DOI: 10.5281/zenodo.13709858
title: Long-term live imaging, cell identification and cell tracking in regenerating crustacean legs

description: Supplementary data and videos for the manuscript 'Long-term live imaging, cell identification and cell tracking in regenerating crustacean legs', by Çevrim, Laplace-Builhé, Sugawara, Rusciano, Labert, Brocard, Almazán and Averof.

The supplementary data include:

Supplementary Data 1 (.csv file);  Live imaging of regenerating Parhyale legs: image acquisition settings

Table with information on the 22 time lapse recordings presented in Figure 3, including image acquisition settings, temperature and duration of the recordings.

Supplementary Data 2 (.zip file);  Live imaging of regenerated Parhyale legs: maximum projections

Compressed folder including maximum projections for each of the 22 time lapse recordings presented in Figure 3. These files were generated by projecting all or a subset of the z slices acquired at each time point. A 20 micron scale bar was added on the first time point. These files serve as a quick way to examine the 22 time lapse recordings.

Supplementary Data 3 (22 .tif files);  Live imaging of regenerated Parhyale legs: complete datasets

Complete image 3D+T hyperstacks for each of the 22 time lapse recordings presented in Figure 3. These files have been generated by concatenating the original image stacks and correcting any image shifts, as described in the Methods section of the paper.

Supplementary Data 4 (.zip file);  Analysis of trade-offs of imaging resolution and image quality

The data used for the analysis of trade-offs in imaging and the results shown in Table 1 are included in two folders. The folder named Image_and_tracking_data includes the image data (.tif, .h5, .xml), ground truth cell tracking files (.mastodon files) and three sets of cell track predictions (.mastodon files) for the original recording (labelled 00), for each of the subsampled datasets (labelled 01 to 05), and for the denoised and deconvoluted datasets. It also includes separate folders containing the Elephant detection and flow model parameters for each set of predictions. The folder named CTC_tracking_results includes the ground-truth data along with three sets of predictions for detection and tracking for each dataset, following the Cell Tracking Challenge format. For each dataset we include label image files (.tif) for every time point along with tracking results in .txt format, and each results directory (01_RES_*) also contains the evaluation results from the Cell Tracking Challenge Evaluation Software. For a detailed explanation of the folder structure, please refer to the Cell Tracking Challenge documentation.

Supplementary Data 5 (.zip file);  Tracking the progenitors of spineless-expressing cells in the distal carpus

The data used to generate Figure 7 are included in this compressed folder, including the live imaging and cell tracking files (.h5, .xml and .mastodon files) and the image stack of the spineless and futsch HCR and DAPI stainings (.tif file). Channel 2 shows spineless expression (mostly nascent transcripts in nuclei), as well as background signal in epidermal nuclei (possibly due to photoconversion of DAPI, see Karg & Golic 2018, Chromosoma 127: 235-245) and strong autofluorescence in granular cells (also visible in channel 1, depicting futsch HCR).

Supplementary Data 6 (.txt file);  Sequences of Parhyale genes targeted by the HCR probes

The sequences are provided in FASTA format.

Supplementary Data 7 (.zip file);  Apoptosis in legs that have not been subjected to live imaging

The data used to generate Figure 2 supplement 2 are contained in this compressed folder, including 9 image stacks of T4 and T5 legs fixed and stained with DAPI 3 days post amputation (with apoptotic nuclei marked) and a .txt file containing the apoptotic cell counts.

Supplementary Data 8 (.zip file);  Analysis of tracking performance in relation to imaging depth

The data used to generate Figure 5 are contained in this compressed folder, including separate folders for the data extracted from the analysis of datasets #1 to #5. Each folder includes data from three replicates (batches 001 to 003), with .csv files listing the z location of nucleus centroids (in µm) for the nuclei that were incorrectly detected by Elephant – either as false positives (FP) or as false negatives (FN) – and the ground truth data (GT). The folder also includes an .xlsx file gathering all the relevant data and the measurements of precision and recall.

Supplementary Data 9 (.zip file);  Detecting the temporal pattern of cell divisions in regenerating legs

The data used to generate Figure 4 are contained in this compressed folder, including the five image datasets (.tif, .h5, .xml), the detected cell divisions (.mastodon files), and an .xlxs file containing all the cell divisions counts and graphs.

Video 1.  Time lapse recording of regeneration in a Parhyale T5 leg (dataset li48-t5)

Live imaging of nuclei labelled with H2B-mREFruby (maximum projection of z slices 3-10). Proximal parts of the leg are to the left and the amputation site is at the right of the frame. For annotations of different features please refer to Figure 2. Shortly after leg amputation (0 hpa) hemocytes adhere to the wound. By 16 hpa the wound has melanized. Up to ~32 hpa epithelial cells can be seen migrating and accumulating at the wound, below the melanized scab (Figure 2A,B). Around 31 hpa, the leg tissues become detached from the scab (Figure 2C). At 43 hpa, the carpus-propodus boundary first becomes visible, and thereafter many cells can be observed dividing at the distal part of the leg stump (Figure 2D). At 56 hpa, the propodus-dactylus boundary first becomes visible (Figure 2E). At later stages, tissues in more proximal parts of the leg retract, making space for the regenerating leg to grow (Figure 2F,G). After ~90 hpa cell proliferation there is less cell proliferation and cell movements, and the nuclear positions within the tissue become fixed. Scale bars, 20 µm.

Video 2.  Time lapse recording of regeneration in a Parhyale T5 leg (dataset li36-t5)

Live imaging of nuclei labelled with H2B-mREFruby (maximum projection of z slices 3-15). Proximal parts of the leg are to the left and the amputation site is at the right of the frame. The sequence of events is similar to that described in Video 1, but the progression is slower: epithelial migration towards the wound is observed up to 40 hpa, tissues detach from the scab at 65 hpa, and the carpus-propodus and propodus-dactylus boundaries first become visible at 78 and 91 hpa. The tissues making up the carpus and propodus can be seen pulsating from 105 to 145 hpa. Scale bars, 20 µm.