Cortical progenitor cells are incorrectly positioned in the tish+/− and tish−/− neocortex
Given recent evidence that radial glial cells (RGCs) and intermediate progenitor cells (IPCs) are neurogenic (Noctor et al., 2001
; Noctor et al., 2002
; Noctor et al., 2004
), we sought to characterize the abnormally-positioned, proliferative cells that have been previously identified in the intermediate zone (IZ) and normally-positioned cortical plate (CP) of the developing tish−/−
neocortex (Lee et al., 1998
). Toward this end, we utilized immunohistochemistry to visualize Pax6+
RGCs and Tbr2+
IPCs (Englund et al., 2005
). Examination of wildtype, tish+/−
, and tish−/−
neocortices at time points corresponding to early, mid, and late cortical plate neurogenesis demonstrated that the heterotopic proliferative zone of the tish−/−
cortex contains both Pax6+
cells (). Early in cortical plate neurogenesis at E15, small groups of inappropriately located Pax6+
cells were found in the tish−/−
preplate region just beneath the pial surface ( arrows, compare with ; arrows, compare with ). These mislocalized cells were present in addition to the normally-positioned Pax6+
cells in the ventricular zone (VZ) and Tbr2+
cells in the subventricular zone (SVZ), but they were not observed in the corresponding preplate region in either wildtype or tish+/−
cortex. As development proceeded, these small groups of heterotopic Pax6+
cells coalesced into a diffuse band in the IZ and CP by E17 ( arrows, compare with ; arrows, compare with ). This band of mislocalized cells persisted through E20 ( arrows, compare with ; arrows, compare with ). Interestingly, at E17 in the tish+/−
neocortex, a few small clusters of Pax6+
cells were present in the IZ, suggesting a partial gene dosage effect for the tish gene in the positioning of Pax6+
progenitor cells during development (, arrowheads). Despite this partial positioning defect in tish+/−
animals, subcortical band heterotopia are observed only in tish−/−
animals and not in tish+/−
animals, suggesting that a greater number of mislocalized cells are required before the adult structural malformation can be established.
Figure 1 Pax6+ and Tbr2+ cells are incorrectly positioned in the tish+/− and tish−/− neocortex. Light microscopic images were taken of coronal sections of embryonic neocortex during timepoints corresponding to early, middle, at late cortical (more ...)
In order to establish whether the abnormally located Pax6+
cells were, in fact, mitotic progenitors or whether they were simply post-mitotic cells that had not yet downregulated a marker of an earlier stage of development (Hevner, 2006
), we employed co-labeling for Pax6 or Tbr2 and BrdU, β-III tubulin (β-tub, an early neuronal marker), and phosphorylated vimentin (pvim, a marker of mitotic cells). Cycling Pax6+
cells (i.e., Pax6+
cells) were observed in an appropriate position in the VZ or SVZ of wildtype, tish+/−
, and tish−/−
neocortices at E17 ( and ). In addition, inappropriately positioned Pax6+
cells were present in the tish−/−
IZ/CP, indicating that these cells were also actively engaged in the cell cycle ( and ). Similarly, mitotic Pax6+
cells were observed in the VZ or SVZ in wildtype, tish+/−
, and tish−/−
neocortices (); however, double-positive cells were also found in the tish−/−
IZ/CP, indicating that these mislocalized Pax6+
cells were undergoing mitosis ( and ).
Figure 2 Incorrectly positioned Pax6+ cells in the tish−/− neocortex are mitotic progenitor cells. Confocal images were taken of coronal sections of embryonic neocortex at E17. In E, J, and O, compressed stacks are presented. Vertical and horizontal (more ...)
Figure 3 Incorrectly positioned Tbr2+ cells in the tish−/− neocortex are mitotic progenitor cells. Confocal images were taken of coronal sections of embryonic neocortex at E17. In E, J, and O, compressed stacks are presented. Vertical and horizontal (more ...)
The positioning of immature neurons relative to RGCs and IPCs was examined using double labeling for Pax6 or Tbr2 and β-III tubulin. Appropriately positioned Pax6+/β-III tubulin- and Tbr2+/β-III tubulin- cells were found in the VZ or SVZ of wildtype, tish+/−, and tish−/− neocortices ( and ). Notably, the mislocalized Pax6+ and Tbr2+ cells in the tish−/− IZ/CP were also β-III tubulin−, indicating that these cells were indeed progenitor cells and not immature neurons ( and ). Although these cells were located in a region of dense β-III tubulin+ neuronal axons, three-dimensional reconstruction of confocal image stacks failed to reveal any colocalization between Pax6 or Tbr2 and β-III tubulin. Rather, β-III tubulin+ processes appeared to course around these Pax6+/β-III tubulin- and Tbr2+/β-III tubulin- cells ( and ). A similar phenotype was observed for the more limited population of Pax6+ and Tbr2+ cells in the IZ of tish+/− neocortex, indicating that these cells were also progenitor cells and not immature neurons (data not shown).
