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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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Gbx2 mutant lineage at E12.5.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).
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pone-0020940-g008: Gbx2 mutant lineage at E12.5.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).

Mentions: Gbx2CreER-ires-eGFP/CreER-ires-eGFP mutants (n = 3) at E12.5 (Figure 8A,B) had Gbx2-mutant (GFP+) neurons that were distributed in a broader morphologically distinct dorsal ventricular zone replete with differentiating mutant neurons at E12.5 (Figure 8C,C′). This finding was consistent with our observations at E10.5. Dorsal Isl1/2+ interneurons and dorsal Pax2-expressing inhibitory interneurons were distributed in a similar pattern as controls (Figure 8D–E′). In contrast, we observed ectopic clusters of Gbx2(GFP) mutant cells in the ventricular and mantle zones of the ventral spinal cord (Figure 8C,C′,*). Ventromedial Isl1/2+ motor neurons of the MMC were depleted in Gbx2 mutants (Figure 8D,D′,*) consistent with our findings at E10.5. The number of Gbx2(GFP) mutant cells that co-expressed Pax2+ in ventral spinal cord (33±12/hemisection) was doubled in comparison to wildtype Gbx2(GFP)+/Pax2+ cells (16±12/hemisection) (Figure 8E,E′,*). The lateral spinal cord of mutants had 2.5 times more Gbx2(GFP)+/Pax2+ cells while the medial spinal cord had a 1.8 fold increase. Finally, we addressed whether the state of mutant lineage derived cells changed over time by administering tamoxifen to Gbx2CreER-ires-eGFP/CreER-ires-eGFP embryos at E9.5. The Gbx2(GFP) mutant cells at E12.5 were not derived from the mutant progenitors (ß-gal+) marked at E9.5, which was similar to control littermates (Figure 8F,F′). Interestingly, the ventral Pax2+ neurons in Gbx2CreER-ires-eGFP/CreER-ires-eGFP embryos were scattered and loosely organized compared to controls (Figure 8G,G′,*1,*2,*3), but the Pax2+ cells were not derived from the Gbx2 mutant lineage marked at E9.5. These findings suggested that a deficiency in Gbx2 did not result in a cell-autonomous fate change in mutant progenitors marked at E9.5 (Figure 8G,G′). Rather, our findings indicated that a cohort of Gbx2(GFP)+/Pax2+ cells in mutant spinal cord resulted from an attempt to re-initiate the expression of Gbx2(GFP) at E12.5 and not from a failure of turning off Gbx2(GFP). Finally, the Gbx2 mutant lineage was aberrantly distributed in ventricular zone of the ventral region of the spinal cord at thoracic and upper limb levels (Figure 8H–H′, arrows). This finding suggested that the lineage boundary was compromised in the absence of Gbx2.


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

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

Gbx2 mutant lineage at E12.5.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).
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pone-0020940-g008: Gbx2 mutant lineage at E12.5.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).
Mentions: Gbx2CreER-ires-eGFP/CreER-ires-eGFP mutants (n = 3) at E12.5 (Figure 8A,B) had Gbx2-mutant (GFP+) neurons that were distributed in a broader morphologically distinct dorsal ventricular zone replete with differentiating mutant neurons at E12.5 (Figure 8C,C′). This finding was consistent with our observations at E10.5. Dorsal Isl1/2+ interneurons and dorsal Pax2-expressing inhibitory interneurons were distributed in a similar pattern as controls (Figure 8D–E′). In contrast, we observed ectopic clusters of Gbx2(GFP) mutant cells in the ventricular and mantle zones of the ventral spinal cord (Figure 8C,C′,*). Ventromedial Isl1/2+ motor neurons of the MMC were depleted in Gbx2 mutants (Figure 8D,D′,*) consistent with our findings at E10.5. The number of Gbx2(GFP) mutant cells that co-expressed Pax2+ in ventral spinal cord (33±12/hemisection) was doubled in comparison to wildtype Gbx2(GFP)+/Pax2+ cells (16±12/hemisection) (Figure 8E,E′,*). The lateral spinal cord of mutants had 2.5 times more Gbx2(GFP)+/Pax2+ cells while the medial spinal cord had a 1.8 fold increase. Finally, we addressed whether the state of mutant lineage derived cells changed over time by administering tamoxifen to Gbx2CreER-ires-eGFP/CreER-ires-eGFP embryos at E9.5. The Gbx2(GFP) mutant cells at E12.5 were not derived from the mutant progenitors (ß-gal+) marked at E9.5, which was similar to control littermates (Figure 8F,F′). Interestingly, the ventral Pax2+ neurons in Gbx2CreER-ires-eGFP/CreER-ires-eGFP embryos were scattered and loosely organized compared to controls (Figure 8G,G′,*1,*2,*3), but the Pax2+ cells were not derived from the Gbx2 mutant lineage marked at E9.5. These findings suggested that a deficiency in Gbx2 did not result in a cell-autonomous fate change in mutant progenitors marked at E9.5 (Figure 8G,G′). Rather, our findings indicated that a cohort of Gbx2(GFP)+/Pax2+ cells in mutant spinal cord resulted from an attempt to re-initiate the expression of Gbx2(GFP) at E12.5 and not from a failure of turning off Gbx2(GFP). Finally, the Gbx2 mutant lineage was aberrantly distributed in ventricular zone of the ventral region of the spinal cord at thoracic and upper limb levels (Figure 8H–H′, arrows). This finding suggested that the lineage boundary was compromised in the absence of Gbx2.

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