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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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Clones derived from subtype-restricted ventral progenitors generate appropriate neuronal subtypes in vitro. (A-I) Immunohistochemical analysis of neuronal subtypes present in clones of ventral restricted progenitors with Chx10 (A), Hb9 (D), Isl1/2 (B, E, H) and Nkx2.2 (G) and Tuj1; (C, F, I) are merged panels with Tuj1 to label neurons. (J) Vertical dot plot of the number of neurons present in each neuronal subtype restricted clone: Chx10+ V2a interneurons (n = 11 clones), Hb9+ and Isl1/2+ MNs (n = 45 clones) and Nkx2.2+ V3 interneurons (n = 8 clones). (K-P) Representative images (bright phase and eGFP) of four- (K, L), six- (M, N) and eight- (O, P) day-old MN clones generated from a pMN-restricted progenitor derived from Hb9::eGFP transgenic mice. (Q) Vertical dot plot of neuronal number present in pMN-restricted clones from Hb9::eGFP mice after four (n = 25 clones), six (n = 32 clones) and eight (n = 45 clones) DIV. The percentage represents the fraction of clones that had more neurons than those counted two days before.
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Figure 5: Clones derived from subtype-restricted ventral progenitors generate appropriate neuronal subtypes in vitro. (A-I) Immunohistochemical analysis of neuronal subtypes present in clones of ventral restricted progenitors with Chx10 (A), Hb9 (D), Isl1/2 (B, E, H) and Nkx2.2 (G) and Tuj1; (C, F, I) are merged panels with Tuj1 to label neurons. (J) Vertical dot plot of the number of neurons present in each neuronal subtype restricted clone: Chx10+ V2a interneurons (n = 11 clones), Hb9+ and Isl1/2+ MNs (n = 45 clones) and Nkx2.2+ V3 interneurons (n = 8 clones). (K-P) Representative images (bright phase and eGFP) of four- (K, L), six- (M, N) and eight- (O, P) day-old MN clones generated from a pMN-restricted progenitor derived from Hb9::eGFP transgenic mice. (Q) Vertical dot plot of neuronal number present in pMN-restricted clones from Hb9::eGFP mice after four (n = 25 clones), six (n = 32 clones) and eight (n = 45 clones) DIV. The percentage represents the fraction of clones that had more neurons than those counted two days before.

Mentions: Next, we analyzed the molecular identity of progenitors and neuronal subtypes within the Nkx6.1-positive clones. These clones were further segregated into three distinct types that expressed either the p3 domain marker Nkx2.2 (Figure 4N–P), the pMN domain marker Olig2 (Figure 4K–M) or neither of these markers (Figure 4H–J) in all progenitors. The third type of clone did not express markers characteristic of more dorsal progenitors such as Nkx6.2, Dbx1/2, or Pax7 (data not shown). We did not find any clones that expressed both Nkx6.1 and Irx3, which is characteristic of the p2 domain (data not shown) [20], nor mixed clones where progenitors expressed markers from two ventral domains. We then asked if the molecular identities of neurons present in these three types of Nkx6.1-positive clones correlated with those of neurons born from these three domains in the spinal cord. We found that the p3 domain clones contained exclusively Nkx2.2-expressing Tuj1+ neurons, but not Isl1/2+ or Hb9+ neurons (Figure 5G–I). In contrast, clones derived from a pMN progenitor had only Hb9+ and Isl1/2+ MNs (Figure 5D–F). The third type of clone, expressing only Nkx6.1 but not Irx3 contained Chx10+ neurons, but not Isl1/2+ neurons (Figure 5A–C) and was, therefore, most similar to a V2a interneuron identity [35]. Based on these findings, we presume that the third class is derived from p2 progenitors that have lost expression of Irx3 during proliferation, which does not affect the generation of V2a neurons in vivo [36]. In addition, we did not find neurons with mixed subtype identities in any Nkx6.1-positive clones that we examined during this analysis. Therefore, there is a complete match between progenitor and neuronal subtype identity within the Nkx6.1-positive clones, indicating that they were derived from lineage-restricted progenitors that are present in the three domains that express Nkx6.1. These three different clone types were present at distinct frequencies, with MN clones being the most frequent (approximately 60%), followed by V2 clones (approximately 22%) and V3 clones (approximately 18%) (Figure 4G). These proportions correspond to the initial abundance of each sorted precursor type (Figure 2K). Therefore, based on the molecular characterization of progenitors and neuronal subtypes, as well as the proportion of clones belonging to each lineage, we conclude that lineage-restricted progenitor subtypes are present in each progenitor domain of the Nkx6.1+ region in the spinal cord, although at a lower frequency than expected assuming that progenitors derived from the same domain are homogeneous at the time of isolation.


