Expression of insulin-like growth factor system genes during the early postnatal neurogenesis in the mouse hippocampus

Abstract

Insulin-like growth factor-1 (IGF-1) is essential to hippocampal neurogenesis and the neuronal response to hypoxia/ischemia injury. IGF (IGF-1 and -2) signaling is mediated primarily by the type 1 IGF receptor (IGF-1R) and modulated by six high-affinity binding proteins (IGFBP) and the type 2 IGF receptor (IGF-2R), collectively termed IGF system proteins. Defining the precise cells that express each is essential to understanding their roles. With the exception of IGFBP-1, we found that mouse hippocampus expresses mRNA for each of these proteins during the first 2 weeks of postnatal life. Compared to postnatal day 14 (P14), mRNA abundance at P5 was higher for IGF-1, IGFBP-2, -3, and -5 (by 71%, 108%, 100%, and 98%, respectively), lower for IGF-2, IGF-2R, and IGFBP-6 (by 65%, 78%, and 44%, respectively), and unchanged for IGF-1R and IGFBP-4. Using laser capture microdissection (LCM), we found that granule neurons and pyramidal neurons exhibited identical patterns of expression of IGF-1, IGF-1R, IGF-2R, IGFBP-2, and -4, but did not express other IGF system genes. We then compared IGF system expression in mature granule neurons and their progenitors. Progenitors exhibited higher mRNA levels of IGF-1 and IGF-1R (by 130% and 86%, respectively), lower levels of IGF-2R (by 72%), and similar levels of IGFBP-4. Our data support a role for IGF in hippocampal neurogenesis and provide evidence that IGF actions are regulated within a defined in vivo milieu.

Fig. 1

Ki67 immunostaining and RT-PCR amplification of IGF system genes in the developing hippocampal formation of early post-natal mice. Hippocampal Ki67-immunostaining was carried out on sagittal brain sections from either P5 (A) or P14 (B) WT mice. Ki67-positive cells shown in green fluorescence were wide spread in the dentate gyrus (DG) region at P5, but restricted into the subgranular layer at P14. Expression of IGF system genes in P5 and P14 hippocampus is represented in (C) and (D). RT-PCR for IGFBP-1 gave rise to a product of 242 bp, instead of the expected size of 354 bp. The rest of the IGF system genes were specifically amplified and of the predicted size: IGFBP-2, 357 bp; IGFBP-3, 420 bp; IGFBP-4, 201 bp; IGFBP-5, 518 bp; IGFBP-6 and IGF-1, 262 bp; IGF-2, 526 bp; IGF-1R, 340 bp; and IGF-2R, 422 bp. PCR products with expected size of 220 bp and 360 bp also were obtained for Ki67 and GAPDH, respectively. For IGFBP-5 and Ki67, non-specific amplification was detected in addition to the predicted bands, likely due to a relatively low annealing temperature used for these two pairs of primer, which failed to amplify their specific targets above 57°C. Consistent with the observation that there are significantly greater numbers of Ki67-positive cells at P5 than at P14, Ki67 mRNA levels were much higher at P5 when compared to that at P14. GAPDH, studied as an internal control for RT-PCR, did not exhibit significant changes between P5 and P14. Scale bar =100 μm. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Fig. 2

Amplification of IGFBP-1 and total IGF-1 by RT-PCR. RT-PCR for IGFBP-1 were carried out with RNA samples from both liver and hippocampus (A). As expected, the specific 354 bp IGFBP-1 product was detected from liver RNA. A smaller product of 242 bp was amplified from hippocampal RNA and the expected 354 bp product was not amplified. Sequencing of the 242 bp DNA showed that it resulted from non-specific amplification, and this confirms that we could not detect IGFBP-1 expression in the hippocampus. B: Duplex PCR of IGF-1 (180 bp) and GAPDH (360 bp) with RNA samples from P5 and P14 hippocampal tissues. This pair of IGF-1 primers detects all IGF-1 isoforms and reflects total IGF-1 mRNA levels, whereas the primers used in Figure 1 only detect IGF-1 mRNA isoforms that contain exon 5.

Fig. 3

Relative quantification of mRNA levels for IGF system genes in P5 and P14 hippocampus. Levels of IGF system gene expression in P5 and P14 hippocampus were quantified by gel densitometry after RT-PCR. P5 hippocampal mRNA levels for IGF-1 (30 cycles), IGFBP-2 (33 cycles), -3 (33 cycles), and -5 (37 cycles) were higher by 71%, 108%, 100%, and 98%, respectively; whereas those for IGF-2 (30 cycles), IGF-2R (36 cycles), and IGFBP-6 (37 cycles) were lower by 65%, 78%, and 44%, respectively, when compared to their mRNA levels at P14. No significant changes were observed for IGF-1R (30 cycles) and IGFBP-4 (32 cycles). For IGF-1, the 180 bp PCR product represents all IGF-1 mRNA isoforms. Data are presented as mean ± SEM; n =4. *P < 0.05; **P < 0.01.

Fig. 4

Profiling of mRNAs for IGF system genes in hippocampal pyramidal neurons and dentate gyrus granule neurons. Dentate granule neurons were isolated from 8 μm sagittal sections from a P14 mouse brain by LCM. Microdissections were carried out deep in the granular layer to avoid immature granule neurons in the subgranular layer (A,B; showing sections before and after microdissection, respectively). From the same sections, pyramidal neurons of hippocampus proper were then dissected (D,E; showing sections before and after microdissection, respectively). RT-PCR were carried out for IGF system genes with RNA samples from granule neurons (C) or pyramidal neurons (F). Identical patterns of IGF system gene expression were observed for both types of neurons, with detection of IGFBP-2 (357 bp), IGFBP-4 (201 bp), IGF-1 (180 bp), IGF-1R (340 bp), IGF-2R (422 bp); but not other components of IGF system. NF-L (414 bp) and β-actin (194 bp) were also detected in both types of neurons. Scale bar =100 μm. GL, granular layer; H, hilus; CA, Cornu Ammonis. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Fig. 5

mRNA expression of IGF system genes in immature and mature granule neurons. Ki67 immunohistochemistry was carried out on 8-μm frozen sagittal sections from P12 mouse brains. Positive cells shown with green fluorescence were clustered along the subgranular layer of dentate gyrus (A), and were considered immature granule neurons. They were first dissected by LCM (B shows where microdissected cells were captured on the section; C shows the same cells that were captured for analysis). Next, Ki67-negative cells in the granular layer (i.e., mature granule neurons) were collected from the same sections (D shows where microdissected cells were captured on the section; E shows the same cells that were captured for analysis). RT-PCR showed detection of both Ki67 (220 bp) and double cortin (DCX, 239 bp) mRNAs exclusively in immature granule neurons (F). Expression of IGF system genes in mature and immature granule neurons were determined by RT-PCR and shown in gels at the bottom (G). Amplification of IGFBP-4 (201 bp), IGF-1 (180 bp), IGF-1R (340 bp), and IGF-2R (422 bp) was observed in both mature and immature granule neurons. Other IGF system genes were not detected. Scale bar = 100 μm. DG, dentate gyrus; SGL, subgranular layer; GL, granular layer; +, Ki67-positive; −, Ki67-negative. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]