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Neogenin is expressed on neurogenic and gliogenic progenitors in the embryonic and adult central nervous system
Gene Expression Patterns 7 (2007) 784–792 www.elsevier.com/locate/modgep
Neogenin is expressed on neurogenic and gliogenic progenitors in the embryonic and adult central nervous system Daniel P. Fitzgerald b
a,1
, DanaKai Bradford a, Helen M. Cooper
a,b,*
a Queensland Brain Institute, The University of Queensland, Brisbane, Qld, Australia School of Biomedical Sciences, The University of Queensland, Brisbane, Qld, Australia
Received 13 November 2006; received in revised form 3 May 2007; accepted 22 May 2007 Available online 26 May 2007
Abstract The Netrin/RGMa receptor, Neogenin, has recently been identified on neuronal and gliogenic progenitors, including radial glia in the embryonic mouse cortex and ganglionic eminences, respectively [Fitzgerald, D.P., Cole, S.J., Hammond, A., Seaman, C., Cooper, H.M., 2006a. Characterization of Neogenin-expressing neural progenitor populations and migrating neuroblasts in the embryonic mouse forebrain. Neuroscience 142, 703–716]. Here we have undertaken a detailed analysis of Neogenin expression in the embryonic mouse central nervous system at key developmental time points. We demonstrate that Neogenin protein is present on actively dividing neurogenic precursors during peak phases of neurogenesis (embryonic days 12.5–14.5) in the forebrain, midbrain and hindbrain. Furthermore, we show that Neogenin protein is localized to the cell bodies and glial processes of neurogenic radial glial populations in all these regions. We have also observed Neogenin on gliogenic precursors within the subventricular zones of the forebrain late in development (embryonic day 17.5). Adult neural stem cells found in the subventricular zone of the lateral ventricle of the rodent forebrain are direct descendants of the embryonic striatal radial glial population. Here we show that Neogenin expression is maintained in the neural stem cell population of the adult mouse forebrain. In summary, this study demonstrates that Neogenin expression is a hallmark of many neural precursor populations (neurogenic and gliogenic) in both the embryonic and adult mammalian central nervous system. Ó 2007 Elsevier B.V. All rights reserved. Keywords: Neogenin; Netrin; Repulsive Guidance Molecule; RGMa; Neurogenesis; Gliogenesis; CNS development; Neural progenitor; Embryonic neural stem cell; Radial glia; Mouse embryo; Adult neural stem cell
1. Results and discussion Neogenin is a multi-functional receptor that regulates several key developmental processes within the embryonic nervous system, including axon guidance, neural migration and cell death (Fitzgerald et al., 2006a; Matsunaga et al., 2004; Mawdsley et al., 2004). Neogenin has been identified
*
Corresponding author. Address: Queensland Brain Institute, The University of Queensland, Brisbane, Qld 4072, Australia. Tel.: +61 7 3365 3155; fax: +61 7 3346 8836. E-mail address: [email protected] (H.M. Cooper). 1 Current address: Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. 1567-133X/$ - see front matter Ó 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.modgep.2007.05.004
as an axon guidance receptor on chick retinal ganglion axons for the chemorepulsive guidance cue, Repulsive Guidance Molecule (RGMa) (Rajagopalan et al., 2004). In the early Xenopus forebrain Neogenin on supraoptic tract axons promotes chemoattraction in response to Netrin1, whereas RGMa–Neogenin interactions promote chemorepulsion (Wilson and Key, 2006). A role for Neogenin in early central nervous system (CNS) development has also been demonstrated in loss-of-function studies in the zebrafish embryo, where loss of Neogenin results in the aberrant formation of the neural tube (Mawdsley et al., 2004). In addition, a possible role for Neogenin in the migration of young neurons in the embryonic day 14.5 (E14.5) telencephalon has been suggested from a recent study demonstrating the presence of Neogenin on young,
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actively migrating interneurons (Fitzgerald et al., 2006a). Neogenin activity is also important in the establishment of the morphological architecture in many other developing organ systems outside the CNS. For example, it has been shown to promote adhesion between the multi-potent progenitor (cap) cells and the adjacent epithelial layer of the developing mammary gland (Srinivasan et al., 2003). Netrin1–Neogenin interactions also promote both adhesion and migration of vascular smooth muscle cells during angiogenesis (Park et al., 2004). In addition, Kang and colleagues (2004) have shown that Neogenin activation by Netrin1 stimulates myotube differentiation in vitro. Neogenin has recently been shown to be present on neuronal progenitors in the E12.5–E14.5 mouse cortex and ganglionic eminences (Fitzgerald et al., 2006a). Along the entire neuraxis of the early CNS (until approximately E12) the expanding neural tube is populated by neuroepithelial progenitor cells (NEPs). The bodies of these cells lie adjacent to the apical surface and extend a radial process to the pial surface. NEPs undergo interkinetic division, dividing either symmetrically to generate two new NEPs or asymmetrically to produce a postmitotic neuron and an NEP (Go¨tz and Huttner, 2005; Guillemot, 2005; Mori et al., 2005). Around E10, NEPs begin to produce radial glial cells (RGs) throughout the developing brain (Edwards et al., 1990; Mori et al., 2005). Like NEPs, the cell bodies of RGs reside within the ventricular zone (VZ) at the apical surface and project a radial process to the pial surface. By E12, RGs have replaced NEPs throughout the forebrain, midbrain and hindbrain and are believed to be the major neurogenic progenitors in most regions except the ventral forebrain, spinal cord and retina (Go¨tz and Huttner, 2005; Hartfuss et al., 2001; Noctor et al., 2002). In the early cortex, RGs divide asymmetrically to generate one new RG and either a neuron or a basal progenitor (Anthony et al., 2004; Heins et al., 2002; Malatesta et al., 2003; Miyata et al., 2001; Noctor et al., 2002, 2004). It is now believed that gliogenic RG populations are probably distinct from the neurogenic population (reviewed in Mori et al., 2005). Thus, in contrast to the dorsal forebrain, RGs in the ventral forebrain are gliogenic, giving rise to new RGs, committed glial precursors and mature glia (Malatesta et al., 2003). Around E16 in the cortex, RGs begin to generate glial restricted precursors, producing astrocytes or oligodendrocytes. Towards the end of neurogenesis (E17 in the forebrain) RGs then transform directly into astrocytes (Li et al., 2004; Qian et al., 2000; Voight, 1989). Many studies have now demonstrated that neuronal progenitors reside within the adult subventricular zone (SVZ), adjacent to the lateral ventricles in rodents and probably also humans (Merkle and Alvarez-Buylla, 2006). Neuroblasts generated in the adult SVZ travel rostrally via tangential migration to the olfactory bulb where they give rise to the granule and periglomerular interneuron populations. The SVZ neural stem cell (Type B cell) is astrocytic in nature, extending long processes and expressing both GFAP (glial fibrillary acidic protein) and
