Role of mef2ca in developmental buffering of the zebrafish larval hyoid dermal skeleton.

Phenotypic robustness requires a process of developmental buffering that is largely not understood, but which can be disrupted by mutations. Here we show that in mef2ca(b1086) loss of function mutant embryos and early larvae, development of craniofacial hyoid bones, the opercle (Op) and branchiostegal ray (BR), becomes remarkably unstable; the large magnitude of the instability serves as a positive attribute to learn about features of this developmental buffering. The OpBR mutant phenotype variably includes bone expansion and fusion, Op duplication, and BR homeosis. Formation of a novel bone strut, or a bone bridge connecting the Op and BR together occurs frequently. We find no evidence that the phenotypic stability in the wild type is provided by redundancy between mef2ca and its co-ortholog mef2cb, or that it is related to the selector (homeotic) gene function of mef2ca. Changes in dorsal-ventral patterning of the hyoid arch also might not contribute to phenotypic instability in mutants. However, subsequent development of the bone lineage itself, including osteoblast differentiation and morphogenetic outgrowth, shows marked variation. Hence, steps along the developmental trajectory appear differentially sensitive to the loss of buffering, providing focus for the future study.

fras1 shapes endodermal pouch 1 and stabilizes zebrafish pharyngeal skeletal development.

Lesions in the epithelially expressed human gene FRAS1 cause Fraser syndrome, a complex disease with variable symptoms, including facial deformities and conductive hearing loss. The developmental basis of facial defects in Fraser syndrome has not been elucidated. Here we show that zebrafish fras1 mutants exhibit defects in facial epithelia and facial skeleton. Specifically, fras1 mutants fail to generate a late-forming portion of pharyngeal pouch 1 (termed late-p1) and skeletal elements adjacent to late-p1 are disrupted. Transplantation studies indicate that fras1 acts in endoderm to ensure normal morphology of both skeleton and endoderm, consistent with well-established epithelial expression of fras1. Late-p1 formation is concurrent with facial skeletal morphogenesis, and some skeletal defects in fras1 mutants arise during late-p1 morphogenesis, indicating a temporal connection between late-p1 and skeletal morphogenesis. Furthermore, fras1 mutants often show prominent second arch skeletal fusions through space occupied by late-p1 in wild type. Whereas every fras1 mutant shows defects in late-p1 formation, skeletal defects are less penetrant and often vary in severity, even between the left and right sides of the same individual. We interpret the fluctuating asymmetry in fras1 mutant skeleton and the changes in fras1 mutant skeletal defects through time as indicators that skeletal formation is destabilized. We propose a model wherein fras1 prompts late-p1 formation and thereby stabilizes skeletal formation during zebrafish facial development. Similar mechanisms of stochastic developmental instability might also account for the high phenotypic variation observed in human FRAS1 patients.

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.

Exposure to valproic acid (VPA) reproduces hdac1 loss of function phenotypes in zebrafish.

Histone deacetylases are enzymes that remove acetyl groups from histone tails and are understood to act as repressors of transcriptional activity. Hdac1 has been previously shown to function in eye, pectoral fin, heart, liver, and pharyngeal skeletal development. We show that high doses of Valproic Acid (VPA) reproduce the hdac1 phenotype. We identify tbx5 genes as potential targets of Hdac1 in eye, pectoral fin, and heart development. Using timed exposures, we show that skeletal structures in the pharyngeal arches are impacted by VPA between 24-36 hours post-fertilization, indicating a role for Hdac1 during post-migration patterning, differentiation, or proliferation of cranial neural crest cells.

Cellular and molecular investigations into the development of the pectoral girdle.

The forelimbs of higher vertebrates are composed of two portions: the appendicular region (stylopod, zeugopod and autopod) and the less prominent proximal girdle elements (scapula and clavicle) that brace the limb to the main trunk axis. We show that the formation of the muscles of the proximal limb occurs through two distinct mechanisms. The more superficial girdle muscles (pectoral and latissimus dorsi) develop by the "In-Out" mechanism whereby migration of myogenic cells from the somites into the limb bud is followed by their extension from the proximal limb bud out onto the thorax. In contrast, the deeper girdle muscles (e.g. rhomboideus profundus and serratus anterior) are induced by the forelimb field which promotes myotomal extension directly from the somites. Tbx5 inactivation demonstrated its requirement for the development of all forelimb elements which include the skeletal elements, proximal and distal muscles as well as the sternum in mammals and the cleithrum of fish. Intriguingly, the formation of the diaphragm musculature is also dependent on the Tbx5 programme. These observations challenge our classical views of the boundary between limb and trunk tissues. We suggest that significant structures located in the body should be considered as components of the forelimb.

