Distributions of ErbB4-expressing interneurons migrating from MGE to cortex show minimum overlap with expression domains of Nrg1 isoforms and Nrg3
Most cortical GABAergic INs originating from the MGE express the receptor tyrosine kinase ErbB4 [29], which binds NRGs and activates their signaling pathways. Therefore, we used ErbB4 as a marker for these INs to study the relationship between their migratory pathways and the expression patterns of NRGs in the telencephalon in both wild-type (WT) and ErbB4 mutant mice. We first determined the expression pattern of NRGs expressed in embryonic telencephalon during the period of IN migration from the MGE to the cortex, including Nrg1-type I (Nrg1-Ig) and Nrg1-type III (Nrg1-CRD) and Nrg3. In the vTel, the expression of Nrg1-type III and Nrg3 is most prominent. At embryonic day (E)12.5, Nrg3 and Nrg1-type III are robustly expressed within the mantle zone of the vTel, outside of the LGE and MGE, in largely complementary patterns, with the expression of Nrg3 being more lateral and ventral to that of Nrg1-type III (Figure 1). These complementary expression patterns persist at E13.5 (Figure 2) and E14.5 (Figure 3). In the cortex, Nrg1-type I and Nrg1-type III expression is mostly restricted to the VZ/SVZ (Figures 2B and 3E for Nrg1-type III, Figure 4C for Nrg1-type I). In contrast, Nrg3 is predominantly expressed in the forming cortical plate (CP) (Figures 2E, 3B, and 4B).
At each age analyzed, which together encompass the migratory period of GABAergic INs from the MGE to cortex, the great majority of the ErbB4-expressing INs within the vTel are found at locations with low or undetectable levels of Nrg1/Nrg3 expression, although a very small portion of ErbB4-expressing INs do overlap with NRG expression domains (Figures 1C, E, 2C, F, and 3C, F). This complementary relationship between NRGs and MGE INs is also observed within the cortex. There are two main migratory pathways for GABAergic cortical INs: the interface of IZ/SVZ and the MZ (Figure 4A). Interestingly, Nrg1 is expressed in the SVZ/VZ just beneath the IZ/SVZ pathway (Figure 4C), whereas Nrg3 is expressed in the CP just beneath the MZ (Figure 4B). In WT cortex, ErbB4 and NRGs are predominantly expressed in different compartments with minimal overlap (Figure 4A-D). The distribution of ErbB4-expressing INs at these ages is strongly focused on the domains of low NRG expression in the IZ and the MZ, and only a small proportion of ErbB4-expressing INs is found within the NRG expression domains in the CP and the VZ (Figures 3A-F and 4).
These strongly complementary relationships between the NRG and ErbB4 expression patterns suggest that NRGs may play important roles during the tangential migration of the ErbB4-expressing cortical INs. Further, if the NRGs have such a role, the mostly non-overlapping patterns of expression of Nrg1/Nrg3 and ErbB4 indicate that NRGs function as inhibitory or repellent guidance cues for migrating ErbB4-expressing INs, and are inconsistent with NRGs acting as attractants for ErbB4-expressing INs.
NRGs repel migrating MGE cells in vitro
To address the influence of Nrg1 and Nrg3 on the migration of MGE-derived INs, and specifically whether they have a repellent/inhibitory influence or, conversely, an attractant effect, we first used the in vitro collagen gel co-culture assay for detecting the effects of secreted molecules such as the NRGs on cell migration. Specifically, we used this co-culture assay to determine the responses of the migrating MGE-derived cells to secreted NRGs. MGE explants isolated from E14.5 mice constitutively expressing enhanced green fluorescent protein (eGFP) [31] were co-cultured at a distance from aggregates of 293T cells transfected with an empty vector as a control, or vectors containing the EGF-like domains of two splice variants of Nrg1 (Nrg1-α and Nrg1-β) or Nrg3, each of which binds ErbB receptors and results in their dimerization and autophosphorylation, as well as activation of the NRG-ErbB4 signaling pathway [21, 24, 26, 27].
