Yaparla A et al. (2019), Myelopoiesis of the Amphibian Xenopus laevis Is...

XB-ART-56687

Front Immunol 2019 Apr 04;10:3015. doi: 10.3389/fimmu.2019.03015.

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Myelopoiesis of the Amphibian Xenopus laevis Is Segregated to the Bone Marrow, Away From Their Hematopoietic Peripheral Liver.

Yaparla A , Reeves P , Grayfer L .

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Across vertebrates, hematopoiesis takes place within designated tissues, wherein committed myeloid progenitors further differentiate toward cells with megakaryocyte/erythroid potential (MEP) or those with granulocyte/macrophage potential (GMP). While the liver periphery (LP) of the Xenopus laevis amphibian functions as a principal site of hematopoiesis and contains MEPs, cells with GMP potential are instead segregated to the bone marrow (BM) of this animal. Presently, using gene expression and western blot analyses of blood cell lineage-specific transcription factors, we confirmed that while the X. laevis LP hosts hematopoietic stem cells and MEPs, their BM contains GMPs. In support of our hypothesis that cells bearing GMP potential originate from the frog LP and migrate through blood circulation to the BM in response to chemical cues; we demonstrated that medium conditioned by the X. laevis BM chemoattracts LP and peripheral blood cells. Compared to LP and by examining a comprehensive panel of chemokine genes, we showed that the X. laevis BM possessed greater expression of a single chemokine, CXCL12, the recombinant form of which was chemotactic to LP and peripheral blood cells and appeared to be a major chemotactic component within BM-conditioned medium. In confirmation of the hepatic origin of the cells that give rise to these frogs' GMPs, we also demonstrated that the X. laevis BM supported the growth of their LP-derived cells.

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Species referenced: Xenopus laevis
Genes referenced: cd4 csf1r ctrl cxcl12 egr1 egr2 fli1 gata1 gata2 gfi1 ighx klf4 nfe2 spi1 tal1

