P1 - Dissecting the role of sialoglycans in embryonic development
The mammalian sialome, particularly cell surface expressed sialoglycans, has essential functions insignaling ‘self’ and ‘foreign’. By targeting CMP-sialic acid synthase (CMAS), the enzyme catalyzing metabolic activation of sialic acid (Sia), we generated an asialo system, exhibiting embryonic (E) lethality at day 8.5, clearly demonstrating that sialoglycans are indispensable during pregnancy. Studies on the cause of fetal demise identified an untamed activation of the alternative branch of the maternal complement system with massive deposition of the complement component 3b (C3b) on the semi-allogeneic trophoblast cells with concurrent inflammation at the feto-maternal interface. However, we could exclude formation of the membrane attack complex and demonstrated that signaling via the anaphylotoxins C3a and C5a has no impact on phenotype development. In contrast, we saw a massive invasion of maternal platelets and in pilot studies were able to largely prevent complement activation and inflammation by inhibition of maternal platelets. Combined with literature data attributing platelets a role in placental inflammation, our preliminary data induced studies on the molecular events that stimulate platelet activation and deleterious consequences in the asialo-background. To uncover Sia functions apart from complement regulation the double knockout Cmas-/-; C3-/- was generated. Cmas-/-; C3-/- mice died at E11.5 and thus revealed a secondary developmental block inthe absence of sialoglycans. A prominent characteristic of Cmas-/-; C3-/- are severe neuronal hemorrhages, which are highly reminiscent to the phenotypes of mice with genetic alterations that impair formation of hematopoietic stem cells (HSC). We thus pursue the goal to understand the role of sialoglycans in hematopoiesis and the differentiation of HSCs. The massive thromboinflammation seen at the fetal-maternal interface of Cmas-/- implants demonstrates that sialylation is essential at the boundary layer between fluid phase and solid tissue. While endothelial expressed sialoglycans have been studied with regard to different aspects and are known to participate in the maintenance of hemostasis and leukocyte recruitment, a model that would allow to study of endothelial functions in the absence of Sia is missing. In the course of the current project we will generate Cdh5-Cre/ERT2; Cmas lox/lox mice that enable the tamoxifen induced and endothelial specific loss of sialylation also in adult animals and will use this model to address Siafunctions in endothelia.