Automated processing of zebrafish imaging data: a survey

Due to the relative transparency of its embryos and larvae, the zebrafish is an ideal model organism for bioimaging approaches in vertebrates. Novel microscope technologies allow the imaging of developmental processes in unprecedented detail, and they enable the use of complex image-based read-outs for high-throughput/high-content screening. Such applications can easily generate Terabytes of image data, the handling and analysis of which becomes a major bottleneck in extracting the targeted information. Here, we describe the current state of the art in computational image analysis in the zebrafish system. We discuss the challenges encountered when handling high-content image data, especially with regard to data quality, annotation, and storage. We survey methods for preprocessing image data for further analysis, and describe selected examples of automated image analysis, including the tracking of cells during embryogenesis, heartbeat detection, identification of dead embryos, recognition of tissues and anatomical landmarks, and quantification of behavioral patterns of adult fish. We review recent examples for applications using such methods, such as the comprehensive analysis of cell lineages during early development, the generation of a three-dimensional brain atlas of zebrafish larvae, and high-throughput drug screens based on movement patterns. Finally, we identify future challenges for the zebrafish image analysis community, notably those concerning the compatibility of algorithms and data formats for the assembly of modular analysis pipelines

Summary graphs comparing the bone formation scores for each structure in the different experiments.

<p>Statistical analysis was performed by X² of Pearson and a logistic regression. In red, the scores are significantly increased. In green, the scores are significantly decreased. (A) PTH. (B) VitD3. (C) 3g hypergravity between 5–6dpf (D) "relative microgravity". For abbreviations see legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126928#pone.0126928.g001" target="_blank">Fig 1</a>.</p

Effect of 3g hypergravity between 5–9dpf on bone formation.

<p>(A,B) Alizarin red staining of 9dpf control larvae (A) and larvae treated for 4 days in 3g hypergravity after 5 days at 1g (B). Ventral view, anterior to the left. (C) Comparison of morphometric measurements for some selected distances within the heads of control and 3g-treated larvae. Mean ± SD and t-test analysis were calculated for each measure on at least 20 individuals. * <i>p <</i> 0.05, ** <i>p <</i> 0.01 and ***<i>p <</i> 0.001. (D) Global score for bone formation in control and 3g treated larvae. (E) Comparison of cumulated frequencies of, respectively light, 1 pair dark or two pairs dark otoliths in control and 3g treated larvae. For abbreviations see legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126928#pone.0126928.g001" target="_blank">Fig 1</a>.</p

Morphometric analysis of bone elements at 6dpf after "relative microgravity”.

<p>The distances are measured in pixels. Mean ± SD and t-test analysis were calculated for each measure on at least 20 individuals. (A) Distances between the different cranial bone elements. (B) Area of the parasphenoid bone. * <i>p <</i> 0.05 and ***<i>p <</i> 0.001. For abbreviations see legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126928#pone.0126928.g001" target="_blank">Fig 1</a>.</p

Morphometric analysis results of bone matrix staining after 5 days chemical treatments.

<p>The distances are measured in pixels. Mean ± SD and t-test analysis were calculated for each measure on at least 20 individuals. * <i>p <</i> 0.05, ** <i>p <</i> 0.01 and ***<i>p <</i> 0.001. (A) Distances after VitD3 treatment. (B) Distances after PTH treatment. (C) Area of the parasphenoid bone results after 5 days PTH or VitD3 treatment. Abbreviations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126928#pone.0126928.g001" target="_blank">Fig 1</a>. <b>A)</b> Analysis of the bone skeleton after VitD3 treatment revealed a significant increase of the distance between maxillae (m), consistent with a broader jaw as already observed by cartilage morphometry. The length of the head skeleton is also increased upon VitD3 treatment with a longer distance between the anterior part of the head (an) and the notochord (n), and between an and the parasphenoid (p) bone. Other measures are not significantly modified (A, C). B) PTH treatment caused an increase of the distance between the anterior part of the head and the summit “a” of the parasphenoid, mainly due to a significant decrease of the size of the parasphenoid (p) (C). Some structures are missing, such as the anguloarticular (aa), branchiostegal ray2 (br2), ceratohyal (ch) and/or maxilla (m). However, a significant broadening of the posterior head skeleton is revealed by the increased distance between left and right ("up" and "down") branchiostegal rays1 (br1), entopterygoids (en) and also the opercula (o) (B).</p

Network of genes affected in "relative microgravity" experiments.

