Emx2 regulates the proliferation of stem cells of the adult mammalian central nervous system
Rossella Galli1,*,
Roberta Fiocco1,
Lidia De Filippis1,
Luca Muzio2,
Angela Gritti1,
Sara Mercurio2,
Vania Broccoli1,
Massimo Pellegrini3,
Antonello Mallamaci2 and
Angelo L. Vescovi1,*
1 Stem Cell Research Institute, H. S. Raffaele, Via Olgettina 58, Milan, I-20132, Italy
2 Department of Biological and Technological Research (DIBIT), Istituto Scientifico H. San Raffaele, via Olgettina 58, Milan, I-20132, Italy
3 Department of Scienze Morfologiche e Medico Legali, University of Modena and Reggio Emilia, Via del Pozzo 71, Modena, I-41100, Italy
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Fig. 1. Emx2 is expressed in progenitors of both the adult dentate gyrus of the hippocampus and the forebrain subventricular zone (SVZ). (A,B) Emx2 mRNA is present in (A) the adult hippocampus and (B) the dentate gyrus. (C) Strong expression of the gene could be observed along the rostral migratory stream (RMS) that extends toward the two olfactory bulbs and (D) in the periventricular region that lines the lateral ventricles. (E) Cells expressing Emx2 messenger appear to be localized in the subventricular layer (arrow). V, ventricle; Str, striatum; SVZ, subventricular zone; dg, dentate gyrus; h, hilus; dgc, dentate granule cells.
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Fig. 2. Emx2 is expressed in acutely isolated stem cells and in ANSCs and is down regulated during differentiation. (A,B) Primary cultures from the adult PVR were obtained by differential adhesion on PDK-treated plastic (Lim and Alvarez-Buylla, 1999 ) in order to establish two distinct cell fractions, the type B/C cell fraction and the PSA-NCAM-positive fraction, called type A. By means of in situ hybridization, Emx2 was shown to be expressed in (A) type B and (B) type A cells. (C) High levels of Emx2 expression were also detected in undifferentiated adult CNS stem/progenitors. (D) A faint signal was observed in the terminally differentiated progeny of ANSCs, restricted to cells morphologically identifiable as immature neurons. Bar in C, 15 µm. (E) A high frequency of cells expressing Emx2 messenger can be observed when cells are highly undifferentiated. The onset of differentiation and maturation is coincident with a decrease in the number of Emx2-positive cells that drops to 8% of the total cell number by 7 days following induction of differentiation. (F) Semi-quantitative RT-PCR shows high levels of Emx2 messenger in undifferentiated multipotent neural stem cells (ANSCs), while in stem cell-derived terminally differentiated progeny (DCs) lower expression could be detected. No such change was observed in the level of the Emx2 paralog Emx1, or of Otx1, Otx2 and Mash1. Quantitative comparison was made with the housekeeper gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
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Fig. 3. Generation and characterization of retroviral vectors. (A) The structure of the retroviral constructs generated for ANSC infection. (B) Emx2 overexpression in ANSCs is stable for up to 3 months in culture as shown by quantitative analysis of HA immunoreactivity. (C) As shown by immunofluorescence, following exposure to G418, 70-80% of the cells displayed nuclear HA immunoreactivity (right panel). (Left panel) DAPI staining of nuclei to determine total cell numbers. Bar: 10 µm.
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Fig. 4. Generation and characterization of transgenic mice and ANSCs. (A) The structure of the transgene driving ectopic-heterochronic expression of Emx2 and the location of both oligos and probes employed for the analysis of transgene expression. These include the two riboprobes, tg-A and tg-B, used for the RNAse protection assay; the three primers, RT, PF and PR, used for the RT-PCR assay; the Emx2 DNA probe, SP, hybridized to RT-PCR products to confirm their specificity. (B,C) Expression of Emx2 in embryonic telencephalon of wild-type and transgenic mice, as measured by the RNAse protection assay. (D) Expression of the Emx2 transgene in the forebrain periventricular tissue dissected from adult wild-type and mutant mice belonging to one of the two lines used, as assayed by RT-PCR-Southern blotting assay. Similar results were obtained for the other transgenic line (not shown). (E) Expression of the Emx2 transgene in ANSCs isolated and propagated from the same tissues described in D is shown.
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Fig. 5. Modulation of Emx2 expression influences growth capacity of neural stem cells. Growth profiles of (A) Emx2-infected ANSCs, (B) Emx2-overexpressing transgenic ANSCs and (C) Emx2 KO-derived stem cell lines as compared to their wild-type controls. Enforced gene expression always gives rise to severe growth impairment, whereas absence of the gene enhances neural stem cell growth. Logarithmic scale on y-axis. Statistical analysis was performed by means of the Bonferroni test. Means ± s.e.m., n=6 for infected ANSCs, n=4 for gain-of-function ANSCs and n=12 for loss-of-function ANSCs.
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Fig. 6. The schematic illustrates the assay by which the frequency of symmetric proliferative cycles that give rise to two stem cells was evaluated in a given ANSCs population. Emx2 loss-of-function ANSCs were established from animals in which Emx2 expression was knocked out; conversely, Emx2 gain-of-function ANSCs were obtained by isolation and expansion from mice constitutively overexpressing Emx2, as well as by infection with a retrovirus carrying the Emx2 sense sequence. Both types of ANSCs were compared with their controls. Single clones were subjected to a clonogenic assay in which the number of secondary clonogenic cells was expressed as percentage of the total cell number in the clone. In the absence of differential cell death – which was not observed in any of these cultures (Table 3) – this value indicates the ratio between symmetric proliferative cycles (generating two stem cells) and symmetric differentiative cycles (generating two more differentiated cells) that contributes to the formation of the clone. If the frequency of the proliferative symmetric divisions exceeds that of the differentiative ones, the number of stem cells in the clone is greater than one and the stem cell population increases. As expected in this culture system, this was observed under all the conditions tested. However, in the Emx2 KO-derived cells, the relative frequency of symmetric proliferative divisions was far greater than in wild-type animals, as shown by the higher frequency of secondary clones generated, whereas this frequency decreased in gain-of-function cells. Asymmetric cycles that may also have occurred during clone formation generated a stem and a more differentiated cell at each division. Thus, alterations in their frequency did not influence the outcome of this assay.
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© The Company of Biologists Ltd 2002
