Combinatorial Expression Patterns of LIM-Homeodomain and Other Regulatory Genes Parcellate Developing Thalamus

Histological organization of dTh and vTh at P2

At P2, thalamic nuclei can be readily identified based on CO histochemistry (Figs. 1, 2) and Nissl staining (data not shown). Within dTh, CO histochemistry distinguishes adjacent nuclei based on their different staining levels, such as VP and Po, and dLG and LP, as well as their separation from one another by lightly stained tissue (Fig.1A–J). The three major nuclei of vTh, the RT, vLG, and ZI, can also be identified by CO staining at P2 (Fig.2A–H). The levels of CO staining vary significantly within vLG, especially at caudal levels (Fig.2G,H). ZI is divided into four subdivisions, rostral (ZIr), dorsal (ZId), ventral (ZIv), and caudal, based on locations and CO staining patterns (Nicolelis et al., 1995). Because of the relative proximity to the TCAs, we focused on the rostrally located subdivisions, ZIr, ZId, and ZIv, in the subsequent analyses of gene expression. The border between vLG and ZI is not as evident as other borders (Fig.2G,H).

Combinatorial expression patterns of LIM-HD and other regulatory genes parcellate postnatal dTh and vTh

The expression patterns of the selected genes were analyzed at P2 to establish their relationship to thalamic nuclei identified by CO histochemistry and Nissl staining. We first examined the expression of the closely related LIM-HD genes Lhx2 and Lhx9(Bertuzzi et al., 1999; Retaux et al., 1999), as well asGbx2 and Ngn2. Lhx2 is expressed mainly in nonprincipal nuclei, including the anteromedial nucleus (AM), centrolateral nucleus (CL), centromedial nucleus (CM), paraventricular nucleus (PV), peripeduncular nucleus (PP), and ventrolateral nucleus (VL) (Fig. 1K–M,O). Among the principal sensory nuclei, only MG is positive, particularly the lateral part of MGv. VP and dLG do not express Lhx2 at detectable levels (Fig. 1L–N). Lhx9 is expressed in most dTh nuclei; however, its expression is low in VP and MGv and almost absent in ventromedial nucleus (VM) (Fig.1P–T). The expression of Gbx2 is distinct from both Lhx2 or Lhx9, although it more closely resembles that of Lhx2. Gbx2 is expressed in MG, AM, CL, CM, mediodorsal nucleus (MD), LP, paratenial nucleus (PT), and PV (Fig. 1U–W,Y), but its expression is not detected in dLG, Po, VL, VM, and VP (Fig.1V–X). Within MG, Gbx2 is expressed most highly in MGv, especially in its lateral part (Fig.1Y). Ngn2 is strongly expressed in VM, laterodorsal nucleus (LD), AV, and weakly in VP, dLG, MGv, CM, and VL (data not shown). Thus, Lhx2, Lhx9,Gbx2, and Ngn2 are expressed in distinct but overlapping patterns in dTh at P2, and their borders of expression often correlate with the borders of dTh nuclei. These results are summarized in Table 2.

Lhx2, Lhx9, and Gbx2 are not expressed in vTh (Fig. 1 and data not shown). Instead, the patterned expression of a different set of LIM-HD genes, Lhx1, Lhx5, and Isl1, the paired-box gene Pax6, and the receptor tyrosine phosphatase gene RPTPδ demarcate vTh nuclei at P2. Lhx1 is expressed in vLG, as well as ZIr, ZIv, and ZId (Fig. 2I–L). Expression in vLG is not uniform; caudally, it is low in the middle and dorsal parts, in which CO activity is high (Fig. 2K,L).Lhx5, which is closely related to Lhx1 (Bertuzzi et al., 1996), is expressed in vLG, but the level is higher in the dorsal and middle parts, in which Lhx1 expression is relatively low (Fig. 2M–P). Lhx5 is expressed at very low levels in ZId and ZIv (Fig.2P). Isl1 is expressed in RT, ZIr, and ZIv (Fig. 2Q–T), and what appears to be a small domain in the medial part of vLG (Fig. 2R). Pax6is expressed in a thin strip of cells located in the rostromedial portion of vLG (Fig. 2V), as well as in ZIr and ZIv but not in RT (Fig. 2U–X). The boundary between RT and ZIr is also clearly defined by the expression ofRPTPδ (Mizuno et al., 1993; Sommer et al., 1997; Tuttle et al., 1999), which is expressed in RT but not in ZIr (Fig.2Y,Z). These results are summarized in Table3.

In summary, the regulatory genes analyzed here have distinct but overlapping expression patterns in subsets of dTh and vTh nuclei; the expression patterns often correlate with the histologically defined borders of nuclei. In addition, potential subdomains within the same nuclei (e.g., MGv and vLG) are suggested by some of the more discrete expression patterns. Thus, the combinatorial expression of these transcription factors may regulate the postnatal development of dTh and vTh nuclei. Because these genes may also serve as markers to define the organization of the embryonic dTh and vTh into nascent nuclei, or cell groups that will later form these nuclei, we examined their expression at embryonic stages.

