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Heterogeneity in the developmental potential of motor neuron progenitors revealed by clonal analysis of single cells in vitro.

Agalliu D, Schieren I - Neural Dev (2009)

Bottom Line: Thus, subtype-restricted progenitors from the Nkx6.1+ region are present in the ventral spinal cord, although at a lower frequency than expected.These findings support a model whereby continuous Shh signaling is required to maintain the identity of ventral progenitors isolated from the spinal cord, including motor neuron progenitors, after in vitro expansion.They also demonstrate that pre-patterned neural progenitors isolated from the central nervous system can change their regional identity in vitro to acquire a broader developmental potential.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, Columbia University Medical Center, New York, NY 10032, USA. dagalliu@stanford.edu

ABSTRACT

Background: The differentiation of neural progenitors into distinct classes within the central nervous system occurs over an extended period during which cells become progressively restricted in their fates. In the developing spinal cord, Sonic Hedgehog (Shh) controls neural fates in a concentration-dependent manner by establishing discrete ventral progenitor domains characterized by specific combinations of transcription factors. It is unclear whether motor neuron progenitors can maintain their identities when expanded in vitro and whether their developmental potentials are restricted when exposed to defined extracellular signals.

Results: We have generated mice expressing the enhanced green fluorescent protein under the control of the Nkx6.1 promoter, enabling fluorescence-activated cell sorting (FACS), purification and culture of individual spinal progenitors at clonal density, and analysis of their progeny. We demonstrate that cells isolated after progenitor domains are established are heterogeneous with respect to maintaining their identity after in vitro expansion. Most Nkx6.1+ progenitors lose their ventral identity following several divisions in culture, whereas a small subset is able to maintain its identity. Thus, subtype-restricted progenitors from the Nkx6.1+ region are present in the ventral spinal cord, although at a lower frequency than expected. Clones that maintain a motor neuron identity assume a transcriptional profile characteristic of thoracic motor neurons, despite some having been isolated from non-thoracic regions initially. Exposure of progenitors to Bone Morphogenetic Protein-4 induces some dorsal cell type characteristics in their progeny, revealing that lineage-restricted progenitor subtypes are not fully committed to their fates.

Conclusion: These findings support a model whereby continuous Shh signaling is required to maintain the identity of ventral progenitors isolated from the spinal cord, including motor neuron progenitors, after in vitro expansion. They also demonstrate that pre-patterned neural progenitors isolated from the central nervous system can change their regional identity in vitro to acquire a broader developmental potential.

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Sorted eGFP+ ventral progenitors from Nkx6.1::IRES::eGFP mice maintain regional identity markers immediately after plating. (A, B) Contour plots of dissociated cells from e9.5 trunks of wild-type (A) or Nkx6.1::IRES::eGFP+/- mice (B). The logarithmic scale of eGFP fluorescence is on the x-axis and cell size on the y-axis (forward scatter). (C-J) Immunohistochemical analysis of sorted eGFP+ cells after attachment (approximately 2 hours after plating) with antisera for eGFP (D) and transcription factors expressed in different ventral progenitor domains, such as Nkx6.1 (C, E, G, I), Olig2 (H), Irx3 (F) and Nkx2.2 (J). Arrows point to progenitors from the p2 or p3 domains. (K) Proportion of sorted eGFP+Nkx6.1+ progenitors that express the pMN, p2, p3 or floor plate markers 2 hours after plating. (n = 20 wells from 2 experiments). (L) Proportions of three progenitor populations and floor plate within the Nkx6.1+ domain of e9.5 neural tube from 6 sections of brachial and thoracic segments (n = 3 animals). Note that the motor neuron progenitor population (pMN) is the most abundant. Bars represent mean ± s.e.m in all plots.
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Figure 2: Sorted eGFP+ ventral progenitors from Nkx6.1::IRES::eGFP mice maintain regional identity markers immediately after plating. (A, B) Contour plots of dissociated cells from e9.5 trunks of wild-type (A) or Nkx6.1::IRES::eGFP+/- mice (B). The logarithmic scale of eGFP fluorescence is on the x-axis and cell size on the y-axis (forward scatter). (C-J) Immunohistochemical analysis of sorted eGFP+ cells after attachment (approximately 2 hours after plating) with antisera for eGFP (D) and transcription factors expressed in different ventral progenitor domains, such as Nkx6.1 (C, E, G, I), Olig2 (H), Irx3 (F) and Nkx2.2 (J). Arrows point to progenitors from the p2 or p3 domains. (K) Proportion of sorted eGFP+Nkx6.1+ progenitors that express the pMN, p2, p3 or floor plate markers 2 hours after plating. (n = 20 wells from 2 experiments). (L) Proportions of three progenitor populations and floor plate within the Nkx6.1+ domain of e9.5 neural tube from 6 sections of brachial and thoracic segments (n = 3 animals). Note that the motor neuron progenitor population (pMN) is the most abundant. Bars represent mean ± s.e.m in all plots.