In control co-cultures, cells migrate out of the MGE explants in a symmetric pattern (15 out of 15 explants; Figure 5A). In contrast, in co-cultures with cell aggregates expressing Nrg1 isoforms or Nrg3, the pattern of cell migration from the MGE explants is asymmetric, with 96% of the explants (45 out of 47 explants) exhibiting diminished migration of MGE cells towards the NRG source relative to the robust migration away from it (Figure 5B-D). This qualitative assessment is supported by quantitative analyses that measured the levels of fluorescence of the eGFP reporter that marked cells migrating from the MGE explants. The histograms in Figure 5 depict the data obtained from the most conservative measurement of fluorescence, in which we measured all fluorescence found immediately outside of the explants' borders, including the dense halo of cells abutting the explants that may be formed by cells that move out of the explants' borders passively or prior to coming under the influence of NRG diffusing from the transfected cell aggregates. Nonetheless, these measurements revealed a statistically significant difference in the levels of eGFP fluorescence between the distal (D) and proximal (P) quadrants, indicating significantly fewer cells in the quadrant emanating from the MGE explant facing the NRG source relative to the opposing quadrant (Figures 5E, F). When the measurement are done at positions progressively further away from the MGE explants, and therefore progressively enriching the sample for cells migrating under the influence of the secreted NRGs, the disparity between the quadrant of the MGE explant proximal to the NRG source relative to the opposing quadrant increases dramatically and quickly reaches the point where essentially all of the MGE cells are present in the quadrant distal to the NRG source (equivalent to a P/D ratio of 0 in Figures 5E, F). In contrast, as expected, in control co-cultures the symmetry in the distribution of fluorescence between the proximal and distal quadrants persists at a distance from the MGE explant. These findings demonstrate that, in vitro, Nrg1 and Nrg3 have a chemorepellent or inhibitory effect on the migration of MGE cells, consistent with the in vivo findings of complementary, non-overlapping distributions of ErbB4-expressing, MGE-derived INs and domains of NRG expression described in the preceding section.
Complementary patterns in distribution of MGE-derived interneurons and NRG expression domains are degraded in mice deficient for NRG-ErbB4 signaling
In WT mice, we find that as ErbB4-expressing, GABAergic INs migrate from the MGE to cortex, they take paths that have low or undetectable levels of NRG expression and tend to avoid the surrounding domains of NRG expression (Figures 1, 2, 3, and 4). These findings, and our findings from the collagen gel co-culture experiments showing that, in vitro, NRGs act as chemorepellents for MGE cells (Figure 5), suggest that most of the ErbB4-expressing, MGE-derived INs avoid NRG expression domains and that NRGs function as inhibitory or repellent guidance cues for them. We have further addressed this issue by analyzing the distributions of ErbB4-expressing, MGE-derived INs relative to NRG expression domains in mice with a targeted deletion of ErbB4, but which express a human ErbB4 transgene under the cardiac-specific α-MHC promoter (HER4heart) to rescue the mid-embryonic lethality of the ErbB4 null mutation due to impaired myocardial trabeculation [30]. In these ErbB4-/- HER4heart mice, NRG signaling through ErbB4 is abolished in INs. Therefore, if NRGs have a repellent effect on migrating MGE-derived INs, we predict that the complementary patterns of NRG expression and the distribution of ErbB4-expressing INs observed during normal development would be degraded in ErbB4 mutant mice.
The ErbB4 riboprobe used to determine the distribution of ErbB4-expressing INs in WT mice is directed against a 5' cDNA fragment of mouse ErbB4 transcript that persists in the ErbB4-/- HER4heart mice. Therefore, as in WT mice, we were able to use in situ hybridization with the ErbB4 riboprobe to visualize MGE-derived INs deficient for ErbB4 in the ErbB4-/- HER4heart mice and directly compare their distributions with the NRG expression patterns. As described in a preceding section (Figures 1, 2, 3, and 4), in WT mice, MGE-derived INs, defined by their expression of ErbB4, exhibit very little overlap with the NRG expression domains, specifically Nrg1-type III and Nrg3. In contrast, ErbB4-expressing, MGE-derived INs are more dispersed in their distribution in ErbB4-/- HER4heart mice (Figures 2G and 3G) compared to their WT littermates (Figures 2A, D and 3A, D), and their distribution overlaps considerably with the expression domains of both Nrg1-type III (Figure 3H, I) and Nrg3 (Figure 2H, I). This degradation in the complementary distributions of ErbB4-expressing INs and NRG expression domains is also observed within the cortex of ErbB4-/- HER4heart mice, as the distribution of ErbB4-expressing INs is more diffuse than in WT and overlaps considerably with the NRG expression domains in the CP and the SVZ/VZ (Figures 2, 3 and 4).
