Figure 1.
Analysis of Plxdc2 in the developing chick brain.
In the chick, Plxdc2 expression was found to be similar to that previously documented in the mouse [9]. a, b, e and f, still images from different external viewing angles of volume rendered specimens showing the orientation of virtual sections in c, d and g–i. c and d, Plxdc2 expression was absent from the region of the cortical hem at HH stage 20 (arrow: b, c and d). g–i, Plxdc2 expression in the cortical hem and surrounding mesoderm was evident by HH stage 21 (arrow: f, g and i). j–l, Plxdc2 in situ hybridisation of coronal sections through the brain of a HH stage 36 chick embryo. m, higher magnification image of indicated region of l showing Plxdc2 expression in the Purkinje cell layer and a lack of Plxdc2 expression in granule cell raphes (arrowheads). Arrowhead in c, floor of midbrain; b, branchial arch expression; ot, otic vesicle; MDB, surrounding mesenchyme of the mesencephalon-diencephalon boundary; MHB, midbrain-hindbrain boundary; Arrow in k and l, Plxdc2 expression in the neuroepithelium; cb, cerebellum; fp, floor plate; med, medulla oblongata; N5n, trigeminal motor nucleus; PCL, Purkinje Cell Layer; Pr5, principle sensory trigeminal nucleus; VI, abducens nucleus.. Scale bar: a–d, 0.4 mm; e–i, 0.5 mm; j–l, 1 mm; m, 250 µm.
Figure 2.
Electroporation of Plxdc2 into the HH stage 10–11 chick brain results in thickening of the neural tube on the experimental side.
a–c, 50 µm transverse sections through the head of a HH stage 17 embryo, electroporated with chPlxdc2-Myc 24 hours previously. d–f, 50 µm horizontal sections through a control embryo, electroporated with pcDNA3.1myc-His(B). Empty pCA-β-EGFPm5-U6 plasmid was routinely co-electroporated (e and f). Scale bar: a and d, 400 µm; b,c,e and f, 100 µm.
Figure 3.
OPT analysis of a HH16 embryo electroporated with chPlxdc2-Myc 24 hours earlier.
ChPlxdc2-Myc was detected by wholemount immunohistochemistry using a myc antibody (DSHB). a and b, still images from different external viewing angles of a volume rendered reconstruction (Supplemental data, movie). c–e, virtual sections through the specimen highlighting thickening of the neural tube on the experimental side (arrowheads). Planes of sectioning are indicated in a and b. f, virtual section through rhombomere 1 of a control specimen electroporated with empty pcDNA3.1myc-His(B) plasmid and pCA-β-EGFPm5-U6 under the same conditions. Thickening of the neural tube is not evident in the electroporated region (arrowheads). In control specimens, wholemount immunohistochemistry was carried out using an EGFP antibody (Invitrogen). MHB, midbrain-hindbrain boundary; R1, rhombomere 1. Scale bar: a–c, 0.4 mm; d and e, 0.25 mm; f, 0.2 mm.
Figure 4.
Statistical analysis of the Plxdc2-induced neural tube thickening phenotype.
a, Representation of the ten measurements taken through each side of the neural tube. b, Plot of the mean thickening ratios across in ovo electroporation experiments. Controls across experimental parameters were found to provide consistent thickening ratios and were pooled. n = number of sections analysed.
Figure 5.
Summary of results from OPT analysis of chPlxdc2-Myc expression and neural tube thickening in seven embryos (A–G), 24 hours post electroporation.
a, Extent of EGFP expressing cells; b, corresponding extent of chPlxdc2-Myc expression (chPlxdc2-Myc data was not obtained for specimen F); c, extent of neural tube thickening in each of seven specimens; d, occurence of thickening in individual brain regions. Grey lines in a-c represent the limits of expression/thickening. IC, inferior colliculus; MHB, midbrain-hindbrain boundary; P1, prosomere 1; P2, prosomere 2; R1, rhombomere 1; R2, rhombomere 2; SC, superior colliculus; ZLI, Zona Limitans Intrathalamica.
Figure 6.
Expression of Plxdc2-AP(CT) in the chick brain causes thickening of the neural tube on the experimental side.
a and d, 20 µm cryostat sections through chick embryos electroporated with Plxdc2-AP, 24 hours previously. b and e, higher magnification images highlighting neural tube thickening on the experimental side. c and f, 30 µm cryostat sections through control specimens electroporated with the empty pAPtag5 plasmid. Scale bar: a and d, 400 µm; b and c, 100 µm; e and f, 200 µm.
