Figure 1.
Dynamic expression of Gbx2 in the developing spinal cord.
Gbx2(GFP) expression detected in whole mount embryo (A). GFP immunolabeling (B, top row) and adjacent sections processed for Gbx2 in situ hybridization (B, bottom row) from E8.5 Gbx2CreER-ires-eGFP embryo; inset in “A” shows wildtype littermate. (C) Gbx2(GFP) expression in lateral view of an E9.5 embryo. (D–E) GFP and Pax7 immunolabeling on E9.5 Gbx2CreER-ires-eGFP/+ sections. (F–G) Lateral (F) and (G) dorsal views of EGFP fluorescence in E10.5 Gbx2CreER-ires-eGFP/+ embryo. (H–J) Antibody labeling of GFP and indicated markers on sagittal sections of E10.5 spinal cord; Note restricted ventral strip of Gbx2(GFP) expression (J, arrows). (K–S) Antibody labeling of GFP and indicated D-V markers on transverse hemi-sections of E10.5 spinal cord at the upper limb level. The insets show a high magnification view of the region indicated by the arrow. (T–U) EGFP fluorescence of E12.5 Gbx2CreER-ires-eGFP/+ embryo showing lateral (T) and dorsal (U) view. (V) GFP antibody labeling on sagittal sections of E12.5 spinal cord. GFP/Pax2 (W–W″) and GFP/Isl1/2 (X–X″) immunolabeling on transverse E12.5 hemi-sections of spinal cord at the upper limb (rostral) level. Abbreviations: mesencephalon (mes), rhombomere 1 (r1), intermediate (int) and posterior (post) neural tube, neuroepithelium (ne), blood vessel (bv), prosencephalon (pros), thalamus (thal), spinal cord (sc).
Figure 2.
Spatial distribution of the Gbx2 lineage in E12.5 spinal cord.
(A–D) Gbx2-derived cells marked at E8.5 (ß-gal+, red) on sagittal sections of E12.5 spinal cord at the indicated levels. (E–G) Transverse sections of E12.5 spinal cord at the indicated levels showing ß-gal+ cells (red) that were marked at E9.5. (H–J) The Gbx2 lineage (ß-gal+, red) marked at E10.5 was confined to the dorsal spinal cord at all axial levels at E12.5. (K–M) Transverse sections showing Gbx2(GFP)+ cells at indicated levels in E12.5 spinal cord. (N–P) Comparison of the Gbx2 lineage (ß-gal+, red) marked at E8.5 (N), E9.5 (O), and E10.5 (P) versus Gbx2 expression (GFP+, green) in E12.5 spinal cord. (N) Four D-V Gbx2-derived sub-populations can be classified by the presence or absence of Gbx2: zones 1 and 3 are Gbx2-derived cells that persisted in Gbx2 expression while zones 2 and 4 have down-regulated Gbx2. (O) Gbx2-derived cells marked at E9.5 continued to express Gbx2(GFP) in dorsal spinal cord at E12.5 in contrast to few ventral cells. (P) The majority of Gbx2(GFP)-expressing cells marked at E10.5 were confined to a dorsal domain and continued to express Gbx2(GFP).
Figure 3.
Molecular identity of the Gbx2 lineage in E12.5 spinal cord.
(A) Sagittal section of E12.5 embryo with nuclear staining (blue) showing regions analyzed. (B) Pax2 expression in dorsal spinal cord (indicated by bracket) in a hemi-transverse section. The box indicates the dorsolateral area of high magnification sampled for panels D–F, M–O (C) Isl1/2 expression in hemi-transverse sections of ventral spinal cord at the upper limb level. Isl1/2 is expressed in all developing motor neurons (MN) and dorsal root ganglia (DRG). Marker analysis of upper (D–L) and lower (M–U) limb levels at E12.5. The Gbx2 lineage (ß-gal+, red) marked at E8.5, E9.5 or E10.5 gave rise to Pax2+ neurons (green) at both upper (D–F) and lower (M–O) limb levels; insets highlight colocalization. (G–I) The Gbx2 lineage (ß-gal+, red) marked at E8.5, but not E9.5 or E10.5, contributed to ventral MNs (Isl1/2+, green) at upper limb level. (P–R) MNs (Is1/2+, green) at lower limb level were derived from the Gbx2 lineage (ß-gal+, red) marked at E8.5 and E9.5, but not E10.5. (J–L) Neurons in upper limb DRG (Isl1/2+, green) were derived from the Gbx2 lineage at E8.5 but not at later stages. (S–U) Caudal DRG (Isl1/2+, green) were derived from the Gbx2 lineage at E8.5 and E9.5.