Patterned expression of regulatory genes in embryonic dTh

Although dTh has undergone considerable architectonic differentiation by E16.5, dTh nuclei cannot be as easily distinguished in CO- or Nissl-stained sections as at P2 (data not shown); for example, the dLG borders with LP and MG do not appear as a cell-free band, and the levels of staining are not clearly different between these nuclei. Nonetheless, it is evident thatLhx2, Lhx9, and Gbx2 are differentially expressed in patterns similar to those at P2 (Fig.3). Robust Lhx2 expression is present in the rostromedial nuclei, i.e., the putative CL, CM, and PV (Fig. 3A,B), as well as in MG and PP (Fig. 3C), whereas its expression in dLG and LP is very low and is undetectable in VP (Fig.3B,C). As at P2, Lhx9 is expressed at high levels in most nuclei except in VM, VP, and MGv (Fig.3D–F). Gbx2 expression is high in MGv and the dorsal subdivision of medial geniculate nucleus (MGd) and is not detected in VP and dLG (Fig. 3G–I). Although the putative border between LP and dLG is not apparent in CO- or Nissl-stained sections at E16.5, the expression pattern ofGbx2 appears to mark it, with moderate expression in LP and undetectable expression in dLG. The expression pattern ofNgn2 in dTh at E16.5 is similar to that at P2 (Fig.3J–L) and is partially complementary to Lhx2 andGbx2 expression. This suggests that, in dTh, Gbx2/Lhx2 and Ngn2 negatively regulate each others expression, or cells expressingGbx2/Lhx2 and cells expressing Ngn2 do not mix with each other and thereby remain as distinct cell groups. In summary, the expression patterns of Lhx2, Lhx9,Gbx2, and Ngn2 observed in dTh at P2 are already evident at E16.5, suggesting that these genes are useful markers to follow the parcellation of dTh into molecularly distinct cell groups that will form specific dTh nuclei.

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Fig. 3.

Gene expression patterns in E16.5 mouse dorsal thalamus are similar to those at P2. Coronal sections of diencephalon showing the expression of Lhx2(A–C), Lhx9(D–F), Gbx2(G–I), and Ngn2(J–L) mRNA. At the rostral level,Lhx9 is strongly expressed in the putative LD (D), in which Lhx2 is only weakly expressed and Gbx2 is undetectable (A,G). Ngn2 is expressed in LD (J). At the middle level, the square-shaped region (asterisks) expresses Lhx9 andNgn2 but not Lhx2 or Gbx2, compatible with dLG (B, E,H, K; asterisks). The putative LP, located immediately dorsal to dLG, expresses high levels of Lhx9 and Gbx2 (E,H; arrows) but only very low levels ofLhx2 and Ngn2 (B,K; arrows). The putative VP is negative for Lhx2 and Gbx2 and weakly positive forLhx9 and Ngn2. The putative VM is positive for Ngn2 and negative for the others (B, E, H,K; arrowheads). At the caudal level, MGv is strongly positive for Lhx2 and Gbx2(C, I; asterisks) and weaker for Lhx9 and Ngn2(F, L; asterisks). The putative PP is positive for Lhx2 and Lhx9(C, F; arrowheads) and negative for Gbx2 and Ngn2(F, L; arrowheads). MGd expresses Lhx2, Lhx9, andGbx2 (E, C,F; arrows) but not Ngn2(L; arrow). Scale bar, 500 μm.

It is not possible to histologically distinguish dTh nuclei at E12.5 or E14.5. However, Lhx2, Lhx9,Gbx2, and Ngn2 already show distinct patterns of expression at these ages, and the overall patterns evident at E14.5 are similar to those at E16.5 and P2. For example, caudally, the putative MG already expresses Lhx2, Lhx9, Gbx2, and Ngn2 in a pattern reminiscent of that observed at later ages (Fig.4C,F,I,L).Lhx2 and Gbx2 are expressed at high levels in the lateral part of the mantle zone, and Lhx9 andNgn2 are expressed at low levels in the putative MGv. More rostrally, the putative dLG expresses Lhx9 andNgn2 but not Lhx2 or Gbx2 (Fig.4B,E,H,K).

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Fig. 4.

