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The lineage contribution and role of Gbx2 in spinal cord development.

Luu B, Ellisor D, Zervas M - PLoS ONE (2011)

Bottom Line: Using lineage tracing and molecular markers to follow Gbx2-mutant cells, we show that the loss of Gbx2 globally affects spinal cord patterning including the organization of interneuron progenitors.Finally, long-term lineage analysis reveals that the presence and timing of Gbx2 expression in interneuron progenitors results in the differential contribution to subtypes of terminally differentiated interneurons in the adult spinal cord.In a broader context, this study provides a direct link between spinal cord progenitors undergoing dynamic changes in molecular identity and terminal neuronal fate.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology and Medicine, Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, Rhode Island, United States of America.

ABSTRACT

Background: Forging a relationship between progenitors with dynamically changing gene expression and their terminal fate is instructive for understanding the logic of how cell-type diversity is established. The mouse spinal cord is an ideal system to study these mechanisms in the context of developmental genetics and nervous system development. Here we focus on the Gastrulation homeobox 2 (Gbx2) transcription factor, which has not been explored in spinal cord development.

Methodology/principal findings: We determined the molecular identity of Gbx2-expressing spinal cord progenitors. We also utilized genetic inducible fate mapping to mark the Gbx2 lineage at different embryonic stages in vivo in mouse. Collectively, we uncover cell behaviors, cytoarchitectonic organization, and the terminal cell fate of the Gbx2 lineage. Notably, both ventral motor neurons and interneurons are derived from the Gbx2 lineage, but only during a short developmental period. Short-term fate mapping during mouse spinal cord development shows that Gbx2 expression is transient and is extinguished ventrally in a rostral to caudal gradient. Concomitantly, a permanent lineage restriction boundary ensures that spinal cord neurons derived from the Gbx2 lineage are confined to a dorsal compartment that is maintained in the adult and that this lineage generates inhibitory interneurons of the spinal cord. Using lineage tracing and molecular markers to follow Gbx2-mutant cells, we show that the loss of Gbx2 globally affects spinal cord patterning including the organization of interneuron progenitors. Finally, long-term lineage analysis reveals that the presence and timing of Gbx2 expression in interneuron progenitors results in the differential contribution to subtypes of terminally differentiated interneurons in the adult spinal cord.

Conclusions/significance: We illustrate the complex cellular nature of Gbx2 expression and lineage contribution to the mouse spinal cord. In a broader context, this study provides a direct link between spinal cord progenitors undergoing dynamic changes in molecular identity and terminal neuronal fate.

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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).
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pone-0020940-g004: 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).

Mentions: An important question in spinal cord development is how molecularly distinct progenitors contribute to adult spinal cord cytoarchitecture. To begin to address this, we marked the Gbx2 lineage in vivo and ascertained the terminal fate of Gbx2-derived neurons marked at E8.5 and E9.5 using biochemical markers for functionally distinct neurons (Figure 4). Calbindin-D28K (CALB) and calretinin (CALR) are calcium-binding proteins that are expressed in a subset of interneurons located in superficial dorsal laminae I/II [36]. Lamina I was sparsely populated with CALB+ and CALR+ interneurons. In contrast, lamina II was densely packed with CALB+ interneurons and moderately populated with loosely arranged CALR+ neurons (Figure 4A–D) [37]. The Gbx2-lineage marked at E8.5 or E9.5 was evenly distributed across laminae I–II and co-localized with CALB+ in lamina II, but was only interspersed with CALB+ cells in laminae I (Figure 4A,B). In contrast, the Gbx2-lineage marked at E8.5 or E9.5 only rarely contributed to CALR+ neurons in layer II (Figure 4C,D). The Gbx2-lineage (ß-gal+) marked at E8.5 or E9.5 contributed to inhibitory neurons in dorsal laminae I–IV expressing glutamic acid decarboxylase (GAD) (Figure 4E,F, insets of 3-D rendered neurons provides clarity of labeling, which was confirmed with single 1 µm thick optical sections in the XZ and YZ plane, not shown). Finally, the Gbx2 lineage marked at E8.5 and E9.5 gave rise to Pax2+ inhibitory interneurons dispersed throughout the dorsal horn (Figure 4G,H).