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Nestin. The rapidly dividing transit-amplifying Type C cell is generated by the Type B stem cell and gives rise to neuroblasts (Type A cells), the precursors of the olfactory bulb interneurons. The neuroblasts migrate through the SVZ in chains encapsulated by the glial processes of the Type B stem cells. Type C intermediate precursors are found in clusters and are closely associated with Type A neuroblasts. It has recently been shown that the Type B stem cell is a direct descendant of the embryonic striatal RG (reviewed in Merkle and Alvarez-Buylla, 2006). These RGs first transform into astrocytes late in embryogenesis and early postnatal life, before differentiating into the astrocytic Type B stem cell found in the adult SVZ. In a previous study we demonstrated that Neogenin is localized to the proliferative zones of the E14.5 mouse cortex, where it is present on the membrane of actively dividing neurogenic RGs (Fitzgerald et al., 2006a). In the ganglionic eminences (GEs) of the E14.5 ventral forebrain Neogenin was found on the dividing gliogenic RG population. These observations prompted us to conduct a more extensive study of Neogenin localization along the embryonic neuraxis at key developmental time points to determine if Neogenin expression is a common feature of dividing neurogenic and gliogenic precursors. Here we demonstrate that Neogenin is not restricted to RG populations in the E14.5 cortex and GEs, but is localized to many neurogenic and gliogenic precursor populations throughout the developing CNS. These observations suggested that Type B stem cells in the adult SVZ may also express Neogenin. We confirm here that Neogenin is indeed present on this neural stem cell population in the adult forebrain. To characterize the spatiotemporal localization of Neogenin protein along the neuraxis of the mouse embryo from E12.5 to E17.5 we employed an antiserum (CT-Neog) raised against the C-terminal peptide of mouse Neogenin which has been previously characterized (Fitzgerald et al., 2006a,b). Throughout this study, preincubation with the immunizing peptide resulted in the loss of immunoreactivity, thereby confirming the specificity of the antiserum (see Supplementary Fig. 1). The CT-Neog antiserum is directed to the C-terminal amino acid sequence of Neogenin and is therefore able to detect all alternatively spliced forms of mouse Neogenin (Keeling et al., 1997). 1.1. Neogenin is expressed by neurogenic progenitors throughout the E14.5 forebrain Our previous study focused on Neogenin expression in the E14.5 cortex and GEs. Here we expanded our study to determine if Neogenin expression is restricted to these specific regions or if it is present on neural progenitor populations in all regions of the E14.5 forebrain. Along the neuraxis, neural progenitors undergo interkinetic division, with their bodies residing within the VZ. During interkinetic division the nuclei migrate to the basal surface of the VZ, where they proceed through S-phase and then move back to the apical surface to complete the cell
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cycle. S-phase cells can be clearly identified at the basal surface by immunolabeling with an antibody specific for Proliferating Cell Nuclear Antigen (PCNA), which is localized to the nucleus during S-phase (Li et al., 1996). Therefore, PCNA-positive cells demarcate the basal boundary of the
VZ within the expanding neural tissue. Co-labeling of the E14.5 forebrain with the CT-Neog antiserum and antiPCNA revealed that Neogenin was expressed in dividing cells at the basal boundary of the VZ in the hippocampus (Figs. 1A–C), olfactory bulb (Figs. 1G–I), and septum
Fig. 1. Neogenin protein is present on dividing neuronal progenitors throughout the VZs of the E14.5 mouse forebrain. Co-labeling with the CT-Neog antiserum (A, G; green) and an antibody specific for the S-phase marker PCNA (B, H; red) demonstrates that Neogenin is present in the VZ of the cortex, hippocampus (A–C; C, CT-Neog, anti-PCNA; merge) and olfactory bulb of the dorsal telencephalon (G–I), as well as the VZ of the septum in the ventral telencephalon. A–C, G–I insets: High power images of the VZ demonstrating that Neogenin (green) is present on PCNA-positive cells (red) (arrows). Colabeling with the CT-Neog (D, J; green) and an anti-Nestin antibody (E, K; red) (F, L; merge) demonstrated that Neogenin is expressed by Nestin-positive progenitors in the hippocampus (D–F), as well as in the olfactory bulb and diencephalon (thalamus) (J–L). A–C, G–L, sagittal sections, rostral-right; D–F, coronal sections; A–L, dorsal-top. cp, cortical plate; di, diencephalon; ge, ganglionic eminence; hp, hippocampus; ob, olfactory bulb; stp, striatal primordium; svz, subventricular zone; th, thalamus; vz, ventricular zone. Scale bars: A–L, 200 lm.
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(data not shown), where it could be seen on the surface of cells containing PCNA-positive nuclei (Figs. 1A–C and G–I; insets). Neogenin was also expressed by cells at the ventricular surface where M-phase cells reside. Neurogenic progenitors, including RGs, express the intermediate neurofilament protein, Nestin, while RGs are further characterized by their expression of GLAST (Frederiksen and McKay, 1988; Hartfuss et al., 2001; Shibata et al., 1997). Neogenin-positive cells within the VZs of the hippocampus and olfactory bulb were Nestin-positive (Figs. 1D–F and J–L) and GLAST-positive (data not shown), indicating that Neogenin is expressed on neurogenic RGs in these regions. Within the ventral forebrain, Neogenin and Nestin were co-expressed on neurogenic progenitors within the VZ of the diencephalon (thalamus; Figs. 1J–L). Co-labeling
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with an antibody recognizing bIII tubulin or MAP2, markers for maturing and mature neurons, respectively, revealed that neurons in the mantle regions of the dorsal and ventral forebrain were Neogenin-negative (Supplementary Fig. 2 and data not shown). In summary, Neogenin protein is not restricted to progenitor populations in the early cortex and GEs but is present on neurogenic progenitors in all regions of the E14.5 forebrain. 1.2. Neogenin expression during neurogenesis in the midbrain and hindbrain We also examined whether Neogenin was present on dividing neuronal progenitors in the midbrain and hindbrain. Neurogenesis in the midbrain commences around
Fig. 2. Neogenin is present on neuronal progenitors in the midbrain and hindbrain. E12.5 (A, B) or E14.5 (C, D) sections co-labeled with CT-Neog (A, C; green) and anti-PCNA (red) (B, D; merge) demonstrate that Neogenin is localized to the radial processes (A, B; arrows) of dividing neuronal progenitors. Neogenin is absent from mature neurons in the midbrain (C; arrow). Co-localization of Neogenin (E, G; green) and Nestin (red) (F, H; merge) is observed on RG processes in the E14.5 midbrain (E, F; arrows) and hindbrain (G, H; arrows). Boxed area in C indicates midbrain region in E and F. Neogenin (green) is localized to the PCNA-positive (red) cell bodies (inset; arrows) and the processes (arrows) of RGs in the ventral E14.5 spinal cord (I). A–H, sagittal sections, rostral-right; I, transverse section; A–I, dorsal-top. cb, cerebellum; dmb, dorsal midbrain; md, medulla oblongata; pn, pons. Scale bars: A, B, 100 lm; C–I, 200 lm.
