Temporal and Spatial Pattern of Olig3 Expression in Spinal Cord Demonstrated by In Situ Hybridization
The expression of Olig3-mRNA in fetal mouse spinal cord was examined by in situ hybridization. Olig3 expression appeared in the neural tube by E9.5 (Takebayashi et al.,2002a). The pattern of expression in spinal cord was examined in detail in coronal sections. The dorsal-most portion expressed Olig3, with the exception of the roof plate (Fig. 1A). No expression was detected in the remaining part of the neural tube at this stage. E10.5 spinal cord also contained Olig3-expressing cells exclusively in its dorsal-most region (Fig. 1B). The dorsal Olig3 domain was reduced in size by E11.5 (Fig. 1C). In addition to the dorsal-most region, Olig3-expressing cells appeared in the ventral half of the spinal cord by this stage, as reported previously (Takebayashi et al.,2002a). Olig3-mRNA-positive cells in the ventral half were grouped into three distinct cell clusters (arrows in Fig. 1C; Takebayashi et al.,2002a). The Olig3-mRNA-positive cells in the dorsal-most region were observed both in the ventricular zone and in the thin mantle layer close to the pial surface, while those in the ventral half were positioned in the border regions between the ventricular zone and mantle layer (Fig. 1C).
Figure 1. Distribution of Olig3-mRNA in fetal mouse spinal cord. A,B: E9.5 and E10.5, respectively. Olig3 expression is restricted to the dorsal-most portion of the neural tube. C: E11.5. Olig3-expressing domain in the dorsal spinal cord is reduced, while new Olig3 domains appear as three distinct spots in the ventral spinal cord (arrows). D: E12.5. Olig3 expression in the dorsal spinal cord remains in a region lateral to the roof plate and in the vertical cell strand (arrows), both of which are magnified in E. Three distinct ventral Olig3 spots are also observed (arrowheads). E: The discontinuous vertical cell strand is magnified. These cells course within the mantle layer. F: E13.5. Olig3-expressing cells are aligned along the outer edge of the ventricular zone. Dorsal-most Olig3 expression is restricted to only few cells. G: E14.5. Olig3 expression is observed in the intermediate and ventromedial parts of spinal cord, and also in the posterior median septum. The dorsal horn (DH) is devoid of Olig3-expressing cells, except for its basal portion (arrowheads). H: Higher magnification of the posterior median septum (arrows). Arrowhead indicates an Olig3-mRNA-containing cell. I: E15.5. Olig3-expressing cells are mostly restricted to the ventromedial part of spinal cord, and only a few cells at the intermediate level express Olig3 (arrowheads). Scales in A–D, F, G, I = 200 μm, and in E, H = 20 μm.
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By E12.5, the Olig3-expressing region had been further reduced in size in the dorsal spinal cord. Cells lateral to the roof plate exhibited Olig3 expression. In addition, Olig3 expression was also observed in a few cells arranged dorsoventrally in the medial part of the mantle layer (Fig. 1D,E, arrows). The ventral Olig3-positive cells formed three distinct clusters (Fig. 1D, arrowheads), as observed at E11.5. The dorsal Olig3 domain was greatly reduced in size in E13.5 spinal cord compared with E12.5, and only a few cells exhibited Olig3 expression in the dorsal-most portion (Fig. 1F). These cells were aligned lateral to the dorsal ventricular zone, in a discontinuous cell strand. Although Olig3-expressing cells were roughly divided into dorsal and ventral groups in the mantle layer, the three distinct clusters in the ventral group were no longer recognized in the ventricular zone (Fig. 1F).
By E14.5, the white matter (or marginal zone) had developed in the peripheral part of the spinal cord and the contour of the gray matter (or mantle layer) had become H-shaped (Fig. 1G), somewhat resembling mature spinal cord. A small number of Olig3-expressing cells were positioned in the base of the dorsal horn (arrowhead in Fig. 1G). At this stage, the posterior median septum developed to separate the dorsal funiculus on both sides. While Olig3-expressing cells disappeared in the dorsal-most portion of the spinal cord, a limited number of such cells were observed in the posterior median septum (Fig. 1H). In addition, the base of the dorsal horn and the ventromedial part of the ventral horn contained Olig3-positive cells. This pattern of expression of Olig3 continued to at least E15.5 in the spinal cord (Fig. 1I), although the number of Olig3-expressing cells decreased.
