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

本研究通过大鼠胚胎全胚培养3天，颅面器官原基培养9天，初步建立了12天培养体系。在WEC中3天的胚胎可以模仿关键分子的外观，即，Lhx 1、Foxa 2、Isl-1、pitx 2、Nkx2.1、NeuroD 1、Tpit、Lhx 3、Shh、Bmp 2、Bmp 4、Fgf 8等是下丘脑和腺垂体发育所必需的。9天后的颅面雏形可以模仿腺垂体激素的出现，即，α亚基、POMC、LHβ和TSHβ。此外，前轴中内胚层（AME）的去除实验的应用到培养系统中表明，AME是必要的Lhx 1，Foxa 2，isl-1，Shh，Fgf 8，Bmp 4，Bmp 2和Nkx 2.1的出现。此外，在培养系统中使用抗Shh抗体的功能丧失导致表达Nkx 2.1的细胞减少，这表明分泌Shh的AME对弓状核的发育是必需的，弓状核是假定的消化中心。培养12 d的弓状核由于缺氧，不能分化为NPY神经元和α MSH神经元。因此，我们开始建立为期6天的大鼠胚胎全培养，以模拟推测的弓状核神经元的发育。在原有培养条件下培养6 d的25枚胚胎中，有19枚没有心跳，而在新的改良培养条件下培养的12枚胚胎中，有8枚心跳维持了6 d。目前，我们正在建立弓状核分化为NPY神经元和α-MSH神经元的器官培养体系。

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.