Sonic Hedgehog Signaling Controls Thalamic Progenitor Identity and Nuclei Specification in Mice

Shh signaling is graded within the thalamic progenitor zone

Within the diencephalon, Shh is expressed in the ZLI and the basal plate, which directly border the thalamus rostrally and ventrally, respectively (Fig. 1A). In this study we asked whether differential Shh signaling is responsible for setting up the expression patterns of the various transcription factors that we described previously (Fig. 1B) and later contributes to the specification of different thalamic nuclei. To determine whether the embryonic thalamus is indeed exposed to graded Shh activity, we analyzed the expression of Gli1 and Ptc1 in mouse embryos from E10.5 to E12.5. Gli1 and Ptc1 are direct target genes of the Shh transduction pathway, and their expression levels reflect the levels of Shh activity in the cell (Agren et al., 2004; Bai et al., 2004). We predicted that if thalamic progenitor cells are differentially patterned by Shh signaling, then both Gli1 and Ptc1 should be expressed in graded manners across pTH-R and pTH-C. Indeed, at both E10.5 and E12.5, Gli1 and Ptc1 were expressed in a high-rostroventral to low-caudodorsal gradient in wild-type brains (Fig. 1D–F,H–J; thalamus is defined by Shh and Olig3 in C and G); both genes were expressed at the highest levels in pTH-R, and their expression gradually tapered off caudally and dorsally away from the ZLI and the basal plate. With this finding, we hypothesized that the highest level of Shh signaling defines pTH-R, and progressively lower levels define different positional identity of progenitor cells within pTH-C depending on the distance from the ZLI and the basal plate. It was also evident that expression of Gli1 and Ptc1 is found only in progenitor cells of the thalamic ventricular zone (Fig. 1E,F,I,J) and not in postmitotic cells in the mantle zone, demonstrating that Shh signaling, whose direct output is gene activation mediated by Gli transcription factors, is restricted to neural progenitor cells.

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Figure 1.

Thalamic progenitor cells are exposed to graded activity of Shh signaling. A, A schematic of the location of the thalamus (TH) in the caudal diencephalon of the mouse embryo. Embryo is facing left. The thalamus is bordered by the pretectum caudally, the habenula dorsally, the basal plate ventrally, and by the ZLI rostrally. The basal plate and ZLI are sources of Shh (blue). Red dashed line shows the curvature of the anterior–posterior (AP) axis of the diencephalon. The two vertical lines (1 and 2) represent the planes of frontal sections shown in C–J. B, Expression patterns of bHLH (Olig2, Olig3, Ngn1/2, Mash1) and homeodomain (Nkx2.2, Dbx1) transcription factors in the thalamic ventricular zone. Olig3 is expressed in the entire thalamic ventricular zone and the ZLI. Expression of Mash1, Nkx2.2, Ngn1, and Ngn2 divides the thalamus into two distinct progenitor domains, a small rostral domain (pTH-R) and a much larger caudal domain (pTH-C). Olig2 and Dbx1 are expressed in opposing gradients within pTH-C. C–J, Graded patterns of Gli1 and Ptc1 expression in thalamic progenitor cells. Frontal sections through the thalamus of E10.5 (C, D, G, H) and E12.5 (E, F, I, J) wild-type brains. Top panels (C–F) are sections at a more dorsal and rostral level (plane 1 in A) and the bottom panels (G–J) are for a more ventral and caudal level (plane 2 in A), showing similar results. C, G, Double immunofluorescence for Olig3 and Shh at E10.5 to show the location of the thalamus. Olig3 expression caudal to the ZLI marks the entire thalamic ventricular zone. D, H, In situ hybridization for Gli1 on sections adjacent to C and G. Gli1 expression is higher closer to the ZLI and decreases in a graded manner throughout the thalamic ventricular zone. Arrowheads indicate dorsal and caudal boundary of the thalamus delineated by Olig3 expression. E, I, In situ hybridization for Gli1 at E12.5, showing a similar gradient to E10.5. F, J, In situ hybridization for Ptc1 on adjacent sections to E and I, respectively, again showing similar gradients. Asterisk and dotted lines in E and F mark the thalamic ventricular zone, indicating that expression of Gli1 and Ptc1 is restricted to progenitor cells. 3V, Third ventricle; LV, lateral ventricle; PT, pretectum; TH, thalamus; PTh, prethalamus. Scale bar, 500 μm.

Ectopic enhancement of Shh signaling strongly induces Gli1 and Ptc1 in the entire thalamic progenitor domains

Based on the above findings, we examined whether increased Shh activity in thalamic progenitor cells can cause a rostroventral shift in their positional identity. More specifically, we predicted that if caudodorsal progenitors are exposed to a higher level of Shh activity than normal, these progenitor cells would express rostroventral molecular markers rather than caudodorsal ones. To test this possibility, we crossed Nestin-Cre (Nes^Cre/+) transgenic mice (Tronche et al., 1999) with ROSA26-stop-SmoM2-EYFP (R26^SmoM2/SmoM2) mice (Jeong et al., 2004) to express the fusion protein of SmoM2-EYFP broadly in neural progenitor cells, including those in the thalamus. SmoM2 carries a missense mutation in the Smoothened (Smo) gene, which encodes a transmembrane Shh effector. SmoM2-expressing cells undergo cell-autonomous activation of Shh signaling independent of ligand binding (Xie et al., 1998). Thus, we expected that mis-expression of SmoM2 would cause elevated Shh signaling in all thalamic progenitor cells. By comparing Nes^Cre/+; R26^SmoM2/+ and Cre-negative, R26^SmoM2/+ controls and wild-type embryos, we found that Cre-negative control and wild-type embryos show the same gene expression and morphology at all stages. In contrast, brains of Nes^Cre/+; R26^SmoM2/+ mice were larger in size than Cre-negative littermates, especially in the dorsal telencephalon.

Analysis of SmoM2-EYFP expression as detected by anti-EGFP antibody showed that in Nes^Cre/+; R26^SmoM2/+ embryos, Cre-mediated recombination starts to occur in the diencephalon by E9.5 (Fig. 2A–C,F–H). At this stage, Shh expression in the ZLI has just started (Fig. 2A,F), and Gli1 and Ptc1 were not clearly detectable in control embryos (Fig. 2D,E). However, in Nes^Cre/+; R26^SmoM2/+ embryos, Gli1 and Ptc1 were already expressed ectopically in thalamic progenitor cells near the ZLI (Fig. 2I,J). Subsequently, recombination progresses through the thalamus into the more caudodorsal region, and by E10.0, SmoM2-EYFP and Gli1 expression covered the entire thalamus labeled by Olig3 (Fig. 2K,L,M,N). By E10.5, most embryos we analyzed showed broad expression of SmoM2-EYFP and Gli1 in the entire diencephalon except the most dorsal portion (Fig. 2P,Q). Analysis of recombination using R26-stop-EYFP reporter mice showed a similar rostroventral to caudodorsal progression of EYFP expression (supplemental Fig. S1A–D, available at www.jneurosci.org as supplemental material). These results demonstrate that in Nes^Cre/+; R26^SmoM2/+ embryos, Shh signaling is elevated in a rostroventral to caudodorsal direction, and by E10.0, there is a uniform enhancement in signaling within the entire thalamic ventricular zone.

