FishFace: interactive atlas of zebrafish craniofacial development at cellular resolution
© Eames et al.; licensee BioMed Central Ltd. 2013
Received: 13 December 2012
Accepted: 21 May 2013
Published: 28 May 2013
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.
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.
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.
KeywordsCraniofacial development Website atlas Zebrafish Bone Cartilage Skeleton Evolution
How do elements of the vertebrate craniofacial skeleton arise, grow, and reshape during development, and how do these processes vary during the course of evolution? Answers to these questions are coming from both molecular-genetic and cell-biological approaches, which rely, first of all, on precise description of the developmental events and processes that comprise skeletogenesis. The availability of developmental atlases facilitates such study. Indeed, many descriptive atlases of craniofacial development in various vertebrate species have been published [1–4]. In today’s world, internet atlases are extremely useful, in part because they are publicly accessible around the world, but also because they can be formatted so that a user interacts with the data in unique ways, such as with freely-rotatable 3D models (e.g., Mouse Limb Atlas: http://www.nimr.mrc.ac.uk/3dlimb/; ).
Zebrafish, with patterns of development conserved among all vertebrates, with favorable attributes for phenotypic analyses of development, and with a sophisticated knowledge by the research community of its genetics and genomics, provides a powerful animal model for learning about craniofacial development. Online zebrafish atlases include the Zebrafish Atlas (zfatlas.psu.edu); 3D Atlas of Zebrafish Vasculature Anatomy (http://uvo.nichd.nih.gov/atlas.html); the Zebrafish Brain Atlas (http://www.ucl.ac.uk/zebrafish-group/zebrafishbrain/index.php); the Atlas of Zebrafish Anatomy (http://www.zebrafish.uni-freiburg.de/anatomy.html); the Atlas of Zebrafish Development (http://bio-imaging.liacs.nl/liacsatlas.html); the Zebrafish Anatomy Portal (http://www.zfap.org); and the FishNet 3D developmental atlas (http://www.fishnet.org.au/index.shtml). None of these atlases, however, currently focus on development of the craniofacial skeleton.
Zebrafish craniofacial development can be detailed with an investigative method unsurpassed in accuracy and sensitivity. Using current transgenic approaches in combination with vital dye staining, one can examine craniofacial skeletal elements in exquisite cellular detail during an extended period of development within living, intact zebrafish embryos and larvae. Here, we describe a high-resolution developmental atlas of the zebrafish craniofacial skeleton, the FishFace Atlas (https://www.facebase.org/fishface/home), to serve as a guide for anatomical and cellular studies of craniofacial development.
Summary of FishFace Atlas website organization
The FishFace Atlas focusses on cellular level anatomy of the developing skeleton in the first two pharyngeal arches. It uses confocal imaging of living specimens. To assist the user unfamiliar with fish craniofacial anatomy, FishFace also provides an overview of the structure of the skull, featuring optical projection tomography (OPT) of preserved material. In this “Skull Anatomy” section, fluorescent OPT images of Alizarin red-stained samples show skull bones of middle and late larval, and adult, zebrafish (https://www.facebase.org/fishface/opt_data; for clarification of the relationship between the FishFace Atlas and FaceBase hub, please see (Additional file 1)). Furthermore, a model of larval craniofacial bones was created by segmentation of 3D reconstructed OPT data, and this model can be freely rotated and virtually dissected in a user-defined fashion (https://www.facebase.org/fishface/Viewer).
The “Arch 1 and 2 Development” section of FishFace complements data from “Skull Anatomy”, including ‘overview’ series of low magnification confocal image stacks through the two most anterior pharyngeal arches and through the craniofacial skeleton derived from these arches. These images show transgenically-labelled cells in living embryos and larvae, and all ontological terms in their descriptions link directly to standardized definitions on the Zebrafish Information Network (http://zfin.org/). One such ‘overview’ is a series of embryonic zebrafish at preskeletal stages showing transgenically-labelled neural crest-derived mesenchymal cells that eventually give rise to craniofacial skeletal elements (https://www.facebase.org/fishface/early_arches). The second ‘overview’ series illustrates developing skeletal elements using transgenically-labelled chondrocytes and Alizarin-red stained bone matrix (https://www.facebase.org/fishface/arch1_and_2).
Meckel’s cartilage (https://www.facebase.org/fishface/meckel);
ceratohyal (https://www.facebase.org/fishface/ceratohyal); and
By studying elements in cellular detail, a much more precise description can be made of the dramatic changes to element growth and shaping that occur in early developmental stages. Most images in FishFace have links to movies playing slice by slice through each confocal stack of the z-series that was used to generate that particular confocal projection. Hence, the FishFace Atlas provides the community with a valuable, interactive resource with which the user can understand not only the complex 3D anatomical relationships of skeletal elements, but also the underlying cellular organization, in the developing zebrafish craniofacial skeleton.
