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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

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).
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pone-0020940-g007: 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).

Mentions: To more fully assess the extent of the patterning defect in spinal cord progenitors, we first determined the total number of molecularly defined progenitors in hemi-transverse spinal cord (n = 3 controls, n = 4 mutants). There were no significant differences of the total number of indicated progenitors between controls (Figure 7A–F) and mutants (Figure 7G–L); see Quantitative approaches in Material and Methods for counts). We then generated quantitative spatial maps of the progenitors in control versus Gbx2 mutants to assess the distribution of mutant progenitors (See Materials and Methods and Figure 7A–L). Our Cartesian coordinate system was comprised of 4 M-L columns (ML1–ML4, most medial to most lateral, respectively) and D-V rows (DV1–DV10, most dorsal to most ventral, respectively) (Figure 7M–R). In Gbx2 mutant embryos at E10.5, there was a reduction of Brn3a+ progenitors along the D-V extent of the off-midline column (ML2), with the largest loss in dorsal ML2 (p<0.05), and an increase in Brn3a+ progenitors in ML3 (Figure 7A,G). We also observed an increase in Brn3a+ progenitors in the ventral half of the mutant spinal cord with the most prominent increase in ML3-DV5 to ML3-DV9 (Figure 7A,G). In control embryos, Isl1/2+ progenitors were primarily located in the MMC (ventral ML2) and LMC (ventral ML3) (Figure 7B). In Gbx2 mutants, there was a significant depletion of Isl1/2+ progenitors in the MMC (p<0.05) (Figure 7H) and a significant increase of Lim1+ progenitors in MMC and LMC (Figure 7C,I). Double immunolabeling revealed a subtle increase Brn3a+/Isl1/2+ progenitors in domains DV3–DV5 in column ML3 in mutants versus controls and a reduction in Brn3a+/Isl1/2+ progenitors in MMC (p<0.05) (Figure 7D,J). Brn3a+/Lim1+ and Pax2+ cells were not significantly different between controls and mutants. Finally, there was a significant depletion of pHH3+ cells in mutant versus control (p<0.05) spinal cords (figure 7F,L). The decrease was seen across the extent of the cord with the largest loss observed in the dorsal half (DV1–DV4) of the spinal cord with an emphasis on the dorsal-lateral cord (ML4-DV2) (Figure 7F,L). This finding and the observation that the spinal cord was not overtly depleted of neurons suggests that proliferating cells prematurely exited the cell cycle and contributed to the general patterning defect resulting from Gbx2 loss.


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

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

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).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3116860&req=5

pone-0020940-g007: 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).
Mentions: To more fully assess the extent of the patterning defect in spinal cord progenitors, we first determined the total number of molecularly defined progenitors in hemi-transverse spinal cord (n = 3 controls, n = 4 mutants). There were no significant differences of the total number of indicated progenitors between controls (Figure 7A–F) and mutants (Figure 7G–L); see Quantitative approaches in Material and Methods for counts). We then generated quantitative spatial maps of the progenitors in control versus Gbx2 mutants to assess the distribution of mutant progenitors (See Materials and Methods and Figure 7A–L). Our Cartesian coordinate system was comprised of 4 M-L columns (ML1–ML4, most medial to most lateral, respectively) and D-V rows (DV1–DV10, most dorsal to most ventral, respectively) (Figure 7M–R). In Gbx2 mutant embryos at E10.5, there was a reduction of Brn3a+ progenitors along the D-V extent of the off-midline column (ML2), with the largest loss in dorsal ML2 (p<0.05), and an increase in Brn3a+ progenitors in ML3 (Figure 7A,G). We also observed an increase in Brn3a+ progenitors in the ventral half of the mutant spinal cord with the most prominent increase in ML3-DV5 to ML3-DV9 (Figure 7A,G). In control embryos, Isl1/2+ progenitors were primarily located in the MMC (ventral ML2) and LMC (ventral ML3) (Figure 7B). In Gbx2 mutants, there was a significant depletion of Isl1/2+ progenitors in the MMC (p<0.05) (Figure 7H) and a significant increase of Lim1+ progenitors in MMC and LMC (Figure 7C,I). Double immunolabeling revealed a subtle increase Brn3a+/Isl1/2+ progenitors in domains DV3–DV5 in column ML3 in mutants versus controls and a reduction in Brn3a+/Isl1/2+ progenitors in MMC (p<0.05) (Figure 7D,J). Brn3a+/Lim1+ and Pax2+ cells were not significantly different between controls and mutants. Finally, there was a significant depletion of pHH3+ cells in mutant versus control (p<0.05) spinal cords (figure 7F,L). The decrease was seen across the extent of the cord with the largest loss observed in the dorsal half (DV1–DV4) of the spinal cord with an emphasis on the dorsal-lateral cord (ML4-DV2) (Figure 7F,L). This finding and the observation that the spinal cord was not overtly depleted of neurons suggests that proliferating cells prematurely exited the cell cycle and contributed to the general patterning defect resulting from Gbx2 loss.

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