Establishment of the culture method to mimic the development of neural nucleus of alimentary center in mammals

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In this study, we initially established the 12 day-culture system by the whole rat embryo culture (WEC) for 3 days followed by the organ culture of craniofacial rudiments for 9days. The embryos in WEC for 3 days could mimic the appearance of crucial molecules, i.e., Lhx1, Foxa2, Isl-1, pitx2, Nkx2.1, NeuroD1, Tpit, Lhx3, Shh, Bmp2, Bmp4, Fgf8, which were necessary for a development of Hypothalamus and adenohypophysis. The craniofacial rudiments following 9 days could mimic the appearance of adenohypophysis hormones, i.e., α subunit, POMC, LHβ and TSHβ. Moreover, the application of removal experiment of anterior axial mesendoderm (AME) to the culture system showed that AME was essential for the appearance of Lhx1, Foxa2, isl-1, Shh, Fgf8, Bmp4, Bmp2 and Nkx2.1. In addition, the loss-of-function using an anti-Shh antibody inthe culture system resulted in the reduction of the cells expressing the Nkx2.1, suggesting that the Shh-secreting AME were essential for the development of arcuate nucleus, the presumptive alimentary center. However, the arcuate nucleus cultured in the 12 day-culture system failed to differentiate into the NPY-neuron and αMSH-neuron due to the oxygen deficiency in the 12day culture system. Therefore, we started to establish the whole rat embryo culture for 6 days in order to mimic the development of neuron in the presumptive arcuate nucleus. The heartbeat were not maintained in 19 of the 25 embryos cultured for 6 days under the previous culture condition, whereas their heartbeat were maintained for 6 days in 8 of the 12 embryos cultured in novel improved condition. At present, we are establishing the organ culture in which the arcuate nucleus in explants differentiates into NPY-neuron and αMSH-neuron.