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Figure 2.

Ectopic expression of SmoM2 in neural progenitor cells elevates the level of Shh signaling throughout the entire thalamic ventricular zone. A–Q, Frontal sections of Nes^Cre/+; R26^SmoM2/+ (F–Q) and Cre− control (A–E) embryos at E9.5 (A–J), E10.0 (K–N), and E10.5 (O–Q). Midline is to the left. A–J, Controls (A–E) are compared with Nes^Cre/+; R26^SmoM2/+ littermates (F–J) at E9.5. A, B, F, G, Double immunofluorescence for Shh and Olig3 to show the locations of the ZLI and thalamus, which are similar between control and Nes^Cre/+; R26^SmoM2/+ embryos. EYFP immunofluorescence shows that Cre-mediated misexpression of SmoM2-EYFP in the thalamus starts to occur at E9.5, but only in the vicinity of the ZLI (C, H, arrowheads). C and H are adjacent to B and G, respectively. In situ hybridization analysis for Gli1 and Ptc1 at E9.5 (D, E, I, J) shows that Gli1 and Ptc1 are not yet expressed in Cre− controls (D, E), but in SmoM2-expressing brain, both Gli1 and Ptc1 are prematurely induced in the thalamus (I, J, arrowheads) as well as in the prethalamus. K–N, In Nes^Cre/+; R26^SmoM2/+ embryos at E10.0, recombination as indicated by SmoM2-YFP expression spans the entire thalamus marked by Olig3 expression. Gli1 expression follows this. Arrowhead indicates the ZLI, and arrow shows the caudodorsal border of the thalamus. O–Q, In Nes^Cre/+; R26^SmoM2/+ embryos at E10.5, recombination has further progressed, and SmoM2-EYFP expression is found in most of the diencephalon. The arrow indicates the most caudal–dorsal part of the diencephalon that has not yet undergone recombination. Triple arrows show that the mantle zone of the thalamus does not express Gli1. Scale bar, 200 μm for A–N, 500 μm for O–Q.

Increased Shh activity induces rostroventral markers in thalamic progenitor cells

We then asked whether the increased Shh signaling in Nes^Cre/+; R26^SmoM2/+ embryos alters the expression profile of transcription factors in the thalamus. In Nes^Cre/+; R26^SmoM2/+ embryos, expression of Shh in the ZLI was not affected (Fig. 3A,B,E,F). Nonetheless, expression of Nkx2.2 and Mash1, two markers for the rostral progenitor domain, pTH-R, was expanded (Fig. 3C,D,G,H). This expansion results in more than two-fold increase in the size of pTH-R and disrupts the normally distinct gene expression boundary between pTH-R and pTH-C. The once distinct boundary is now replaced by a “mixed zone (pTH-R/C)” in which some cells expressing only pTH-R markers (Nkx2.2 and Mash1) are intermingled with cells expressing only pTH-C markers (Ngn2 and Olig2) (Fig. 3G,H). Such a zone of mixed pTH-R and pTH-C identity was not seen in control embryos (Fig. 3C,D). Although expression of SmoM2-EYFP appeared ubiquitous and homogeneous throughout the thalamic ventricular zone of Nes^Cre/+; R26^SmoM2/+ embryos at E11.5 (supplemental Fig. S1E–H, available at www.jneurosci.org as supplemental material), it is possible that only a small number of cells in this zone expressed ectopic SmoM2 early enough to take on the pTH-R fate, whereas the remaining cells were irreversibly determined to become pTH-C progenitor cells.

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Figure 3.

Ectopic SmoM2 expression causes caudodorsal expansion of pTH-R marker genes Nkx2.2 and Mash1 in thalamic progenitors. A–H, Frontal sections of Nes^Cre/+; R26^SmoM2/+ (E–H) and Cre− control (A–D) embryos at E12.5. Midline is to the left. Immunofluorescence for Shh shows the location of the ZLI, which is similar between Nes^Cre/+; R26^SmoM2/+ (A) and control brains (E). B, F, High-magnification views of the boxed areas in A and E, respectively. These are adjacent to C/D and G/H, respectively. Double immunofluorescence for Nkx2.2 and Olig2 (C, G) and double immunofluorescence for Mash1 and Ngn2 (D, H) show that in the control brain, Nkx2.2 and Mash1 are expressed in pTH-R and form a distinct boundary with pTH-C, which expresses Olig2 and Ngn2 (C, D). In Nes^Cre/+; R26^SmoM2/+ brains, both Nkx2.2 and Mash1 are expanded caudodorsally, increasing the size of pTH-R. Only in Nes^Cre/+; R26^SmoM2/+ embryos, there is an additional domain between pTH-R and pTH-C that contain both pTH-R markers Nkx2.2/Mash1 and pTH-C markers Olig2/Ngn2. These two sets of markers are not colocalized (arrows in G, H). Dotted lines outline the ZLI in each panel. Arrowheads indicate the domain with both pTH-R and pTH-C markers, which we refer to as pTH-R/C. I, J, Analysis of E12.5 embryos electroporated with pCAG-SmoM2-EYFP plasmid at E11.5 in the thalamus. Double immunofluorescence of Nkx2.2 and YFP on frontal sections. Right side was transfected. Left side is control. Nkx2.2 is ectopically induced in the thalamic progenitor cells only on the transfected side (double arrows in I). Transfection occurred only in the thalamus, not in the prethalamus, as shown by YFP expression (J). K, Strategy for making the Olig3^Cre mice. The exon encoding for Olig3 was replaced by the Cre-frt-neo-frt (Cre-FNF) cassette. L–O, Analysis of recombination in Olig3^Cre mice. Olig3^Cre/+ mice were bred with ROSA26-stop-EYFP mice and embryos were analyzed at E11.5. Double immunofluorescence of YFP (L) and Olig3 (M) shows that YFP is expressed in the thalamus, where endogenous Olig3 is expressed. Low-magnification images of the same embryo indicate the specificity of Cre-mediated recombination in the forebrain (N, O). Neither other regions of the diencephalon or the neocortex have undergone recombination (arrows). P–S, Frontal sections of Olig3^Cre/+; R26^SmoM2/+ embryos at E12.5, showing that thalamus-specific, ectopic expression of SmoM2 causes the same marker changes in progenitor cells as seen in Nes^Cre/+; R26^SmoM2/+ embryos (A–H). pTH-R is expanded (bracket), and the mixed zone (pTH-R/C) emerges between pTH-R and pTH-C (arrowheads). Scale bar, 200 μm for A–M and P–S, 500 μm for N and O.

