Circulating primitive erythroblasts establish a functional, protein 4.1R-dependent cytoskeletal network prior to enucleating

Yu-Shan Huang,Kathleen E Mcgrath,Luis F Delgadillo,Richard E Waugh,Jiandi Wan,Paul D Kingsley,John G Conboy,Narla Mohandas,Kathryn H Cyr,James Palis,Xiuli An

doi:10.1038/s41598-017-05498-4

Abstract

Hematopoietic ontogeny is characterized by distinct primitive and definitive erythroid lineages. Definitive erythroblasts mature and enucleate extravascularly and form a unique membrane skeleton, composed of spectrin, 4.1R-complex, and ankyrinR-complex components, to survive the vicissitudes of the adult circulation. However, little is known about the formation and composition of the membrane skeleton in primitive erythroblasts, which progressively mature while circulating in the embryonic bloodstream. We found that primary primitive erythroblasts express the major membrane skeleton genes present in similarly staged definitive erythroblasts, suggesting that the composition and formation of this membrane network is conserved in maturing primitive and definitive erythroblasts despite their respective intravascular and extravascular locations. Membrane deformability and stability of primitive erythroblasts, assayed by microfluidic studies and fluorescence imaged microdeformation, respectively, significantly increase prior to enucleation. These functional changes coincide with protein 4.1 R isoform switching and protein 4.1R-null primitive erythroblasts fail to establish normal membrane stability and deformability. We conclude that maturing primitive erythroblasts initially navigate the embryonic vasculature prior to establishing a deformable cytoskeleton, which is ultimately formed prior to enucleation. Formation of an erythroid-specific, protein 4.1R-dependent membrane skeleton is an important feature not only of definitive, but also of primitive, erythropoiesis in mammals.

Highlights

In the adult, definitive red blood cells require a functional erythroid-specific membrane skeletal network to maintain cell integrity while repeatedly deforming in the microcirculation
Having previously determined that primitive erythroblasts at E12.5, which are at the orthochromatic erythroblasts (OrthoE) stage of maturation, are highly deformable[12], we asked when during murine embryogenesis does this membrane skeleton become functional
Since the development of membrane skeleton function is associated with changes in the shape of adult red cells, we first examined the shape of primary primitive erythroblasts isolated at progressive days of embryogenesis

Summary

Introduction

Definitive red blood cells require a functional erythroid-specific membrane skeletal network to maintain cell integrity while repeatedly deforming in the microcirculation. We have previously determined that primitive erythropoiesis in the mouse embryo first emerges as a transient wave of colony-forming progenitors in the E7.5-E9.0 yolk sac that generate a single cohort of circulating erythroblasts that progressively mature in the bloodstream[6, 7] Morphologic examination of these cells indicate that they progress from immature proerythroblasts (ProE) at E9.5 to basophilic erythroblasts (BasoE) at E10.5 and to late-stage orthochromatic erythroblasts (OrthoE) by E12.5 (Fig. 1a). We have recently determined that late stage primitive erythroblasts in the E12.5 mouse embryo are highly deformable[12], suggesting that they form a functional membrane skeleton prior to enucleating to cope with the vicissitudes of the fetal circulation. Protein 4.1R isoform switching and the development of a functional, protein 4.1R-dependent membrane skeleton are conserved in the primitive and definitive erythroid lineages of mammals

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Results

Conclusion