A Critical Role for Dorsal Progenitors in Cortical Myelination

Abstract

Much controversy regarding the anatomical sources of oligodendrocytes in the spinal cord and hindbrain has been resolved. However, the relative contribution of dorsal and ventral progenitors to myelination of the cortex is still a subject of debate. To assess the contribution of dorsal progenitors to cortical myelination, we ablated the basic helix-loop-helix transcription factor Olig2 in the developing dorsal telencephalon. In Olig2-ablated cortices, myelination is arrested at the progenitor stage. Under these conditions, ventrally derived oligodendrocytes migrate dorsally into the Olig2-ablated territory but cannot fully compensate for myelination deficits observed at postnatal stages. Thus, spatially restricted ablation of Olig2 function unmasks a contribution of dorsal progenitors to cortical myelination that is much greater than hitherto appreciated.

Introduction

Cortical myelination by oligodendrocytes facilitates the formation of a highly complex but organized brain structure in vertebrates (Zalc and Colman, 2000). Failure of oligodendrocyte differentiation may contribute to many neurological diseases such as the demyelinating disease multiple sclerosis (Noseworthy et al., 2000; Franklin, 2002). In the developing brain, oligodendrocyte precursor cells (OPCs) are derived from neural progenitors present in the ventricular and subventricular zones at early developmental stages with their maturation and myelination following at postnatal stages (Sauvageot and Stiles, 2002; Miller and Reynolds, 2004; Goldman, 2005).

Much of the controversy regarding the origins of oligodendrocytes in the spinal cord and hindbrain has been recently resolved with the discovery of a late-arising pool of dorsal progenitors that contribute to a larger population of oligodendrocytes derived from progenitors in the ventral neural tube (Cai et al., 2005; Fogarty et al., 2005; Miller, 2005; Vallstedt et al., 2005). However, the relative contribution of dorsal and ventral progenitor cells to oligodendrocyte myelination in the cortex is highly contended in the brain development field (Spassky et al., 2000; Woodruff et al., 2001; Marshall et al., 2003; Miller, 2005). A current view suggests that the majority of cortical oligodendrocytes are initially derived from the ventral telencephalon, populating the dorsal regions by migration and proliferation (Woodruff et al., 2001). Intriguingly, the fate-mapping analysis of progenitor cells in the forebrain of rodent neonates and in vitro cortical progenitor culture data suggest that oligodendrocytes can be derived from the dorsal telencephalon (Levison and Goldman, 1993; Gorski et al., 2002; Ivanova et al., 2003). Nonetheless, the extent and timing that dorsal progenitor cells contribute to cortical oligodendrogenesis during brain development remain unknown.

Olig2, a member of the basic helix-loop-helix Olig transcription factor family, plays a critical regulatory role in oligodendroglial cell fate specification in the developing spinal cord (Rowitch, 2004). Olig gene expression in the CNS persists through adulthood (Lu et al., 2000; Zhou et al., 2000), wherein it may participate in the glial response to injury and in brain repair (Arnett et al., 2004; Fancy et al., 2004; Balabanov and Popko, 2005). Gene-targeting studies show that Olig2 is essential for the formation of oligodendrocytes and motor neurons in the spinal cord (Lu et al., 2002; Takebayashi et al., 2002; Zhou and Anderson, 2002). However, neonatal lethality of Olig2 knock-out mice impedes the study of Olig2 function in oligodendrocyte myelination during postnatal brain development. In this study, we generated a conditional Olig2 allele using the Cre/lox system by flanking the Olig2 coding region on mouse chromosome 16 with loxP sites (see Fig. 1A–D). Utilizing the critical requirement of Olig2 in oligodendrocyte specification, we examined the role of dorsal progenitors in cortical myelination by deleting Olig2 in these progenitors. Olig2 ablation mediated by cortical progenitor-restricted Cre recombinases results in severe myelination defects in the cortex, and the myelination deficit cannot be fully compensated for by ventrally derived oligodendrocytes. Thus, spatially restricted ablation of Olig2 function unmasks an important contribution of dorsal progenitors to cortical myelination.

Figure 1.