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E10 and peaks at E14, coincident with neurogenesis in the forebrain (Go¨tz and Huttner, 2005; Jacobsen, 1991). At E12.5 the neural tube of the presumptive midbrain still comprises NEPs, some of which have begun to produce RGs (reviewed in Go¨tz and Huttner, 2005; Guillemot, 2005). Co-labeling with antibodies to Neogenin and PCNA demonstrated that Neogenin was present on dividing cells within the E12.5 midbrain, being clearly visible along the radial processes of these cells (Figs. 2A and B; arrows). As the midbrain matured, Neogenin expression was maintained in dividing (PCNA-positive) cells within the E14.5 dorsal midbrain VZ, but was absent from maturing neurons residing in the mantle regions (Fig. 2C; arrow). Neogenin-positive cells within the VZs of the dorsal midbrain also co-labeled for Nestin (Figs. 2E and F) and GLAST (data not shown), indicating their RG identity. The RG processes spanning the more immature dorso-caudal E14.5 midbrain were also Neogenin- and Nestin-positive (Figs. 2E and F; arrows). Neurogenesis in the hindbrain, including the cerebellum, begins around E10, but does not reach its peak until E14 (reviewed in Go¨tz and Huttner, 2005). Neurogenesis then continues into the postnatal period in these regions. Within the E14.5 hindbrain, Neogenin was observed on the Nestin-positive processes of the neurogenic RGs spanning the expanding medulla oblongata (Figs. 2G and H; arrows) and cerebellum (data not shown). Therefore, in the early forebrain, midbrain and hindbrain, Neogenin is expressed on actively dividing neurogenic progenitors, including NEPs and RGs, where it is localized to both the cell bodies and radial processes extending to the pial surface. In contrast, Neogenin is absent from the maturing neuronal populations in the E14.5 CNS. 1.3. Gliogenic precursors express Neogenin After E16 in the mouse telencephalon, gliogenic RGs begin producing astrocyte and oligodendrocyte precursors which come to reside within the SVZs (Cameron and Rakic, 1991; Mission et al., 1991; Parnavelas, 1999; Qian et al., 2000). By E17–E18, neurogenesis is largely completed in the forebrain, and the neurogenic RG population has disappeared (Bayer and Altman, 1991; Hartfuss et al., 2001), resulting in the disappearance of the VZ, which is replaced by the ependymal lining of the ventricles (Takahashi et al., 1995). From this developmental stage into postnatal life, progenitors continue to produce the SVZ-based gliogenic precursors (Hartfuss et al., 2001; Takahashi et al., 1995). In addition, gliogenic RGs also transform directly into astrocytes (Edwards et al., 1990; Voight, 1989). To determine if Neogenin is expressed by dividing gliogenic precursors in the E17.5 forebrain we co-labeled forebrain sections with the CT-Neog antiserum and antibodies specific for PCNA. Neogenin was restricted to the proliferative zones (i.e., the SVZs) of the E17.5 dorsal and ventral forebrain (including those of the cortex, hippocampus and striatum), where the dividing PCNA-positive cells were sit-
uated (Figs. 3A–C). It has been clearly demonstrated that the majority of RGs isolated at this late embryonic stage generate only gliogenic progeny and can be identified by their expression of Nestin and GLAST (Hartfuss et al., 2001; Mori et al., 2005). To verify that Neogenin is expressed on these gliogenic precursors, we co-labeled with either an anti-Nestin (Figs. 3D–F) or an anti-GLAST antibody (Figs. 3G–I). As expected, both Nestin-positive and GLAST-positive precursors were found within the SVZs of the E17.5 cortex, hippocampus and striatum (Figs. 3D–F and G–I). Moreover, Neogenin protein was localized to the surface of these Nestin- and GLAST-positive precursors (Figs. 3D–F and G–I; insets), confirming that Neogenin is present on gliogenic precursors in the late embryonic forebrain. In contrast to most regions of the embryonic brain, the neural progenitors within the spinal cord VZ display both NEP and RG characteristics, and can be distinguished by their morphology and expression of Nestin (Hartfuss et al., 2001; Li et al., 2004; Liu et al., 2002; Go¨tz and Huttner, 2005; Guillemot, 2005). Neurogenesis occurs early (E8– E12.5) in the developing mouse spinal cord. After neurogenesis is completed in the rodent spinal cord, progenitors become gliogenic, producing both astrocytes and oligodendrocytes (Li et al., 2004; Liu et al., 2002; Zhou et al., 2000). Neogenin mRNA has been observed in the VZ of the ventral spinal cord during the neurogenic period (Gad et al., 1997; Keeling et al., 1997; Keino-Masu et al., 1996), indicating that spinal cord neurogenic progenitors express Neogenin. Here we examined Neogenin protein localization in the E14.5 mouse spinal cord during gliogenesis. Neogenin was localized to the cells bodies (Fig. 2I; inset) and radial processes (Fig. 2I; arrows) of dividing gliogenic progenitors in the ventral VZ of the E14.5 spinal cord. It is the ventral spinal cord that is believed to be the origin of the oligodendrocyte–astrocyte precursors (Liu et al., 2002; Orentas and Miller, 1996; Zhou et al., 2000). In summary, Neogenin is expressed by gliogenic precursors in the E17.5 cortex, hippocampus, striatum and E14.5 spinal cord. We have previously demonstrated that Neogenin is present on RGs within the E14.5 GEs (Fitzgerald et al., 2006a), which have recently been shown to be gliogenic in nature (Li et al., 2004; Malatesta et al., 2003). Therefore, Neogenin expression appears to be a characteristic of many gliogenic precursor populations throughout the developing CNS. 1.4. Neural stem cells in the adult mouse forebrain express Neogenin Since the neural stem cells (Type B cells) found in the SVZ of the lateral ventricle of the adult rodent forebrain are direct descendants of the embryonic striatal RG population (reviewed in Merkle and Alvarez-Buylla, 2006), we determined whether these stem cells also expressed Neogenin. Fig. 4A demonstrates that the CT-Neog antiserum immunolabels cells within the SVZ surrounding the lateral ventri-
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Fig. 3. Neogenin is present on gliogenic progenitors in the E17.5 forebrain. Co-labeling with the CT-Neog (A, D; green) and the anti-PCNA antibody (B; red) (C; merge), or the anti-Nestin antibody (E; red) (F; merge), demonstrates that Neogenin is expressed by dividing gliogenic progenitors within the SVZs. Co-labeling with the CT-Neog (G; green) and the anti-GLAST antibody (H; red) (I; merge) demonstrates that Neogenin is present on gliogenic RGs. High power images show Neogenin and Nestin co-localization (D–F, insets; arrows) or GLAST co-localization (G–I, insets; arrows) on these progenitors. A–I, coronal sections, dorsal-top. ctx, cortex; hp, hippocampus, st, striatum; svz, subventricular zone. Scale bars: A–I, 200 lm.
cles of the adult forebrain. In contrast, preincubation with the immunizing peptide removed all immunoreactivity in an adjacent section (Fig. 4B). The Type B stem cell expresses both the astrocytic marker, GFAP, and the neural progenitor marker, Nestin, and extends long processes that encapsulate the Type A neuroblast (GFAP-negative). The intermediate precursors (Type C cells) giving rise to these neuroblasts are also GFAP-negative (Doetsch et al., 1997). CT-Neog immunolabeling of the anterior corner of the lateral ventricle (Figs. 4C and F) revealed that Neogenin was localized to cell bodies (arrowheads) within the SVZ in a punctate pattern, as well as on the long processes extending from these cells (arrows). In addition, co-labeling with antiNestin (Figs. 4C–E) or anti-GFAP (Figs. 4F–H) further revealed that both the Neogenin-positive cell bodies and their processes were also Nestin- and GFAP-positive. Together these observations demonstrate that Neogenin is expressed by the neural stem cell (Type B cell) in the adult SVZ, which has been shown to give rise to both interneurons and oligodendrocytes (Merkle and Alvarez-Buylla, 2006).
1.5. Conclusion Here we show, that throughout the developing mouse CNS, Neogenin is present on neurogenic progenitor cells actively undergoing division. Neogenin is found on the earliest neural progenitors at E12.5 (NEPs) when they are undergoing both symmetric (producing two new NEPs) and asymmetric division (producing one NEP and either a neuron, RG, or basal progenitor). As neurogenesis peaks, Neogenin is expressed on neurogenic RG populations throughout the neuraxis, as these cells divide asymmetrically to generate neurons and new RGs. We also observed strong Neogenin expression on glial precursors within the SVZs of the E17.5 forebrain, and in the E14.5 spinal cord. The adult neural stem cell within the SVZ of the mouse forebrain derives from the gliogenic RG population in the embryonic striatum (Merkle and Alvarez-Buylla, 2006), which we have previously shown to be Neogeninpositive (Fitzgerald et al., 2006a). Here we show that Neogenin expression is maintained in the adult neural stem cell.
