Embryos were raised according to standard protocols and staged in hours post fertilization (hpf) [51, 52].
mRNA injection and cell labeling
Embryos were labeled by injection of RNA encoding histone H2B/red fluorescent protein fusion (H2B-RFP) (100 pg), H2B-GFP (100 pg), membrane-GFP (memGFP, 100 pg), membrane-Cherry (100 pg) or Pard3-GFP (100 pg). Donor embryos for transplantation were injected at the 1 to 2 cell stage to ensure ubiquitous labeling. For mosaic labeling, RNA was injected into a single cell at the 16 to 64 cell stage.
Heterochronic cell transplantation
For control isochronic transplants, embryos of the same age collected at the same time were used for donor and hosts. For heterochronic transplants, donor and host embryos were obtained with a 3-h time difference. All embryos were subject to the same temperature conditions until transplantation to maintain the original time difference between donors and hosts. Transplantations were carried out at blastula and gastrula stages (between 3 to 7 hpf) in agarose-lined dishes containing fish water supplemented with penicillin and streptomycin (Sigma, Gillingham, UK) [52–54]. Approximately 10 to 20 cells were extracted from the donor embryo using a micropipette and expelled into the host embryo in the prospective hindbrain region. Host embryos were unlabeled except for timelapse imaging of young cell divisions when the older host was ubiquitously labeled with H2B-GFP.
For cell polarization analyses, transplanted host embryos were fixed in 4% paraformaldehyde. Old hosts were fixed at 17.5 to 18 hpf when Pard3-GFP should be strongly polarized, and young hosts were fixed at 14.5 to 15 hpf when Pard3-GFP polarization should be weak or absent.
Midline crossing analysis following heterochronic transplantation
For the midline crossing analysis of heterochronic cells, the location of transplanted cells was assessed at 10 to 11 hpf, and only unilateral distributions were imaged later. Midline crossing was used as an indirect measure of whether cells have undergone C-division at the normal time and place, and was assessed by counting the number of integrated cells on each side of the neural tube at 22 to 24 hpf. At this stage all transplanted cells should have undergone a C-division, and most cells should not have divided again. As all transplanted cells were initially unilateral, and cells are only able to cross the midline after division , the percentage of cells crossing the midline was calculated by dividing the number of cells on the contralateral side by the total number of cells × 100. Assuming that all cells will have attempted a C-division and produced two daughter cells, then doubling this percentage gives the percentage of successful C-divisions that have occurred in each embryo.
Ectopic yolk transplants
Approximately 20 to 30 neural plate cells were taken from a labeled wild-type or HuC-GFP donor embryo at 11 hpf using a glass micropipette and immediately expelled just under the enveloping layer above the yolk in a central lateral location in an unlabeled host embryo. Host embryos were incubated overnight in fish water supplemented with penicillin and streptomycin until analysis at 28 to 30 hpf.
Matrigel three-dimensional culture
For this experiment we used cells from the neural plates of MZoep mutant embryos as their anterior neural plate is enlarged and it is easier to harvest large numbers of cells. These donor embryos were ubiquitously labeled with Pard3-GFP and H2B-RFP. When donor embryos were 11 hpf, a 30 μl drop of Matrigel culture medium (50% Matrigel Phenol Red-free (BD Biosciences, Oxford, UK), 39% L-15 media (Gibco, Life Technologies, Paisley, UK), 10% fetal calf serum, 1% penicillin/streptomycin (Sigma, Gillingham, UK)) was placed on a glass coverslip or petri dish on ice. Then 20 to 30 cells were removed from the donor neural plate using a capillary needle and micromanipulator and immediately but slowly expelled into the Matrigel drop. The Matrigel drops were left to gel for 30 to 60 minutes at room temperature, and then covered with a solution of 2% Matrigel in L15 (including 5% FCS and 1% 150 μl penicillin/streptomycin) and incubated at 28.5°C until imaging.
Blocking cell division
To block cell division during neurulation, a combination of the cell division inhibitors aphidicolin and hydroxyurea was used [11, 12, 19–21]. First, both experimental and control embryos were injected at the 1 to 2 cell stage with 1 nl of 1 mM p53 morpholino (p53MO, 5’-GCGCCATTGCTTTGCAAGAATTG-3’, Genetools Inc., Philomath, OR, USA) to reduce cell death. Experimental embryos were then treated from 6 to 11 hpf with 150 μM aphidicolin (Enzo Lifesciences, Exeter, UK) and 20 mM hydroxyurea (Sigma, Gillingham, UK) in 2.5% dimethylsulfoxide (DMSO) in embryo medium to inhibit division for at least one cell cycle, and control embryos were incubated in 2.5% DMSO in embryo medium for the same time period.
Embryos were fixed for 2 h at room temperature or overnight at 4°C. Primary antibodies used were anti-ZO-1 (1:500, Life Technologies, Paisley, UK), anti-GFAP (1:500, Z0344 Dako, Ely, UK), anti-phosphohistone H3 (1:500, Millipore, Billerica, MA, USA), and anti-laminin (1:500, L9393 Sigma, Gillingham, UK). Secondary antibodies were fluorescently conjugated anti-mouse, anti-rabbit or anti-chick alexa-488, alexa-546, alexa-633 or alexa-647 (1:400, Life Technologies, Paisley, UK). Embryos were counterstained with nuclear To-Pro or Sytox stains (1:1,000, T3605 or S7020, Life Technologies, Paisley, UK,) for 1 h after secondary antibody incubation when required.
Confocal and timelapse imaging
Images were acquired on a SP5 Leica confocal microscope. Embryos were mounted in low-melting-point agarose and oriented for dorsal or transverse views of the neural primordium, or oriented laterally for imaging of ectopic transplanted cells. Z-slices were acquired with a z-interval of 2 to 5 μm. For timelapse imaging, a z-stack was captured every 5 to 7 minutes. When imaging embryos at 22 to 30 hpf, embryos were anaesthetized with MS222 (Sigma, Gillingham, UK) and treated with 0.003% 1-phenyl-2-thiourea (w/v) to prevent pigmentation. To analyze Pard3-GFP polarization of transplanted cells, all settings on the confocal were standardized and images were analyzed without subsequent processing by an independent investigator who was ‘blind’ to the embryos experimental status. Imaging of cells in Matrigel was carried out live, by dipping a water immersion lens into the L-15 medium.
The Student’s t test was used to test for a significant difference between the percentages of successful C-divisions in isochronic transplants compared to division-blocked isochronic transplants, as the data were of a Gaussian distribution. The Kolmogorov-Smirnov (KS) test was used to test for a significant difference between the percentages of C-divisions in isochronic transplants and each type of heterochronic transplant because the data were of a skewed distribution. This test was performed online using the ‘Statistics to Use’ website (http://www.physics.csbsju.edu/stats/). The χ2 test (GraphPad Prism) was used to test for a significant difference between the cell division orientations of young transplanted and host cells from a 3 × 2 contingency table of C, D or oblique angled divisions for the two ages of cells.
Separate χ2 (GraphPad Prism) or Fishers tests were used to test for significant differences between the Pard3-GFP polarization state of heterochronic transplanted cells compared to isochronic transplanted cells at 15 hpf and 18 hpf.
The Kruskal-Wallis test (GraphPad Prism) was used to test for a significance difference between the orientation of cell divisions in control and 14th/15th cycle embryos.