Pax6 modulates the dorsoventral patterning of the mammalian telencephalon

Abstract

The Pax6 gene encodes a transcription factor with a restricted expression in the ventricular zone of the pallium and subpallium. We tested whether the function of Pax6 is necessary for the correct patterning and morphogenesis of the vertebrate telencephalon. Homozygous embryos of the Pax6/Small eye mutant lack functional PAX6 protein because of a point mutation of the gene. In the mutant Small eye embryos we detected a ventralization of the molecular patterning of the telencephalon at two borders, the pallium/subpallium and the lateral/medial ganglionic eminence. The results indicate that Pax6 controls the lateral limit of the expression of Nkx2.1, Shh, and Lhx6 in the prechordal neural tube, the telencephalon. This finding is in agreement with previous studies and supports a model for a common genetic mechanism for modulation of the dorsoventral patterning of the prechordal and epichordal CNS. The pattern defects caused by the loss of Pax6 function result in multiple morphological abnormalities in the Small eye brain: dysgenesis of the piriform, insular, and lateral cortices, the claustrum-endopiriform nucleus, and a failure in the differentiation of a subpopulation of the cortical precursors. Together the results demonstrate that Pax6 has an essential role for the modulation of the dorsoventral patterning of the embryonic telencephalon, influencing thereby the forebrain morphogenesis.

Fig. 1.

Ventralization of the neuroepithelium at the pallio/subpallial border at stage E12.5 in the Sey/Seytelencephalon. Adjacent coronal sections from the brain of wild-type (WT; A–D; F,H; E, G) and homozygous (Sey/Sey;A′–D′; F′,H′; E′, G′) littermates at E12.5 were hybridized with RNA probes for region-specific markers as indicated. The empty arrowhead inA points to the morphological corticostriatal sulcus, whereas the arrows in A,C, D, and E,H point to the pallial/subpallial border.A, The region of the ventral pallium is located between the arrowhead and the arrow (also in Figs. 5, 7). The thin arrows in A and A′) point to early-born Pax6+ cells possibly generated from the VP and migrating toward the presumptive anlage of the piriform cortex and anterior amygdala. B, B′,Emx1 is dorsally retracted from the LP in the mutant as compared with the WT brain. The subpallial markers for the VZ–SVZ are ectopically expressed in the Ne of the VP, LP, and DP of theSey/Sey as illustrated for: Dlx1(C′), Vax1 (D′), andMash1 (G′). The arrowheadsin C′ and D′ point to the extension of the Dlx1 and Vax1 expression into a more dorsal domain within the septum in the mutant brain. InH′, note that the normal expression ofSix3 in the striatal mantle extends laterally into the mantle of the VP. In E and E′, note that Ngn2 expression is abolished in the VZ of the VP, strongly suppressed in the LP and in a part of the DP, but appeared unaffected in the MP (designated by the asterisk below the MP). The two arrowheads in F point to the medial limit of Tbr1 expression along the pallial/subpallial border, thus including in its expression domain the mantle zone of the VP (the anlage of the ventromedial claustrum;Puelles et al., 1999). The expression of Tbr1 in this domain is abolished in the Sey/Sey brain (F′).

Fig. 2.

Ventralization of the neuroepithelium of the LGE in Sey/Sey. In situ hybridization on coronal (A–D′) and cross (E, E′) sections from the WT and Sey/Sey brains. Different markers for the MGE were tested at stages: E13.5 (A–C′), E12.0 (D,D′), and E14.5 (E,E′). In A, note the strikingly different level of Pax6 expression in the VZ of the VP and LGE. The arrowheads in A andA′ point to Pax6+ cells that appear to stream out from the Ne of the VP toward the basolateral telencephalon.B–C′, In the mutant telencephalon, the lateral limit of the expression of Shh(B,B′) andNkx2.1 (C,C′) extends from the MGE into the adjacent territory of the LGE. In D′, thelarge and smallarrowspoint to the lateral limit of the strong and the faint ectopic expression of Nkx2.1 within the VZ of the mutant LGE, respectively. In E and E′, note the enlarged MGE (which includes at this late stage the adjacent LGE domain with a ventralized identity) and the differentiating globus pallidus, labeled by the Nkx2.1 probe (open arrowhead). F, F′, Coronal sections from E15.5 WT (F) andSey/Sey (F′) brain at the level of the preoptic area stained with neutral red, illustrating the enlarged MGE in the Sey/Sey telencephalon.

Fig. 3.

Expression of Lhx6 in the basolateral telencephalon in wild-type and Sey/Seybrain. A, A′, Coronal sections from E12.5 wild-type (A) and mutant (A′) brain. Note the enhanced number of Lhx6+ cells in the mutant LGE. B–D′, Coronal sections from E14.0 wild-type (B–D) and mutant (B′–D′) brain at different rostrocaudal levels. The open and the thin arrows in B point to a Lhx6+layer of cells in the intermediate and marginal zones of the CP, respectively. In all levels note the enhanced expression ofLhx6 in the LGE in the mutant as compared with the WT brain and the lack of Lhx6+ cells in the lower part of the Sey/Sey CP. B, B′, Note the increased expression of Lhx6 in the rostral septum inSey/Sey (open arrowhead) from where moreLhx6+ cells seem to populate directly the LGE.

Fig. 4.