SmoM2 acts cell autonomously to enhance Shh signaling (Xie et al., 1998). Thus, the observed changes in marker expression are likely to be caused by intrinsic changes in thalamic patterning. Nonetheless, since most CNS progenitor cells are forced to express SmoM2 in Nes^Cre/+; R26^SmoM2/+ embryos, it is possible that some secondary, indirect effects from outside the thalamus may have contributed to the expansion of pTH-R and/or the formation of the “mixed zone.” To investigate this possibility, we took two additional approaches to further restrict the SmoM2 expression spatially and temporally within the thalamus.

First, we performed in utero electroporation of SmoM2 plasmid (pCAG-SmoM2-EYFP) at E11.5 and analyzed the embryos a day later. We found that similar to Nes^Cre/+; R26^SmoM2/+ embryos, both Nkx2.2 (Fig. 3I,J) and Mash1 (data not shown) are expanded and are ectopically expressed more caudally on the electroporated side compared with the control side. As a second approach, we generated Olig3^Cre mice (Fig. 3K), so that we can express Cre recombinase in pTH-R, pTH-C, and the ZLI, but not in surrounding regions of the forebrain such as the prethalamus, the habenula, the pretectum, the diencephalic basal plate or the telencephalon. Cre-mediated recombination as assessed with R26-stop-EYFP reporter mice (Fig. 3L–O) and ROSA26-stop-SmoM2-EYFP mice (supplemental Fig. S1I,J, available at www.jneurosci.org as supplemental material) faithfully followed the expression of the endogenous Olig3 gene. Thus, Olig3^Cre mice are useful in thalamus-specific gene manipulation. We found that brains of Olig3^Cre/+; R26^SmoM2/+ are morphologically indistinguishable from control littermates, and some of these pups survive postnatally. Neuronal differentiation as determined by the expression of TuJ1, a neuronal marker, was also not affected in these embryos (supplemental Fig. S2A,B, available at www.jneurosci.org as supplemental material). Additionally, analysis of cell proliferation in Olig3^Cre/+; R26^SmoM2/+ embryos at E11.5 revealed that BrdU-incorporation and phospho-histone H3 (PH3), an M-phase marker, also appeared normal (supplemental Fig. S2A,B, available at www.jneurosci.org as supplemental material). Quantification of PH3-positive cells in Olig3-expressing thalamic progenitor cells showed that there was no significant difference in number between Olig3^Cre/+; R26^SmoM2/+ and control embryos (4440 ± 206.0, n = 2 for Olig3^Cre/+; R26^SmoM2/+, 4906 ± 449.5, n = 2 for Cre− controls; p = 0.45).

Analysis of progenitor markers in Olig3^Cre/+; R26^SmoM2/+ embryos showed similar changes in gene expression compared with Nes^Cre/+; R26^SmoM2/+ embryos; pTH-R was expanded, and a “mixed zone (pTH-R/C)” was formed between pTH-C and the expanded pTH-R (Fig. 3P–S). The results of these two additional approaches, in vivo electroporation and the use of Olig3^Cre mice, further reinforce our conclusion that it is the intrinsic increase in Shh signaling that causes the caudodorsal shift of pTH-R markers in thalamic progenitor cells.

We next examined how the pTH-C domain was affected by the increase in Shh signaling. First, in Nes^Cre/+; R26^SmoM2/+ embryos, Olig3 was expanded caudodorsally into the pretectal/habenular region at E12.5 (Fig. 4A,G). At this stage, Gbx2 and RORα, markers of the postmitotic thalamus derived from the pTH-C domain (Vue et al., 2007), were also expanded accordingly (supplemental Fig. S3A–C,F–H, available at www.jneurosci.org as supplemental material). In contrast, Pax7, which is normally expressed in the pretectum and habenula, but not in thalamic progenitor cells, was not detected in Nes^Cre/+; R26^SmoM2/+ embryos (Fig. 4E,K). Moreover, expression of Mash1 in the caudal pretectum was replaced by Ngn2, which normally spans from pTH-C only into the rostral pretectum (Fig. 4F,L). Irx2 and BHLHB4, early postmitotic markers of the pretectum, were also reduced at E12.5 (supplemental Fig. S3D,E,I,J, available at www.jneurosci.org as supplemental material). These results show that a high Shh signal in the caudal diencephalon induces the thalamic progenitor domain, pTH-C, at the expense of the pretectum and habenula. Olig2, which is normally expressed in a rostral–ventral to caudal–dorsal gradient within pTH-C, was homogeneously expressed in this expanded pTH-C domain of Nes^Cre/+; R26^SmoM2/+ embryos (Fig. 4B,H). Another marker gene, Pdlim3, which is normally expressed in pTH-R as well as the rostroventral part of pTH-C (Fig. 4C) (Gray et al., 2004), also expanded (Fig. 4C,I). These results demonstrate that enhanced Shh signaling in the entire diencephalon transformed it into the rostral–ventral thalamus, which is composed of the pTH-R domain and the rostral–ventral part of the pTH-C domain (summarized in Fig. 9A,B1,B2,C1,C2).

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Figure 4.

