A Cdx4-Sall4 regulatory module controls the transition from mesoderm formation to embryonic hematopoiesis. Danio rerio

Deletion of caudal/cdx genes alters hox gene expression and causes defects in posterior tissues and hematopoiesis. Yet, the defects in hox gene expression only partially explain these phenotypes. To gain deeper insight into Cdx4 function, we performed ChIP-seq combined with gene expression profiling in zebrafish, and identified the transcription factor spalt-like 4 (sall4) as a Cdx4 target. ChIP-seq revealed that Sall4 bound to its own gene locus and the cdx4 locus. Expression profiling showed that Cdx4 and Sall4 co-regulate genes such as hox, scl, and lmo2 that initiate hematopoiesis. Combined cdx4/sall4 gene knock-down impairs erythropoiesis, and overexpression of the Cdx4 and Sall4 target genes scl and lmo2 together rescued the erythroid program. These findings suggest that auto- and cross- regulation of Cdx4 and Sall4 establish a stable molecular circuit in mesoderm that facilitates the activation of the blood-specific program as development proceeds. Overall design: ChIP-seq was performed against Cdx4, Sall4, H3K27ac, and H3K4me3 in bud-stage zebrafish embryos. Input material was sequenced as controls.

尾型同源框基因（caudal/Cdx genes）的缺失会改变同源异形框基因（Hox genes）的表达，并引发后部组织发育异常及造血功能缺陷。然而，Hox基因表达异常仅能部分解释上述表型。为深入解析Cdx4的生物学功能，本研究在斑马鱼模型中开展了染色质免疫共沉淀测序（ChIP-seq）联合基因表达谱分析，鉴定出转录因子SPALT样蛋白4（spalt-like 4, SALL4）为Cdx4的下游靶标。ChIP-seq结果显示，SALL4可结合自身基因位点及cdx4基因位点。基因表达谱分析表明，Cdx4与SALL4共同调控造血起始相关的Hox基因、干细胞白血病基因（stem cell leukemia, SCL）及LIM域仅含2（LIM domain only 2, LMO2）等靶基因。联合敲低cdx4与sall4基因会损害红细胞生成过程，而同时过表达Cdx4与SALL4的靶基因SCL及LMO2则可挽救红细胞生成程序。上述研究结果表明，Cdx4与SALL4的自身调控与交叉调控可在中胚层中构建稳定的分子环路，进而在胚胎发育进程中促进血液特异性基因程序的激活。实验整体设计：在胚盾期斑马鱼胚胎中，针对Cdx4、SALL4、组蛋白H3赖氨酸27乙酰化（H3K27ac）及组蛋白H3赖氨酸4三甲基化（H3K4me3）开展ChIP-seq实验，并以输入样本作为测序对照。