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doi: 10.1242/10.1242/dev.00184

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Fgfr3 expression by astrocytes and their precursors: evidence that astrocytes and oligodendrocytes originate in distinct neuroepithelial domains

¹ Wolfson Institute for Biomedical Research and Department of Biology, University College London, Gower Street, London WC1E 6BT, UK
² Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609
³ Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110, USA

View larger version (68K): [in a new window]   Fig. 4. Different populations of Fgfr3+ and Pdgfra+ cells in the newborn spinal cord. We hybridised sections of P2 mouse cervical spinal cord simultaneously with a DIG-labelled Fgfr3 probe together with an FITC-labelled Pdgfra probe to visualise OLPs. The Fgfr3 signal (red) was visualised with rhodamine-tyramide reagent and the Pdgfra signal (green) with fluorescein-tyramide. Scattered individual Fgfr3+ and Pdgfra+ cells can be seen throughout both white and grey matter of the cord, but these are separate and discrete cell populations. We conclude that the great majority of Fgfr3+ cells in the cord are not OLPs.

View larger version (145K): [in a new window]   Fig. 7. Co-expression of Fgfr3 and glutamine synthetase (Glns) in the VZ and parenchyma of the embryonic mouse spinal cord. There was considerable overlap between the in situ hybridisation signals for Fgfr3 and Glns in the E15 mouse spinal cord, strongly suggesting that Fgfr3+ cells correspond to glial (presumably astrocyte) precursors. Arrows indicate cells that express both Fgfr3 and Glns.

View larger version (69K): [in a new window]   Fig. 1. Fgfr3 expression in transverse sections of embryonic chick and mouse cervical spinal cords. (A) Chick stage 22-24 (E3.5-4); (B) chick stage 34 (E8); (C) chick stage 35 (E9); (D) chick stage 37 (E11); (E) chick stage 35 (E9); (F) mouse E13.5; and (G) mouse E14.5. Initially, Fgfr3 is expressed in the floor plate and the ventral two-thirds of the VZ (A) and is later downregulated in part of the ventral VZ (B). Starting around stage 34 (E8) Fgfr3+ cells are visible in the parenchyma of the cord. By stage 37 (E11) the floor plate and VZ no longer express Fgfr3 but scattered Fgfr3+ cells are present throughout most of the cross-section of the cord, including both grey and white matter (D). (E) A magnified image of the ventral VZ from a stage 35 (E9) cord, showing the two spatially separated domains of Fgfr3 expression. A similar progression occurs in mouse (F,G). However, the ventral `gap' is not so pronounced in mouse (arrow in G). Scale bars: 200 µm (A-D), 100 µm (F,G), 50 µm (E).

View larger version (98K): [in a new window]   Fig. 2. Expression of Fgfr3 and Olig2. Transverse sections through stage 35 (E9) chicken spinal cords were subjected to in situ hybridisation for Fgfr3 (A) or double in situ for Fgfr3 and Olig2 (B). At this age, Fgfr3 expression is confined to the VZ and a few scattered cells outside the VZ. The two spatially separated domains of Fgfr3 expression are clearly visible (A). Olig2 is expressed predominantly within the ventral `gap' of Fgfr3 expression (B). This suggests that pMN (brackets), which generates Pdgfra+ oligodendrocyte progenitors (OLPs), does not also generate Fgfr3+ putative astrocyte progenitors. Scale bar: 50 µm.

View larger version (71K): [in a new window]   Fig. 3. Incorporation of BrdU by Fgfr3-expressing cells. We labelled E18 embryos by two intra-peritoneal injections of BrdU, 6 hours apart, into the mother. We harvested the embryos 3 hours later and performed in situ hybridisation for Fgfr3 followed by immunohistochemistry for BrdU. The Fgfr3 (A) and BrdU (B) images were superimposed using Adobe Photoshop (C). Many Fgfr3-expressing cells incorporated BrdU (C, arrows), confirming that they can divide after exiting the VZ and are therefore unlikely to be neurones. Arrowheads in B,C indicate Fgfr3-negative cells that have incorporated BrdU.

View larger version (125K): [in a new window]   Fig. 5. Fgfr3-positive cells are unaffected in Pdgfa null spinal cords. Consecutive sections of newborn wild-type or Pdgfa knockout mouse cervical spinal cords were hybridised in situ with probes to Fgfr3 (A,B) or Pdgfra (C,D). The number of Pdgfra+ OLPs is strongly reduced in the Pdgf-A knockout (compare C with D) but neither the number nor the distribution of Fgfr3+ cells is changed noticeably (A,B). Again, we conclude that the Fgfr3+ cells and Pdgfra+ OLPs are different cells.

View larger version (52K): [in a new window]   Fig. 6. Newly differentiating white matter astrocytes express Fgfr3. We simultaneously hybridised sections of E18 mouse cervical spinal cord with an FITC-labelled Gfap mRNA probe (A) and a DIG-labelled Fgfr3 probe (B). The Gfap and Fgfr3 hybridisation signals were visualised and photographed sequentially (see Materials and Methods). All the Gfap-expressing astrocytes also expressed Fgfr3 (e.g. arrows). In general, Fgfr3+ cells in the grey matter (arrowheads) did not co-express Gfap.

View larger version (145K): [in a new window]   Fig. 8. Cultured cells from E17 rat spinal cord double-labelled for Fgfr3 and Gfap. Cells were hybridised in situ with a 35S-labelled RNA probe for Fgfr3, then immunolabelled for Gfap followed by autoradiography (see Materials and Methods). The Fgfr3 signal (black silver grains) is present over most Gfap-positive cells (brown DAB reaction product; arrows) (also see Table 1). Scale bar: 10 µm. Arrohead indicates an Fgfr3-positive, Gfap-negative cell.

View larger version (76K): [in a new window]   Fig. 9. Gfap upregulation in grey matter astrocytes in Fgfr3-null mice. Transverse sections through the cervical spinal cords of 2-month-old wild-type (A) and Fgfr3-null mice (B) were immunolabeled with anti-Gfap. In the wild-type cord, white matter (fibrous) astrocytes express Gfap but there is little or no Gfap immunoreactivity in the grey matter. By contrast, the Fgfr3-null mouse (B) shows extensive Gfap labelling of grey matter (protoplasmic) astrocytes. Scale bar: 100 µm.

View larger version (120K): [in a new window]   Fig. 10. Neutralising Shh activity in explant cultures of ventral spinal cord. Stage 12/13 (E2) chick neural tube was dissected into dorsal, intermediate and ventral thirds. The ventral thirds were cultured in collagen gels in the presence of either control antibody (A,C) or with neutralising anti-Shh antibody (B,D). Explants were double-labelled with O4 monoclonal antibody (C,D) and anti-Gfap (A,B). Anti-Shh blocks the formation of O4-positive OLPs but not Gfap-positive astrocytes. Scale bar: 10 µm.