Cerebral organoids derived from mouse Pax6-/- ES cells recapitulate key developmental phenotypes of Pax6-/ mutant mouse forebrain

1. Centre for Integrative Physiology, Hugh Robson Building, University of Edinburgh, Edinburgh EH8 9XD, UK.
2. MRC Centre for Regenerative Medicine-Institute for Stem Cell Research, School of Biological Sciences, 5 Little France Drive, University of Edinburgh, Edinburgh EH16 4UU, UK.

The transcription factor Pax6 is a key regulator of embryonic forebrain development in both mice and humans1. It is expressed in cortical progenitor cells, and, among other activities, it maintains the cortical progenitor pool via controlling cell proliferation, in part by directly repressing Cdk62. Most studies of embryonic forebrain development have used animal models, particularly the mouse. The recent description of cerebral organoids, stem cell-derived structures that display the three-dimensional architecture and physiology of forebrain tissue, offers the potential to model spatial and temporal aspects of cortical development in a more accessible environment, in vitro3.

We aimed to determine the extent to which cerebral organoids accurately recapitulate early cortical development by comparing the structure of Pax6-/- mutant mouse cerebral organoids to that of Pax6-/- embryos. We used a previously established Pax6-/- mouse embryonic stem (ES) cell line4 to generate cerebral organoids using an established protocol5. Organoids were collected at day 8 and day 9 and analysed by immunohistochemistry.

We found that both mutant (Pax6-/-) and control (Pax6+/+) mouse organoids formed N-cadherin-expressing neuroepithelium (NE) with the apical side organized around lumen-like structures. Organoid neuroepithelium showed clear spatial separation of progenitor (Sox2+) and neuronal (Tuj1+) layers. Pax6-/- mutant organoids contained a higher proportion of Tuj1+ cells at later stages - the neuronal layer became thicker with a concurrent decrease in the progenitor layer, suggesting precocious neural differentiation. Basal progenitor cells (Tbr2+) and deep layer neurons (Tbr1+) were present in both wild type and mutant organoids, organized in the same way as is found in vivo. However, Pax6-/- organoids contained a substantially lower proportion of Tbr2+ cells. Interestingly, both precocious neuronal differentiation and reduced Tbr2+ cells are characteristic phenotypes of Pax6-/- mouse embryonic cortex at E12.5, indicating that Pax6-/- cerebral organoids successfully recapitulate key phenotypes of Pax6-/- mutant embryos.

References

1. Ypsilanti, A. R. & Rubenstein, J. L. R. Transcriptional and epigenetic mechanisms of early cortical development: An examination of how Pax6 coordinates cortical development. J. Comp. Neurol. 524, 609–29 (2016).
2. Mi, D. et al. Pax6 Exerts regional control of cortical progenitor proliferation via direct repression of Cdk6 and Hypophosphorylation of pRb. Neuron 78, 269–284 (2013).
3. Lancaster, M. a et al. Cerebral organoids model human brain development and microcephaly. Nature 501, 373–9 (2012).
4. Quinn, J. C., Molinek, M., Nowakowski, T. J., Mason, J. O. & Price, D. J. Novel lines of Pax6-/- embryonic stem cells exhibit reduced neurogenic capacity without loss of viability. BMC Neurosci. 11, 26 (2010).
5. Nasu, M. et al. Robust Formation and Maintenance of Continuous Stratified Cortical Neuroepithelium by Laminin-Containing Matrix in Mouse ES Cell Culture. PLoS One 7, 13–14 (2012).

Funded by:  Ministry of Higher Education, Malaysia (Grant G32486)

* entered into the PhD student poster competition