Figure 7.
Examination of BrdU-positive cells in the neural tube of a HH stage 18 chick ectopically expressing chPlxdc2-myc illustrates an increased number of BrdU positive cells on the electroporated side.
a, low magnification image of a transverse section of the neural tube in the region of rhombomere 1/2 highlighting the regions shown in b–g. b–g, compressed confocal z-stack images through the neural tube at higher magnification. b and e, overlay of EGFP expression and BrdU incorporation on the experimental side of the neural tube showing examples of cells co-expressing EGFP and BrdU (arrows). Apparent ectopic proliferation in the VZ evident in this section was not evident in other sections (this specimen was chosen as the clearest example of increased BrdU incorporation). Ectopic proliferating cells in the c, d, f and g, BrdU incorporation on either side of the neural tube. Scale bar: a, 200 µm; b–g, 50 µm.
Figure 8.
Plxdc2 misexpression in the neural tube of the chick results in disruption of the normal expression pattern of Cash1.
a–c, immunohistochemistry of coronal sections illustrating Plxdc2-Myc and EGFP expression (double headed arrows highlight neural tube thickening on the experimental side). d, adjacent coronal section through the same embryo following Cash1 in situ hybridisation (Arrows highlight the displacement of Cash1 expression on the experimental side). e, ‘open book’ preparation of the neural tube following wholemount Cash1 in situ hybridisation (Arrowheads, ventral midline). Arrows in f, mesencephalic regions lacking Cash1 expression. Arrows in g, displacement of regions marked in f by expression of chPlxdc2-Myc. Scale bar: b, c, and d 115 µm; e, 300 µm; f and g, 200 µm.
Figure 9.
Expression of Plxdc2 in regions of restricted proliferation.
a–h, horizontal sections through the head of an E10.5 Plxdc2GFP heterozygous mutant showing Plxdc2GFP expression (c, e and h; in green) in the floorplate (fp), cortical hem and MHB. Fewer BrdU-positive cells are present in regions of Plxdc2 expression relative to surrounding regions. hb, hindbrain; mb, midbrain; tel, telencephalon. Scale bar: a, 500 µm; b, c, d and e, 100 µm; g and h, 200 µm; f, 750 µm.
Figure 10.
Homozygous Plxdc2GFP mutants do not exhibit reduced rates of proliferation in regions of the neural tube surrounding the floorplate and cortical hem.
a and b, representative images of horizontal sections through a heterozygous Plxdc2GFP mouse illustrating areas of analysis of the proportion of BrdU-positive cells within the dotted borders on both sides of the region of normal Plxdc2 expression in the floorplate (fp) and cortical hem. (a) In order to carry out regional investigation, the floorplate was divided into two regions, fpA and fpB. c) Statistical analysis illustrated no difference in the proportion of BrdU positive cells in homozygous Plxdc2GFP mice in regions of the neural tube surrounding the floorplate and cortical hem. Data were Arc Sign Transformed and a Mann Whitney U-test carried out due to the non-parametric nature of BrdU data sets. Scale Bar: 100 µm.
Figure 11.
Characterisation of ENCs cultured from the mouse E9.5 neural tube.
In accordance with previously published data, ENCs cultured from the mouse neural tube were shown to express Nestin and Sox2 (a & b). AP binding assays demonstrated that msPlxdc2-AP (d) but not AP alone (c) bound to ENCs. Scale bar: a and b, 200 µm; c and d 100 µm.
Figure 12.
Treatment with extracellular msPlxdc2-AP results in an increase in proliferation in ENCs cultured from the E9.5 neural tube.
Immunocytochemistry with an antibody to BrdU illustrated an increase in the number of BrdU-positive cells following treatment with msPlxdc2-AP (a and b). c) Total number of BrdU-positive cells observed in msPlxdc2-AP cultures originating from four individual neural tubes (A–D). Observed numbers are shown alongside expected values calculated from the proportion of BrdU-positive cells in APtag5 treated controls. Times stated refer to the period of treatment with conditioned media. Chi-squared analyses showed a significant increase in the proportion of BrdU-positive cells in msPlxdc2-AP treated cultures (Chi square results: A, χ2 = 41.37 (43 hrs), 20.52 (65.5 hrs) and 27.73 (73 hrs); B, χ2 = 44.31(43 hrs) and 24.49(65.5 hrs); Cχ2 = 29.03; D, χ2 = 16.39; df = 1, p<0.001 in all instances). Scale bar: 300 µm.