MSI2 mediates WNT/β-Catenin pathway function in hematopoietic stem cells

Abstract

First, we sorted out hematopoietic stem cells (HSCs) and hematopoietic stem and progenitor cells (HSPCs) from wildtype (WT) and Apc knockout (Apc KO, Apc^−/−) mice, which are Apc^fl/fl and Apc^fl/flMx1Cre respectively. The Apc deletion was induced by Poly(I:C) injection. Quantitative PCR (qPCR) demonstrated a significant decrease of Msi2 expression in the HSCs (lin⁻c-Kit⁺ Sca1⁺CD150⁺CD48⁻) and HSPCs (lin⁻c-Kit⁺) of Apc KO mice (Fig. 1B, C). To investigate the role of MSI2 in the WNT signaling pathway, MSI2 was re-expressed in Apc^−/− HSPCs by retrovirus infection, and western blot (WB) confirmed Msi2 was successfully overexpressed (Supplementary Fig. 1). The colony-forming assay showed that restoring MSI2 expression could partially rescue the diminished colony-forming ability observed in Apc^+/− and Apc^−/− cells (Fig. 1D, E). Next, we collected colony cells from the above colony-forming assay, and found that restoration of MSI2 expression can significantly reduce the high apoptosis rate induced by Apc KO (Fig. 1F and Supplementary Fig. 2).

To further investigate the relationship between MSI2 and WNT signaling pathway, we performed in vivo transplantation experiments following the restoration of MSI2 expression in Apc KO cells. Five days after the 5-FU injection, HSPCs from WT (Apc^fl/fl) or Apc^fl/fl Mx1Cre mice were infected with MigR1 vector or MSI2, and then were transplanted into CD45.1 recipient mice irradiated lethally (10 Gray). One month later, pIpC was injected intra-peritoneally at bi-daily intervals to induce the expression of Mx1Cre to knock out Apc. Seven days after the third pIpC injection, the mice were euthanized, and bone marrow cells were gathered for detection (Fig. 1G). The restoration of MSI2 expression considerably increased the populations of HSCs (Lin⁻c-Kit⁺Sca1⁺CD48⁻CD150⁺), LSK (Lin⁻c-Kit⁺Sca1⁺) and HPCs (Lin⁻c-Kit⁺Sca1⁻) compared to cells from Apc KO mice (Fig. 1H and Supplementary Fig. 3). Furthermore, the proportion of granulocyte-monocyte progenitors (GMP) and common myeloid progenitors (CMP) in the HPC population returned to normal. The megakaryocyte-erythroid progenitors (MEP) also changed, but not significantly (Fig. 1I and Supplementary Fig. 4). Apc deletion can induce a significant increase in apoptosis, which is one of the main reasons for the decline of HSPCs [7]. We found that the restoration of MSI2 expression can significantly inhibit the increase of apoptosis caused by Apc deletion (Fig. 1J and Supplementary Fig. 5A, B), suggesting that MSI2 can partially rescue the decline of HSPCs induced by Apc loss through inhibiting apoptosis. In addition to HSPCs, we also analyzed the changes in mature cells and found that the restoration of MSI2 expression significantly increased myeloid cells in both bone marrow and spleen (Fig. 1K and Supplementary Fig. 6A, B). Conversely, B cells in bone marrow (Supplementary Fig. 7A, B) and T cells in the thymus (Supplementary Fig. 8A, B) showed no significant alterations.