Conditional ablation of Olig2 in cortical progenitors. A, Olig2 conditional knock-out mice are generated through homologous recombination in embryonic stem cells with the Olig2-coding region flanked by two loxP sites. B, Generation of Olig2-ablated alleles by Cre-mediated loxP site recombination preceded by Flpase-mediated FRT site recombination of the neomycin selection cassette in the targeted genome. C, Olig2 mutant alleles were confirmed by Southern blot with a 5′ external probe (wild-type and mutant fragment sizes; 5 and 7 kb, respectively). D, Olig2 mutant alleles are confirmed by PCR analysis [wt +/+, 100 bp; Olig2 mutant (c/+ and c/c), 150 bp]. E, Coronal sections of hGFAPCre forebrain at E14.5 were immunostained with a Cre antibody. Red and white arrows indicate the subventricular zone of the dorsal telencephalon and the ganglionic eminence, respectively. F, β-Galactosidase activity in the forebrain of hGFAPCre;Rosa26 mice at E14.5. Red and green arrows indicate the dorsal and ventral of forebrain, respectively. G–N, Expression of Olig2 and PdgfαR mRNAs (arrows) in the forebrain of Olig2c/-;hGFAP^Cre (Cko^G) and control mice (ctrl, Olig2c/+;hGFAP^Cre) was analyzed by in situ hybridization at E14.5 and E16.5 as indicated.

Materials and Methods

Generation of Olig2 conditional mutant mice.

To construct the floxed Olig2 targeting vector, we inserted two loxP sites to flank the Olig2 coding region, followed by a neomycin selection cassette, into a 10 kb SacI–SacII genomic fragment in the pKO-915 vector (Stratagene, La Jolla, CA). The targeting vector was linearized with NotI and electroporated into mouse J1 embryonic stem cells and G418-resistant cells were selected. Genomic DNA from drug-resistant cells was digested with HindIII and analyzed by Southern blot using a 0.5 kb or HindIII–SacI fragment as a 5′ probe for Olig2. Transmission of the target allele through the germline was confirmed by Southern blot. Subsequently a PCR strategy was used to identify the mutants. PCR primers 5′ and 3′ are agc cag ccc tca ctt gga gaa ctg ggc ctg and cgc tag agc ctg ttt tgc acg ttc acc, respectively. The sizes of PCR products for conditional mutants (cko) and wild-types are 350 and 150 bp respectively. Olig2 mutant mice, Cre activator lines, and Rosa26 reporter lines were maintained on a C57BL6/J and 129 SVJ hybrid background. All protocols involving the use of animals were approved by the Institutional Animal Care and Research Advisory Committee at University of Texas Southwestern Medical Center at Dallas.

RNA in situ hybridization.

Olig2 control and mutant brains from embryonic and postnatal stages were harvested from anesthetized mice. They were fixed in 4% paraformaldehyde at 4°C overnight, cryoprotected in 20% sucrose in PBS overnight, embedded in OCT, and cryosectioned at 16 μm. Digoxigenin-labeled riboprobes were used to perform RNA in situ hybridization as described in Lu et al. (2000). Probes used were as follows: Olig2, PdgfαR, Plp, and Mbp (Lu et al., 2002).

Immunostaining and histochemistry.

Immunostaining and β-galactosidase staining methods with tissue sections from mouse brains were as described previously (Lu et al., 2002). The following antibodies were used: Cre (1:500; Covance, Berkeley, CA), O4 (1:10; gift from Dr. Alex Gow, Wayne State University, Detroit, MI). Goat anti-mouse secondary antibodies conjugated to Cy2 or Cy3 (Jackson ImmunoResearch, West Grove, PA) were used for double-labeling experiments. Microscopy was performed on a Zeiss (Oberkochen, Germany) LSM 510 confocal microscope.

Electron microscopy.

Electron microscopy was performed as described previously. Briefly, the corpus callosum of forebrains from control and Olig2-ablated mice were dissected and fixed overnight by a fixative containing 2% glutaraldehyde in 0.1 m cacodylate, pH 7.2. Cross sections of these tissues were processed and thin sections were cut for electron microscopy.

