CC BY
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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: robert.storm@charite.de
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: ingo.bormuth@charite.de
Section MOLECULAR NEUROSCIENCE Aims
The closely related neuronal bHLH transcription factors Neurod1, Neurod2 and Neurod6 are expressed by differentiating pyramidal neurons in the developing cere-bral cortex and have long been suspected to regulate the maturation of these cells. Each of the three genes was genetically inactivated in mice, but studies of single-deficient animals failed to identify important functions in embryonic pyramidal neu-rons. High sequence similarity and overlapping expression patterns suggest func-tional redundancy. We used double and triple deficient mice to identify NeuroD-family dependent functions and downstream molecular mechanisms.
Methods
We bred Neurod2/6 double-deficient and Neurod1/2/6 triple deficient mice and analyzed cerebral cortex development with an emphasis on pyramidal neuron identity and neocortical connectivity. We use in situ hybridization to visualize and in utero electroporation to manipulate the expression of possible target genes in the cerebral cortex.
Results
Neurod2 and Neurod6 indeed share several hitherto unknown functions and compensate for each other's loss. At least one of the two genes is necessary for: (1) the control of radial migration in a subset of pyramidal neurons; (2) area determination in the neocortex; and (3) the formation of fiber tracts connecting the neocortex to the striatum, to the thalamus, and to the contralateral hemisphere. In Neurod2/6 double-deficient mice, callosal axons form fasciculated fiber bundles that grow tan-gentially into the medial neocortex, but stall and defasciculate before reaching the ipsilateral cingulum or any midline associated structure. This new variant of callosal agenesis implies the presence of a so far identified axon guidance mechanism in the medial neocortex. We present EphrinA signaling as possible mechanism. Neurod1 shares additional functions with Neurod2 and Neurod6. At least one of the three genes is necessary for hippocampal pyramidal neuron differentiation and the prevention of developmental cell death in the medial cortex. While the simulta-neous inactivation of Neurod1/2/6 results in the complete loss of archicortical pyramidal neurons, many neocortical pyramidal cells survive, migrate radially and settle in the cortical plate. However, terminal pyramidal neuron differentiation is incomplete and neocortical connectivity is dramatically reduced in triple-deficient mice.
Conclusion
NeuroD-family transcription factors cooperatively regulate pyramidal neuron differentiation, survival, migration, specification and axon growth in the developing cerebral cortex.
References
1. I. Bormuth et al., J Neurosci., 2013, 33(2), 641-51.
Defining the Role of Ciliary Proteins BBS (Bardet-Biedl Syndrome) in Neuroplasticity
Sophia Christou-Savina*
London's Global University, UK. * Presenting e-mail: s.christou-savina@ucl.ac.uk
Bardet-Biedl syndrome (BBS) is a genetically heterogeneous, autosomal recessive disorder characterised by early-onset retinal degeneration, obesity, polydactyly, and renal malformation/dysfunction. It affects approximately 1:100,000 people in Northern Europe reaching 1:13,500 in more isolated populations. More than half of all individuals with BBS also experience developmental disabilities ranging from mild learning impairment to severe mental retardation independent of gene mutation. Many patients display obsessive/compulsive tendencies and a preference for fixed routines and 37% of children attending our national BBS clinics have autism spectrum disorder. Poor memory and cognition in Bardet-Biedl syndrome lead to inability to live independently and few are in employment (9%).
OM&P