Ectopic SmoM2 expression in neural progenitor cells causes the caudal diencephalon to acquire rostral thalamic identity. A–L, Frontal sections of Nes^Cre/+; R26^SmoM2/+ (G–L) and Cre− control (A–F) embryos at E12.5. Midline is to the left. In Nes^Cre/+; R26^SmoM2/+ thalamus, Olig3 expression maintains its rostral boundary between the ZLI and the prethalamus, but is expanded caudodorsally (A, G; arrows in G indicate the area of expansion). Similarly, Olig2 and Pdlim3, both of which are expressed in the rostroventral part of pTH-C in Cre− embryos (B, C), are significantly expanded in Nes^Cre/+; R26^SmoM2/+ embryos (H, I; arrows show the extent of expansion). Dbx1, which is normally expressed in the caudal pTH-C and the rostral pretectum (D, arrow), is not significantly altered in Nes^Cre/+; R26^SmoM2/+ embryos (J, arrows). Pax7 is expressed in the pretectum and the habenula of Cre− controls (E, arrow), but is reduced in Nes^Cre/+; R26^SmoM2/+ embryos (K, no expression between arrowheads). Ngn2 is expressed in pTH-C and the rostral pretectum, whereas Mash1 is expressed in the caudal pretectum (F, arrows to show Mash1 expression). In Nes^Cre/+; R26^SmoM2/+ brains, Ngn2 is induced in the caudal pretectum and Mash1 is repressed (L, no expression between the arrows). M–R, Olig3^Cre/+; R26^SmoM2/+ (P–R) and Cre− control (M–O) embryos at E12.5. Midline is to the left. In Olig3^Cre/+; R26^SmoM2/+ embryos, Olig3 expression is not expanded, unlike in Nes^Cre/+; R26^SmoM2/+ embryos (M, P, arrow showing the caudodorsal boundary of pTH-C), indicating that SmoM2 expression is indeed limited to the thalamus. Within the thalamus, however, Ptc1 expression is increased and now occupies the entire pTH-R and pTH-C (N, Q). Olig2 is also induced more caudodorsally within pTH-C (O, R). Arrow in M–R indicates the caudal–dorsal limit of gene expression, showing that unlike in the Cre− control, Ptc1 and Olig2 are now expressed in the entire pTH-C in Olig3^Cre/+; R26^SmoM2/+ embryos. S, T, E12.5 embryo in which pCAG-SmoM2-EYFP was electroporated into two focal diencephalic regions at E11.5. The right side was electroporated. Olig2 is ectopically expressed in the caudal group of electroporated cells in an isolated manner (arrow). Scale bar, 200 μm.

Surprisingly, the expression of Dbx1, which is normally expressed in the caudodorsal part of pTH-C as well as in the pretectum and habenula, was not significantly reduced in Nes^Cre/+; R26^SmoM2/+ embryos (Fig. 4D,J), suggesting that Dbx1 is positively regulated by some unknown mechanisms operating in the caudodorsal diencephalon. Expression of Dlx2, a prethalamic marker, was not ectopically induced in the thalamic progenitor cells in Nes^Cre/+; R26^SmoM2/+ embryos (data not shown), consistent with a previous study showing that the thalamus and the prethalamus are distinctly prepatterned and express different sets of genes in response to Shh signaling (Kiecker and Lumsden, 2004).

To ensure that the above changes were also not the product of indirect effects, we again analyzed embryos electroporated with the pCAG-SmoM2-EYFP plasmid, as well as Olig3^Cre/+; R26^SmoM2/+ embryos. In electroporated embryos where an isolated population of progenitor cells in the pretectum were transfected, Olig2 was ectopically induced (Fig. 4S,T). In Olig3^Cre/+; R26^SmoM2/+ embryos, although the pretectum and the habenula are spared and the extent of Olig3 expression was similar to controls (Fig. 4M,P), Ptc1 and Olig2 were homogeneously elevated within the entire pTH-C domain (Fig. 4N,O,Q,R), demonstrating that thalamus-specific elevation of Shh signaling intrinsically induced the rostral pTH-C markers.

Expression of rostral postmitotic thalamic markers is expanded in SmoM2-expressing embryos

Does increased Shh signaling in thalamic progenitor cells lead to reorganization of postmitotic thalamus? To address this question, we examined the expression of postmitotic markers that are differentially expressed within nuclei of the late embryonic thalamus (Fig. 5).

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Figure 5.

Ectopic SmoM2 expression causes a rostroventral shift of postmitotic thalamus. A–L, Frontal sections of Nes^Cre/+; R26^SmoM2/+ (G–L) and Cre− control embryos (A–F) at E16.5. Midline is to the left. In control embryos, Nkx2.2 and Sox14 expression delineates the lateral vLG and IGL (A–C, arrowhead), which are derived from pTH-R. B, High-magnification image of the boxed region in A. In Nes^Cre/+; R26^SmoM2/+ embryos, Nkx2.2 and Sox14 expression is expanded caudally and dorsally (G–I, arrowhead). H, High-magnification image of the boxed areas in G, showing that some cells ectopically expressing Nkx2.2 (H, arrowheads) are intermingled with Sox2-expressing cells (J). In Cre− controls, Sox2 is highly expressed in rostroventral part of the postmitotic thalamus, including VP and dLG, but not in more caudodorsal part (D, asterisk). In Nes^Cre/+; R26^SmoM2/+ embryos, Sox2 is robustly expanded caudodorsally (J, arrowheads). Similarly, RORα expression, which corresponds to rostrolateral nuclei such as VP and dLG (E), is also expanded caudodorsally in Nes^Cre/+; R26^SmoM2/+ embryos (K, arrowheads). Note that the medial part of the thalamus that is negative for RORα in control is also negative in SmoM2 embryos (E, K, asterisk). The caudodorsal expression border of Gbx2 is elongated near the midline in SmoM2 embryos (F, L, arrow), but overall, Gbx2 expression is reduced, especially from the medial dorsal part (F, L, asterisk). M, N, E17.5 embryo in which pCAG-SmoM2-EYFP plasmid was electroporated at E11.5 into the thalamus. The right side was electroporated, and the left side is the control. On the electroporated side, Sox2 is ectopically induced in a caudodorsal region (M, arrow), while this induction did not occur on the control side. Conversely, Gbx2 is downregulated where Sox2 is induced (N, arrow). O–T, Olig3^Cre/+; R26^SmoM2/+ and Cre− control embryos at E18.5. Midline is to the left. In Olig3^Cre/+; R26^SmoM2/+ embryos, Sox2 expression is expanded and now occupies the entire thalamic mantle zone (R). The Sox2-negative, caudodorsal region (asterisk in O) is undetectable in Olig3^Cre/+; R26^SmoM2/+ embryos. The caudodorsal boundary of the thalamus is outlined by a dotted line. Similarly, expression of RORα is expanded (P, S). Unlike Sox2, RORα is not induced in the medial part of the thalamic mantle zone (P, S, asterisk). Gbx2 expression is dramatically reduced in Olig3^Cre/+; R26^SmoM2/+ embryos (Q, T). U–X, Postnatal day 1 thalamus of Olig3^Cre/+; R26^SmoM2/+ (W, X) and Cre− control (U, V) pups injected with CTB in the contralateral retina at P0. dLG nucleus is delineated as dashed lines by CTB immunohistochemistry (V, X) and DAPI staining (U, W). dLG is expanded by ∼3.6 times (see Results). Scale bar, 200 μm for B, H; 500 μm for other panels.