Figure 4.
Terminal neuronal fate of the Gbx2 lineage.
The Gbx2 lineage (ß-gal+, red) marked at E8.5 (A) or E9.5 (B) contributed to dorsal spinal cord. Calbindin+ interneurons (green) were derived from the Gbx2-lineage marked at both stages; Insets reveal colocalization. (C–D) Gbx2-derived cells (ß-gal+, blue) marked at E8.5 (C) or E9.5 (D) were interspersed and only rarely co-localize with calretinin+ (CALR) interneurons; Insets show lack of overlap in lamina II. (E–F) GABAergic inhibitory neurons (GAD6+, green) were derived from Gbx2-expressing progenitors marked at E8.5 (E) or E9.5 (F). Diffuse GAD6 labeling in axonal and dendritic projections engulfs ß-gal labeling in neuronal cell bodies (insets E–F). (G–H) Gbx2-derived cells (ß-gal+, red) marked at E8.5 (G) or E9.5 (H) contributed to Pax2+ (green) interneurons; arrowheads show co-localization. (I–K) Choline-Acetyl-Transferase (ChAT, red) or CALR (red) expression compared to ß-gal immunolabeling (blue) shows that the Gbx2 lineage marked at E8.5 (I, K) but not E9.5 (J) contributed to both cholinergic motor neurons and interneurons in ventral horn at the upper limb level (Insets in I, K show colocalization; inset in J shows lack of contribution). (L) The Gbx2 lineage (ß-gal+, red) marked at E8.5 contributed to brain lipid binding protein (BLBP)+ glial cells in white matter. (M) Summary schematic of Gbx2 lineage (red circles) contribution to distinct laminae in the adult spinal cord. The summary is based on data presented in this figure and in Figure 6. The Gbx2 lineage marked at E8.5 gave rise to motor neurons and interneurons in ventral spinal cord (blue) as well as dorsal lamina interneurons (orange). The Gbx2 lineage marked at E9.5 occupied distinct D-V spinal cord domains depending on the A-P location in adult spinal cord. At the upper limb level (anterior, A), the Gbx2 lineage marked at E9.5 gave rise to dorsal interneurons (green) including superficial lamina (orange), but not ventral motor neurons (blue). At the lower limb level (posterior, P), the Gbx2 lineage marked at E9.5 spanned the D-V axis and gave rise to ventral motor neurons (blue) and dorsal interneurons (green, orange).
Figure 5.
Distribution and identity of Gbx2 mutant cells at E10.5.
Gbx2CreER-ires-eGFP/+ control heterozygote (A) and mutant Gbx2CreER-ires-eGFP/CreER-ires-eGFP embryos (A′) showing intact (r1) and reduced r1 (r1*), respectively at E10.5. Gbx2 in situ with an RNA probe on E10.5 transverse spinal cord sections from control (B) versus Gbx2 mutant embryos (B′). Note that Gbx2 was expressed in a broad dorsal (d) and in a restricted ventral (v) domain in wildtype embryos and absent in mutants. E10.5 control (C,D) and mutant (C′,D′) transverse sections immunolabeled with GFP (green) and indicated markers (red). Gbx2-deficient cells (GFP+, green) were co-localized with Brn3a in a wider swath of cells dorsally (*1). Gbx2(GFP) was broader and co-localized with Pax7 ventrally (*v) and in some cases dorsally (*d). Brn3a+ cells were in an ectopic ventral domain (*2); arrows indicate regions shown in insets. (E–E″) Compared to control Gbx2CreER-ires-eGFP/+ sections (E′), those obtained from Gbx2CreER-ires-eGFP/− mutants (E″) showed a significantly broader Gbx2(GFP) medial-lateral domain∶spinal cord width (E, inset). Notably in E,E′ both embryos have only one copy of GFP.
Figure 6.
Gbx2 loss affects spinal cord progenitor patterning.
