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

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. 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. 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