First, we analyzed nuclei that are derived from the pTH-R domain. We previously found that progenitor cells in pTH-R generate part of the vLG as well as the IGL, two nuclei that do not contain neurons projecting to the neocortex (Vue et al., 2007). Consistent with this fate analysis and the expanded pTH-R markers in Nes^Cre/+; R26^SmoM2/+ as well as Olig3^Cre/+; R26^SmoM2/+ embryos, vLG/IGL was expanded caudodorsally along the lateral surface of the diencephalon (Fig. 5A,G for Nes^Cre/+; R26^SmoM2/+, and data not shown for Olig3^Cre/+; R26^SmoM2/+). These expanded nuclei are marked by expression of Nkx2.2, Sox14 (Fig. 5A–C,G–I), and Sox1 (data not shown). The expanded Nkx2.2 and Sox14 expression is consistent with previous studies using chick and mouse embryos (Hashimoto-Torii et al., 2003; Kiecker and Lumsden, 2004; Vieira et al., 2005) and establishes that IGL and the lateral part of vLG are specified by high Shh activity in pTH-R. Nkx2.2-positive cells were also found in a more caudolateral region that is away from the large cluster of expanded vLG/IGL cells (Fig. 5H, arrowheads). These cells did not express Sox2 but intermingled with Sox2-expressing cells (Fig. 5, compare G, H, J). This intermingling of two molecularly different cell types reflects our earlier observation that two completely separate progenitor cell populations, one with pTH-R identity and the other with pTH-C identity, are mixed in the SmoM2-expressing, thalamic ventricular zone (Fig. 3G,H).

Next, we tested whether markers of thalamic nuclei that are derived from the pTH-C domain are also shifted in SmoM2-expressing embryos. In control embryos, Sox2 is expressed in the rostroventral part of the pTH-C-derived thalamic mantle zone (Fig. 5D,M,O) (Vue et al., 2007). In E16.5 Nes^Cre/+; R26^SmoM2/+ brains, Sox2 expression was expanded caudally and dorsally, occupying essentially the entire diencephalic mantle zone (Fig. 5D,J). Similarly, another rostral nucleus marker, RORα, which is expressed more specifically in ventral posterior nucleus (VP) and dLG (Nakagawa and O'Leary D, 2003), was expanded caudally and dorsally in Nes^Cre/+; R26^SmoM2/+ brains compared with controls (Fig. 5E,K). To determine whether the expansion of these rostral nucleus markers is accompanied by the repression of caudal nucleus markers, we analyzed the expression of Gbx2. Although Gbx2 is initially expressed in the entire postmitotic thalamus (supplemental Fig. S3A,B,F,G, available at www.jneurosci.org as supplemental material), its expression later becomes restricted to medial and caudodorsal nuclei and is nearly complementary to that of Sox2 and RORα (Fig. 5F,N,Q). In Nes^Cre/+; R26^SmoM2/+ brains, Gbx2 expression was dramatically reduced, particularly in regions where both Sox2 and RORα are ectopically induced (Fig. 5L).

To better demonstrate the local effects of enhanced Shh signal on postmitotic thalamic development, we again analyzed SmoM2-electroporated embryos and Olig3^Cre/+; R26^SmoM2/+ embryos. When caudodorsal thalamus was electroporated at E11.5 and analyzed at E17.5, Sox2 was highly induced in the cohort of cells on the transfected side (Fig. 5M). Gbx2 was specifically repressed in these Sox2-expressing cells, but its expression on the control side was unaffected (Fig. 5M,N). In Olig3^Cre/+; R26^SmoM2/+ brains at E18.5, the overall morphology of the thalamic mantle zone was normal. However, Sox2 was ubiquitously expressed in the entire thalamus, not just in rostroventral nuclei as seen in the control (Fig. 5O,R). RORα was also significantly expanded caudodorsally in the lateral part of the thalamus (Fig. 5P,S). Furthermore, Gbx2 expression is almost completely absent in Olig3^Cre/+; R26^SmoM2/+ brains (Fig. 5Q,T). These data demonstrate that in Olig3^Cre/+; R26^SmoM2/+ embryos, the entire postmitotic thalamus is dominated by rostroventral identity.

Although the expansion of RORα implicates that rostroventrally located nuclei, VP, and dLG are increased in size in Olig3^Cre/+; R26^SmoM2/+ embryos, we used criteria other than molecular markers that would definitively prove that individual nuclei are altered. For this purpose, we used anatomical criteria to define thalamic nuclei. Specifically, we injected anterograde axon tracer CTB to the retina of the neonatal Olig3^Cre/+; R26^SmoM2/+ mice to label retinogeniculate axons in the dLG nucleus. Because Olig3 is not expressed in the retina or along the retinogeniculate pathway to the thalamus, Olig3^Cre/+; R26^SmoM2/+ mice provide a particularly useful model to evaluate the specific effects of enhanced Shh signal in thalamic progenitor cells on the size of postnatal thalamic nuclei. We found that the volume of the dLG nucleus as estimated by the CTB-labeled region in the thalamus, was increased by ∼3.6 times in Cre+ pups (0.086 ± 0.008 mm³ in Cre+ pups, n = 3; 0.024 ± 0.003 mm³ in Cre− pups, n = 2; p = 0.0091) (Fig. 5U–X).

In summary, we demonstrated that increasing Shh signaling in CNS progenitor cells results in expansion of rostral–ventral thalamic progenitor pools, pTH-R, and rostroventral pTH-C, at the expense of more caudal–dorsal thalamus as well as the pretectum and habenula (summarized in Fig. 9B1,B2,C1,C2). When the manipulation of the Shh signal was restricted to thalamic progenitor cells, pTH-R and rostroventral pTH-C domain still expanded and caudal–dorsal pTH-C was shrunken. These results strongly indicate that the high Shh signal biases thalamic progenitor cells toward more rostral–ventral fates in a cell-intrinsic manner. This conclusion is consistent with our observation that the number of PH3+ cells in the entire Olig3-expressing domain was similar between Olig3^Cre/+; R26^SmoM2/+ and control embryos (supplemental Fig. S2, available at www.jneurosci.org as supplemental material). Compatible with changes in progenitor identity, postmitotic thalamic nuclei are expanded or shrunken based on their putative locations of origin (summarized in Fig. 9B3,C3).

Because Gli1 and Ptc1 are not ectopically expressed in the mantle zone of SmoM2-expressing brains, the altered organization of the postmitotic thalamus is unlikely to be due to the direct activation of Shh signaling in postmitotic cells. Instead, we conclude that shifted identity of thalamic progenitor cells resulted in corresponding alteration of the identity of postmitotic thalamic nuclei.

