons lacking Satb2 (Satb2neg
) express Ctip2. In contrast, nearly 80% of the E17-born neurons express Satb2 () and none express Ctip2 alone. These results are consistent with observations in vivo
(Britanova et al., 2008
). Thus, cortical neurons express and continue to develop key cell-type specific characteristics when grown in culture, providing a tractable model system in which cell biological mechanisms contributing to cell-type specific responses can be identified and evaluated.
Satb2neg neurons respond more robustly to Sema3A
We asked whether Sema3A differentially affects axonal growth from cortical neuron populations defined by Satb2. Layer 5 neurons were labeled with YFP using EUE at E15, dissociated and exposed to Sema3A for 72h, and then immunolabeled for Satb2. Axonal growth was compared quantitatively by tracing axons in their entirety. The data show that E15-born Satb2neg
neurons have significantly shorter axons when grown in the presence of Sema3A compared to control conditions (), and consistent with previous work (Dent et al., 2004
) axon branching is diminished (30% reduction in branch number relative to control). In contrast, Sema3A has no significant effect on the length of axons originating from E17-born neurons, the vast majority of which express Satb2 (ANOVA, p > 0.05; n=50).
Sema3A triggers distinct responses in two populations of cortical neurons
Acute exposure to Sema3A can produce growth cone collapse (Luo et al., 1993
). To compare growth cone collapse in Satb2pos
neurons, axonal growth cones were imaged for thirty minutes before and following addition of Sema3A-Fc, after which cell bodies were immunolabeled post-hoc for Satb2. A significantly greater proportion of Satb2neg
neurons show growth cone collapse ( and legend).
Sema3A binds chondroitin sulfate proteoglycans and thus may be presented to neurons as part of the extracellular matrix rather than as a soluble cue (de Wit and Verhaagen, 2003
). To test whether cortical axon populations respond differentially to a substrate of Sema3A, we plated neurons on stripes of Sema3A-Fc alternating with control Fc stripes. A comparison of the number of border crossings shows that axons of Satb2neg
neurons avoid Sema3A stripes while axons of Satb2pos
neurons grow equally well on Sema3A and control stripes ().
The data indicate that axons of Satb2neg
neurons respond more robustly to the repulsive effects of Sema3A than axons of Satb2pos
neurons. We next tested whether this difference is dictated by the restricted expression of transcription factors. Overexpression of Satb2 would be predicted to reduce responsiveness to Sema3A, but it also increases cell death and could not be used for this. In mice lacking Satb2, Ctip2 expression is permitted in layer 2/3 neurons, many of which send axons inappropriately to subcortical targets (Alcamo et al., 2008
; Britanova et al., 2008
). With this in mind, we asked whether Ctip2 overexpression in a mixed population of cortical neurons (Satb2pos
) would increase responsiveness to Sema3A. The data show that Ctip2-overexpression increases axon outgrowth over 72h and that Sema3A abrogates this effect (Supplementary Fig. 1
). Moreover, in a stripe assay we see that axons of Ctip2 overexpressing neurons avoid Sema3A stripes (). Thus, Ctip2 expression enhances axon responsiveness to Sema3A.
All cortical neurons express similar levels of Sema3A receptors
Guidance cue responsiveness can be regulated by selective expression of receptors and co-receptors (Hong et al., 1999
; Liu et al., 2005
; Chauvet et al., 2007
). While all cortical neurons express Npn1, PlexA4 and L1CAM mRNAs, we asked whether subcellular distribution of the receptors could alter responsiveness. Immunocytochemistry () and mean fluorescence intensity measurements (not shown) show that growth cones of Satb2pos
neurons express similar levels of Npn1, PlxA4, and L1CAM receptors.
Subunits of the Sema3A receptor complex are expressed at similar levels in cortical axonal growth cones
Although the total pool of receptors is similar, their availability on the growth cone surface may differ. However, surface labeling for Npn1 and L1CAM was also similar in Satb2pos and Satb2neg axons (). It remains possible that PlexinA4 is differentially maintained at the surface, but we were unable to detect reliable PlexinA4 labeling under non-permeabilizing conditions.