Taken together, these results indicate that a combination of mitotic RGCs and IPCs are present in the region of heterotopic proliferation in the tish−/− neocortex and, to a lesser extent, in the corresponding region of the neocortex of tish+/− animals as well. Furthermore, these results demonstrate that the number of heterotopic progenitors in the developing neocortex is a key determinant of SBH formation.
Progenitor cell density is increased in the tish−/− neocortex
Qualitative observations of the Pax6 and Tbr2 immunohistochemistry results suggested that tish−/−
neocortex contained more progenitor cells than wildtype cortex. We therefore sought to quantify these observations by measuring Pax6+
cell density in radial columns extending from the ventricular surface to the pial surface at each embryonic day from E15-E20. Pax6+
cell density across the depth of the cortical wall was increased in tish−/−
animals compared with wildtype or tish+/−
animals beginning on E17 and proceeding through E20 (). It is known that alterations in the size of the progenitor population can lead to changes in the size of the adult neocortex (Caviness et al., 2003
); however, previous work from our laboratory has shown that adult tish−/−
neocortex (normotopic cortex + SBH) is similar in volume to control neocortex (Lee et al., 1999
). We therefore hypothesized that the observed increase in progenitor cell density in tish−/−
neocortex must be offset by an increased amount of apoptosis during development. Indeed, activated caspase 3 immunohistochemistry at E17 revealed a significant increase in apoptotic cell death in the IZ/CP of tish−/−
neocortex compared with either tish+/−
or wildtype cortex. There was also a trend toward an increase in apoptosis in the tish−/−
VZ/SVZ (), although this effect did not achieve statistical significance. These data suggest that, in addition to a defect in progenitor cell positioning, a disruption in progenitor cell density also occurs in the developing tish−/−
neocortex. This alteration in cell density is offset during development by an increased level of cell death, leading to an adult neocortex whose volume is similar to that of wildtype neocortex.
Figure 4 Progenitor cell density and cellular apoptosis are increased in the tish−/− neocortex. A, Pax6+ cell density, measured in radial columns from the ventricular to pial surfaces, is increased in tish−/− neocortex as compared (more ...)
Cell cycle kinetics are altered in tish−/− neocortical progenitor cells
In an effort to identify an underlying basis for the increase in progenitor cell density at E17 and E20 in the tish−/−
neocortex, we employed an IdU/BrdU labeling assay to assess the cell cycle kinetics of normally- and abnormally-positioned progenitor populations at these times. This assay has been shown previously to yield values for cell cycle kinetic parameters that are comparable to those calculated using serial BrdU injection methods (Quinn et al., 2007
). We reasoned that, inasmuch as longer cell cycle times are associated with neurogenic rather than self-renewing divisions (Calegari et al., 2005
; Calegari and Huttner, 2003
), an increase in progenitor cell density at E17 and E20 could result from a shortening of the cell cycle in these populations and, thus, an increase in self-renewing divisions.
We considered first the population of Pax6+ progenitors, which corresponds to RGCs. Pax6+ cells were identified in wildtype, normotopic tish−/− (n-tish−/−) and heterotopic tish−/− (h-tish−/−) neocortices. This analysis allows a differential assessment of cell cycle kinetics in the appropriately- and inappropriately-positioned proliferative cells in tish−/− neocortex. Unexpectedly, at E17, no significant differences were detected among groups for Pax6+ cells in terms of the percentage of time spent in S phase (Ts/Tc), the total cell cycle length (Tc), or the lengths of S phase (Ts) or G2+M+G1 phases (Tc-Ts) (, ). In contrast, at E20, multiple differences were noted in the Pax6+ cell populations among the various groups. Pax6+ cells in the n-tish−/− neocortex did not differ from wildtype in terms of their cell cycle kinetics, demonstrating that, even in tish−/− neocortex, appropriate positioning of Pax6+ progenitors serves as a prerequisite for proper control of cell cycle behavior. However, E20 Pax6+ cells in h-tish−/− neocortex demonstrated an increased Ts/Tc and a decreased Tc-Ts compared with wildtype or n-tish−/− Pax6+ cells (, ). Interestingly, Tc was not significantly different among groups. Although a trend toward a decreased Tc was observed in h-tish−/− cells, this effect did not achieve statistical significance (, ). Taken together, these results indicate that heterotopic Pax6+ progenitors in tish−/− neocortex at E20 possess shorter G2+M+G1 phases than wildtype cells with a trend toward a decrease in the length of the cell cycle.
