A potent neuroepithelium-promoting activity of Otx2 during gastrulation as demonstrated by its exogenous epiblast-wide expression in chicken embryos

Despite the history of Otx2 research over three decades, the role of Otx2 expressed in the brain-forming anterior epiblast during gastrulation stages has not been clarified. We paid attention to the fact epiblast regions exist where Otx2 expression is absent or downregulated, namely the posterior epiblast and tissue belts along the neural plate bending. Developmental events that follow filling these expression gaps with exogenous Otx2 expression would be informative concerning the roles of Otx2 during gastrulation. The effects were pleiotropic: (1) Development of open and flat midbrain and hindbrain, which occurred via unzippering of once closed neural tube; (2) Precocious neural tube development at the cervical level that gave rise to thick and unclosed neural tissue; (3) The absence of the sinus rhomboidalis where NMP stem cells multiply to provide a cell source for the trunk posterior extension, resulting in the trunk posterior extension; (4) Inhibition of somite development. To elucidate principles underlying these developmental abnormalities, the cellular events were analyzed using live imaging of epiblasts, cell states were characterized via transcriptome analysis, and spatial organization of transcription factor expression was determined by immunohistology. Common denominating Otx2 activities that account for these pleiotropic effects were identified: (1) Significant boosts of neuroepithelium development, which even overrides the bipotential nature of NMPs, resulting in the loss of paraxial mesoderm precursor and loss of multiplying NMP stem cell population, and (2) The development of bending-refractory neural tissues. By virtue of these Otx2 functions, Otx2-expressing anterior epiblasts develop into the vast brain tissue in advance of the trunk neurogenesis that occurs in much smaller scale. We did not observe the exogenous Otx2 expression affecting the CNS regional specification. RNA-seq profiling of two chicken embryo portions, the midbrain-hindbrain portion and the trunk sinus rhomboidalis-equivalent portion, at st. 10-11, which were electroporated with pCAGGS-mOtx2 and pCAGGS-EGFP on the epiblast side at st.4/5 and cultured ex ovo. Control embryos were electroporated with pCAGGS-EGFP only or without electroporation.