DOI: 10.5061/dryad.tqjq2bw8x
title: Data from: Nerve-dependent regulation of H3K27me3 during the induction of patterning competency in regenerating axolotl limb cells

description: Limb regeneration in the Mexican axolotl requires the dedifferentiation of
 mature limb cells into progenitor cells known as blastema cells. Blastema
 cells become responsive to the signals that define the blueprint, or
 pattern, of the regenerating cells organizing into complex limb
 structures. Signaling downstream of the limb nerves plays a significant
 role in inducing patterning competency; however, the foundations of this
 process remain poorly understood. Studying patterning competency in
 amputated axolotl limbs is challenging because it occurs within the
 context of an intricate network of signals and tissue interactions that
 are activated simultaneously during regeneration. Here, we develop a
 simplified limb injury model assay called the competency accessory limb
 model (CALM), which we used as a platform to characterize the specific
 timing of competency induction and maintenance and identify the H3K27me3
 chromatin signatures associated with this cellular state. We also
 discovered that a combination of FGF and BMP ligands is sufficient to
 induce patterning competency in limb wound cells and identified the ErBB
 pathway among the regulatory epigenetic targets downstream of these
 signals.
description: Tissues were harvested, fixed overnight in 4% formaldehyde,
 decalcified in 10% EDTA (Amresco) for 5 days on an orbital shaker, and
 then moved into 30% sucrose (Fisher Scientific) for 3 days on an orbital
 shaker at 4 °C. Samples were then flash frozen in OCT (Sakura) and cut
 into 10 μm longitudinal tissue sections for immunodetection. After
 sectioning, the samples were warmed and then washed twice in PBS and once
 in 1X PBS-Tween to remove OCT. Samples for H3K27me3 detection were then
 pressure cooked for 2 minutes in sodium citrate buffer. Samples are then
 washed with diH<sub>2</sub>O. After blocking with 0.5% BSA,
 the samples were incubated overnight in a 1:300 solution of rabbit
 anti-histone H3K27me3 polyclonal antibody (A4039050, Epigentek) in
 PBS-Tween at 4 °C. Slides were washed and blocked for an additional 10
 minutes before being incubated for 2 hours at 4 °C in a 1:200 dilution of
 goat anti-rabbit IgG H&L secondary antibody (ab150080, Abcam).
 Samples were then stained with DAPI in PBS-Tween for 10 minutes prior to
 being washed in PBS-Tween and mounted with VECTASHIELD Antifade Mounting
 Medium. Tissue sections were imaged on an Axio Observer
 Z1 inverted fluorescence microscope equipped with an EXCELITAS
 Technologies X-Cite 120 LED lamp, a 63X oil immersion objective with a
 Hamamatsu digital camera C11440 and an Apotome2 unit. The imaging was
 controlled by the Zen Pro 2.0 software package. Z-stacks were imaged using
 0.24 μm intervals. Raw z-stacks were then converted into apotome
 calculation stacks using Zen software and exported to TIFF format files
 for analysis. Images were preprocessed using the custom ImageJ plug-in
 <em>Crop&Snip</em> as previously reported in
 <em>Sosnik et al.</em> and uploaded here as the raw
 images. In the paper, these images are then run through
 <em>H3K_CbyC,</em> a new algorithm and MATLAB code that
 calculates the relative amount of fluorescently tagged protein present
 within a cell nucleus in large 3-dimensional image datasets (Supplemental
 Figure 3). This works by Gaussian blurring the individual images within
 the DAPI channel, binarizing, setting the image to a black background,
 filling holes, and then recombining the individual layers back into a
 matrix. Small objects and under segmented nuclei are removed by volume
 filtering, and the number of nuclei is detected using a connected
 component. From the red channel, (specific to AB detection) pixels that
 overlap with the identified nucleus locations are counted, and the total
 fluorescence per nucleus within an image stack is calculated. Outlier
 z-stacks were removed from subsequent analysis. 50-100 nuclei were
 included in the calculation of averages for each biological sample.
  Analyzed data are saved as a single csv format file for the entire data
 set. <strong>References:</strong>
 Sosnik, J., Vieira, W. A., Webster, K. A., Siegfried, K. R.
 & McCusker, C. D. A new and improved algorithm for the
 quantification of chromatin condensation from microscopic data shows
 decreased chromatin condensation in regenerating axolotl limb
 cells. <em>PLoS One</em> <strong>12</strong>,
 e0185292 (2017).
description: # Data from: Nerve-dependent regulation of H3K27me3 during the induction
 of patterning competency in regenerating axolotl limb cells
 [https://doi.org/10.5061/dryad.tqjq2bw8x](https://doi.org/10.5061/dryad.tqjq2bw8x) ## Description of the data and file structure This data set supports Supplemental Figure 3 of the publication *Nerve-dependent regulation of H3K27me3 during the induction of patterning competency in regenerating axolotl limb cells*. The images of the compressed folder are the selected cell nuclei images that were stained with DAPI and H3K27me3 (Epigentek A4039050), were capture as Z-stacks using Axio Observer Z1 inverted fluorescence microscope equipped with an EXCELITAS Technologies X-Cite 120 LED lamp, a 40X oil immersion objective with a Hamamatsu digital camera C11440 and an Apotome2 unit. The imaging was controlled by the Zen Pro 2.0 software package. Z-stacks were imaged using 0.24 μm intervals. Raw z-stacks were then converted into apotome calculation stacks using Zen software and exported to TIF format files for analysis. These image files were subsequently cropped using the the Crop_Split_Kate.ijm ImageJ plug-in, which is provided. *Supplemental Figure 3 H3K27me3 