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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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Related in: MedlinePlus

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.
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pone-0020940-g003: 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.

Mentions: We next ascertained the intermediate fate of the Gbx2 lineage marked at different stages by analyzing ß-gal+ cells and well-characterized markers at E12.5 (Figure 3A–U). Pax2, a transcription factor that distinguishes a subset of differentiating inhibitory dorsal horn interneurons (Figure 3B) [34] was expressed in Gbx2-derived cells marked at E8.5, E9.5, or E10.5 at upper (Figure 3D–F) and lower (Figure 3M–O) limb levels. Therefore, Gbx2(GFP)-expressing progenitors continually gave rise to dorsal Pax2+ inhibitory neurons. Because Gbx2(GFP) was expressed throughout the neural tube at E8.5 and contributed to the entire cord, we hypothesized that the Gbx2 lineage would contribute to motor neurons at E12.5. Comparing ß-gal expression with Isl1/2 expression, which defines all differentiating motor neurons in ventral spinal cord [33], demonstrated that progenitors expressing Gbx2(GFP) at E8.5 gave rise to Isl1/2+ motor neurons at all A-P levels of the maturing spinal cord (Figure 3G,P). In contrast, Gbx2-derived progenitors marked at E9.5 no longer contributed to Isl1/2+ motor neurons at the upper limb level (Figure 3H), although the Gbx2 lineage marked at E9.5 gave rise to motor neurons at the posterior lower limb level (Figure 3Q). We observed a complete exclusion of the Gbx2 lineage marked at E10.5 from contributing to motor neurons at all A-P levels (Figure 3I,R). The dorsal root ganglia (DRG), which contain Isl1/2+ post-mitotic neurons [35], was derived from the Gbx2 lineage (ß-gal+) marked at E8.5 along the full length of the maturing spinal cord (Figure 3J,S). Similar to motor neurons, ß-gal+ cells from E9.5 marking were not detected in upper limb DRG (Figure 3K), but were observed at lower limb level of spinal cord (Figure 3T). Finally, the Gbx2 lineage marked at E10.5 did not contribute to Isl1/2+ DRG neurons at any A-P level (Figure 3L,U). Therefore, both dorsal and ventral populations in spinal cord are derived from a progenitors expressing Gbx2 for twenty-four hours beginning at E8.5. Rapidly though, at the upper limb level, ventral motor neurons and DRG neurons were no longer derived from the Gbx2 lineage. In contrast, motor neurons and DRG neurons at the lower limb level were derived from the Gbx2 lineage twenty-four hours longer than those located rostrally.


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

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

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.
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Related In: Results  -  Collection

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pone-0020940-g003: 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.
Mentions: We next ascertained the intermediate fate of the Gbx2 lineage marked at different stages by analyzing ß-gal+ cells and well-characterized markers at E12.5 (Figure 3A–U). Pax2, a transcription factor that distinguishes a subset of differentiating inhibitory dorsal horn interneurons (Figure 3B) [34] was expressed in Gbx2-derived cells marked at E8.5, E9.5, or E10.5 at upper (Figure 3D–F) and lower (Figure 3M–O) limb levels. Therefore, Gbx2(GFP)-expressing progenitors continually gave rise to dorsal Pax2+ inhibitory neurons. Because Gbx2(GFP) was expressed throughout the neural tube at E8.5 and contributed to the entire cord, we hypothesized that the Gbx2 lineage would contribute to motor neurons at E12.5. Comparing ß-gal expression with Isl1/2 expression, which defines all differentiating motor neurons in ventral spinal cord [33], demonstrated that progenitors expressing Gbx2(GFP) at E8.5 gave rise to Isl1/2+ motor neurons at all A-P levels of the maturing spinal cord (Figure 3G,P). In contrast, Gbx2-derived progenitors marked at E9.5 no longer contributed to Isl1/2+ motor neurons at the upper limb level (Figure 3H), although the Gbx2 lineage marked at E9.5 gave rise to motor neurons at the posterior lower limb level (Figure 3Q). We observed a complete exclusion of the Gbx2 lineage marked at E10.5 from contributing to motor neurons at all A-P levels (Figure 3I,R). The dorsal root ganglia (DRG), which contain Isl1/2+ post-mitotic neurons [35], was derived from the Gbx2 lineage (ß-gal+) marked at E8.5 along the full length of the maturing spinal cord (Figure 3J,S). Similar to motor neurons, ß-gal+ cells from E9.5 marking were not detected in upper limb DRG (Figure 3K), but were observed at lower limb level of spinal cord (Figure 3T). Finally, the Gbx2 lineage marked at E10.5 did not contribute to Isl1/2+ DRG neurons at any A-P level (Figure 3L,U). Therefore, both dorsal and ventral populations in spinal cord are derived from a progenitors expressing Gbx2 for twenty-four hours beginning at E8.5. Rapidly though, at the upper limb level, ventral motor neurons and DRG neurons were no longer derived from the Gbx2 lineage. In contrast, motor neurons and DRG neurons at the lower limb level were derived from the Gbx2 lineage twenty-four hours longer than those located rostrally.

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