CC BY
25
Section MOLECULAR NEUROSCIENCE
MOLECULAR NEUROSCIENCE
Lineage Tracing of Cortical Progenitors by Long-Term Live Imaging
R.Storm*, T. Schaub, V. Tarabykin
Charité Berlin, Berlin, Germany. Presenting e-mail: [email protected]
During development of the six-layered mammalian cortex, genereation of the different excitatory pyramidal neuron subtypes proceeds sequentially with deep layer (DL) neurons being produced first, followed by upper layer (UL) neuronsl. Nevertheless, during mid-neurogenesis, both DL -and UL neuron types are produced simultaneously but their lineage relationship is yet unclear.
Aims
To investigate the lineage relationship of DL - and UL subtypes, we set out to visualize and monitor single progentiors in primary cortical cultures over time and determine DL -and UL marker expression of their progeny.
Methods
GFP expressing cells derived from dissociated cortices of E12.5 to E13.5 RosaGFP mice were mixed and cultured with an excess of isocronic wild-type cells. In an alternative approach progenitors were targeted by ex-utero electroporation to exclusively induce stable expression of fluorescent markers in progenitors and their offspring. Subsequently, cortical cultures containing single targeted progenitors among an excess of unlabeled cells were subjected to spinning disk con-focal live imaging for five days. Cultures were fixed and the expression of Ctip2 (DL marker) and Satb2 (UL marker) was analyzed by immunohistochemistry to elucidate clonal composition.
Results
Contrary to culture of single isolated progenitors2, our mixed cortical cultures yielded substantial amounts of UL type neurons in the time frame analyzed. Clonal size ranged from 2 up to 40 cells. The majority of clones contained neurons expressing either Ctip2 or Satb2. A proportion of clones included neurons that expressed no marker or were weakly positive for both, Ctip2 or Satb2. Interestingly, no two-cell clones consisting of both, a DL -and UL type neuron were found.
Conclusions
Long-term live imaging of primary cortical cultures containing single genetically labeled progenitors represents a valuable method to investigate cortical progenitor output. Our results suggest that individual progenitors preferentially produce either DL -or UL subtypes during mid-neurogenesis.
References
1. J. B. ANGEVINE, & R. L. SIDMAN, Nature 1961, 192, 766-768.
2. Q. SHEN et al., Nature Neuroscience 2006, 9 (6), 743-751.
Transcriptional Control of Cortical Pyramidal Neuron Differentiation and Axonal Growth by Neurod 1/2/6
I. Bormuth*, K. Yan, O. Grishinaand V. Tarabykin
Institute of Cell Biology and Neurobiology, Charité - Medical University of Berlin, Germany. * Presenting e-mail: [email protected]
18 Opera Med Physiol 2016 Vol. 2 (S1)