The fact that the patterned distribution of the INs is not completely lost is evidence of the action of other guidance activities that have been reported to affect the migration of MGE-derived INs (see Introduction). Thus, the complementary patterns of NRG expression and the distribution of ErbB4-expressing, MGE derived INs are degraded in ErbB4-/- HER4heart mice, consistent with our in vitro findings that NRGs function as inhibitory or repellent guidance cues for ErbB4-expressing, MGE-derived INs.
Cells migrating from WT MGE but not ErbB4-deficient MGE avoid endogenous WT NRG expression domains
To provide additional evidence that INs avoid domains of NRG expression as they migrate from the MGE to the cortex, we compared the distributions of cells that migrate from explants of MGE from E14.5 WT and ErbB4-/- HER4heart mice placed on living coronal slices of E14.5 WT forebrain (Figure 6). The MGE explants and all cells that migrate from them were marked with an eGFP reporter by breeding ErbB4+/- HER4heart mice with mice constitutively expressing eGFP [31]; the distributions of eGFP-marked cells were compared after 48 hours of culture for MGE explants isolated from ErbB4-/- HER4heart eGFP mice or, as a control, ErbB4+/+ HER4heart eGFP mice.
WT cells migrating out of MGE explants from ErbB4+/+ HER4heart eGFP mice are distributed in a pattern that exhibits little overlap with NRG expression domains (n = 4; compare Figure 6B, B' with 6A). These WT eGFP-labeled, MGE-derived cells exhibit patterned distributions with a tendency to avoid domains of high NRG expression in the slices of WT forebrain - for example, the domain of high Nrg3 expression in the CP and the domains of robust Nrg1 expression in the vTel mantle zone. This patterned, complementary distribution is similar to that of ErbB4-expressing INs relative to NRG expression domains in WT mice (Figures 1, 2, and 3). In contrast, on similar living sections of WT forebrain, eGFP-labeled, ErbB4-null cells migrate out of MGE explants from ErbB4-/- HER4heart eGFP mice in a more or less uniform, radial pattern that does not show the preference exhibited by eGFP-labeled, WT MGE-derived cells to avoid NRG expression domains (n = 5; compare Figure 6C, C' with 6A). Thus, in summary, WT MGE-derived cells show a preference to avoid NRG expression domains not seen for ErbB4-null, MGE-derived cells when migrating under the same conditions on living slices of WT forebrain. Further, these findings demonstrate that the avoidance behavior exhibited by WT MGE-derived cells is due to their expression of ErbB4 and provide evidence that the migratory defects of MGE-derived INs in ErbB4-/- HER4heart mice are due to an absence of NRG-ErbB4 signaling autonomous to the MGE-derived INs. These findings support the conclusion that the NRG expression domains in embryonic forebrain inhibit or repel ErbB4-expressing INs as they migrate from the MGE and function during normal development as barriers that funnel migrating INs from the MGE to the cortex.
Migration of MGE-derived interneurons is blocked in vivo by ectopic NRG expression domains and results in a reduction of interneurons in cortex
The evidence that we have obtained from a variety of analyses, including the distributions of ErbB4-expressing INs relative to NRG expression domains in WT mice and mice deficient for NRG-ErbB4 signaling, from in vitro co-cultures, and explant migration assays on brain slices, demonstrate that NRGs act as repellents to direct the migration of MGE-derived ErbB4-expressing INs. As an additional assessment of the function of NRGs in influencing the migration of MGE-derived ErbB4-expressing INs, we carried out in utero electroporations to produce focal ectopic domains of NRG expression targeted for the migratory path of MGE-derived INs. We predicted that if NRGs are repellents for migrating MGE-derived INs, the INs would avoid a domain of NRG expression ectopically positioned within their migration path, and their migration should be at least partially blocked. On the other hand, if NRGs are attractants for ErbB4-expressing INs, we would predict that MGE-derived INs would not avoid ectopic NRG expression domains and would either accumulate within them and/or would pass through them, similar to the published results of electroporation of the cytokine Cxcl12, a putative chemoattractant for migrating INs [18, 32].