Overexpression of the bHLH transcription factor Olig2 partially mimics the effect of SmoM2 expression in the thalamus

If Shh signal directly controls the identity of thalamic progenitor cells and indirectly influences the specification of postmitotic thalamic nuclei, it is likely that transcription factors that are regulated by Shh signal in progenitor cells translate their positional information onto their postmitotic progeny. Olig2 is normally expressed in the rostroventral portion of pTH-C, and is induced in progenitor cells that ectopically express SmoM2. These SmoM2-expressing embryos also showed induction of Sox2 and RORα caudally and dorsally throughout the thalamic mantle zone. Thus, it is possible that Olig2 functions to specify rostroventrally located thalamic nuclei that express Sox2 and RORα. To test this possibility, we first performed genetic lineage tracing of Olig2-expressing progenitor cells (supplemental Fig. S4A–C, available at www.jneurosci.org as supplemental material). We crossed Olig2^Cre/+ mice (Dessaud et al., 2007) with ROSA26-stop-EGFP reporter mice, and analyzed E14.5 compound heterozygous embryos. At E11.5, GFP was expressed in the rostroventral part of pTH-C, as well as pTH-R (supplemental Fig. S4A, available at www.jneurosci.org as supplemental material). Expression of GFP in pTH-R suggests that there is transient expression of Olig2 in this progenitor domain before E10.5, which is similar to the observation in ventral spinal cord (Dessaud et al., 2007). At E14.5, EGFP was expressed in the rostroventral portion of the postmitotic thalamus, which largely overlapped with Sox2 expression (supplemental Fig. S4B,C, available at www.jneurosci.org as supplemental material). VP and the ventral part of medial geniculate (MGv) nuclei were two nuclei that had particularly strong EGFP expression. In contrast, EGFP was not found in more caudodorsal nuclei. Thus, Olig2-expressing thalamic progenitor cells give rise to rostroventral part of the postmitotic thalamus, consistent with the possibility that Olig2 plays a role in the specification of rostroventral nuclei such as VP and MGv.

We then tested whether Olig2 is necessary and/or sufficient for the specification of these nuclei. When we ectopically expressed Olig2 by in utero electroporation at E10.5 in the caudodorsal diencephalon, Sox2 was induced in the mantle zone only on the transfected side at E13.5 and E14.5 (supplemental Fig. S4E,F, available at www.jneurosci.org as supplemental material), which partially mimicked the effect of expressing SmoM2. Cell proliferation and neuronal differentiation did not seem to be affected in Olig2-transfected cells, and overexpressing EGFP alone did not induce Sox2 expression (data not shown). These results suggest that Olig2 mediates the Shh signal and confers at least part of the rostroventral identity to the postmitotic thalamus. In contrast, Olig2 null mice did not show altered expression of Sox2 or progenitor markers such as Mash1, Ngn2, and Dbx1 (data not shown). Thus, Olig2 is not necessary but is sufficient to induce at least some molecular features of the rostroventral thalamic nuclei.

Attenuation of Shh signal in thalamic progenitor cells reduces the expression of rostroventral markers and increases caudodorsal markers

Previous studies on chick embryos showed that ectopic expression of Ptc1Δloop2, a dominant-negative form of the Shh receptor, Ptc1, reduced the expression of Nkx2.2 and Gbx2, and increased the expression of Pax6, which is normally expressed highly in the caudal diencephalon (Kiecker and Lumsden, 2004). It was also reported that Gli2/3 double knock-out mice showed reduced expression of Gbx2, whereas Gli1 mutant mice showed reduced Sox14 expression (Hashimoto-Torii et al., 2003). These studies demonstrated the requirement of Shh signaling in establishing the identity of the thalamus as a whole. However, it is still unknown whether Shh is required for the patterned expression of progenitor markers in pTH-R and pTH-C, or for the normal organization of postmitotic thalamic nuclei. Shh null mice show pronounced reduction in size of the diencephalon by E9.0, which reflects the early role of Shh in cell proliferation and survival (Ishibashi and McMahon, 2002). Therefore, we analyzed conditional Shh knock-out mice by using the Nes^Cre allele to avoid such early effects. Conditional Shh mutant mice were smaller than control littermates, but they were postnatally viable and have relatively normal gross morphology of the brain (data not shown). The smaller brain size is reflected in part by a significant decrease in the number of PH3-positive progenitor cells in the thalamic ventricular zone of conditional Shh knock-out (Nes^Cre/+; Shh^c/c) embryos (3437 ± 146.0, n = 2 for Nes^Cre/+; Shh^c/c, 6605 ± 472.0, n = 2 for Cre− controls; p = 0.024) (supplemental Fig. S2C,D, available at www.jneurosci.org as supplemental material).

In Nes^Cre/+; Shh^c/c embryos, Shh expression in the forebrain was lost by E10.5 (data not shown; Machold et al., 2003). Consistent with the loss of Shh in the ZLI and the basal plate, Ptc1 (data not shown) and Gli1 mRNA was undetectable at E10.5 and E12.5 in Nes^Cre/+; Shh^c/c embryos (Fig. 6B,G and not shown). Furthermore, markers for the pTH-R domain were not induced. At E12.5, we observed a complete loss of Nkx2.2 (Fig. 6C,H) and Mash1 (Fig. 6D,I) within the thalamic ventricular zone, although their expression in the prethalamic progenitor domain remained at very low levels (Fig. 6H,I). Thus, Nes^Cre/+ mice are useful in deleting the Shh gene in the brain when Shh signal is still critically required for the induction and/or maintenance of the expression of pTH-R markers. In addition to Nkx2.2 and Mash1, expression of Olig2, a rostroventral pTH-C marker, was also lost in pTH-C of Nes^Cre/+; Shh^c/c embryos, although it was weakly detected in the prethalamus (Fig. 6C,H, arrowhead for the prethalamus). In contrast, markers that are expressed throughout the pTH-C domain, such as Olig3 (Fig. 6A,F) and Ngn2 (Fig. 6D,I) were still expressed in Nes^Cre/+; Shh^c/c embryos at E12.5 and earlier stages, although the size and intensity of the expression of Olig3 seemed to be reduced. Dbx1, which is normally restricted to the caudodorsal pTH-C, was expanded rostroventrally in Nes^Cre/+; Shh^c/c thalamus (Fig. 6E,J). A pretectal marker, Pax7, was still expressed (data not shown). Together, these findings demonstrate that Shh activity is required for progenitor cell identity in pTH-R and rostroventral pTH-C and for suppressing the caudodorsal pTH-C identity (summarized in Fig. 9B1,B2,D1,D2).

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Figure 6.