Axonal internalization of Sema3A correlates with responsiveness
Sema3A induces endocytosis (Fournier et al., 2000
; Jurney et al., 2002
; Castellani et al., 2004
; Piper et al., 2005
), so we asked whether different cortical neuron populations could be differentiated based on their ability to internalize Sema3A. In order to examine Sema3A internalization directly, we tagged recombinant Sema3A-hFc with a quantum (Q) dot conjugated Fab fragment that recognizes the hFc-tail. Neurons were incubated with Sema3A-Qdots and the internalized pool was visualized selectively by stripping surface ligands with an acidic wash. Growth cones, dendrites and cell bodies all show internalized fluorescent puncta which are heterogeneous in shape and size. In order to compare populations, we used EUE to label layer 5 or layer 2/3 neurons and then quantified the level of internalized Sema3A in growth cones. E15-born neurons internalize twice the level of Sema3A-Qdots as E17-born neurons (). To confirm that the enhanced internalization capacity resides in layer 5 neurons lacking Satb2, neurons were immunolabeled post-hoc for Satb2, Ctip2, or Tbr1. As expected, Satb2neg
neurons internalize significantly more Sema3A than Satb2pos
neurons (Supplementary Fig.2A,B
), and these Satb2neg
neurons are either corticospinal or corticothalamic projecting neurons as they express Ctip2 or Tbr1, respectively (Supplementary Fig.2C,D
Sema3A internalization is distinct between deep and superficial layer neurons
Despite the similar distribution of Sema3A receptors in all cortical growth cones, it is possible that Satb2neg neurons can bind more Sema3A. To test this Sema3A-Qdot binding was assessed in neurons that were cooled to 12ºC, which prevents internalization. Satb2neg growth cones bind a modestly greater amount of Sema3A than Satb2pos growth cones, but this difference is not significant (1.0 ± 0.12 vs. 0.71 ± 0.2, t-test, p = 0.17; n= 9 per group). More notable is that surface bound Sema3A appears more diffuse in Satb2neg compared to Satb2pos neurons (). This observation suggests that Sema3A receptors might be differentially clustered and/or distributed within membrane domains of growth cones.
Raft-mediated endocytosis is the principal mode of Sema3A internalization in responsive growth cones
Mammalian cells employ distinct endocytic paths in order to compartmentalize cargo within contexts that are significant for downstream signaling, recycling, or degradation (Nichols and Lippincott-Schwartz, 2001
; Pelkmans and Helenius, 2002
; Conner and Schmid, 2003
). In order to identify the route taken by Sema3A, we compared the two most prominent pathways: Clathrin-mediated endocytosis (CME) and raft-mediated endocytosis (RME). We first confirmed that the two paths are present in cortical neurons by comparing internalization of rhodamine-tagged Transferrin, a marker for CME with FITC-tagged Cholera-toxin B, which binds GM1 gangliosides and is used to tag sphingolipid enriched membrane rafts. As expected, there are endosomes tagged with Transferrin alone, or Cholera toxin alone, in addition to a population tagged with both (Pelkmans et al., 2004
) (Supplementary Fig. 3
We next tested the impact of RME or CME blockade on Sema3A internalization. In axonal growth cones of E15-born neurons, filipin, an inhibitor of RME, dramatically reduces levels of internalized Sema3A (), while monodansyl-cadaverin (MDC), an inhibitor of CME, has virtually no effect (). In contrast, in growth cones of E17-born neurons, which internalize much less Sema3A overall, both filipin and MDC decrease levels of internalized Sema3A suggesting that both endocytic routes contribute equally ().
These findings suggest that RME may be responsible for the repelling effects of Sema3A. To test this, we assayed Sema3A-mediated growth cone collapse in neurons exposed to filipin. Consistent with a critical role for RME, filipin prevents collapse (, Guirland et al, 2004
Since CME contributes to Sema3A internalization in layer2/3 neurons, we asked whether it counters the impact of RME. However, the data show that blocking CME in the less responsive Satb2pos neurons does not enhance Sema3A-mediated growth cone collapse (). Taken together, these data indicate that Sema3A internalization via RME is required for its repulsive effects in Satb2neg neurons.
