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The vertebrate central nervous system is characterized by regional specialization, which arises during early development and contributes to patterning the emerging central nervous system (CNS). In the hindbrain, rhombomeres demarcate nonoverlapping regions of the CNS that give rise to distinct neural structures. The cellular structures that define boundaries between adjacent rhombomeres are as yet unclear. However, in certain species the boundary regions between discrete CNS regions appear to be defined by specialized glial cells. Here, we show that in developing Xenopus, DM gamma, a membrane protein of the proteolipid protein family, is expressed in a subset of radial glia. During development, DM gamma transcripts are first expressed in presumptive glial cells throughout the hindbrain, but later become confined to the ventricular zone at rhombomere centers, whereas the protein is exclusively expressed in radial glial cell processes that occupy the rhombomere boundary regions. Likewise, early in development vimentin and glial fibrillary acidic protein are extensively coexpressed in hindbrainradial glia but subsequently define distinct rhombomere domains: vimentin remains localized in radial glia at the rhombomere boundary regions, whereas expression of glial fibrillary acidic protein becomes restricted to the centers. Moreover, radial glial processes at the boundary region are distinguishable from those at the center region; the processes of the boundary region radial glia extend upward in a fan-shaped arrangement and are encircled by the processes from the center glia. These data suggest that an early event in determining rhombomere topology is the specification of both morphologically and biochemically distinct subsets of radial glia.
Fig. 1. A: DMg immunoblot of Xenopus brain. In brain homogenates from stage 55/56, DMg antibody detects a single band of 35 kDa and a faint lower molecular band. This lower molecular weight band as well as a very weak band in stage 63 were not absorbed by DMg–glutathione-S-transferase fusion proteins, revealing that these bands were nonspecific. In stage 42 forebrain (B,C) and hindbrain (D,E), DMg immunoreactivity is detected in radially oriented cells, extending from ventral surface to pial surface. At higher magnification (C, shown in B by box), cells are observed, which have their cell bodies facing the ventricle and which extend their processes toward the pial surface. At the pial surface of the hindbrain (E, shown in D by box), the endfeet and connected processes are positive for DMg. F–K: Double immunolabelling of DMg and vimentin in stage 53 hindbrain. DMg and vimentin are coexpressed in some laterally extending processes (arrowheads). On the other hand, the processes extending both at the floor and roof plates, in which vimentin is strongly positive, do not show DMg immunoreactivity (arrows). Scale bars 5 50 mm in B,D; 20 mm in C,E; 100 mm in H (applies to F–H); 50 mm in K (applies to I–K).
Fig. 2. DMg transcript distribution by in situ hybridization.A: Stage 29/30 embryo. DMg transcripts are seen in the eyes, forebrain, and hindbrain. DMg signal is uniformly distributed in the hindbrain. B,C: Stage 42/43 tadpole and isolated brain. DMg transcripts appear to be segmentally distributed in the center of each rhombomere. D: Nomarsky optics of the stage 42/43 isolated brain.E: At stage 54/55, DMg transcripts are detected segmentally in the center of each rhombomere as they are at stage 42/43. F: Transverse section shows the DMg localization at the rhombomere centers. White lines and black arrowheads indicate center regions, black dotted lines indicate boundary regions. Scale bars 5 20mm in D,F.
Fig. 3. (Overleaf) Double labelling by in situ hybridization and 5-bromo-29-deoxyuridine (BrdU) antibody. A,B: Stage 40 Xenopus tadpolehindbrain. DMg is expressed strongly in the ventricular zone (A) and BrdU incorporation is mainly seen in the ventricular zone (B). C,D: Metamorphosed froglet (stage 59) forebrain. BrdU-positive cells are clustered around the ventricular zone (D) and distributed similarly as the DMg-positive cells (C). Arrows indicate a cell positive for both DMg and BrdU. Scale bars 5 50 mm in A,B; 25 mm in C,D.
Fig. 4. DMg, vimentin, and glial fibrillary acidic protein (GFAP) localization on stage 45/46 hindbrain. A: Hoechst nuclear staining. Arrowheads indicate the rhombomere centers. B: DMg-positive radial glial processes are observed throughout the hindbrain. Segmental distribution of radial glia, if any, is not evident. C: Vimentin-positive cells start arranging in a segmental manner. D: GFAP-positive cells are extensively colocalized with vimentin, except the longitudinal fibers are strongly positive for GFAP (arrow). Scale bar 5 20 mm in A (applies to A–D).
Fig. 5. DMg mRNA, DMg, vimentin, and glial fibrillary acidic protein (GFAP) protein localization in stage 53/54 hindbrain longitudinal sections. A: DMg transcript is concentrated in the center of rhombomere (arrowheads). B: In contrast, DMg-positive radial glial processes distribute in the boundaries of the rhombomere and are occasionally seen in the center region. C: Vimentin-positive radial glial processes distribute in a similar manner as that of DMg. D: However, GFAP immunoreactivity is strongly concentrated in the center of each rhombomere and distribute throughout the white matter. Scale bars 5 20 mm in A–D.
Fig. 6. A: DiI is injected into the ventricle of the stage 53/54
hindbrain. DiI uptake is exclusively seen in the radial glia at the
centers (A). B: These processes are almost exclusively derived from
the region where DMg-positive processes are found. The processes of
the boundary radial glia extend upward in a fan-shaped manner (B).
C,D: Vimentin and glial fibrillary acidic protein (GFAP) localization
in stage 60 hindbrain. Vimentin is mainly detected at the borders (C),
whereas GFAP is localized in the center (D). Arrowheads indicate
center regions. Scale bars 5 20 mm in B (also applies to A) and D (also
applies to C).
gpm6b (glycoprotein M6B) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 29 and 30, lateral view, anteriorleft, dorsal up.