Shh is required for the normal organization of thalamic progenitor domains. Frontal sections of Nes^Cre/+; Shh^c/c forebrain (F–J) and Nes^Cre/+; Shh^c/+ controls at E12.5 (A–E). Midline is to the left. Shh is expressed in the ZLI of control embryos (A, arrow) but not in the Nes^Cre/+; Shh^c/c mice (F, arrow). Olig3 expression is maintained in Nes^Cre/+; Shh^c/c embryos, but the domain is smaller and density of Olig3-positive cells in this domain also seems reduced (F). Gli1, a target gene of Shh signaling, is lost in the thalamic progenitor cells of Nes^Cre/+; Shh^c/c embryos (G), while it is expressed in a graded manner in the control (B). Expression of Nkx2.2 and Olig2 is found in controls (C, arrow for Nkx2.2, double arrows for Olig2), but is completely lost in Nes^Cre/+; Shh^c/c embryos (H). A reduced number of Olig2-expressing cells are still found in the prethalamus (C, H, arrowhead). Mash1 expression in the pTH-R domain is detected in the control but is lost in Nes^Cre/+; Shh^c/c embryos (D, I, arrowhead). Mash1 expression in the posterior pretectum is maintained (D, I, arrow). Expression of Ngn2 is intact and now occupies the entire thalamus, including the most rostroventral region (I, arrowhead). Dbx1 expression is limited to the rostral pretectum and caudodorsal part of pTH-C in control embryos (E). The expression domain of Dbx1 is expanded rostroventrally in Nes^Cre/+; Shh^c/c embryos (E, J, arrow). Scale bar, 200 μm.

Next, to more directly test the intrinsic requirement of Shh signaling in the thalamus, we conditionally knocked out the gene encoding Smo, a transmembrane effector of Shh signaling. Two Cre drivers, Nes^Cre/+ and Olig3^Cre/+, were used. Both Nes^Cre/+; Smo^c/c and Olig3^Cre/+; Smo^c/c mice showed normal gross morphology of the brain and survived postnatally. We found that Nes^Cre/+; Smo^c/c embryos show lack of Ptc1 expression in the thalamus at E12.5 (Fig. 7B,G) despite the normal expression of Shh in the ZLI (Fig. 7A,F). In these embryos, we also found that Olig3 expression was reduced in the thalamic ventricular zone (Fig. 7A,F), and Mash1 and Olig2 were undetectable in pTH-R and pTH-C, respectively (Fig. 7C,D,H,I). Furthermore, Dbx1 expression was expanded rostroventrally (Fig. 7E,J). However, unlike in Nes^Cre/+; Shh^c/c embryos, Nkx2.2 was still detectable though not as robustly as in the control (Fig. 7C,H).

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Figure 7.

Smo is required for the normal organization of thalamic progenitor domains. Frontal sections of Nes^Cre/+; Smo^c/c and Nes^Cre/+; Smo^c/+ control embryos (A–J) and Olig3^Cre/+; Smo^c/c and Olig3^Cre/+; Smo^c/+ control embryos (K–T) at E12.5. Midline is to the left. A–J, Nes^Cre/+; Smo^c/c embryos show normal expression of Olig3 and Shh (A, F), but Ptc1 (B, G, arrow) and Olig2 (C, H, arrow) are lost in thalamic progenitor cells. Nkx2.2 is still detectable in a reduced number (C, H double arrows). Mash1 expression in pTH-R is undetectable in Nes^Cre/+; Smo^c/c embryo (D, I, arrow), whereas it is still detectable in the prethalamus (D, I, double arrows). Dbx1 expression is expanded in Nes^Cre/+; Smo^c/c embryos (E, J). Arrows in E and J indicate the boundaries of the expression domain of Dbx1. K–T, Olig3^Cre/+; Smo^c/c embryos show normal expression of Olig3 and Shh (K, P). Ptc1 expression is reduced in the thalamus (L, Q, arrow) but not in the prethalamus (L, Q, double arrows). Mash1 expression in the thalamus is reduced in Olig3^Cre/+; Smo^c/c embryos (M, R, double arrow). In high-magnification views of M and R, Ngn2, a pTH-C marker, is now expressed more rostroventrally. Ngn2-expressing cells are intermingled with Mash1-expressing cells, forming a mixed zone of pTH-R/C at the expense of pTH-R (N, S, bracket). Expression of Dbx1 is expanded in Olig3^Cre/+; Smo^c/c embryos, reaching near the rostroventral border of the thalamus (O, T, arrowhead). Olig2 expression is still detected in the pTH-C domain of Olig3^Cre/+; Smo^c/c embryos (O, T). Scale bar, 100 μm for N, S; 200 μm for other panels.

Olig3^Cre/+; Smo^c/c embryos at E12.5 showed normal expression of Shh in the ZLI (Fig. 7K,P) and reduction of Ptc1 expression in the thalamus but not in the prethalamus (Fig. 7L,Q), compatible with the restricted deletion of Smo and reduced Shh signal specific to the thalamus. The reduction of Ptc1 expression in the thalamus was less significant than in Nes^Cre/+; Smo^c/c embryos, where Ptc1 was undetectable in the thalamus and the prethalamus (Fig. 7G). Nevertheless, Olig3^Cre/+; Smo^c/c embryos showed reduction of Mash1-expressing cells in the thalamus (Fig. 7M,N,R,S). In addition, the decreased number of Mash1-expressing cells were intermingled with Ngn2-expressing cells, giving rise to the mixed zone (pTH-R/C) instead of pure pTH-R as we found in the control (Fig. 7N,S). Although Nkx2.2 and Olig2 were not consistently reduced in expression, Dbx1 expression was grossly expanded rostroventrally (Fig. 7O,T), as in the Nes^Cre/+; Shh^c/c and Nes^Cre/+; Smo^c/c embryos. Together, these results indicate that the Shh signal is intrinsically required for the induction and/or maintenance of molecular characteristics of the pTH-R domain and for the suppression of caudodorsal part of the pTH-C domain.

The dramatic changes in the progenitor cell identity with conditional deletion of the Shh or Smo gene prompted us to examine which populations of postmitotic thalamus are affected in the absence of Shh or Smo. In E17.5 Nes^Cre/+; Shh^c/c embryos, as expected from the loss of pTH-R markers at E12.5 (Fig. 6C,D,H,I), Nkx2.2 (Fig. 8A,C,F,H) and Sox14 (data not shown) in vLG/IGL were both dramatically reduced, confirming the requirement of Shh signaling for the vLG/IGL lineage. Similar changes were found in Nes^Cre/+; Smo^c/c embryos (Fig. 8K–N). We next analyzed the pTH-C derivative. Expression of Sox2 (Fig. 8B,G) and RORα (Fig. 8D,I) was profoundly reduced in Nes^Cre/+; Shh^c/c embryos. In contrast, Gbx2 expanded and occupied almost the entire thalamic mantle zone (Fig. 8E,J). Again, similar changes were observed in Nes^Cre/+; Smo^c/c embryos (Fig. 8K,M and data not shown). In Olig3^Cre/+; Smo^c/c mice, IGL, as marked by Sox1 and neuropeptide Y (NPY) expression, was dramatically reduced in size and cell number. Sox1-positive cells in this nucleus was reduced by ∼70% (Olig3^Cre/+; Smo^c/+: 337.5 ± 13.80 cells/slide, Olig3^Cre/+; Smo^c/c: 103 ± 24.93 cells/slide, n = 4; p = 0.0002). This reduction likely reflects the reduction of Mash1-positive cells in the pTH-R at E12.5 (Fig. 7M,N,R,S).

