Dlx1&2 and Mash1 transcription factors control striatal patterning and differentiation through parallel and overlapping pathways

Abstract

Here we define the expression of approximately 100 transcription factors in progenitors and neurons of the developing basal ganglia. We have begun to elucidate the transcriptional hierarchy of these genes with respect to the Dlx homeodomain genes, which are essential for differentiation of most GABAergic projection neurons of the basal ganglia. This analysis identified Dlx-dependent and Dlx-independent pathways. The Dlx-independent pathway depends in part on the function of the Mash1 b-HLH transcription factor. These analyses define core transcriptional components that differentially specify the identity and differentiation of the striatum, nucleus accumbens, and septum.

Figure 1. Dlx1&2-Dependent Expression of Transcription Factors in LGE Progenitors

In situ hybridization of coronal hemisections through the E15.5 telencephalon of control and Dlx1&2^−/− mutant animals demonstrates Dlx1&2-dependent expression in the SVZ and MZ of the LGE. For some genes the effect is either exclusive, or predominant, to the dLGE, leaving expression in the vLGE relatively normal (indicated by arrows). In other cases, however, vLGE expression is reduced. (a-d′) Note the loss of Dlx5&6 expression in the LGE, but not in the septum. Arrowheads mark the defects in the dSe, despite the maintenance of several transcription factors. dLGE, dorsal lateral ganglionic eminence; dSe, dorsal septum; vLGE, ventral lateral ganglionic eminence; vSe, ventral septum. Scale bars a-r′, 500 μm

Figure 2. Ectopic Expression of Ventrolateral Cortical Markers in the dLGE

In situ hybridization of coronal hemisections through the E15.5 telencephalon of control and Dlx1&2^−/− mutants. (a-c′) Arrows mark the ectopic expression of three ventrolateral cortical markers (Ebf3, Id2, NHLH2) in the dLGE of the Dlx1&2^−/− mutant. (d-e′); Id2 is also ectopically expressed in the septum (arrowhead). Two cortical markers (Tbr1, Vglut2) do not show ectopic expression. VPs, ventral pallidum superficial; VPd, ventral pallidum deep. Scale bars a-e′, 500 μm.

Figure 3. Dlx1&2 Repress Expression of Transcription Factors in LGE Progenitors

In situ hybridization of coronal hemisections through the E15.5 telencephalon of control and Dlx1&2^−/− mutants show transcription factors whose expression is up-regulated in the LGE, particularly in the SVZ. (a-f′): Genes whose expression is normally detected only in the VZ. (g-o′): Genes whose expression is normally detected in the VZ and/or SVZ of the LGE. Scale bars a-o′, 500 μm.

Figure 4. Dlx1&2^−/− Mutants Have Ectopic LGE and Septal Expression of Transcription Factors That Normally Mark the MGE and/or Diencephalon

In situ hybridization of coronal hemisections through the E15.5 telencephalon of control and Dlx1&2^−/− mutants. (a-e′) Several transcription factors that are normally not expressed in the LGE or Septum are ectopically expressed in the Dlx1&2^−/− mutant animals. This includes markers of the MGE (Gbx1, Gbx2, and Gsh1) and diencephalon (Otp). Scale bars a-d′, 500 μm.

Figure 5. Dlx-Dependent and Independent Transcription Factor Expression in Maturing Striatal Neurons

In situ hybridization of coronal hemisections through the E15.5 telencephalon of control and Dlx1&2^−/− mutants. (a-f′) Transcription factors whose expression is severely reduced in the Dlx1&2^−/− mutants. (g-x′) Transcription factors whose expression is partially reduced or maintained. (h′,i′) Arrowheads show reduced expression in the dorsal septum. dStr, dorsal striatum; OT, olfactory tubercle, Se, septum; vStr, ventral striatum. Scale bars a-x′, 500 μm.

Figure 6. Dlx-Dependent and Independent Expression of Non-Transcription Factor Markers of Striatal Differentiation and Migration

In situ hybridization of coronal hemisections through the E15.5 telencephalon of control and Dlx1&2^−/− mutants. (a-n′) Non transcription factors whose expression is decreased in the striatum. (o-u′) Non transcription factors whose expression is maintained or slightly increased in the striatum. dStr, dorsal striatum; OT, olfactory tubercle; PPT, preprotachykinin; Se, septum; vStr, ventral striatum. Scale bars a-u′, 500 μm.

Figure 7. Dlx1&2^−/−;Mash1^−/− Compound Mutants Define Epistatic Relationships in dLGE, vLGE and Septal Differentiation

In situ hybridization of coronal hemisections through the E15.5 telencephalon of control, Dlx1&2^−/−, Mash1^−/− mutants and Dlx1&2^−/−;Mash1^−/− mutants. (a′-p′) The Dlx1&2^−/− mutant shows severe dLGE differentiation defects and mild vLGE and septum defects (e.g. Sp8 n, n′). (a″-p″) The Mash1^−/− mutant animal shows severe septal vLGE defects and mild dLGE defects (e.g. Sp9 b,b″). (a‴-p‴) The Dlx1&2^−/−;Mash1^−/− mutant animal demonstrates the cooperative roles of by Dlx1&2 and Mash1 in dLGE, vLGE and septal development due to the aggravation of the individual mutant phenotypes. Arrows mark remnants of normal LGE gene expression in the Dlx1&2^−/−;Mash1^−/− mutants. dStr, dorsal striatum; OT, olfactory tubercle, Se, septum; vStr, ventral striatum. Scale bars a-ppp‴, 500 μm.