Mentions: We determined the purity and molecular profile of sorted eGFP+ cells after FACS purification by analyzing the expression of various transcription factors that are normally found within the Nkx6.1+ region. We isolated eGFP+ cells from neural tubes and somites (trunks) at e9.5, when dorsoventral patterning of neural progenitors is established [20] and MN generation has begun [32]. We found that 14.6% of cells from e9.5 forelimb and thoracic regions expressed eGFP (Figure 2A,B). We analyzed the identities of these cells 2 hours after plating with the following markers: Irx3 (p2), Olig2 (pMN), Nkx2.2 (p3) and Hnf3β (floor plate). We found that 96% of sorted cells were eGFP+ and approximately 65% of the eGFP+ cells expressed the progenitor markers Sox3 and Nkx6.1 (Figure 2C,D; data not shown). The remaining 35% of the GFP+ cells were Hb9+ and Isl1/2+ MNs (data not shown), isolated due to the perdurance of eGFP protein in these cells. The majority of sorted Nkx6.1+ progenitors were pMN progenitors (approximately 58%) that expressed the bHLH protein Olig2 (Figure 2G,H,K). The rest of the progenitors fell into three classes: Irx3+ p2 progenitors (approximately 22%), Nkx2.2+ p3 progenitors (approximately 15%) and HNF3β+ floor plate cells (approximately 5%; Figure 2E–K; data not shown). Therefore, the majority of sorted eGFP+ cells were precursors (65%), of which pMN progenitors comprised the largest population (37%), followed by p2 (14%), and p3 (10%) progenitors (Figure 2K). The fractions of sorted progenitor subtypes were similar to the relative sizes of the p2, pMN and p3 domains within the Nkx6.1+ region in vivo (Figure 2L). Therefore, the progenitor populations derived from the Nkx6.1+ domain were faithfully represented in our culture system.


Heterogeneity in the developmental potential of motor neuron progenitors revealed by clonal analysis of single cells in vitro.

Agalliu D, Schieren I - Neural Dev (2009)