Differential gene expression patterns already exist in the dorsal thalamus at E12.5. Coronal sections of diencephalon showing the expression of Lhx2 (A–C,M, N), Lhx9(D–F, O, P), andGbx2 (G–I, Q,R), and Ngn2 (J–L,S, T) mRNA. A–Lare for E14.5, and M–T are for E12.5. At E14.5,Lhx9 and Ngn2 are expressed in the lateral–ventral part of dTh at the rostral level, corresponding to LD, but Lhx2 and Gbx2 are not (A, D, G,J; arrows). A band of strongLhx2 and Gbx2 expression (A, G, asterisks) is located more medially to a band of strong Lhx9expression (D; cross).Ngn2 is weak in the band of Lhx2 andGbx2 expression (J;asterisk) but strong in the putative ventricular zone (J, K; double-headed arrow), in which Lhx2, Lhx9, andGbx2 are negative. More caudally, a region expressesLhx9 and Ngn2 but not Lhx2or Gbx2, which is likely to be dLG (B,E, H, K;asterisks). The putative LP, located dorsally to dLG (B, E, H,K; arrows), expresses high levels ofLhx9 and Gbx2 but only low levels ofLhx2 and Ngn2. The putative VP is negative for Lhx2 and Gbx2 and weakly positive for Lhx9 and Ngn2(B, E, H,K). The putative PP is positive forLhx2 and Lhx9 and negative forGbx2 and Ngn2 (C,F, I, L;arrowheads), whereas MGv strongly expressesLhx2 and Gbx2 and weakly expressesLhx9 and Ngn2 (C,F, I, L;asterisks). These patterns of differential expression are not still apparent at E12.5, but the band with high levels ofLhx2/Gbx2 expression is already located medial to the band with the high Lhx9 expression (L, N–R;asterisks), similar to E14.5. Ngn2 is expressed in the ventricular zone, as well as the mantle zone, but is weak in the band with strong Lhx2/Gbx2expression (S, T;asterisk). Scale bars, 200 μm.

Although the similarities in gene expression patterns at early and late stages are striking, some differences are also apparent. At E16.5, the expression domains of Lhx2 overlap with those ofLhx9 except for MGv, in which Lhx2 is highly expressed but Lhx9 expression is low. However, at E14.5, a rostromedial part of dTh exhibits a high level of Lhx2expression but a very low level of Lhx9 expression (Fig.4A,D), which is not found at E16.5. This band of cells is located just outside of the ventricular zone and expresses Gbx2 at a high level and Ngn2 at a very low level (Fig. 4G,J). An approximately similar pattern is already apparent at E12.5 (Fig.4M–T). Another example of a difference in the patterns of gene expression is found between E12.5 and E14.5 in the putative dLG; the overall pattern characteristic of dLG after E14.5 is not evident at the putative location of this nucleus in the caudolateral portion of dTh (Fig. 4N–T).

The band of Gbx2-expressing cells located just outside of the ventricular zone (Fig. 4G,Q,asterisks) has been described to be in the thalamic subventricular zone (Bulfone et al., 1993; Miyashita-Lin et al., 1999). Lhx2 is expressed in a similar band. However, pulse labeling with BrdU ∼1 hr before fixation shows that this expression domain of Gbx2 and Lhx2 is BrdU-negative at these ages (data not shown; also see below and Fig. 8), suggesting that this domain is not a proliferative zone and that these cells are postmitotic. A similar study in rat using tritiated thymidine reported the labeling of a few scattered cells in this domain just lateral to the ventricular zone, leading the authors to term this zone the subependymal layer, but noted that a large proportion of the cells in this layer must be postmitotic (Altman and Bayer, 1989a).

In summary, the differential expression patterns of genes that mark dTh nuclei at later ages are already evident in dTh as early as E12.5. These patterns undergo some changes until E16.5, but then the patterns appear to be stable to P2. It is unclear whether changes in these expression patterns between E12.5 and E16.5 are attributable to changes in expression per se or whether the expressing population is the same but the patterns change as a result of cell movements (see Discussion).

Patterned expression of regulatory genes in embryonic vTh

At E16.5, CO histochemistry suggests that the organization of vTh is similar to that at P2 (Fig.5A–D). RT is identified as a sheet of cells interposed between dTh, vLG, ZIr, and the internal capsule (ic), and the subdivisions of ZI are evident by their positions and patterns of CO staining. The expression patterns ofLhx1, Lhx5, Isl1, Pax6, andRPTPδ in vTh at E16.5 are also similar to P2 (Fig.5E–X). The only difference observed is that, at E16.5, a thin band of cells expressing Lhx1 (Fig.5E–G), Lhx5 (Fig. 5I), andPax6 (Fig. 5R) delineates the dTh–vTh border and extends laterally to vLG. Isl1 expression also appears to overlap with this band (Fig. 5N). The location of this band of cells is consistent with its identification as the zona limitans intrathalamica (ZLI), which has been defined in earlier embryos as abutting dTh and vTh and expressing Shh (Sonic hedgehog) (Shimamura et al., 1995).

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Fig. 5.

