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doi: 10.1242/10.1242/dev.00328

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Tlx and Pax6 co-operate genetically to establish the pallio-subpallial boundary in the embryonic mouse telencephalon

¹ Division of Developmental Biology, Children's Hospital Research Foundation, Cincinnati, OH 45229-3039, USA
² Wallenberg Neuroscience Center, Division of Neurobiology, Lund University, Solvegatan 17, BMC A11, S-221 84 Lund, Sweden
³ Howard Hughes Medical Institute, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA

View larger version (125K): [in a new window]   Fig. 1. Forebrain expression of Tlx. (A) At E12.5, Tlx is expressed at high levels in the ventricular zone of the lateral telencephalon, whereas lower levels are present in the dorsal-medial telencephalon and in the medial ganglionic eminence (MGE). (B) The Tlx expression pattern is similar at E14.5. The asterisk indicates the remnant of the MGE. Note also that Tlx is also expressed in the ventricular zone of the diencephalon surrounding the third ventricle (3v).

View larger version (90K): [in a new window]   Fig. 2. Dorsal expansion of GSH2 expression in the Tlx mutant telencephalon. (A,E) GSH2 expression in the E12.5 wild-type (A) and Tlx mutant (E) LGE. (B,F) PAX6 expression in wild type (B) and Tlx mutants (F). (C,D,G,H) Merged confocal images of GSH2 and PAX6 expression; D and H are higher magnification of C and G, respectively. Note that in wild type (C,D) a small overlap of cells (approximately 2-3 cell diameters) expressing GSH2 and PAX6 is evident (yellows cells). In the Tlx mutant (G,H), this overlap is broader than in wild type (C,D). In D and H the filled arrows point to the domain of overlap; unfilled arrows point to the broader domain of PAX6-positive cells in the SVZ of the mutant (H) compared to the wild type (D).

View larger version (100K): [in a new window]   Fig. 3. Alterations in gene expression at the pallio-subpallial boundary of Tlx mutants at E14.5. In the wild-type telencephalon, GSH2 (A) and MASH1 (B) expression stops short of the LGE-cortex angle (i.e. ventral pallium). Expression of these subpallial markers in the Tlx mutant is shifted dorsally (arrows in E and F) into the LGE-cortex angle. Ngn2 (C) and PAX6 (D) are normally expressed throughout the ventricular zone of the pallium with a ventral limit of expression in the LGE-cortex angle. However, in the Tlx mutant expression of these markers is retracted from this region (G,H). Unfilled arrows in E-H point to the approximate position where the pallio-subpallial boundary would be in a wild type. Note that the PAX6-expressing SVZ cells (marked by filled arrows), which normally emanate from the pallio-subpallial boundary, are shifted dorsally and are more numerous in the Tlx mutant (H) than in the wild type (D).

View larger version (131K): [in a new window]   Fig. 4. Loss of ventral pallial markers in the Tlx mutant telencephalon. (A) Dbx1 is normally expressed in the LGE-cortex angle (i.e. ventral pallium, arrows in A and B). (B) In theTlx mutant, however, Dbx1 expression is lost in this region but not in the diencephalon (Di). Asterisk in A marks Dbx1 expression in a portion of the diencephalon that is in close contact with the ventral telencephalon. (C) Sfrp2 is also expressed in the LGE-cortex angle (arrows in C and D). Expression of this gene is also missing in this region of the Tlx mutant (D). Note that Sfrp2 expression remains in the Tlx mutant diencephalon around the third ventricle, which also normally expresses Tlx (see Fig. 1B).

View larger version (79K): [in a new window]   Fig. 5. Dorsal shift in SVZ and mantle markers in the Tlx mutant telencephalon at E14.5. (A) DLX proteins are normally expressed in the germinal zones and mantle regions of the ventral telencephalon, including the LGE. Arrows in A show the dorsal limit of DLX expression in the LGE SVZ. (B) In the Tlx mutant, the LGE is smaller, which is reflected in the smaller DLX-expressing domain. In addition, the normal limit of DLX expression (indicated by broken line) is shifted dorsally in the mutant (asterisk in B). (C) Islet1 (ISL1) is expressed in the LGE SVZ and developing striatum (arrows point to the dorsal limit of expression). (D) Although this domain is smaller in the Tlx mutant, it is not shifted dorsally (arrows) as is the case with DLX (B). (E) Er81 is normally expressed in a small domain of the LGE SVZ at the dorsal limit of DLX expression, as well as in the pallial ventricular zone (see also inset of pallio-LGE angle in E), excluding the medial pallium and part of the dorsal pallium. (F) In the Tlx mutant, the LGE SVZ domain of Er81 appears to be selectively expanded (normal limit marked by broken line) and shifted dorsally (asterisk) similar to that for DLX (B). Note the strong reduction of Er81 expression in the LGE-cortex angle (i.e. ventral pallium) of the Tlx mutant (arrow in F inset). Er81 expression in the lower portion of E and F reflect cells in the globus pallidus and developing striatum.

