FishFace: interactive atlas of zebrafish craniofacial development at cellular resolution

Background: The vertebrate craniofacial skeleton may exhibit anatomical complexity and diversity, but its genesis and evolution can be understood through careful dissection of developmental programs at cellular resolution. Resources are lacking that include introductory overviews of skeletal anatomy coupled with descriptions of craniofacial development at cellular resolution. In addition to providing analytical guidelines for other studies, such an atlas would suggest cellular mechanisms underlying development. Description We present the Fish Face Atlas, an online, 3D-interactive atlas of craniofacial development in the zebrafish Danio rerio. Alizarin red-stained skulls scanned by fluorescent optical projection tomography and segmented into individual elements provide a resource for understanding the 3D structure of the zebrafish craniofacial skeleton. These data provide the user an anatomical entry point to confocal images of Alizarin red-stained zebrafish with transgenically-labelled pharyngeal arch ectomesenchyme, chondrocytes, and osteoblasts, which illustrate the appearance, morphogenesis, and growth of the mandibular and hyoid cartilages and bones, as viewed in live, anesthetized zebrafish during embryonic and larval development. Confocal image stacks at high magnification during the same stages provide cellular detail and suggest developmental and evolutionary hypotheses. Conclusion: The FishFace Atlas is a novel learning tool for understanding craniofacial skeletal development, and can serve as a reference for a variety of studies, including comparative and mutational analyses

Modes of developmental outgrowth and shaping of a craniofacial bone in zebrafish.

The morphologies of individual bones are crucial for their functions within the skeleton, and vary markedly during evolution. Recent studies have begun to reveal the detailed molecular genetic pathways that underlie skeletal morphogenesis. On the other hand, understanding of the process of morphogenesis itself has not kept pace with the molecular work. We examined, through an extended period of development in zebrafish, how a prominent craniofacial bone, the opercle (Op), attains its adult morphology. Using high-resolution confocal imaging of the vitally stained Op in live larvae, we show that the bone initially appears as a simple linear spicule, or spur, with a characteristic position and orientation, and lined by osteoblasts that we visualize by transgenic labeling. The Op then undergoes a stereotyped sequence of shape transitions, most notably during the larval period occurring through three weeks postfertilization. New shapes arise, and the bone grows in size, as a consequence of anisotropic addition of new mineralized bone matrix along specific regions of the pre-existing bone surfaces. We find that two modes of matrix addition, spurs and veils, are primarily associated with change in shape, whereas a third mode, incremental banding, largely accounts for growth in size. Furthermore, morphometric analyses show that shape development and growth follow different trajectories, suggesting separate control of bone shape and size. New osteoblast arrangements are associated with new patterns of matrix outgrowth, and we propose that fine developmental regulation of osteoblast position is a critical determinant of the spatiotemporal pattern of morphogenesis

Morphometric analysis of opercle morphogenesis between 4 and 58 dpf reveals separate nonlinear trajectories of growth (A) and shape change (B-D).

<p>The data points in the bivariate plots indicate individual samples, color-coded by age: blue, 4–12 dpf, red, 13–18 dpf, green, 20–58 dpf. (A) Centroid size (CS) versus dpf. (B, C) Shape summary variables PC1 and PC2 versus dpf. (D) PC1 versus PC2. PC1 explains 73% and PC2 explains 15% of the total shape variation in the dataset (n = 176). The diagrams that accompany the bivariate plot in D show the Op shapes at the indicated stages as average thin plate spline deformations from the consensus configuration for the entire dataset (not shown). For the 4 dpf average n = 7, for 15 dpf n =  10, and for 58 dpf n =  7. Asterisks and large arrowheads point out prominent developmental changes described in the main text.</p

Osteoblast arrangements change dynamically during opercle morphogenesis.

<p>Confocal imaging of live preparations. (A-C) Two-color merged images showing Alizarin Red A labeling of the matrix and <i>osx</i>:eGFP labeling of the bone forming cells. (A) At 3 dpf osteoblasts line up along the developing bony spur. (B, C) At 4 and 7 dpf a new arrangement is present with cells especially concentrated along the rapidly outgrowing vp edge. The newly forming posterior branchiostegal ray (br) is included in C. (D) Red channel, and (E) merge at 11 dpf. Arrows indicate the vj veil. In E the outline of the Alizarin Red labeled bone (from D) is superimposed. Very flattened and compact looking <i>osx:eGFP</i>-expressing cells line the very slowly growing jp edge and are present in outer rows of the very rapidly growing vp edge. Small round cells are present at the j apex spur. Larger diffusely labeled cells are present along the vj veil and in the innermost row along the vp edge, where the cells immediately contact new mineralized matrix (a portion of this edge is enlarged in the inset). Scale bar: 50 µm.</p

The Op of the young adult shows a high rate of incremental banding outgrowth of the vp edge.