TABLE 2. Expression Levels of Transcription Factors Studied in the Dlx1&2^−/− Mutants

Table alphabetically lists the transcription factor gene name, basal ganglia/cortex (BG/Ctx) ratio of expression in wild type E15.5 embryos, the expression levels in the wild type cortex (Ctx) and basal ganglia (BG), and the expression level in the BG of the Dlx1&2^−/− mutants (BG −/−) (expression levels are in arbitrary units generated by the analysis of gene expression array data). Also shown are the investigators from who we received the plasmid used for in situ hybridization (Origin of Plasmid). Genes shown in green are expressed primarily in the basal ganglia. Genes shown in aqua are expressed both in the basal ganglia and cortex, but have a 2-fold bias towards the basal ganglia. Genes shown in yellow are expressed in both the basal ganglia and cortex at roughly the same level. Genes shown in lavender are expressed primarily in the cortex. The gene in orange is expressed in the diencephalon and only a small part of the amygdala. The columns on the left indicate whether we performed in situ hybridization (ISH) at E12.5 and E15.5. NR1H4 (FXR), FoxO1, NR4A1, and Nolz1 are genes expressed in the striatum at E18.5 (Chang et al., 2004; Gray et al., 2004); our in situ analysis did not detect E15.5 expression.

TABLE 2. Expression Levels of Transcription Factors Studied in the Dlx1&2^−/− Mutants

Table alphabetically lists the transcription factor gene name, basal ganglia/cortex (BG/Ctx) ratio of expression in wild type E15.5 embryos, the expression levels in the wild type cortex (Ctx) and basal ganglia (BG), and the expression level in the BG of the Dlx1&2^−/− mutants (BG −/−) (expression levels are in arbitrary units generated by the analysis of gene expression array data). Also shown are the investigators from who we received the plasmid used for in situ hybridization (Origin of Plasmid). Genes shown in green are expressed primarily in the basal ganglia. Genes shown in aqua are expressed both in the basal ganglia and cortex, but have a 2-fold bias towards the basal ganglia. Genes shown in yellow are expressed in both the basal ganglia and cortex at roughly the same level. Genes shown in lavender are expressed primarily in the cortex. The gene in orange is expressed in the diencephalon and only a small part of the amygdala. The columns on the left indicate whether we performed in situ hybridization (ISH) at E12.5 and E15.5. NR1H4 (FXR), FoxO1, NR4A1, and Nolz1 are genes expressed in the striatum at E18.5 (Chang et al., 2004; Gray et al., 2004); our in situ analysis did not detect E15.5 expression.

TABLE 2. Expression Levels of Transcription Factors Studied in the Dlx1&2^−/− Mutants

Table alphabetically lists the transcription factor gene name, basal ganglia/cortex (BG/Ctx) ratio of expression in wild type E15.5 embryos, the expression levels in the wild type cortex (Ctx) and basal ganglia (BG), and the expression level in the BG of the Dlx1&2^−/− mutants (BG −/−) (expression levels are in arbitrary units generated by the analysis of gene expression array data). Also shown are the investigators from who we received the plasmid used for in situ hybridization (Origin of Plasmid). Genes shown in green are expressed primarily in the basal ganglia. Genes shown in aqua are expressed both in the basal ganglia and cortex, but have a 2-fold bias towards the basal ganglia. Genes shown in yellow are expressed in both the basal ganglia and cortex at roughly the same level. Genes shown in lavender are expressed primarily in the cortex. The gene in orange is expressed in the diencephalon and only a small part of the amygdala. The columns on the left indicate whether we performed in situ hybridization (ISH) at E12.5 and E15.5. NR1H4 (FXR), FoxO1, NR4A1, and Nolz1 are genes expressed in the striatum at E18.5 (Chang et al., 2004; Gray et al., 2004); our in situ analysis did not detect E15.5 expression.

TABLE 3. Expression of Transcription Factors in the VZ, SVZ and MZ of the LGE in the Dlx1&2^−/− Mutants

Table depicts the ventricular zone (VZ), subventricular zone (SVZ) and mantle zone (MZ) of the LGE of an E15.5 embryo as discrete boxes. The effect of the Dlx1&2^−/− mutation on gene expression in each box is indicated using a color code: Gray represents unchanged gene expression. White represents no detectable expression. Magenta represents severe reduction in expression. Orange represents moderate/mild reduction in expression. Blue represents an increase of gene expression. Green represents ectopic expression. The genes are ordered as follows: left column are genes expressed in the proliferative zones (VZ and SVZ); right column are genes expressed at later developmental stages (SVZ, SVZ&MZ, MZ). The genes are arranged alphabetically within each grouping. A ‘d’ represents the effect is primarily in the dorsal part of the LGE and a ‘v’ represents the ventral part.

TABLE 4. Expression of Genes in the VZ, SVZ and MZ of the LGE and Septum in Dlx1&2^−/−, Mash1^−/− and Dlx1&2^−/−;Mash1^−/− Mutants

Table depicts the ventricular zone (VZ), subventricular zone (SVZ) and mantle zone (MZ) of the LGE and Septum of an E15.5 embryo as discrete boxes. The effect of the Dlx1&2^−/− (D), Mash1^−/− (M) and Dlx1&2^−/−;Mash1^−/− (DM) mutations on gene expression in each box is indicated using a color code: Gray represents unchanged gene expression. White represents no detectable expression. Magenta represents severe reduction in expression. Orange represents moderate/mild reduction in expression. Blue represents an increase of gene expression. Green represents ectopic expression. The genes are ordered according to when their expression begins; genes at the beginning are expressed in the VZ, whereas genes at the end are expressed only in the MZ. The genes are arranged alphabetically within each generalized grouping. The left columns correspond to the LGE and the right columns correspond to the septum. A ‘d’ represents the effect is primarily in the dorsal part of the structure and a ‘v’ represents the ventral part.