<p>A network was constructed using the genes common to all three experiments, or the genes common only to 3g>1g and 3g>axe. Color overlay indicates the fold change relative to the 3g sample taken as control. Genes up-regulated (red), down-regulated (green), (*) indicates that the gene is represented by two or more probes on the microarray.</p

Schematic overview of the different hypergravity experiments.

<p>(A) larvae are placed at hypergravity at 5dpf until 9dpf (3g), while (control) larvae are kept at normal gravity for 9 days. Total mRNA was extracted at 6dpf and batches of larvae were fixed at 9dpf for Alizarin red staining of bone matrix. (B) Experiment in which the control larvae were placed at 3g and kept at 3g until 6dpf (3g), or returned at 5dpf to 1g outside (3g>1g) or on the axis of the centrifuge (3g>axe) for one day. An additional batch of larvae was kept at normal gravity until 6dpf (1g). RNA extraction and Alizarin red staining are performed at 6dpf. For abbreviations see legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126928#pone.0126928.g001" target="_blank">Fig 1</a>.</p

Comparison of genes affected after PTH or VitD3 treatment between 5–6dpf.

<p>(A) List of common genes and their respective log2(fold change) in the two conditions. (B) Comparison of the number of genes affected by PTH or VitD3 treatment. The number of probes resulting in different hybridization signals is given, with the numbers in parenthesis and the graph showing the numbers of IPA-annotated genes. (C) Network constructed using the common genes and extended using the genes affected in one of the two conditions. The color overlay indicates the fold change after VitD3 (left) or PTH (right) treatment. Genes up-regulated (red), down-regulated (green), (*) indicates that the gene is represented by two or more probes on the microarray.</p

Number of genes affected in the various hypergravity experiments.

<p>The absolute number of probes resulting in a statistically significant hybridization signal is given for each condition. In parentheses, the corresponding number of genes with an annotation in IPA is given, while the Venn diagrams represent the number of genes unique to each condition and genes common to two or three conditions.</p

(A-D) Cartilage and bone elements of the head skeleton in 10dpf zebrafish.

<p>(A) Alcian blue staining of head cartilage representing the landmarks used for morphometry. (B) Schematic representation of the different head cartilage elements. anterior limit (an), articulation (ar), ceratobranchial pairs 1 to 4 (cb1-4), ceratohyal (ch), ethmoid plate (et), hyosymplectic (h), Meckel's cartilage (mk), palatoquadrate (pq), posterior limit (po). (C) Alizarin red staining of cranial bones representing the landmarks used for morphometry. (D) Schematic representation of the different cranial bone elements with 29 landmarks used for chemicals treatments and 15 landmarks for the 3g and the relative-hypergravity. The 15 landmarks are anguloarticular (aa), anterior (an), branchiostegal ray1 (br1), entopterygoid (en), maxilla (m), notochord (n), opercle (o), parasphenoid (p). Note that the parasphenoid is a triangular bone defined by its anterior summit (a) and two posterior summits (b,c). The 29 landmarks include the 15 named before with branchiostegal ray2 (br2), cleithrum (c), ceratobranchial 5 (cb), ceratohyal (ch), dentary (d), hyomandibular (hm). (<b>E-J</b>) <b>10dpf zebrafish larvae after 5 days chemical treatments.</b> (E-G) Alcian blue staining of cartilage. (H-J) Alizarin red staining of bone. (E,H) Controls in DMSO. (F,G) no significant effect of, respectively VitD3 and PTH on cartilage development, nor on chondrocyte shape or size (inlays showing close-up). I: increase of bone development after VitD3 treatment. (J) decrease of bone development after PTH treatment. Ventral views, anterior to the left, (E-J) scale bar = 250μm.</p