Role of Trim71 in embryonic cardiovascular development and hematopoiesis

Abstract

The circulatory system consists of blood cells, vasculature and the heart, and it enables the exchange of oxygen, nutrients, metabolic waste products and cells throughout the body. These functions make prenatal cardiovascular development and hematopoiesis essential for mid-gestational embryonic survival, as the embryo becomes too large for molecule exchange to occur exclusively via passive diffusion. The hematoendothelial lineage comprises blood cells and endothelial cells (EC), which make up the inner lining of blood vessels. This cell lineage is derived from the mesodermal germ layer that arises from gastrulation. We investigated the role of Trim71, an RNA-binding protein that is widely expressed at early embryonic stages, in the development of the mammalian circulatory system. Previous studies showed that mouse embryos with a global Trim71 knockout (Trim71-KO) die at the onset of organogenesis, but the reasons for this were unknown. <br /> The present work identifies Trim71 as an essential factor for cardiovascular development and primitive hematopoiesis. The onset of growth retardation in Trim71-KO embryos at organogenesis coincided with the stage at which the circulatory system normally develops. Trim71-KO embryos had a global reduction in primitive erythroid cells (EryP) and showed substantial vascular impairments, which were most pronounced in the extraembryonic yolk sac. While the initial formation of blood vessels (vasculogenesis) was not affected, a defect in angiogenesis was apparent from the unremodeled vascular network of Trim71-KO yolk sacs and a lack of large vitelline vessels. This was primarily driven by the decreased expression of angiogenic genes in EC, as shown by single-cell RNA-sequencing (scRNA-seq). A subset of Trim71-KO embryos was also absent of a heartbeat, and the remaining embryos had a reduced heart rate. The cardiovascular defects led to the inability of premacrophages (pMac), that originate from erythro-myeloid progenitors (EMP) in the yolk sac, to colonize the embryo proper and seed intraembryonic macrophages populations. Conditional knockout (cKO) of Trim71 in EMP using the Csf1riCre line showed that this phenotype was independent of EMP-intrinsic Trim71 expression, indicating that defective blood circulation is the reason for the impaired translocation upon global Trim71-KO. At later fetal stages, Trim71 expression in EMP limited the generation of myeloid cells. The combined defects in primitive erythropoiesis, cardiovascular development and blood circulation of Trim71-KO embryos provide a conclusive explanation for their lethality. <br /> We further explored the origins of circulatory system defects in Trim71-KO embryos. Mice with Tie2^Cre Trim71 cKO in the hematoendothelial progenitors (HEP) of EryP and EC were viable and had a mild reduction in yolk sac angiogenesis, while primitive erythropoiesis was unaffected. This demonstrated that Trim71 expression in HEP is dispensable for circulatory system development. Broad inactivation of Trim71 in the mesoderm using the TCre line resulted in embryonic lethality with a variable embryonic phenotype. TCre Trim71 cKO had no clear effects on vascular development and erythropoiesis, which could in part be explained by an insufficient activity of the TCre line in the hematoendothelial lineage, as shown by fate mapping experiments. By performing scRNA-seq of embryos at gastrulation stage, we detected widespread transcriptional changes in Trim71-KO embryos that precede the onset of morphological defects. The expression of the mesodermal pioneer transcription factor Eomes was highly increased in Trim71-KO mesodermal progenitors. Using differentiated mouse embryonic stem cells, we discovered an NHL domain-dependent interaction of Trim71 with the Eomes mRNA, presumably mediated by a stem-loop motif present in the 3' untranslated region. This suggested that Eomes is a direct target of canonical Trim71-mediated post-transcriptional mRNA repression. Altogether, our results show a novel function of Trim71 at gastrulation and indicate that defects in the generation of the circulatory system of Trim71-KO embryos are initiated in mesodermal progenitors.