MELK is expressed by neural progenitors
Because MNP characteristics depend upon the age at which they are isolated, producing neurons earlier and glia at later developmental times (Qian et al., 2000
; Irvin et al., 2003
), we analyzed MELK expression in neurospheres derived from different aged animals. MELK was expressed by NS from embryonic day 12 (E12) telencephalon, as well as E17 and P0 cortex ( A, a). After growth factor withdrawal, MELK mRNA levels declined dramatically, to <10% of the original expression after 24 h ( A, b). MELK was also expressed in NS derived from adult striatal SVZ (unpublished data).
Figure 1. MELK is highly enriched in cultures containing multipotent progenitors. (A, a) MELK expression as determined by semiquantitative RT-PCR using GAPDH as a standard in neurospheres (NS) and differentiated sister cultures generated by the withdrawal of bFGF (more ...)
MELK expression declined as progenitor differentiation proceeded, whether the differentiation was induced by growth factor withdrawal or addition of retinoic acid ( B). NS differentiation was confirmed by increased expression of neurofilament heavy chain (NFH), GFAP, and proteolipid protein (PLP)—markers for neuronal, astrocytic, and oligodendroglial differentiation, respectively.
MELK mRNA expression in GZs in vivo
RT-PCR analysis shows that MELK mRNA was expressed in the developing brain during early and mid-embryonic periods with a dramatic decline between E15 and E17, with no detectable expression in adult whole brain or lung (used as a control tissue) ( A). MELK expression in ES cells was relatively high.
Figure 2. Developmental and regional expression of MELK mRNA in vivo. (A, a) MELK expression in ES cells and during brain development analyzed by RT-PCR. The triangle indicates increasing cycle number. (B) In situ hybridization with radiolabeled antisense MELK (more ...)
In situ hybridization ( B) demonstrated that MELK mRNA was expressed throughout the central nervous system (CNS) within periventricular GZs as early as E9. This general pattern of expression persisted through early postnatal periods to adulthood, including cells of the anterior subventricular zone (SVZa) and rostral migratory stream ( B, b–h). No specific hybridization was detectable in the CNS outside of GZs, indicating that MELK is not expressed by mature cell types. In the adult brain, the only hybridization found was in the SVZ lining the lateral ventricle ( B, h) along its entire rostrocaudal extent, but within only a minority of SVZ cells along the lateral side of the lateral ventricle ( D, arrows). No labeling was detected in adult hippocampus (HC) ( D, a and b) or other GZs. Lack of detection of MELK in hippocampus was further confirmed by RT-PCR ( C).
To further define cell types that express MELK, we performed double labeling with in situ hybridization and immunohistochemistry (). In the brain, MELK was expressed by proliferating cell nuclear antigen (PCNA)–positive cells ( A, a–e). Outside the brain (in the same sections) we did not detect MELK mRNA in PCNA-positive cells, indicating that MELK is not universally expressed by dividing cells ( A, f).
Figure 3. MELK expression in proliferating CNS progenitors in vivo. The photomicrographs in A–D are of dipped sections sampled from the regions identified in the brain section at the top. Sections were hybridized with MELK cRNA, and then stained by immunohistochemistry. (more ...)
MELK was also expressed by GFAP-containing cells, although the extent of this colocalization was dependent on the developmental stage. Throughout embryonic and early postnatal ages, MELK-expressing cells were GFAP-negative ( B, a and b, insets) because there is little or no SVZ GFAP expression at these ages (Imura et al., 2003
; Fox et al., 2004
). Subsequently, as GFAP expression increased in the SVZ, MELK mRNA was detected in some SVZ GFAP-expressing cells. In the adult SVZ, MELK expression was also detectable in GFAP-positive cells ( B, inset in c). MELK, unlike the adult case, was expressed in the hippocampus during early postnatal ages, at least up to P7, within GFAP-positive cells at the hilar border of the dentate gyrus ( C, inset in a). TuJ1-positive neurons in the dentate gyrus or anywhere else did not express MELK ( C, inset in b).
MELK mRNA was also identified within the external granule cell layer (EGL) of the developing cerebellum ( D) within the outer proliferative EGL with no expression in the inner, premigratory, TuJ1-positive zone ( D, c). Expression in the EGL was detectable as early as the EGL could be distinguished clearly at E13 (unpublished data), and disappeared along with the EGL during later postnatal development.