Olig3lacZ expression in developing spinal cord
We generated lacZ knock-in mice as shown in Figure 2A–C, and carried out short-term lineage tracing. The reporter gene, lacZ, was knocked-in to the Olig3 locus (Fig. 2A). To confirm correct expression of lacZ in the Olig3 lineage cells, we compared localization of LacZ in the neural tube with that of Olig3-mRNA. Figure 2D shows E9.25 mouse with whole-mount X-gal staining. Olig3 signals were detected in the paramedian dorsal midline, similar to the pattern of localization of Olig3-mRNA (Takebayashi et al.,2002a). Expression of LacZ was further examined in coronal sections of E10.5 spinal cord. Almost all Olig3-positive cells (Fig. 3B) were immunoreactive to β-galactosidase (Fig. 3A,C), indicating correct expression of the reporter gene.
Figure 2. Construction of the Olig3lacZ knock-in targeting vector. A: Targeted mutation of the Olig3 locus. Strategy for targeted replacement of Olig3 with lacZ. The open box indicates the Olig3 exon and the filled box the bHLH domain-encoding region. The lacZ and floxed PGK-neo cassette were inserted using an EcoRI site and XbaI site in the exon. The 5′ probes used for Southern-blot analysis are shown below the wild-type genomic map. B: Southern-blot analyses of genomic DNA isolated from mouse tails. BamHI-digested tail DNA was hybridized with 5′ probe. The 5′ probe detects 17.5-kb wild-type and 12.5-kb mutant bands. C: PCR analysis of tail DNA extracted from wild-type or Olig3+/- mice, using primers specific for wild-type Olig3 allele and Olig3lacZ allele. The positions of primers are shown in A. D: Whole-mount X-gal staining of E9.25 mouse. Paramedian dorsal portion (arrowheads) in the hindbrain (Hb) and spinal cord/neural tube (SpC/NT) show X-gal staining. Scale bar = 500 μm.
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Figure 3. Presence of LacZ in Olig3-immunoreactive cells. E11.5 Olig3lacZ mouse spinal cord was double-labeled with anti-β-galactosidase (A) and anti-Olig3 antibodies (B) and observed under a confocal laser scanning microscope. C: The images have been digitally merged. Olig3-immunoreactive cells (B, red) are simultaneously positive for β-galactosidase (A, green). Note that the roof plate is immunoreactive for β-galactosidase but not for Olig3 (arrows). Scale bar = 100 μm.
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We next performed a short-term lineage tracing experiment using X-gal staining. As mentioned above, the dorsal-most portion of the spinal cord expressed LacZ in early, e.g., E9.5, neural tube (Fig. 4A). In addition to the X-gal-positive cells in the Olig3-expressing domain, the labeled cells were scattered in the subpial portion of the dorsolateral and lateral parts of the neural tube (arrows in Fig. 4A). Since we could not detect these cells by in situ hybridization (Fig. 1A), they had probably ceased Olig3 expression and had, therefore, become undetectable by in situ hybridization, though they were still detectable by sensitive X-gal staining. These X-gal-positive cells appeared to be derived from the dorsal Olig3-expressing domain and to be migrating ventrally.
Figure 4. X-gal staining of developing Olig3lacZ mouse spinal cord. Coronal sections were subjected to X-gal histochemistry and counterstained with Nuclear Fast Red. A: E9.5. The dorsal-most portion of the neural tube exhibits intense X-gal reaction. In addition, a few X-gal-positive cells were sporadically observed in the subpial portion in the dorsolateral and lateral parts of spinal cord (arrows). B: E10.5. X-gal-positive cells are densely packed in the dorsal-most spinal cord. The dorsal-to-ventral cell strand (DVS) is formed from the dorsal Olig3 domain to the ventral-most level. Dorsally in the strand, the cells are positioned in the subpial portion, while in the ventral part of the strand X-gal-positive cells course medially to the ventral horn. Note that X-gal-positive cells reach the ventral-most part of the spinal cord (arrows). C: E11.5. The pattern of expression of lacZ is essentially the same as that at E10.5. In addition, weak X-gal-positive cells appear in the ventral ventricular zone, which are magnified in D. D: A higher magnification of the ventral ventricular zone. Weakly Olig3-positive cell clusters are indicated by arrows, and probably correspond to ventral Olig3-mRNA-expressing cells. The most ventrally located X-gal-positive cell cluster may include both ventrally- and dorsally-derived Olig3 lineage cells. E: E12.5. X-gal-positive cells are aligned along the outer edge of the ventricular zone and have accumulated in the intermediate level of spinal cord, which is magnified in F. Note that nearly all the roof plate cells are X-gal-positive. F: Higher magnification of the intermediate level. The mediolateral X-gal-positive cell cluster (curved arrow) is continuous with the DVS (smaller arrows). G: E14.5. X-gal cells are roughly grouped into three clusters, i.e., the posterior median septum (arrow), the mediolateral cell cluster, and the ventromedial cell group. H: E18.5. Three cell clusters are still observed in the dorsal midline (arrow), intermediate level, and the ventromedial part, although the number of labeled cells is decreased. I–K: E10.5. Double-immunofluorescence staining of β-galactosidase (I) and Islet1/2 (J) observed under a confocal laser scanning microscope. The images have been digitally merged in K. Note that double-labeled cells (arrowheads) are observed in the DVS. L,M: E10.5. X-gal staining followed by in situ hybridization of Math1 (L) and Brn3a (M). Light blue staining is X-gal reaction product while dark blue represents mRNA. Arrowheads indicate double-labeled cells in the dorsal spinal cord. Scale bars = 100 μm in A–C, E, G, H; = 50 μm in D, F, I–M.
