In this study, we provide evidence that the pallial meninges regulate tangential migration of interneuron precursors. In mice homozygous for a Foxc1 mutation (hith) meningeal integrity and function is impaired, which has profound, negative effects on the ability of GABA-ergic interneurons to migrate along the adjacent cortical MZ. The migrating interneurons are absent from the dorsolateral cortex, which raises the question as to where these cells accumulate and/or migrate. Careful analysis of cortical interneuron distribution in the developing brain using the Dlx5/6-lacZ transgenic marker and Gad67 and Lhx6 in situ hybridization analysis failed to show any region of increased presence of labeled cells. Interestingly, the most dorsal aspect of the SVZ/IZ migratory stream of homozygous mutants appears thicker as a result of a more loose packing of migrating interneurons. This finding may parallel the developmental lamination defects observed in mice deficient for the Cxcl12 receptors Cxcr4 and Cxcr7, which are also characterized by a less dense packing of the streams of migrating intereneurons [17, 18]. The appearance of the migration defects in the Foxc1
mutants likely also precludes alternative explanations for their causes, like defects centered on the pallial-subpallial boundary, an important signaling center regulating tangential migration. In such an instance, we would expect a more global phenotype affecting tangential migration of medial ganglionic eminence-generated interneurons in all cortical aspects and not only the dorsolateral MZ, which correlates with both Cxcl12 downregulation and meningeal defects in this area.
Our data, in conjunction with other work, indicate that the meningeally expressed factor guiding tangential migration along the MZ is very likely Cxcl12. We show that meningeal Cxcl12 expression is severely decreased or entirely absent in Foxc1
mutants during the period of tangential interneuron migration. At E13.5 Cxcl12 expression is reduced throughout the meninges while at E17.5 basolateral aspects show intact expression levels but dorsal aspects show reduced ones, which might point towards a developmental delay in Cxcl12 expression of the basolateral meninges. Support for the hypothesis that Cxcl12 is the chemo-attractant factor for MZ interneurons comes from several recent studies. For instance, it has been shown that Tbr2+ cells of the cortical SVZ express Cxcl12 and forced expression of Tbr2 by in vivo electroporation leads to concomitant Cxcl12 upregulation associated with interneuron accumulating foci at the electroporation sites . Further, Cxcl12 signaling in responsive cells is initiated upon binding to the chemokine receptors Cxcr4 and Cxcr7 and both receptors are coexpressed in migrating interneurons. Interestingly, inactivation of either receptor leads to comparable interneuron migration defects due to their distinct functions in these cells [17, 18]. While both receptors bind Cxcl12, Cxcr7 is internalized upon ligand binding and essential for the modulation of Cxcr4 function by regulating Cxcr4 protein levels at the cell surface . Thereby by controlling the amount of chemokine receptor protein present, Cxcr7 modulates chemokine responsiveness in migrating interneurons. In addition, it has been shown that meningeally expressed Cxcl12 directs migrating neurons not only in the forebrain but also in the cerebellum. Here, it has been demonstrated that neuronal precursors of the external germinal layer are attracted by meningeally expressed Cxcl12, which regulates their positioning in the external granule cell layer until they differentiate and lose Cxcl12 responsiveness . Thus, the attractive interaction between Cxcl12-expressing meningeal cells and the Cxcr4-expressing neurons appears to be a conserved and reappearing mode in the regulation of neuronal migration.
We considered the idea that expression of Cxcl12 in the meninges might be directly regulated by Foxc1 and tested this hypothesis through ChIP analysis. We identified a Foxc1 consensus-binding site within the third intron of the Cxcl12 gene and verified complex formation between Foxc1 and this fragment. In addition, forced overexpression of Foxc1 in transfected meningeal cells leads to a significant upregulation of Cxcl12. This analysis allowed us to add the Cxcl12 gene to the list of direct targets of Foxc1 transcriptional activity and, to our knowledge, the first one expressed in the meninges. Interestingly, although Foxc1 and Cxcl12 expression also overlap in the facial mesenchyme, we do not see any downregulation of Cxcl12 in the facial mesenchyme.
CRCs in the dorsal MZ, although also dependent on Cxcl12 signaling for their proper migration, were considerably less disturbed in their positioning than interneurons in the Foxc1
mutants. This finding, in combination with the fact that Foxc1 directly activates meningeal Cxcl12 transcription, points towards tangential migration defects being a primary defect rather than the consequence of a disrupted trophic role of the meninges leading to more generic defects in corticogenesis and CRCs and/or interneuron survival as previously reported . CRCs that originate in the cortical hem express the Cxcl12 receptor Cxcr4 and in Cxcr4-deficient mice a fraction of CRCs becomes ectopically positioned by leaving the MZ and invading the cortical plate . The fact that, despite reduced Cxcl12 signaling from the meninges in Foxc1
mice, we do not see any major disruption of CRC distribution might be best explained by the hypomorphic nature of the hith allele and incomplete loss of Cxcl12 signaling in the meninges early in development. There is also the possibility that additional factors regulate CRC positioning in the MZ as there are three focal sources of CRCs that have been identified in mice - the cortical hem, the ventral pallium, and the septum - and Cxcl12 has been demonstrated to influence only the migration of hem-derived CRCs . The subpopulations derived from the ventral pallium and septum do not express Cxcr4, and are therefore largely insensitive to Cxcl12.
Surprisingly, developmental migration defects in interneurons of Foxc1
mutants do not lead to obvious differences in adult distribution compared to the WT, although this later finding remains difficult to evaluate in great detail considering the overlying massive cortical dysplasia seen in the mutants. However, we undertook a quantitative evaluation of the distribution of interneurons by comparing two aspects of the adult cortex. A medial aspect corresponding to the most severe developmental migration defects and a lateral aspect without obvious migration defects during development. Confirming our initial impression that neither medial nor lateral cortical aspects show any significant differences between homozygote mutants and WT in the number of interneurons present. In addition, the laminar distribution of interneuron subtypes, positive for the expression of calbindin, calretinin, and parvalbumin, is also not affected in the adult cortex of hith/hith mice, suggesting a corrective mechanism in postnatal development, which guides interneurons to their correct location. This result is also consistent with recent work showing that the laminar interneuron distribution in the postnatal cortex is almost completely restored in Tbr2 mutants lacking developmentally the deep stream of tangential interneuron migration along the SVZ/IZ .
The fact that at postnatal stages no obvious differences can be seen in the distribution of cortical interneurons of Foxc1
mice compared to the WT may be best explained by the movements these cells undertake later in development. While their initial route of migration is tangential, once interneurons reach their approximate lateral position within the pallium they undergo extensive radial migration [22–25]. These cell movements, perpendicular to the overall tangential mode of migration, can be directed both from the MZ to the ventricular surface and from the intermediate zone to the pial surface. Apparently, these radial movements of GABA-ergic interneurons provide for correct layer positioning and proper integration into the cortical circuitry and likely present the source of cells compensating for the observed deficiency in interneurons in the MZ of Foxc1
mice. In addition, it appears likely that layer positioning of interneurons perinatally occurs independently of Cxcl12, since recent work in our laboratory demonstrated a developmental loss of interneuron response to Cxcl12 signaling . Such a postnatal loss of responsiveness to meningeally expressed Cxcl12 has been also shown for cerebellar granule cells as they differentiate and radially migrate through the Purkinje cell layer to the internal granule cell layer .