Regulation of neuroblast mitosis is determined by PACAP receptor isoform expression

Abstract

Although neurogenesis in the embryo proceeds in a region- or lineage-specific fashion coincident with neuropeptide expression, a regulatory role for G protein-coupled receptors (GPCR) remains undefined. Pituitary adenylate cyclase activating polypeptide (PACAP) stimulates sympathetic neuroblast proliferation, whereas the peptide inhibits embryonic cortical precursor mitosis. Here, by using ectopic expression strategies, we show that the opposing mitogenic effects of PACAP are determined by expression of PACAP receptor splice isoforms and differential coupling to the phospholipase C (PLC) pathway, as opposed to differences in cellular context. In embryonic day 14 (E14) cortical precursors transfected with the hop receptor variant, but not cells transfected with the short variant, PACAP activates the PLC pathway, increasing intracellular calcium and eliciting translocation of protein kinase C. Ectopic expression of the hop variant in cortical neuroblasts transforms the antimitotic effect of PACAP into a promitogenic signal. Furthermore, PACAP promitogenic effects required PLC pathway function indicated by antagonist U-73122 studies in hop-transfected cortical cells and native sympathetic neuroblasts. These observations highlight the critical role of lineage-specific expression of GPCR variants in determining mitogenic signaling in neural precursors.

Figure 1

Cortical cultures consist of neural precursors and differentiating neurons. Cortical cells at DIV2 express (a) nestin (FITC labeling, arrows) or tau (Texas Red labeling, arrowheads) or (b) TuJ1 (FITC) and tau (Texas Red). (c) The proliferative population, labeled with BrdUrd (arrows, red chromophore), is nestin-positive (blue chromophore). Solid arrowhead indicates a nestin-positive cell, which did not incorporate BrdUrd. (d) Summary of immunocytochemical markers exhibited by cortical cells at 2 DIV. (Scale bars = 10 μm.)

Figure 2

Characterization of GFP-transfected cells. Cells were transfected with vector 12 h after plating and were analyzed by double immunocytochemistry at 2 DIV. GFP is expressed in both nestin-IR cells (a and a′: arrowhead) and TuJ1-IR cells (b and b′). In another experiment, cells were pulsed for 4 h with 10 μM BrdUrd and processed for GFP/BrdUrd double immunostaining. Cells expressing GFP (FITC labeling) exhibit (c, arrows) or lack (d, arrowheads) BrdUrd labeling (Texas red). (Scale bars = 10 μm.)

Figure 3

Ectopic expression of PAC1 receptor in cortical precursors. (a) The majority of normal, untransfected cortical precursors express PAC1 immunoreactivity (as previously reported in ref. 10) revealed by extended peroxidase incubation time (5 min, DAB substrate). Arrows, PAC1-IR cells exhibiting intense cytoplasmic signal and a negatively stained, eccentric nucleus; arrowheads, negative cell exhibiting only background signal. (b and b′) Following a brief (1 min) reaction time, this oblong cell cotransfected with GFP and PAC1 expression vectors exhibits intense cytoplasmic immunostaining for PAC1 (b, arrow) and prominent GFP autofluorescence in the eccentric nucleus (b′), although weaker fluorescence is present in the cytoplasm. (c and c′) In contrast, cells transfected with GFP vector alone exhibit intense GFP signal (c′), but only background DAB signal (c, arrowhead). Note that, in contrast to the GFP immunostaining procedure (Figs. 1 and 2), GFP autofluorescence is predominantly observed in the nucleus. (Scale bar = 10 μm.)

Figure 4

PAC1hop activation increases intracellular calcium levels in cortical cells. Following transfection with different PAC1 isoforms, cells were analyzed by using Fura-2 calcium ratio imaging at steady state and after 10 nM PACAP (arrow) and 25 mM KCl (arrowhead). Traces show individual plots of F340/F380 ratio in (a) three representative GFP/PAC1hop-transfected cells and (b) three nontransfected cells from the same dish. (c) Ratio of transfected cells that responded to PACAP with an increase in intracellular calcium to total GFP positive cells assessed.

Figure 5

Phorbol ester- and PACAP-induced translocation of PKCγ-GFP fusion protein in cortical cells. (a) PKCγ-GFP fusion protein was visualized throughout the cytoplasm in transfected cortical cells. Note the lack of staining over the eccentric nuclei as indicated by open arrowheads. This pattern was observed in most unstimulated cortical cells transfected with PKCγ-GFP alone, PKCγ-GFP/PAC1short, or PKCγ-GFP/PAC1hop. (b) Activation of PKCγ by 0.2 μM PMA for 15 min induced the translocation of PKCγ-GFP from cytosol to the nucleus (indicated by solid arrowheads). (c) PKCγ-GFP was also visualized throughout the cytoplasm in PKCγ-GFP/PAC1short-transfected cortical cells 45 s after 10 nM PACAP treatment. Open arrowhead indicates absence of prominent nuclear staining. (d) In contrast, PKCγ translocation to the membrane (arrows) and nucleus (solid arrowheads) occurred in PKCγ-GFP/PAC1hop-transfected cells 45 s after 10 nM PACAP. (e) Quantification of PKCγ-GFP nuclear/membrane localization in cells transfected with PKCγ-GFP alone, PKCγ-GFP/PAC1short, or PKCγ-GFP/PAC1hop after 45 s PACAP treatment. Data were obtained from three experiments; *, P < 0.001 vs. GFP-PKC-transfected cells. (Scale bar, 10 μm.)

Figure 6

PAC1 isoforms differentially regulate DNA synthesis and proliferation. At 2 DIV, transfected cortical precursors were incubated for 6 h with 10 nM PACAP. Cultures were pulsed with BrdUrd (10 μM) for the final 2 h and processed for double GFP/BrdUrd labeling. (a) BrdUrd labeling index of transfected cells. PACAP treatment decreased DNA synthesis in GFP- or GFP/PAC1short-transfected cells, whereas the peptide increased mitosis in GFP/PAC1hop-transfected cells. Values are expressed as % ± SEM (n = 9). Differs from vehicle-treated group: *, P < 0.05; **, P < 0.01. (b) Transfection rates in corresponding cultures. PACAP treatment resulted in an overall increase in GFP-IR cell number (P < 0.05). Values are expressed as % of GFP-IR cells/total ± SEM. (c and d) GFP/PAC1short- or GFP/PAC1hop-transfected cells were treated daily with PACAP for 5 days and processed for GFP immunostaining. (c) Example of a clone at 5 DIV. (Scale bar = 10 μm.) (d) Quantification of GFP-IR clones (≥4 cells) at 5 DIV. Data are expressed as mean number of clones per ten fields ± SEM (n = 8, four experiments). *, unpaired t test, P < 0.02.