Raw Images.tar* The compressed master directory "Supplemental Figure 3 H3K27me3 Raw Images", contains a total of 1061 directories and sub-directories with 28282 files total. Each subdirectory of the main folder (i.e. *Apoptome_D14apical1_1_00* within the Blastemas directory) was generated by using the Crop_Split_Kate.ijm plug-in in imageJ to crop a region of interest out of a corresponding Z-stack image. The directory tree structure is visible below: ``` Supplemental Figure 3 H3K27me3 Raw Images +---Blastemas | +---Apotome_D14apical1_1_00 | | +---C1_Apotome_D14apical1_1_00 | | \---C2_Apotome_D14apical1_1_00 | +---Apotome_D14apical1_1_01 | | +---C1_Apotome_D14apical1_1_01 | | \---C2_Apotome_D14apical1_1_01 | +---Apotome_D14apical1_2_00 | | +---C1_Apotome_D14apical1_2_00 | | \---C2_Apotome_D14apical1_2_00 | +---Apotome_D14apical1_2_01 | | +---C1_Apotome_D14apical1_2_01 | | \---C2_Apotome_D14apical1_2_01 | +---Apotome_D14apical1_3_00 | | +---C1_Apotome_D14apical1_3_00 | | \---C2_Apotome_D14apical1_3_00 | +---Apotome_D14apical1_3_01 | | +---C1_Apotome_D14apical1_3_01 | | \---C2_Apotome_D14apical1_3_01 | +---Apotome_D14apical2_1_00 | | +---C1_Apotome_D14apical2_1_00 | | \---C2_Apotome_D14apical2_1_00 | +---Apotome_D14apical2_1_01 | | +---C1_Apotome_D14apical2_1_01 | | \---C2_Apotome_D14apical2_1_01 | +---Apotome_D14apical2_1_02 | | +---C1_Apotome_D14apical2_1_02 | | \---C2_Apotome_D14apical2_1_02 | +---Apotome_D14apical2_2_00 | | +---C1_Apotome_D14apical2_2_00 | | \---C2_Apotome_D14apical2_2_00 | +---Apotome_D14apical2_2_01 | | +---C1_Apotome_D14apical2_2_01 | | \---C2_Apotome_D14apical2_2_01 | +---Apotome_D14apical2_3_00 | | +---C1_Apotome_D14apical2_3_00 | | \---C2_Apotome_D14apical2_3_00 | +---Apotome_D14apical2_3_01 | | +---C1_Apotome_D14apical2_3_01 | | \---C2_Apotome_D14apical2_3_01 | +---Apotome_D14apical3_1_00 | | +---C1_Apotome_D14apical3_1_00 | | \---C2_Apotome_D14apical3_1_00 | +---Apotome_D14apical3_1_01 | | +---C1_Apotome_D14apical3_1_01 | | \---C2_Apotome_D14apical3_1_01 | +---Apotome_D14apical3_2_00 | | +---C1_Apotome_D14apical3_2_00 | | \---C2_Apotome_D14apical3_2_00 | +---Apotome_D14apical3_2_01 | | +---C1_Apotome_D14apical3_2_01 | | \---C2_Apotome_D14apical3_2_01 | +---Apotome_D14apical3_3_00 | | +---C1_Apotome_D14apical3_3_00 | | \---C2_Apotome_D14apical3_3_00 | +---Apotome_D14basal1_1_00 | | +---C1_Apotome_D14basal1_1_00 | | \---C2_Apotome_D14basal1_1_00 | +---Apotome_D14basal1_1_01 | | +---C1_Apotome_D14basal1_1_01 | | \---C2_Apotome_D14basal1_1_01 | +---Apotome_D14basal1_2_00 | | +---C1_Apotome_D14basal1_2_00 | | \---C2_Apotome_D14basal1_2_00 | +---Apotome_D14basal1_2_01 | | +---C1_Apotome_D14basal1_2_01 | | \---C2_Apotome_D14basal1_2_01 | +---Apotome_D14basal1_3_00 | | +---C1_Apotome_D14basal1_3_00 | | \---C2_Apotome_D14basal1_3_00 | +---Apotome_D14basal1_3_01 | | +---C1_Apotome_D14basal1_3_01 | | \---C2_Apotome_D14basal1_3_01 | +---Apotome_D14basal1_4_00 | | +---C1_Apotome_D14basal1_4_00 | | \---C2_Apotome_D14basal1_4_00 | +---Apotome_D14basal1_4_01 | | +---C1_Apotome_D14basal1_4_01 | | \---C2_Apotome_D14basal1_4_01 | +---Apotome_D14basal2_1_00 | | +---C1_Apotome_D14basal2_1_00 | | \---C2_Apotome_D14basal2_1_00 | +---Apotome_D14basal2_1_01 | | +---C1_Apotome_D14basal2_1_01 | | \---C2_Apotome_D14basal2_1_01 | +---Apotome_D14basal2_2_00 | | +---C1_Apotome_D14basal2_2_00 | | \---C2_Apotome_D14basal2_2_00 | +---Apotome_D14basal2_2_01 | | +---C1_Apotome_D14basal2_2_01 | | \---C2_Apotome_D14basal2_2_01 | +---Apotome_D14basal2_3_00 | | +---C1_Apotome_D14basal2_3_00 | | \---C2_Apotome_D14basal2_3_00 | +---Apotome_D14basal2_4_00 | | +---C1_Apotome_D14basal2_4_00 | | \---C2_Apotome_D14basal2_4_00 | +---Apotome_D14basal2_4_01 | | +---C1_Apotome_D14basal2_4_01 | | \---C2_Apotome_D14basal2_4_01 | +---Apotome_D14basal2_5_00 | | +---C1_Apotome_D14basal2_5_00 | | \---C2_Apotome_D14basal2_5_00 | +---Apotome_D14basal2_5_01 | | +---C1_Apotome_D14basal2_5_01 | | \---C2_Apotome_D14basal2_5_01 | +---Apotome_D14basal3_1_00 | | +---C1_Apotome_D14basal3_1_00 | | \---C2_Apotome_D14basal3_1_00 | +---Apotome_D14basal3_1_01 | | +---C1_Apotome_D14basal3_1_01 | | \---C2_Apotome_D14basal3_1_01 | +---Apotome_D14basal3_2_00 | | +---C1_Apotome_D14basal3_2_00 | | \---C2_Apotome_D14basal3_2_00 | +---Apotome_D14basal3_2_01 | | +---C1_Apotome_D14basal3_2_01 | | \---C2_Apotome_D14basal3_2_01 | +---Apotome_D14basal3_3_00 | | +---C1_Apotome_D14basal3_3_00 | | \---C2_Apotome_D14basal3_3_00 | +---Apotome_D14basal3_3_01 | | +---C1_Apotome_D14basal3_3_01 | | \---C2_Apotome_D14basal3_3_01 | +---Apotome_D14basal3_4_00 | | +---C1_Apotome_D14basal3_4_00 | | \---C2_Apotome_D14basal3_4_00 | +---Apotome_D14basal3_4_01 | | +---C1_Apotome_D14basal3_4_01 | | \---C2_Apotome_D14basal3_4_01 | +---Apotome_D21apical1_1_00 | | +---C1_Apotome_D21apical1_1_00 | | \---C2_Apotome_D21apical1_1_00 | +---Apotome_D21apical1_1_01 | | +---C1_Apotome_D21apical1_1_01 | | \---C2_Apotome_D21apical1_1_01 | +---Apotome_D21apical1_2_00 | | +---C1_Apotome_D21apical1_2_00 | | \---C2_Apotome_D21apical1_2_00 | +---Apotome_D21apical1_2_01 | | +---C1_Apotome_D21apical1_2_01 | | \---C2_Apotome_D21apical1_2_01 | +---Apotome_D21apical1_3_00 | | +---C1_Apotome_D21apical1_3_00 | | \---C2_Apotome_D21apical1_3_00 | +---Apotome_D21apical1_3_01 | | +---C1_Apotome_D21apical1_3_01 | | \---C2_Apotome_D21apical1_3_01 | +---Apotome_D21apical2_1_00 | | +---C1_Apotome_D21apical2_1_00 | | \---C2_Apotome_D21apical2_1_00 | +---Apotome_D21apical2_1_01 | | +---C1_Apotome_D21apical2_1_01 | | \---C2_Apotome_D21apical2_1_01 | +---Apotome_D21apical2_1_02 | | +---C1_Apotome_D21apical2_1_02 | | \---C2_Apotome_D21apical2_1_02 | +---Apotome_D21apical2_2_00 | | +---C1_Apotome_D21apical2_2_00 | | \---C2_Apotome_D21apical2_2_00 | +---Apotome_D21apical2_2_01 | | +---C1_Apotome_D21apical2_2_01 | | \---C2_Apotome_D21apical2_2_01 | +---Apotome_D21apical2_3_00 | | +---C1_Apotome_D21apical2_3_00 | | \---C2_Apotome_D21apical2_3_00 | +---Apotome_D21apical2_3_01 | | +---C1_Apotome_D21apical2_3_01 | | \---C2_Apotome_D21apical2_3_01 | +---Apotome_D21apical3_1_00 | | +---C1_Apotome_D21apical3_1_00 | | \---C2_Apotome_D21apical3_1_00 | +---Apotome_D21apical3_1_01 | | +---C1_Apotome_D21apical3_1_01 | | \---C2_Apotome_D21apical3_1_01 | +---Apotome_D21apical3_1_02 | | +---C1_Apotome_D21apical3_1_02 | | \---C2_Apotome_D21apical3_1_02 | +---Apotome_D21apical3_2_00 | | +---C1_Apotome_D21apical3_2_00 | | \---C2_Apotome_D21apical3_2_00 | +---Apotome_D21apical3_2_01 | | +---C1_Apotome_D21apical3_2_01 | | \---C2_Apotome_D21apical3_2_01 | +---Apotome_D21apical3_2_02 | | +---C1_Apotome_D21apical3_2_02 | | \---C2_Apotome_D21apical3_2_02 | +---Apotome_D21apical3_3_00 | | +---C1_Apotome_D21apical3_3_00 | | \---C2_Apotome_D21apical3_3_00 | +---Apotome_D21apical3_3_01 | | +---C1_Apotome_D21apical3_3_01 | | \---C2_Apotome_D21apical3_3_01 | +---Apotome_D21apical3_3_02 | | +---C1_Apotome_D21apical3_3_02 | | \---C2_Apotome_D21apical3_3_02 | +---Apotome_D21basal1_1_00 | | +---C1_Apotome_D21basal1_1_00 | | \---C2_Apotome_D21basal1_1_00 | +---Apotome_D21basal1_1_01 | | +---C1_Apotome_D21basal1_1_01 | | \---C2_Apotome_D21basal1_1_01 | +---Apotome_D21basal1_2_00 | | +---C1_Apotome_D21basal1_2_00 | | \---C2_Apotome_D21basal1_2_00 | +---Apotome_D21basal1_2_01 | | +---C1_Apotome_D21basal1_2_01 | | \---C2_Apotome_D21basal1_2_01 | +---Apotome_D21basal1_3_00 | | +---C1_Apotome_D21basal1_3_00 | | \---C2_Apotome_D21basal1_3_00 | +---Apotome_D21basal1_3_01 | | +---C1_Apotome_D21basal1_3_01 | | \---C2_Apotome_D21basal1_3_01 | +---Apotome_D21basal1_4_00 | | +---C1_Apotome_D21basal1_4_00 | | \---C2_Apotome_D21basal1_4_00 | +---Apotome_D21basal1_4_01 | | +---C1_Apotome_D21basal1_4_01 | | \---C2_Apotome_D21basal1_4_01 | +---Apotome_D21basal2_1_00 | | +---C1_Apotome_D21basal2_1_00 | | \---C2_Apotome_D21basal2_1_00 | +---Apotome_D21basal2_1_01 | | +---C1_Apotome_D21basal2_1_01 | | \---C2_Apotome_D21basal2_1_01 | +---Apotome_D21basal2_1_02 | | +---C1_Apotome_D21basal2_1_02 | | \---C2_Apotome_D21basal2_1_02 | +---Apotome_D21basal2_1_03 | | +---C1_Apotome_D21basal2_1_03 | | \---C2_Apotome_D21basal2_1_03 | +---Apotome_D21basal2_2_00 | | +---C1_Apotome_D21basal2_2_00 | | \---C2_Apotome_D21basal2_2_00 | +---Apotome_D21basal2_2_01 | | +---C1_Apotome_D21basal2_2_01 | | \---C2_Apotome_D21basal2_2_01 | +---Apotome_D21basal2_3_00 | | +---C1_Apotome_D21basal2_3_00 | | \---C2_Apotome_D21basal2_3_00 | +---Apotome_D21basal2_3_02 | | +---C1_Apotome_D21basal2_3_02 | | \---C2_Apotome_D21basal2_3_02 | +---Apotome_D21basal2_4_00 | | +---C1_Apotome_D21basal2_4_00 | | \---C2_Apotome_D21basal2_4_00 | +---Apotome_D21basal2_4_01 | | +---C1_Apotome_D21basal2_4_01 | | \---C2_Apotome_D21basal2_4_01 | +---Apotome_D21basal3_1_00 | | +---C1_Apotome_D21basal3_1_00 | | \---C2_Apotome_D21basal3_1_00 | +---Apotome_D21basal3_1_01 | | +---C1_Apotome_D21basal3_1_01 | | \---C2_Apotome_D21basal3_1_01 | +---Apotome_D21basal3_1_02 | | +---C1_Apotome_D21basal3_1_02 | | \---C2_Apotome_D21basal3_1_02 | +---Apotome_D21basal3_2_00 | | +---C1_Apotome_D21basal3_2_00 | | \---C2_Apotome_D21basal3_2_00 | +---Apotome_D21basal3_2_01 | | +---C1_Apotome_D21basal3_2_01 | | \---C2_Apotome_D21basal3_2_01 | +---Apotome_D21basal3_3_00 | | +---C1_Apotome_D21basal3_3_00 | | \---C2_Apotome_D21basal3_3_00 | +---Apotome_D21basal3_3_01 | | +---C1_Apotome_D21basal3_3_01 | | \---C2_Apotome_D21basal3_3_01 | +---Apotome_D4B1_1_00 | | +---C1_Apotome_D4B1_1_00 | | \---C2_Apotome_D4B1_1_00 | +---Apotome_D4B1_1_01 | | +---C1_Apotome_D4B1_1_01 | | \---C2_Apotome_D4B1_1_01 | +---Apotome_D4B1_2_00 | | +---C1_Apotome_D4B1_2_00 | | \---C2_Apotome_D4B1_2_00 | +---Apotome_D4B1_2_01 | | +---C1_Apotome_D4B1_2_01 | | \---C2_Apotome_D4B1_2_01 | +---Apotome_D4B1_3_00 | | +---C1_Apotome_D4B1_3_00 | | \---C2_Apotome_D4B1_3_00 | +---Apotome_D4B1_3_01 | | +---C1_Apotome_D4B1_3_01 | | \---C2_Apotome_D4B1_3_01 | +---Apotome_D4B1_3_02 | | +---C1_Apotome_D4B1_3_02 | | \---C2_Apotome_D4B1_3_02 | +---Apotome_D4B1_4_00 | | +---C1_Apotome_D4B1_4_00 | | \---C2_Apotome_D4B1_4_00 | +---Apotome_D4B1_4_01 | | +---C1_Apotome_D4B1_4_01 | | \---C2_Apotome_D4B1_4_01 | +---Apotome_D4B1_5_00 | | +---C1_Apotome_D4B1_5_00 | | \---C2_Apotome_D4B1_5_00 | +---Apotome_D4B1_5_01 | | +---C1_Apotome_D4B1_5_01 | | \---C2_Apotome_D4B1_5_01 | +---Apotome_D4B1_6_00 | | +---C1_Apotome_D4B1_6_00 | | \---C2_Apotome_D4B1_6_00 | +---Apotome_D4B2_1_00 | | +---C1_Apotome_D4B2_1_00 | | \---C2_Apotome_D4B2_1_00 | +---Apotome_D4B2_1_01 | | +---C1_Apotome_D4B2_1_01 | | \---C2_Apotome_D4B2_1_01 | +---Apotome_D4B2_2_00 | | +---C1_Apotome_D4B2_2_00 | | \---C2_Apotome_D4B2_2_00 | +---Apotome_D4B2_2_01 | | +---C1_Apotome_D4B2_2_01 | | \---C2_Apotome_D4B2_2_01 | +---Apotome_D4B2_3_00 | | +---C1_Apotome_D4B2_3_00 | | \---C2_Apotome_D4B2_3_00 | +---Apotome_D4B2_3_01 | | +---C1_Apotome_D4B2_3_01 | | \---C2_Apotome_D4B2_3_01 | +---Apotome_D4B2_4_00 | | +---C1_Apotome_D4B2_4_00 | | \---C2_Apotome_D4B2_4_00 | +---Apotome_D4B2_4_01 | | +---C1_Apotome_D4B2_4_01 | | \---C2_Apotome_D4B2_4_01 | +---Apotome_D4B3_1_00 | | +---C1_Apotome_D4B3_1_00 | | \---C2_Apotome_D4B3_1_00 | +---Apotome_D4B3_1_01 | | +---C1_Apotome_D4B3_1_01 | | \---C2_Apotome_D4B3_1_01 | +---Apotome_D4B3_2_00 | | +---C1_Apotome_D4B3_2_00 | | \---C2_Apotome_D4B3_2_00 | +---Apotome_D4B3_2_01 | | +---C1_Apotome_D4B3_2_01 | | \---C2_Apotome_D4B3_2_01 | +---Apotome_D4B3_2_02 | | +---C1_Apotome_D4B3_2_02 | | \---C2_Apotome_D4B3_2_02 | +---Apotome_D4B3_3_00 | | +---C1_Apotome_D4B3_3_00 | | \---C2_Apotome_D4B3_3_00 | +---Apotome_D4B3_3_01 | | +---C1_Apotome_D4B3_3_01 | | \---C2_Apotome_D4B3_3_01 | +---Apotome_D6B1_1_00 | | +---C1_Apotome_D6B1_1_00 | | \---C2_Apotome_D6B1_1_00 | +---Apotome_D6B1_1_01 | | +---C1_Apotome_D6B1_1_01 | | \---C2_Apotome_D6B1_1_01 | +---Apotome_D6B1_2_00 | | +---C1_Apotome_D6B1_2_00 | | \---C2_Apotome_D6B1_2_00 | +---Apotome_D6B1_2_01 | | +---C1_Apotome_D6B1_2_01 | | \---C2_Apotome_D6B1_2_01 | +---Apotome_D6B1_3_00 | | +---C1_Apotome_D6B1_3_00 | | \---C2_Apotome_D6B1_3_00 | +---Apotome_D6B1_4_00 | | +---C1_Apotome_D6B1_4_00 | | \---C2_Apotome_D6B1_4_00 | +---Apotome_D6B2_1_00 | | +---C1_Apotome_D6B2_1_00 | | \---C2_Apotome_D6B2_1_00 | +---Apotome_D6B2_1_01 | | +---C1_Apotome_D6B2_1_01 | | \---C2_Apotome_D6B2_1_01 | +---Apotome_D6B2_2_00 | | +---C1_Apotome_D6B2_2_00 | | \---C2_Apotome_D6B2_2_00 | +---Apotome_D6B2_2_01 | | +---C1_Apotome_D6B2_2_01 | | \---C2_Apotome_D6B2_2_01 | +---Apotome_D6B2_3_00 | | +---C1_Apotome_D6B2_3_00 | | \---C2_Apotome_D6B2_3_00 | +---Apotome_D6B2_3_01 | | +---C1_Apotome_D6B2_3_01 | | \---C2_Apotome_D6B2_3_01 | +---Apotome_D6B2_4_00 | | +---C1_Apotome_D6B2_4_00 | | \---C2_Apotome_D6B2_4_00 | +---Apotome_D6B2_5_00 | | +---C1_Apotome_D6B2_5_00 | | \---C2_Apotome_D6B2_5_00 | +---Apotome_D6B2_5_01 | | +---C1_Apotome_D6B2_5_01 | | \---C2_Apotome_D6B2_5_01 | +---Apotome_D6B3_1_00 | | +---C1_Apotome_D6B3_1_00 | | \---C2_Apotome_D6B3_1_00 | +---Apotome_D6B3_1_01 | | +---C1_Apotome_D6B3_1_01 | | \---C2_Apotome_D6B3_1_01 | +---Apotome_D6B3_2_00 | | +---C1_Apotome_D6B3_2_00 | | \---C2_Apotome_D6B3_2_00 | +---Apotome_D6B3_2_01 | | +---C1_Apotome_D6B3_2_01 | | \---C2_Apotome_D6B3_2_01 | \---Apotome_D6B3_3_00 | +---C1_Apotome_D6B3_3_00 | \---C2_Apotome_D6B3_3_00 +---Denervated Blastemas | +---Apotome_D6Den1_1_00 | | +---C1_Apotome_D6Den1_1_00 | | \---C2_Apotome_D6Den1_1_00 | +---Apotome_D6Den1_1_01 | | +---C1_Apotome_D6Den1_1_01 | | \---C2_Apotome_D6Den1_1_01 | +---Apotome_D6Den1_2_00 | | +---C1_Apotome_D6Den1_2_00 | | \---C2_Apotome_D6Den1_2_00 | +---Apotome_D6Den1_2_01 | | +---C1_Apotome_D6Den1_2_01 | | \---C2_Apotome_D6Den1_2_01 | +---Apotome_D6Den1_3_00 | | +---C1_Apotome_D6Den1_3_00 | | \---C2_Apotome_D6Den1_3_00 | +---Apotome_D6Den1_3_01 | | +---C1_Apotome_D6Den1_3_01 | | \---C2_Apotome_D6Den1_3_01 | +---Apotome_D6Den1_4_00 | | +---C1_Apotome_D6Den1_4_00 | | \---C2_Apotome_D6Den1_4_00 | +---Apotome_D6Den2_1_00 | | +---C1_Apotome_D6Den2_1_00 | | \---C2_Apotome_D6Den2_1_00 | +---Apotome_D6Den2_2_00 | | +---C1_Apotome_D6Den2_2_00 | | \---C2_Apotome_D6Den2_2_00 | +---Apotome_D6Den2_3_00 | | +---C1_Apotome_D6Den2_3_00 | | \---C2_Apotome_D6Den2_3_00 | +---Apotome_D6Den2_4_00 | | +---C1_Apotome_D6Den2_4_00 | | \---C2_Apotome_D6Den2_4_00 | +---Apotome_D6Den2_5_00 | | +---C1_Apotome_D6Den2_5_00 | | \---C2_Apotome_D6Den2_5_00 | +---Apotome_D6Den2_5_01 | | +---Apotome_Mat1_2_01 | | +---C1_Apotome_D6Den2_5_01 | | \---C2_Apotome_D6Den2_5_01 | +---Apotome_D6Den3_1_00 | | +---C1_Apotome_D6Den3_1_00 | | \---C2_Apotome_D6Den3_1_00 | +---Apotome_D6Den3_1_01 | | +---C1_Apotome_D6Den3_1_01 | | \---C2_Apotome_D6Den3_1_01 | +---Apotome_D6Den3_2_00 | | +---C1_Apotome_D6Den3_2_00 | | \---C2_Apotome_D6Den3_2_00 | +---Apotome_D6Den3_2_01 | | +---C1_Apotome_D6Den3_2_01 | | \---C2_Apotome_D6Den3_2_01 | +---Apotome_D6Den3_3_00 | | +---C1_Apotome_D6Den3_3_00 | | \---C2_Apotome_D6Den3_3_00 | +---Apotome_D6Den3_3_01 | | +---C1_Apotome_D6Den3_3_01 | | \---C2_Apotome_D6Den3_3_01 | +---Apotome_D6Den3_4_00 | | +---C1_Apotome_D6Den3_4_00 | | \---C2_Apotome_D6Den3_4_00 | +---Apotome_D6Den3_4_01 | | +---C1_Apotome_D6Den3_4_01 | | \---C2_Apotome_D6Den3_4_01 | +---Apotome_D6Den3_5_00 | | +---C1_Apotome_D6Den3_5_00 | | \---C2_Apotome_D6Den3_5_00 | +---Apotome_D6Den3_5_01 | | +---C1_Apotome_D6Den3_5_01 | | \---C2_Apotome_D6Den3_5_01 | +---Apotome_D6Den4_1_00 | | +---C1_Apotome_D6Den4_1_00 | | \---C2_Apotome_D6Den4_1_00 | +---Apotome_D6Den4_2_00 | | +---C1_Apotome_D6Den4_2_00 | | \---C2_Apotome_D6Den4_2_00 | +---Apotome_D6Den4_3_00 | | +---C1_Apotome_D6Den4_3_00 | | \---C2_Apotome_D6Den4_3_00 | +---Apotome_D6Den4_3_01 | | +---C1_Apotome_D6Den4_3_01 | | \---C2_Apotome_D6Den4_3_01 | +---Apotome_D6Den4_4_00 | | +---C1_Apotome_D6Den4_4_00 | | \---C2_Apotome_D6Den4_4_00 | \---Apotome_D6Den4_5_00 | +---C1_Apotome_D6Den4_5_00 | \---C2_Apotome_D6Den4_5_00 +---Lateral Wounds | +---Apotome_D14W1_1_00 | | +---C1_Apotome_D14W1_1_00 | | \---C2_Apotome_D14W1_1_00 | +---Apotome_D14W1_2_00 | | +---C1_Apotome_D14W1_2_00 | | \---C2_Apotome_D14W1_2_00 | +---Apotome_D14W1_3_00 | | +---C1_Apotome_D14W1_3_00 | | \---C2_Apotome_D14W1_3_00 | +---Apotome_D14W1_4_00 | | +---C1_Apotome_D14W1_4_00 | | \---C2_Apotome_D14W1_4_00 | +---Apotome_D14W1_5_00 | | +---C1_Apotome_D14W1_5_00 | | \---C2_Apotome_D14W1_5_00 | +---Apotome_D14W1_6_00 | | +---C1_Apotome_D14W1_6_00 | | \---C2_Apotome_D14W1_6_00 | +---Apotome_D14W1_6_01 | | +---C1_Apotome_D14W1_6_01 | | \---C2_Apotome_D14W1_6_01 | +---Apotome_D14W1_7_00 | | +---C1_Apotome_D14W1_7_00 | | \---C2_Apotome_D14W1_7_00 | +---Apotome_D14W1_8_00 | | +---C1_Apotome_D14W1_8_00 | | \---C2_Apotome_D14W1_8_00 | +---Apotome_D14W1_8_01 | | +---C1_Apotome_D14W1_8_01 | | \---C2_Apotome_D14W1_8_01 | +---Apotome_D14W1_9_00 | | +---C1_Apotome_D14W1_9_00 | | \---C2_Apotome_D14W1_9_00 | +---Apotome_D14W2_1_00 | | +---C1_Apotome_D14W2_1_00 | | \---C2_Apotome_D14W2_1_00 | +---Apotome_D14W2_1_01 | | +---C1_Apotome_D14W2_1_01 | | \---C2_Apotome_D14W2_1_01 | +---Apotome_D14W2_2_00 | | +---C1_Apotome_D14W2_2_00 | | \---C2_Apotome_D14W2_2_00 | +---Apotome_D14W2_3_00 | | +---C1_Apotome_D14W2_3_00 | | \---C2_Apotome_D14W2_3_00 | +---Apotome_D14W2_4_00 | | +---C1_Apotome_D14W2_4_00 | | \---C2_Apotome_D14W2_4_00 | +---Apotome_D14W2_5_00 | | +---C1_Apotome_D14W2_5_00 | | \---C2_Apotome_D14W2_5_00 | +---Apotome_D14W2_6_00 | | +---C1_Apotome_D14W2_6_00 | | \---C2_Apotome_D14W2_6_00 | +---Apotome_D14W2_7_00 | | +---C1_Apotome_D14W2_7_00 | | \---C2_Apotome_D14W2_7_00 | +---Apotome_D14W2_8_00 | | +---C1_Apotome_D14W2_8_00 | | \---C2_Apotome_D14W2_8_00 | +---Apotome_D14W2_9_00 | | +---C1_Apotome_D14W2_9_00 | | \---C2_Apotome_D14W2_9_00 | +---Apotome_D14W3_1_00 | | +---C1_Apotome_D14W3_1_00 | | \---C2_Apotome_D14W3_1_00 | +---Apotome_D14W3_1_01 | | +---C1_Apotome_D14W3_1_01 | | \---C2_Apotome_D14W3_1_01 | +---Apotome_D14W3_2_00 | | +---C1_Apotome_D14W3_2_00 | | \---C2_Apotome_D14W3_2_00 | +---Apotome_D14W3_2_01 | | +---C1_Apotome_D14W3_2_01 | | \---C2_Apotome_D14W3_2_01 | +---Apotome_D14W3_3_00 | | +---C1_Apotome_D14W3_3_00 | | \---C2_Apotome_D14W3_3_00 | +---Apotome_D14W3_4_00 | | +---C1_Apotome_D14W3_4_00 | | \---C2_Apotome_D14W3_4_00 | +---Apotome_D14W3_5_01 | | +---C1_Apotome_D14W3_5_01 | | \---C2_Apotome_D14W3_5_01 | +---Apotome_D14W3_6_00 | | +---C1_Apotome_D14W3_6_00 | | \---C2_Apotome_D14W3_6_00 | +---Apotome_D14W3_6_01 | | +---C1_Apotome_D14W3_6_01 | | \---C2_Apotome_D14W3_6_01 | +---Apotome_D14W3_8_00 | | +---C1_Apotome_D14W3_8_00 | | \---C2_Apotome_D14W3_8_00 | +---Apotome_D14W3_8_01 | | +---C1_Apotome_D14W3_8_01 | | \---C2_Apotome_D14W3_8_01 | +---Apotome_D14W3_8_02 | | +---C1_Apotome_D14W3_8_02 | | \---C2_Apotome_D14W3_8_02 | +---Apotome_D14W3_8_03 | | +---C1_Apotome_D14W3_8_03 | | \---C2_Apotome_D14W3_8_03 | +---Apotome_D14W3_9_00 | | +---C1_Apotome_D14W3_9_00 | | \---C2_Apotome_D14W3_9_00 | +---Apotome_D14W3_9_01 | | +---C1_Apotome_D14W3_9_01 | | \---C2_Apotome_D14W3_9_01 | +---Apotome_D21W1_1_00 | | +---C1_Apotome_D21W1_1_00 | | \---C2_Apotome_D21W1_1_00 | +---Apotome_D21W1_1_01 | | +---C1_Apotome_D21W1_1_01 | | \---C2_Apotome_D21W1_1_01 | +---Apotome_D21W1_2_00 | | +---C1_Apotome_D21W1_2_00 | | \---C2_Apotome_D21W1_2_00 | +---Apotome_D21W1_2_02 | | +---C1_Apotome_D21W1_2_02 | | \---C2_Apotome_D21W1_2_02 | +---Apotome_D21W1_3_00 | | +---C1_Apotome_D21W1_3_00 | | \---C2_Apotome_D21W1_3_00 | +---Apotome_D21W1_3_01 | | +---C1_Apotome_D21W1_3_01 | | \---C2_Apotome_D21W1_3_01 | +---Apotome_D21W1_3_02 | | +---C1_Apotome_D21W1_3_02 | | \---C2_Apotome_D21W1_3_02 | +---Apotome_D21W1_4_00 | | +---C1_Apotome_D21W1_4_00 | | \---C2_Apotome_D21W1_4_00 | +---Apotome_D21W1_4_01 | | +---C1_Apotome_D21W1_4_01 | | \---C2_Apotome_D21W1_4_01 | +---Apotome_D21W1_4_02 | | +---C1_Apotome_D21W1_4_02 | | \---C2_Apotome_D21W1_4_02 | +---Apotome_D21W1_4_03 | | +---C1_Apotome_D21W1_4_03 | | \---C2_Apotome_D21W1_4_03 | +---Apotome_D21W1_5_00 | | +---C1_Apotome_D21W1_5_00 | | \---C2_Apotome_D21W1_5_00 | +---Apotome_D21W1_5_01 | | +---C1_Apotome_D21W1_5_01 | | \---C2_Apotome_D21W1_5_01 | +---Apotome_D21W1_5_02 | | +---C1_Apotome_D21W1_5_02 | | \---C2_Apotome_D21W1_5_02 | +---Apotome_D21W1_6_00 | | +---C1_Apotome_D21W1_6_00 | | \---C2_Apotome_D21W1_6_00 | +---Apotome_D21W1_6_01 | | +---C1_Apotome_D21W1_6_01 | | \---C2_Apotome_D21W1_6_01 | +---Apotome_D21W1_6_02 | | +---C1_Apotome_D21W1_6_02 | | \---C2_Apotome_D21W1_6_02 | +---Apotome_D21W1_8_00 | | +---C1_Apotome_D21W1_8_00 | | \---C2_Apotome_D21W1_8_00 | +---Apotome_D21W1_8_01 | | +---C1_Apotome_D21W1_8_01 | | \---C2_Apotome_D21W1_8_01 | +---Apotome_D21W1_8_02 | | +---C1_Apotome_D21W1_8_02 | | \---C2_Apotome_D21W1_8_02 | +---Apotome_D21W1_8_03 | | +---C1_Apotome_D21W1_8_03 | | \---C2_Apotome_D21W1_8_03 | +---Apotome_D21W1_8_04 | | +---C1_Apotome_D21W1_8_04 | | \---C2_Apotome_D21W1_8_04 | +---Apotome_D21W2_10_00 | | +---C1_Apotome_D21W2_10_00 | | \---C2_Apotome_D21W2_10_00 | +---Apotome_D21W2_1_00 | | +---C1_Apotome_D21W2_1_00 | | \---C2_Apotome_D21W2_1_00 | +---Apotome_D21W2_1_01 | | +---C1_Apotome_D21W2_1_01 | | \---C2_Apotome_D21W2_1_01 | +---Apotome_D21W2_1_02 | | +---C1_Apotome_D21W2_1_02 | | \---C2_Apotome_D21W2_1_02 | +---Apotome_D21W2_2_00 | | +---C1_Apotome_D21W2_2_00 | | \---C2_Apotome_D21W2_2_00 | +---Apotome_D21W2_2_01 | | +---C1_Apotome_D21W2_2_01 | | \---C2_Apotome_D21W2_2_01 | +---Apotome_D21W2_3_00 | | +---C1_Apotome_D21W2_3_00 | | \---C2_Apotome_D21W2_3_00 | +---Apotome_D21W2_4_00 | | +---C1_Apotome_D21W2_4_00 | | \---C2_Apotome_D21W2_4_00 | +---Apotome_D21W2_4_01 | | +---C1_Apotome_D21W2_4_01 | | \---C2_Apotome_D21W2_4_01 | +---Apotome_D21W2_8_00 | | +---C1_Apotome_D21W2_8_00 | | \---C2_Apotome_D21W2_8_00 | +---Apotome_D21W2_8_01 | | +---C1_Apotome_D21W2_8_01 | | \---C2_Apotome_D21W2_8_01 | +---Apotome_D21W2_9_00 | | +---C1_Apotome_D21W2_9_00 | | \---C2_Apotome_D21W2_9_00 | +---Apotome_D21W3_1_01 | | +---C1_Apotome_D21W3_1_01 | | \---C2_Apotome_D21W3_1_01 | +---Apotome_D21W3_2_00 | | +---C1_Apotome_D21W3_2_00 | | \---C2_Apotome_D21W3_2_00 | +---Apotome_D21W3_2_01 | | +---C1_Apotome_D21W3_2_01 | | \---C2_Apotome_D21W3_2_01 | +---Apotome_D21W3_3_00 | | +---C1_Apotome_D21W3_3_00 | | \---C2_Apotome_D21W3_3_00 | +---Apotome_D21W3_3_01 | | +---C1_Apotome_D21W3_3_01 | | \---C2_Apotome_D21W3_3_01 | +---Apotome_D21W3_4_00 | | +---C1_Apotome_D21W3_4_00 | | \---C2_Apotome_D21W3_4_00 | +---Apotome_D21W3_4_01 | | +---C1_Apotome_D21W3_4_01 | | \---C2_Apotome_D21W3_4_01 | +---Apotome_D21W3_5_00 | | +---C1_Apotome_D21W3_5_00 | | \---C2_Apotome_D21W3_5_00 | +---Apotome_D21W3_5_01 | | +---C1_Apotome_D21W3_5_01 | | \---C2_Apotome_D21W3_5_01 | +---Apotome_D21W3_6_00 | | +---C1_Apotome_D21W3_6_00 | | \---C2_Apotome_D21W3_6_00 | +---Apotome_D21W3_6_01 | | +---C1_Apotome_D21W3_6_01 | | \---C2_Apotome_D21W3_6_01 | +---Apotome_D21W3_7_00 | | +---C1_Apotome_D21W3_7_00 | | \---C2_Apotome_D21W3_7_00 | +---Apotome_D21W3_8_00 | | +---C1_Apotome_D21W3_8_00 | | \---C2_Apotome_D21W3_8_00 | +---Apotome_D4W1_1_00 | | +---C1_Apotome_D4W1_1_00 | | \---C2_Apotome_D4W1_1_00 | +---Apotome_D4W1_2_00 | | +---C1_Apotome_D4W1_2_00 | | \---C2_Apotome_D4W1_2_00 | +---Apotome_D4W1_3_00 | | +---C1_Apotome_D4W1_3_00 | | \---C2_Apotome_D4W1_3_00 | +---Apotome_D4W1_4_00 | | +---C1_Apotome_D4W1_4_00 | | \---C2_Apotome_D4W1_4_00 | +---Apotome_D4W1_5_00 | | +---C1_Apotome_D4W1_5_00 | | \---C2_Apotome_D4W1_5_00 | +---Apotome_D4W1_6_00 | | +---C1_Apotome_D4W1_6_00 | | \---C2_Apotome_D4W1_6_00 | +---Apotome_D4W1_7_00 | | +---C1_Apotome_D4W1_7_00 | | \---C2_Apotome_D4W1_7_00 | +---Apotome_D4W2_1_00 | | +---C1_Apotome_D4W2_1_00 | | \---C2_Apotome_D4W2_1_00 | +---Apotome_D4W2_1_01 | | +---C1_Apotome_D4W2_1_01 | | \---C2_Apotome_D4W2_1_01 | +---Apotome_D4W2_2_00 | | +---C1_Apotome_D4W2_2_00 | | \---C2_Apotome_D4W2_2_00 | +---Apotome_D4W2_3_00 | | +---C1_Apotome_D4W2_3_00 | | \---C2_Apotome_D4W2_3_00 | +---Apotome_D4W2_4_00 | | +---C1_Apotome_D4W2_4_00 | | \---C2_Apotome_D4W2_4_00 | +---Apotome_D4W2_5_00 | | +---C1_Apotome_D4W2_5_00 | | \---C2_Apotome_D4W2_5_00 | +---Apotome_D4W2_6_00 | | +---C1_Apotome_D4W2_6_00 | | \---C2_Apotome_D4W2_6_00 | +---Apotome_D4W2_7_00 | | +---C1_Apotome_D4W2_7_00 | | \---C2_Apotome_D4W2_7_00 | +---Apotome_D4W2_8_00 | | +---C1_Apotome_D4W2_8_00 | | \---C2_Apotome_D4W2_8_00 | +---Apotome_D4W3_1_00 | | +---C1_Apotome_D4W3_1_00 | | \---C2_Apotome_D4W3_1_00 | +---Apotome_D4W3_1_01 | | +---C1_Apotome_D4W3_1_01 | | \---C2_Apotome_D4W3_1_01 | +---Apotome_D4W3_2_00 | | +---C1_Apotome_D4W3_2_00 | | \---C2_Apotome_D4W3_2_00 | +---Apotome_D4W3_2_01 | | +---C1_Apotome_D4W3_2_01 | | \---C2_Apotome_D4W3_2_01 | +---Apotome_D4W3_3_00 | | +---C1_Apotome_D4W3_3_00 | | \---C2_Apotome_D4W3_3_00 | +---Apotome_D4W3_3_01 | | +---C1_Apotome_D4W3_3_01 | | \---C2_Apotome_D4W3_3_01 | +---Apotome_D4W3_4_00 | | +---C1_Apotome_D4W3_4_00 | | \---C2_Apotome_D4W3_4_00 | +---Apotome_D4W3_5_00 | | +---C1_Apotome_D4W3_5_00 | | \---C2_Apotome_D4W3_5_00 | +---Apotome_D4W3_5_01 | | +---C1_Apotome_D4W3_5_01 | | \---C2_Apotome_D4W3_5_01 | +---Apotome_D4W3_6_00 | | +---C1_Apotome_D4W3_6_00 | | \---C2_Apotome_D4W3_6_00 | +---Apotome_D4W3_7_00 | | +---C1_Apotome_D4W3_7_00 | | \---C2_Apotome_D4W3_7_00 | +---Apotome_D6W1_1_00 | | +---C1_Apotome_D6W1_1_00 | | \---C2_Apotome_D6W1_1_00 | +---Apotome_D6W1_2_00 | | +---C1_Apotome_D6W1_2_00 | | \---C2_Apotome_D6W1_2_00 | +---Apotome_D6W1_3_00 | | +---C1_Apotome_D6W1_3_00 | | \---C2_Apotome_D6W1_3_00 | +---Apotome_D6W1_4_00 | | +---C1_Apotome_D6W1_4_00 | | \---C2_Apotome_D6W1_4_00 | +---Apotome_D6W1_5_00 | | +---C1_Apotome_D6W1_5_00 | | \---C2_Apotome_D6W1_5_00 | +---Apotome_D6W1_6_00 | | +---C1_Apotome_D6W1_6_00 | | \---C2_Apotome_D6W1_6_00 | +---Apotome_D6W2_1_00 | | +---C1_Apotome_D6W2_1_00 | | \---C2_Apotome_D6W2_1_00 | +---Apotome_D6W2_1_01 | | +---C1_Apotome_D6W2_1_01 | | \---C2_Apotome_D6W2_1_01 | +---Apotome_D6W2_2_00 | | +---C1_Apotome_D6W2_2_00 | | \---C2_Apotome_D6W2_2_00 | +---Apotome_D6W2_2_01 | | +---C1_Apotome_D6W2_2_01 | | \---C2_Apotome_D6W2_2_01 | +---Apotome_D6W2_3_00 | | +---C1_Apotome_D6W2_3_00 | | \---C2_Apotome_D6W2_3_00 | +---Apotome_D6W2_3_01 | | +---C1_Apotome_D6W2_3_01 | | \---C2_Apotome_D6W2_3_01 | +---Apotome_D6W2_4_00 | | +---C1_Apotome_D6W2_4_00 | | \---C2_Apotome_D6W2_4_00 | +---Apotome_D6W2_4_01 | | +---C1_Apotome_D6W2_4_01 | | \---C2_Apotome_D6W2_4_01 | +---Apotome_D6W2_5_00 | | +---C1_Apotome_D6W2_5_00 | | \---C2_Apotome_D6W2_5_00 | +---Apotome_D6W2_6_00 | | +---C1_Apotome_D6W2_6_00 | | \---C2_Apotome_D6W2_6_00 | +---Apotome_D6W3_1_00 | | +---C1_Apotome_D6W3_1_00 | | \---C2_Apotome_D6W3_1_00 | +---Apotome_D6W3_1_01 | | +---C1_Apotome_D6W3_1_01 | | \---C2_Apotome_D6W3_1_01 | +---Apotome_D6W3_2_00 | | +---C1_Apotome_D6W3_2_00 | | \---C2_Apotome_D6W3_2_00 | +---Apotome_D6W3_3_00 | | +---C1_Apotome_D6W3_3_00 | | \---C2_Apotome_D6W3_3_00 | +---Apotome_D6W3_3_01 | | +---C1_Apotome_D6W3_3_01 | | \---C2_Apotome_D6W3_3_01 | +---Apotome_D6W3_4_00 | | +---C1_Apotome_D6W3_4_00 | | \---C2_Apotome_D6W3_4_00 | +---Apotome_D6W3_5_00 | | +---C1_Apotome_D6W3_5_00 | | \---C2_Apotome_D6W3_5_00 | +---Apotome_D6W3_6_00 | | +---C1_Apotome_D6W3_6_00 | | \---C2_Apotome_D6W3_6_00 | +---Apotome_D6W3_7_00 | | +---C1_Apotome_D6W3_7_00 | | \---C2_Apotome_D6W3_7_00 | \---Apotome_D6W3_8_00 | +---C1_Apotome_D6W3_8_00 | \---C2_Apotome_D6W3_8_00 \---Mature Tissue - D0 +---Apotome_Mat1_1_00 | +---C1_Apotome_Mat1_1_00 | \---C2_Apotome_Mat1_1_00 +---Apotome_Mat1_2_00 | +---C1_Apotome_Mat1_2_00 | \---C2_Apotome_Mat1_2_00 +---Apotome_Mat1_2_01 | +---C1_Apotome_Mat1_2_01 | \---C2_Apotome_Mat1_2_01 +---Apotome_Mat1_3_00 | +---C1_Apotome_Mat1_3_00 | \---C2_Apotome_Mat1_3_00 +---Apotome_Mat1_4_00 | +---C1_Apotome_Mat1_4_00 | \---C2_Apotome_Mat1_4_00 +---Apotome_Mat1_5_00 | +---C1_Apotome_Mat1_5_00 | \---C2_Apotome_Mat1_5_00 +---Apotome_Mat1_6_00 | +---C1_Apotome_Mat1_6_00 | \---C2_Apotome_Mat1_6_00 +---Apotome_Mat1_7_00 | +---C1_Apotome_Mat1_7_00 | \---C2_Apotome_Mat1_7_00 +---Apotome_Mat1_8_00 | +---C1_Apotome_Mat1_8_00 | \---C2_Apotome_Mat1_8_00 +---Apotome_Mat1_9_00 | +---C1_Apotome_Mat1_9_00 | \---C2_Apotome_Mat1_9_00 +---Apotome_Mat2_1_00 | +---C1_Apotome_Mat2_1_00 | \---C2_Apotome_Mat2_1_00 +---Apotome_Mat2_2_00 | +---C1_Apotome_Mat2_2_00 | \---C2_Apotome_Mat2_2_00 +---Apotome_Mat2_3_00 | +---C1_Apotome_Mat2_3_00 | \---C2_Apotome_Mat2_3_00 +---Apotome_Mat2_4_00 | +---C1_Apotome_Mat2_4_00 | \---C2_Apotome_Mat2_4_00 +---Apotome_Mat2_5_00 | +---C1_Apotome_Mat2_5_00 | \---C2_Apotome_Mat2_5_00 +---Apotome_Mat2_6_00 | +---C1_Apotome_Mat2_6_00 | \---C2_Apotome_Mat2_6_00 +---Apotome_Mat2_7_00 | +---C1_Apotome_Mat2_7_00 | \---C2_Apotome_Mat2_7_00 +---Apotome_Mat2_8_00 | +---C1_Apotome_Mat2_8_00 | \---C2_Apotome_Mat2_8_00 +---Apotome_Mat2_9_00 | +---C1_Apotome_Mat2_9_00 | \---C2_Apotome_Mat2_9_00 +---Apotome_Mat3_1_00 | +---C1_Apotome_Mat3_1_00 | \---C2_Apotome_Mat3_1_00 +---Apotome_Mat3_1_01 | +---C1_Apotome_Mat3_1_01 | \---C2_Apotome_Mat3_1_01 +---Apotome_Mat3_2_00 | +---C1_Apotome_Mat3_2_00 | \---C2_Apotome_Mat3_2_00 +---Apotome_Mat3_2_01 | +---C1_Apotome_Mat3_2_01 | \---C2_Apotome_Mat3_2_01 +---Apotome_Mat3_3_00 | +---C1_Apotome_Mat3_3_00 | \---C2_Apotome_Mat3_3_00 +---Apotome_Mat3_4_00 | +---C1_Apotome_Mat3_4_00 | \---C2_Apotome_Mat3_4_00 +---Apotome_Mat3_4_01 | +---C1_Apotome_Mat3_4_01 | \---C2_Apotome_Mat3_4_01 +---Apotome_Mat3_5_00 | +---C1_Apotome_Mat3_5_00 | \---C2_Apotome_Mat3_5_00 +---Apotome_Mat3_6_00 | +---C1_Apotome_Mat3_6_00 | \---C2_Apotome_Mat3_6_00 +---Apotome_Mat3_7_00 | +---C1_Apotome_Mat3_7_00 | \---C2_Apotome_Mat3_7_00 +---Apotome_Mat3_8_00 | +---C1_Apotome_Mat3_8_00 | \---C2_Apotome_Mat3_8_00 +---Apotome_Mat3_9_00 | +---C1_Apotome_Mat3_9_00 | \---C2_Apotome_Mat3_9_00 +---Apotome_Mat4_10_01 | +---C1_Apotome_Mat4_10_01 | \---C2_Apotome_Mat4_10_01 +---Apotome_Mat4_10_02 | +---C1_Apotome_Mat4_10_02 | \---C2_Apotome_Mat4_10_02 +---Apotome_Mat4_11_00 | +---C1_Apotome_Mat4_11_00 | \---C2_Apotome_Mat4_11_00 +---Apotome_Mat4_11_01 | +---C1_Apotome_Mat4_11_01 | \---C2_Apotome_Mat4_11_01 +---Apotome_Mat4_1_00 | +---C1_Apotome_Mat4_1_00 | \---C2_Apotome_Mat4_1_00 +---Apotome_Mat4_3_00 | +---C1_Apotome_Mat4_3_00 | \---C2_Apotome_Mat4_3_00 +---Apotome_Mat4_3_02 | +---C1_Apotome_Mat4_3_02 | \---C2_Apotome_Mat4_3_02 +---Apotome_Mat4_4_00 | +---C1_Apotome_Mat4_4_00 | \---C2_Apotome_Mat4_4_00 +---Apotome_Mat4_4_01 | +---C1_Apotome_Mat4_4_01 | \---C2_Apotome_Mat4_4_01 +---Apotome_Mat4_5_00 | +---C1_Apotome_Mat4_5_00 | \---C2_Apotome_Mat4_5_00 +---Apotome_Mat4_5_01 | +---C1_Apotome_Mat4_5_01 | \---C2_Apotome_Mat4_5_01 +---Apotome_Mat4_6_00 | +---C1_Apotome_Mat4_6_00 | \---C2_Apotome_Mat4_6_00 +---Apotome_Mat4_7_01 | +---C1_Apotome_Mat4_7_01 | \---C2_Apotome_Mat4_7_01 +---Apotome_Mat4_8_00 | +---C1_Apotome_Mat4_8_00 | \---C2_Apotome_Mat4_8_00 +---Apotome_Mat4_8_01 | +---C1_Apotome_Mat4_8_01 | \---C2_Apotome_Mat4_8_01 +---Apotome_Mat4_8_02 | +---C1_Apotome_Mat4_8_02 | \---C2_Apotome_Mat4_8_02 +---Apotome_Mat4_9_00 | +---C1_Apotome_Mat4_9_00 | \---C2_Apotome_Mat4_9_00 +---Apotome_Mat4_9_01 | +---C1_Apotome_Mat4_9_01 | \---C2_Apotome_Mat4_9_01 \---Apotome_Mat4_9_02 +---C1_Apotome_Mat4_9_02 \---C2_Apotome_Mat4_9_02 ``` It is divided into four main directories that were used to perform the analyses (Blastemas, Denervated Blastemas, Lateral Wounds, and Mature Tissue - T0). Each if these directories contains a nested subdirectory format that contains multiple file TIF format files. An example directory tree of the folder and file structure within a subdirectory (ex. *Apoptome_D14apical1_1_00* within the Blastemas directory) can be seen below: ``` Apoptome_D14apical1_1_00 | C1-Apotome_D14apical1_1_00_cropped.tif | C2-Apotome_D14apical1_1_00_cropped.tif | +---C1_Apotome_D14apical1_1_00 | C1-Apotome_D14apical1_1_00_cropped-0001.tif | C1-Apotome_D14apical1_1_00_cropped-0002.tif | C1-Apotome_D14apical1_1_00_cropped-0003.tif | C1-Apotome_D14apical1_1_00_cropped-0004.tif | C1-Apotome_D14apical1_1_00_cropped-0005.tif | C1-Apotome_D14apical1_1_00_cropped-0006.tif | C1-Apotome_D14apical1_1_00_cropped-0007.tif | C1-Apotome_D14apical1_1_00_cropped-0008.tif | C1-Apotome_D14apical1_1_00_cropped-0009.tif | C1-Apotome_D14apical1_1_00_cropped-0010.tif | C1-Apotome_D14apical1_1_00_cropped-0011.tif | C1-Apotome_D14apical1_1_00_cropped-0012.tif | C1-Apotome_D14apical1_1_00_cropped-0013.tif | C1-Apotome_D14apical1_1_00_cropped-0014.tif | C1-Apotome_D14apical1_1_00_cropped-0015.tif | C1-Apotome_D14apical1_1_00_cropped-0016.tif | C1-Apotome_D14apical1_1_00_cropped-0017.tif | C1-Apotome_D14apical1_1_00_cropped-0018.tif | C1-Apotome_D14apical1_1_00_cropped-0019.tif | C1-Apotome_D14apical1_1_00_cropped-0020.tif | C1-Apotome_D14apical1_1_00_cropped-0021.tif | C1-Apotome_D14apical1_1_00_cropped-0022.tif | C1-Apotome_D14apical1_1_00_cropped-0023.tif | C1-Apotome_D14apical1_1_00_cropped-0024.tif | C1-Apotome_D14apical1_1_00_cropped-0025.tif | C1-Apotome_D14apical1_1_00_cropped-0026.tif | C1-Apotome_D14apical1_1_00_cropped-0027.tif | C1-Apotome_D14apical1_1_00_cropped-0028.tif | C1-Apotome_D14apical1_1_00_cropped-0029.tif | C1-Apotome_D14apical1_1_00_cropped-0030.tif | C1-Apotome_D14apical1_1_00_cropped-0031.tif | C1-Apotome_D14apical1_1_00_cropped-0032.tif | C1-Apotome_D14apical1_1_00_cropped-0033.tif | C1-Apotome_D14apical1_1_00_cropped-0034.tif | C1-Apotome_D14apical1_1_00_cropped-0035.tif | C1-Apotome_D14apical1_1_00_cropped-0036.tif | C1-Apotome_D14apical1_1_00_cropped-0037.tif | C1-Apotome_D14apical1_1_00_cropped-0038.tif | C1-Apotome_D14apical1_1_00_cropped-0039.tif | C1-Apotome_D14apical1_1_00_cropped-0040.tif | C1-Apotome_D14apical1_1_00_cropped-0041.tif | C1-Apotome_D14apical1_1_00_cropped-0042.tif | C1-Apotome_D14apical1_1_00_cropped-0043.tif | C1-Apotome_D14apical1_1_00_cropped-0044.tif | C1-Apotome_D14apical1_1_00_cropped-0045.tif | \---C2_Apotome_D14apical1_1_00 C2-Apotome_D14apical1_1_00_cropped-0001.tif C2-Apotome_D14apical1_1_00_cropped-0002.tif C2-Apotome_D14apical1_1_00_cropped-0003.tif C2-Apotome_D14apical1_1_00_cropped-0004.tif C2-Apotome_D14apical1_1_00_cropped-0005.tif C2-Apotome_D14apical1_1_00_cropped-0006.tif C2-Apotome_D14apical1_1_00_cropped-0007.tif C2-Apotome_D14apical1_1_00_cropped-0008.tif C2-Apotome_D14apical1_1_00_cropped-0009.tif C2-Apotome_D14apical1_1_00_cropped-0010.tif C2-Apotome_D14apical1_1_00_cropped-0011.tif C2-Apotome_D14apical1_1_00_cropped-0012.tif C2-Apotome_D14apical1_1_00_cropped-0013.tif C2-Apotome_D14apical1_1_00_cropped-0014.tif C2-Apotome_D14apical1_1_00_cropped-0015.tif C2-Apotome_D14apical1_1_00_cropped-0016.tif C2-Apotome_D14apical1_1_00_cropped-0017.tif C2-Apotome_D14apical1_1_00_cropped-0018.tif C2-Apotome_D14apical1_1_00_cropped-0019.tif C2-Apotome_D14apical1_1_00_cropped-0020.tif C2-Apotome_D14apical1_1_00_cropped-0021.tif C2-Apotome_D14apical1_1_00_cropped-0022.tif C2-Apotome_D14apical1_1_00_cropped-0023.tif C2-Apotome_D14apical1_1_00_cropped-0024.tif C2-Apotome_D14apical1_1_00_cropped-0025.tif