Results

Olig2 ablation in cortical progenitor cells does not prevent OPC formation

Olig2 ablation in the dorsal telencephalon is initiated by an hGFAP^Cre deleter line (Zhuo et al., 2001) (Fig. 1B). At embryonic stages, the Cre recombinase is predominantly expressed in cortical multipotent progenitor cells (Fig. 1E), which give rise to neurons, oligodendrocytes, and astrocytes in the developing cortex (Zhuo et al., 2001; Malatesta et al., 2003; Zhu et al., 2005). When this Cre line was crossed with the Rosa26-LacZ reporter line (Soriano, 1999), expression of β-galactosidase in the progeny of recombined progenitor cells was found in most of neural cell types in the cortex (Fig. 1F, red arrow), but is essentially absent in the ganglionic eminence in the ventral forebrain at embryonic day 14.5 (E14.5) (Fig. 1F, green arrow), suggesting that the Cre activity is mainly confined to dorsal progenitor cells of the developing telencephalon (Fig. 1E). Thus, the initial Olig2 expression in the ganglionic eminence is not removed by Cre recombinase in Olig2c/-;hGFAP^Cre embryos at E14.5 (Fig. 1, compare G, H). This is consistent with the formation of PdgfαR+ OPCs in the ventral forebrain of these embryos to an extent comparable with the control (Fig. 1J, I). At E16.5, when Olig2 expression appears in the cortex of control embryos (Fig. 1K), hGFAP-Cre activity completely ablates Olig2 expression in this region (Fig. 1L). However, even with Olig2 ablation, OPCs that express PdgfαR are formed in the cortex (Fig. 1N) at levels similar to those seen in controls (Fig. 1M).

We found that there are essentially no Olig2+ cells in the cortex of the Olig2-ablated embryos. Thus, the Olig2-ablated cortex does not appear to be populated by ventral to dorsal migration of unablated Olig2+ OPCs from the ventral forebrain. We considered that Olig2 expression might be ablated by hGFAP-Cre activity en route while migrating into the cortex tangentially. However, hGFAP-Cre recombinase expression is confined to the dorsal subventricular zone (Fig. 1E), suggesting the Olig2-negative OPCs present in the Olig2-ablated cortex are mainly derived from dorsal progenitor cells of the telencephalon.

Cortical ablation of Olig2 results in severe myelination deficits in the cortex

Pups with the Olig2Cko;hGFAP^Cre genotype are born at normal Mendelian ratios; however, they develop an abnormal limb-clasping reflex and tremors beginning at postnatal week 2 (data not shown), reminiscent of the phenotypes described for dysmyelinating mutants (Sidman et al., 1964; Dupouey et al., 1979; Popko et al., 1987; Nave, 1994; Xin et al., 2005).

Because Olig2 ablation does not affect cortical OPC proliferation, we examined whether OPCs lacking Olig2 could undergo myelinogenesis postnatally. In the Olig2-ablated cortex, a series of premyelinating and myelinating oligodendroglial markers are reduced (Fig. 2A–N). Olig2 expression is essentially absent in the cortex (Fig. 2H), despite the presence of residual Olig2+ cells in the cerebral white matter (Fig. 2H, arrowhead). Expression of premyelinating oligodendrocyte markers such as O4 (Pfeiffer et al., 1993) is undetectable in the cortex at P14 and adulthood except for a small population of these cells present in the white matter (Fig. 2N) (data not shown). At stage P7, no expression of a myelin gene Mbp (myelin basic protein) is detectable in mutants (Fig. 2B). From P14 onward, expression of mature oligodendrocyte markers Mbp and Plp (proteolipid protein) is not observed in the cortex but is detectable in the white matter of the Olig2-ablated brain at markedly reduced levels (Fig. 2D, F, J). In contrast, the number of PdgfαR+ and NG2+ OPCs in the cortex appears unaltered (Fig. 2L) (data not shown). These data suggest that the loss of Olig2 causes the oligodendrocyte maturation process to be arrested at a progenitor cell stage before the formation of myelinating oligodendrocytes in the developing cortex.

Figure 2.

Oligodendrocyte myelination deficit in the Olig2-ablated cortex. A–N, Expression of oligodendroglial markers in coronal cortical sections from control and Olig2-ablated (Cko^G) mice at neonatal P7 (A, B), perinatal P14 (C, D, G–N), and adult stage P79 (E, F) was analyzed by in situ hybridization with antisense probes Olig2, Mbp, Plp/dm20, or PdgfaR, as well as O4 immunostaining as indicated by arrows. Arrowhead in H indicates residual Olig2+ cells in the cerebral white matter. O, P, Electron micrographs of cross-sections of the corpus callosum from control (O) and Cko^G (P) animals at P21. Red and blue arrows indicate myelin sheaths and dysmyelinated axons, respectively. Q–R, Expression of Mbp mRNA in the ventral forebrain of Olig2 mutant (Cko^G) mice at P14 (Q) and adult P79 (R). Arrows and arrowheads indicate Mbp expression in the cortex and the ventral forebrain, respectively. Scale bar: P, 500 nm.