Skull Anatomy: an interactive model of zebrafish craniofacial skeletal anatomy
Arch 1 and 2 Development: arch ectomesenchyme to cartilages and bones
To understand development of the first two pharyngeal arches, mandibular and hyoid, the FishFace Atlas uses confocal imaging in living preparations. Generated from transgenic zebrafish, the images illustrate cellular details not only of the developing pharyngeal arches, but also of the cartilages and bones that form within them. As such, the confocal data provide a nice complement to the OPT data, which visualizes mineralized matrix of the developing zebrafish craniofacial skeleton. Two sets of low-magnification confocal images help the user transition from the OPT dataset and show how craniofacial morphology changes with time. One covers six stages before, and the other eight stages after, skeletal elements become individually recognizable. Both generally include ventral and lateral views.
Element Development: detailed single-cell resolution of imaging suggests developmental and evolutionary hypotheses of individual skeletal elements
The final component of the FishFace Atlas, and the most novel, is a set of ‘element’ pages. This resource includes abundant images at high magnification that provide cellular resolution to accompany the lower magnification ‘overview’ images just discussed. By using the ‘overview’ and ‘element’ parts of the atlas together, the viewer is able to directly connect developing element morphologies with changes in cellular patterning, such as cellular arrangements, shapes, and sizes. Together, these help to elucidate the cellular basis of morphogenesis and growth. Here we use the mandible, the lower jaw, to show that imaging with cellular resolution allows one to support hypotheses that then can be tested by more directed and analytically based study. The mandible represents an interesting case study, due to its complex developmental and evolutionary history [1, 8–10].
Cellular imaging also provides support for hypotheses explaining growth in size of individual cartilages. As is well known, chondrocytes greatly enlarge in size, especially during early stages of differentiation morphogenesis, and the number of chondrocytes in growing cartilages increases as well (e.g., as quantified for the zebrafish symplectic cartilage ). New chondrocytes can come from the perichondrium (resulting in appositional growth), and they can come from cell divisions of the chondrocytes themselves (resulting in interstitial growth). Interstitial growth yields distinctive chondrocyte doublets, which at first occupy single lacunae within the matrix. FishFace images support interstitial growth of Meckel’s cartilage (but do not exclude appositional growth). Compared to arrangements of chondrocytes and their separating matrix in Meckel’s cartilage at 4 dpf (Figure 4C,C’), images at 6 dpf capture what seems to be a nest of doublets (Figure 4D,D’; essentially all of the cells in Figure 4D’ are included in this nest), and suggest that cell-cycles are locally synchronous.
We emphasize that the Fishface images by themselves motivate, but do not establish, the validity of the above hypotheses. All three hypotheses we propose here are amenable to rigorous testing. For example, in time-lapse recordings of appropriate strains of transgenic zebrafish, one can observe directly, in the intact fish, cell intercalations mediating convergence and extension , waves of cell division , and whether osteoblasts are initially recruited to two adjacent bone primordia that quickly coalesce, or to a single one .
The FishFace Atlas is an interactive, 3D tool for understanding aspects of the complex anatomy of the developing zebrafish craniofacial skeleton at cellular resolution. FishFace builds on and elucidates the foundation set out primarily in Cubbage and Mabee (1996), who comprehensively described skeletal elements in the zebrafish skull from fixed preparations stained for cartilage and bone through adult stages, but also in other studies that included cartilage development  and more detailed investigations of both cartilage  and bone [16, 18] morphogenesis and growth. FishFace first provides overviews of the whole skull and then focusses on the first and second pharyngeal arches, the region that probably has been the most thoroughly examined region of the zebrafish skull, and which promises to continue to yield fundamental information on its genetics, patterning, morphogenesis and growth . Therefore, the FishFace Atlas should be particularly useful in comparative and mutational analyses where there is interest in understanding not only the genetic basis, but also the cellular basis of skeletogenesis and patterning.
