Figure 1.
The Gbx2-lineage exclusively gives rise to neurons in the thalamus.
(A–G) Anti-GFP staining of YFP, which is expressed from the R26RYFP locus and marks the descendants of Gbx2-expressing cells labeled at E10.5 (A–D) or E14.5 (F–G), and neuronal or glial markers as indicated on coronal sections of P21 Gbx2creER/+; R26RYFP mice. Asterisks indicate the significantly large number of GAFP+ cells in the IGL. (H–I) Double labeling of BLBP and NeuN or CC1 on sections of P21 thalamus. Nuclei are stained with TOPO3 (blue). Scale bars: A 100 µm; B (for B–I) 20 µm.
Figure 2.
Conditional deletion of Gbx2 at different embryonic stages.
(A) Diagram of temporal cohorts of Gbx2-expressing cells giving rise to different groups of thalamic nuclei. Gbx2 was deleted following administration of tamoxifen at E10.5, E14.5, or E16.5, as indicated by red arrows and the histology of thalamic nuclei was examined at P4 or P21 (Fig. 3 and 4). (B) Schematic representation of breeding scheme and “self-deletion” of Gbx2 by creER-mediated recombination in Gbx2creER/F embryos. (C–N) ISH for Gbx2 on coronal sections corresponding to the rostral (C–H) and caudal level (I–N) of the thalamus in control and Gbx2-CKO embryos between E14.5 and E18.5. The age of embryos and the stage of tamoxifen administration are indicated to the left. The RNA probe recognizes the Gbx2 sequence that will be deleted by creER-mediated recombination. Note that the Gbx2 transcripts are missing in the presumptive MG (red asterisk) following tamoxifen administration at E10.5, E14.5, or E16.5. By contrast, Gbx2 expression in the rostral-medial thalamus (blue asterisk) is mostly unaffected by deletion at E10.5. Abbreviations of thalamic nuclei are listed in table I. Scale bar in A: 250 µm (for A–L); 237 µm (for G–N).
Table 1.
Summary of defects of individual nuclei with Gbx2 deletion at different embryonic stages.
Figure 3.
Deletion of Gbx2 at E10.5 or E14.5, but not at E16.5, disrupts distinct thalamic nuclei.
(A–P) CO histochemistry on coronal brain sections of control (A–D) and Gbx2creER/F (E–P) mice at P4 that were given tamoxifen at E10.5 (E–H), E14.5 (I–L) or E16.5 (M–P). Thalamic nuclei are outlined in control sections and the affected thalamic nuclei are listed in blue at the lower left corner. Identification and nomenclature of thalamic nuclei are based on Caviness and Frost [28] and Jones [1]. The asterisk marks the loss of tissue corresponding to the anterior-medial group of nuclei (I), and the double arrow indicates the reduced medal-lateral dimension of VB (G). Note that thalamic nuclei are indistinguishable between control and Gbx2-CKO mice receiving tamoxifen at E16.5. Scale bar in P: 500 µm (for A–P).
Figure 4.
Altered expression of thalamic markers in P21 thalamus following temporal deletion of Gbx2.
(A–L) ISH for Tnnt1 on coronal sections of P21control (A–C) and Gbx2creER/F brains that was given tamoxifen at E10.5 (D–F), E14.5 (G–I) and E16.5 (J–L), respectively. Insets in (C) and (F) show ISH for Prkcd. The affected nuclei are listed in the lower left corner or indicated by an asterisk. The double arrow indicates the reduced size of VB; red arrowheads show residual Tnnt1+ and Prkcd+ cells in the presumptive MG nucleus; the white arrowhead indicates LP in G (this section corresponds a slightly more caudal position than those in A, D, and J). Abbreviations: mt, mamillothalamic tract; S, submedial nucleus. Scale bar in L: 500 µm (for A–L).
Figure 5.
The VB and dLG nuclei are disrupted when Gbx2 is deleted at E10.5, but not at E14.5.
(A–C) Double immunofluorescence for Sox2 and GFP, which reflects the transcription of the Gbx2 locus, on coronal sections of E18.5 brains of indicated genotypes. Boxed areas are magnified in E–G'. The asterisk indicates increased GFP expression in MD, and reduction in size of the VB; the dashed lines mark the border between Sox2 and GFP corresponding to the dLG and LP border; the brackets indicate the width of a band of strong Sox2-expressing cells along the lateral side of dLG and LP. (D) A schematic diagram indicates the position and the plane of sections shown in the figure. (H–M) Immunofluorescence for Nkx2.2 and NPY on coronal sections of E18.5 brains of indicated genotypes. Asterisks indicate the reduction of NPY+ neurites and the smaller VB in I; arrows mark NPY+ cells in the IL. Scale bars: A (A–C) 200 µm; E (for E–G'') 50 µm.
Figure 6.
Gbx2 is transiently required for cell survival for a subset of thalamic nuclei.
(A–D) Immunofluorescence of activated Casp3 on coronal sections of the thalamus of indicated genotypes. GFP signals in A and B were derived from the Gbx2cereER allele. MG (A and B) and the dorsomedial thalamus (C and D) are outlined by the dashed lines. Arrows indicate apoptotic cells. (E) Histogram representation of the average number of apoptotic cells in different thalamic nuclei. Error bars indicate standard deviations. Asterisks * and ** indicate p< 0.05 and p<0.001, respectively (Student's t-test). (F–I) X-gal histochemistry on coronal brain sections of P4 mice that were given tamoxifen at E10.5. The double arrow indicates reduced size of VB in mutants. Scale bars: B (for A–B) 100 µm; D (for C–D) 200 µm; I (F–I) 250 µm.
Figure 7.
Parcellation of the cerebral cortex is unaffected due to the disruption of thalamic nuclei.
(A–B) Immunohistochemistry for 5-HTT on tangential sections of P7 brains of control and Gbx2-CKO mice that received tamoxifen at E10.5. The 5-HTT immunohistochemistry reveals the body map in the cortex: a, auditory; v, visual; fl, florelimeb; hl, hindlimb; ll, lower lip; t, trunk; lw, large whiskers; sw, small whiskers [21]. (C–F) Red fluorescence (tdTomato) on coronal sections of P21 brains of Gbx2creER/+; R26RRFP/+ (C and E) and Gbx2creER/F; R26RRFP/+ (D and F) mice that were given tamoxifen at E10.5. The primary auditory area is indicated by arrowheads and cortical layers IV and VI were marked by white and green arrows, respectively. Note that the thickness (bracket) of the auditory cortex is reduced in Gbx2-CKO mutants. (E'–F') Nissl histology of the auditory cortex of control (E') and Gbx2-CKO mice (F'). (G–I) ISH for Lmo4 on sagittal sections of E18.5 control and Gbx2-CKO mutants that received tamoxifen at E10.5 or E14.5 as indicated. Black arrowheads demarcate the borders of the somatosensory area. Abbreviations: FC frontal; Occ, occipital; Par, parietal. Scale bar in A: 250 µm (A–B); 154 µm (for C–D); 307 µm (for E–F); 189 µm (for G–I).