Histone deacetylase-4 is required during early cranial neural crest development for generation of the zebrafish palatal skeleton.

BACKGROUND: Histone deacetylase-4 (Hdac4) is a class II histone deacetylase that inhibits the activity of transcription factors. In humans, HDAC4 deficiency is associated with non-syndromic oral clefts and brachydactyly mental retardation syndrome (BDMR) with craniofacial abnormalities. RESULTS: We identify hdac4 in zebrafish and characterize its function in craniofacial morphogenesis. The gene is present as a single copy, and the deduced Hdac4 protein sequence shares all known functional domains with human HDAC4. The zebrafish hdac4 transcript is widely present in migratory cranial neural crest (CNC) cells of the embryo, including populations migrating around the eye, which previously have been shown to contribute to the formation of the palatal skeleton of the early larva. Embryos injected with hdac4 morpholinos (MO) have reduced or absent CNC populations that normally migrate medial to the eye. CNC-derived palatal precursor cells do not recover at the post-migratory stage, and subsequently we found that defects in the developing cartilaginous palatal skeleton correlate with reduction or absence of early CNC cells. Palatal skeletal defects prominently include a shortened, clefted, or missing ethmoid plate, and are associated with a shortening of the face of young larvae. CONCLUSIONS: Our results demonstrate that Hdac4 is a regulator of CNC-derived palatal skeletal precursors during early embryogenesis. Cleft palate resulting from HDAC4 mutations in human patients may result from defects in a homologous CNC progenitor cell population.

ZebraShare: a new venue for rapid dissemination of zebrafish mutant data.

BACKGROUND: In the past decade, the zebrafish community has widely embraced targeted mutagenesis technologies, resulting in an abundance of mutant lines. While many lines have proven to be useful for investigating gene function, many have also shown no apparent phenotype, or phenotypes not of interest to the originating lab. In order for labs to document and share information about these lines, we have created ZebraShare as a new resource offered within ZFIN. METHODS: ZebraShare involves a form-based submission process generated by ZFIN. The ZebraShare interface (https://zfin.org/action/zebrashare) can be accessed on ZFIN under "Submit Data". Users download the Submission Workbook and complete the required fields, then submit the completed workbook with associated images and captions, generating a new ZFIN publication record. ZFIN curators add the submitted phenotype and mutant information to the ZFIN database, provide mapping information about mutations, and cross reference this information across the appropriate ZFIN databases. We present here examples of ZebraShare submissions, including phf21aa, kdm1a, ctnnd1, snu13a, and snu13b mutant lines. RESULTS: Users can find ZebraShare submissions by searching ZFIN for specific alleles or line designations, just as for alleles submitted through the normal process. We present several potential examples of submission types to ZebraShare including a phenotypic mutants, mildly phenotypic, and early lethal mutants. Mutants for kdm1a show no apparent skeletal phenotype, and phf21aa mutants show only a mild skeletal phenotype, yet these genes have specific human disease relevance and therefore may be useful for further studies. The p120-catenin encoding gene, ctnnd1, was knocked out to investigate a potential role in brain development or function. The homozygous ctnnd1 mutant disintegrates during early somitogenesis and the heterozygote has localized defects, revealing vital roles in early development. Two snu13 genes were knocked out to investigate a role in muscle formation. The snu13a;snu13b double mutant has an early embryonic lethal phenotype, potentially related to a proposed role in the core splicing complex. In each example, the mutants submitted to ZebraShare display phenotypes that are not ideally suited to their originating lab's project directions but may be of great relevance to other researchers. CONCLUSION: ZebraShare provides an opportunity for researchers to directly share information about mutant lines within ZFIN, which is widely used by the community as a central database of information about zebrafish lines. Submissions of alleles with a phenotypic or unexpected phenotypes is encouraged to promote collaborations, disseminate lines, reduce redundancy of effort and to promote efficient use of time and resources. We anticipate that as submissions to ZebraShare increase, they will help build an ultimately more complete picture of zebrafish genetics and development.

Zebrafish sp7:EGFP: a transgenic for studying otic vesicle formation, skeletogenesis, and bone regeneration.

