MRTF-SRF signaling is required for seeding of HSC/Ps in bone marrow during development

Abstract

Chemokine signaling is important for the seeding of different sites by hematopoietic stem cells (HSCs) during development. Serum response factor (SRF) controls multiple genes governing adhesion and migration, mainly by recruiting members of the myocardin-related transcription factor (MRTF) family of G-actin-regulated cofactors. We used vav-iCre to inactivate MRTF-SRF signaling early during hematopoietic development. In both Srf- and Mrtf-deleted animals, hematopoiesis in fetal liver and spleen is intact but does not become established in fetal bone marrow. Srf-null HSC progenitor cells (HSC/Ps) fail to effectively engraft in transplantation experiments, exhibiting normal proximal signaling responses to SDF-1, but reduced adhesiveness, F-actin assembly, and reduced motility. Srf-null HSC/Ps fail to polarize in response to SDF-1 and cannot migrate through restrictive membrane pores to SDF-1 or Scf in vitro. Mrtf-null HSC/Ps were also defective in chemotactic responses to SDF-1. Srf-null HSC/Ps exhibit substantial deficits in cytoskeletal gene expression. MRTF-SRF signaling is thus critical for expression of genes required for the response to chemokine signaling during hematopoietic development.

Figure 1

Early hematopoietic inactivation of Srf causes perinatal lethality and lack of bone marrow cellularity. (A) Embryos or animals were genotyped at the indicated stages and proportion of SrfKO^H (vav-iCre; Srf^f/f) scored; n = total embryos/animals genotyped. (B) Hemorrhage into skin, bladder, or eye in newborn SrfKO^H compared with Srf⁺ (Srf^f/f, Srf^f/+) animals. (C) P1 femurs stained with hematoxylin and eosin (H&E) (left) or endomucin (right). (D) Colony-formation assays with cells from the P1 femur of SrfKO^H animals. Data are from 3 Srf⁺ and 3 SrfKO^H animals, each assay was performed in triplicate. See supplemental Figure 1A. (E) Left, reduced colony-formation activity in hind-limb long bones of E18.5 SrfKO^H animals. Data are from 5 Srf⁺ and 3 SrfKO^H embryos; each assay was performed in triplicate. Right, Gr-1⁺ cellularity in E18.5 long bones. Data are from 6 embryos of each genotype (P < .0001; unpaired Student t test).

Figure 2

Hematopoietic progenitor cells in SrfKO^H fetal liver. (A) Cellularity of SrfKO^H and Srf⁺ fetal liver. (B) Fetal liver LSK cells (see also supplemental Figure 1B). Panels Bi-ii, elevated numbers of LSK cells in SrfKO^H fetal liver. Panel Biii, BrdU labeling indicates that SrfKO^H LSK cells are more proliferative than those from Srf⁺ embryos. (C) Similar proportions of CD150^hi cells in SrfKO^H fetal liver. (D) SrfKO^H and Srf⁺ fetal liver cells generate similar numbers of colonies in colony-formation assays. Data are from 6 Srf⁺ and 4 SrfKO^H embryos; each assay was performed in triplicate. See supplemental Figure 1C. (E) Although SrfKO^H and Srf⁺ colony morphologies are different (i), the total cell numbers are similar (ii).

Figure 3

SrfKO^H fetal liver cells fail to engraft durably. 10⁶ total CD45.2⁺ Srf⁺ or SrfKO^H fetal liver cells were used to reconstitute wild-type CD45.1⁺ animals after high-dose (2 × 6 Gy) irradiation as indicated. Donor Srf⁺ and SrfKO^H cells were distinguished by CD45.2 marker or use of the mT/mG system (mT, Srf⁺; mG, SrfKO^H; see supplemental Figure 3). Solid symbols, Srf⁺; open symbols, SrfKO^H. For raw data and results after low-dose irradiation (2 × 4.5 Gy), see supplemental Figure 4. (A) Peripheral blood analysis. Left, total donor and host cells were distinguished using CD45.2 (donor) and CD45.1 (host); middle and right, donor cells were analyzed for contribution of Srf⁺ (mT) or SrfKO^H (mG) cells to B220 (B cell) or Gr-1 (granulocyte) lineages by gating on mT or mG as appropriate. Mean values are shown (n = 4-6 per condition). (B) Srf⁺ (mT) and SrfKO^H (mG) cells in reconstituted bone marrow (left) and spleen (right) at 18 weeks. (C) Srf⁺ (mT) and SrfKO^H (mG) LSK cells in reconstituted bone marrow at 18 weeks. Left, LSK-gated bone marrow; right, proportion of CD150⁺ cells. (D) Secondary transplants of bone marrow cells from animals reconstituted for 18 weeks with Srf⁺ or SrfKO^H fetal liver cells. Reconstitutions were performed using 2 × 10⁵ Srf⁺ (mT) or SrfKO^H (mG) bone marrow cells; irradiation was at 2 × 6 Gy. Analysis was done as in (A). See also supplemental Figure 4C. (E) Proportion of LSK cells in bone marrow 16 weeks after the secondary transplant in (C). (F) SrfKO^H cells compete ineffectively with Srf⁺ cells for bone marrow engraftment. Donor Srf⁺ and SrfKO^H cells were engrafted either alone (left and center panels) or in 1:19 Srf⁺:SrfKO^H ratio (right panel) after 2 × 6 Gy irradiation, and the proportions of LSK-gated Srf⁺ (mT), SrfKO^H (mG), and host cells were measured 12 weeks later.