Molecular mechanisms controlling the raft-mediated internalization of Sema3A
To identify proteins that could contribute to Sema3A internalization, we first assembled a list of proteins known to function downstream of Sema3A, Npn1, L1CAM, and PlexinAs (PlxA) (). Using Genes2networks (Berger et al 2007), we employed this seed list to find previously reported protein-protein interactions identified experimentally in mammalian cells that would “connect” the seed list proteins through additional intermediate proteins. This approach generated a list of potential one step-intermediate interactions between members of the seed list. The candidate proteins () were ordered according to their calculated z-scores, which is computed based on the candidate protein links in the seed sub-network compared to the candidate's known previously reported direct physical protein interactions (Supplementary Table 1
). The higher a protein's z-score, the more likely the protein is to function specifically downstream of Sema3A. Of the 33 proteins identified having z-scores higher than 2.5, two proteins stood out because of their known role in endocytosis: Flotillin-1 and -2, also known as Reggie-2 and -1 (Bickel et al., 1997
; Lang et al., 1998
) (z-score = 3.9, 6.2, respectively). Flotillins are members of the SPFH-domain family of proteins, which bind lipid rafts. Significantly, Glebov and colleagues have shown that in HeLa cells Flotillins define and are required for a caveolin-independent RME pathway (Nichols and Lippincott-Schwartz, 2001
; Glebov et al., 2006
; Frick et al., 2007
Flotillin-1 and -2 are novel candidates downstream of Sema3A and are immunodetected in cortical axons in vivo and in vitro
To determine whether developing cortical axons express Flotillins, we immunolabeled E14.5 neocortex for Flotillin-1 or -2. Immunolabeling for both Flotillins can be seen throughout the neocortex. The overlying pia and blood vessels show the highest levels of immunolabeling, while lower levels are observed throughout the cortical plate, intermediate zone, and ventricular zone. In the intermediate zone and internal capsule, where corticocortical and corticofugal axons travel, Flotillin labeling colocalizes with F-actin-labeled fibers (). In axonal growth cones of dissociated neurons, where Flotillin localization can be examined at a higher resolution, labeling is primarily punctate ().
Flotillin-1 responds to Sema3A stimulation
In non-neuronal cells, Flotillins relocalize from smaller patches on the plasmalemma to larger and brighter clusters in endosomal compartments in response to EGF (Neumann-Giesen et al., 2007
; Riento et al., 2009
). Thus, to determine whether Flotillins respond similarly to Sema3A, we used an imaging based approach. In Satb2neg
neurons, Flotillin-1 appears less clustered than Flotillin-2 in unexposed growth cones. In response to Sema3A, only Flotillin-1 responds, becoming more clustered (). In axons from Satb2pos
neurons, the distribution of both Flotillins appears similar to Satb2neg
growth cones, but neither changes in response to Sema3A (). These observations were analyzed quantitatively by plotting the mean intensity vs. the area labeled in growth cones, and the data show that Sema3A selectively increases the appearance of low to medium intensity clusters of Flotillin-1 in satb2neg
Sema3A increases Flotillin-1 clustering in growth cones of Satb2neg but not Satb2pos neurons
The Sema3A-stimulated increase in fluorescence is prevented by preincubation with filipin () and unchanged by MDC (), consistent with Flotillin-1 recruitment to cholesterol enriched membrane microdomains. Based on previous work, the increased Flotillin-1 labeling most likely arises from the clustering of a pre-existing pool (Neumann-Giesen et al., 2007
; Riento et al., 2009
), but it is also possible that Flotillin is newly synthesized. To test this we assayed Sema3A-dependent clustering when protein translation was blocked by anisomycin. Anisomycin had no effect (Supplementary Fig. 4A,B,E,F
In HeLa cells, EGF-mediated Flotillin recruitment requires the activation of fyn kinase (Neumann-Giesen et al., 2007
; Riento et al., 2009
). Since fyn activation is also essential for Sema3A function (Morita et al., 2006
), we asked whether exposure to the Src family kinase inhibitor SU6656 would alter Flotillin-1 recruitment in response to Sema3A. Flotillin-1, but not Flotillin-2, acquires a diffuse expression in cortical axons in response to SU6656, and exposure to Sema3A only modestly increases its clustered appearance from this low baseline level (Supplementary Fig. 4A-D
). These data suggest that the higher capacity of Satb2neg
neurons to internalize Sema3A via a raft-mediated pathway likely results from their ability to cluster Flotillin-1 in a src-dependent manner in response to Sema3A.