The imaging resolution of skeletal elements in the FishFace Atlas illuminates not only cellular behaviors that drive early craniofacial morphogenesis in living embryos and larvae, but also potential evolutionary mechanisms. Previous studies of craniofacial development have shown that cell migration, aggregation, intercalation, proliferation, enlargement, recruitment, and death, all play crucial roles in generating proper morphology [4, 16, 19–22]. As we point out for the mandible, the FishFace images support developmental hypotheses explaining reshaping and organized growth of its cartilaginous element, Meckel’s cartilage. Cellular intercalations may drive convergence and extension, and interstitial growth, perhaps including mitotic waves, might contribute to growth. The images also support a compound nature of the dentary, a posterior dermal element fused with an anterior chondral one. We hypothesize that the fusion is present in the osteoblast primordium (i.e., condensation) that initially makes the bone, which is consistent with a mechanism of skeletal evolution expounded by Atchley and Hall . Furthermore, Patterson, in his important review of skeletal element evolution, proposes that often bone ‘loss’ is actually fusion and may occur in evolutionary sequences . The primitive condition is multiple bones: two in the case of anterior mandibular bones, with independent mentomeckelian and dentary bones apparent in teleost outgroups, such as the bowfin Amia. Then a step occurs that Patterson calls “ontogenetic fusion”, in which separate bone rudiments are present in early ontogeny and then fuse (as for the trout and pike; ). Finally, as a yet more derived condition, there is “phylogenetic fusion” in Patterson’s words; where the ancestor had two (or more) bones, the descendant at all developmental stages has but a single one. Patterson notes that convincing evidence for phylogenetic fusion is lacking . As pointed out above, time-lapse studies in zebrafish could provide this evidence, an analysis motivated by imaging in the FishFace Atlas.
In order to increase understanding of the development of the zebrafish craniofacial skeleton at cellular resolution, the FishFace Atlas is a website that presents images generated by confocal microscopy and optical projection tomography (OPT) in a user-interactive format. We anticipate its utility as a reference for mutational and evolutionary analyses.
Construction and Content
Larval rearing and quality control
All zebrafish lines were maintained, and zebrafish embryos and larvae were raised, according to established protocols [4, 25] with IACUC approval (see http://zfin.org/zf_info/zfbook/chapt3/3.2.html). Briefly, up to 20 larvae in 150 ml EM were fed paramecia in 250 ml beakers from 4 days post-fertilization (dpf) to 9 dpf, when they were shifted to mouse cages and fed paramecia and baby brine shrimp until 15 dpf, when they were fed only brine shrimp. Regarding quality control of larval rearing, co-authors followed their strict published methods  to monitor general hallmarks of larval health (e.g., swim bladder, prey capture) in our nursery, and standard length measurements were taken for each imaged specimen. Regarding quality control of images included in FishFace, co-authors of the paper collected the images, which were selected for Atlas inclusion by consensus among co-authors, based upon 1) how well the images reflected average development of multiple specimens, often from multiple independent clutches; and 2) how consistent the data were with relevant published literature (e.g., [4, 6, 17]).
Alizarin red staining
For OPT imaging of mineralized matrix, fixed zebrafish specimens were stained as described previously . Tissues were fixed overnight in 4% PFA, washed for an hour in 1% KOH, bleached in 3% H2O2/0.5% KOH for 40 min. with lids open, washed in 1% KOH, stained overnight in 0.003% Alizarin red in 1% KOH, and de-stained in 1% KOH. After eyes were removed, heads were rinsed in water, embedded in low melting point agarose (Invitrogen), washed twice in methanol, and cleared in benzyl alcohol:benzyl benzoate (1:2). For confocal imaging of mineralized matrix in live specimens, zebrafish larvae were maintained in Embryo Medium supplemented with 0.005% Alizarin red and 0.01 M HEPES at least two hours, and often overnight, prior to confocal imaging .
OPT imaging and processing
Images of craniofacial bones of 14 dpf, 18 dpf, and adult zebrafish were captured using Bioptonics OPT Scanner 3001 M (MRC Technology). 3D reconstructions of raw data were made using NRecon (MRC Technology). Segmentations of skeletal elements in reconstructions were made using Amira 5.2.2 (Visage Imaging), and movies highlighting specific skeletal elements were created in Amira and QuickTime Player. Movies showing progression through virtual sections of the reconstruction were created in QuickTime Player (Apple). For information on how to download movies from FishFace for importation into ImageJ, please see the Additional file 1.
All transgenic fish were maintained on the AB background.
The transgenic zebrafish line sox9a zc81Tg , which we refer to here as sox9a:EGFP, was created using Gateway technology and includes a portion of the foxp2 promoter driving EGFP expression (Tg(foxp2.A:EGFP); ). Please see Construction and content and Additional file 2 for details on how sox9a:EGFP was isolated from the transgenic line Tg(foxp2.A:EGFP)zc42.
The transgenic zebrafish line Tg(sp7:EGFP)b1212, which we refer to here as sp7:EGFP, was created using BAC transgenesis and includes a large portion of the sp7 promoter driving EGFP expression .
The transgenic zebrafish line Tg(fli1a:EGFP)y1, which we refer to here as fli1a:EGFP, was created by micro-injecting a construct including a portion of the fli1a promoter driving EGFP expression  and has been used widely as a marker of neural crest cells and their derivatives, as well as of endothelial cells (e.g., [19, 30, 31]).
