Loss of Gsx1 and Gsx2 function rescues distinct phenotypes in Dlx1/2 mutants

Abstract

Mice lacking the Dlx1 and Dlx2 homeobox genes (Dlx1/2 mutants) have severe deficits in subpallial differentiation, including overexpression of the Gsx1 and Gsx2 homeobox genes. To investigate whether Gsx overexpression contributes to the Dlx1/2 mutant phenotypes, we made compound loss-of-function mutants. Eliminating Gsx2 function from the Dlx1/2 mutants rescued the increased expression of Ascl1 and Hes5 (Notch signaling mediators) and Olig2 (oligodendrogenesis mediator). In addition, Dlx1/2;Gsx2 mutants, like Dlx1/2;Ascl1 mutants, exacerbated the Gsx2 and Dlx1/2 patterning and differentiation phenotypes, particularly in the lateral ganglionic eminence (LGE) caudal ganglionic eminence (CGE), and septum, including loss of GAD1 expression. On the other hand, eliminating Gsx1 function from the Dlx1/2 mutants (Dlx1/2;Gsx1 mutants) did not severely exacerbate their phenotype; on the contrary, it resulted in a partial rescue of medial ganglionic eminence (MGE) properties, including interneuron migration to the cortex. Thus, despite their redundant properties, Gsx1 and -2 have distinct interactions with Dlx1 and -2. Gsx2 interaction is strongest in the LGE, CGE, and septum, whereas the Gsx1 interaction is strongest in the MGE. From these studies, and earlier studies, we present a model of the transcriptional network that regulates early steps of subcortical development.

Figure 1

LGE expression of ASCL1, DLX2, and GSX2 proteins using two-color immunofluoresence, at E10.5 (A–C) and E12.5 (D–F). Cells (nuclei) expressing both proteins are yellow; green and red correspond to specific transcription factors defined at the top of each panel. The boxed areas in each panel are shown at higher magnification in A′–F′. E12.5 data showing previously published information about the relationship between Gsx and Dlx expression in Gsx2 and Dlx1/2 mutants. Dlx1/2 mutants overexpress Gsx1 (G,H) and Gsx2 (I,J). Gsx2 mutants express less Dlx1, especially in the dorsal LGE (K,L). LGE, lateral ganglionic eminence; MZ, mantle zone; SE, septum; SVZ1 and SVZ2, subventricular zones 1 and 2. Scale bars = 20 μm in F (applies to A–F); 20 μm in F′ (applies to A′–F′); 500 μm in L (applies to G–L).

Figure 2

A–P: Combined functions of Gsx2 and Dlx1/2 define regional identity of LGE progenitor cells. In situ hybridization analysis of Ascl1, Dlx1, Vax1, and Ngn2 expression at E12.5 in the rostral telencephalon, highlighting the septum and LGE, in wild-type (WT), Gsx2^−/−, Dlx1/2^−/−, and Gsx2^−/−;Dlx1/2^−/−. Note that the LGE and septum of the mutant have lost subpallial properties and show expression of Ngn2 (pallial marker). See Figure 3 for E15.5 data. Hemisections of the telencephalon are shown. LGE, lateral ganglionic eminence; NCx, neocortex; SE, septum; SVZ1, subventricular zone 1; VZ, ventricular zone. Scale bar = 500 μm.

Figure 3

A–P: Combined functions of Gsx2 and Dlx1/2 define regional identity of LGE progenitor cells. In situ hybridization analysis of Ascl1, Dlx1, Vax1, Ngn2 expression at E15.5 in the rostral telencephalon, highlighting the septum and LGE, in wildp-type (WT), Gsx2^−/−, Dlx1/2^−/−, and Gsx2^−/−;Dlx1/2^−/−. Note that by this age there is nearly full recovery of the wild-type phenotype in Gsx2^−/−, whereas in Gsx2^−/−;Dlx1/2^−/− there remains ectopic Ngn2 expression in the septum and the dorsal LGE (arrows in P), as reduced expression of Vax1 and Dlx1 in the dLGE. Hemisections of the telencephalon are shown. LGE, lateral ganglionic eminence; NCx, neocortex; MZ, mantle zone; SE, septum; SVZ1, subventricular zone 1; VZ, ventricular zone. Scale bar = 1 mm.