The MELK promoter lies upstream of its first exon, and is active only in undifferentiated neural progenitors
The isolation and initial characterization of a 3.5-kb mouse and human MELK promoter (PMELK) is described in the online supplemental data (available at http://www.jcb.org/cgi/content/full/jcb.200412115/DC1
). To investigate the specificity of the PMELK sequence, cells were transfected with PMELK-EGFP or control vectors and then sorted based on EGFP expression. RT-PCR analysis was used to detect MELK expression both in EGFP-positive and -negative populations ( A). The PMELK-EGFP–positive fraction was highly enriched for MELK mRNA as compared with the EGFP-negative fraction or unsorted cells ( A, c).
Figure 4. The MELK promoter is active only in undifferentiated neural progenitors. (A) FACS analysis of UD cells transfected with the MELK promoter–containing (a) and control (b) EGFP clones. (c) RT-PCR for MELK after separation of the fluorescence-positive (more ...)
Using the PMELK-EGFP construct, we characterized the cellular specificity of MELK expression in cortical progenitors derived from E12 embryos ( B). Cells expressing EGFP driven by the CMV promoter were morphologically heterogeneous, whereas MELK promoter-driven EGFP-positive cells were relatively homogeneous with a fusiform shape ( B). MELK-positive cells expressed the neural progenitor markers nestin, NG2, RC2, BLBP, and SOX2 in proliferating cultures ( B, a–o), but no PMELK-driven EGFP was detected in cells expressing differentiation markers (TuJ1, neurons, GFAP, astrocytes and O4, oligodendrocytes), even in proliferating cultures ( B, p–v). These data indicate that the MELK promoter is active only in neural progenitors, and not in more differentiated cells. Furthermore, the data are consistent with, and support the findings of, native MELK expression described above.
MELK is a marker for tripotent, self-renewing progenitors in embryonic cortical cultures
MNPs have the fundamental properties of self-renewal and multipotency. Therefore, we tested the ability of MELK-expressing cells to form primary and secondary neurospheres and examined the differentiation capacity of these spheres. The LeX antigen is expressed by neural progenitors, and LeX-positive cells form neurospheres (Capela and Temple, 2002
). Immunocytochemistry shows that virtually all EGFP-expressing cells also expressed LeX ( A). Cultures from E12 telencephalon were then separated by FACS using an anti-LeX antibody ( B). Approximately 65% of the cells in the cultures were LeX-positive ( B, a and b). RT-PCR analysis demonstrated that MELK mRNA was completely restricted to the LeX-positive fraction ( B, c). LeX sorting also resulted in enrichment of other neural stem cell–associated genes, including nucleostemin (NCS) and SOX2. In contrast, musashi1 (Msi1) and GFAP were not enriched in the LeX-positive fraction ( B, c), consistent with previous observations of their expression in both progenitor and nonprogenitor populations (Kaneko et al., 2000
Figure 5. MELK-expressing progenitors are neurosphere-initiating MNPs. (A) PMELK-EGFP expression overlaps with LeX immunofluorescence. Arrows/arrowheads indicate LeX-positive, MELK-negative cells in the same culture. Arrowhead is negative, arrow is positive. (B) (more ...)
We next tested the capacity of MELK-expressing cells to form neurospheres. MELK-positive E15 progenitors generated ~5 times more primary neurospheres than LeX-positive cells at a density (2,000 cells/ml) where most spheres form from a single cell (Tropepe et al., 1999
) ( C). Given that virtually all MELK-positive cells express LeX, these data suggest that the MELK-positive fraction of LeX-expressing cells is more highly enriched for sphere-initiating cells. LeX-negative populations did not generate neurospheres under these conditions. Primary spheres derived from MELK-positive progenitors formed “secondary” neurospheres when dissociated and replated ( C, g), indicating self-renewal capacity. Control cultures transfected with cytomegalovirus promoter (PCMV)-EGFP yielded equivalent percentages of neurospheres in EGFP-positive and -negative fractions (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200412115/DC1
), indicating that the present findings are not simply due to a general preference for transfection of sphere-forming cells.
