Embryonic lethality upon targeted disruption of a glycerophosphodiesterase: unravelling the role of Gpcpd1 in foetal mouse erythropoiesis

• Applicant: Rosemarie Marchan, Ph.D.
• Subject Area: Hematology, Oncology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 554295864

GPCPD1 hydrolyses glycerophosphocholine (GPC) into two intermediate metabolites, choline and glycerol-3-phosphate (G3P). Choline is important for the de novo production of phosphatidylcholine, an abundant phospholipid in eukaryotic membranes. Conversely, G3P is the backbone molecule for all cellular glycerophospholipids, which are components of cellular and vesicular membranes, as well as noted signalling molecules capable of controlling cellular function. Despite the importance of GPCPD1’s metabolic products, little is known about its physiological role. Therefore, to characterize the physiological function of GPCPD1, we generated a constitutive knockout mouse model using CRISPR/Cas9 technology. Breeding heterozygous mice resulted in no homozygote offspring, suggesting embryonic lethality. At E16.5, obvious differences were observed between Gpcpd1+/+ and Gpcpd1-/- embryos; specifically, embryos lacking Gpcpd1 were smaller, paler, and weighed less. Histological analysis of haematoxylin-eosin-stained whole-embryo sections showed that a large fraction of the red blood cells retained their nuclei at E16.5, suggesting defective erythropoiesis. In support, markers of mature erythrocytes, such as Cd71 and Cd235a that normally increase during mouse erythropoiesis, were downregulated in E16.5 liver tissue of Gpcpd1-/- mice. Moreover, RNA-seq performed on bulk foetal liver from E16.5 embryos revealed many changes after Gpcpd1 KO related to key cellular processes, including energy metabolism. The proposed project aims to clarify the role of Gpcpd1 in erythrocyte development. In the first work package (WP1), we will verify Gpcpd1’s importance in foetal erythropoiesis by investigating the effect of knocking out Gpcpd1 on erythropoiesis markers in the foetal liver. We will then examine how Gpcpd1 loss affects the differentiation capacity of lineage negative cells also isolated from foetal mouse liver. Rescue experiments via Gpcpd1 transfection and choline supplementation will then be done to confirm that Gpcpd1 is directly responsible for the observed phenotypes. WP2 aims to identify further novel factors downstream of Gpcpd1 that will help elucidate mechanisms by which Gpcpd1 influences erythropoiesis. Here, several experimental approaches will be used - transcriptomics, metabolomics, lipidomics, MALDI-mass spectrometry imaging, and global methylation profiling - followed by experimental verification in vitro with rescue experiments. To investigate if the findings in mice are relevant to human erythropoiesis, in WP3 we will measure the expression of GPCPD1 during the differentiation of human induced pluripotent stem cells (hiPSCs) to erythrocytes and then investigate how knocking out GPCPD1 influences the differentiation process. We anticipate that the findings of the proposed study will introduce a new player in erythrocyte development and clarify why mice lacking Gpcpd1 do not survive postnatally.

• DFG Programme: Research Grants
• Co-Investigator: Karolina Edlund, Ph.D.