Gene expression patterns in E16.5 mouse ventral thalamus are similar to those at P2. Coronal sections of diencephalon showing the CO histochemistry (A–D) and the expression of Lhx1 (E–H),Lhx5 (I–L), Isl1(M–P), Pax6(Q–T), and RPTPδ (U–X) mRNA. Within each column, serial sections are aligned in a rostral-to-caudal order fromLhx1 to RPTPδ and then CO. CO staining shows similar patterns to P2 and delineates the nuclei of vTh (A–D). Lhx1 is expressed in vLG, ZIr, ZId, and ZIv. Expression in vLG is high in the ventromedial (E–G; arrows) and ventrolateral (F, G; double arrows) parts. In addition, Lhx1 is expressed in ZLI (E, F). Lhx5 is expressed in vLG, most highly in the dorsal part (I–L), and in the most rostral part of ZLI (I). It is also weakly expressed in ZId and ZIv (L). Part of hypothalamus is positive forLhx5 (I, J;asterisk). Isl1 is expressed in RT, ZIr, ZIv (M–P), and possibly in a small, rostroventral part of vLG (N; arrow).Pax6 is expressed in ZIr, ZIv (S,T), and a rostroventral part of vLG (R; arrow). RPTPδ is expressed in RT and ZIv. Scale bar, 500 μm.

Patterns of gene expression reveals several distinct cell groups within ZLI; rostrally, it expresses both Lhx1 andLhx5 (Fig. 5E,I), whereas caudally, Lhx1 is expressed but Lhx5 is not (Fig. 5F,J). In addition, overlaying Figure 5, F and R, as well as double-immunostaining shows that the population of ZLI cells expressing Lhx1 and those expressing Pax6 are distinct; the former is located immediately dorsal to the latter (data not shown). Therefore, at least three different populations of cells exist in ZLI; one expresses both Lhx1 and Lhx5but not Pax6, one expresses Lhx1 but notLhx5 or Pax6, and the third one expressesPax6 but not Lhx1 or Lhx5. We find that vLG also exhibits heterogeneity in gene expression patterns. As at P2, at E16.5, the expression of Lhx1 and Lhx5 is partially complementary. Expression of Pax6 andLhx1 is also exclusive to each other in the ventromedial part of vLG in both in situ hybridization (Fig.5F,R) and immunostaining (data not shown). The expression of Lhx5 in dorsal parts of both the ZLI and vLG closely resembles that of Nkx2.2 (data not shown). Based on the relative locations of ZLI and vLG and the disappearance of the ZLI by P2, we assume that the ZLI constitutes migratory streams for cells that eventually form vLG. A similar assumption has been made by Kitamura et al. (1997), in which they suggested that Brx1and Nkx2.2 mark dorsal ZLI and vLG, whereas Dlx1and Arx mark ventral ZLI and vLG.

At E14.5, nuclei in vTh cannot be distinguished by Nissl or CO staining (data not shown). However, the positional relationships between the gene expression domains of Lhx1,Lhx5, Isl1, Pax6, andRPTPδ appear to be similar to E16.5 (Fig.6). Two separate bands of ZLI can be clearly distinguished; the dorsal one extends laterally to the dorsal part of vLG and expresses Lhx5 as well as Lhx1(Fig.6A,B,E,F). The ventral one extends laterally to the ventral vLG and expressesLhx1 and Pax6 (Fig.6A,B,M,N), although cells expressing Lhx1and Pax6 rarely overlap (data not shown). The dorsal part of theIsl1-expressing domain also appears to overlap with the ventral band (Fig. 6J). These results suggest that the cell types of vLG are already specified as they initiate their migration from the ventricular zone.

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Fig. 6.

Differential gene expression patterns in E14.5 mouse ventral thalamus is similar to those at E16.5 and P2. Coronal sections of the mouse diencephalon showing the expression ofLhx1(A–D), Lhx5(E–H), and Isl1(I–L), Pax6(M–P), and RPTPδ (Q–T) mRNA. Lhx1 is expressed in vLG, ZIr, ZId, and ZIv. Expression in vLG is high ventrally (A, B). Lhx1 is also expressed in two bands of ZLI (A, B;arrows and double arrows).Lhx5 is expressed in dorsal vLG, as well as the dorsal band of ZLI (E, F; double arrows). Isl1 is expressed in RT, ZIr, and ZIv. Its expression domain appears to extend dorsally to the ventral part of vLG (J; arrow). Pax6 is expressed in ZLI (M, arrow), part of vLG (N; arrow), ZIr, and ZIv.RPTPδ is expressed in RT and ZIv.Asterisks in C, G,K, O, and S appear to be the lateral part of ZIr or ZIv. Scale bar, 200 μm.