The absence of myelin-related antigens is accompanied by morphological changes in myelinogenesis as assessed by electron microscopy. Ultrastructural examination of cross sections from the corpus callosum reveals a severe reduction in the number of myelinated axons as well as in the thickness of the myelin sheath formed in Olig2-ablated mice (Fig. 2P).

We observed considerable myelinating oligodendrocytes formed postnatally in the ventral forebrain of Olig2 mutant mice (Fig. 2Q, R, arrowheads), suggesting that a small population of oligodendrocytes observed in the white matter tract of Olig2 mutants is derived from ventral-to-dorsal migratory oligodendrocytes at postnatal stages.

Populations of dorsally and ventrally derived oligodendrocytes dynamically contribute to cortical myelination

Residual hGFAP-Cre activity detected in the ventral forebrain at postnatal stages (Malatesta et al., 2003) could contribute to cortical myelination deficits in the Olig2 mutant mice. To derive independent evidence for the role of cortical progenitors in oligodendrocyte myelination in the cortex, we used a second Cre deleter line, Foxg1-Cre, where Cre activity is restricted to telencephalon progenitors (Hebert and McConnell, 2000; Ferguson et al., 2002). Strikingly, Olig2 ablation mediated by Foxg1-Cre mainly occurs in the dorsal region of the cortex (Fig. 3G, red arrow) but not in the ventral forebrain at neonatal stages such as postnatal day 5 (P5) (Fig. 3H). As with Olig2 ablation by hGFAP-Cre, expression of myelin genes Mbp and Plp in the cortex of Olig2Cko;Foxg1^Cre mice is undetectable at perinatal stages P12 and P18 in dorsal cortical regions (Fig. 3D, E, I, J, red arrows), in sharp contrast to intense myelin gene expression in control cortices (Fig. 3A, B).

Figure 3.

Contribution of dorsal and ventral progenitors to cortical myelination in Olig2Cko;Foxg1^Cre mice. A–L, In situ hybridization labeling of Mbp (A–F) and Plp (I–L) in the forebrain of control (A–C) and Olig2Cko;Foxg1^Cre (Cko^F) (D–K) mice at P12, P18, and P28 as indicated. Arrows in D–F and I–K indicate the dorsal regions of the cortex. Blue arrows in G (dorsal) and H (ventral) indicate Olig2 labeling in the Olig2 mutant forebrain at P5. M–P, Mbp expression is shown in the ventral forebrain of control (M, N) and Olig2 mutant (O, P) mice. The square areas in M and O are shown at a larger magnification in N and P, respectively. Arrows in M and O indicate the dorsal cortical regions.

Despite the defect in cortical oligodendrocyte formation, a ventral-to-dorsal gradient of Mbp expression appears in the cortex at perinatal stages (Fig. 3D, E). By a young adult stage P28, a large area of the dorsal region of the cortex is populated by myelinating oligodendrocytes expressing Mbp (Fig. 3F), giving the impression that ventrally derived oligodendrocytes progressively migrate toward the dorsal telencephalon beginning at the perinatal stage. A similar ventral-to-dorsal migratory trend exhibited by ventral oligodendrocytes is observed with another oligodendrocyte differentiation marker Plp (Fig. 3I–K). Nonetheless, the absence of myelinating oligodendrocytes in the dorsal region of the cortex persists in young adult Olig2 mutants (Fig. 3F, K), in contrast to the well distributed oligodendrocytes in cortical regions seen in control mice (Fig. 3C, L).

Strikingly, despite the defect in cortical myelination (Fig. 3O, arrow), in the ventral forebrain, the amount of myelin gene expression (Fig. 3O, P) and the number of Plp+ oligodendrocytes (data not shown) are maintained in Olig2 mutants at levels similar to those of control littermates (Fig. 3, compare N, P), suggesting that dorsally restricted Olig2 ablation by Foxg1-Cre does not affect ventral oligodendrocyte formation, and partial myelination observed in the Olig2-ablated cortex is likely contributed by ventral migrating oligodendrocytes.

The critical role of dorsal progenitors in cortical myelination can be further confirmed by using an additional cortex-restricted Cre activator, Emx1-Cre, for Olig2 ablation (Fig. 4A–C) (Iwasato et al., 2000; Gorski et al., 2002). Cortical ablation of Olig2 mediated by Emx1-Cre results in severe dysmyelination in the cortex (Fig. 4, compare D, E), whereas the formation of myelinating oligodendrocytes in the ventral forebrain appears to be normal (Fig. 4F). The cortical dysmyelination phenotypes obtained with independent cortex-restricted Olig2 ablations are in agreement with each other, providing evidence that the dorsal progenitors of the forebrain play a critical role in cortical myelination.