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In E10.5 spinal cord, in addition to the dorsal-most cells, a “dorsal-to-ventral cell strand” (DVS) of X-gal-positive cells was formed from the dorsal-most portion to ventral spinal cord (Fig. 4B). Unexpectedly, X-gal-positive cells were observed in the ventral-most part of the spinal cord at this stage (arrows in Fig. 4B). Since ventral Olig3-mRNA was not yet detected in E10.5 spinal cord (see Fig. 1B), these X-gal-positive cells were probably immigrating Olig3-expressing cells in the dorsal-most spinal cord. The DVS was aligned along the pial surface in the dorsal spinal cord and then coursed between the ventricular zone and the ventral horn in the ventral spinal cord.
The E11.5 spinal cord of the Olig3lacZ mice also exhibited the DVS (Fig. 4C). The cell strand was thicker and the number of labeled cells higher at this stage than at E10.5. X-gal-positive cells in the most ventral level were positioned just dorsal to the floor plate and lateral to the ventricular zone. At this stage, Olig3-mRNA expression had begun in the ventral ventricular zone (Fig. 1C), and weakly X-gal-labeled cells in the ventricular zone (arrows in Fig. 4D) probably corresponded to the ventral Olig3-expressing groups.
In E12.5 spinal cord, the DVS coursed between the ventricular zone and the mantle layer in the dorsal spinal cord as the mantle layer developed lateral to the strand (Fig. 4E). At this stage, X-gal-labeled cells began to accumulate in the intermediate level of the spinal cord, forming the mediolateral cell cluster (Fig. 4E,F). This cell cluster was continuous with the DVS (arrows in Fig. 4F). In the ventral spinal cord, X-gal-positive cells were distributed mainly in the ventromedial portion. Although the roof plate cells did not express Olig3-mRNA (Fig. 1), LacZ was labeled immunohistochemically or with X-gal histochemistry in these cells (Figs. 3A,C and 4C,E).
E14.5 Olig3lacZ mouse spinal cord contained more X-gal-positive cells in the intermediate and ventral levels. In addition, almost all of the cells in the posterior median septum were X-gal-positive (Fig. 4G). In contrast, few or no labeled cells were detected in the dorsal horn, except in its base. During the next three days, the number of X-gal-positive cells decreased greatly, and in the E18.5 Olig3lacZ mouse such cells were distributed in the intermediate region, probably including the base of the dorsal horn and the anterior horn (Fig. 4H). The posterior median septum also contained a small number of X-gal-positive cells (arrow in Fig. 4H).
Recently, eight types of dorsal interneurons have been distinguished based on combinatorial expression of various transcription factors (Bermingham et al.,2001; Gross et al.,2002; Helms and Johnson2003). To characterize dorsal Olig3 lineage cells in the early spinal cord, we used three transcription factors as dorsal interneuron markers: Islet1/2 (homeobox transcription factor for dI3), Math1 (bHLH transcription factor for dI1), and Brn3a (POU domain transcription factor for dI1, dI2, and dI3) (Bermingham et al.,2001; Gross et al.,2002; Helms and Johnson2003). E10.5 spinal cord was double-labeled with anti-β-galactosidase antibody and with anti-Islet1/2 antibody, and observed under confocal laser scanning microscopy.
Several LacZ-positive cells were also immunoreactive to Islet1/2 (Fig. 4I–K), and nearly half of Olig3 lineage cells were double-labeled with Islet1/2 in the DVS (44.4 ± 7.47%). In addition, Olig3 lineage cells appeared to express Math1 (Fig. 4L) or Brn3a (Fig. 4M) in the dorsal part of the spinal cord, though the sensitivity of in situ hybridization was decreased after X-gal staining. These patterns of expression indicate that Olig3 lineage cells differentiate into dI1, dI2, and dI3 dorsal interneurons, consistent with recent findings by Müller et al. (2005).