Figure 5 Cell cycle kinetics are altered in tish−/− neocortical progenitor cells. A, No significant differences in the percentage of time spent in S phase (Ts/Tc) were found among Pax6+ cells at E17 in the wildtype, n-tish−/−, and (more ...)
Cell cycle parameters (mean ± SEM) for RGC and IPC populations in wildtype, normotopic tish−/− (n-tish−/−), and heterotopic tish−/− (h-tish−/−) proliferative zones.
We next considered the possibility that changes in cell cycle kinetics could be occurring in the IPC population. For Tbr2+ cells at E17 and E20, n-tish−/− progenitors possessed cell cycle kinetics that were not significantly different from wildtype, highlighting that, even in tish−/− neocortex, appropriate positioning of Tbr2+ progenitors may contribute to proper control of cell cycle behavior. In contrast, Ts/Tc was increased in h-tish−/− cells compared to wildtype, while both Tc and Ts-Tc were significantly decreased (, ). Taken together, these results indicate that heterotopic Tbr2+ progenitors in tish−/− neocortex at E17 and E20 possess a shorter cell cycle length than wildtype cells due to shortened G2+M+G1 phases.
Radial glial adherens junctions and apical polarity domains are intact in tish−/− neocortex
In light of the finding that some RGCs and IPCs are located heterotopically in the tish−/−
neocortex, we sought to identify a mechanism by which these cells might become mislocalized. Considering that the RGC population appeared to be affected most severely at E15 (), we hypothesized that the observed positioning defect might result from a population of VZ RGCs losing its attachments to the ventricular surface and migrating into the IZ/CP to seed the heterotopic proliferative zone. In order to test this possibility, we employed immunohistochemistry and in utero
electroporation techniques to assess the status of adherens junctions and apical polarity markers at the ventricular surface. We reasoned that if RGCs were losing their attachments to the ventricular surface and seeding a new proliferative zone, then we would observe disruptions in the F-actin components of VZ adherens junctions and in the apical polarity proteins aPKC-λ and PAR3 (Cappello et al., 2006
; Costa et al., 2008
). We also reasoned that we would observe a greater percentage of RGCs with retracted apical processes following in utero
electroporation of a pCAGGS-GFP construct.
Examination of adherens junctions using Alexa 488 conjugated phalloidin to identify F-actin demonstrated no obvious differences between wildtype and tish−/−
neocortices at E13, E15, or E17 (). Had a loss of adherens junctions been responsible for the heterotopic mitoses in tish−/−
neocortex, one would have anticipated an interruption in phalloidin staining at the ventricular surface as has been described previously (Cappello et al., 2006
). Such an interruption was not observed. Similarly, aPKC-λ and PAR3 staining revealed no obvious disruptions of apical polarity within the endfeet of RGCs at the ventricular surface at E13, E15, or E17 (). Moreover, examination of RGC apical processes at E17, 12h after electroporation with a pCAGGS-GFP construct, revealed that the percentage of electroporated GFP+
cells maintaining an apical process with ventricular contact did not differ between wildtype and tish−/−
neocortex (wt 67.2±2.84%, tish−/−
69.3±1.23%, p > 0.05). Thus, we conclude that adherens junctions and apical polarity within RGC endfeet are maintained in tish−/−
neocortex and that progenitor cells do not become mislocalized as a result of losing their apical attachments to the ventricular surface.
Figure 6 Radial glial adherens junctions and apical polarity domains are intact at the ventricular surface of E13, E15, and E17 tish−/− neocortex. Confocal images were taken of coronal sections of embryonic neocortex at E13, E15, and E17. Immunohistochemistry (more ...)