Sorted eGFP+ ventral progenitors from Nkx6.1::IRES::eGFP mice maintain regional identity markers immediately after plating. (A, B) Contour plots of dissociated cells from e9.5 trunks of wild-type (A) or Nkx6.1::IRES::eGFP+/- mice (B). The logarithmic scale of eGFP fluorescence is on the x-axis and cell size on the y-axis (forward scatter). (C-J) Immunohistochemical analysis of sorted eGFP+ cells after attachment (approximately 2 hours after plating) with antisera for eGFP (D) and transcription factors expressed in different ventral progenitor domains, such as Nkx6.1 (C, E, G, I), Olig2 (H), Irx3 (F) and Nkx2.2 (J). Arrows point to progenitors from the p2 or p3 domains. (K) Proportion of sorted eGFP+Nkx6.1+ progenitors that express the pMN, p2, p3 or floor plate markers 2 hours after plating. (n = 20 wells from 2 experiments). (L) Proportions of three progenitor populations and floor plate within the Nkx6.1+ domain of e9.5 neural tube from 6 sections of brachial and thoracic segments (n = 3 animals). Note that the motor neuron progenitor population (pMN) is the most abundant. Bars represent mean ± s.e.m in all plots.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 2: Sorted eGFP+ ventral progenitors from Nkx6.1::IRES::eGFP mice maintain regional identity markers immediately after plating. (A, B) Contour plots of dissociated cells from e9.5 trunks of wild-type (A) or Nkx6.1::IRES::eGFP+/- mice (B). The logarithmic scale of eGFP fluorescence is on the x-axis and cell size on the y-axis (forward scatter). (C-J) Immunohistochemical analysis of sorted eGFP+ cells after attachment (approximately 2 hours after plating) with antisera for eGFP (D) and transcription factors expressed in different ventral progenitor domains, such as Nkx6.1 (C, E, G, I), Olig2 (H), Irx3 (F) and Nkx2.2 (J). Arrows point to progenitors from the p2 or p3 domains. (K) Proportion of sorted eGFP+Nkx6.1+ progenitors that express the pMN, p2, p3 or floor plate markers 2 hours after plating. (n = 20 wells from 2 experiments). (L) Proportions of three progenitor populations and floor plate within the Nkx6.1+ domain of e9.5 neural tube from 6 sections of brachial and thoracic segments (n = 3 animals). Note that the motor neuron progenitor population (pMN) is the most abundant. Bars represent mean ± s.e.m in all plots.
Mentions: We determined the purity and molecular profile of sorted eGFP+ cells after FACS purification by analyzing the expression of various transcription factors that are normally found within the Nkx6.1+ region. We isolated eGFP+ cells from neural tubes and somites (trunks) at e9.5, when dorsoventral patterning of neural progenitors is established [20] and MN generation has begun [32]. We found that 14.6% of cells from e9.5 forelimb and thoracic regions expressed eGFP (Figure 2A,B). We analyzed the identities of these cells 2 hours after plating with the following markers: Irx3 (p2), Olig2 (pMN), Nkx2.2 (p3) and Hnf3β (floor plate). We found that 96% of sorted cells were eGFP+ and approximately 65% of the eGFP+ cells expressed the progenitor markers Sox3 and Nkx6.1 (Figure 2C,D; data not shown). The remaining 35% of the GFP+ cells were Hb9+ and Isl1/2+ MNs (data not shown), isolated due to the perdurance of eGFP protein in these cells. The majority of sorted Nkx6.1+ progenitors were pMN progenitors (approximately 58%) that expressed the bHLH protein Olig2 (Figure 2G,H,K). The rest of the progenitors fell into three classes: Irx3+ p2 progenitors (approximately 22%), Nkx2.2+ p3 progenitors (approximately 15%) and HNF3β+ floor plate cells (approximately 5%; Figure 2E–K; data not shown). Therefore, the majority of sorted eGFP+ cells were precursors (65%), of which pMN progenitors comprised the largest population (37%), followed by p2 (14%), and p3 (10%) progenitors (Figure 2K). The fractions of sorted progenitor subtypes were similar to the relative sizes of the p2, pMN and p3 domains within the Nkx6.1+ region in vivo (Figure 2L). Therefore, the progenitor populations derived from the Nkx6.1+ domain were faithfully represented in our culture system.

Bottom Line: Thus, subtype-restricted progenitors from the Nkx6.1+ region are present in the ventral spinal cord, although at a lower frequency than expected.These findings support a model whereby continuous Shh signaling is required to maintain the identity of ventral progenitors isolated from the spinal cord, including motor neuron progenitors, after in vitro expansion.They also demonstrate that pre-patterned neural progenitors isolated from the central nervous system can change their regional identity in vitro to acquire a broader developmental potential.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, Columbia University Medical Center, New York, NY 10032, USA. dagalliu@stanford.edu

ABSTRACT

Background: The differentiation of neural progenitors into distinct classes within the central nervous system occurs over an extended period during which cells become progressively restricted in their fates. In the developing spinal cord, Sonic Hedgehog (Shh) controls neural fates in a concentration-dependent manner by establishing discrete ventral progenitor domains characterized by specific combinations of transcription factors. It is unclear whether motor neuron progenitors can maintain their identities when expanded in vitro and whether their developmental potentials are restricted when exposed to defined extracellular signals.

Results: We have generated mice expressing the enhanced green fluorescent protein under the control of the Nkx6.1 promoter, enabling fluorescence-activated cell sorting (FACS), purification and culture of individual spinal progenitors at clonal density, and analysis of their progeny. We demonstrate that cells isolated after progenitor domains are established are heterogeneous with respect to maintaining their identity after in vitro expansion. Most Nkx6.1+ progenitors lose their ventral identity following several divisions in culture, whereas a small subset is able to maintain its identity. Thus, subtype-restricted progenitors from the Nkx6.1+ region are present in the ventral spinal cord, although at a lower frequency than expected. Clones that maintain a motor neuron identity assume a transcriptional profile characteristic of thoracic motor neurons, despite some having been isolated from non-thoracic regions initially. Exposure of progenitors to Bone Morphogenetic Protein-4 induces some dorsal cell type characteristics in their progeny, revealing that lineage-restricted progenitor subtypes are not fully committed to their fates.

Conclusion: These findings support a model whereby continuous Shh signaling is required to maintain the identity of ventral progenitors isolated from the spinal cord, including motor neuron progenitors, after in vitro expansion. They also demonstrate that pre-patterned neural progenitors isolated from the central nervous system can change their regional identity in vitro to acquire a broader developmental potential.

Show MeSH
Related in: MedlinePlus