Figure 4

Homing to bone is defective in SrfKO^H cells. (A) Srf⁺ and SrfKO^H fetal liver LSK cells were mixed 1:1 and 10⁵ cells injected into the tail vein of C57Bl6 mice, with prior irradiation where indicated. The ratio of SrfKO^H to Srf⁺ cells present in the hind-limb long bones was evaluated at the indicated times. (B) Absolute numbers of cells homed to the long bones at different times after injection in the experiment shown in (A). (C) Proliferation of CFSE-labeled cells homed to long bones at different times after injection. (D) Homing to calvaria. Animals were injected with fetal liver LSK cells from Srf⁺ and SrfKO^H animals, labeled with CFSE and SNARF, respectively, to increase detection sensitivity, mixed 1:1. Left, relative proportions of Srf⁺ and SrfKO^H cells in calvaria 16 hours after injection. Right, distance of each homed LSK cell from the nearest bone or endothelial cell. Error bars = standard error of the mean.

Figure 5

SrfKO^H HSC/P cells exhibit defective adhesion, polarization, and motile responses to SDF-1. (A) Adhesion analysis. Srf⁺ and SrfKO^H fetal liver LSK cells were mixed 1:1 and seeded on fibronectin-coated substrate (i) or on monolayers of MBA-2.1 endothelial cells (ii), in the presence or absence of 100 ng/mL SDF-1. Solid symbols, Srf⁺; open symbols, SrfKO^H. See supplemental Figure 6D. (B) Morphology analysis. Cells were plated as in (A) on fibronectin-coated or MBA-2.1 endothelial cell monolayers, allowed to settle, and then stimulated for 45 minutes with SDF-1 before fixing and visualization with either Texas Red phalloidin (FN) or by prestaining with CFSE (MBA-2.1). Cell shape, defined as circularity = 4π (area/perimeter) was measured. A perfect circle has circularity 1.0, which decrease with increasing elongation. Solid symbols, Srf⁺; open symbols, SrfKO^H. See supplemental Figure 7A. (C) Transendothelial migration. CFSE-stained LSK cells were plated on SNARF-stained MBA-2.1 monolayers and treated as in (B). Panel Ci, cell locations were displayed by cumulative CFSE pixel intensity (LSKs, black lines) and [1-cumulative pixel intensity] (MBA-2.1, red line) relative to the membrane surface. Panel Cii, representative images: LSK cells in green and MBA-2.1 cells in semitransparent red. (D) Cell migration. LSK cells were plated on MBA2.1 cells and allowed to adhere for 20 minutes, and SDF-1 was added, followed by tracking for 20 minutes. The rose plots map the movement of each cell during the course of the experiment relative to its position at time zero. Data from 243 Srf⁺ and 228 SrfKO^H LSK cells tracked in 3 independent experiments are summarized.

Figure 6

Srf and the Mrtfs are required for the chemotactic response to SDF-1. (A) Srf⁺ (mT) and SrfKO^H (mG) fetal liver LSK cells were mixed 1:1, plated onto fibronectin-coated 5-µM pore transwells, and allowed to migrate across the membrane toward SDF-1 or SCF for 4 hours. See supplemental Figure 7B. (B) Srf⁺ or SrfKO^H fetal liver LSK cells prelabeled with CFSE were plated onto the upper chamber of a fibronectin-coated 5-µM pore transwell and allowed to migrate toward SDF-1 for 45 minutes. After fixation, cells were stained with Texas Red phalloidin and imaged by confocal microscopy, and the cells associated with pores in 12 to 13 fields were counted. (C) Srf⁺ or SrfKO^H fetal liver LSK cells were plated and processed as in (B), and Z-stacks acquired. Top panels, proportions of cells at different locations. Black and red arrows, upper and lower membrane surfaces. Bottom, cell numbers: NP, not associated with pores; PA, pore-entrance–associated; TM, transiting pore. (D) RNA-seq analysis of fetal liver LSK cells. Volcano plots of fold-change in RNA expression upon Srf inactivation vs statistical significance (left) and of SDF-1 induced transcripts in Srf⁺ cells. (E) Comparison of positively-regulated Srf-dependent gene sets in LSK and MEFs at significance P < .05. When a fold-change threshold of 2 is set for Srf dependence, 245 genes are positively regulated in LSKs, 657 in MEFs, and only 12 in both cell types. 1951 genes were negatively regulated by Srf in LSKs, of which 155 were shared with MEFs.

Figure 7

Inactivation of both Mrtfs phenocopies SRF deletion. (A) E18.5 femurs stained with H&E (left) or endomucin (right). (B) Gr-1⁺ cellularity in mutant Mrtf mutant E18.5 long bones. Data are from 6 embryos of each genotype (P < .0001; unpaired Studen t test). (C) Colony-formation assays by cells from Mrtf-mutant MrtfabKO^H E18.5 long bones. Data are from 3 embryos of each genotype, and each assay was performed in triplicate. (D) Proportion of LSK cells in Mrtf E14.5–mutant fetal livers. (E) Colony-formation assays with cells from Mrtf-mutant E14.5 fetal livers. Data are from 3 embryos of each genotype, and each assay was performed in triplicate. (F) Mrtf⁺, Mrtfa^−/−, MrtfbKO^H, or MrtfabKO^H fetal liver LSK cells were mixed and plated onto fibronectin-coated 5-µM pore transwells and allowed to migrate across the membrane toward SDF-1 for 4 hours. (G) Roles of MRTF-SRF and chemokine signaling in tissue colonization by HSC/P. See “Discussion” for details.