Single cell analysis of the Common Lymphoid Progenitor compartment reveals functional and molecular heterogeneity. Mus musculus

In order to investigate molecular events involved in the regulation of lymphoid lineage commitment, we crossed lamda5 reporter transgenic mice to mice where the GFP gene is inserted into the Rag1 locus. This allowed us to sub-fractionate common lymphoid progenitors (CLPs) and pre-pro-B cells into lamda5-Rag1low, lamda5-Rag1high and lamda5+Rag1high cells. Clonal in vitro differentiation analysis demonstrated that Rag1low cells gave rise to B/T and NK cells. Rag1high cells displayed reduced NK-cell potential with preserved capacity to generate B- and T-lineage cells while the lamda5+ cells were B-lineage restricted. Ebf1 and Pax5 expression was largely confined to the Rag1high populations. These cells also expressed a higher level of the surface protein LY6D providing an additional tool for the analysis of early lymphoid development. These data suggest that the classical CLP compartment composes a mixture of cells with more or less restricted lineage potentials opening new possibilities to investigate early hematopoiesis. Overall design: Two newly identified subpopulations of common lymphoid progenitors (CLP), hereafter called CLP_RAGhigh and CLP_RAGlow, have been sorted in 2 replicates. RNA was extracted from 2,000 purified adult BM cells using the RNAeasy microkit. RNA was labeled and amplified by dual amplification and hybridized to Affymetrix microarray MOE430_2, according to AffymetrixTM GeneChip Expression Analysis Technical Manual. Probe level expression values were calculated using the RMA algorithm.

为探究参与淋巴系细胞谱系定向调控的分子事件，我们将λ5报告基因转基因小鼠（lamda5 reporter transgenic mice）与绿色荧光蛋白（Green Fluorescent Protein, GFP）基因插入Rag1基因座（Rag1 locus）的小鼠进行杂交。该策略使得我们能够将共同淋巴系祖细胞（common lymphoid progenitors, CLPs）与前前B细胞（pre-pro-B cells）细分为λ5-Rag1low、λ5-Rag1high与λ5+Rag1high三类细胞群。克隆性体外分化分析结果显示，Rag1low细胞可分化产生B细胞、T细胞与自然杀伤细胞（Natural Killer, NK）。Rag1high细胞的NK细胞分化潜能降低，但仍保留生成B系与T系细胞的能力；而λ5+细胞则仅具备B系限制性分化潜能。Ebf1与Pax5的表达主要局限于Rag1high细胞群中。此类细胞还高表达表面蛋白LY6D，可为早期淋巴发育的研究提供额外的分选标记工具。上述数据表明，经典的共同淋巴系祖细胞池实际由谱系分化潜能各异的细胞混合组成，这为早期造血发生的研究开辟了新的方向。
实验整体设计：我们分选了两群新鉴定的共同淋巴系祖细胞亚群，下文将其分别命名为CLP_RAGhigh与CLP_RAGlow，每组设置2次生物学重复。使用RNAeasy微量RNA提取试剂盒从2000个纯化的成年骨髓（bone marrow, BM）细胞中提取总RNA。参照Affymetrix™ GeneChip表达分析技术手册，通过双扩增法对RNA进行标记与扩增，并将其与Affymetrix微阵列芯片MOE430_2进行杂交。采用RMA算法计算探针水平的基因表达量。