We report the expression pattern and construction of a transgenic zebrafish line for a transcription factor involved in otic vesicle formation and skeletogenesis. The zinc finger transcription factor sp7 (formerly called osterix) is reported as a marker of osteoblasts. Using bacterial artificial chromosome (BAC)-mediated transgenesis, we generated a zebrafish transgenic line for studying skeletal development, Tg(sp7:EGFP)b1212. Using a zebrafish BAC, EGFP was introduced downstream of the regulatory regions of sp7 and injected into one cell-stage embryos. In this transgenic line, GFP expression reproduces endogenous sp7 gene expression in the otic placode and vesicle, and in forming skeletal structures. GFP-positive cells were also detected in adult fish, and were found associated with regenerating fin rays post-amputation. This line provides an essential tool for the further study of zebrafish otic vesicle formation and the development and regeneration of the skeleton.

Evolution and development of the fish jaw skeleton.

The evolution of the jaw represents a key innovation in driving the diversification of vertebrate body plans and behavior. The pharyngeal apparatus originated as gill bars separated by slits in chordate ancestors to vertebrates. Later, with the acquisition of neural crest, pharyngeal arches gave rise to branchial basket cartilages in jawless vertebrates (agnathans), and later bone and cartilage of the jaw, jaw support, and gills of jawed vertebrates (gnathostomes). Major events in the evolution of jaw structure from agnathans to gnathostomes include axial regionalization of pharyngeal elements and formation of a jaw joint. Hox genes specify the anterior-posterior identity of arches, and edn1, dlx, hand2, Jag1b-Notch2 signaling, and Nr2f factors specify dorsal-ventral identity. The formation of a jaw joint, an important step in the transition from an un-jointed pharynx in agnathans to a hinged jaw in gnathostomes involves interaction between nkx3.2, hand2, and barx1 factors. Major events in jaw patterning between fishes and reptiles include changes to elements of the second pharyngeal arch, including a loss of opercular and branchiostegal ray bones and transformation of the hyomandibula into the stapes. Further changes occurred between reptiles and mammals, including the transformation of the articular and quadrate elements of the jaw joint into the malleus and incus of the middle ear. Fossils of transitional jaw phenotypes can be analyzed from a developmental perspective, and there exists potential to use genetic manipulation techniques in extant taxa to test hypotheses about the evolution of jaw patterning in ancient vertebrates. This article is categorized under: Comparative Development and Evolution > Evolutionary Novelties Early Embryonic Development > Development to the Basic Body Plan Comparative Development and Evolution > Body Plan Evolution.

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.

BACKGROUND: 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. RESULTS: 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 http://www.nimr.mrc.ac.uk/3dlimb/. 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. CONCLUSION: 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.

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 >90%). 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.

A scanning electron microscopy study of idiopathic external tooth resorption in the cat.

BACKGROUND: Multiple idiopathic root resorption (MIRR) is a rare condition in man characterized by cervical resorption leading to significant tooth loss. A similar condition, feline osteoclastic resorptive lesions (FORL), affects up to 70% of domestic cats and thus provides a valuable model for investigating the etiopathogenesis of MIRR. The aim of the present study was to establish changes in the surface microanatomy of the tooth in late stage FORL and to identify whether its location has a surface bias. METHODS: Scanning electron microscopy (SEM) was used to analyze the surface features of enamel and cementum of feline teeth affected with advanced FORL. RESULTS: Resorption involved the coronal root at the cementoenamel junction (CEJ) in 95% of teeth and focal resorption of intact enamel was observed in 14% of teeth. In 55% of teeth, the main lesion was on the buccal surface and a distinct circumferential resorption "front" was present at the apical margin of resorption. The root surfaces of most affected teeth either lacked extrinsic fibers or cellular lacunae or featured evidence of cementum remodeling. Woven bone-like tissue was found within lesions, on resorbed dentin, or on the root surface in 27% of teeth. CONCLUSIONS: This study demonstrates that most FORL involve the CEJ, and the presence of focal lesions at this site suggests that this is where resorption is initiated. This implies that local factors in the oral microenvironment play a role in the etiopathogenesis of this condition. The study also shows that FORL are more likely to occur on buccal surfaces and are associated with changes in the microarchitecture of the root surface consistent with destruction of the normal periodontal attachment and stimulation of a reparative response. These findings may be relevant to understanding the etiopathogenesis of multiple idiopathic resorption areas in man.

Comparing phototoxicity during the development of a zebrafish craniofacial bone using confocal and light sheet fluorescence microscopy techniques.

