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First published online 20 August 2003
doi: 10.1242/dev.00717

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Combinatorial function of the homeodomain proteins Nkx2.1 and Gsh2 in ventral telencephalic patterning

¹ Developmental Genetics Program and the Department of Cell Biology, The Skirball Institute of Biomolecular Medicine, New York University Medical Center, 540 First Avenue, New York, NY 10016, USA
² Department of Neurology, Institute for Cell Engineering, Johns Hopkins School of Medicine, Baltimore, MD 21287, USA

View larger version (62K): [in a new window]   Fig. 1. Expression of patterning genes between E9.5 and E10.5. (A) As shown in coronal sections, at 23 somites (E9.5), expression of Pax6 (green) and Nkx2.1 (red) forms a boundary in the ventral telencephalon (arrow). (B) Higher power view shows Shh (blue) expression in the ventral-most telencephalon. (C) The border of Pax6 and Nkx2.1 expression, and nested Shh expression, is also shown. (D) By 27 somites (E10.0), a slight gap (arrows) appears between Pax6 (green) and Nkx2.1 (red) expression; (E,F) Gsh2 is (blue) expressed laterally (arrows in E). (G) By E10.5, the gap in the expression of Pax6 (green) and Nkx2.1 (red) expands (arrow). (H,I) Gsh2 (blue) is expressed in this gap (I) and is partially overlapping (white) with Pax6 expression (green; arrow; H). (I) At this stage, expression of Gsh2 (blue) is also no longer restricted to the lateral domain, but expands more ventrally into the Nkx2.1-positive (red) domain. Ctx, cerebral cortex; LGE, presumptive future lateral ganglionic eminence; MGE, presumptive future medial ganglionic eminence. Scale bars: 50 µm (A,D,E,F); 25 µm (B,C).

View larger version (71K): [in a new window]   Fig. 2. Telencephalic expression of Gsh2 and Nkx2.1 at 36-38 somites. Expression of Gsh2 (green) and Nkx2.1 (red) on coronal telencephalic sections is shown from anterior (A) to posterior (F). (A) In the anterior telencephalon, Gsh2 is expressed in the lateral domain; Nkx2.1 is not expressed at this level. (B) The most anterior expression of Nkx2.1 is at the level of the septum, where Gsh2 expression is non-overlapping with Nkx2.1 expression. (C-E) More posteriorly, some Gsh2-positive cells (yellow) begin to be observed within the Nkx2.1-positive domain. (F) At the most posterior level of the telencephalon, Gsh2 is highly expressed in the region of the presumptive CGE, whereas Nkx2.1 is expressed only in the diencephalon. CGE, presumptive future caudal ganglionic eminence; Di, diencephalon; Sep, septum. Scale bar: 50 µm (A-F).

View larger version (71K): [in a new window]   Fig. 3. Analysis of potential cross-repressive interactions in the early telencephalon. (A-H) Immunohistochemical analysis of Nkx2.1, Gsh2 and Pax6 expression on coronal sections in Pax6–/– (Sey/Sey), Nkx2.1–/– and Gsh2–/– mutant mice, respectively. Arrows mark the ventral or dorsal limit of expression of each protein. (A,B) At 22 somites, when expression of Pax6 and Nkx2.1 form a distinct border, Nkx2.1 expression does not expand dorsally in Sey/Sey mutants. (C,D) At this same stage, Pax6 expression also does not expand ventrally in Nkx2.1–/– mutants. (E,F) Between 26 and 30 somites, in Nkx2.1–/– mutants, Gsh2 expression in the lateral domain does not expand ventrally into the presumptive MGE region. (G,H) Conversely, in Gsh2–/– mutants at 31-32 somites, Nkx2.1 expression remains restricted ventrally. Scale bars: 50 µm (A-H).

View larger version (109K): [in a new window]   Fig. 4. Retroviral expression of Gsh2 represses Nkx2.1 expression in the MGE. Gsh2-expressing retroviruses were delivered to the developing telencephalon at E9.5 and analyzed at E14.5. Expression of bi-cistronic retroviral inserts was assayed by PLAP reporter expression. (A,D) Retrovirally infected, PLAP-expressing cells (red) are shown in the MGE (arrows) in coronal sections. (B,E) Nkx2.1 expression (green) in the MGE is repressed in clusters of retroviral expression. (C,F) Overlay of PLAP and Nkx2.1 shows a direct correspondence between retrovirally infected cells (red) and repression of Nkx2.1 expression (green). Scale bar in A: 25 µm (A-F).

View larger version (85K): [in a new window]   Fig. 5. Comparison of ventral patterning defects in Nkx2.1–/–, Gsh2–/– and Shh–/– single mutants, and Nkx2.1–/–;Gsh2–/– double mutants. At E12.5, Nkx2.1–/–;Gsh2–/– double-mutant mice (D,I,N,S) display a notable decrease in the size of ventral telencephalic structures and a reduction in the expression of Dlx2, Mash1, Gsh1 and Lhx6 compared with controls (A,F,K,P). Comparison with single Gsh2–/– mutants (B,G,L,Q) and single Nkx2.1–/– mutants (C,H,M,R) is also shown. Arrows (A-N) show the dorsal limits of Dlx2, Mash1 and Gsh1 gene expression. In less severely affected Shh–/– mutants, expression of Dlx2 and Mash1 (arrows) persists in the ventral telencephalon (E,J). By contrast, expression of Gsh1 and Lhx6 is never detected in these animals (O,T). Scale bar in A: 200 µm for A-D,F-I,K-N,P-S; 275 µm for E,J,O,T.

