Function of the chemokine receptor CXCR4 in haematopoiesis and ...

More documents

Recommendations

Info

letters to nature result from deficiency in commitment and/or proliferation of Bprogenitor cells. CXCR4 is highly expressed in the thymus, particularly in immature CD4 + CD8 + cells 4 . However, T lymphopoiesis occurred normally in the mutant embryos at E17.5 (not shown). At this stage, most thymocytes are immature CD4 + CD8 + cells; few CD4 + CD8 − or CD4 − CD8 + mature T lymphocytes are present, and there is little or no emigration of thymocytes to the peripheral lymphoid organs. Emigration commences only after birth through a process in which the Gi proteins are important 14,15 . As CXCR4 expressed in resting T cells is coupled to G�i2 (refs 4, 10) and as mature T cells can migrate in response to SDF-1 stimulation 9 , migration and recirculation of mature T cells might require signals delivered through CXCR4. To determine whether maturation and emigration of thymocytes requires CXCR4, we engrafted the thymuses from E17.5 CXCR4 −/− embryos under the kidney capsule of mice lacking T-cell antigen receptor (TCR)-�. As the recipient mice are deficient in ��-T-cell development, any TCR-�� T cells detected in the periphery are derived from the donor thymus. Our results indicate that mature CD4 + CD8 − and CD4 − CD8 + T cells developed normally in the implanted thymuses. Moreover, these T cells efficiently populated the peripheral lymphoid organs, including peripheral blood 8 (Fig. 2d), spleen, and mesenteric lymph nodes (results not shown). Thus, CXCR4 has a negligible effect on thymocyte maturation and subsequent migration to lymphoid organs. As CXCR4 messenger RNA is expressed in brain 4 , we used in situ hybridization to study the embryonic mouse brain at E13, E15 and E18. CXCR4 mRNA transcripts were found in many regions of the Figure 2 Impaired haematopoiesis and SDF-1-induced chemotaxis, but normal development of T cells, in CXCR4 −/− mice. a, Flow-cytometric analysis of fetal liver cells and bone-marrow cells isolated from E17.5 embryos. Cells were stained with antibodies against the indicated cell-surface markers (Gr1, CD11b, CD61 and TER119), and the percentages of cells in the marked gates are indicated. b, Transwell assay measurement of SDF-1-induced chemotaxis of fetal liver cells from E17.5 mutant embryos (open circles) and wild-type littermates (filled circles). Flow-cytometric analysis showed that �90% of cells migrating into the bottom chamber expressed myeloid-lineage markers. Results from two independent experiments are shown. c, In vitro clonal assay of pro-B-cell colony formation. Black bar, wild-type; white bar, CXCR4 −/− . Results represent the mean of four separate experiments. d, Flow-cytometric analysis of thymocytes isolated from transplanted thymi of mutant and control embryos. B and T lymphocytes from the peripheral blood of the recipient mice were detected using antibodies against the lineage-specific markers CD45 (B220) and TCR-��. The percentage of cells in the given quadrants is indicated. Nature © Macmillan Publishers Ltd 1998 596 NATURE | VOL 393 | 11 JUNE 1998
developing brain, including the retina, olfactory epithelium, olfactory bulb, hippocampus, cerebellum and spinal cord (Fig. 3, and data not shown). A similar pattern of CXCR4 mRNA expression was found in rat brain 5 . Expression was observed in regions of the cerebellum enriched in proliferating cells, including neuroepithelium, rhombic lip and the external granule layer (EGL) (Fig. 3). We then compared brains of the mutant mice with those of wild-type littermates. Grossly, the structures of brains from CXCR4 −/− mice were comparable with those from wild-type animals (data not shown). However, closer examination showed that the laminar structure of the cerebellum of CXCR4 −/− mice was aberrant. Although the EGL was present, clusters of granule cells were found in ectopic positions, beneath the Purkinje cell layer or intermingled with Purkinje cells (Fig. 4a, b). These abnormally placed cells expressed the Math 1 protein, a marker of external granule cells 16 (Fig. 4c, d). All 20 mutant cerebella examined showed normal patterning of neuronal layers in the caudal part close to the rhombic lip, indicating that the defect in granule cell migration is probably a result of premature migration from the EGL rather than of abnormal laminar migration from the rhombic lip. The descending migration of EGL cells to form the internal granule layer (IGL) is normally a postmitotic event that occurs primarily after birth. In the mutant mice, migration of the granule cells from the EGL was observed as early as E17.5, and involved cells that continued to proliferate, as assessed by incorporation of bromodeoxyuridine during a 2-h pulse labelling (Fig. 4e, f). In contrast to the granule cells, the Purkinje cells were situated correctly in mutant embryos, as shown by staining with an anti-calbinding antibody (not shown). The perpendicular migration of EGL cells may be directed by a Figure 3 Localization of CXCR4 mRNA in the wild-type developing brain as shown by in situ hybridization. A sense-chain RNA probe used as a negative control gave no significant signal above background. a, Sagittal section showing CXCR4 expression (darker shading) in neuronal precursors on the surface of the cerebellar anlage of E13.5 embryo (original magnification, ×50). b, Sagittal section showing increased CXCR4 expression in E15.5 cerebellum (×100). c, Transverse section of the lateral aspect of the E18.5 cerebellum (original magnification, ×100). cb, Cerebellum; cp, choroid plexus; EGL, external granule layer; IV, fourth ventricle; IC, inferior colliculus; NE, neuroepithelium; PM, pia mater; PO, pons. letters to nature reciprocal interaction between the granule neurons and Bergmann cells (the cerebellar radial glial cells). Although the postmitotic granule cells undergo differentiation and induce Bergmann cell fibre extension, Bergmann cells provide a scaffold for neuronal migration and positioning 17,18 . Therefore, the dislocation of EGL in the mutant embryos early in cerebellar development could be a consequence of the malformation of Bergmann cells. Staining with brain-specific lipid-binding protein (BLBP) 19 , which labels radial glial cells, 8 showed that these cells were properly localized with their fibres arranged in a normal configuration and orientation in the CXCR4 −/− mice (Fig. 4g, h). In some mutant embryos, there was an increase in the number of neuronal cells along the Bergmann glial fibres (Fig. 4h). These results indicate that CXCR4-mediated signalling is required to prevent premature migration of proliferating granule cells inwards from the EGL. This could involve the induction of Figure 4 Abnormal migration of cerebellar EGL cells in CXCR4 −/− embryos. a, b, Haematoxylin-and-eosin-stained sagittal sections, showing dislocated cell aggregates underneath the Purkinje cell layer (arrow heads) in postnatal day 0 mutant animals. c–h, Sagittal sections prepared from E17.5 embryos. Sections were stained with antibodies against Math1 (c, d), BrdU (e, f) or BLBP (g, h; counterstained with haematoxylin). Arrowheads indicate ectopic granule cells. Migrating granule cells from the EGL are viewed by higher magnification (arrows, f). CP, choroid plexus. PCL, Purkinje cell layer; RL, rhombic lip. Anterior is to the right and dorsal to the top in each photograph. Original magnifications: a, b, ×100; c, d, ×200; e–h, ×400. Nature © Macmillan Publishers Ltd 1998 NATURE | VOL 393 | 11 JUNE 1998 597
Page 1: Function of the chemokine receptor
Page 5: 25. Connor, R. I., Sheridan, K. E.,

Function of the chemokine receptor CXCR4 in haematopoiesis and ...

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?