Figure 4

A–P: Gsx2 promotes and Dlx1/2 represses the Notch signaling pathway and oligodendrocyte progenitors in subpallial SVZ cells. In situ hybridization analysis of Ascl1, Hes5, Olig2, and Six3 expression at E12.5 in the middle telencephalon, highlighting the LGE, along with MGE in wild-type (WT), Gsx2^−/−, Dlx1/2^−/−, and Gsx2^−/−;Dlx1/2^−/−. Note that Ascl1, Hes5, and Olig2 MGE expression in the Gsx2^−/−;Dlx1/2^−/− is restored toward WT levels (compare with the phenotype of Dlx1/2^−/−). See Figure 12 for E15.5 data. Hemisections of the telencephalon are shown. LGE, lateral ganglionic eminence; MGE; medial ganglionic eminence; NCx, neocortex; SE, septum; SVZ1 and SVZ2, subventricular zones 1 and 2; VZ, ventricular zone. Scale bar = 500 μm.

Figure 5

A–X: Gsx2 and Dlx1/2 have central roles in driving expression of Arx, Ebf1, Gad1, and Isl1 in striatal progenitors and neurons. In situ hybridization analysis of Arx, Ebf1, Foxp4, Gad1, and Isl1 expression at E12.5 (and FoxP4 at E15.5) in the middle telencephalon, highlighting the LGE (striatum) and MGE in wild-type (WT), Gsx2^−/−, Dlx1/2^−/−, and Gsx2^−/−;Dlx1/2^−/−. Note that LGE/striatal expression of Arx, Ebf1, Gad1, and Isl1 is greatly decreased in all of the mutants, whereas striatal FoxP4 expression is relatively well preserved at E12.5 and E15.5. See Figure 12 for E15.5 data. Hemisections of the telencephalon are shown. LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; NCx, neocortex; Str; striatum. Scale bars = 500 μm in T (applies to A–T); 1 mm in X (applies to U–X).

Figure 6

A–AB: Combined functions of Gsx2 and Dlx1/2 define regional identity of CGE progenitor cells. In situ hybridization analysis of Arx, Ascl1, Dlx1, Gsx1, FoxP4, Six3, and Sp9 expression at E12.5 in the caudal telencephalon, highlighting the CGE in wild-type (WT), Gsx2^−/−, Dlx1/2^−/−, and Gsx2^−/−;Dlx1/2^−/−. Note that the CGE of the Gsx2^−/−;Dlx1/2^−/− mutant has lost most of its subpallial properties. See Figure 10 for E15.5 data. Hemisections of the telencephalon are shown. CGE, caudal ganglionic eminence. Scale bar = 500 μm.

Figure 7

A–AB: Combined functions of Gsx2 and Dlx1/2 define regional identity and differentiation of CGE progenitor cells. In situ hybridization analysis of Arx, Ascl1, Dlx1, Gsx1, FoxP4, Six3, and Sp9 expression at E15.5 in the caudal telencephalon, highlighting the CGE in wild-type (WT), Gsx2^−/−, Dlx1/2^−/−, and Gsx2^−/−; Dlx1/2^−/−. Note that the CGE of the Gsx2^−/−;Dlx1/2^−/− mutant has lost most of its subpallial properties, except for residual Ascl1, Dlx1, Gsx1, and Sp9 expression in progenitor cells. Hemisections of the telencephalon are shown. CGE, caudal ganglionic eminence; Hyp, hypothalamus; RT, reticular thalamus. Scale bar = 1 mm.

Figure 8

A–L: Gsx2 is required for Dlx1/2-mediated repression of Gbx1, but not of Gsx1 and Otp expression, in subpallial progenitors. In situ hybridization analysis of Gsx1 and Otp expression at E12.5 and Gbx1 at E15.5, in the middle telencephalon, highlighting the LGE and MGE in wild-type (WT), Gsx2^−/−, Dlx1/2^−/−, and Gsx2^−/−;Dlx1/2^−/−. Abnormal expression of Gbx1 in the Dlx1/2^−/− LGE is reversed in Gsx2^−/−;Dlx1/2^−/−, whereas the Gsx1 and Otp phenotypes are not reversed. See Figure 7 for E15.5 Gsx1 data. Hemisections of the telencephalon are shown. LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; MZ, mantle zone; NCx, neocortex; SE, septum; SVZ1 and SVZ2, subventricular zones 1 and 2; VZ, ventricular zone. Scale bars = 500 μm in H (applies to A–H); 1 mm in L (applies to I–L).