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Fig. 4. Neogenin is expressed on neural stem cells (Type B cells) in the SVZ of the adult forebrain. (A) Immunolabeling with the CT-Neog antiserum shows that cells within the SVZ surrounding the lateral ventricles of the adult forebrain express Neogenin. (B) Preincubation with the immunizing peptide removes all immunoreactivity in an adjacent section. Co-labeling of the anterior corner of the lateral ventricle with the CT-Neog antiserum (C, F; green) and the anti-Nestin antibody (D; red) (E; merge), or the anti-GFAP antibody (G; red) (H; merge) demonstrates that Neogenin is co-expressed with Nestin and GFAP on the cell bodies (arrowheads) within the SVZ, as well as on the long processes extending from these cells (arrows). A, B, coronal sections; C– H, sagittal sections, rostral-right; A–H, dorsal-top. lv, lateral ventricle; svz, subventricular zone. Scale bars: A, B, 200 lm; C–H, 10 lm.
Together, this evidence indicates that Neogenin expression is a hallmark of many dividing neural precursor populations (neurogenic and gliogenic) in both the embryonic and adult mammalian CNS. 2. Experimental procedures 2.1. Antiserum The CT-Neog rabbit polyclonal antiserum was raised against a unique C-terminal peptide (VQETTRMLEDSESS) of mouse Neogenin corresponding to amino acids 1454–1467 of the published sequence (Keeling et al., 1997) and has been previously described in detail (Fitzgerald et al., 2006a,b). For immunohistochemistry on frozen sections, CT-Neog was diluted 1:250. To demonstrate specificity, diluted antiserum was preincubated with the immunizing peptide (100 lg/ml) for 1 h prior to addition to sections.
2.2. Tissue preparation and immunohistochemical analysis C57Bl/6 embryos were dissected from the uterine horn and immediately flash-frozen in O.C.T. Compound (Tissue-Tek, CA). Adult brains were dissected from C57Bl/6 males at 9 weeks of age. Twelve micron sagittal and coronal sections were cut using a Leica CM3050 Cryostat
and mounted onto SuperFrost Plus-coated slides (Microm, Germany). The use of animals as described here was approved by the Animal Ethics Committee of the University of Queensland in accordance with the guidelines stipulated by the National Health and Medical Research Council of Australia. Sections were post-fixed in absolute ethanol for 10 min, rehydrated in 70%, then by 30% ethanol-PBS, and washed in PBS. The specific immunoreactivity demonstrated in this study was achieved by the use of a very mild fixative (ethanol) after cryostat sectioning without prior exposure to any other fixation protocol. Sections were pre-blocked and permeabilized for several hours at room temperature in blocking solution (PBS; 2% fetal bovine serum; 2% goat serum; 0.2% Triton X-100). Sections were incubated in primary antibodies in blocking solution overnight at 4 °C. Primary antibodies were mouse anti-bIII tubulin (1:500, Promega, WI), mouse anti-Nestin (1:250, Chemicon, CA), mouse anti-PCNA (1:500, Sigma, MO), mouse antiGFAP (1:250, Chemicon), and guinea-pig anti-GLAST (astrocyte-specific glutamate transporter) (1:250, Chemicon). Neogenin immune complexes were detected by amplification with goat anti-rabbit IgG conjugated to biotin (1:500, Chemicon), followed by incubation with streptavidin conjugated to AlexaFluor 488 (1:1000, Molecular Probes, OR). Other secondary antibodies were: goat anti-mouse IgG-AlexaFluor 568 (1:1000), and goat anti-guinea-pig IgG-AlexaFluor 568 (1:1000). Slides were mounted in DakoCytomation Fluorescent Mounting Medium (DAKO, CA). Images were acquired on an Olympus IX81 microscope using AnalySIS software, or on a Zeiss Axioplan 2 microscope using Axiovision software.
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Acknowledgements We thank Ms Rowan Tweedale and Ms Stacey Cole for critical reading of the manuscript. D. Bradford is supported by an Australian Postgraduate Award. This work was supported by the National Health and Medical Research Council of Australia. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.modgep. 2007.05.004. References Anthony, T.E., Klein, C., Fishell, G., Heintz, N., 2004. Radial glia serve as neuronal progenitors in all regions of the central nervous system. Neuron 41, 881–890. Bayer, S.A., Altman, J., 1991. Neocortical Development. Raven Press, New York. Cameron, R.S., Rakic, P., 1991. Glial cell lineage in the cerebral cortex, a review and synthesis. Glia 4, 124–137. Doetsch, F., Garcia-Verdugo, J.M., Alvarez-Buylla, A., 1997. Cellular composition and three-dimensional organization of the subventricular germinal zone in the adult mammalian brain. J. Neurosci. 17, 5046–5061. Edwards, M.A., Yamamoto, M., Caviness, V.S., 1990. Organization of radial glia and related cells in the developing murine CNS, an analysis based on a new monoclonal antibody marker. Neuroscience 36, 121–144. Fitzgerald, D.P., Cole, S.J., Hammond, A., Seaman, C., Cooper, H.M., 2006a. Characterization of neogenin-expressing neural progenitor populations and migrating neuroblasts in the embryonic mouse forebrain. Neuroscience 142, 703–716. Fitzgerald, D.P., Seaman, C., Cooper, H.M., 2006b. Localization of neogenin protein during morphogenesis in the mouse embryo. Dev. Dynam. 235, 1720–1725. Frederiksen, K., McKay, R.D., 1988. Proliferation and differentiation of rat neuroepithelial precursor cells in vivo. J. Neurosci. 8, 1144–1151. Gad, J.M., Keeling, S.L., Wilks, A.F., Tan, S.-S., Cooper, H.M., 1997. The expression patterns of the guidance receptors, DCC and Neogenin, are spatially and temporally distinct throughout mouse embryogenesis. Dev. Biol. 192, 258–273. Go¨tz, M., Huttner, W.B., 2005. The cell biology of neurogenesis. Nat. Revs. Mol. Cell Biol. 6, 777–788. Guillemot, F., 2005. Cellular and molecular control of neurogenesis in the mammalian telencephalon. Curr. Opin. Cell Biol. 17, 639–647. Hartfuss, E., Galli, R., Heins, N., Go¨tz, M., 2001. Characterization of CNS precursor subtypes and radial glia. Dev. Biol. 229, 15–30. Heins, N., Malatesta, P., Cecconi, F., Nakafuku, M., Tucker, K.L., Hack, M.A., Chapouton, P., Barde, Y.-A., Go¨tz, M., 2002. Glial cells generate neurons, the role of the transcription factor Pax6. Nat. Neurosci. 5, 308–315. Jacobsen, M., 1991. Developmental Neurobiology, third ed. Plenum Press, New York. Kang, J.-S., Yi, M.-J., Zhang, W., Feinleib, J.L., Cole, F., Krauss, R.S., 2004. Netrins and neogenin promote myotube formation. J. Cell Biol. 167, 493–594. Keeling, S.L., Gad, J.M., Cooper, H.M., 1997. Mouse Neogenin, a DCC-like molecule, has four splice variants and is expressed widely in the adult mouse and during embryogenesis. Oncogene 15, 691–700.