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	Figure 1. Analysis of lineage-specific transcription factor gene expression in X. laevis liver periphery and bone marrow cells. X. laevis liver periphery and bone marrow cells were isolated and examined by qPCR for their expression of (A) HSC-associated, (B) myeloid lineage, (C) erythroid-lineage, and (D) lymphoid-lineage transcription factor genes. All gene expressions were quantified relative to the gapdh endogenous control and normalized against the lowest observed expression. 30 μg of total cell lysate proteins from LP and BM cells was used for western blot analysis to determine the protein levels of (E) Tal1, Egr1, and Gfi1 with beta actin as a loading control. The protein levels were quantified by (F) densitometry analyses using ImageJ software. Results are means ± SEM (A–D: N = 5; E,F: N = 3) and asterisk overhead of horizontal lines denotes statistical significance, P < 0.05.
	Figure 2. Analysis of lineage-specific transcription factor gene expression in liver periphery and bone marrow cells from rCSF-1- and rEPO-stimulated X. laevis. Adult X. laevis frogs were injected intraperitoneally with 5 μg of rCSF-1, rEPO, or equal volumes of r-ctrl, and 3 days later their liver periphery and bone marrow cells were assessed for lineage-specific transcription factor gene expression in (A) rCSF-1-stimulated and (B) rEPO-stimulated cells. The examined genes included hematopoietic-associated TFs: tal1, klf4, gata2, nfe2; myeloid-lineage TFs: pu1, egr1, egr2; and erythroid lineage TFs: gata1, nfe2. All gene expressions was quantified relative to the gapdh endogenous control and the gene expression is presented relative to the respective gene expression in r-ctrl-treated animals; denoted by dashed lines. Results are means ± SEM (N = 5) and asterisk overhead of horizontal lines denote statistical significance between the two cell types and (*) denotes statistical differences between the r-ctrl and respective r-growth factor stimulation, within respective cell types, P < 0.05.
	Figure 3. X. laevis bone marrow-conditioned medium chemoattracts peripheral liver and blood leukocytes. Tenfold concentrated bone marrow-conditioned medium was serially diluted and examined for its ability to chemo-attract liver periphery (LP) cells and peripheral blood leukocytes (PBLs). Chemokinesis of LP cells and PBLs was measured by adding the tenfold concentrated BM-medium to both the upper and the lower chemotaxis chambers (denoted as 10/10). All chemotaxis/chemokinesis experiments were performed using cells from five individual animals (N = 5), enumerating 10 random fields of view per chemotaxis filter per animal. Results are means ± SEM. Above-head letters denote statistical designations: experimental groups described by distinct letters are statistically different (P < 0.05), while those marked by the same letters are not.
	Figure 4. Analysis of chemokine gene expression in X. laevis liver periphery and bone marrow. Frog liver periphery (LP) and bone marrow (BM) from five individual animals (N = 5) were examined for their expression of a panel of chemokine genes by qPCR. All gene expressions were quantified relative to the gapdh endogenous control and normalized against the lowest observed expression. Results are means ± SEM and (*) overhead of horizontal lines denote statistical significance, P < 0.05.
	Figure 5. X. laevis rCXCL12 is chemotactic to liver periphery and peripheral blood leucocytes. A recombinant X. laevis CXCL12 (rCXCL12, 10−7-101 ng/ml) was examined for its capacity to chemoattract (A) liver periphery (LP) cells or peripheral blood leucocytes (PBLs; 104 cells/well) with chemokinesis assessed by adding 10−3 ng/ml of rCXCL12 (optimal dose) to both the upper and the lower chemotaxis chambers. (B) The relative capacities of tenfold concentrated bone marrow conditioned medium (BM-med) and rCXCL12 (10−3 ng/ml) to chemoattract LP cells and PBLs (104 cells/well) were compared through chemokinesis experiments by adding either BM-med or rCXCL12 and rCXCL12 or BM-med to the upper and lower chemotaxis chambers, respectively (denoted as BM-med/rCXCL12; rCXCL12/BM-med) and compared to BM-med or rCXCL12 in only bottom wells. All chemotaxis/chemokinesis experiments were performed using cells from 5 individual animals (N = 5), enumerating 10 random fields of view per chemotaxis filter per animal. Results are means ± SEM. Above-head letters denote statistical designations: experimental groups described by distinct letters are statistically different (P < 0.05), while those marked by the same letters are not.
	Figure 6. X. laevis rCXCL12 chemoattracts myeloid-lineage cells. Chemotaxis assay using the optimal concentration of rCXCL12 (10−3 ng/ml) was performed on liver periphery (LP) cells and peripheral blood leucocytes (PBLs; 104 LP cells or PBLs/well, cell from five individual frogs, N = 5), and the chemoattracted cells were examined for their gene expression of crcr4 (receptor for CXCL12); lineage specific markers for myeloid: csf1r (macrophage), csf3r (granulocyte), pu1, gfi1; HSC-associated: tal1, klf4, gata2; erythroid: fli1, gata1, nfe2, and lymphoid cell populations: igm (B cell); cd4 (T helper cell); and cd8 (cytotoxic T cell) by qPCR. All gene expressions was quantified relative to the gapdh endogenous control and normalized against the corresponding gene expression observed in the LP cells or PBLs (input, indicated by the dashed line) used in these chemotaxis experiments. Results are means ± SEM, () denotes statistical differences from the gene expression in total input LP or PBL population (indicated by the dashed line) and () above horizontal bars denote statistical differences between LP cells and PBLs, P < 0.05.
	Figure 7. The X. laevis bone marrow supports the survival of liver periphery cells. The frog femurs were isolated and cut at the condyles on one side of each bone to create an opening. One femur from each animal was flushed with saline and the other with methanol to fix the stromal/supportive cells. Liver periphery (LP) cells (105 cells per femur from the same respective animals) were introduced into each of the femurs and placed in semi-solid medium with the open-end facing up. After 3 days of incubation, the viable cells in these femurs were enumerated. Results represent combined data derived from three independent such experiments, each experiment assessing tissues/cells from six individual frogs (N = 6 per experiment; N = 18). Results are means ± SEM and (*) overhead of horizontal lines denote statistical significance, P < 0.05.

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