Figure 9

A–AC: Relatively mild MGE phenotypes in the Gsx2^−/−, Dlx1/2^−/−, and Gsx2^−/−;Dlx1/2^−/− mutants. In situ hybridization analysis of Arx, Dlx1, Gad1, Nkx2-1, Nkx6-2, Gbx1, FoxP4, Pbx1, and Sp9 expression at E12.5 in the middle telencephalon, highlighting the LGE and MGE in wild-type (WT), Gsx2^−/−, Dlx1/2^−/−, and Gsx2^−/−;Dlx1/2^−/−. Although MGE differentiation is abnormal in the Dlx1/2 mutant (e.g., small globus pallidus, reduced Arx and Gad1 expression), the phenotype is not strongly altered by removing Gsx2. See Figure 10 for E15.5 data. Hemisections of the telencephalon are shown. LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; MZ, mantle zone; NCx, neocortex; GP, globus pallidus; SVZ1 and SVZ2, subventricular zones 1 and 2; VP, ventral pallidum; VZ, ventricular zone. Scale bar = 500 μm.

Figure 10

A–AE: Relatively mild MGE phenotypes in the Gsx2^−/−, Dlx1/2^−/−, and Gsx2^−/−;Dlx1/2^−/− mutants. In situ hybridization analysis of Arx, Dlx1, Gad1, Lhx6, Nkx2-1, Nkx6-2, Gbx1, FoxP4, Pbx1, and Sp9 expression at E15.5 in the middle telencephalon, highlighting the LGE and MGE in wild-type (WT), Gsx2^−/−, Dlx1/2^−/−, and Gsx2^−/−;Dlx1/2^−/−. Although MGE differentiation is abnormal in the Dlx1/2 mutant (e.g., small globus pallidus, reduced Arx and Gad1 expression), the phenotype is not strongly altered by removing Gsx2. Expression of Lhx6 in the Gsx2^−/− is not shown; previously, we found it to be normal at E11.5 and E18.5 (Yun et al., 2003). Hemisections of the telencephalon are shown. LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; GP, globus pallidus; VP, ventral pallidum. Scale bar = 1 mm.

Figure 11

A–AN: Dlx1/2 and Gsx2 are critical for septal development. In situ hybridization analysis of Arx, Ascl1, Dlx1, Gad1, Gsx1, Isl1, Ngn2, Olig2, Six3, and Vax1 at E12.5 in the rostral telencephalon, highlighting the LGE and septum in wild-type (WT), Gsx2^−/−, Dlx1/2^−/−, and Gsx2^−/−;Dlx1/2^−/−. Each single mutant shows gene expression defects, which are amplified in Gsx2^−/−;Dlx1/2^−/−. See Figure 12 for E15.5 data. Hemisections of the telencephalon are shown. LGE, lateral ganglionic eminence; NCx, neocortex; SE, septum. Scale bar = 500 μm.

Figure 12

A–AN: Gsx2 and Dlx1/2 have central roles in maintaining expression of Gad1 and other features of septal and striatal development. In situ hybridization analysis of Arx, Dlx1, Gad1, Gsx1, Isl1, Ngn2, Olig2, Six3, and Vax1 expression at E15.5 in the rostral telencephalon, highlighting the LGE (striatum) and septum in wild-type (WT), Gsx2^−/−, Dlx1/2^−/−, and Gsx2^−/−;Dlx1/2^−/−. Note the loss of Arx, Gad1, and Isl1 expression in the LGE/striatum and septum. Hemisections of the telencephalon are shown. LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; NCx, neocortex; Str, striatum. Scale bar = 1 mm.

Figure 13

A–AV: Gsx1, in combination with Dlx1/2, regulates septal development. In situ hybridization analysis of Ascl1, Dlx1, Dlx2, Gad1, Gbx1, Gsx2, Hes5, Lhx6, Nkx2-1, Otp, Sall3, and Sp9 at E15.5 in the rostral telencephalon, highlighting the LGE and septum in wild-type (WT), Gsx1^−/−, Dlx1/2^−/−, and Gsx1^−/−;Dlx1/2^−/−. The Gsx1 septum had more obvious phenotypes than the LGE (see Results); arrows point to reduced Dlx1, Dlx2, and Lhx6 expression in the septum (G,H,K,L,AE,AF). Hemisections of the telencephalon are shown. LGE, lateral ganglionic eminence; NCx, neocortex; SE, septum. Scale bar = 1 mm.

