Optimizing sperm collection procedures in zebrafish

Zebrafish are a highly-valued model organism used for developmental biology research. Zebrafish can be used for genetic manipulation and hence, many mutant and transgenic lines exist. It is impractical to maintain lines of adult zebrafish, due to resource constraints and the need to continuously produce new generations. Therefore, a practical way to preserve zebrafish lines is to freeze sperm and retrieve lines using in vitro fertilization of fresh eggs. Most existing in vitro protocols used by research labs have a wide variety of fertilization rates (ranging from 0% to \u3e90%). Due to this variability, lines may be at risk of not being regenerated, and may be permanently lost. For this project, aspects of existing published sperm collection protocols were tested and modified, with the goal of improving the proportion of males giving quality ejaculate. Males were tested for production of ejaculate by housing fish either in groups or in separate, individual tanks the night before sperm collection. The effect of age of male zebrafish and genetic background (5D and AB lines) on production of quality ejaculate was also tested. Isolating males before sperm collection significantly increased the proportion of individuals producing quality ejaculate. The proportion of fish that gave quality ejaculate samples did not co-vary with age between 17-68 weeks. Overall, AB fish were significantly more likely to give quality ejaculate samples compared to 5D fish. Based on this study, we strongly recommend separating male fish before sperm collection to improve the likelihood of obtaining samples. Our results indicate that AB fish give proportionately better samples than 5D fish, and this does not vary with age between 17-68 weeks

hdac4 mediates perichondral ossification and pharyngeal skeleton development in the zebrafish

Background Histone deacetylases (HDACs) are epigenetic factors that function to repress gene transcription by removing acetyl groups from the N-terminal of histone lysines. Histone deacetylase 4 (HDAC4), a class IIa HDAC, has previously been shown to regulate the process of endochondral ossification in mice via repression of Myocyte enhancer factor 2c (MEF2C), a transcriptional activator of Runx2, which in turn promotes chondrocyte maturation and production of bone by osteoblasts. Methods & Materials In this study, we generated two zebrafish lines with mutations in hdac4 using CRISPR/Cas9 and analyzed mutants for skeletal phenotypes and expression of genes known to be affected by Hdac4 expression. Results Lines have insertions causing a frameshift in a proximal exon of hdac4 and a premature stop codon. Mutations are predicted to result in aberrant protein sequence and a truncated protein, eliminating the Mef2c binding domain and Hdac domain. Zygotic mutants from two separate lines show a significant increase in ossification of pharyngeal ceratohyal cartilages at 7 days post fertilization (dpf) (p < 0.01, p < 0.001). At 4 dpf, mutant larvae have a significant increase of expression of runx2a and runx2b in the ceratohyal cartilage (p < 0.05 and p < 0.01, respectively). A subset of maternal-zygotic (mz) mutant and heterozygote larvae (40%) have dramatically increased ossification at 7 dpf compared to zygotic mutants, including formation of a premature anguloarticular bone and mineralization of the first and second ceratobranchial cartilages and symplectic cartilages, which normally does not occur until fish are approximately 10 or 12 dpf. Some maternal-zygotic mutants and heterozygotes show loss of pharyngeal first arch elements (25.9% and 10.2%, respectively) and neurocranium defects (30.8% and 15.2%, respectively). Analysis of RNA-seq mRNA transcript levels and in situ hybridizations from zygotic stages to 75–90% epiboly indicates that hdac4 is highly expressed in early embryos, but diminishes by late epiboly, becoming expressed again in larval stages. Discussion Loss of function of hdac4 in zebrafish is associated with increased expression of runx2a and runx2b targets indicating that a role for hdac4 in zebrafish is to repress activation of ossification of cartilage. These findings are consistent with observations of precocious cartilage ossification in Hdac4 mutant mice, demonstrating that the function of Hdac4 in skeletal development is conserved among vertebrates. Expression of hdac4 mRNA in embryos younger than 256–512 cells indicates that there is a maternal contribution of hdac4 to the early embryo. The increase in ossification and profound loss of first pharyngeal arch elements and anterior neurocranium in a subset of maternal-zygotic mutant and heterozygote larvae suggests that maternal hdac4 functions in cartilage ossification and development of cranial neural crest-derived structures

The Mouse Limb Anatomy Atlas: An interactive 3D tool for studying embryonic limb patterning

<p>Abstract</p> <p>Background</p> <p>The developing mouse limb is widely used as a model system for studying tissue patterning. Despite this, few references are available that can be used for the correct identification of developing limb structures, such as muscles and tendons. Existing textual references consist of two-dimensional (2D) illustrations of the adult rat or mouse limb that can be difficult to apply when attempting to describe the complex three-dimensional (3D) relationship between tissues.</p> <p>Results</p> <p>To improve the resources available in the mouse model, we have generated a free, web-based, interactive reference of limb muscle, tendon, and skeletal structures at embryonic day (E) 14.5 <url>http://www.nimr.mrc.ac.uk/3dlimb/</url>. The Atlas was generated using mouse forelimb and hindlimb specimens stained using immunohistochemistry to detect muscle and tendon. Limbs were scanned using Optical Projection Tomography (OPT), reconstructed to make 3D models and annotated using computer-assisted segmentation tools in Amira 3D Visualisation software. The annotated dataset is visualised using Java, JAtlasView software. Users click on the names of structures and view their shape, position and relationship with other structures within the 3D model and also in 2D virtual sections.</p> <p>Conclusion</p> <p>The Mouse Limb Anatomy Atlas provides a novel and valuable tool for researchers studying limb development and can be applied to a range of research areas, including the identification of abnormal limb patterning in transgenic lines and studies of models of congenital limb abnormalities. By using the Atlas for "virtual" dissection, this resource offers an alternative to animal dissection. The techniques we have developed and employed are also applicable to many other model systems and anatomical structures.</p

The Mouse Limb Anatomy Atlas: An interactive 3D tool for studying embryonic limb patterning

<p>Abstract</p> <p>Background</p> <p>The developing mouse limb is widely used as a model system for studying tissue patterning. Despite this, few references are available that can be used for the correct identification of developing limb structures, such as muscles and tendons. Existing textual references consist of two-dimensional (2D) illustrations of the adult rat or mouse limb that can be difficult to apply when attempting to describe the complex three-dimensional (3D) relationship between tissues.</p> <p>Results</p> <p>To improve the resources available in the mouse model, we have generated a free, web-based, interactive reference of limb muscle, tendon, and skeletal structures at embryonic day (E) 14.5 <url>http://www.nimr.mrc.ac.uk/3dlimb/</url>. The Atlas was generated using mouse forelimb and hindlimb specimens stained using immunohistochemistry to detect muscle and tendon. Limbs were scanned using Optical Projection Tomography (OPT), reconstructed to make 3D models and annotated using computer-assisted segmentation tools in Amira 3D Visualisation software. The annotated dataset is visualised using Java, JAtlasView software. Users click on the names of structures and view their shape, position and relationship with other structures within the 3D model and also in 2D virtual sections.</p> <p>Conclusion</p> <p>The Mouse Limb Anatomy Atlas provides a novel and valuable tool for researchers studying limb development and can be applied to a range of research areas, including the identification of abnormal limb patterning in transgenic lines and studies of models of congenital limb abnormalities. By using the Atlas for "virtual" dissection, this resource offers an alternative to animal dissection. The techniques we have developed and employed are also applicable to many other model systems and anatomical structures.</p

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