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First published online 30 June 2004
doi: 10.1242/dev.01209

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HGF regulates the development of cortical pyramidal dendrites

Humberto Gutierrez^1,*,, Xavier Dolcet^1,, Mary Tolcos^1, and Alun Davies^1,*

¹ Department Preclinical Veterinary Sciences, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Summerhall Square, Edinburgh EH9 1QH, UK

View larger version (71K): [in a new window]   Fig. 1. (A) Epifluorescence photomicrograph of layer 2 and III pyramidal neurons showing stable labeling with GFP 24 hours after biolistic transfection with a GFP expression plasmid. (B) Z stack projection of a representative layer 2 pyramidal neuron after 48 hours in culture constructed from 10 confocal images of the neuron obtained at 10 µm intervals through the dendritic arbor. Scale bars: 50 µm. (C) RT-PCR analysis of HGF and MET expression in freshly prepared cortical slices and in cortical slices that had been cultured for 2 days. The PCR products for HGF, MET and GAPDH are indicated. No amplification products are observed in control reactions in which the reverse transcription (RT) step was omitted.

View larger version (29K): [in a new window]   Fig. 2. Representative sample of reconstructed layer 2 pyramidal neurons after 48 hours in culture. The neurons shown correspond to percentiles 25, 50, 75 and 100 of the sampled populations in terms of total dendritic length. Control, no additives; HGF, cultures treated with 200 ng/ml HGF; anti-HGF, cultures treated with function blocking anti-HGF antibodies (1 µg/ml); BDNF, cultures treated with 200 ng/ml BDNF. Scale bar: 50 µm.

View larger version (54K): [in a new window]   Fig. 3. Quantitative analysis of the effect of different treatments on the number of primary dendrites. Cortical slices were grown under control conditions (Ctr), or in medium supplemented with 200 ng/ml HGF (HGF), 1 µg/ml anti-HGF (aHGF), 200 ng/ml HGF plus 1 µg/ml anti-HGF pre-incubated for 1 hour before addition to the cultures (HGF + a1), 200 ng/ml HGF plus 3 µg/ml anti-HGF pre-incubated for 1 hour before addition to the cultures (HGF + a2), 200 ng/ml BDNF (BDNF) or HGF + BDNF. The bar charts illustrate the total number of primary dendrites. *P<0.05. Between 50 and 60 individual neurons were analysed under each condition.

View larger version (21K): [in a new window]   Fig. 4. Quantitative analysis of the effect of different treatments on the apical and basal compartment of dendritic arbors. Cortical slices were grown under control conditions (Ctr), or in medium supplemented with 200 ng/ml HGF (HGF), 1 µg/ml anti-HGF (aHGF), 200 ng/ml HGF plus 1 µg/ml anti-HGF pre-incubated for 1 hour before addition to the cultures (HGF+a1), 200 ng/ml HGF plus 3 µg/ml anti-HGF pre-incubated for 1 hour before addition to the cultures (HGF+a2), 200 ng/ml BDNF (BDNF) or HGF+BDNF. The bar charts illustrate percent change on either dendrite length or number of branches relative to untreated controls. *P<0.05, **P<0.01. Between 50 and 60 individual neurons were analysed under each condition.

View larger version (28K): [in a new window]   Fig. 5. Sholl analysis of dendritic morphology. The number of dendrite intersections on concentric rings spaced at 15 µm intervals from the cell soma are shown for neurons grown under control conditions compared with cultures grown with 200 ng/ml HGF (A), 200 ng/ml BDNF (B), 1 µg/ml anti-HGF (C) and HGF plus BDNF (D). Between 50 and 60 individual neurons were analysed under each condition.

View larger version (46K): [in a new window]   Fig. 6. Effects of HGF and anti-HGF treatment on the growth rate of the same layer 2 pyramidal neurons imaged at two time points in culture. Cultures were treated with either 200 ng/ml HGF or 1 µg/ml anti-HGF function-blocking antibodies. A number of sampled neurons per condition were each scanned at two different time points: 18 and 42 hours of incubation in either treatment. Control cultures did not receive any treatment. (A) Pictures of representative cells taken from each condition at each time point. Within this time frame an apparent difference in the rate of change of both the total dendritic length and number of branching points was observed (5B,D, respectively). (C,E) The differences in the actual rate expressed as percentage of increase in length and number of branching points, respectively, with respect to the measurements taken at 18 hours (lgt F(2,27)=15.86, P<0.0001; NB F(2,27)=11.51, P=0.0002).

View larger version (30K): [in a new window]   Fig. 7. Representative confocal images of GFP expressing layer 2 pyramidal neurons 48 hours after biolistic co-transfection with either an empty vector (A) or the MET KD vector (B). (C) A representative sample of reconstructed control transfected neurons (top row) and MET KD-transfected neurons (bottom row) corresponding to percentiles 25, 50, 75 and 100 of the sampled populations in terms of total dendritic length. Scale bar: 50 µm.

View larger version (22K): [in a new window]   Fig. 8. Quantitative analysis of the effect of MET KD transfection on the apical and basal compartment of dendritic arbors. Cortical slices were bombarded with gold particles carrying a combination of either pEGFP and a control empty vector (Ctr) or pEGFP and a vector expressing the kinase-dead MET receptor (METKD). We additionally tested the effect of the dominant negative transfection on cultures supplemented with 200 ng/ml HGF (HGF). The bar charts illustrate percent change in dendrite length and branch number relative to control-transfected neurons. *P<0.05, **P<0.01 versus control transfections; P<0.001 post-hoc significance between control and MET KD transfections in HGF-treated cultures. Between 40 and 50 individual neurons were analysed under each condition.

View larger version (23K): [in a new window]   Fig. 9. Sholl analysis of dendritic morphology of MET KD-transfected pyramidal neurons. (A) Number of dendrite intersections on concentric rings spaced at 15 µm intervals from the cell soma for neurons transfected with the control vector compared with cells transfected with the MET KD vector. (B) The same analysis carried out on cultures grown in the presence of 200 ng/ml HGF. *P<0.05, **P<0.001 in post-hoc comparisons between control and MET KD transfections at each ring.

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