<p>(A) Image at 54 dpf with green monochromatic (to increase resolution) transmitted light, and crossed-polarizing filters to reveal birefringence and the incremental banding pattern. The arrow indicates the vp edge of the bone. (B) Epiflorescence of Alizarin Red S, applied as a pulse at 43 dpf, in the same field as in A. The labeling front (arrow) shows where the vp edge was located at the 43 dpf stage. Sites (Howship's lacunae) of likely remodeling (bone resorption by osteoclasts, followed by replacement with new, unlabeled bone) in the old bone behind this front are indicated by asterisks. (C) Merge of A and B. The Alizarin Red front is indicated by the red line, the bone vp edge by the green line, and the double-headed arrow shows the approximate extent of outgrowth during the 11 day interval after labeling (an average 333 µm, from several measurements along the bone). (D) Detail of the banding pattern between the labeling front and the bone edge (double-headed arrow). Widths of prominent bands are about 30 µm; the three linked arrows show two 30 µm intervals. We note more finely spaced bands are also present, and that we measured a substantial variation in bandwidths among different preparations. Scale bars: 200 µm.</p

Arrangements of neural crest-derived mesenchymal cells associated with the opercle developing in the young larva.

<p>Two-color confocal imaging of live preparations. (A, C) Red channel at 3 and 5 dpf showing the Alizarin Red S labeled bone. (B, D) Merge of the red channel and the green channel showing cells expressing the <i>fli1:eGFP</i> transgene. Endothelial cells of capillary tubules also brightly express this transgene. The dense condensation of Op-associated cells present at 3 dpf thins out considerably by 5 dpf, particularly along the very slowing growing jp edge of the bone (arrow in D). Abbreviations and orientations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009475#pone-0009475-g001" target="_blank">Figure 1</a>. Scale bar: 50 µm.</p

Comparison of opercle shapes of (A) zebrafish, <i>Danio rerio</i>, (B) sucker, <i>Castostomus sp</i>., and (C) northern pike, <i>Esox lucius.</i>

<p>Letters along the edges indicate hypothetically homologous locations along the bones. Shape differences along the upper (jp) edge and the j apex are discussed in the text. The arrow in (A) indicates a prominent Howship's lacunae, a site of osteoclast-mediated bone resorption. The vp edge appears to outgrow differentially in all three species, as indicated by the incremental bands being wider in region c. The region between c and p shows the narrowest banding for both zebrafish and sucker, resulting in the concavity here shared by these two species but not by the pike. Zebrafish and the sucker are placed in separate families within the order Cypriniformes, the pike is in the order Esociformes and therefore is an outgroup to the other two species. Scale bars: 0.5 mm (A), 10 mm (B,C).</p

Mineralized matrix addition occurs in a stereotyped spatiotemporal pattern.

<p>Live confocal imaging as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009475#pone-0009475-g001" target="_blank">Figure 1</a>. The larvae were vitally stained successively with two Ca²⁺-binding dyes, first Alizarin Red S, then Calcein (green), with a washout period between the two applications. They were imaged just after the second staining period. (A-D) The same Op, stained first for 2 hr at 3 dpf (A, red channel), and then overnight between 5 and 6 dpf (B, green channel). C shows the merge and D the outlines of the two colors. The vj edge (arrowhead in D) and jp edge (asterisk) show only a thin layer of new (green) matrix on top of the older (red) matrix. In contrast, the vp edge, showing short spurs at the time of the first pulse, grows out prominently (arrows in D). (E) Another preparation in which a larva was stained with overnight first at 6–7 dpf and then at 12–13 dpf. The jp edge, as at the earlier stage, shows only a very thin layer of new (green) bone (asterisk). The veil along the vj edge (arrowhead), and the upward pointing short j apex spur are made of new bone. Note that this two-color matrix staining method also reveals that mineralization of the branchiostegal ray (br) began before 7 dpf (since the br is doubly labeled), but that subopercle (sop) mineralization is initiated only after day 7 (since the sop is not Alizarin Red-labeled). (F) A preparation stained overnight first at 12–13 dpf and then at 18–19 dpf. In contrast to the earlier stages, there is now an elaborate outgrowing veil, made of new bone, along dorsal jp edge. The anterior strut along the vj edge is new (arrowhead), and the j apex has elongated by new bone addition. Note that in both E and F the outgrowth of the vp edge is differential, more rapid near the p apex than near the v apex, as indicated by the lengths of the arrows. Abbreviations and orientations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009475#pone-0009475-g001" target="_blank">Figure 1</a>. Scale bars: 40 µm.</p