The role of neuregulin-ErbB4 interactions on the proliferation and organization of cells in the subventricular zone

Abstract

Coordinated regulation of neuronal progenitor differentiation in the subventricular zone (SVZ) is a fundamental feature of adult neurogenesis. However, the molecular control of this process remains mostly undeciphered. Here, we investigate the role of neuregulins (NRGs) in this process and show that a NRG receptor, ErbB4, is primarily expressed by polysialylated neural cell adhesion molecule immature neuroblasts but is also detected in a subset of GFAP+ astroglial cells, ependymal cells, and Dlx2+ precursors in the SVZ. Of the NRG ligands, both NRG1 and -2 are expressed by immature polysialylated neural cell adhesion molecule neuroblasts in the SVZ. NRG2 is also expressed by some of the GFAP+ putative stem cells lining the ventricles. Infusion of exogenous NRG1 leads to rapid aggregation of Dlx2+ cells in the SVZ and affects the initiation and maintenance of organized neuroblast migration from the SVZ toward the olfactory bulb. In contrast, the infusion of NRG2 increased the number of Sox2 and GFAP+ precursors in the SVZ. An outcome of this NRG2 effect is an increase in the number of newly generated migrating neuroblasts in the rostral migratory stream and GABAergic interneurons in the olfactory bulb. The analysis of conditional null mice that lack NRG receptor, ErbB4, in the nervous system revealed that the observed activities of NRG2 require ErbB4 activation. These results indicate that different NRG ligands affect distinct populations of differentiating neural precursors in the neurogenic regions of the mature forebrain. Furthermore, these studies identify NRG2 as a factor capable of promoting SVZ proliferation, leading to the formation of new neurons in vivo.

Fig. 1.

Distribution of ErbB receptors and NRGs in the SVZ. (A–D) In the SVZ of hGFAP promoter–GFP mice, ErbB4 (A) is expressed in a subset of GFP-expressing SVZ astrocytes (green; open arrow), and EGFR⁺ (blue) cells (filled arrow, A). (B) ErbB4 is also expressed by a subset of CD24⁺ ependymal cells (blue). (C and D) ErbB2 is coexpressed in occasional ErbB4⁺ cells in the SVZ, whereas ErbB3 is expressed mainly by hGFAP-GFP⁺ astrocytes (arrow). (E–G) PSA-NCAM⁺ cells in the SVZ express NRG1 and -2. NRG2 also labels some hGFAP-GFP⁺ cells that line the ventricle (G, arrowheads). [Scale bar: 8 μm (A), 25 μm (B), 20 μm (C), 15 μm (D), and 25 μm (E–G)].

Fig. 2.

Effects of short-term NRG infusion. NRG1, but not NRG2, induced BrdUrd⁺ cell clustering along the walls of the ventricle (arrowheads in B, Insets in A–C). However, no changes in the density of BrdUrd⁺ or total number of nuclei (Nissl label) were noticed (D). (E–H) Cells aggregating in response to NRG1 are Dlx2⁺ progenitors (arrows, F). (I–L) SVZ sections from Sox2-EGFP mice that received NRG infusions indicate that NRG2 induced the expression of the transcription factor Sox2 without a significant increase in BrdUrd incorporation (L). White asterisks denote ventricles. Number of days of NRG and BrdUrd administration is indicated (Left). Data shown are mean ± SEM; ∗, P < 0.01, Student’s t test. [Scale bar: 100 μm (A–C), 65 μm (E–G), and 110 μm (I–K)]. Also see Table 2 and Supporting Text.

Fig. 3.

Long-term effects of NRG infusion on SVZ cell proliferation. Both single (A–D) and continuous (E–G) injections of NRG2 increased the number of BrdUrd⁺ cells significantly (C and D), whereas NRG1 induced clustering of BrdUrd⁺ cells (arrowheads, B and F). Similarly, when BrdUrd was administered during the last 24 h of the 7-day NRG infusion period to preferentially label fast-dividing population of SVZ precursors, both NRG1 and -2 increased the number of BrdUrd⁺ cells. NRG1, as before, induced BrdUrd⁺ cell clustering (arrowhead, J). White asterisks indicate the position of the ventricles. Number of days of NRG and BrdUrd administration is indicated (Left). Data shown are mean ± SEM; ∗, P < 0.01, Student’s t test. [Scale bar: 50 μm (A, C, E, G, I, K); 65 μm (B, F, J)]. Also see Tables 2, 4, and 5 and Supporting Text.