To more accurately determine the frequency of neurosphere-initiating cells (NS-ICs), sorted progenitors from E15 telencephalon cultures were serially diluted. At each density, MELK-positive progenitors gave rise to significantly greater numbers of spheres than did LeX-positive progenitors. Approximately 1 out of 10 MELK-positive progenitors were NS-IC, whereas 1 out of 29 LeX-positive cells was NS-IC ( C, h and i). Thus, even at an extremely low seeding density, MELK-expressing cells were highly enriched for NS-IC.
Staining of undifferentiated MELKP-EGFP–derived neurospheres revealed that virtually all cells expressed nestin and LeX ( D, a and b). After differentiation of primary or secondary spheres, staining revealed that the spheres formed neurons, astrocytes, and oligodendrocytes ( D, c–e). These data demonstrate that MELKP-derived cells are indeed multipotent, self-renewing progenitors.
MELK is expressed by self-renewing multipotent progenitors in vivo
To examine whether MELK can be expressed by MNP in vivo, we constructed transgenic reporter mice using the MELK promoter to drive EGFP expression. In general, EGFP expression recapitulated the expression pattern of endogenous MELK mRNA being largely restricted to developing GZ, including the GZ surrounding the lateral ventricles and the rostral migratory stream, the inner granule zone of the early postnatal hippocampus, and external granule cells of the neonatal cerebellum ( A, b–e).
Figure 6. MELK-positive cells are self-renewing multipotent progenitors in vivo. (A, a) Experimental design of long-term passage of neurospheres from transgenic reporter mice. Expression of EGFP in a P8 transgenic mouse demonstrating specific signals in the SVZ (more ...)
Cortical progenitors from P1 transgenic mice were cultured as neurospheres according to the schemes shown in A (a). Primary neurospheres were all EGFP positive ( A, f). After up to 12 clonal passages over 4 mo, neurospheres remained EGFP positive ( A, g and h). EGFP-positive neurospheres derived from the MELK-EGFP transgenic mice were multipotent, containing neurons, astrocytes, and oligodendrocytes after induction of differentiation at each passage ( A, f). These findings indicate that MELK expression persists in progenitors within clonally passaged neurospheres throughout multiple rounds of self-renewal. To determine whether sphere-initiating progenitors are EGFP positive, we performed FACS for EGFP and then grew neurospheres at high and clonal densities from P1 forebrain. As is shown in B (b), EGFP-expressing cells yielded neurospheres both in clonal and high density conditions. In contrast, MELK-negative progenitors failed to form neurospheres even in high density conditions. Thus, neurosphere-forming cells derived from the developing brain express MELK, and MELK expression persists throughout multiple passages, suggesting that it is expressed by long-term, self-renewing progenitors.
MELK regulates MNP proliferation
The studies thus far demonstrate that MELK is expressed by MNPs. To determine the function of MELK in these cells, we assessed the effects of overexpression and knockdown according to the scheme shown in A. Neurospheres were generated from the following: E12 telencephalon as a stage of neurogenesis, E15 and P0 cerebral cortex as stages of transition and gliogenesis, respectively. Adherent cultures of progenitors derived from neurospheres were transduced with expression vectors or appropriate double-strand RNA designed to be small inhibitory RNA (siRNA) or controls. Using PCMV-EGFP we estimated transfection efficiency at ~70% (unpublished data). Specificity and efficacy of the overexpression and siRNA vectors used is described in the online supplemental data, and illustrated in Fig. S3 (available at http://www.jcb.org/cgi/content/full/jcb.200412115/DC1
). In addition to mock transfection, we used NCS and CRT1 siRNAs as positive and negative controls, due to previous studies demonstrating that NCS promotes MNP proliferation, whereas CRT1 does not (Rauch et al., 2000
; Tsai and McKay, 2002
). These adherent cultures varied in their characteristics, depending on age. E12 telencephalic cells largely contained nestin/LeX-positive cells, with few cells bearing differentiation markers, whereas cultures from older animals contained more cells expressing differentiation markers ( B, a–g). Spheres were generated from transfected cultures and propagated. To assay sphere potency, we differentiated E12-derived spheres by removal of growth factor and plating on substrate, and found that they reliably and readily formed neurons, astrocytes, and oligodendrocytes ( B, h–j).
