Transcription Factors Sp8 and Sp9 Regulate Medial Ganglionic Eminence-Derived Cortical Interneuron Migration

Cortical interneurons are derived from the subpallium and reach the developing cortex through long tangential migration. Mature cortical interneurons are characterized by remarkable morphological, molecular, and functional diversity. The calcium-binding protein parvalbumin (PV) and neuropeptide somatostatin (SST) identify most medial ganglionic eminence (MGE)-derived cortical interneurons. Previously, we demonstrated that Sp9 plays a curial transcriptional role in regulating MGE-derived cortical interneuron development. Here, we show that SP8 protein is weekly expressed in the MGE mantle zone of wild type mice but upregulated in Sp9 null mutants. PV+ cortical interneurons were severely lost in Sp8/Sp9 double conditional knockouts due to defects in tangential migration compared with Sp9 single mutants, suggesting that Sp8/9 coordinately regulate PV+ cortical interneuron development. We provide evidence that Sp8/Sp9 activity is required for normal MGE-derived cortical interneuron migration, at least in part, through regulating the expression of EphA3, Ppp2r2c, and Rasgef1b.


INTRODUCTION
The cerebral cortex plays an irreplaceable role in many advanced functions, such as learning, movement, emotion memory and decision-making. The normal execution of these functions depends on the subtle ratio of excitatory projection neurons to inhibitory interneurons in the cerebral cortex and the appropriate functional circuits formed between them (Rubenstein and Merzenich, 2003;Del Pino et al., 2018;Lim et al., 2018). Glutamatergic cortical excitatory projection neurons are generated by pallial (cortical) neuroepithelial and radial glial cells (Kriegstein and Alvarez-Buylla, 2009). On the other hand, GABAergic cortical inhibitory interneurons, both in primates and rodents, are derived from the subpallium and reach their final position in the cortex through tangential migration (Wonders and Anderson, 2006;Hansen et al., 2013;Ma et al., 2013). During development, the subpallium is composed of four major proliferative zones: the medial (MGE), lateral (LGE) and caudal (CGE) ganglionic eminences and the preoptic area (POA); the MGE generates 60% of all cortical interneurons and includes parvalbumin (PV + ) and somatostatin (SST + ) subtypes (Gelman and Marín, 2010;Hu et al., 2017;Wamsley and Fishell, 2017).
The Sp9 zinc finger transcription factor is widely expressed in the ganglionic eminences (Long et al., 2009;Zhang et al., 2016). Sp8 and Sp9 are two closely related button head-like transcription factors and have many redundant functions in regulating GABAergic neuronal development Xu et al., 2018). Previously, we showed that Sp9 has a curial transcriptional role in regulating MGE-derived cortical interneuron development (Liu et al., 2018). While Sp8 is highly expressed in the dorsal LGE and the CGE (Ma et al., 2012), it is also weekly expressed in the MGE . However, the function of Sp8 in the MGE remains largely unknown.
In the present study, we found that SP8 protein expression was upregulated in the MGE mantle zone of Sp9 null mutants. There were more MGE-derived cortical interneurons that failed to migrate into the cortex in Sp8/Sp9 double conditional knockouts than in Sp9 single conditional knockouts. Furthermore, fewer Erbb4 + cells (immature PV + cortical interneurons) migrated in the cortical marginal zone (MZ), whereas more migrated in the cortical subventricular zone (SVZ). In the postnatal cortex, the most prominent phenotype was that approximately 80% of PV + interneurons were lost in Sp8/Sp9 double conditional knockouts. We provide evidence that Sp8 and Sp9 mediate these effects by regulating the expression of several genes that regulate cortical interneuron migration and development, such as EphA3, Ppp2r2c and, Rasgef1b.

Mice
All animal experiments described in this study were approved in accordance with institutional guidelines, and the institutional review board (Ethics Committee) of Shanghai Medical College of Fudan University approved the study design. The strains used in this study have been previously reported: Nkx2-1-Cre (Xu et al., 2008), Rosa26-YFP (R26R-YFP; Srinivas et al., 2001), Sp9 LacZ/+ (Zhang et al., 2016), Sp9 F/+ (Zhang et al., 2016), Sp8 F/+ (Bell et al., 2003) and Lhx6-Cre (Fogarty et al., 2007). All lines were maintained in a mixed genetic background of C57BL/6J, 129 and CD1. Pregnancy of mated mice was determined by vaginal plug detection, which was defined as embryonic day 0.5 (E0.5), and the day of birth was defined as postnatal day 0 (P0). The range of embryos used had a ±0.5day deviation.