Differently patterned structures byPax6 and Tbr1 are distorted in theSey/Sey basolateral telencephalon. A, B, D, E, Adjacent coronal sections from the E18.5 WT brain were hybridized with probes for Tbr1 (A, B) and Pax6 (D, E). C is a bright-field picture of an adjacent section to the section (B) after hematoxylin–eosin (HE) staining. F is a close-up of C for the indicated field. A, B,Tbr1 expression is detected in the differentiating claustrum proper (Cl) and in the dense layer II (B, open arrowhead) of the piriform cortex. D, E,Pax6 is expressed in the olfactory tuberculum (Tu), in the ventral part (presumptive layer I) of the piriform cortex (E, arrowhead), and in the presumptive anlage of the anterior amygdala–endopiriform nucleus, a thin arrow in E and F. InE, note that the dense layer of the piriform cortex isPax6-negative (open arrowhead).G–J are adjacent coronal sections from the E18.5Sey/Sey brain, hybridized with Tbr1(G) and Pax6 (I) probes or stained with HE (H, J). In C,F, H, and Jnote that in the mutant brain, the piriform cortex, the claustrum proper, the endopiriform nucleus–anterior amygdala and the reservoir (r) are not distinguishable. The dark-stained structures in H and J are cells from the pallial germinative neuroepithelium that form clumps (or a thick band at other levels) located all along the pathway of the lateral migratory stream (Fig. 5) in the Sey/Sey pallium.

Fig. 5.

Inhibition of Ngn2 activity in a subpopulation of the cortical progenitors in Sey/Sey.Coronal sections from E16.5 wild-type (A, B) andSey/Sey (A′, B′) brain hybridized with Ngn2 (A,A′) and Pax6 (B,B′) probes. In A note that the region of the VP in the VZ of the LGE is still distinguishable at this late developmental stage. In the mutant, the expression ofNgn2 is completely abolished from the region of the VP and LP, severely repressed in the DP, but appears unaffected in the MP. In B′ note that the enlarged VZ–SVZ in theSey/Sey pallium is expressing abundantly the mutantPax6 mRNA (B′). C, D,Coronal sections from E13.5 WT brain were double-labeled by in situ histochemistry with the Ngn2 antisense RNA probe (blue cytoplasmic staining) and by immunohistochemistry with the Pax6 antibody (red nuclear stain). The enlarged inserts are higher magnifications showing that some Pax6-immunoreactive cortical progenitors expressNgn2 mRNA (open arrowheads). Thefilled arrowheads point to progenitors that are onlyNgn2+. D is a composite picture ofC and the Pax6 immunostaining; the overlay has been done in Adobe Photoshop.

Fig. 6.

Failure in the differentiation of the cortical plate in Sey/Sey. Coronal sections at a rostral level from E18.5 WT (B–D) and Sey/Sey(B′–D′) brain were hybridized with different cortical markers, as indicated. A andA′ are Nissl-stained sections from a WT and a mutant brain illustrating the severe abnormalities of the pallium in the mutant: an enlarged germinative zone (VZ–SVZ), a lack of a delineated intermediate zone (IZ) and subplate (SP), a thin cortical plate (CP) without radial alignment of the cells, and a wide and hypercellular marginal zone (MZ). The asterisks inE′ and F′ point to the germinative Ne of the VP + LP that are compressed by the growing striatum and therefore not easily distinguishable; the thin arrow inB points to the lateral migratory stream (LMS). InB′ note the Pax6-positive VZ–SVZ in the mutant pallium, which expands within the domain of the VP, LP, DP, in the hilus (h) of the LMS, but not in the MP. InC′ and G′ note that the expression ofEmx1 and Otx1 in the pallial VZ–SVZ (arrowheads) of Sey/Sey is detectable.E, F, In the WT brain, Tbr1 andmSorLa are expressed in the SVZ (empty arrowhead) of the entire pallium and their expression outline the piriform cortex as well. E′, F′, InSey/Sey, the expression of Tbr1 andSorLA is abolished in the enlarged SVZ of the VP–LP (asterisks), except for the region of the MP. In the basolateral telencephalon note the disorganization of the lateral insular and piriform cortex. C′,F′, G′, Emx1, Otx1, andmSorLa show diffuse expression in the abortive mutant CP, whereas the Tbr1 transcripts are accumulated in the lower part of the CP (E′). InD′ note the stronger expression of reelinin the mutant MZ. The arrowhead in Dpoints to a layer of reelin+ cells in the CP that is not detectable in the Sey/Sey cortex (D′).

Fig. 7.

Schematic representation of the DV pattern defects in the telencephalon of the Pax6/Small eye mutant. The scheme illustrates that in the absence of functional Pax6 protein, the molecular patterning of the embryonic telencephalon is ventralized at the level of the pallial/subpallial and MGE/LGE borders. The drawing is based on the proposed subdivision of the telencephalic Ne (Puelles et al., 1999, 2000) and the results obtained from the expression analysis performed on coronal sections at a rostral level of the E12.5 wild-type (WT) and homozygous Small eye (Sey/Sey) brain. The pallial and subpallial markers have been color-coded as indicated. Thearrow points to the morphological corticostriatal sulcus. The filled arrowhead points to the pallial/subpallial border, from where a of Pax6+ stream of cells (red dots) and Tbr1+ (black dots) cells migrate toward the basolateral telencephalon as discussed in the text. Noteworthy, results from a very recent homology study in chick and mouse suggest that thePax6+ cells migrate within the striatal territory (Puelles et al., 2000). The open arrowhead points to the boundary between the MGE (pallidum) and the LGE (striatum).