At E12.5, the differential expression patterns ofLhx1, Lhx5, Isl1, and Pax6appear to be approximately similar to those at E14.5 and later (Fig.7). The ZLI expresses Lhx1,Lhx5, and Pax6 and extends laterally to the putative vLG; the dorsal part of vLG is characterized by a high level of Lhx5 and low level of Lhx1 expression (Fig.7B,E), whereas the ventral part expresses Lhx1 (Fig. 7B). More ventrally, a domain expressing Isl1 but not Lhx1,Lhx5, or Pax6 is located just medial to the bundle of TCAs forming the internal capsule (Fig.7B,E,H,J). Based on the position of this domain and its pattern of gene expression, which is only found in RT at E14.5 and later, this domain is likely the nascent RT. Between the putative vLG and RT, another domain expressing Lhx1, Isl1, and Pax6exists and is likely the nascent ZIr or ZIv based on the pattern of gene expression and its relative position (Fig.7B,H,J). Positioned more caudally are four distinct domains with gene expression patterns that match those of dorsal vLG, ventral vLG, ZId, and ZIv/ZIr (moving from dorsal to ventral) (Fig.7C,F,I,K).

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Fig. 7.

The differential expression ofLhx1, Lhx5, Isl1, andPax6 in the E12.5 ventral thalamus and the region surrounding the tuning TCAs. Coronal sections of the mouse diencephalon showing the expression ofLhx1(A–C), Lhx5(D–F), Isl1(G–I), and Pax6(J, K) mRNA and Lhx1/5 (L–N), Pax6 (O,P), and Isl1 (L–P) protein.A′, B′, D′,E′, G′, and H′ are the DAPI counterstaining of A, B,D, E, G, andH, respectively. Note the dark parts in the DAPI staining, which come from the signal of in situhybridization. L is approximately at the same level asA, D, and G, whereasM/O and N/P are at similar levels toB/E/H/J and C/F/I/K, respectively. The level of A/D/G is at the level at which TCAs make a lateral turn at the diencephalic–telencephalic border. The TCA bundle is seen as a cell-sparse area (A, A′,D, D′, G,G′; arrow). This bundle is surrounded by the domain expressing Lhx1 and Lhx5 on its lateral, dorsal (A, D), and rostral (data not shown) aspects. The medioventral part of the TCA bundle contains cells expressing Isl1 (G;arrowhead). This domain does not express Lhx1 or Lhx5 (L; arrow) and continues caudally to the putative RT (H; asterisk). At the more caudal level (B, E, H,J, M, O,Q), the TCA is on the lateral surface of vTh (B′, E′, H′;arrows), and the putative RT is located immediately medial to it, which express Isl1 but notLhx1, Lhx5, or Pax6(B, E, H,J; asterisks). Just dorsal to RT, there is a domain that expresses Lhx1, Isl1, and Pax6 but not Lhx5 (B,E, H, J;arrows), which is compatible with ZIr or ZIv. Immunostaining of the boxed areas inB/E/H/J shows that in this domain Isl1-expressing cells rarely overlap with Lhx1/5-expressing cells, but they do with Pax6-expressing cells (M, O;arrows). More dorsally, ZLI expressesLhx1 and Lhx5 but not Isl1(B, E, H;arrowheads). The putative vLG expressesLhx1, Lhx5, and Pax6(B, E, J; double arrows). Expression is high dorsally for Lhx5and ventrally for Lhx1 and Pax6. These results are summarized in Q. The nomenclaturesVTh-1d, VTh-1v, and VTh-2were used in our previous study based on the expression ofPax6 and RPTPδ at E13.5 (Tuttle et al., 1999). At the most caudal level, vLG is again evident as a domain expressing Lhx1, Lhx5, andPax6 (C, F,K; double arrows), as is ZLI (C, F; arrowheads). More ventrally, a domain with high level of Lhx1 and lowLhx5 expression but no Isl1 orPax6 expression is detected, which corresponds to ZId (arrows). The putative ZIv is ventral to ZId and expresses high levels of Lhx1, Isl1, andPax6 and very low levels of Lhx5(asterisks). Immunostaining of the boxed areas in C/F/I/K shows that the ZIv contains heterogeneous populations of cells for expression of Isl1, Lhx1/5, and Pax6 (N, P). These results are summarized in R. Scale bar, 50 μm.

Results of immunostaining confirm the above assignments and directly show the molecular heterogeneity of cells within the same nucleus (Fig. 7L–P); in the putative ZIr/ZIv, cells that express Isl1 and Lhx1/5 exhibit virtually no overlap rostrally (Fig.7M) but considerable overlap caudally (Fig.7N), whereas those that express Isl1 and Pax6 overlap rostrally and caudally (Fig. 7O,P). Similar patterns are also observed at E14.5 (data not shown), suggesting that the differential expression of these genes not only parcellates and specifies the vTh at the level of presumptive nuclei but is also likely involved in specification of distinct cell types within these nuclei.

