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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

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).
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pone-0020940-g001: 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).

Mentions: To study Gbx2 expression, lineage contribution, and function we utilized the mouse line, Gbx2CreER-ires-eGFP that was generated by targeting CreERT2-ires-eGFP to the 5′ untranslated region of Exon 1 in the Gbx2 locus by homologous recombination [24]. In this configuration, the eGFP element allowed us to monitor Gbx2 expression at the time of analysis by GFP whole mount fluorescence or by GFP antibody labeling of sections. Thus, we operationally defined Gbx2-expressing cells as being Gbx2(GFP)+. We validated that GFP accurately reflected endogenous Gbx2 expression in the spinal cord by comparing anti-GFP antibody labeling to in situ hybridization with a labeled RNA probe specific to Gbx2 on adjacent transverse sections from embryonic (E)8.5–E12.5 Gbx2CreER-ires-eGFP/+ embryos (Figures 1 and S1). The CreERT2 element [28], indicated as CreER, in the Gbx2CreER-ires-eGFP allele allowed us to perform GIFM [27], [29] (Figure S2). GIFM and tamoxifen administration indelibly marked the Gbx2 lineage at distinct time points. With this approach and molecular marker analysis, we fate mapped and tracked the Gbx2-derived progeny, assessed their current state of Gbx2 expression, and determined their molecular identity during development and in the adult. We also took advantage of this line to determine the functional requirement of Gbx2 in spinal cord development by analyzing Gbx2CreER-ires-eGFP/CreER-ires-eGFP homozygous mutant embryos.


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

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

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).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3116860&req=5

pone-0020940-g001: 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).
Mentions: To study Gbx2 expression, lineage contribution, and function we utilized the mouse line, Gbx2CreER-ires-eGFP that was generated by targeting CreERT2-ires-eGFP to the 5′ untranslated region of Exon 1 in the Gbx2 locus by homologous recombination [24]. In this configuration, the eGFP element allowed us to monitor Gbx2 expression at the time of analysis by GFP whole mount fluorescence or by GFP antibody labeling of sections. Thus, we operationally defined Gbx2-expressing cells as being Gbx2(GFP)+. We validated that GFP accurately reflected endogenous Gbx2 expression in the spinal cord by comparing anti-GFP antibody labeling to in situ hybridization with a labeled RNA probe specific to Gbx2 on adjacent transverse sections from embryonic (E)8.5–E12.5 Gbx2CreER-ires-eGFP/+ embryos (Figures 1 and S1). The CreERT2 element [28], indicated as CreER, in the Gbx2CreER-ires-eGFP allele allowed us to perform GIFM [27], [29] (Figure S2). GIFM and tamoxifen administration indelibly marked the Gbx2 lineage at distinct time points. With this approach and molecular marker analysis, we fate mapped and tracked the Gbx2-derived progeny, assessed their current state of Gbx2 expression, and determined their molecular identity during development and in the adult. We also took advantage of this line to determine the functional requirement of Gbx2 in spinal cord development by analyzing Gbx2CreER-ires-eGFP/CreER-ires-eGFP homozygous mutant embryos.

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