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. 2009 Jan 5;4:2.
doi: 10.1186/1749-8104-4-2.

Heterogeneity in the Developmental Potential of Motor Neuron Progenitors Revealed by Clonal Analysis of Single Cells in Vitro

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Free PMC article

Heterogeneity in the Developmental Potential of Motor Neuron Progenitors Revealed by Clonal Analysis of Single Cells in Vitro

Dritan Agalliu et al. Neural Dev. .
Free PMC article

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.

Figures

Figure 1
Figure 1
Generation of Nkx6.1::IRES::eGFP mice and embryonic expression of eGFP in the spinal cord. (A) Targeting strategy for the generation of Nkx6.1::IRES::eGFP knock-in mice. (B) Southern blotting to determine wild type (12.5 kb) and targeted (15.5 kb) alleles at the Nkx6.1 locus. (C-F) Expression of Sox3, Nkx6.1, eGFP and Isl1/2 proteins in the e9.5 neural tube of Nkx6.1::IRES::eGFP+/- mice. Nkx6.1 and eGFP proteins are expressed in Sox3+ progenitors (C-E). eGFP is also expressed by MNs (F; ventral Isl1/2+ cells). (G-J) eGFP is expressed in Nkx6.1+ neural progenitors (G, H) and in mature ventral neuronal subtypes such as V2a interneurons (I; Chx10+ cells), and MNs (J; ventral Isl1/2+ cells) at e10.5.
Figure 2
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.
Figure 3
Figure 3
Sorted Nkx6.1+ progenitors that immediately differentiate in vitro generate the correct ventral neuronal subtypes. (A) Schematic diagram of the fluorescence-activated cell sorting (FACS), isolation and culture of Nkx6.1+ spinal progenitors. (B-G) Immunohistochemical analysis of neurons that differentiate in vitro without proliferation using Chx10 (V2a interneurons; B-C); Isl1/2 (MNs; D-E); and Nkx2.2 (V3 interneurons; F-G) antibodies. Tuj1 stains the neuron-specific βIII tubulin. (H, I) No Lim1/2+ neurons were present in cultures from sorted eGFP+ cells, although these neurons were born from sorted eGFP-negative progenitors (J, K). (L) Frequencies of three different neuronal subtypes generated in vitro from sorted Nkx6.1+ precursors. Bars represent mean ± s.e.m (n = 16 wells from 3 experiments).
Figure 4
Figure 4
Ventral subtype-restricted progenitors are present at low frequencies in the spinal cord. (A) Diagram of sorted Nkx6.1+ progenitor fates after culture for several days. (B-D) Immunohistochemical analysis for Sox3 and Nkx6.1 in clones derived from single Nkx6.1+ proliferating progenitors. The progenitor state of the cell is revealed by Sox3 (green). Negative clones have no expression of Nkx6.1 (B), patchy clones have some cells that express Nkx6.1 (C; yellow cells) and positive clones have more than 95% of cells that express Nkx6.1 (D). (E) Fractions of three different types of clones observed in cultures from sorted e9.0 (red), e9.5 (green) or e10.0 (blue) Nkx6.1+ progenitors. (n = 420 clones from 6 experiments at e9.5; n = 234 clones at e9.0; and n = 124 clones at e10.0 from 3 experiments for the latter two time points). (F) Fraction of eGFP+ cells that forms clones in culture at three developmental stages. (G) Frequency plot for the three classes of subtype-restricted clones isolated from Nkx6.1+ progenitors (n = 52 Nkx6.1+ clones). Bars represent mean ± s.e.m in all plots. (H-P) Expression of Nkx6.1, Olig2 and Nkx2.2 in subtype-restricted progenitors derived from a presumed p2 (H-J), pMN (K-M) or p3 (N-P) progenitor. No clones displayed a mixture of Olig2+ and Nkx2.2+ progenitors.
Figure 5
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.
Figure 6
Figure 6
pMN-restricted and pMN-patchy clones acquire thoracic identity after in vitro expansion, regardless of their origin of isolation. (A, M) Contour plots of dissociated cells from e9.5 brachial (A) or thoracic (M) trunks from Nkx6.1::IRES::eGFP+/- mice. (B, C) Immunohistochemistry with HoxC6 (B) and Hb9 (red) and Tuj1 (green) (C) of MNs born from non-proliferating brachial Nkx6.1+ progenitors. These motor neurons express low levels of HoxC6. (D-I) Immunohistochemistry for HoxC6 (D) or HoxC9 (G) of brachial pMN-restricted clones that contain either Hb9+Tuj1+ (E, F) or Isl1/2+Tuj1+ (H, I) neurons. Brachial-derived pMN-restricted clones lose expression of HoxC6 and now express HoxC9. (J-L) Brachial-derived pMN-patchy clones express HoxC9 (J) even in neurons that have lost their ventral identity (white arrows, K, L). (N, O) Immunohistochemistry with HoxC9 (N) and Isl1/2 (red) and Tuj1 (green) (O) of MNs born from non-proliferating thoracic Nkx6.1+ progenitors. These MNs express HoxC9. (P-U) Immunohistochemistry for HoxC9 (P) or HoxD10 (S) of thoracic pMN-restricted clones containing Isl1/2+ Tuj1+ neurons. These neurons do not express HoxD10, a marker of lumbar MNs. (V-X) Immunohistochemistry for HoxC9 (V) of thoracic pMN-patchy clones that express Isl1/2 (W, X; red) in a subset of Tuj1+ neurons (X; green). Thoracic-derived pMN-patchy clones (white arrows) express HoxC9 even in neurons that have lost their ventral identity.
Figure 7
Figure 7
Clones derived from a pMN-restricted progenitor are not committed to the motor neuron fate. (A) Diagram of the experimental strategy used to test the commitment of pMN-restricted clones. (B-G) Staining of pMN-restricted clones for Nkx6.1 (B, E), Olig2 (C, F) and merge panels with Nkx6.1 (red), Olig2 (blue), and eGFP (green) (D, G). Nkx6.1 and Olig2 label motor neuron progenitors in control (B-D) and BMP4-treated (E-G) clones. Both Nkx6.1 and Olig2 are downregulated in BMP4-treated motor neurons clones. eGFP labels MNs. (H-J) Staining of MNs for Isl1/2 and eGFP in control (H) and BMP4-treated clones (I, J). Several Isl1/2+ Tuj1+ neurons are not labeled with eGFP in BMP4-treated cultures (I, J; white arrows). (K-M) Immunhistochemistry for Brn3a (K), Isl1/2 (L) and merge panel (M) of BMP4-treated MN clones. Some Isl1/2+ neurons also express Brn3a (M; yellow cells, white arrow).

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