C2-Apotome_D14apical1_1_00_cropped-0026.tif C2-Apotome_D14apical1_1_00_cropped-0027.tif C2-Apotome_D14apical1_1_00_cropped-0028.tif C2-Apotome_D14apical1_1_00_cropped-0029.tif C2-Apotome_D14apical1_1_00_cropped-0030.tif C2-Apotome_D14apical1_1_00_cropped-0031.tif C2-Apotome_D14apical1_1_00_cropped-0032.tif C2-Apotome_D14apical1_1_00_cropped-0033.tif C2-Apotome_D14apical1_1_00_cropped-0034.tif C2-Apotome_D14apical1_1_00_cropped-0035.tif C2-Apotome_D14apical1_1_00_cropped-0036.tif C2-Apotome_D14apical1_1_00_cropped-0037.tif C2-Apotome_D14apical1_1_00_cropped-0038.tif C2-Apotome_D14apical1_1_00_cropped-0039.tif C2-Apotome_D14apical1_1_00_cropped-0040.tif C2-Apotome_D14apical1_1_00_cropped-0041.tif C2-Apotome_D14apical1_1_00_cropped-0042.tif C2-Apotome_D14apical1_1_00_cropped-0043.tif C2-Apotome_D14apical1_1_00_cropped-0044.tif C2-Apotome_D14apical1_1_00_cropped-0045.tif ``` Within these directories **C1 **refers to z-stacks and images that are in the blue channel (DAPI) and C2 refers to images that are in the red channel (H3K27me3). The two files directory within this directory (C1-Apotome_D14apical1_1_00_cropped.tif and C2-Apotome_D14apical1_1_00_cropped.tif) are the split cropped z-stacks of the original object that was analyzed to generate all files within this directory. The sub-directories that bear the same names contain each layer of the Z-stack saved as an individual file. These files are separated for later use in running the H3K_CbyC.m code in MATLAB. *Blastemas Directory* The subdirectory (and corresponding files) within this directory are broken into the individual timepoints of 4 days post amputation (dpa), 6dpa, 14dpa, and 21dpa (**D4B**, **D6B**, **D14**, and **D21**). The D14 and D21 subdirectories additionally will be **apical** or **basal** (ex. C2-Apotome_D14apical1... or C2-Apotome_D14basal1...) which indicate whether the images come from the apical (top/distal) or basal (bottom) region of the blastema. This was only done in this folder, and for the purposes of analysis, apical and basal were combined together in both the D14 and D21 timepoints. These images were used in the generation of the following Supplemental Figures: * Supplemental Figure 3g Time-course * Supplemental Figure 3h Innervated vs. Denervated (D6 subdirectories only) *Denervated Blastemas Directory* The subdirectory (and corresponding files) within this directory only contains 6dpa blastemas that were denervated 4 days prior (**D6Den**). These images were only used in the generation of Supplemental Figure 3h Innervated vs. Denervated. *Lateral Wounds Directory* The subdirectory (and corresponding files) within this directory are broken into the individual timepoints of 4, 6, 14, and 21 days post wounding (**D4W**, **D6W**, **D14W**, and **D21W**). These images were only used in the generation of Supplemental Figure 3g Time-course *Mature Tissue - D0 Directory* The subdirectory (and corresponding files) within this directory only contains uninjured mature tissue (**Mat**) collected at the start of lateral wounding (day 0). This tissue was used as time point 0 in all analyses and it's average across biological replicates was used to normalize values during the final analysis. These images were used in the generation of the following Supplemental Figures: * Supplemental Figure 3g Time-course * Supplemental Figure 3h Innervated vs. Denervated *Sub-Directory Naming Conventions* Each sub-directory has three numbers appended to the end (ex. Apoptome_D14apical1_1_00). The first number (...**1***1_00) refers to which biological replicate these images came from - all sub-directories from the same sample type (i.e. D14apical) will have all of the outputs from that biological replicate averaged together. The second number (1***1***00) refers to a different position imaged in that same biological replicate and the third number 1_1***00**) references different elevations positions at the same position (00 is the first). ## Code/software **Crop_Split_Kate.ijm** Previously reported in *Sosnik et al*, this ImageJ macro allows for the cropping of a multi-channel z-stack image, in this case to crop out nuclei of interest. *Reference:* Sosnik, J., Vieira, W. A., Webster, K. A., Siegfried, K. R. & McCusker, C. D. A new and improved algorithm for the quantification of chromatin condensation from microscopic data shows decreased chromatin condensation in regenerating axolotl limb cells. *PLoS One* **12**, e0185292 (2017). **H3K_CbyC.m** A MATLAB algorithm in which directories of nuclei z-stacks are chosen by the user to determine the abundance of a protein of interest (in this case H3K27me3) within a cell nuclei. Directories will need to be generated for each experiment and formatted as shown in these truncated examples: *Time-course Experiment* ``` Example for Time-Course +---Blue Channel | +---C1_Apotome_D4B1_1_00 | +---C1_Apotome_D4B1_1_01 | +---C1_Apotome_D4W1_1_00 | +---C1_Apotome_D4W1_2_00 | +---C1_Apotome_Mat1_1_00 | \---C1_Apotome_Mat1_2_00 \---Red Channel +---C2_Apotome_D4B1_1_00 +---C2_Apotome_D4B1_1_01 +---C2_Apotome_D4W1_1_00 +---C2_Apotome_D4W1_2_00 +---C2_Apotome_Mat1_1_00 \---C2_Apotome_Mat1_2_00 ``` *Innervated vs. Denervated Experiment* ``` Example for Innervated vs Denervated +---Blue Channel | +---C1_Apotome_D6B1_1_01 | +---C1_Apotome_D6B1_2_00 | +---C1_Apotome_D6Den1_1_00 | +---C1_Apotome_D6Den1_1_01 | +---C1_Apotome_Mat1_1_00 | \---C1_Apotome_Mat1_2_00 \---Red Channel +---C2_Apotome_D6B1_1_01 +---C2_Apotome_D6B1_2_00 +---C2_Apotome_D6Den1_1_00 +---C2_Apotome_D6Den1_1_01 +---C2_Apotome_Mat1_1_00 \---C2_Apotome_Mat1_2_00 ``` Compatible with MATLAB 2022b and previous. May be compatible with more recent MATLAB releases. Refer to Supplemental Figure 3f for a graphical representation of how the code operates.