Figure 7. MELK regulates neural progenitor proliferation. (A) Experimental design. (B) Characterization of adherent progenitors from neurospheres generated from E12 telencephalon and P0 cerebral cortices (a–f). Monolayer progenitor cultures from neurospheres (more ...)
Overexpression of MELK in neural progenitors yielded increased numbers of spheres after transfection. MELK knockdown resulted in the opposite effect: a diminished number of spheres compared with controls, indicating that MELK regulates the proliferation of sphere-forming progenitors ( C, a–c). As expected, knockdown of NCS had effects similar to that of MELK siRNA, whereas knockdown of CRT1 had no effect. The total number of cells within cultures was affected as well, with MELK overexpression resulting in a greater number of cells and knockdown in fewer cells. MELK overexpression resulted in significantly larger spheres, compared with control conditions or siRNA for MELK ( C, e). This latter finding suggests that MELK overexpression influences not only sphere-initiating cells, but also cells that contribute to overall sphere size.
MELK knockdown inhibited (whereas overexpression enhanced) BrdU labeling indices after pulse labeling, indicating a direct effect on proliferation ( C, f). The number of dead or dying cells was not affected by siRNA treatment ( C, g). These data suggest that MELK influenced proliferation itself rather than survival of proliferating cells.
Spheres generated after MELK knockdown or overexpression were multipotent, yielding neurons, astrocytes ( D), and oligodendrocytes (not depicted). The neurogenic capacity was not significantly altered by the change of MELK expression, indicating that endogenous MELK likely regulated the proliferation of sphere-forming cells, which were, in turn, multipotent, without influencing the relative numbers of differentiated cells (i.e., the proliferation of committed progenitors). To determine whether MELK directly influences differentiation, we analyzed the effects of MELK knockdown and overexpression in adherent E12 cortical progenitors that were then differentiated on the coverslip for 5 d by withdrawal of bFGF, and found no effect on the formation of neurons, astrocytes ( D, c and d), or oligodendrocytes (not depicted). Together, these functional experiments indicate that MELK regulates MNP proliferation and their capacity to self-renew, at least in the short term, without a major effect on the proliferation of committed progenitors or on cell fate decisions.
MELK is necessary for the production of GFAP-negative MNPs from neonatal astrocyte cultures
Recent studies have documented the ability of GFAP-positive cells of the adult forebrain SVZ to form rapidly amplifying progenitors in the presence of bFGF (Imura et al., 2003
; Morshead et al., 2003
). These transition processes can be monitored by RT-PCR and immunocytochemistry (). 24 h of bFGF treatment resulted in diminished GFAP mRNA expression and increased NCS expression. MASH1 mRNA was up-regulated after 7 d, but not 24 h of treatment ( A). On d 0, virtually all the cells in culture were GFAP immunoreactive, whereas a minority (~5%) was strongly LeX positive (). 5 d after placement in bFGF, GFAP immunoreactivity had dramatically declined, and ~30% of the total cell numbers were strongly LeX positive ( B and C, b). Most of these LeX-positive cells were either GFAP negative or weakly GFAP positive. These LeX-positive cells function as progenitors, as the number of neurospheres produced from the LeX-positive fraction, after 2 d of bFGF treatment, was markedly higher than the number from the LeX-negative fraction ( C, a), and the LeX-positive cell-derived spheres were competent to produce neurons in addition to glia (unpublished data). Together, these findings are consistent with the hypothesis that the addition of bFGF to these cultures results in the production of highly proliferative, GFAP-negative, LeX-positive MNPs from GFAP-positive cells.
Figure 8. MELK is necessary for the transition from GFAP-positive into GFAP-negative highly proliferative progenitors in vitro. (A) RT-PCR of cortical astrocyte cultures after addition of bFGF on d 1 and 7. (B and C) Effects of MELK siRNA on the formation of multipotent (more ...)
After bFGF treatment MELK mRNA expression was up-regulated, whereas GFAP expression declined ( A). These observations suggest that high levels of MELK expression is either a reflection of the MNP state or that MELK regulates the production of, or transition to, GFAP-negative/LeX-positive cells. To determine whether this transition was dependent on MELK, we decreased MELK expression during bFGF stimulation. Strikingly, siRNA for MELK, but not for NCS, resulted in diminished numbers of neurospheres ( C, e) and prevented the increase in numbers of LeX-positive cells ( B and C, c). Instead, there was a relative persistence of GFAP-positive cells ( C, d). Knockdown of MELK also resulted in the reduced expression of nestin and SOX2 during bFGF treatment ( D). However, knockdown did not influence cell survival ( C, f). These data show that MELK is necessary for the production of GFAP low or negative, LeX-positive MNPs from progenitors that highly express GFAP.