Ventral Migration of Cells Derived From Dorsal Spinal Cord
To verify ventral migration and differentiation of the cells derived from the dorsal-most region, including the Olig3 domain, in the spinal cord, we carried out electroporation of the EGFP gene to the dorsal-most portion of spinal cord and examined the migration and morphology of EGFP-expressing cells.
Prior to the electroporation experiment, we examined Olig3 expression in chick embryonic spinal cord with an in situ hybridization method. The E2 neural tube, the youngest stage examined, expressed Olig3-mRNA exclusively in its dorsal-most portion (Fig. 5A). No signals were detected in other parts of the neural tube. In E3 spinal cord, the dorsal-most portion exhibited intense expression of Olig3. In addition, three distinct spots of Olig3-mRNA were located in the ventral half of the spinal cord (Fig. 5B). This pattern of expression lasted until E4 in the spinal cord (not shown). Dorsal expression weakened with embryonic development (Fig. 5C). In E6 spinal cord, Olig3-expressing cells in the dorsal-portion were observed lateral to the roof plate. A short vertical cell strand also exhibited Olig3-mRNA expression (arrowheads in Fig. 5C). By contrast, at this stage the ventral spinal cord contained more Olig3-expressing cells in the ventral part than in the previous stages, as a result of which the ventral three Olig3 spots had become continuous. The spatio-temporal pattern of expression of Olig3 was thus similar in fetal mouse and early chick embryo, especially in dorsal spinal cord. The pattern of expression of Olig3 in E2 chick embryo neural tube appeared to correspond to that in E10.5 mouse spinal cord, while those in E3 and E4 chick spinal cord were similar to that in E11.5 mouse spinal cord.
Figure 5. Dorsal-to-ventral migration of the cells derived from dorsal-most spinal cord in chick embryo. A–C: Olig3-mRNA distribution in chick embryo neural tube. A: E2. Olig3 expression is restricted to the dorsal-most portion of the neural tube. B: E3. The dorsal-most portion of the spinal cord exhibits intense expression of Olig3-mRNA, while weak expression is found in the ventral spinal cord as three distinct clusters (arrowheads). C: E6. Dorsal Olig3 expression is weak while ventral Olig3-expressing cells are increased in number. Arrowheads indicate the vertical Olig3 cell strand in the dorsal spinal cord. D-I: Electroporation of EGFP gene-containing plasmid to the dorsal-most spinal cord and subsequent distribution of EGFP-expressing cells. Brown color represents EGFP expression and dark blue in H and I represents Nkx2.2 immunoreactivity. D,F,H: Lower-magnification pictures showing sites of electroporation (larger arrowheads). Areas indicated by larger arrows in D, F, and H are magnified in E, G, and I, respectively. Smaller arrows indicate commissural fibers in the ventral commissure. Note that the EGFP-expressing cells have accumulated at the intermediate level. Smaller arrowheads in H indicate radial migration of cells which are crossing the DVS. E,G, I: Higher magnifications of D, F, and H, respectively. E and G are of the intermediate level, while I shows the ventral-most level. Arrowheads in G indicate EGFP-expressing cells in the intermediate level, and the arrow in G indicates a cell with fusiform shape, which appears to be migrating ventrally. Arrows in I indicate EGFP-expressing cells with commissural fibers located in the ventral-most level of the spinal cord, adjacent to Nkx2.2-positive p3 domain. Scale bars = 100 μm in A; = 20 μm in B–D, F, H; = 50 μm E, G, I.
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We then performed electroporation to transfect the EGFP gene into the dorsal neural tube at E3 or E4, and the distribution of EGFP-expressing cells was observed three days after electroporation. Portions of spinal cord with electroporation into restricted regions were selected and analyzed. Of over 100 cases of electroporation examined, in at least 5 cases the EGFP gene was successfully transfected to a restricted dorsal region (larger arrowheads in Fig. 5D,F,H), which included the dorsal Olig3 domain. In these cases, EGFP-expressing cells can be considered as progeny of the dorsal spinal cord cells. In such cases of restricted electroporation, EGFP-expressing cell bodies were mostly located in the intermediate level of the mantle layer (larger arrows in Fig. 5E,G). The labeled cells extended fibers crossing the ventral commissure, which finally reached the contralateral ventral funiculus (smaller arrows in Fig. 5D,F,H). Thus, some dorsally derived cells differentiated into commissural interneurons at the intermediate level. Some EGFP-expressing cells were fusiform in shape (Fig. 5G, arrow), and appeared still to be migrating ventrally. Furthermore, EGFP-expressing cell bodies were also observed in the ventral-most level of the spinal cord, adjacent to the Nkx2.2 domain (Fig. 5H). These cells also extended commissural fibers (Fig. 5H,I). These findings indicate that dorsal cells contribute to commissural interneurons at the intermediate and ventral-most levels.