Abstract
BackgroundThe 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.ResultsWe 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.ConclusionThese 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.
Highlights
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
A small subset of progenitors, including pMN progenitors, is able to maintain its identity, suggesting that subtype-restricted progenitors from the Nkx6.1+ region are present in the ventral spinal cord, at a lower frequency than expected from the apparently uniform expression of transcription factors within these progenitors
We determined the purity and molecular profile of sorted enhanced green fluorescent protein (eGFP)+ cells after fluorescence-activated cell sorting (FACS) purification by analyzing the expression of various transcription factors that are normally found within the Nkx6.1+ region
Summary
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. Neural Development 2009, 4:2 http://www.neuraldevelopment.com/content/4/1/2 nals that initiate cell fate decisions by regulating expression of transcription factors These transcription factors, in turn, impose developmental restrictions on multipotent progenitor cells before effecting their final differentiation [1,2,3]. Clonal analysis has revealed the importance of cell-intrinsic mechanisms for regulating progenitor cell fate decisions, the degree to which the intrinsic program can be modified by extrinsic cues has not been rigorously tested because the identities of the inductive signals as well as molecular markers for distinct progenitors are poorly defined
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