哺乳动物的大脑皮质是一次进化上的创新，但我们对其进化机制知之甚少。鸟类和哺乳动物同属高等脊椎动物，都具有复杂的神经系统，但鸟类却有着不同于哺乳动物六层大脑皮质的核团式结构。对五百多种脊椎动物皮质发育相关基因（Emx1/2、Tbr1和Pax6）和皮质下区域发育相关基因（Dlx2和Nkx1/2）进行比较分析，发现这些基因在染色体重复、丢失、基因共线性以及达尔文选择（Ka/Ks<1）等方面没有明显差别。与序列较为保守的皮质下区域表达基因相比，哺乳动物的Emx1多出一段可以编码20个氨基酸的片段，Emx2多出一段编码6-8个多聚丙氨酸的序列。前期研究表明，在鸡胚端脑中表达小鼠Emx2（mEmx2），会引起鸡胚端脑皮质各区域中表达Reelin、Vimentin以及GABA的阳性细胞数量有不同程度增多，GABA能中间神经元迁移模式转变，行为学实验表明雏鸡的被动回避率有所增强。但与哺乳动物大脑新皮层相比，还是差异较大，说明除了Emx2外，还有其他基因或因素的参与才导致哺乳类和鸟类大脑的差异。

       基于以上研究背景，本研究以小鸡为研究对象，对处于两个不同发育时期的鸡胚（E8和E10）和鼠胚（E12.5和E14.5）端脑同源皮质区域进行转录组测序分析，以检测相关差异基因。结果在E8鸡胚中检测到15,294个表达基因，E10鸡胚中检测到15,159个表达基因，E12.5鼠胚中检测到16,276个表达基因，E14.5鼠胚中检测到16,384个表达基因。在两个不同发育时期，正常鸡胚端脑有744个基因发生变化，通过慢病毒（Lentiviruses, Lv）表达鼠Emx2（Lv-mEmx2），鸡胚端脑有773个基因发生变化，正常鼠胚端脑有586个基因发生变化。将发生变化的基因注释到相关信号通路后发现，Notch、Wnt、Hh、FGF、及TGF-beta等对发育起重要调控作用的信号通路都发生了变化，Wnt信号通路在所有组中都呈下调趋势，变化幅度位于所有通路的前20位（Top 20）。同时，表达mEmx2的鸡胚相比于同时间段表达对照病毒Cv的鸡胚，Wnt信号通路变化程度也位于显著下降的前20位。

       本研究利用慢病毒为载体，在鸡胚第三天将表达Ctnnb1的慢病毒（Lv-Ctnnb1）注射到端脑脑室，以激活Wnt信号通路, 或同时将Lv-Ctnnb1慢病毒与Lv-mEmx2慢病毒一起注射到端脑脑室，发育至12或16天后检测鸡脑皮质所发生的变化。结果如下：

1）单独注射Lv-Ctnnb1慢病毒

       与脑发育相关的神经元重要骨架分子Reelin、Vimentin和MAP2，以及不同类型神经元如GABA能中间神经元、Ctip2和Satb2的数量与分布均发生改变，GABA能神经元中PV神经元和CB神经元的迁移模式也发生了变化。鸡脑Wulst、M和N内外侧Reelin、MAP2和GABA能中间神经元细胞数量有不同程度增多，放射状胶质纤维Vimentin平均长度增加。PV和CB神经元的迁移模式均表现为由内向外，并且这两类神经元与BrdU双标的细胞表达峰值向后推迟。此外，大脑皮质中深、浅层标志性神经元Ctip2和Satb2产生的顺序和时间虽然没有变化，但数量和分布都发生改变。对新生雏鸡的学习记忆能力以及探索能力等行为探究发现，Ctnnb1的过表达没有引起小鸡相关行为变化。

2）同时注射Lv-Ctnnb1及Lv-mEmx2慢病毒

       与前面相应的脑发育相关骨架分子Reelin、Vimentin和MAP2及不同类型神经元GABA能中间神经元、Ctip2和Satb2的数量与分布都有不同程度的变化。鸡脑Wulst、M和N内外侧Reelin、MAP2和GABA能中间神经元细胞数量均有增加，Vimentin平均长度增加。PV和CB神经元的迁移模式均表现为由内向外，并且与BrdU双标的细胞表达峰值也向后推迟。Ctip2和Satb2的产生时间没有变化，但数量和分布均发生改变。雏鸡的学习能力和记忆能力有所提高，而探索能力则没有明显改变。

       新皮质结构及功能的完整性是在神经元的增殖、分化及迁移等活动的协同调控下实现的。本研究提示了哺乳类新皮层与非哺乳类大脑皮层间的差异可能与Wnt信号通路及Emx2在早期胚胎神经系统发育中的作用有关。