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)

Clones derived from subtype-restricted ventral progenitors generate appropriate neuronal subtypes in vitro. (A-I) Immunohistochemical analysis of neuronal subtypes present in clones of ventral restricted progenitors with Chx10 (A), Hb9 (D), Isl1/2 (B, E, H) and Nkx2.2 (G) and Tuj1; (C, F, I) are merged panels with Tuj1 to label neurons. (J) Vertical dot plot of the number of neurons present in each neuronal subtype restricted clone: Chx10+ V2a interneurons (n = 11 clones), Hb9+ and Isl1/2+ MNs (n = 45 clones) and Nkx2.2+ V3 interneurons (n = 8 clones). (K-P) Representative images (bright phase and eGFP) of four- (K, L), six- (M, N) and eight- (O, P) day-old MN clones generated from a pMN-restricted progenitor derived from Hb9::eGFP transgenic mice. (Q) Vertical dot plot of neuronal number present in pMN-restricted clones from Hb9::eGFP mice after four (n = 25 clones), six (n = 32 clones) and eight (n = 45 clones) DIV. The percentage represents the fraction of clones that had more neurons than those counted two days before.
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Figure 5: Clones derived from subtype-restricted ventral progenitors generate appropriate neuronal subtypes in vitro. (A-I) Immunohistochemical analysis of neuronal subtypes present in clones of ventral restricted progenitors with Chx10 (A), Hb9 (D), Isl1/2 (B, E, H) and Nkx2.2 (G) and Tuj1; (C, F, I) are merged panels with Tuj1 to label neurons. (J) Vertical dot plot of the number of neurons present in each neuronal subtype restricted clone: Chx10+ V2a interneurons (n = 11 clones), Hb9+ and Isl1/2+ MNs (n = 45 clones) and Nkx2.2+ V3 interneurons (n = 8 clones). (K-P) Representative images (bright phase and eGFP) of four- (K, L), six- (M, N) and eight- (O, P) day-old MN clones generated from a pMN-restricted progenitor derived from Hb9::eGFP transgenic mice. (Q) Vertical dot plot of neuronal number present in pMN-restricted clones from Hb9::eGFP mice after four (n = 25 clones), six (n = 32 clones) and eight (n = 45 clones) DIV. The percentage represents the fraction of clones that had more neurons than those counted two days before.
Mentions: Next, we analyzed the molecular identity of progenitors and neuronal subtypes within the Nkx6.1-positive clones. These clones were further segregated into three distinct types that expressed either the p3 domain marker Nkx2.2 (Figure 4N–P), the pMN domain marker Olig2 (Figure 4K–M) or neither of these markers (Figure 4H–J) in all progenitors. The third type of clone did not express markers characteristic of more dorsal progenitors such as Nkx6.2, Dbx1/2, or Pax7 (data not shown). We did not find any clones that expressed both Nkx6.1 and Irx3, which is characteristic of the p2 domain (data not shown) [20], nor mixed clones where progenitors expressed markers from two ventral domains. We then asked if the molecular identities of neurons present in these three types of Nkx6.1-positive clones correlated with those of neurons born from these three domains in the spinal cord. We found that the p3 domain clones contained exclusively Nkx2.2-expressing Tuj1+ neurons, but not Isl1/2+ or Hb9+ neurons (Figure 5G–I). In contrast, clones derived from a pMN progenitor had only Hb9+ and Isl1/2+ MNs (Figure 5D–F). The third type of clone, expressing only Nkx6.1 but not Irx3 contained Chx10+ neurons, but not Isl1/2+ neurons (Figure 5A–C) and was, therefore, most similar to a V2a interneuron identity [35]. Based on these findings, we presume that the third class is derived from p2 progenitors that have lost expression of Irx3 during proliferation, which does not affect the generation of V2a neurons in vivo [36]. In addition, we did not find neurons with mixed subtype identities in any Nkx6.1-positive clones that we examined during this analysis. Therefore, there is a complete match between progenitor and neuronal subtype identity within the Nkx6.1-positive clones, indicating that they were derived from lineage-restricted progenitors that are present in the three domains that express Nkx6.1. These three different clone types were present at distinct frequencies, with MN clones being the most frequent (approximately 60%), followed by V2 clones (approximately 22%) and V3 clones (approximately 18%) (Figure 4G). These proportions correspond to the initial abundance of each sorted precursor type (Figure 2K). Therefore, based on the molecular characterization of progenitors and neuronal subtypes, as well as the proportion of clones belonging to each lineage, we conclude that lineage-restricted progenitor subtypes are present in each progenitor domain of the Nkx6.1+ region in the spinal cord, although at a lower frequency than expected assuming that progenitors derived from the same domain are homogeneous at the time of isolation.

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