MELK expression is cell cycle-regulated and MELK function is likely mediated through the B-myb proto-oncogene
Our data thus far indicate that MELK plays an important role in neural progenitor proliferation. This was somewhat surprising because other members of the snf1/AMPK family appear to function in cell survival (Kato et al., 2002
; Inoki et al., 2003
; Suzuki et al., 2003a
). To further explore potential roles that MELK may play in cellular function, we used a large microarray dataset derived from human brain tumors to identify genes whose expressions were coregulated with MELK. MELK expression was highly and significantly correlated with genes known to play roles in the cell cycle, especially those associated with the mitosis (M) phase. ( A, a). Genes whose expression was not correlated with MELK functioned in other processes as determined by Gene Ontology, including metabolism, transcription, and protein modification. Thus, this genome-wide analysis of coregulation supports a role for MELK in cell cycle regulation.
Figure 9. Cell cycle regulation and the B-myb proto-oncogene in MELK function. (A, a) Functional grouping of genes most and least correlated with MELK expression; P < 0.001. (b) RT-PCR after separation of P0 progenitors into apoptotic (A), G0/G1 (more ...)
Many genes that play roles in the cell cycle show phase-dependent transcriptional regulation. Therefore, we sorted progenitors based on their DNA content and evaluated the expression of MELK and other progenitor genes. MELK, like nestin, Sox2, and bmi-1, but unlike Msi1, was most highly expressed during phases of the cell cycle with 4n DNA content (S, G2, and M) rather than at G0–G1, indicating that MELK expression varies with the cell cycle ( A, b). To examine the cell cycle characteristics of MELK-expressing cells, we transfected progenitor cultures with the PMELK-EGFP or control construct and analyzed cell cycle parameters by FACS ( A, c). Greater numbers of MELK-expressing cells were found to be in the S and G2/M phases, whereas fewer were in the G0/G1 phase compared with the total or putative non-MELK–expressing cells. On the other hand, LeX-positive cells were not different from the total cell fraction or LeX-negative cells in their cell cycle parameters.
The data described in this section and those above indicate that MELK functions in the regulation of the cell cycle in MNPs. However, its mechanism is unknown. Previous studies have demonstrated the importance of the PTEN/AKT/MTOR pathway in MNP proliferation (Groszer et al., 2001
; Sinor and Lillien, 2004
). However, as described in Fig. S4 (available at http://www.jcb.org/cgi/content/full/jcb.200412115/DC1
), we did not find evidence that MELK interacts with this pathway. Recent studies have implicated the protein ZPR9 in the function of MELK and, in turn, ZPR9 in the function of the cell cycle regulatory proto-oncogene B-myb (Seong et al., 2002
). To determine whether MELK function could be mediated by B-myb in MNP, we first examined Zpr9 and B-myb expression in cultured neural progenitors ( B, a). As is the case for MELK, ZPR9 and B-myb were highly enriched in NS from P0 cortex compared with DC ( B, a, top). Also, like MELK, B-myb was enriched in the LeX-positive fraction of neurospheres ( B, a, bottom). B-myb, like MELK, was also expressed during phases of the cell cycle with 4n DNA content ( A, b). The brain expression pattern of B-myb was similar to that of MELK throughout development, with the exception of the adult hippocampus, where B-myb mRNA was found to be expressed ( B, b).
MELK siRNA treatment resulted in a down-regulation of both B-myb and ZPR9, without significantly influencing other stem cell–related genes such as nestin or SOX2 ( B, c) 48 h after transfection. Knockdown of B-myb produced similar effects to MELK, resulting in a dose-dependent decrease in neurosphere formation from progenitors ( C, d). Thus, these data suggest that inhibition of endogenous MELK expression down-regulates B-myb, which, in turn, results in the reduction of neurosphere numbers and is consistent with the hypothesis that MELK exerts some or all of its actions via regulation of B-myb expression.