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Keino-Masu, K., Masu, M., Hinck, L., Leonardo, E.D., Chan, S.S.-Y., Culotti, J.G., Tessier-Lavigne, M., 1996. Deleted in Colorectal Cancer (DCC) encodes a netrin receptor. Cell 87, 175–185. Li, H., Babiarz, J., Woodbury, J., Kane-Goldsmith, N., Grumet, M., 2004. Spatiotemporal heterogeneity of CNS radial glial cells and their transition to restricted precursors. Dev. Biol. 271, 225–238. Li, R., Hannon, G.J., Beach, D., Stillman, B., 1996. Subcellular distribution of p21 and PCNA in normal and repair-deficient cells following DNA damage. Curr. Biol. 6, 189–199. Liu, Y., Wu, Y., Lee, J.C., Xue, H., Pevny, L.H., Kaprielian, Z., Rao, M.S., 2002. Oligodendrocyte and astrocyte development in rodents, an in situ and immunohistological analysis during embryonic development. Glia 40, 25–43. Malatesta, P., Hack, M., Hartfuss, E., Kettenmann, H., Klinkert, W., Kirchoff, F., Go¨tz, M., 2003. Neuronal or glial progeny, regional differences in radial glia fate. Neuron 37, 751–764. Matsunaga, E., Tauszig-Delamasure, S., Monnier, P.P., Mueller, B.K., Strittmatter, P.M., Mehlen, P., Che´dotal, A., 2004. RGM and its receptor neogenin regulate neuronal survival. Nat. Cell Biol. 6, 749–755. Mawdsley, D., Cooper, H.M., Hogan, B., Cody, S., Lieschke, G., Heath, J., 2004. The netrin receptor, neogenin, is required for neural tube formation and somitogenesis in zebrafish. Dev. Biol. 269, 302–315. Merkle, F., Alvarez-Buylla, A., 2006. Neural stem cells in mammalian development. Curr. Opin. Cell Biol. 18, 704–709. Mission, J.P., Takahashi, T., Caviness, V.S.J., 1991. Ontogeny of radial glia and other astroglial cells in murine cerebral cortex. Glia 4, 138–148. Miyata, T., Kawaguchi, A., Okano, H., Ogawa, M., 2001. Asymmetric inheritance of radial glial fibers by cortical neurons. Neuron 31, 727–741. Mori, T., Buffo, A., Go¨tz, M., 2005. The novel roles of glial cells revisited, the contribution of radial glia and astrocyte to neurogenesis. In: Schatten, G.P. (Ed.), Neural Development. Elsevier Academic Press, San Diego, pp. 67–99. Noctor, S.C., Flint, A.C., Weissman, T.A., Wong, W.S., Clinton, B.K., Kriegstein, A.R., 2002. Dividing precursor cells of the embryonic cortical ventricular zone have morphological and molecular characteristics of radial glia. J. Neurosci. 22, 3161–3173. Noctor, S.C., Martı´nez-Cerden˜o, V., Ivic, L., Kriegstein, A.R., 2004. Cortical neurons arise in symmetric and asymmetric division zones and migrate through specific phases. Nat. Neurosci. 7, 136–144. Orentas, D.M., Miller, R.H., 1996. The origin of spinal cord oligodendrocytes is dependent on local influences from the notochord. Dev. Biol. 177, 43–53. Park, K.-W., Crouse, D., Lee, M., Karnik, S., Sorensen, L., Murphy, K., Kuo, C., Li, D., 2004. The axonal attractant netrin-1 is an angiogenic factor. Proc. Natl. Acad. Sci. USA 101, 16210–16215. Parnavelas, J.G., 1999. Glial cell lineages in the rat cerebral cortex. Exp. Neurol. 156, 418–429. Qian, X., Shen, Q., Goderie, S.K., He, W., Capela, A., Davis, A.A., Temple, S., 2000. Timing of CNS cell generation, a programmed sequence of neuron and glial cell production from isolated murine cortical stem cells. Neuron 28, 69–80. Rajagopalan, S., Deitinghoff, L., Davis, D., Conrad, S., Skutella, T., Che´dotal, A., Mueller, B., Strittmatter, S., 2004. Neogenin mediates the action of repulsive guidance molecule. Nat. Cell Biol. 6, 755–762. Shibata, T., Yamada, K., Ikenaka, K., Wada, K., Tanaka, K., Inoue, Y., 1997. Glutamate transporter GLAST is expressed in the radial gliaastrocyte lineage of developing mouse spinal cord. J. Neurosci. 17, 9212–9219. Srinivasan, K., Strickland, P., Valdes, A., Shin, G., Hinck, L., 2003. Netrin-1/neogenin interaction stabilizes multipotent progenitor cap cells during mammary gland morphogenesis. Dev. Cell 4, 371–382.
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Wilson, N.H., Key, B., 2006. Neogenin interacts with RGMa and Netrin-1 to guide axons within the embryonic vertebrate forebrain. Dev. Biol. 15, 485–498. Zhou, Q., Wang, S.-Y., Anderson, D.J., 2000. Identification of a novel family of oligodendrocyte lineage-specific basic helix-loop-helix transcription factors. Neuron 25, 331–343.
Neogenin is expressed on neurogenic and gliogenic progenitors in the embryonic and adult central nervous system Daniel P. Fitzgerald b
a,1
, DanaKai Bradford a, Helen M. Cooper
a,b,*
a Queensland Brain Institute, The University of Queensland, Brisbane, Qld, Australia School of Biomedical Sciences, The University of Queensland, Brisbane, Qld, Australia
Received 13 November 2006; received in revised form 3 May 2007; accepted 22 May 2007 Available online 26 May 2007
Abstract The Netrin/RGMa receptor, Neogenin, has recently been identified on neuronal and gliogenic progenitors, including radial glia in the embryonic mouse cortex and ganglionic eminences, respectively [Fitzgerald, D.P., Cole, S.J., Hammond, A., Seaman, C., Cooper, H.M., 2006a. Characterization of Neogenin-expressing neural progenitor populations and migrating neuroblasts in the embryonic mouse forebrain. Neuroscience 142, 703–716]. Here we have undertaken a detailed analysis of Neogenin expression in the embryonic mouse central nervous system at key developmental time points. We demonstrate that Neogenin protein is present on actively dividing neurogenic precursors during peak phases of neurogenesis (embryonic days 12.5–14.5) in the forebrain, midbrain and hindbrain. Furthermore, we show that Neogenin protein is localized to the cell bodies and glial processes of neurogenic radial glial populations in all these regions. We have also observed Neogenin on gliogenic precursors within the subventricular zones of the forebrain late in development (embryonic day 17.5). Adult neural stem cells found in the subventricular zone of the lateral ventricle of the rodent forebrain are direct descendants of the embryonic striatal radial glial population. Here we show that Neogenin expression is maintained in the neural stem cell population of the adult mouse forebrain. In summary, this study demonstrates that Neogenin expression is a hallmark of many neural precursor populations (neurogenic and gliogenic) in both the embryonic and adult mammalian central nervous system. Ó 2007 Elsevier B.V. All rights reserved. Keywords: Neogenin; Netrin; Repulsive Guidance Molecule; RGMa; Neurogenesis; Gliogenesis; CNS development; Neural progenitor; Embryonic neural stem cell; Radial glia; Mouse embryo; Adult neural stem cell
1. Results and discussion Neogenin is a multi-functional receptor that regulates several key developmental processes within the embryonic nervous system, including axon guidance, neural migration and cell death (Fitzgerald et al., 2006a; Matsunaga et al., 2004; Mawdsley et al., 2004). Neogenin has been identified
*
Corresponding author. Address: Queensland Brain Institute, The University of Queensland, Brisbane, Qld 4072, Australia. Tel.: +61 7 3365 3155; fax: +61 7 3346 8836. E-mail address: [email protected] (H.M. Cooper). 1 Current address: Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. 1567-133X/$ - see front matter Ó 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.modgep.2007.05.004
as an axon guidance receptor on chick retinal ganglion axons for the chemorepulsive guidance cue, Repulsive Guidance Molecule (RGMa) (Rajagopalan et al., 2004). In the early Xenopus forebrain Neogenin on supraoptic tract axons promotes chemoattraction in response to Netrin1, whereas RGMa–Neogenin interactions promote chemorepulsion (Wilson and Key, 2006). A role for Neogenin in early central nervous system (CNS) development has also been demonstrated in loss-of-function studies in the zebrafish embryo, where loss of Neogenin results in the aberrant formation of the neural tube (Mawdsley et al., 2004). In addition, a possible role for Neogenin in the migration of young neurons in the embryonic day 14.5 (E14.5) telencephalon has been suggested from a recent study demonstrating the presence of Neogenin on young,