Cloning genomic locus of sox9azc81Tg
Genomic DNA was isolated from fins of three adult sox9a zc81Tg , which we refer to here as sox9a:EGFP, zebrafish with cartilage expression and of one non-glowing sibling adult zebrafish (QIAGEN DNeasy, QIAGEN Inc.). 1 μg gDNA from each of these four samples was digested with AluI or HaeIII in 40 μl at 37C for 3 hours. 0.5 μg of digested gDNA was ligated with 4 μl T4 DNA ligase (Epicentre) in 500 μl overnight at 16 C. The reaction was concentrated using Zymo-5 column (Zymo Research Corp.) and resuspended in 20 μl for use as PCR template. Inverse PCR was carried out according to published protocols for cloning Tol2 insertion sites into the zebrafish genome [32, 33]. First-round PCR primers were either [Tol2-3’invf2 + Tol2-3’/r1] or [Tol2-3’invf1 + Tol2-3’invr1], while second-round PCR primers were either [Tol2-3’/f2 + Tol2-3’invr2] or [Tol2-3’invf2 + Tol2-3’invr2] [32, 33]. No PCR products were obtained from non-transgenic fish. Many clones from products of the different PCR primer pairs were sequenced from each transgenic fish, and sequences BLASTed to the same location (Chr12: 2278311; Zv9, http://www.ensembl.org).
Imaging of live zebrafish specimens at various time points on 1, 2, 3, 4, 6, 8, 10, 14, and 21 dpf was conducted using either a Zeiss LSM 5 Pascal confocal or Leica SD6000 spinning disk confocal with Borealis illumination technology. In order to visualize Alizarin red staining with most sensitivity, pinhole and/or detector gain settings were adjusted by hand to levels just below those that showed red fluorescence in surrounding, non-mineralized tissues. Maximum projections were made from stacks of images that demonstrated the entire depth of the element(s) under focus. Movies showing progression through all images of the stack were created in Pascal (Carl Zeiss, Inc.) or Metamorph (Leica Microsystems) and sometimes were edited in ImageJ (NIH) for orientation and cropping.
Comments on developmental generalizations
The FishFace Atlas illustrates a generalized developmental sequence of the appearance of specific skeletal elements, but there are caveats to such generalizations. First, development is a dynamic, variable process. Apart from time-lapse movies, temporal series of images are merely snapshots of this process and thus cannot represent the full dynamics of developmental processes. Second, considerable clutch variation exists within a given “wild-type” stock, let alone between “wild-type” stocks under use around the world. We have attempted to normalize such differences to some extent by noting the standard length of each specimen and also by comparing morphological features of specimens to standardized staging series [34, 35]. In addition, we tried to select specimens that were representative of the clutch at each timepoint. Finally, imaging always is subject to limits of detection, so we try to avoid making declarative statements that a given skeletal element “appears” at a given time. That said, we have refined our methods to detect skeletal elements with the most sensitive techniques available. Our confocal imaging illustrates specifically skeletogenic cells prior to their secretion of abundant extracellular matrix. Also, we incubate and image our specimens with the vital dye Alizarin red while they are alive. As such, we avoid the severe reduction in Alizarin red binding that occurs after even brief periods in fixative and artificial buffers, for these chemicals leech mineral from skeletal tissues in the specimen. In summary, the FishFace Atlas addresses potential pitfalls of a variable developmental process and limitations of skeletal detection to provide the user an understanding of development of the pharyngeal arches and its skeleton in the embryonic and larval zebrafish.
Availability and requirements
The data sets supporting the results of this article are included within the article (and its Additional files 1 and 2) or are available in the FishFace Atlas (https://www.facebase.org/fishface/home). There are no restrictions on use by non-academics. All users are free to download images or movies (please see Additional file 1).
Terminology and abbreviations used in the FishFace Atlas are based largely upon Cubbage and Mabee (1996) and Zebrafish Information Network ontology (http://zfin.org).
We thank members of the FaceBase hub for establishing and maintaining the FishFace Atlas on their website; Josh Bonkowsky and the laboratory of the late Chi-Bin Chien for providing Tg(foxp2.A:EGFP)zc42, in which they noticed unexpected pharyngeal arch transgene expression; all members of the University of Oregon Fish Facility; and Richard Baldock, Bill Hill, and Nicholas Burton from the Edinburgh Mouse Atlas Project (EMAP), MRC Human Genetics Project for their generous assistance with converting OPT data so it could be viewed in JatlasViewer. This work was supported by NIH grants F32DE019345 to AD, F32GM086027 to JTN, F32DE019056 to MMS, and RC1DE020655, R01DE13834, and PO1HD22486 to CBK.
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