Relationship between expression domains and the TCA path

TCAs extend ventrally from dTh into vTh and, at approximately E12.5, make a sharp lateral–rostral turn at the vTh–telencephalic border and enter ventral telencephalon (Braisted et al., 1999; Tuttle et al., 1999). The turning TCA bundle is identified as a cell-sparse area in 4′,6′-diaminino-2-phenylindole (DAPI) staining (Fig. 7A′,D′,G′). This bundle is surrounded dorsally and laterally by cell domains expressingLhx5 and both Lhx1 and Lhx5, respectively (Fig. 7A,D). These cells are likely to be in ventral telencephalon. TheIsl1-expressing cell domain interpreted to be a rostral extension of the putative RT occupies the dorsomedial part of the TCA bundle (Fig. 7G,H). The lack of overlap between Isl1- and Lhx1/5-expressing cells is confirmed by immunostaining (Fig. 7L).Lhx1/5-expressing, hypothalamic domain is located medial to the Isl1-expressing RT (Fig. 7L). Thus, the path taken by the TCA bundle near the diencephalic–telencephalic border is outlined by cell groups that express distinct combinations of LIM-HD genes, suggesting that these genes may differentially regulate the expression of guidance molecules that control TCA pathfinding.

Onset of differential gene expression in dTh and vTh

To determine whether the regulatory genes analyzed here exhibit patterned expression at the onset of neurogenesis in thalamus, we analyzed their expression at E10.5 when the first thalamic neurons are generated. At this stage, Lhx2, Lhx9, andGbx2 are expressed in thin overlapping bands that extend dorsoventrally along the lateral edge of dTh (Fig.8A–C). Some expressing cells are scattered in the ventricular zone. In contrast to later ages,Ngn2 is expressed only in the ventricular zone at E10.5 (Fig. 8D). Lhx9 shows a longer expression domain than Lhx2 and Gbx2. Double-labeling with Lhx2/9 antibodies and BrdU (injected 1.5 hr before fixation) shows that virtually all of the Lhx2/9-positive cells are BrdU-negative, suggesting that they are postmitotic (Fig. 8E); only a small number of cells near the lateral border of BrdU-positive region are double-labeled with both antibodies. Lhx2/9-positive cells scattered in the ventricular zone are all BrdU-negative. Double-labeling with an Lhx2/9 antibody and antibodies against class III β-tubulin and microtubule-associated protein-2 shows that a subpopulation of Lhx2/9-positive cells express these neuronal markers (data not shown). These findings suggest that Lhx2 and/orLhx9 begin to be expressed around the time the first dTh neurons become postmitotic, and the onset of expression precedes the appearance of neuronal markers.

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Fig. 8.

Expression of Lhx2,Lhx9, Gbx2, and Ngn2 in dTh and Lhx1, Lhx5, Isl1, and Pax6 in vTh is apparent at E10.5. Coronal sections of the mouse dTh (A–E) and vTh (F–L). Lhx2, Lhx9, and Gbx2 are expressed in the lateral part of dTh (A–C; arrows). Patterns ofLhx2 and Gbx2 are indistinguishable, and expression of Lhx9 extends further dorsally compared with Lhx2 and Gbx2. Ngn2 is expressed only in the ventricular zone and not in the lateral part in whichLhx2, Lhx9, and Gbx2 are expressed (D; arrow). Lhx2/9 is mostly localized in BrdU-negative cells at the mantle zone, and only a small number of Lhx2/9-positive cells are BrdU-positive (E;arrowheads). Lhx1, andIsl1 are expressed in the mantle zone of vTh, whereasLhx5 and Pax6 are expressed both in the mantle and ventricular zones (F–I;arrow). Isl1 is expressed only in BrdU-negative cells (J). The mantle zone is already composed of heterogeneous cell populations for the expression of Lhx1, Isl1, and Pax6 (K, L). Scale bars:A–D, F–I, 100 μm; E,J–L, 50 μm.

In the vTh, Lhx1, Lhx5, Isl1, and Pax6 are all expressed at E10.5. Lhx1 andIsl1 are expressed in a thin band of cells in the mantle zone (Fig. 8F,H). In contrast, Lhx5 and Pax6 are highly expressed throughout both the mantle and ventricular zones (Fig.8G,I). The great majority of cells in the ventricular zone that are immunopositive for Lhx5 or Pax6 are also BrdU-positive (data not shown). The expression of these two genes does not become restricted to the mantle zone until E12.5. In contrast, only a few cells in the ventricular zone immunostain for Isl1, and only a small subset of these cells are BrdU-positive (Fig.8L). Immunostaining also shows that as early as E10.5, the heterogeneity in vLG cells expressing Lhx1/5, Isl1, and Pax6 observed at later ages is evident in the mantle zone (Fig.8J,K).

In summary, the LIM-HD genes Lhx1, Lhx2,Lhx9, and Isl1, as well as the homeodomain geneGbx2, are expressed by postmitotic cells in the mantle zone of dTh and vTh as it first begins to form. Lhx5 andPax6 are expressed in postmitotic as well as progenitor cells in the ventricular zone, suggesting that these genes may define specific vTh cell types even at the progenitor cell stage.