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actively migrating interneurons (Fitzgerald et al., 2006a). Neogenin activity is also important in the establishment of the morphological architecture in many other developing organ systems outside the CNS. For example, it has been shown to promote adhesion between the multi-potent progenitor (cap) cells and the adjacent epithelial layer of the developing mammary gland (Srinivasan et al., 2003). Netrin1–Neogenin interactions also promote both adhesion and migration of vascular smooth muscle cells during angiogenesis (Park et al., 2004). In addition, Kang and colleagues (2004) have shown that Neogenin activation by Netrin1 stimulates myotube differentiation in vitro. Neogenin has recently been shown to be present on neuronal progenitors in the E12.5–E14.5 mouse cortex and ganglionic eminences (Fitzgerald et al., 2006a). Along the entire neuraxis of the early CNS (until approximately E12) the expanding neural tube is populated by neuroepithelial progenitor cells (NEPs). The bodies of these cells lie adjacent to the apical surface and extend a radial process to the pial surface. NEPs undergo interkinetic division, dividing either symmetrically to generate two new NEPs or asymmetrically to produce a postmitotic neuron and an NEP (Go¨tz and Huttner, 2005; Guillemot, 2005; Mori et al., 2005). Around E10, NEPs begin to produce radial glial cells (RGs) throughout the developing brain (Edwards et al., 1990; Mori et al., 2005). Like NEPs, the cell bodies of RGs reside within the ventricular zone (VZ) at the apical surface and project a radial process to the pial surface. By E12, RGs have replaced NEPs throughout the forebrain, midbrain and hindbrain and are believed to be the major neurogenic progenitors in most regions except the ventral forebrain, spinal cord and retina (Go¨tz and Huttner, 2005; Hartfuss et al., 2001; Noctor et al., 2002). In the early cortex, RGs divide asymmetrically to generate one new RG and either a neuron or a basal progenitor (Anthony et al., 2004; Heins et al., 2002; Malatesta et al., 2003; Miyata et al., 2001; Noctor et al., 2002, 2004). It is now believed that gliogenic RG populations are probably distinct from the neurogenic population (reviewed in Mori et al., 2005). Thus, in contrast to the dorsal forebrain, RGs in the ventral forebrain are gliogenic, giving rise to new RGs, committed glial precursors and mature glia (Malatesta et al., 2003). Around E16 in the cortex, RGs begin to generate glial restricted precursors, producing astrocytes or oligodendrocytes. Towards the end of neurogenesis (E17 in the forebrain) RGs then transform directly into astrocytes (Li et al., 2004; Qian et al., 2000; Voight, 1989). Many studies have now demonstrated that neuronal progenitors reside within the adult subventricular zone (SVZ), adjacent to the lateral ventricles in rodents and probably also humans (Merkle and Alvarez-Buylla, 2006). Neuroblasts generated in the adult SVZ travel rostrally via tangential migration to the olfactory bulb where they give rise to the granule and periglomerular interneuron populations. The SVZ neural stem cell (Type B cell) is astrocytic in nature, extending long processes and expressing both GFAP (glial fibrillary acidic protein) and
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Nestin. The rapidly dividing transit-amplifying Type C cell is generated by the Type B stem cell and gives rise to neuroblasts (Type A cells), the precursors of the olfactory bulb interneurons. The neuroblasts migrate through the SVZ in chains encapsulated by the glial processes of the Type B stem cells. Type C intermediate precursors are found in clusters and are closely associated with Type A neuroblasts. It has recently been shown that the Type B stem cell is a direct descendant of the embryonic striatal RG (reviewed in Merkle and Alvarez-Buylla, 2006). These RGs first transform into astrocytes late in embryogenesis and early postnatal life, before differentiating into the astrocytic Type B stem cell found in the adult SVZ. In a previous study we demonstrated that Neogenin is localized to the proliferative zones of the E14.5 mouse cortex, where it is present on the membrane of actively dividing neurogenic RGs (Fitzgerald et al., 2006a). In the ganglionic eminences (GEs) of the E14.5 ventral forebrain Neogenin was found on the dividing gliogenic RG population. These observations prompted us to conduct a more extensive study of Neogenin localization along the embryonic neuraxis at key developmental time points to determine if Neogenin expression is a common feature of dividing neurogenic and gliogenic precursors. Here we demonstrate that Neogenin is not restricted to RG populations in the E14.5 cortex and GEs, but is localized to many neurogenic and gliogenic precursor populations throughout the developing CNS. These observations suggested that Type B stem cells in the adult SVZ may also express Neogenin. We confirm here that Neogenin is indeed present on this neural stem cell population in the adult forebrain. To characterize the spatiotemporal localization of Neogenin protein along the neuraxis of the mouse embryo from E12.5 to E17.5 we employed an antiserum (CT-Neog) raised against the C-terminal peptide of mouse Neogenin which has been previously characterized (Fitzgerald et al., 2006a,b). Throughout this study, preincubation with the immunizing peptide resulted in the loss of immunoreactivity, thereby confirming the specificity of the antiserum (see Supplementary Fig. 1). The CT-Neog antiserum is directed to the C-terminal amino acid sequence of Neogenin and is therefore able to detect all alternatively spliced forms of mouse Neogenin (Keeling et al., 1997). 1.1. Neogenin is expressed by neurogenic progenitors throughout the E14.5 forebrain Our previous study focused on Neogenin expression in the E14.5 cortex and GEs. Here we expanded our study to determine if Neogenin expression is restricted to these specific regions or if it is present on neural progenitor populations in all regions of the E14.5 forebrain. Along the neuraxis, neural progenitors undergo interkinetic division, with their bodies residing within the VZ. During interkinetic division the nuclei migrate to the basal surface of the VZ, where they proceed through S-phase and then move back to the apical surface to complete the cell
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cycle. S-phase cells can be clearly identified at the basal surface by immunolabeling with an antibody specific for Proliferating Cell Nuclear Antigen (PCNA), which is localized to the nucleus during S-phase (Li et al., 1996). Therefore, PCNA-positive cells demarcate the basal boundary of the
VZ within the expanding neural tissue. Co-labeling of the E14.5 forebrain with the CT-Neog antiserum and antiPCNA revealed that Neogenin was expressed in dividing cells at the basal boundary of the VZ in the hippocampus (Figs. 1A–C), olfactory bulb (Figs. 1G–I), and septum
Fig. 1. Neogenin protein is present on dividing neuronal progenitors throughout the VZs of the E14.5 mouse forebrain. Co-labeling with the CT-Neog antiserum (A, G; green) and an antibody specific for the S-phase marker PCNA (B, H; red) demonstrates that Neogenin is present in the VZ of the cortex, hippocampus (A–C; C, CT-Neog, anti-PCNA; merge) and olfactory bulb of the dorsal telencephalon (G–I), as well as the VZ of the septum in the ventral telencephalon. A–C, G–I insets: High power images of the VZ demonstrating that Neogenin (green) is present on PCNA-positive cells (red) (arrows). Colabeling with the CT-Neog (D, J; green) and an anti-Nestin antibody (E, K; red) (F, L; merge) demonstrated that Neogenin is expressed by Nestin-positive progenitors in the hippocampus (D–F), as well as in the olfactory bulb and diencephalon (thalamus) (J–L). A–C, G–L, sagittal sections, rostral-right; D–F, coronal sections; A–L, dorsal-top. cp, cortical plate; di, diencephalon; ge, ganglionic eminence; hp, hippocampus; ob, olfactory bulb; stp, striatal primordium; svz, subventricular zone; th, thalamus; vz, ventricular zone. Scale bars: A–L, 200 lm.