Tissue Preparation
The pregnant mice were killed by cervical dislocation on designated dates. Each embryo was separated from the placenta, and the brain was dissected out and then fixed in 4% diethylpyrocarbonate and paraformaldehyde (DEPC-PFA) for several hours or overnight. Postnatal mice were deeply anesthetized and perfused adequately with cold 0.01 M PBS and 4% DEPC-PFA, and brains were then removed and postfixed overnight. Cryosectioning (Leica, CM 1950) was performed on brains embedded in optimal cutting temperature (OCT; Sakura); brains were immersed in OCT for 5 min at 4 • C and subsequently transferred to a plastic mold filled with OCT and frozen in dry ice-chilled ethanol.

SP8 Expression Is Upregulated in the MGE Mantle Zone of Sp9 Null Mutants
SP9 is strongly expressed in the SVZ of the MGE, but very few SP9 + cells are observed in the MGE VZ (Liu et al., 2018).
SP8 is also not expressed in the MGE VZ and SVZ, but we observed that a subset of cells in the MGE mantle zone weakly expressed SP8 protein at E13.5 and E15.5 (Figures 1A-C This finding is consistent with a previous report, which showed that Sp8 mRNA is weakly expressed in the MGE . In the Sp9 LacZ/LacZ null mutant, however, SP8 expression was greatly upregulated in the MGE mantle zone and in a subset of MGE-derived cells (Figures 1D-F ,J-L ; Liu et al., 2018). We next quantified the number of NKX2-1 + /SP8 + cells in the MGE mantle zone. The Sp9 mutants showed >120% FIGURE 3 | In situ RNA hybridization of Sst, Erbb4 and Npas1. (A-D) Sst mRNA expression was increased in the E15.5 neocortex (arrows) and ventral telencephalon (arrows) of mutants. More Sst + cells were found in the ventral telencephalon of double mutants than single mutants and controls (arrows). (E-I) Abnormal migration of Erbb4 + cells (PV + immature cortical interneurons) in mutant mice. Double mutants appeared to have relatively more Erbb4 + cells in the cortical SVZ (arrows) and in the ventral telencephalon (arrows) and relatively fewer Erbb4 + cells in the cortical marginal zone (MZ; arrowheads). (J-M) Decreased Npas1 + cells in the cortical MZ (arrowheads) and increased Npas1 + cells in the cortical SVZ (arrows) were observed in double mutants compared to single mutants and controls. * P < 0.05; * * P < 0.01; * * * P < 0.001. Scale bar: 200 µm in (A) for (A-C,E-G,J-L).
At E15.5, in control, Nkx2-1-Cre; Sp9-CKO and Nkx2-1-Cre; Sp8/9-DCKO mice, migration of cortical interneurons appears to occur primarily in two streams, in the cortical MZ and the SVZ, but Nkx2-1-Cre; Sp9-CKO and Nkx2-1-Cre; Sp8/9-DCKO mice had fewer GFP + cells in the cortex (Figures 2D-F ,G). We then analyzed the distribution of MGE-derived (GFP + ) cortical interneurons. The number of GFP + cells in the MZ, CP, SP, IZ and SVZ of the cortex were greatly reduced in single and double mutants compared with controls ( Figure 2G), as was the percentage of GFP + cells in the SP and the IZ (Figure 2H). In contrast, the percentage of mutant GFP + cells in the cortical SVZ was increased ( Figure 2H). Notably, the percentage of GFP + cells was notably higher in the SVZ of Sp8/9 double mutants than in Sp9 single mutants, and the percentage of GFP + cells in the SP and the IZ was much lower (Figure 2H). Again, very few GFP + cells were found in the LGE SVZ of mutants ( Figures 2D -F ). The ectopic accumulation of MGE-derived GFP + cells was observed in Nkx2-1-Cre; Sp9-CKO mice (Liu et al., 2018), but the loss of both Sp8 and Sp9 function led to severe increases in GFP + cells in the ventral telencephalon (Figures 2D-F). Taken together, both Sp9 and Sp8 promote the tangential migration of MGE-derived cortical interneurons from the MGE to the cortex and control the migration of cortical interneurons that follow distinct pathways in the cortex. In addition, loss of Sp8 function in Sp9 mutants further

