Single cell transcriptomic analysis of endoderm derived cell lineages contribution in pituitary

Single cell transcriptomic analysis of pituitary gland from adult zebrafish that had specifically cells labeled with endoderm lineage by different strategies. The goal of this study is to examine the contribution of endoderm derived cell in the pituitary of vertebrate animals. Overall design: We applied two strategies for labeling the endoderm derived cell in the pituitaries of zebrafish. The contribution of endoderm derived cells and the cell heterogeneity was later analyzed and resolved through constructing the single cell transcriptome through 10X Genomic pipeline. The first strategy is through endodermal lineage tracing techniques by Sox17 driven Cre recombinase conjuncted with an estrogen receptor (Sox17:CreERT2), which allows us to induce the conversion by addition of tamoxifen. We are able to identify the endoderm derived cell by microscopic observation with expression of mCherry gene from EGFP background. We made two single cell transcriptomic libraries by this strategy: the males library (file names started with M_Pituitary_Sox17creERUbiGtR) was derived by pooling 6 adult zebrafish pituitaries with moderate Sox17:CreERT2 conversion rate. The male1 library (file names started with M1_Pituitary_Sox17creERUbiGtR) was derived from the pituitary from a male zebrafish with high conversion rate. The second strategy is through transplant of taram-a mRNA injected GFP+ embryo to limit the cell into endoderm lineage. The treated embryonic cells were then injected into a BFP+ wild type embryo for tracing. The endoderm derived cells can be identify by expression of GFP. We made one single cell transcriptomic library by this strategy: the ubeGFP_transplanted (file names started with ubieGFP_Pit-Endo-transp) library was derived from pooling of pituitaries from adult transplanted fish.

本数据集针对通过不同策略标记内胚层谱系（endoderm lineage）细胞的成年斑马鱼垂体开展单细胞转录组分析。本研究旨在探究内胚层来源细胞在脊椎动物垂体中的贡献。
总体实验设计：我们采用两种策略对斑马鱼垂体内的内胚层来源细胞进行标记。后续通过10X Genomics单细胞转录组建库流程构建单细胞转录组文库，以此解析内胚层来源细胞的贡献及细胞异质性。
第一种策略为采用基于Sox17驱动的Cre重组酶与雌激素受体融合蛋白（Sox17:CreERT2）的内胚层谱系示踪技术：通过添加他莫昔芬（tamoxifen）可诱导重组酶激活，实现细胞标记的特异性转换。我们可在EGFP背景下通过观察mCherry基因的表达，鉴定内胚层来源细胞。基于该策略我们构建了两个单细胞转录组文库：其一为雄性文库（文件前缀为M_Pituitary_Sox17creERUbiGtR），由6只中等转化率的成年斑马鱼垂体混合制备；其二为雄性1号文库（文件前缀为M1_Pituitary_Sox17creERUbiGtR），由单只高转化率雄性斑马鱼的垂体制备。
第二种策略为：将注射了taram-a mRNA的GFP阳性胚胎细胞移植至BFP阳性野生型胚胎中，以此限定细胞为内胚层谱系并进行示踪。通过观察GFP的表达即可鉴定内胚层来源细胞。基于该策略我们构建了一个单细胞转录组文库：ubeGFP_transplanted文库（文件前缀为ubieGFP_Pit-Endo-transp），由多只经移植处理的成年斑马鱼垂体混合制备。