The combination of genetically encoded fluorescent proteins and three-dimensional imaging enables cell-type-specific studies of embryogenesis. Light sheet microscopy, in which fluorescence excitation is provided by a plane of laser light, is an appealing approach to live imaging due to its high speed and efficient use of photons. While the advantages of rapid imaging are apparent from recent work, the importance of low light levels to studies of development is not well established. We examine the zebrafish opercle, a craniofacial bone that exhibits pronounced shape changes at early developmental stages, using both spinning disk confocal and light sheet microscopies of fluorescent osteoblast cells. We find normal and aberrant opercle morphologies for specimens imaged with short time intervals using light sheet and spinning disk confocal microscopies, respectively, under equivalent exposure conditions over developmentally-relevant time scales. Quantification of shapes reveals that the differently imaged specimens travel along distinct trajectories in morphological space.

Observation of miRNA gene expression in zebrafish embryos by in situ hybridization to microRNA primary transcripts.

MicroRNAs (miRNAs) add a previously unexpected layer to the post-transcriptional regulation of protein production. Although locked nucleic acids (LNAs) reveal the distribution of mature miRNAs by in situ hybridization (ISH) experiments in zebrafish and other organisms, high cost has restricted their use. Further, LNA probes designed to recognize mature miRNAs do not distinguish expression patterns of two miRNA genes that produce the same mature miRNA sequence. Riboprobes are substantially less expensive than LNAs, but have not been used to detect miRNA gene expression because they do not bind with high affinity to the short, 22-nucleotide-long mature miRNAs. To solve these problems, we capitalized on the fact that miRNAs are initially transcribed into long primary transcripts (pri-mRNAs). We show here that conventional digoxigenin-labeled riboprobes can bind to primary miRNA transcripts in zebrafish embryos. We tested intergenic and intronic miRNAs (miR-10d, miR-21, miR-27a, miR-126a, miR-126b, miR-138, miR-140, miR-144, miR-196a1, miR-196a2, miR-196a2b [miR-196c], miR-196b, miR-196b1b [miR-196d], miR-199, miR-214, miR-200, and miR-222) in whole mounts and some of these in histological sections. Results showed that pri-miRNA ISH provides an attractive and cost-effective tool to study miRNA expression by ISH. We use this method to show that miR-126a and miR-126b are transcribed in the caudal vasculature in the pattern of their neighboring gene ci116 or host gene egfl7, respectively, and that the chondrocyte miRNA mir-140 lies downstream of Sox9 in development of the craniofacial skeleton.

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.

Tbx4 and tbx5 acting in connective tissue are required for limb muscle and tendon patterning.

Proper functioning of the musculoskeletal system requires the precise integration of bones, muscles, and tendons. Complex morphogenetic events ensure that these elements are linked together in the appropriate three-dimensional configuration. It has been difficult, however, to tease apart the mechanisms that regulate tissue morphogenesis. We find that deletion of Tbx5 in forelimbs (or Tbx4 in hindlimbs) specifically affects muscle and tendon patterning without disrupting skeletal development, thus suggesting that distinct cues regulate these processes. We identify muscle connective tissue as the site of action of these transcription factors and show that N-Cadherin and beta-Catenin are key downstream effectors acting in muscle connective tissue and regulating soft-tissue morphogenesis. In humans, TBX5 mutations lead to Holt-Oram syndrome, which is characterized by forelimb musculoskeletal defects. Our results suggest that a focus on connective tissue is required to understand the etiology of diseases affecting soft tissue formation.

Pitx1 determines the morphology of muscle, tendon, and bones of the hindlimb.

The vertebrate forelimb and hindlimb are serially homologous structures; however, their distinctive morphologies suggest that different mechanisms are associated with each limb type to give rise to limb-type identity. Three genes have been implicated in this process; T-box transcription factors Tbx5 and Tbx4, which are expressed in the forelimb and hindlimb, respectively, and a paired-type homeodomain transcription factor Pitx1, expressed in the hindlimb. To explore the roles of Pitx1 and Tbx4 in patterning the hindlimb, we have ectopically misexpressed these genes in the mouse forelimb using transgenic methods. We have developed a novel technique for visualising the structure and organisation of tissues in limbs in 3D using optical projection tomography (OPT). This approach provides unparalleled access to understanding the relationships between connective tissues during development of the limb. Misexpression of Pitx1 in the forelimb results in the transformation and translocation of specific muscles, tendons, and bones of the forelimb so that they acquire a hindlimb-like morphology. Pitx1 also upregulates hindlimb-specific factors in the forelimb, including Hoxc10 and Tbx4. In contrast, misexpression of Tbx4 in the forelimb does not result in a transformation of limb-type morphology. These results demonstrate that Pitx1, but not Tbx4, determines the morphological identity of hindlimb tissues.