Brain lipid-binding protein is a direct target of Notch signaling in radial glial cells

Abstract

Radial glia function during CNS development both as neural progenitors and as a scaffolding supporting neuronal migration. To elucidate pathways involved in these functions, we mapped in vivo the promoter for Blbp, a radial glial gene. We show here that a binding site for the Notch effector CBF1 is essential for all Blbp transcription in radial glia, and that BLBP expression is significantly reduced in the forebrains of mice lacking the Notch1 and Notch3 receptors. These results identify Blbp as the first predominantly CNS-specific Notch target gene and suggest that it mediates some aspects of Notch signaling in radial glia.

Figure 1.

The 766-bp Blbp promoter recapitulates endogenous BLBP expression in the E12 forebrain and P6 cerebellum of transgenic mice. (A) The construct used to assay promoter activity is similar to 9TM-0.8 (Feng and Heintz 1995) and contains Blbp genomic sequence from –766 to +53, an intron containing a splice donor and acceptor (SD/SA), the E. coli β-galactosidase gene (LacZ), and a polyadenylation site (pA). This construct drove efficient expression throughout the embryonic (B,C) and post-natal (D) CNS. Confirmation of the specificity of this construct was obtained by using the 766-bp fragment to drive eGFP; double labeling for BLBP and eGFP demonstrated that transgene expression is restricted to BLBP⁺ cells in both the E12.5 forebrain (E–G) and P6 cerebellum (H). Arrows in E and F point to region shown at higher magnification in G.

Figure 2.

Elements between –230 and –400 are necessary and sufficient to drive Blbp transcription in the E12 forebrain and P6 cerebellum. A schematic of deletion (1–7) and transfer (8) constructs is shown. The ratios to the right indicate the number of independent transgenic founders that had detectable LacZ expression over the total number of founders obtained for a given construct. Of the constructs assayed, only deletions between –230 and –400 were observed to ablate expression (arrows). Representative whole-mount histochemical staining for β-galactosidase activity of founders are shown at the bottom; the construct used and assay time point is indicated in the upper left of each panel. (*) The transfer construct expressed very strongly at E12 but rather weakly in the P6 cerebellum (histochemical staining is shown), suggesting that additional elements are required in the latter site to obtain normal transcriptional levels. (Tg) Transgenic founder; (nd) not determined.

Figure 3.

The sequence between –230 and –265 is essential for embryonic and post-natal transcription of Blbp. Finer deletions within the –230 to –400 interval defines the sequence between –230 and –265 (deletion 12, arrow) as critical for expression in both the E12 forebrain and P6 cerebellum. Note that the other three constructs drove expression at one of the two locations assayed, suggesting that elements in those intervals are mostly involved in regional and/or temporal control of Blbp transcription. Representative whole-mount histochemical staining for β-galactosidase activity of founders is shown at the bottom; the construct used and assay time point are indicated in the upper left of each panel. The strong expression in the developing olfactory bulbs seen at E12.5 for construct 9 was observed in two independent founders. (*) Construct 10 drove complete embryonic CNS expression but was noticeably weaker than the full-length 766-bp promoter.

Figure 4.

A consensus CBF1-binding site in the critical portion of the Blbp promoter is required for transcriptional activity. (A) The sequence of the critical interval between –230 and –265 contains a putative binding site for CBF1 (boxed); the homology between the Blbp CBF1-binding site and the published consensus sequence is indicated. (B) Mutating the element in the context of the 766 promoter rendered the reporter construct completely inactive in both the E12 forebrain and P6 cerebellum; mice transgenic for the mutant CBF1-binding site were essentially identical in appearance (i.e., no detectable LacZ histochemical staining) to mice transgenic for construct 12 (Fig. 3).

Figure 5.

Radial glial expression of BLBP requires Notch1 and Notch3 signaling. Coronal sections of wild-type (A–H) and cNotch1;Notch3 E14.5 mice (I–P) immunostained for BLBP (A,B,D–J,L–P) and nestin (C,D,K,L); sections in A–D, G, H, I–L, O, and P are from the rostral forebrain, whereas those in E, F, M, and N are at a more caudal level. BLBP expression is significantly reduced in the forebrains of cNotch1;Notch3 mice compared with wild type. (J–L) This decrease is not due to an absence of BLBP-expressing progenitors, as nestin⁺ cells containing low levels of BLBP can still be detected. In addition, cells with long radial processes are observed in mutant brains and stain weakly for BLBP (arrows in E,M point to radial fibers shown at higher magnification in F,N, respectively), confirming that Blbp transcription in radial glia requires Notch1 and Notch3 signaling. (G,H,O,P) Note that the reduction in BLBP expression is restricted to Foxg1-expressing regions (such as the telencephalon), whereas olfactory ensheathing glia contain equivalent levels of BLBP in both wild-type and mutant mice. Arrows in H and P point to olfactory ensheathing cells. (oe) Olfactory epithelium; (tel) telencephalon. Bars: A,E,G,I,M,O, 200 μm; F,N, 80 μm; B–D,H,P, 50 μm; J–L, 40 μm.