Fig. 4.

Long-term effects of NRG infusion on hGFAP-GFP⁺ astrocytes in the SVZ. NRG2, but not NRG1 or control GST, infusion for 7 days increased the number of GFAP-GFP⁺ astrocytes (green) in the SVZ of hGFAP-GFP mice. NRG2 also increased the number of GFAP-GFP⁺/BrdUrd⁺ astrocytes in the SVZ (D). Number of days of NRG and BrdUrd administration is indicated (Left). White asterisks indicate the position of the ventricles. Data shown are mean ± SEM; ∗, P < 0.01, Student’s t test. (Scale bar: 30 μm.)

Fig. 5.

NRG1 inhibits the initiation of migration from the SVZ into the RMS. Single ventricular injections of the cell tracker dye CMFDA (green) were followed by 3 (A–C, G), or 7 days (E–F, H) of continuous infusion of NRGs. Sagittal sections containing the SVZ, RMS, and the olfactory bulbs were Nissl-counterstained (red). SVZ cells and migrating neuroblasts labeled with CMFDA are green. NRG1 infusion for 3 (B and G) or 7 (E and H) days inhibits the emigration of neuroblasts from the SVZ into the RMS. The presence of migrating cells in the RMS (arrowheads, B and E) or the olfactory bulb (asterisks, B and E) is reduced after NRG1 infusion. (G and H) Quantification of the immunofluorescence of CMFDA⁺ cells from the SVZ to the olfactory bulb (OB) indicates a significant decrease in the number of migrating cells in the RMS or the olfactory bulb after NRG1 infusion. NRG2, however, increased the number of CMFDA⁺ cells in the RMS and in the olfactory bulb. Boxes in A i–iii depict the regions on the SVZ-RMS-OB axis, where immunofluorescence intensity was measured. Data shown are mean ± SEM; ∗, P < 0.01, Student’s t test. (Scale bar: 500 μm.)

Fig. 6.

Conditional deletion of ErbB4 retards NRG2-mediated increase in SVZ cell proliferation. In control mice (ErbB4^lox/+ hGFAP-cre), 7-day infusion of NRG2 resulted in a significant increase in the number of BrdUrd⁺ cells in the SVZ (A–D). In ErbB4 mutant mice (ErbB4^lox/− hGFAP-cre), infusion of NRG2 did not increase SVZ cell proliferation (A′–D′). NRG1, as in controls, did not change SVZ cell proliferation (B, B′, and D′). Data shown are mean ± SEM; ∗, P < 0.01, Student’s t test. (Scale bar: 130 μm.) Also see Table 5 and Supporting Text.

Fig. 7.

Conditional deletion of ErbB4 disrupts the cellular organization of the SVZ. Distribution of CD24⁺ ependymal cells, Dlx2⁺ progenitors, PSA-NCAM⁺ neuroblasts, Lex/SSEA1⁺ stem cells, and GFAP⁺ astrocytic cells in the SVZ of control (A–E) and conditional mutant mice lacking the ErbB4 receptor (ErbB4^lox/− hGFAP-cre; A′–E′). The characteristic elongated GFAP⁺ processes oriented radially away from the ventricular surface (arrowheads, E) are shorter and disrupted in their normal orientation in ErbB4 mutants (arrowheads, E′). (F) Quantification of changes in density (number of cells per mm³) of distinct cell types in control and ErbB4 mutant SVZ indicates reduced density of CD24⁺, PSA-NCAM⁺, and GFAP⁺ cells in the SVZ. (G–I) Quantification of GFAP⁺ process orientation, relative to the ventricular surface indicates that that orientation of GFAP⁺ processes is widely dispersed in ErbB4 conditional mutants compared with controls (G). In addition, the length and number of “bends” (red arrowhead, E′) of GFAP⁺ processes are also altered in ErbB4 mutants (H and I). Data shown are mean ± SEM; ∗, P < 0.01, Student’s t test. [Scale bar: 240 μm (A,A′–C,C′); 140 μm (D,D′); 20 μm (E,E′).]