Mislocalized tish−/− neocortical progenitor cells are not produced by the pallial ventricular zone at mid-neurogenesis
Based on our finding that tish−/− RGCs maintain their ventricular attachments during a time of extensive heterotopic proliferation, it appears that a different causative mechanism underlies the mislocalization of progenitor cells in the tish−/− IZ/CP. One possibility is that a population of VZ RGCs could have suffered a disruption in interkinetic nuclear migration such that their nuclei failed to return to the ventricular surface to undergo mitosis. Instead, the nuclei of these cells may have continued toward the pial surface after completing S phase, dividing at some location within the IZ/CP while maintaining a ventricle-contacting radial process. Alternatively, daughter cells produced by RGC mitoses in the VZ might initiate migration and fail to undergo cell cycle arrest, instead continuing to cycle as they migrate into the IZ/CP.
In order to test these possibilities, we electroporated a pCAGGS-GFP construct into the lateral ventricles of E14.5 and E16.5 wildtype and tish−/−
embryos and examined the neocortex 3 days later, after a single BrdU pulse 2h prior to embryo collection. We chose a 3 day survival period after initial experiments demonstrated no GFP+ cells in the heterotopic proliferative zone at 24 hrs post-electroporation (data not shown). At this earlier time point, GFP+
cells were found only in the VZ/SVZ, consistent with the latent period during which radial glial progeny reside in the SVZ for a 24 hr period before resuming migration toward the CP (Noctor et al., 2004
). Due to a high rate of post-electroporation mortality of E14.5 tish−/−
embryos secondary to amniotic fluid leakage caused by polyhydramnios and increased intra-amniotic pressure, we were unable to assess whether heterotopic progenitors were produced by the VZ/SVZ at the onset of cortical plate neurogenesis. Consequently, we present here the data from embryos electroporated at E16.5, an age corresponding to the middle stages of cortical plate neurogenesis.
In wildtype neocortex electroporated at E16.5, GFP+ cells were detected in the IZ and CP 3 days post-electroporation. Those cells in the IZ maintained a migratory morphology with a long pial-directed process characteristic of migrating neurons (). GFP+ cells in the CP elaborated branched apical dendrites and a basal axonal projection, which, in some cases, could be followed into the IZ (). While there were some BrdU incorporating cells in these regions, our immunohistochemical analysis revealed no colocalization between BrdU and GFP, indicating that VZ daughter cells born at E16.5 in wildtype neocortex were able to arrest their cell cycles before migrating into the IZ.
Figure 7 Mislocalized tish−/− progenitor cells are not produced by the pallial ventricular zone during the middle stage of cortical plate neurogenesis. Wildtype and tish−/− embryos were electroporated with a pCAGGS-GFP construct (more ...)
In tish−/− neocortex electroporated at E16.5, GFP+ cells were also detected in the IZ and CP 3 days post-electroporation. Some cells within the IZ maintained a morphology characteristic of migrating neurons; however, other cells appeared to be extending dendrites and axons as part of the growing SBH ( and ). Similar to wildtype, GFP+ cells in the CP elaborated apical dendrites and basal axons. Surprisingly, despite the abundance of BrdU+ cells in the IZ and CP of tish−/− neocortex, no GFP+ cells were observed to incorporate BrdU (, n = 3–5 sections from each of 4 embryos). These results indicate that VZ daughter cells born at E16.5 in tish−/− neocortex were able to arrest their cell cycles before migrating into the IZ, suggesting that VZ born cells at this age do not seed the heterotopic proliferative zone in the IZ/CP. These data also suggest that errors in interkinetic nuclear migration at mid-neurogenesis do not underlie the progenitor cell positioning defect, because BrdU+ proliferative cells in the tish−/− IZ do not maintain ventricle-contacting processes that can be electroporated at E16.5. Instead, heterotopic proliferative cells are either produced from a source other than the pallial VZ, or they are produced by the pallial VZ early in neurogenesis (before E16.5) and migrate into the IZ/CP without exiting the cell cycle, thus seeding the new proliferative zone.
Figure 8 The normally-positioned proliferative zone in tish−/− neocortex produces neurons for both the cortical plate and heterotopia. Wildtype and tish−/− embryos were electroporated with a pCAGGS-GFP construct at E16.5 and examined (more ...)
The normally-positioned proliferative zone in tish−/− neocortex produces neurons for both the cortical plate and heterotopia
In light of the finding that GFP+
cells were found in the CP of tish−/−
neocortex at 3 days post-electroporation, we decided to examine the distribution of electroporated cells across the cortical wall in greater depth. In previous work from our laboratory, we had hypothesized that normally-positioned progenitors in the tish−/−
neocortex produce neurons destined for the heterotopia, while abnormally-positioned progenitors produce neurons for both the CP and the heterotopia (Lee et al., 1998
). Our goal was to test this hypothesis using in utero
electroporation to trace the origins of CP and SBH neurons.
