Person:

Paik, Elizabeth Jae-Eun

During embryogenesis, hematopoietic cells arise from the lateral plate mesoderm (LPM) following gastrulation. The transcriptional program required for this LPM to blood switch is not fully understood. Previous work on a zebrafish mutant with a deletion in the cdx4 gene demonstrated the importance of this caudal transcription factor in the LPM to blood transition. To explain how cdx4 regulates embryonic hematopoiesis, two main approaches were taken in this thesis. The first part of the thesis describes a chemical genetics screen that identified cdx4 interacting pathways. To find small molecules that could rescue the loss of red blood cells caused by the cdx4 deletion, cdx4 mutant embryos were incubated with 2640 compounds from the beginning of the gastrula stage to the 10-somite stage. Two related psoralen compounds, Bergapten (Ber) and 8-methoxypsoralen (8-MOP), rescued the erythroid progenitors in the cdx4 mutants. This rescue is closely linked to the compounds' effects on anteriorposterior patterning, reminiscent of retinoic acid pathway compounds. The second part of my thesis identifies a Cdx4-Sall4 transcriptional module in the LPM. Chromatin-immunoprecipitation coupled to sequencing (ChIP-seq) and microarray analysis revealed that Cdx4 directly regulates cdx4 and a zinc finger transcription factor spalt-like 4 (sall4) transcription. Sall4 ChIP-seq showed that Sall4 also binds to its own locus and to the cdx4 locus, suggesting an auto- and cross-regulation between two transcription factors. In addition, Cdx4 and Sall4 bind to common genomic regions proximal to mesodermal progenitor (tbx16 and mespa) and hematopoietic genes (scl, gata2a, and ldb1a), indicating Cdx4 and Sall4 co-regulate key genes that are required for LPM and blood specification. sall4 knockdown in the cdx4 mutants demonstrated that Sall4 synergizes with Cdx4 in regulating embryonic hematopoiesis. These findings suggest that auto- and cross-regulation of Cdx4 and Sall4 establish a stable circuit in the LPM that facilitates the activation of blood-specific program as development proceeds. How undifferentiated germ layers transition into various tissues is a key question in developmental biology. My thesis establishes a model based on LPM to blood transition, which is also applicable to other studies on germ layer specification.

cdx4, a caudal-related homeodomain-containing transcription factor, functions as a regulator of hox genes, thereby playing a critical role in anterior–posterior (A-P) patterning during embryogenesis. In zebrafish, homozygous deletion of the cdx4 gene results in a mutant phenotype known as kugelig, with aberrant A-P patterning and severe anemia characterized by decreased gata1 expression in the posterior lateral mesoderm. To identify pathways that interact with cdx4 during primitive hematopoiesis, we conducted a chemical genetic screen in the cdx4 mutant background for compounds that increase gata1 expression in cdx4 mutants. Among 2640 compounds that were tested, we discovered two compounds that rescued gata1 expression in the cdx4-mutant embryos. The strongest rescue was observed with bergapten, a psoralen compound found in bergamont oil. Another member of the psoralen family, 8-methoxypsoralen, was also found to rescue gata1 expression in cdx4-mutant embryos. The psoralen compounds also disrupted normal A-P patterning of embryos. These compounds modify the cdx4-mutant phenotype and will help elucidate signaling pathways that act downstream or parallel to the cdx4-hox pathway.