Figure 14

A–AV: Removal of Gsx1 partially rescues MGE differentiation in Dlx1/2 mutants. In situ hybridization analysis of Ascl1, Dlx1, Dlx2, Gad1, Gbx1, Gsx2, Hes5, Lhx6, Nkx2-1, Otp, Sall3, and Sp9 at E15.5 in the middle telencephalon, highlighting the LGE and MGE in wild-type (WT), Gsx1^−/−, Dlx1/2^−/−, and Gsx1^−/−;Dlx1/2^−/−. Note the partial rescue of Lhx6⁺ cells in the neocortex (higher magnifications in AC–AF) and the increased expression of Gad1 and Lhx6 in the SVZ of the MGE. Hemisections of the telencephalon are shown. GP, globus pallidus; MGE, medial ganglionic eminence; NCx, neocortex; POA, preoptic area. Scale bar = 1 mm.

Figure 15

A–AN: Although loss of Gsx1 partially rescues the Dlx1/2^−/− MGE, Lhx6⁺, Nkx2-1⁺, Gad1⁺, and Sp9⁺ cells continue to migrate ectopically in the CGE (arrows in O,P,AE,AF,AI,AJ,AU,AV). In situ hybridization analysis of Ascl1, Dlx1, Dlx2, Gad1, Gbx1, Gsx2, Hes5, Lhx6, Nkx2-1, Otp, Sall3, and Sp9 at E15.5 in the caudal telencephalon, highlighting the CGE in wild-type (WT), Gsx1^−/−, Dlx1/2^−/−, and Gsx1^−/−;Dlx1/2^−/−. Hemisections of the telencephalon are shown. CGE, caudal ganglionic eminence. Scale bar = 1 mm.

Figure 16

Expression of transcription factors in the ventricular zone (VZ), subventricular zone (SVZ), and mantle zone (MZ) of the LGE, MGE, and CGE in the Gsx2^−/− (Gsx2), Dlx1/2^−/− (Dlx), and Gsx2^−/−;Dlx1/2^−/− (Gsx2/Dlx) mutants at E12.5 and E15.5. This figures depicts, as discrete boxes, the VZ, SVZ, and MZ of the CGE, LGE, MGE, and septum. The genes are listed alphabetically. The effect of each mutation on transcription factor expression in each box is indicated using a color code. Black indicates that expression was not analyzed (if no squares are listed, this also means that this analysis was not performed). Gray indicates that expression was not clearly changed in the mutant. White indicates no detectable expression. Red indicates severe reduction in expression. Orange indicates moderate/mild reduction in expression. Green indicates ectopic expression. Blue indicates increased expression. If the box is subdivided diagonally, the top part correspond to the dorsal region, the bottom to the ventral region.

Figure 17

Expression of transcription factors in the ventricular zone (VZ), subventricular zone (SVZ), and mantle zone (MZ) of the LGE, MGE, and CGE in the Gsx1^−/− (Gsx1), Dlx1/2^−/− (Dlx), and Gsx1^−/−;Dlx1/2^−/− (Gsx1/Dlx) mutants at E15.5. This figure depicts, as discrete boxes, the VZ, SVZ, and MZ of the CGE, LGE, MGE, and septum. The genes are listed alphabetically. The effect of each mutation on transcription factor expression in each box is indicated using a color code. Gray indicates that expression was not clearly changed in the mutant. White indicates no detectable expression. Red indicates severe reduction in expression. Orange indicates moderate/mild reduction in expression. Green indicates ectopic expression. Blue indicates increased expression. If no squares are listed, this means that this analysis was not performed. If the box is subdivided diagonally, the top part correspond to the dorsal region, the bottom to the ventral region. Asterisk indicates that diagonal band GAD1 expression was absent.

Figure 18

Model of transcription factor network interactions in the developing LGE based largely on loss-of-function analyses (see Discussion). Green arrows indicate activation; magenta squares indicate inhibition. Genes activated by Dlx1 and -2 that have asterisks correspond to genes whose expression is most strongly reduced in the Dlx1/2^−/− mutant. At the top of the hierarchy is Gsx2; Gsx2(1) indicates that Gsx1 can compensate for loss of Gsx2. For comparison see Long et al. (2009a,b) for summaries of gene expression changes in the Dlx1/2, Ascl1, and Dlx1/2;Ascl1 mutants, using the same schemata.