Embryos were electroporated at E16.5 and examined three days post-electroporation. In wildtype embryos, GFP+
cells were detected in developmental zones across the depth of the neocortex, and many cells could be identified largely on the basis of their morphology. GFP+
cells in the VZ maintained a radial morphology with apical and basal processes characteristic of parental RGCs. GFP+
cells in the SVZ possessed a multipolar morphology, indicative of neurons in phase two of radial migration, which are known to arrest in the SVZ before continuing toward the CP (Noctor et al., 2004
), and IPCs ( and data not shown). Within the IZ, GFP+
cells possessed a bipolar morphology with a long leading process, indicative of migrating neurons (, open arrowheads). In the CP, GFP+
cells were arranged in laminae beneath the pial surface, and they extended a ramified apical dendrite as well as a basal axon that could, in many cases, be traced into the IZ (, arrows).
In the tish−/− neocortex, similar to wildtype, GFP+ cells were detected across the depth of the neocortex. GFP+ cells within the VZ maintained a radial morphology with apical and basal processes characteristic of parental RGCs. GFP+ cells within the SVZ possessed a multipolar morphology, indicative of either neurons in phase two of radial migration or IPCs ( and data not shown). Interestingly, GFP+ cells were also located within the developing heterotopia amid the axons of the IZ and within the normally positioned CP (). Many of these cells in both locations could be identified as neurons based on the presence of a ramified apical dendrite and a basal axonal projection that, in some cases, could be traced into the IZ (, arrows). Some GFP+ neurons within the heterotopia deviated from a normal orientation. Instead, these cells were oriented at angles which, in the most severe cases, caused them to align themselves parallel to the ventricular surface (, closed arrowheads). In one instance, a misaligned, heterotopic neuron was observed to project a ramified dendrite medially, while its axon coursed laterally before looping back medially and entering the white matter directed toward the contralateral hemisphere (, a * indicates the cell body, closed arrowheads trace the axon). Moreover, GFP+ cells that resembled migrating cells with a long leading process and trailing cell body were observed in the heterotopia and CP (, open arrowheads). In some instances, these cells appeared to be migrating as closely apposed clusters rather than as individual, spread-out entities. Taken together, these data suggest that the normally-positioned proliferative zone produces neurons that are destined for both the CP and the heterotopia. Given that abnormally-located tish−/− progenitors are not generated by the VZ compartment at E16.5 and thus could not have produced neurons for the CP and heterotopia, it seems most plausible to conclude that, at E16.5, normally-positioned RGCs produced daughter cells either directly or indirectly via IPCs that differentiated into neurons and migrated into both locations.
Radial glial fibers are disrupted in the tish−/− SBH
In light of the finding that the normally-positioned proliferative zone in tish−/− neocortex produces neurons that can arrive successfully in the CP, we reasoned that the radial glial scaffold must remain at least partially intact in order for neurons to traverse the growing SBH and reach the CP. In order to investigate the integrity of the radial glial scaffold, we utilized immunohistochemistry against the intermediate filament protein vimentin, which is present in progenitor cells of the neocortex as well as in the long radial fibers of radial glial cells. We utilized sections from E19.5 neocortex from wildtype and tish−/− animals for examination, which corresponds to the same developmental timepoint examined in the previous electroporation experiments (see ). In wildtype neocortex, vimentin positive cells were concentrated in the VZ/SVZ and their radial fibers extended through the IZ to the pial surface (). These fibers were organized into dense, radial arrangements in the IZ (), which terminated in punctate endfeet at the pial surface (). In tish−/− neocortex, vimentin positive cells were concentrated in the VZ/SVZ as in wildtype; however, radial fibers were less dense in the region of the SBH. Vimentin-positive fibers within the SBH also lacked the tight radial arrangement of wildtype fibers, appearing more wavy and disorganized, with interspersed vimentin-positive cell bodies corresponding to mislocalized progenitor cells (, cell bodies indicated by arrows). Interestingly, despite the disorganization present in the region of the SBH, radial fibers in the tish−/− CP appeared more similar to wildtype in terms of density, organization, and endfoot formation (). These results indicate that radial fibers in tish−/− neocortex are capable of penetrating the SBH and reaching the pial surface, albeit after a disorganized course through the developing SBH ().
Figure 9 Radial glial fibers are disrupted in the tish−/− neocortex. Confocal images were taken of 60µm thick coronal sections of wildtype and tish−/− neocortex at E19.5, and compressed stacks are presented. A, Wildtype (more ...)