Cell lineage analyses for histogenesis and diversification of the telencephalon

据信，在脑的各个区域中发现的细胞多样性主要来源于神经祖细胞的区域性不同特性。为了阐明在成熟脑结构中发现的区域差异在多大程度上取决于早期神经上皮区域性，我们决定使用Cre/loxP重组系统进行基于基因表达的命运图研究。我们已经产生了转基因小鼠品系，其中他莫昔芬诱导形式的Cre重组酶（CreERT 2）的Six 3基因座的控制下驱动，在前前脑原基中表达的基因在一个临时的动态模式。我们采用敲入策略精确再现了内源Six 3基因的表达。建立了三个新霉素抗性ES克隆以及来自这些克隆的小鼠品系。将Cre-报告基因系Rosa 26 R与Six 3-CreERT 2neo的杂合子杂交，并将他莫昔芬递送给母亲，这使得Cre能够切除转录激活lacZ报告基因的floxed终止盒。虽然LacZ表达细胞被检测为在给定时间表达Six 3的祖细胞的后代，但我们在前脑的Six 3表达结构域内发现了非常少的X-gal细胞，其在样本之间是可变的，并且由于Cre表达水平非常低，有时在样本内（横向不对称）。即使在通过与CAGGS-Flpw小鼠杂交切除FRT侧翼的pgk-neo-pA盒后，其基本上相同，尽管LacZ阳性细胞的数量显著增加。因此，我们得出结论，这些细胞系不用于Six 3表达结构域的阶段特异性谱系分析。虽然这些小鼠被证明不适合我们最初的目的，但在这些领域中的小细胞亚群中的重组可能对详细分析有用（例如。G. mosaic）作为条件敲除研究的Cre-driver线。

It is believed that cellular diversity found in the various regions of the brain is primarily derived from regionally distinct properties of the neural progenitors. To elucidate the extent to which the regional differences found in the mature brain structures depend upon the early neuroepithelial regionality, we decided to conduct a fate map study based upon gene expression, using Cre/loxP recombination system. We have generated transgenic mouse lines in which a tamoxifen-inducible form of Cre recombinase (CreERT2) was driven under the control of Six3 locus, a gene expressed in the anterior forebrain primordium in a temporary dynamic pattern. We took a knock-in strategy to recapitulate precisely the expression of endogenous Six3 gene. Three neomycin-resistant ES clones as well as mouse lines derived from those clones were established. A Cre-reporter line Rosa26R was crossed with the heterozygous of Six3-CreERT2neo and tamoxifen was delivered to the mother which enables Cre to excise the floxed stop cassette that transcriptionally activates lacZ reporter. While the LacZ-expressing cells were to be detected as the descendant of the progenitors that expressed Six3 at a given time, we found very few X-gal cells within the Six3-expressing domains of the forebrain which were variable among the specimens and within them sometimes (laterally asymmetric) due to very low level of Cre-expression. It was essentially the same, even after the FRT-flanked pgk-neo-pA cassette was excised by crossing with the CAGGS-Flpw mice, although the number of LacZ-positive cells was significantly increased. We thus concluded that these lines are not to be used for the stage-specific lineage analysis of the Six3-expressing domains. Although these mice turned out to be inappropriate for our original purpose, recombination in small subsets of cells in those domains could potentially be useful for detailed analysis (e. g. mosaic) as a Cre-driver line for the conditional knock out studies.