       Little is known about the mechanism underlying the innovation of the mammalian neocortex. Mammals and birds both have complex brains, but mammals have a six-layered cerebral cortex which is formed in an inside-out pattern, whilst birds have a number of pallial nuclear masses which are less stratified than the mammalian homologues, and formed in an outside in pattern. Our preliminary studies on comparative analysis of genes involved in the development of the pallium (Emx1/2、Tbr1 and Pax6) and sub pallium (Dlx2 and Nkx1/2) among 500 vertebrate species have shown that these genes have no clear variations in chromosomal duplication/loss, gene locus synteny or Darwinian selection (Ka/Ks<1). However, there is an additional fragment of approximately 20 amino acids in mammalian Emx1 and a poly-(Ala)6–7 in Emx2. Further studies have demonstrated that expressing mouse Emx2 (mEmx2) in the pallium of chicken embryos can cause a change in the migration pattern of GABAergic intermediate neurons, as well as the increases in the number of cells that express GABA and Reelin, Vimentin in most parts of pallium. Nevertheless, the changed pallium of chick is still largely different from mammalian pallium. These studies suggest that more genes should have been involved in the development of avian or mammalian pallium rather than Emx2.

       To obtain more genes concerned with the development of avian or mammalian pallium, we analyzed and compared the transcriptomic changes in the mouse brain at embryonic (E) day E12.5 and E14.5, and in the chicken brain at E8 and E10. The results indicated that 15,294 genes were expressed in the brain of chicken embryo at E8, and 15,159 genes were expressed at E10. In contrast, 16,276 genes were expressed in the brain of mouse embryo at E12.5, and 16,384 genes were expressed at E14.5. There were 744 genes changed between the two developmental stages in the chick, 773 genes changed in the chick infected with lentiviruses expressing mEmx2, and 586 genes in the mouse. Of these changed genes, some were annotated into some signaling pathways, and the top 20 included Notch, Wnt, Hh, FGF, and TGF-beta signaling pathways which are crucial during the development of pallium. During the two developmental stages, only the Wnt signaling pathway always indicated decreases in the studied groups, or compared between the chicken embryos infected with control lentiviruses (Cv) and the chicken embryos infected with lentiviruses expressing mEmx2. Thus, the Wnt signaling pathway is involved in the development of pallium and it might be also related to the differences in the development of pallium between birds and mammals.

       To address the above issue, chick Ctnnb1 was cloned into the lentiviruses, and cells infected with the lentiviruses expressing chick Ctnnb1 can cause the activation of the Wnt signaling pathway. The lentiviruses expressing chick Ctnnb1 solely or jointly with the lentiviruses expressing mouse Emx2 (1:1) were injected into the ventricle of the chick telencephalon at embryonic Day 3 (E3), and the embryos were allowed to develop to E12-16 or to post hatchling. The results are as follows:

1) Infection only with the lentiviruses expressing chick Ctnnb1

       Following the injection of the lentiviruses expressing chick Ctnnb1 into the ventricle of the chick telencephalon at E3, cells expressing Reelin or Vimentin increased, and the distribution pattern of MAP2 and GABA changed significantly. The neurons expressing Reelin, MAP2 or GABA increased in most parts of pallium. The size of the areas shaped by the labeled ends of the Vimentin fibers extending farthest and the ventricle were compared in W, M and N. The results indicated that the areas increased significantly in W, M and N. The numbers and distribution of Ctip2 and Satb2 which are generally located in the deep or upper locations of the pallium changed, although the time when they were generated did not change. Neurogenetic pattern of parvalbumin or calbindin followed an inside to outside fashion in W and M. Moreover, the expression peak of BrdU with parvalbumin or calbindin double target cells was delayed in W and M. In addition, Open-field tests were performed for the chicks to assess their locomotor activities, and curious or exploratory behaviors on three successive days (from post-hatching day 5 to 7). The results indicated that the passive avoidance ratio, novel exploration, and locomotor activities did not change obviously.

2）Joint infections with the lentiviruses expressing chick Ctnnb1 or mouse Emx2

       Following the joint injection of the lentiviruses expressing chick Ctnnb1 or mouse Emx2 into the ventricle of the chick telencephalon at E3, cells expressing Reelin or Vimentin increased, and the distribution pattern of MAP2 and GABA varied to different degrees. The neurons expressed Reelin, MAP2 and GABAergic interneurons increased in Wulst, M and N, and Vimentin fibers became longer and occupied larger areas. The numbers and distribution of Ctip2 and Satb2 were changed, but the time when they were generated did not change. Neurogenetic pattern of parvalbumin or calbindin followed an inside to outside fashion. Besides, behavior test indicated that the passive avoidance ratio and novel exploration increased, but locomotor activities did not change significantly.

       Our results indicated that cell proliferation, cell differentiation, and migration pattern of some cells such as CB and PV were changed after the activation of the Wnt signaling pathway solely or jointly with the overexpression of mouse Emx2, and there was a coregulation for the Wnt signaling pathway and Emx2. Thus, both the Wnt signaling pathway and mouse Emx2 are both involved in the development of the pallium, as well as the differences between the birds and mammals.