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Figure 8.

Shh signaling is required for the normal organization of postmitotic thalamus. A–P, Frontal sections of Nes^Cre/+; Shh^c/c and Nes^Cre/+; Shh^c/+ control embryos at E17.5 (A–J), Nes^Cre/+; Smo^c/c and Nes^Cre/+; Smo^c/+ control embryos at E17.5 (K–N), and Olig3^Cre/+; Smo^c/c and Olig3^Cre/+; Smo^c/+ control embryos at E18.5 (O, P). Midline is to the left. A–J, Immunohistochemistry for Nkx2.2 (A, F), Sox2 (B, G), and in situ hybridization of RORα (D, I) and Gbx2 (E, J). C, H, High-magnification images of A/B and F/G, respectively, showing the double immunofluorescence of Nkx2.2 and Sox2. Nkx2.2 is expressed in vLG/IGL in the control but is undetectable in Nes^Cre/+; Shh^c/c brains (A, C, F, H; see arrowhead in C, H). Sox2 is expressed in the rostroventral part of the postmitotic thalamus, including VP and dLG in control embryos (B). In Nes^Cre/+; Shh^c/c thalamus, Sox2 expression is severely reduced to a small, lateral region (G). Expression of RORα shows an even more dramatic reduction in Nes^Cre/+; Shh^c/c mice (D, I). Gbx2 is expressed in many thalamic nuclei, but is excluded from the rostroventral nuclei such as VP and dLG in control embryos (E). In Nes^Cre/+; Shh^c/c brain, Gbx2 is expanded, reaching the most rostroventral part of the thalamus in these mice (J, arrowheads). K–N, Immunohistochemistry for Nkx2.2 and Sox2 shows that Sox2 expression is dramatically reduced in Nes^Cre/+; Smo^c/c embryos at E17.5. High-magnification images of the boxed areas in K and M clearly show the loss of Nkx2.2 in the vLG/IGL region of Nes^Cre/+; Smo^c/c embryos (L, N). O, P, Immunohistochemistry for the two IGL markers, Sox1 and NPY, shows that both markers are severely reduced in Olig3^Cre/+; Smo^c/c embryos at E18.5. Po, Posterior nucleus; LP, lateral posterior nucleus. Scale bar, 500 μm for C, H; 500 μm for other panels.

In summary, decreasing Shh signaling resulted in reduction of rostral–ventral progenitor pools, pTH-R, and the rostroventral part of the pTH-C domain, and expansion of caudodorsal progenitor pools (summarized in Fig. 9B1,B2,D1,D2). This change in thalamic progenitor cell identity corresponds to alterations in postmitotic populations (Fig. 9B3,D3). Although conditional deletion of Shh or Smo with two different Cre lines caused different degrees of reduction in Shh signal, we observed consistent changes in the progenitor cell populations as well as their postmitotic derivatives. These observations confirm the cell-intrinsic requirement of Shh signal in the specification of rostral–ventral progenitor cells in the thalamus.

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Figure 9.

Summary of the current study. A, Side view of the embryonic thalamus. The curved anterior–posterior (A–P) axis of the brain is shown in a red dotted line. The black vertical line indicates the orientation of frontal sections used throughout this study. In a given section, the top part is more dorsal and caudal, while the bottom part is more ventral and rostral. B1, C1, D1, Schematic frontal sections showing the progenitor domains at early embryonic stages in wild type (WT), Nes^Cre/+; R26^SmoM2/+ (SmoM2), and Nes^Cre/+; Shh^c/c (Shh cko) mice. Color gradients within pTH-C indicate the graded differential patterns of gene expression within this domain, which are altered when Shh signal is increased (C1) or decreased (D1). B2, C2, D2, Differential gene expression patterns in each genotype. B3, C3, D3, Schematic frontal sections indicating the postmitotic thalamus at late embryonic stages. The graded color of pTH-C-derived thalamus corresponds to the overall lineage relationship with the progenitor domains shown in B1, C1, and D1. Alterations in SmoM2-expressing mice and Shh cko mice indicate that the changes in the mantle zone are consistent with those observed in progenitor cells.

Relationship between Shh and Fgf signaling pathways in thalamic patterning

A recent study by Kataoka and Shimogori (2008) reported that Fgf8 is expressed in the dorsal midline of the caudal diencephalon and that this expression domain continues to a region immediately rostral to the dorsal tip of the ZLI, suggesting that Fgf signaling may have a role in thalamic patterning. Focal in vivo electroporation of Fgf8 near the ZLI increased the size of pTH-R and its derivative, “Rim domain,” although overexpression of Fgf8 did not change the expression pattern of Shh or Ptc1 (Kataoka and Shimogori, 2008). These results suggest that Fgfs may play a patterning role independent of Shh signaling in the thalamus by regulating gene expression in pTH-R progenitor cells. Alternatively, Fgf8 may be downstream of the Shh pathway and mediates part of the roles that Shh plays. In fact, Shh induces the expression of Fgf4 in early limb development (Laufer et al., 1994), and complete Shh null embryos show reduced expression of Fgf15 in mouse diencephalon at E8.5 (Ishibashi and McMahon, 2002). To assess the role of Shh in Fgf8 expression and Fgf signal in the thalamus, we analyzed the expression of Fgf8 and its downstream targets, Spry1 and Spry2 (Liu et al., 2003) in Nes^Cre/+; R26^SmoM2/+ and Nes^Cre/+; Shh^c/c embryos (supplemental Fig. S4, available at www.jneurosci.org as supplemental material). We found that in both control and Nes^Cre/+; R26^SmoM2/+ embryos, Fgf8 is expressed in the dorsal midline as well as in the region immediately rostral to the dorsal part of the ZLI (supplemental Fig. S4B,F, available at www.jneurosci.org as supplemental material). Expression of Spry1 and Spry2 was also similar in the control and SmoM2-expressing embryos (supplemental Fig. S4C,D,G,H, available at www.jneurosci.org as supplemental material). In addition, Nes^Cre/+; Shh^c/c embryos at E12.5 showed no significant reduction of expression of Fgf8, Spry1, or Spry2 (supplemental Fig. S4J–L, available at www.jneurosci.org as supplemental material) near the ZLI. These results indicate that the changes to the pTH-R domain observed in Nes^Cre/+; R26^SmoM2/+ and Nes^Cre/+; Shh^c/c embryos are not due to alterations in Fgf8 expression or Fgf signal.