The N-glycome regulates the endothelial-to-hematopoietic transition

Abstract

Definitive hematopoietic stem and progenitor cells (HSPCs) arise from the transdifferentiation of hemogenic endothelial cells (hemECs). The mechanisms of this endothelial-to-hematopoietic transition (EHT) are poorly understood. We show that microRNA-223 (miR-223)-mediated regulation of N-glycan biosynthesis in endothelial cells (ECs) regulates EHT. miR-223 is enriched in hemECs and in oligopotent nascent HSPCs. miR-223 restricts the EHT of lymphoid-myeloid lineages by suppressing the mannosyltransferase alg2 and sialyltransferase st3gal2, two enzymes involved in protein N-glycosylation. ECs that lack miR-223 showed a decrease of high mannose versus sialylated sugars on N-glycoproteins such as the metalloprotease Adam10. EC-specific expression of an N-glycan Adam10 mutant or of the N-glycoenzymes phenocopied miR-223 mutant defects. Thus, the N-glycome is an intrinsic regulator of EHT, serving as a key determinant of the hematopoietic fate.

Fig. 1.. miR-223 is expressed in hemECs undergoing EHT.

(A) (Top) Schematic of miR-223:GFP reporter. (Bottom) Lateral Z-projections of the zebrafish AGM. White and yellow arrows point to flat and budding miR-223:GFP⁺ kdrl:mCH⁺ cells, respectively. (B to D) Plots of wild-type kdrl:mCH⁺ trunk ECs 27 hours after fertilization showing expression of (B) EHT, (C) blood lineage, and (D) miR-223:gfp genes (n = 6227 cells). Arterial (branch 3) and EHT (branches 8, 11, and 4) trajectories are indicated. (E) During EHT, a flattened EC (red outline) gains hemogenic potential (orange outline) and buds as a oligopotent nascent HSPC (yellow outline) from the DA wall. Gradients for endothelial and EHT markers are colored on the basis of their expression in the indicated cell types. The green gradient and text represent miR-223 expression. DA, dorsal aorta; hpf, hours post fertilization; PCV, posterior cardinal vein

Fig. 2.. miR-223 is an endothelial inhibitor of EHT and lymphoid-myeloid–oligopotent HSPC production.

(A and B) (Top) Z-projections or (bottom) mean ± SEM number of gata2b:GFP⁺ kdrl:mCH⁺ hemECs [n = 24 or 25 embryos, (A)] or cmyb:GFP⁺ kdrl:mCH⁺ nascent HSPCs [n = 13 to 18, 32 hours after fertilization; n= 34 to 36, 36 hours after fertilization (8), (B)]. (C and D) (Top) Representative images or (bottom) mean ± SEM area of (C) T-lymphocyte marker rag1 expression (n = 18 to 35 embryos) and Sudan black⁺ neutrophil clusters (n = 36 or 37 embryos) and (D) cmyb expression (n = 18 to 21 embryos). The EC miR-223 rescuing construct is described in fig. S4E. DA, dorsal aorta; dpf, days post fertilization; hpf, hours post fertilization; PCV, posterior cardinal vein. Significance is represented as not significant (n.s.) P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.01, ****P ≤ 0.0001, unpaired, two-tailed Mann-Whitney U test.

Fig. 3.. miR-223 limits EHT and lymphoid-myeloid–oligopotent HSPC production through repression of alg2 and st3gal2.

(A) (Top) Arrows indicate cmyb:GFP⁺ kdrl:mCh⁺ nascent HSPCs in the zebrafish AGM. (Bottom) Mean ± SEM of cmyb:GFP⁺ kdrl:mCH⁺ cells (n = 28 to 48 embryos). (B) (Top) Images or (bottom) mean ± SEM area of T-lymphocyte marker rag1 expression (n = 28 to 35) embryos and Sudan black⁺ neutrophil clusters (n = 21 or 37 embryos). Abbreviations and significance calculations are as in Fig. 2.

Fig. 4.. miR-223 regulates the endothelial glycome.

(A) Extraction procedure to assess endothelial N-glycan profiles 27 hours after fertilization. Extracted N-glycans were analyzed by means of mass spectrometry (MS). (B) MS chromatograms for ECs showing relative abundance of N-glycan subtypes. A subset of the identified N-glycan structures are shown (monosaccharide legend is provided in fig. S8A). (C and D) Relative abundance of N-glycan subtypes in (C) ECs 27 hours after fertilization or (D) whole embryos. N-glycan subtypes are colored as in (B). EC GOF, endothelial cell gain of function.

Fig. 5.. A distinct N-glycan repertoire regulates protein function to limit EHT.

(A) Log₂ (fold change) of N-glycan relative abundance for miR-223–regulated N-glycopeptides (n = 3 replicates). (B) White arrows indicate nascent HSPCs in the zebrafish AGM of embryos injected with a gRNA that diminishes adam10a or with EC gain of function (GOF) adam10a constructs. (C) Mean ± SEM number of cmyb:GFP⁺ kdrl:mCH⁺ HSPCs (n = 13 to 18 embryos). Abbreviations and significance calculations are as in Fig. 2. (D) miR-223–dependent regulation of the N-glycome restricts EHT and lymphoid-myeloid HSPC production. In ECs, miR-223 represses alg2 and st3gal2 N-glycoenzymes, leading to high mannose-modified (green gradient) versus sialylated-modified (pink and blue gradients) proteins, which restricts (opacity) their signaling (black gradient). EHT cell types and N-glycan subtypes are colored according to Fig. 1E and Fig. 4B, respectively.