Molecular Defects in Tangentially Migrating Sp8/9 Double Mutant MGE-Derived Cortical Interneurons
We next examined the molecular profile of tangentially migrating cells in these mutants at E15.5. We previously demonstrated using RNA-Seq and ChIP-Seq (chromatin coimmunoprecipitation followed by high-throughput DNA sequencing) that Sp9 has a curial transcriptional role in regulating MGE-derived cortical interneuron development (Liu et al., 2018). Loss of Sp9 function resulted in upregulation of Sst expression in a subset of MGE-derived cells; however, Sst expression was further upregulated in Sp8/9 double mutants (Figures 3A-C). Indeed, although Sp8/9 double mutants and Sp9 single mutants had almost same numbers of GFP + cells in the cortex at E15.5 (Figure 2G), double mutants had many more Sst + cells than Sp9 single mutants ( Figure 3D). Moreover, more ectopic Sst + cells accumulated in the ventral telencephalon in double mutants compared with single mutants (Figures 3B,C). Erbb4 is mainly expressed in migratory and mature PV + cortical interneurons (Fazzari et al., 2010;Mayer et al., 2018). We found that the percentage of Erbb4 + cells was reduced in the mutant cortical MZ, whereas it was greatly increased in the cortical SVZ (Figures 3E-I). Again, Nkx2-1-Cre; Sp8/9-DCKO mice had relative more Erbb4 + cells in the cortical SVZ and in the ventral telencephalon than Nkx2-1-Cre; Sp9-CKO mice (Figures 3E-I). This finding suggests that loss of Sp8/9 function results in relatively more immature PV + cortical interneurons that migrate in the cortical SVZ and that ectopically accumulate in the ventral telencephalon. Npas1 is mainly expressed in cortical interneurons in the MZ, although a small number of interneurons in the cortical SVZ also express Npas1 (Cobos et al., 2006;Stanco et al., 2014). We found that the percentage of Npas1 + cells in the cortical MZ were reduced, whereas it was greatly increased in the cortical SVZ of mutants compared with controls (Figures 3J-M).
Epha3 expression was increased in the mutant MGE at E15.5 (Figures 4A-A ), which explained why the LGE had very few MGE-derived cells, as enhanced Eph/ephrin signaling in the LGE VZ/SVZ increases the repulsive effect on migrating interneurons (Zimmer et al., 2008;Rudolph et al., 2010;Villar-Cerviño et al., 2015;Liu et al., 2018). Intracellular signaling molecule Ppp2r2c is mainly expressed in cortical interneurons in the MZ, whereas Rasgef1b is mainly expressed in cortical interneurons in the SVZ (Colasante et al., 2009;Antypa et al., 2011;Friocourt and Parnavelas, 2011). We found that the expression of Ppp2r2c in the cortical MZ was reduced, whereas the expression of Rasgef1b was greatly increased in the cortical SVZ of mutants compared with controls (Figures 4B-D ; Antypa et al., 2011). Notably, these phenotypes were more prominent in Sp8/9 double mutants than in Sp9 single mutants (Figures 4A-D ), further suggesting that Sp8 supplements the role of Sp9 in regulating MGE-derived cortical interneuron migration. Quantified data showed that the density of PV + /GFP + cells was severely reduced in double mutants compared with single mutants and controls. The density of SST + /GFP + and NPY + /GFP + cells was also reduced in the mutant somatosensory cortex, whereas CR + /GFP + cells were less affected. * P < 0.05; * * P < 0.01; * * * P < 0.001. Scale bars: 200 µm in PV + Cortical Interneurons Are Severely Reduced in Sp8/Sp9 Double Mutants Analysis of the P30 neocortex showed profound MGE-derived cortical interneuron defects, especially PV + cortical interneurons. We found that the density of MGE-derived cortical interneurons (GFP + cells) in the somatosensory cortex was significantly reduced in Nkx2-1-Cre; Sp8/9-DCKO mice compared within Nkx2-1-Cre; Sp9-CKO mice and controls (Figures 5A-D). The density of GFP + cells was also greatly reduced in each cortical layer (Figure 5E), but the percentage of GFP + cells in cortical layers II/III and V was relatively increased in single and double mutants (Figure 5F).
cortical interneurons were also reduced in double mutants compared with single mutants or controls (Figures 6S-X), whereas GFP + /SST + cells and GFP + /CR + cells were less affected in double mutants (Figures 6G-K,M-R). In Sp9 single and Sp8/9 double mutants, the percentage of GFP + /PV + cells was higher in cortical layer V, whereas the percentage of GFP + /PV + cells and GFP + /SST + cells was lower in cortical layer IV (Figures 6F,L), further indicating that Sp8/9 not only regulates migration but also affects the layer distribution of MGE-derived cortical interneurons. Nkx2-1-Cre recombination occurs from the MGE VZ, the SVZ and the mantle zone (Xu et al., 2008). We, therefore, used the Lhx6-Cre transgenic line to conditionally knock out Sp8/9 in postmitotic MGE-derived neurons (Fogarty et al., 2007;Nóbrega-Pereira et al., 2008) and quantified the density of GFP + , GFP + /PV + and GFP + /SST + interneurons in the somatosensory cortex. In general, loss of Sp8/9 function in postmitotic MGE cells resulted in greatly reduced GFP + and GFP + /PV + cortical interneuron density (Figures 7A-F, 8A-L). Furthermore, an altered allocation of MGE-derived interneurons in the cortical layers was also observed in the mutants (Figures 7A-F, 8A-L), consistent with results of the Nkx2-1-Cre line knockouts.