View larger version (29K): [in a new window]   Fig. 6. Schematic diagram of patterning defects in the ventricular zone of the Tlx mutant telencephalon. These mutants lack ventral pallial markers and exhibit a dorsal shift in the expression limits of genes that normally abut at the pallio-subpallial boundary.

View larger version (142K): [in a new window]   Fig. 7. Alterations in the radial glial palisade of Tlx mutants. (A,B) Nestin staining of radial glial fibers in the lateral telencephalon of E14.5 wild type (A) and Tlx mutant (B). In the wild type (A) the radial glial fibers fasciculate to form the radial glial palisade characteristic of the pallio-subpallial boundary (arrows). (B) The Tlx mutant shows fewer stained fibers and a lack of fasciculation of these fibers. (C,D) Parvalbumin-labeled radial glial fibers in the lateral telencephalon. Again, the parvalbumin-positive radial fibers fasciculate to form the glial palisade in wild type (arrows in C), whereas this does not occur in the mutant (D).

View larger version (109K): [in a new window]   Fig. 8. Tlx and Pax6 genetically interact to pattern the pallio-subpallial boundary. GSH2 (A,D,G,J), MASH1 (B,E,H,K) and Ngn2 (C,F,I,L) in the telencephalon of E14.5 Tlx–/– (A-C), Sey/+;Tlx–/– (D-F), Sey/Sey (G-I) and Sey/Sey; Tlx–/– mutants (J-L). Note the dorsal shift of GSH2 and MASH1 expression in the Sey/+;Tlx–/– (D,E) mutant as compared to the Tlx–/– mutant (A,B). This dorsal shift in subpallial markers also results in a greater retraction of Ngn2 expression in the Sey/+;Tlx–/– mutant (F) as compared to the Tlx–/– mutant (C). The Sey/Sey mutant displays a more significant dorsal shift in GSH2 (G) and MASH1 (H) expression as well as retraction of Ngn2 expression (I) than in the Sey/+;Tlx–/– mutant (D-F). Interestingly, removal of both TLX alleles on the Sey/Sey background (i.e. Sey/Sey; Tlx–/–) does not further exacerbate the pallial misspecification (J-L) over that seen in the Sey/Sey mutant alone (G-I).

View larger version (33K): [in a new window]   Fig. 9. Schematic diagram illustrating gene dosage requirements for Tlx and Pax6 in regulating gene expression at the pallio-subpallial boundary. Removal of one allele of Pax6 (i.e. Sey/+) results in a dorsal shift of the expression limits of the genes that normally abut at the pallio-subpallial boundary, which is more severe than that seen in homozygous Tlx mutants but less so than in Sey/Sey mutants.

View larger version (61K): [in a new window]   Fig. 10. Amygdalar defects in adult Tlx mutant brains. (A,B) Er81 is expressed in cells of the basolateral amygdala (BLA) in the wild type (A), whereas only scattered Er81-expressing cells are found in the amygdalar region of the Tlx mutant (B). (C) Acetylcholine esterase (AChE) staining reveals the lateral (LA), BLA and the central (Ce) nuclei of the wild type. (D) Although the staining in the Ce of Tlx mutants appears similar to that of wild type, the staining in the BLA and particularly the LA is greatly reduced. (E) DARPP-32 expression delineates the LA and BLA as a `tear drop' shape in the wild type, by virtue of its expression in portions of the Ce and in the interstitial nucleus (I). (F) No evidence of the BLA or LA is apparent in the DARPP-32-stained Tlx mutant brain, however, the interstitial nucleus (I) seems to be present. (G,H) MEIS2 expression is also seen in the interstitial nucleus (I) in both the wild type (G) and mutant (H). (I,J) Fate mapping the subpallial contribution to the LA and BLA using a Dlx5/6-cre crossed with the gtROSA reporter mouse shows that only a few subpallial cells (i.e. blue X-gal-positive cells) contribute to these amygdalar nuclei. Few, if any, of the Er81-positive cells in the BLA are X-gal positive, supporting the notion that the BLA and LA are largely derived from the pallium. Conversely, many cells in the Ce and medial (Me) amygdalar nuclei are labeled, supporting a largely subpallial origin for these nuclei.

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