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(data not shown), where it could be seen on the surface of cells containing PCNA-positive nuclei (Figs. 1A–C and G–I; insets). Neogenin was also expressed by cells at the ventricular surface where M-phase cells reside. Neurogenic progenitors, including RGs, express the intermediate neurofilament protein, Nestin, while RGs are further characterized by their expression of GLAST (Frederiksen and McKay, 1988; Hartfuss et al., 2001; Shibata et al., 1997). Neogenin-positive cells within the VZs of the hippocampus and olfactory bulb were Nestin-positive (Figs. 1D–F and J–L) and GLAST-positive (data not shown), indicating that Neogenin is expressed on neurogenic RGs in these regions. Within the ventral forebrain, Neogenin and Nestin were co-expressed on neurogenic progenitors within the VZ of the diencephalon (thalamus; Figs. 1J–L). Co-labeling
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with an antibody recognizing bIII tubulin or MAP2, markers for maturing and mature neurons, respectively, revealed that neurons in the mantle regions of the dorsal and ventral forebrain were Neogenin-negative (Supplementary Fig. 2 and data not shown). In summary, Neogenin protein is not restricted to progenitor populations in the early cortex and GEs but is present on neurogenic progenitors in all regions of the E14.5 forebrain. 1.2. Neogenin expression during neurogenesis in the midbrain and hindbrain We also examined whether Neogenin was present on dividing neuronal progenitors in the midbrain and hindbrain. Neurogenesis in the midbrain commences around
Fig. 2. Neogenin is present on neuronal progenitors in the midbrain and hindbrain. E12.5 (A, B) or E14.5 (C, D) sections co-labeled with CT-Neog (A, C; green) and anti-PCNA (red) (B, D; merge) demonstrate that Neogenin is localized to the radial processes (A, B; arrows) of dividing neuronal progenitors. Neogenin is absent from mature neurons in the midbrain (C; arrow). Co-localization of Neogenin (E, G; green) and Nestin (red) (F, H; merge) is observed on RG processes in the E14.5 midbrain (E, F; arrows) and hindbrain (G, H; arrows). Boxed area in C indicates midbrain region in E and F. Neogenin (green) is localized to the PCNA-positive (red) cell bodies (inset; arrows) and the processes (arrows) of RGs in the ventral E14.5 spinal cord (I). A–H, sagittal sections, rostral-right; I, transverse section; A–I, dorsal-top. cb, cerebellum; dmb, dorsal midbrain; md, medulla oblongata; pn, pons. Scale bars: A, B, 100 lm; C–I, 200 lm.
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E10 and peaks at E14, coincident with neurogenesis in the forebrain (Go¨tz and Huttner, 2005; Jacobsen, 1991). At E12.5 the neural tube of the presumptive midbrain still comprises NEPs, some of which have begun to produce RGs (reviewed in Go¨tz and Huttner, 2005; Guillemot, 2005). Co-labeling with antibodies to Neogenin and PCNA demonstrated that Neogenin was present on dividing cells within the E12.5 midbrain, being clearly visible along the radial processes of these cells (Figs. 2A and B; arrows). As the midbrain matured, Neogenin expression was maintained in dividing (PCNA-positive) cells within the E14.5 dorsal midbrain VZ, but was absent from maturing neurons residing in the mantle regions (Fig. 2C; arrow). Neogenin-positive cells within the VZs of the dorsal midbrain also co-labeled for Nestin (Figs. 2E and F) and GLAST (data not shown), indicating their RG identity. The RG processes spanning the more immature dorso-caudal E14.5 midbrain were also Neogenin- and Nestin-positive (Figs. 2E and F; arrows). Neurogenesis in the hindbrain, including the cerebellum, begins around E10, but does not reach its peak until E14 (reviewed in Go¨tz and Huttner, 2005). Neurogenesis then continues into the postnatal period in these regions. Within the E14.5 hindbrain, Neogenin was observed on the Nestin-positive processes of the neurogenic RGs spanning the expanding medulla oblongata (Figs. 2G and H; arrows) and cerebellum (data not shown). Therefore, in the early forebrain, midbrain and hindbrain, Neogenin is expressed on actively dividing neurogenic progenitors, including NEPs and RGs, where it is localized to both the cell bodies and radial processes extending to the pial surface. In contrast, Neogenin is absent from the maturing neuronal populations in the E14.5 CNS. 1.3. Gliogenic precursors express Neogenin After E16 in the mouse telencephalon, gliogenic RGs begin producing astrocyte and oligodendrocyte precursors which come to reside within the SVZs (Cameron and Rakic, 1991; Mission et al., 1991; Parnavelas, 1999; Qian et al., 2000). By E17–E18, neurogenesis is largely completed in the forebrain, and the neurogenic RG population has disappeared (Bayer and Altman, 1991; Hartfuss et al., 2001), resulting in the disappearance of the VZ, which is replaced by the ependymal lining of the ventricles (Takahashi et al., 1995). From this developmental stage into postnatal life, progenitors continue to produce the SVZ-based gliogenic precursors (Hartfuss et al., 2001; Takahashi et al., 1995). In addition, gliogenic RGs also transform directly into astrocytes (Edwards et al., 1990; Voight, 1989). To determine if Neogenin is expressed by dividing gliogenic precursors in the E17.5 forebrain we co-labeled forebrain sections with the CT-Neog antiserum and antibodies specific for PCNA. Neogenin was restricted to the proliferative zones (i.e., the SVZs) of the E17.5 dorsal and ventral forebrain (including those of the cortex, hippocampus and striatum), where the dividing PCNA-positive cells were sit-
uated (Figs. 3A–C). It has been clearly demonstrated that the majority of RGs isolated at this late embryonic stage generate only gliogenic progeny and can be identified by their expression of Nestin and GLAST (Hartfuss et al., 2001; Mori et al., 2005). To verify that Neogenin is expressed on these gliogenic precursors, we co-labeled with either an anti-Nestin (Figs. 3D–F) or an anti-GLAST antibody (Figs. 3G–I). As expected, both Nestin-positive and GLAST-positive precursors were found within the SVZs of the E17.5 cortex, hippocampus and striatum (Figs. 3D–F and G–I). Moreover, Neogenin protein was localized to the surface of these Nestin- and GLAST-positive precursors (Figs. 3D–F and G–I; insets), confirming that Neogenin is present on gliogenic precursors in the late embryonic forebrain. In contrast to most regions of the embryonic brain, the neural progenitors within the spinal cord VZ display both NEP and RG characteristics, and can be distinguished by their morphology and expression of Nestin (Hartfuss et al., 2001; Li et al., 2004; Liu et al., 2002; Go¨tz and Huttner, 2005; Guillemot, 2005). Neurogenesis occurs early (E8– E12.5) in the developing mouse spinal cord. After neurogenesis is completed in the rodent spinal cord, progenitors become gliogenic, producing both astrocytes and oligodendrocytes (Li et al., 2004; Liu et al., 2002; Zhou et al., 2000). Neogenin mRNA has been observed in the VZ of the ventral spinal cord during the neurogenic period (Gad et al., 1997; Keeling et al., 1997; Keino-Masu et al., 1996), indicating that spinal cord neurogenic progenitors express Neogenin. Here we examined Neogenin protein localization in the E14.5 mouse spinal cord during gliogenesis. Neogenin was localized to the cells bodies (Fig. 2I; inset) and radial processes (Fig. 2I; arrows) of dividing gliogenic progenitors in the ventral VZ of the E14.5 spinal cord. It is the ventral spinal cord that is believed to be the origin of the oligodendrocyte–astrocyte precursors (Liu et al., 2002; Orentas and Miller, 1996; Zhou et al., 2000). In summary, Neogenin is expressed by gliogenic precursors in the E17.5 cortex, hippocampus, striatum and E14.5 spinal cord. We have previously demonstrated that Neogenin is present on RGs within the E14.5 GEs (Fitzgerald et al., 2006a), which have recently been shown to be gliogenic in nature (Li et al., 2004; Malatesta et al., 2003). Therefore, Neogenin expression appears to be a characteristic of many gliogenic precursor populations throughout the developing CNS. 1.4. Neural stem cells in the adult mouse forebrain express Neogenin Since the neural stem cells (Type B cells) found in the SVZ of the lateral ventricle of the adult rodent forebrain are direct descendants of the embryonic striatal RG population (reviewed in Merkle and Alvarez-Buylla, 2006), we determined whether these stem cells also expressed Neogenin. Fig. 4A demonstrates that the CT-Neog antiserum immunolabels cells within the SVZ surrounding the lateral ventri-
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Fig. 3. Neogenin is present on gliogenic progenitors in the E17.5 forebrain. Co-labeling with the CT-Neog (A, D; green) and the anti-PCNA antibody (B; red) (C; merge), or the anti-Nestin antibody (E; red) (F; merge), demonstrates that Neogenin is expressed by dividing gliogenic progenitors within the SVZs. Co-labeling with the CT-Neog (G; green) and the anti-GLAST antibody (H; red) (I; merge) demonstrates that Neogenin is present on gliogenic RGs. High power images show Neogenin and Nestin co-localization (D–F, insets; arrows) or GLAST co-localization (G–I, insets; arrows) on these progenitors. A–I, coronal sections, dorsal-top. ctx, cortex; hp, hippocampus, st, striatum; svz, subventricular zone. Scale bars: A–I, 200 lm.