DISCUSSION
The function of Sp9 in the MGE has been described (Liu et al., 2018), but the function of Sp8 in the MGE, which is highly homologous with Sp9 and weakly expressed in the MGE mantle zone, has not yet been studied. Here, we found that the expression of SP8 in the MGE was significantly increased in Sp9 null mutants. When we conditionally deleted both Sp8 and Sp9 in the MGE, the tangential migration of MGE-derived cortical interneurons was greatly inhibited, and the density of MGE-derived cortical interneurons, especially PV + interneurons, was greatly reduced. These results suggest that Sp8 plays a supplementary role to Sp9 in regulating MGE-derived cortical interneuron development.
Previous studies have mainly focused on the function of Sp8 in the LGE, which show that Sp8 is important for the development of olfactory bulb interneurons and striatal medium spiny neurons (Waclaw et al., 2006;Liu et al., 2009;Li et al., 2011Xu et al., 2018). SP8 is very weakly expressed in the MGE mantle zone (Ma et al., 2012;Vogt et al., 2014), but its function remains to be investigated. In the present study, we found that SP8 expression was upregulated in the Sp9 mutant MGE, indicating that SP8 could compensate for SP9 function. Indeed, when we used the Nkx2-1-Cre transgenic line to conditionally knock out Sp8/9, the tangential migration of MGE-derived cortical interneurons was blocked, resulting in ectopic accumulation of interneuron-like cells in the ventral telencephalon. We propose that the loss of 80% of PV + cortical interneurons was mainly due to defects in interneuron migration, as conditional deletion of Sp8/9 in MGE neural stem cells using Nkx2-1-Cre lines and conditionally delete Sp8/9 in MGE-derived postmitotic cells using Lhx6-Cre lines show a similar reduction of cortex interneurons. Furthermore, we observed that the apoptotic cell death in the control, single and double mutant cortices were at the same levels (data not shown), suggesting that Sp8/9 do not affect the survival of MGE-derived cortical interneurons.
We suggest that this migratory deficit results from multiple mechanisms. In the double mutant neocortex, Erbb4 + immature PV interneurons mainly migrated in the cortical SVZ, whereas only a few migrated in the cortical MZ. Furthermore, we observed that more Erbb4 + cells ectopically accumulated in the ventral telencephalon. This finding explains why PV + cortical interneurons were severely reduced in the double mutant cortex compared with the single mutant cortex and indicate that Sp8 indeed has an important function in promoting MGE-derived cortical interneuron migration. The Ppp2r2c gene, encoding a subunit of protein phosphatase 2A, has a unique expression pattern in the embryonic mouse neocortex; interneurons in the cortical MZ express Ppp2r2c, but interneurons in the cortical SVZ do not (Colasante et al., 2009;Antypa et al., 2011;Friocourt and Parnavelas, 2011). Rasgef1b expression appeared to be limited to the cortical SVZ interneuron stream (Colasante et al., 2009;Antypa et al., 2011;Friocourt and Parnavelas, 2011). The Rasgef1b gene encodes a guanine nucleotide exchange factor for Ras family proteins and is a downstream target of Arx (Friocourt and Parnavelas, 2011). We found that the expression of Rasgef1b was significantly upregulated after removal of Sp8/9, whereas the expression of Ppp2r2c in the cortical MZ was significantly decreased. The dysregulation of these two genes may explain the great increase in the proportion of MGE-derived GFP + cells in the cortical SVZ. Notably, loss of Sp8/9 in the dorsal LGE also induced a block in tangential migration from the LGE to the olfactory bulb Guo et al., 2019). Thus, we propose that Sp8/9 genes might play a general role in regulating the tangential migration of telencephalic interneurons.

AUTHOR CONTRIBUTIONS
GT and ZL performed all experiments and analyses. YW, XS, SW and HD helped conduct some experiments and analyze the data.
ZY and YY helped guide the project and discussed the results. ZY, ZX, YY and GT wrote and edited the manuscript.