Parcellation of dTh and vTh into nuclei

At P2, when thalamic nuclei are readily distinguished, the regulatory genes studied here are expressed in unique, overlapping patterns that mark specific nuclei or subsets of nuclei. An important issue for assessing the roles of these genes is whether they mark the same cells at earlier times. The expression patterns observed at P2 are also evident at E16.5, when thalamic nuclei are still differentiating but can be defined histologically. Although at E14.5 the borders between prospective thalamic nuclei are often not histologically distinguishable, the locations of the nuclei can be readily approximated, and the expression patterns resemble those at E16.5 and P2. It is more difficult to make this assessment at earlier ages. E12.5 is during thalamic neurogenesis, but a majority of neurons have been generated and many have completed their migration and established a mantle zone within which nuclei will differentiate. Even at this early developmental stage, the expression patterns of the eight genes relative to each other are approximately similar to those seen later.

Thus, thalamic neurons appear to express the same subset of these regulatory genes between E12.5 and P2. Each of these genes is expressed in a unique pattern as early as E10.5, when the first thalamic neurons are generated; thus, they may mark distinct subsets of thalamic neurons beginning around the time they are generated through the time they form nuclei. Based on the expression patterns and known functions of these genes, they are good candidates to act in a combinatorial manner to control the specification of nuclei-specific properties of thalamic cells and the differentiation of nuclei. This suggestion is supported by analyses of Gbx2-deficient mice (Miyashita-Lin et al., 1999). Although the mutant exhibits a severe disorganization of dTh, some parts that correspond to VP, dLG, Po, and MG persist. These results are consistent with our finding that Gbx2 is not expressed in VP, dLG, and Po, and only in part of MG. Furthermore, the “sparing” of these nuclei in the mutant suggests that the cell populations that express Gbx2 do not significantly change over embryonic development.

The expression patterns observed in dTh do not appear to change between E16.5 and P2. However, at E14.5 and E12.5, a band of cells in rostromedial dTh exhibits a Lhx2/Gbx2-high,Lhx9/Ngn2-low expression not seen later. One possible explanation for the difference is that this population is still present after E14.5 but is mixed with other populations. This possibility is supported by the fact that neurons of rostromedial dTh nuclei are born later than caudolateral dTh nuclei (Altman and Bayer, 1979; Altman and Bayer, 1988) and may mingle with each other during migration. Another possibility is that these cells express a different combination of genes at E16.5 than at E14.5, similar to the finding that some spinal motor neurons initially express Lhx3 and Lhx4 but later do not (Sharma et al., 1998).

Heterogeneity in gene expression within a nucleus

Because thalamic nuclei contain multiple types of neurons, regulatory genes might exhibit not only nuclei-specific expression but also differential expression within a nucleus. For example, each subdivision of ZI contains heterogeneous populations of cells that express different neurotransmitters and calcium binding proteins (Kolmac and Mitrofanis, 1998). In the putative ZI at E12.5, our immunostaining for Lhx1/5, Isl1, and Pax6 reveals at least four (and possibly 18) different subsets of cells; in rostral ZI, cells only express Isl1 or Lhx1/5, whereas in caudal ZI, many cells express Isl1 and Lhx1/5. Therefore, the differential expression of Lhx1/5, Isl1, and Pax6 may have a role in regulating the differentiation of specific cell types and subdivisions within this nucleus.

Lhx1, Lhx5, and Pax6 may have a similar role within vLG, because it is subdivided by their differential expression. This heterogeneity of gene expression is also evident in the ZLI, which at this stage of development appears to be a migratory stream containing vLG neurons. Based on the expression of the regulatory genes Dlx1, Arx, Brx1, andNkx2.2, Kitamura et al. (1997) have suggested that ZLI is composed of two cell groups that give rise to different parts of vLG. Together our findings show that vLG is subdivided into multiple domains based on the differential expression of regulatory genes and that this molecular heterogeneity is already evident while vLG neurons are migrating within the ZLI.

Potential roles in control of TCA axon pathfinding

Combinations of LIM-HD genes expressed by subsets of motor neurons in vertebrates and Drosophila constitute a “LIM-HD combinatorial code” that dictates their axonal pathfinding (Tsuchida et al., 1994; Sharma et al., 1998; Thor et al., 1999). In addition, theDrosophila LIM-HD gene apterous, the ortholog ofLhx2 and Lhx9, is required for the axonal pathfinding of a subset of interneurons (Lungdren et al., 1995). Similarly, the differential expression of LIM-HD genes in dTh nuclei might regulate TCA pathfinding in a cell-autonomous manner.

