Receptor tyrosine kinase ErbB4 modulates neuroblast migration and placement in the adult forebrain

Supplementary Fig. 1 (jpg 60K)
In situ localization of ErbB receptors and their ligands in the adult rat brain. Darkfield photomicrographs of 30 m sagittal sections of the forebrain region, which includes the rostral migratory stream (RMS) and subventricular zone (SVZ) are shown. All probes were amplified from rat brain cDNA. Emulsion dipped slides were developed 2-5 weeks post-dipping. (A) ErbB4, (B) ErbB3, (C) ErbB2, (D) Epidermal growth factor receptor (EGFR), (E) Nissl stained section, (F) Neuregulin-1, SMDF/type III isoform, (G) Neuregulin-1, Ig-specific probe (types I/II), (H) Neuregulin-2, (I) Neuregulin-3, (J) immunocytochemical staining using an affinity-purified rabbit polyclonal anti-ErbB4 serum (#0616; arrows indicate the RMS), (K) Epidermal growth factor (EGF) (L), Epiregulin, (M) Betacellulin (BTC), (N) Transforming growth factor- (TGF-), (O) Heparin-binding epidermal growth factor (HB-EGF). In Nissl stained section (E), the numbers indicate the following structures: 1- anterior olfactory nucleus; dorsal, 2-anterior olfactory nucleus; ventral, 3-rostral migratory stream; 4-corpus callosum; 5-subventricular zone; 6-lateral ventricle; 7-striatum. Sections were prepared from between 2-5 rats per probe.

Supplementary Fig. 2 (jpg 25K)
Lack of ErbB4 expression in nestin-cre; F/- and hGFAP-cre; F/- mutants. Nestin-cre or hGFAP-cre mediated recombination was used to inactivate ErbB4 expression in the RMS. (A) Immunohistochemical localization of ErbB4 in the RMS of control mice (cre -; F/F). ErbB4 was readily detected throughout the RMS, but was not detectable in the RMS following hGFAP-cre (B) or nestin-cre (C) mediated recombination of ErbB4 floxed alleles. ∆ denotes ventral boundaries of the RMS. Sections were prepared from between 3-5 adult mice per group. Scale bar: 400µm.

Supplementary Fig. 3 (jpg 75K)
Deficits in chain migration in nestin-cre; F/- and hGFAP-cre; F/- mutants in vitro. (A) The observed cell migration from SVZ explants could be divided into 3 categories: explants that gave rise to compact chains of migrating neuroblasts, explants that exhibited mixed cell outgrowth [i.e., both individual cells migrating and cells migrating in chains], and explants that only exhibited individual cell migration. Quantification of types of migration observed in explant cultures prepared from the SVZ of control (hGFAP-cre; F/+ [n=4 mice] , nestin-cre; F/+[n=4 mice]), or conditional knockout (nestin-cre; F/- [n=4 mice], hGFAP-cre; F/-[n=3 mice]) mice indicates that chain migration in vitro from SVZ explants is impaired in the ErbB4 deficient SVZ. (B-E) hGFAP-cre; F/- (D) or nestin-cre; F/- (E) explant outgrowth is scattered, with occasional chain formation, when compared to controls, hGFAP-cre; F/+ (B), nestin-cre; F/+ (C). * (C), ^ (D), (E) indicate compact chains of migrating neuroblasts, mixed cell outgrowth [i.e., both individual cells and cells in chains], and scattered cell migration, respectively. Data shown are mean SEM; asterisk, significant when compared with controls at p0.05 (Student's t test). Scale bar: 60µm.

Supplementary Fig. 4 (jpg 23K)
Lack of accumulation of BrdU positive cells in the ventricular zone region adjacent to the anterior ventricle of hGFAP-cre; F/- mice after one month. (A, B) In both control and ErbB4 mutant mice, immunohistochemical localization of BrdU positive cells indicates no accumulation of BrdU labeled cells in the anterior ventricular zone, where the cells began their migration, after one month of BrdU pulsing. Sections were prepared from 5 adult mice per group. Asterisk denotes ventricles. Scale bar: 35µm.

Supplementary Methods (pdf 129KB)

Supplementary Video 1 (avi 1,950K)
CMTMR labeled neuroblasts in the RMS were repeatedly imaged at 5-10 minute intervals using a Zeiss inverted microscope (attached to a PASCAL confocal laser scanning system and a live cell incubation chamber) for 132 (control) or 150 (ErbB4 mutant) minutes. These images were compiled as AVI movie files to illustrate the differences in migration between control and ErbB4 deficient neuroblasts. In the case of control RMS shown, lower right corner is towards the SVZ, upper left corner is towards the olfactory bulb. For ErbB4 mutant RMS, right side is towards the SVZ, left side is towards the olfactory bulb.

Supplementary Video 2 (avi 7,431K)
CMTMR labeled neuroblasts in the RMS were repeatedly imaged at 5-10 minute intervals using a Zeiss inverted microscope (attached to a PASCAL confocal laser scanning system and a live cell incubation chamber) for 132 (control) or 150 (ErbB4 mutant) minutes. These images were compiled as AVI movie files to illustrate the differences in migration between control and ErbB4 deficient neuroblasts. In the case of control RMS shown, lower right corner is towards the SVZ, upper left corner is towards the olfactory bulb. For ErbB4 mutant RMS, right side is towards the SVZ, left side is towards the olfactory bulb.

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