Figure 4.

Cortical myelination deficit in Olig2Cko;Emx1^Cre mice. A, β-Galactosidase activity was examined in coronal brain sections of Emx1-Cre;Rosa26 reporter mice at P14. The red arrow indicates the cortex-restricted β-galactosidase expression. The green arrowhead indicates the ventral forebrain region such as the striatum. B–F, In situ hybridization labeling of Olig2 (B, C) and Mbp (D–F) is shown in the forebrain of control and Olig2Cko;Emx1^Cre (Cko^E) mice at P14. Arrows in B–E indicate Olig2 and Mbp expression in the cortex, respectively. Arrow in D indicates the formation of Mbp+ oligodendrocytes in the ventral forebrain of Olig2 mutants. Magnifications: A, 10×; B–E, 100×; D, 40×

Discussion

Oligodendrocytes are widely distributed in the brain, however, their specification and differentiation are tightly regulated in a spatially and temporally specified manner (Woodruff et al., 2001; Rowitch, 2004). Previous studies suggest that oligodendroglial populations initially appear in the ventral brain structures (Spassky et al., 2000; Tekki-Kessaris et al., 2001; Woodruff et al., 2001). However, the contribution of dorsal progenitors to cortical myelination remains controversial. This gap in our knowledge may present a challenge for repairing myelin damage in diseases such as multiple sclerosis, in which cortical demyelination is prominent (Peterson et al., 2001; Franklin, 2002).

In the spinal cord and hindbrain, the dorsal progenitors for oligodendrocytes emerge subsequent to a more abundant population of ventral progenitors and they appear to play a relatively minor role in total oligodendrogenesis there (Cai et al., 2005; Fogarty et al., 2005; Vallstedt et al., 2005). By analogy, dorsal progenitors might not play a major role in cortical oligodendrogenesis. In this study, we analyze cortical myelination by ablating an essential oligodendrocyte specification gene Olig2 in dorsal progenitor cells of the developing cortex. Spatially restricted Olig2 ablation leads to a nearly complete absence of myelination in the cortex at early postnatal stages and severe dysmyelination even at adulthood, suggesting that dorsal progenitor cells are a critical source for oligodendrocyte myelination in the developing cortex.

Despite the deficits of cortical myelination, the number of PdgfαR-expressing cells appears to be normal in the Olig2-ablated cortex, suggesting that differentiation of cortical progenitors up to the OPC stage and proliferation of OPCs do not require Olig2 function. However, absence of O4 expression in the cortical layers continuing through adulthood suggests that Olig2 is required for either the formation of O4+ premyelinating oligodendrocytes beyond the PdgfαR+ OPC stage or O4 expression in OPCs in the developing cortex.

In contrast to the cortex, Olig2 ablation in cortical progenitors does not prevent the formation of myelinating oligodendrocytes in the postnatal ventral forebrain. The presence of a small population of Olig2+ cells and oligodendrocytes expressing O4, Mbp, and Plp in the white matter of Olig2 mutants is likely attributable to the ongoing migration or maturation of premyelinating oligodendrocytes derived from the ventral forebrain, such as the ganglionic eminence (He et al., 2001). These ventrally derived oligodendrocytes contribute to cortical myelination by progressively migrating into the dorsal region of the Olig2-ablated cortex where myelination is limited, however, they cannot fully compensate for the cortical myelination deficits. Contribution of dorsal and ventral progenitors to cortical myelination may occur in a temporally specified manner, however, because the final number and distribution of mature oligodendrocytes are tightly controlled (Barres and Raff, 1994; Calver et al., 1998), the dynamics and extent of the contribution for ventrally derived oligodendrocytes to cortical myelination during normal brain development remain unclear at present. Interestingly, all three mutants with Olig2 ablation develop tremors to a variable extent perinatally, suggesting cortical dysmyelination contributes to this phenotype. The severity of dysmyelination in Cre-mediated Olig2 mutant cortices from different Cre lines may depend on the extent of Olig2 ablation in cortical regions. Nonetheless, Olig2 ablation in the cortical progenitors mediated by independent cortex-restricted Cre activator lines results in consistent myelination deficits in the developing cortex, suggesting that oligodendrocytes of the cortex are largely derived from dorsal progenitors rather than ventral-to-dorsal migrating oligodendrocytes. Unmasking an important dorsal source of myelinating cells for the cortex may illuminate avenues for myelin repair in demyelinating diseases.