The lineage contribution and role of Gbx2 in spinal cord development.

Luu B, Ellisor D, Zervas M - PLoS ONE (2011)

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).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3116860&req=5

pone-0020940-g004: 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).
Mentions: An important question in spinal cord development is how molecularly distinct progenitors contribute to adult spinal cord cytoarchitecture. To begin to address this, we marked the Gbx2 lineage in vivo and ascertained the terminal fate of Gbx2-derived neurons marked at E8.5 and E9.5 using biochemical markers for functionally distinct neurons (Figure 4). Calbindin-D28K (CALB) and calretinin (CALR) are calcium-binding proteins that are expressed in a subset of interneurons located in superficial dorsal laminae I/II [36]. Lamina I was sparsely populated with CALB+ and CALR+ interneurons. In contrast, lamina II was densely packed with CALB+ interneurons and moderately populated with loosely arranged CALR+ neurons (Figure 4A–D) [37]. The Gbx2-lineage marked at E8.5 or E9.5 was evenly distributed across laminae I–II and co-localized with CALB+ in lamina II, but was only interspersed with CALB+ cells in laminae I (Figure 4A,B). In contrast, the Gbx2-lineage marked at E8.5 or E9.5 only rarely contributed to CALR+ neurons in layer II (Figure 4C,D). The Gbx2-lineage (ß-gal+) marked at E8.5 or E9.5 contributed to inhibitory neurons in dorsal laminae I–IV expressing glutamic acid decarboxylase (GAD) (Figure 4E,F, insets of 3-D rendered neurons provides clarity of labeling, which was confirmed with single 1 µm thick optical sections in the XZ and YZ plane, not shown). Finally, the Gbx2 lineage marked at E8.5 and E9.5 gave rise to Pax2+ inhibitory interneurons dispersed throughout the dorsal horn (Figure 4G,H).

Bottom Line: Using lineage tracing and molecular markers to follow Gbx2-mutant cells, we show that the loss of Gbx2 globally affects spinal cord patterning including the organization of interneuron progenitors.Finally, long-term lineage analysis reveals that the presence and timing of Gbx2 expression in interneuron progenitors results in the differential contribution to subtypes of terminally differentiated interneurons in the adult spinal cord.In a broader context, this study provides a direct link between spinal cord progenitors undergoing dynamic changes in molecular identity and terminal neuronal fate.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology and Medicine, Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, Rhode Island, United States of America.

ABSTRACT

Background: Forging a relationship between progenitors with dynamically changing gene expression and their terminal fate is instructive for understanding the logic of how cell-type diversity is established. The mouse spinal cord is an ideal system to study these mechanisms in the context of developmental genetics and nervous system development. Here we focus on the Gastrulation homeobox 2 (Gbx2) transcription factor, which has not been explored in spinal cord development.

Methodology/principal findings: We determined the molecular identity of Gbx2-expressing spinal cord progenitors. We also utilized genetic inducible fate mapping to mark the Gbx2 lineage at different embryonic stages in vivo in mouse. Collectively, we uncover cell behaviors, cytoarchitectonic organization, and the terminal cell fate of the Gbx2 lineage. Notably, both ventral motor neurons and interneurons are derived from the Gbx2 lineage, but only during a short developmental period. Short-term fate mapping during mouse spinal cord development shows that Gbx2 expression is transient and is extinguished ventrally in a rostral to caudal gradient. Concomitantly, a permanent lineage restriction boundary ensures that spinal cord neurons derived from the Gbx2 lineage are confined to a dorsal compartment that is maintained in the adult and that this lineage generates inhibitory interneurons of the spinal cord. Using lineage tracing and molecular markers to follow Gbx2-mutant cells, we show that the loss of Gbx2 globally affects spinal cord patterning including the organization of interneuron progenitors. Finally, long-term lineage analysis reveals that the presence and timing of Gbx2 expression in interneuron progenitors results in the differential contribution to subtypes of terminally differentiated interneurons in the adult spinal cord.

Conclusions/significance: We illustrate the complex cellular nature of Gbx2 expression and lineage contribution to the mouse spinal cord. In a broader context, this study provides a direct link between spinal cord progenitors undergoing dynamic changes in molecular identity and terminal neuronal fate.

Show MeSH
Related in: MedlinePlus