Candidate genes regulated by the potential “combinatorial transcription factor code” include Eph receptor tyrosine kinases (Gao et al., 1998; Mackarehtschian et al., 1999; Vanderhaeghen et al., 2000) and cadherins such as Cad6, Cad8, andCad11, which have matching expression between dTh nuclei and their target cortical areas (Suzuki et al., 1997; Inoue et al., 1998). Interestingly, Lhx2 expression also shows a correlation between dTh and neocortex; the auditory area expresses the highest level of Lhx2 in the cortical plate (Nakagawa et al., 1999), the target of MGv axons, the only principal sensory nucleus that highly expresses Lhx2 (present study). The visual and somatosensory areas express lower levels of Lhx2 (Nakagawa et al., 1999), and the principal sensory nuclei that project to them, VP and dLG, do not express Lhx2 (present study). Because Lhx2expression in cortex is established independent of TCAs (Nakagawa et al., 1999), the matching of Lhx2 expression between dTh and cortex may independently regulate the expression of molecules involved in TCA targeting from MGv to the auditory area.

Our results also suggest a role for these genes in controlling TCA pathfinding through a non-cell-autonomous mechanism. At E12.5,Lhx1, Lhx5, Isl1, and Pax6are expressed in distinct patterns that mark domains along the TCA pathway through vTh to ventral telencephalon and may influence the expression of guidance molecules. For example, RT expressesRPTPδ (Tuttle et al., 1999; present study), which could function as both a ligand and a receptor for axon guidance (Wang and Bixby, 1999), and Slit1 (J. E. Braisted, T. Ringstedt, and D. D. M. O'Leary, unpublished observations), a repulsive axon guidance molecule and a ligand for robo receptors (Brose et al., 1999;Kidd et al., 1999; Li et al., 1999), which are expressed in dTh (Braisted, Ringstedt, and O'Leary, unpublished observations). The perireticular nucleus, which is apposed to the TCA path near the border of vTh and ventral telencephalon (Clemence and Mitrofanis, 1992;Earle and Mitrofanis, 1996), has been proposed to be a guidepost for TCAs (Mitrofanis and Guillery, 1993) (but see Coleman and Mitrofanis, 1999). Although molecular markers defining this nucleus have not been reported for early stages, it appears to coincide with the rostral part of the Isl1-positive, putative RT (present study), and/or ventral telencephalic cells expressing Nkx2.1 (Tuttle et al., 1999).

Regionalization of diencephalon into dTh and vTh

The sets of genes that we show to be expressed in dTh and vTh are distinct from one another and similar to those expressed in dorsal and ventral spinal cord, respectively. This similarity suggests that the expression patterns in thalamus might be established by mechanisms similar to those in spinal cord. In spinal cord, inductive signals from the roof plate and floor plate control neuronal fate along the dorsoventral axis (Tanabe and Jessell, 1996; Lee and Jessell, 1999). Signals from the roof plate, such as TGFβ family members, are required in dorsal spinal cord for the induction of Lhx2 andLhx9, which define D1A and D1B interneurons, respectively (Liem et al., 1997; Lee and Jessell, 1999; Lee et al., 2000). In ventral spinal cord, distinct classes of motor neurons and ventral interneurons are generated by a graded signaling activity of Shh (Briscoe et al., 1999, 2000). Shh controls these neural fates by establishing different progenitor cell populations defined by their expression of Pax6 and Nkx2.2. Pax6 establishes distinct populations of ventral progenitor cells and controls the identity of motor neurons and V1 and V2 interneurons (Ericson et al., 1997), whereas Nkx2.2 specifies the identity of V3 interneurons at a more ventral location (Briscoe et al., 1999). These genes appear to be essential intermediaries for Shh to regulate the differential expression of LIM-HD proteins, including Lhx1, Lhx3, Lhx4, Lhx5, Isl1, and Isl2.

In diencephalon, Shh is transiently expressed as early as E9.5 in the ZLI, which at this stage is a narrow cell domain interposed between prospective dTh and vTh (Shimamura et al., 1995; Kitamura et al., 1997). Similar to ventral spinal cord, Nkx2.2 and Pax6 are also expressed in progenitor cells in vTh. Shh induces in vitrothe expression of Isl1 in chick forebrain explants and neuroepithelial cells from rat forebrain (Ericson et al., 1995; Nakagawa et al., 1996). Therefore, ZLI-derived Shh may specify progenitor cell types in vTh to produce different neuronal subtypes, which are determined by the subset of LIM-HD and other transcription factors expressed by these neurons. Interestingly, dTh, which is adjacent to the ZLI, does not express any of the LIM-HD genes induced by Shh and expressed in vTh.Ngn2, which is expressed by progenitor cells of dTh but not vTh, could act to limit the responsiveness of dTh to an Shh-mediated induction of vTh-type LIM-HD genes, which may be a crucial step in regionalization of the diencephalon.