View larger version (111K): [in a new window]   Fig. 6. Expansion of dorsal gene expression and reduction of Ebf1-positive cells in Nkx2.1–/–;Gsh2–/– mice. In situ hybridization and immunohistochemical analysis on coronal sections at E12.5. Ngn2 and Pax6 expression normally extends just across the cortical-striatal sulcus into the dorsal LGE (A,E,M), and CRPB1 expression marks LGE radial glia (I). In Gsh2–/– mutants, expression of Ngn2 (B) and Pax6 (F,N) expands ventrally into the CRBP1-positive (J,N) mutant LGE. By contrast, in Nkx2.1–/– mutants, Ngn2 (C) and Pax6 (G,O) expression does not expand into the subpallium, but CRBP1 expression (K,O) expands ventrally into the mutant MGE. Similar to Gsh2–/– mutants, Nkx2.1–/–;Gsh2–/– double mutants display a significant expansion of Ngn2 (D) and Pax6 (H,P) into the ventral-most telencephalon. Arrows show the ventral-most limit of Ngn2 (A-D) and Pax6 (E-H,M-P) expression in control and mutant brains. Similar to Nkx2.1–/– mutants, CRBP1 expression also expands ventrally Nkx2.1–/–;Gsh2–/– double mutants (L,P). Ebf1 is expressed in differentiating cells of the LGE (Q). Ebf1 expression is reduced in the LGE in Gsh2–/– mutants (R) and expanded ventrally in Nkx2.1–/– mutants (S). In Nkx2.1–/–;Gsh2–/– mutants, Ebf1 expression is both reduced and expanded ventrally (T). Scale bar in A: 200 µm for A-D,Q-T; 100 µm for E-P.

View larger version (84K): [in a new window]   Fig. 7. Interneuron and oligodendrocyte specification. The status of developing interneurons and oligodendrocytes was examined on coronal sections at E12.5. Gad67 marks developing inhibitory neuron populations and is significantly reduced in Nkx2.1–/–;Gsh2–/– double-mutant mice (D) compared with control (A), Gsh2–/– (B) or Nkx2.1–/– (C) single mutant mice. Gad67 expression, although reduced and restricted to the ventral midline, persists in Shh–/– mutants (E). Olig2 is expressed in the VZ of the LGE and MGE (F). In Gsh2–/– mutants, Olig2 expression is reduced in the mutant LGE (G). By contrast, Olig2 expression appears unaffected in Nkx2.1–/– mutants (H). Olig2 expression is reduced in Nkx2.1–/–;Gsh2–/– mutants (I), and is absent in Shh–/– mutants (J). Arrows show dorsal limit of expression of Gad67 (A-D) and Olig2 (F-I) in control and mutant brains. Expression of Pdgfra is normally observed in the MGE as punctate staining (K; arrow). In Gsh2–/– mutants, Pdgfra is ectopically expressed in the VZ of the MGE and the ventral LGE (L), but is completely absent in Nkx2.1–/– mutants (M). Pdgfra is ectopically expressed in the lateral domain in Nkx2.1–/–;Gsh2–/– double mutants (N), and is absent in Shh–/– mutants (O). By contrast, Plp/DM20 expression (P; arrows) is unaffected in Gsh2–/– (Q) or Nkx2.1–/– (R) single, or Nkx2.1–/–;Gsh2–/– double (S), mutants. However, in Shh–/– mutants expression of Plp/DM20 is lost (T). Scale bar in A: 200 µm for A-D,F-I,K-N,P-S; 275 µm for E,J,O,T.

View larger version (106K): [in a new window]   Fig. 8. Reduction of cortical interneurons in Nkx2.1–/–;Gsh2–/– mutants. E18.5 coronal sections were immunostained for the interneuronal markers Calbindin and GABA. A marked reduction in both Calbindin-positive (A,B) and GABA-positive cortical interneurons (C,D) is observed in Nkx2.1–/–;Gsh2–/– mutants (B,D) compared with controls (A,C). Arrows show individual interneurons. Scale bar: 25 µm (A-D).

View larger version (32K): [in a new window]   Fig. 9. Schematic of homeodomain interactions that pattern the telencephalon. Diagram represents a coronal hemisection of an E12.5 telencephalon showing domains of homeodomain gene expression. The outline of the major genetic interactions governing telencephalic development is incorporated from the results of this study and others (Wilson and Rubenstein, 2000; Schuurmans and Guillemot, 2002; Rallu et al., 2002b; Campbell, 2003). Shh, via repression of the repressive action of Gli3, is required for normal ventral patterning. Shh is necessary and sufficient for the expression of Nkx2.1, which functions to repress LGE character in the MGE. However, this function of Nkx2.1 is not mediated through repression of Gsh1 and/or Gsh2. Conversely, Gsh1 and Gsh2 are not required to repress Nkx2.1 expression. By contrast, Gsh2, whose expression is regulated both via Shh-dependent and Shh-independent pathways, functions to repress dorsal character in all but the ventral-most one third of the LGE via cross-repression with Pax6. Patterning of the ventral-most one third of the LGE is dependent on Gsh1 gene function, whose expression, similar to Nkx2.1, is dependent on Shh. Expression of Dlx2, Mash1 and Olig2 is mediated either directly through Gsh1 and Gsh2 and/or indirectly through Pax6. Residual expression of Dlx2, Mash1, Olig2 and Gad67 in Nkx2.1–/–;Gsh2–/– mutants is hypothesized to be attributable to the persistence of Gsh1 expression.