cles of the adult forebrain. In contrast, preincubation with the immunizing peptide removed all immunoreactivity in an adjacent section (Fig. 4B). The Type B stem cell expresses both the astrocytic marker, GFAP, and the neural progenitor marker, Nestin, and extends long processes that encapsulate the Type A neuroblast (GFAP-negative). The intermediate precursors (Type C cells) giving rise to these neuroblasts are also GFAP-negative (Doetsch et al., 1997). CT-Neog immunolabeling of the anterior corner of the lateral ventricle (Figs. 4C and F) revealed that Neogenin was localized to cell bodies (arrowheads) within the SVZ in a punctate pattern, as well as on the long processes extending from these cells (arrows). In addition, co-labeling with antiNestin (Figs. 4C–E) or anti-GFAP (Figs. 4F–H) further revealed that both the Neogenin-positive cell bodies and their processes were also Nestin- and GFAP-positive. Together these observations demonstrate that Neogenin is expressed by the neural stem cell (Type B cell) in the adult SVZ, which has been shown to give rise to both interneurons and oligodendrocytes (Merkle and Alvarez-Buylla, 2006).
1.5. Conclusion Here we show, that throughout the developing mouse CNS, Neogenin is present on neurogenic progenitor cells actively undergoing division. Neogenin is found on the earliest neural progenitors at E12.5 (NEPs) when they are undergoing both symmetric (producing two new NEPs) and asymmetric division (producing one NEP and either a neuron, RG, or basal progenitor). As neurogenesis peaks, Neogenin is expressed on neurogenic RG populations throughout the neuraxis, as these cells divide asymmetrically to generate neurons and new RGs. We also observed strong Neogenin expression on glial precursors within the SVZs of the E17.5 forebrain, and in the E14.5 spinal cord. The adult neural stem cell within the SVZ of the mouse forebrain derives from the gliogenic RG population in the embryonic striatum (Merkle and Alvarez-Buylla, 2006), which we have previously shown to be Neogeninpositive (Fitzgerald et al., 2006a). Here we show that Neogenin expression is maintained in the adult neural stem cell.
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Fig. 4. Neogenin is expressed on neural stem cells (Type B cells) in the SVZ of the adult forebrain. (A) Immunolabeling with the CT-Neog antiserum shows that cells within the SVZ surrounding the lateral ventricles of the adult forebrain express Neogenin. (B) Preincubation with the immunizing peptide removes all immunoreactivity in an adjacent section. Co-labeling of the anterior corner of the lateral ventricle with the CT-Neog antiserum (C, F; green) and the anti-Nestin antibody (D; red) (E; merge), or the anti-GFAP antibody (G; red) (H; merge) demonstrates that Neogenin is co-expressed with Nestin and GFAP on the cell bodies (arrowheads) within the SVZ, as well as on the long processes extending from these cells (arrows). A, B, coronal sections; C– H, sagittal sections, rostral-right; A–H, dorsal-top. lv, lateral ventricle; svz, subventricular zone. Scale bars: A, B, 200 lm; C–H, 10 lm.
Together, this evidence indicates that Neogenin expression is a hallmark of many dividing neural precursor populations (neurogenic and gliogenic) in both the embryonic and adult mammalian CNS. 2. Experimental procedures 2.1. Antiserum The CT-Neog rabbit polyclonal antiserum was raised against a unique C-terminal peptide (VQETTRMLEDSESS) of mouse Neogenin corresponding to amino acids 1454–1467 of the published sequence (Keeling et al., 1997) and has been previously described in detail (Fitzgerald et al., 2006a,b). For immunohistochemistry on frozen sections, CT-Neog was diluted 1:250. To demonstrate specificity, diluted antiserum was preincubated with the immunizing peptide (100 lg/ml) for 1 h prior to addition to sections.
2.2. Tissue preparation and immunohistochemical analysis C57Bl/6 embryos were dissected from the uterine horn and immediately flash-frozen in O.C.T. Compound (Tissue-Tek, CA). Adult brains were dissected from C57Bl/6 males at 9 weeks of age. Twelve micron sagittal and coronal sections were cut using a Leica CM3050 Cryostat
and mounted onto SuperFrost Plus-coated slides (Microm, Germany). The use of animals as described here was approved by the Animal Ethics Committee of the University of Queensland in accordance with the guidelines stipulated by the National Health and Medical Research Council of Australia. Sections were post-fixed in absolute ethanol for 10 min, rehydrated in 70%, then by 30% ethanol-PBS, and washed in PBS. The specific immunoreactivity demonstrated in this study was achieved by the use of a very mild fixative (ethanol) after cryostat sectioning without prior exposure to any other fixation protocol. Sections were pre-blocked and permeabilized for several hours at room temperature in blocking solution (PBS; 2% fetal bovine serum; 2% goat serum; 0.2% Triton X-100). Sections were incubated in primary antibodies in blocking solution overnight at 4 °C. Primary antibodies were mouse anti-bIII tubulin (1:500, Promega, WI), mouse anti-Nestin (1:250, Chemicon, CA), mouse anti-PCNA (1:500, Sigma, MO), mouse antiGFAP (1:250, Chemicon), and guinea-pig anti-GLAST (astrocyte-specific glutamate transporter) (1:250, Chemicon). Neogenin immune complexes were detected by amplification with goat anti-rabbit IgG conjugated to biotin (1:500, Chemicon), followed by incubation with streptavidin conjugated to AlexaFluor 488 (1:1000, Molecular Probes, OR). Other secondary antibodies were: goat anti-mouse IgG-AlexaFluor 568 (1:1000), and goat anti-guinea-pig IgG-AlexaFluor 568 (1:1000). Slides were mounted in DakoCytomation Fluorescent Mounting Medium (DAKO, CA). Images were acquired on an Olympus IX81 microscope using AnalySIS software, or on a Zeiss Axioplan 2 microscope using Axiovision software.
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