Hemi-transverse sections from E10.5 wildtype (A–H) and Gbx2CreER-ires-eGFP/CreER-ires-eGFP mutant embryos (A′–H′) triple immunolabeled with indicated markers. (A, A′) Broader Brn3a domain dorsally (*1) and Brn3a expressing cells in the ventral domain (*2) in Gbx2 mutants. (B, B′) Brn3a+/Isl1/2+ neurons (arrows) and depletion of the medial-ventral domain of Isl1/2+ neurons (medial motor column,*) in Gbx2 mutants. Qualitatively, some ventral Isl1/2+ neurons inappropriately expressed Brn3a (yellow overlap). (C,C′) Ectopic Brn3a expressing cells in close proximity to the ventral Nkx2.2 population (*2). (D,D′) Medial-lateral expansion of early differentiating neurons in Gbx2 mutant embryos (*1, brackets). In addition, ectopic Brn3a+/Lim1/2+ neurons were seen ventral to their normal position (arrow). (E,E′) Pax3/Pax2 showing that the Pax3 domain is unchanged. (F,F′) Immunolabeling for phosphorylated-Histone H3 (pH3) showing fewer mitotic dorsally in mutant littermates. Wnt1 expression in the roofplate (RP) was subtly expanded (G,G′,*) while Shh expression in the floor plate (FP) and notochord (NC) was unaffected in mutants (H,H′).
Figure 7.
Quantitative assessment of aberrantly distributed spinal cord progenitors in Gbx2 mutant embryos.
Quantitative spatial analysis of control Gbx2CreER-ires-eGFP/+ (A–F) and mutant Gbx2CreER-ires-eGFP/CreER-ires-eGFP (G–L) spinal cords at E10.5. The average number of progenitors was assessed by counting cells with expressing the indicated markers in two sections at the upper limb level from control embryos (n = 3) and mutant embryos (n = 4). To facilitate a clear comparison of the spatial distribution across samples, we a Cartesian coordinate system where ML1-DV1 represented the most medial-dorsal quadrant, ML1-DV10 the most medial-ventral quadrant, ML4-DV1 the most lateral-dorsal quadrant, and ML4-DV10 the most lateral-ventral quadrant (M–R). The yellow boxes in panels M–R are shown at higher magnification with white dots used to track counted cells. The yellow boxes also correlate with the domains that were highlighted in the graphs with a yellow arrow. Quantitative spatial mapping revealed the distribution of Brn3a+ cells (A,G,M), Isl1/2+ (B,H,N), Lim1+ (C,I,O), Brn3a+/Isl1/2+ (D,J,P), Brn3a+/Lim1+ (E,K,Q), and pHH3 (F,L,R).
Figure 8.
Control Gbx2CreER-ires-eGFP/+ (A) and mutant Gbx2CreER-ires-eGFP/CreER-ires-eGFP (B) embryos at E12.5; mutants have reduced r1 (r1*). (C,C′) GFP immunolabeling on level-matched hemi-transverse sections of E12.5 heterozygous control (C) versus Gbx2 mutant embryos (C′). Ectopic clusters of Gbx2 mutant (GFP+) cells (*) in the ventricular zone. (D,D′) Isl1/2 immunolabeling on transverse sections of E12.5 heterozygous (D) versus Gbx2 mutant embryos (D′) showing loss of medial motor neurons in Gbx2 mutant embryos (*). (E, E′) Immunolabeling for GFP and Pax2 showing ectopically located ventral Gbx2(GFP)-mutant/Pax2+ interneurons (*) in mutant embryos; arrows indicate regions shown in insets. (F–G′) GIFM of thoracic sections from wildtype control (F,G) versus mutant (F′,G′) spinal cord. (F) ß-gal and GFP immunolabeling showing the wildtype Gbx2 lineage (ß-gal+, red) marked at E9.5 and Gbx2-expressing neurons at E12.5. (F′) The Gbx2-mutant lineage marked at E9.5 (ß-gal+, red) and Gbx2-mutants cells (GFP+, green) analyzed at E12.5. (G, G′) ß-gal expression resulting from lineage marking at E9.5 versus Pax2 expression in E12.5 control (G) versus Gbx2 mutant embryos (G′). Note that some ventral Pax2+ cells are disorganized (*1, *2), and others are ectopically located (*3). (H–H′) Cells of the Gbx2 mutant lineage marked at E9.5 reside in ectopic locations (arrows) that are ventral to the lineage boundary seen in controls (arrowheads).