In utero electroporations of a CAG expression vector containing the EGF domains of mouse Nrg1α, Nrg1β or Nrg3, and eGFP were targeted to the vTel migratory path of MGE-derived INs at E12.5, and the distributions of ErbB4-expressing INs relative to the ectopic NRG expression domains were assessed at E17.5. The CAG promoter drives robust expression in essentially all cell types [33] and resulted in a strong focal expression of NRGs at ectopic sites in the electroporated brains. Ectopic expression domains were initially assessed by visualizing eGFP, and subsequently confirmed using in situ hybridization for the relevant NRG; the distribution of MGE-derived INs was assessed by their expression of ErbB4 (Figure 7). We performed Nissl staining on sections of the Nrg1α, Nrg1β or Nrg3, and/or eGFP electroporated brains and did not observe any significant cytoarchitectural changes in these brains (data not shown).
We find that ectopic expression domains of NRGs positioned in the migratory path of MGE-derived INs ventrolaterally within the telencephalon blocked the migration of ErbB4-expressing INs (Figure 7). The majority of ErbB4-expressing INs accumulates at a position in their migratory path proximal to the ectopic expression domain of Nrg1-α (Figure 7A-A'''; n = 4), Nrg1-β (Figure 7B-B'''; n = 4), or Nrg3 (data not shown; n = 3) and relatively few enter it. In contrast, ErbB4-expressing INs migrate into and through the electroporation domain in the control cases electroporated with CAG-eGFP alone (Figure 7C-C'''; n = 5). The distribution of the ErbB4-expressing INs is reminiscent of the distribution of cells migrating from WT MGE explants on living forebrain slices, with both exhibiting a tendency to avoid entering the NRG expression domain, whether the ectopic NRG expression for the in vivo electroporation experiments presented here (Figure 7) or the endogenous NRG expression as for the WT MGE explant-forebrain slice migration experiments presented in the preceding section (Figure 6).
These in vivo electroporation experiments indicate that NRGs are repellents for migrating MGE-derived INs and that the ectopic domains of NRGs block their migration to the cortex. Consistent with this interpretation, we find that the cortex distal to the ectopic NRG expression domain is virtually devoid of ErbB4-expressing INs (Figure 7A'', A''', B'', B'''). This 'shadow effect' indicates that the ectopic NRG expression domain indeed blocked the migration of MGE-derived, ErbB4-expressing INs, resulting in not only their aberrant accumulation within their subpallial pathway but also in their failure to reach the cortex distal to the NRG blockade. In contrast, the control transfections have no detectable effect on the migration of MGE-derived, ErbB4-expressing INs along either their subpallial path or their distribution in the cortex distal to the control transfection domain (Figure 7C'', C'''). As expected, the migration of ErbB4-expressing INs into caudomedial cortex and the hippocampus is largely unaffected even in the brains electroporated with NRG expression constructs (Figure 7A'', A''', B'', B'''), consistent with their origin in the CGE and their caudal migratory path, which is distinct from the path of MGE-derived INs blocked by the ectopic NRG expression domain [5, 7]. In summary, the most straightforward interpretation of these findings is that Nrg1 and Nrg3 inhibit or repel INs migrating from the MGE to the cortex in vivo.
Cortex of ErbB4-/- HER4heart mice has reduced numbers of interneurons
Using in situ hybridizations with an array of markers for GABAergic INs, including Dlx1/2 [3], GAD67 [34], EGFR/ ErbB1 [35] and Reelin [36], we studied at postnatal day (P)0 the cortical distribution of INs in ErbB4-/- HER4heart mice (Figure 8). Each marker indicated a considerable decrease in the number of INs in the ErbB4-/- HER4heart mice relative to WT littermates (Figure 8). Further, each marker showed that the density of INs in the ErbB4-/- HER4heart mice is diminished along the entire rostral to caudal axis, with a drastic decrease in density and near absence of INs in posterior cortex (Figure 8). In WT P0 mice, Reelin-expressing INs are preferentially located in anterior CP (Figure 8D). In the ErbB4-/- HER4heart mice, the presence of Reelin-expressing cells within the CP is significantly reduced, while the presence of Reelin-expressing cells in the MZ, indicative of Reelin-positive Cajal-Retzius neurons that are distinct from ErbB4-expressing, MGE-derived INs, remains relatively normal (Figure 8D'). In conclusion, these results are consistent with our findings presented in the preceding sections that NRG-ErbB4 signaling plays an important role in directing the tangential migration of GABAergic INs through the vTel and to their cortical destinations.