Flotillin-1 is required for Sema3A internalization
Since Flotillin-1 responds robustly to Sema3A in Satb2neg
neurons, we asked whether it is required for Sema3A internalization. To test this, we knocked down Flotillin-1 using an equal mix of three shRNAs, each of which target different regions of the mRNA (see methods), and together significantly decrease Flotillin-1 levels to 48% of that in neurons expressing control shRNAs (Supplementary Fig. 5A-C
). Flotillin-1 knockdown significantly decreases Sema3A internalization in growth cones to 35% of control values ().
Flotillin-1 is required for the Dynamin-independent, ERM-dependent endocytosis of Sema3A
Previous work suggests that Flotillin-defined RME does not require dynamin-mediated fission (Glebov et al., 2006
). To test whether the RME pathway utilized by Sema3A is also dynamin-independent, we treated neurons with a specific and rapidly acting small molecule inhibitor of dynamin, dynasore (Macia et al., 2006
), and then quantified Sema3A-Qdot internalization. Dynasore treatment produced a modest, but insignificant decrease in Sema3A internalization ( and data not shown), supporting that this pathway is largely independent of dynamin. When dynasore was combined with Flotillin-1 shRNAs, there was a further, but not statistically signficant reduction in Sema3A internalization (). Together these data support that Sema3A utlizes a Flotillin-dependent, but dynamin-indendent endocytic pathway in growth cones from Satb2neg
We have previously shown that ERMs can also regulate internalization of Npn1 and L1CAM in response to Sema3A, most likely via their association with the intracellular tail of L1CAM (Mintz et al., 2008
). To test whether Flotillin-1 mediated endocytosis functions in the same pathway as ERMs, we compared Sema3A internalization in growth cones expressing an ERM dominant negative, NEz, which acts as a pan-ERM dominant negative (Algrain et al., 1993
; Dickson et al., 2002
), alone or together with Flotillin-1 shRNAs. As expected, NEz greatly reduces Sema3A internalization (). Combined with Flotillin knockdown, there is a similar reduction in Sema3A internalization. The absence of an additive effect suggests that Flotillin and ERMs function in the same pathway controlling the magnitude of Sema3A internalization ().
Flotillin-1 mediates signaling to the cytoskeleton and axonal responsiveness to Sema3A
The data suggest that Flotillin-1 mediated Sema3A internalization could be important for mediating the repulsive effects of Sema3A in axons from Satb2neg neurons. To test this, we assayed axonal growth inhibition in response to Sema3A in Satb2neg neurons expressing Flotillin-1 shRNAs. The data show that axonal growth inhibition is prevented (). Flotillin-1 knockdown also significantly inhibited Sema3A-mediated growth cone collapse (57.8% ± 3% vs. 38.5% ± 3%; n = 2 experiments; t-test, p < 0.01), and in neurons growing on alternating stripes of Sema3A and control substrates, flotillin-1 knock down produced a modest increase in the frequency of border crossings per axon (1.7 ±0.4 vs. 2.1 ± 0.3; n = 2 experiments;15 per group).
Flotillin-1 knockdown decreases Sema-mediated axon responses and LIMK activation
To mediate repulsion, Flotillin-based RME would be anticipated to initiate signalling pathways to the growth cone cytoskeleton. It has been shown previously that Sema3A-mediated growth cone collapse in dorsal root ganglia requires LIM kinase (LIMK)-1 mediated inactivation of cofilin (Aizawa et al., 2001
). LIM kinases are positively regulated by phosphorylation, so we first asked whether Sema3A stimulates LIMK phosophorylation in growth cones from Satb2neg
cortical neurons. As expected, 5min exposure to Sema3A increases phospho-LIMK labeling by nearly 3-fold, while showing no change in labeling for total LIMK (). Flotillin-1-knockdown reduces this response by a third. Taken together, the data show that in presumptive corticofugal axons, Flotillin-1 is required for the internalization of Sema3A generating endosomes that signal to the cytoskeleton via LIMK activation and thus can initiate axonal responsiveness.