Identification of conserved skeletal enhancers associated with craniosynostosis risk genes.

Craniosynostosis, defined by premature fusion of one or multiple cranial sutures, is a common congenital defect affecting more than 1/2000 infants and results in restricted brain expansion. Single gene mutations account for 15-20% of cases, largely as part of a syndrome, but the majority are nonsyndromic with complex underlying genetics. We hypothesized that the two noncoding genomic regions identified by a GWAS for craniosynostosis contain distal regulatory elements for the risk genes BMPER and BMP2. To identify such regulatory elements, we surveyed conserved noncoding sequences from both risk loci for enhancer activity in transgenic Danio rerio. We identified enhancers from both regions that direct expression to skeletal tissues, consistent with the endogenous expression of bmper and bmp2. For each locus, we also found a skeletal enhancer that also contains a sequence variant associated with craniosynostosis risk. We examined the activity of each enhancer during craniofacial development and found that the BMPER-associated enhancer is active in the restricted region of cartilage closely associated with frontal bone initiation. The same enhancer is active in mouse skeletal tissues, demonstrating evolutionarily conserved activity. Using enhanced yeast one-hybrid assays, we identified transcription factors that bind each enhancer and observed differential binding between alleles, implicating multiple signaling pathways. Our findings help unveil the genetic mechanism of the two craniosynostosis risk loci. More broadly, our combined in vivo approach is applicable to many complex genetic diseases to build a link between association studies and specific genetic mechanisms.

Osteoclast activity sculpts craniofacial form to permit sensorineural patterning in the zebrafish skull.

Efforts to understand the morphogenesis of complex craniofacial structures have largely focused on the role of chondrocytes and osteoblasts. Along with these bone-creating cells, bone-resorbing osteoclasts are critical in homeostasis of adult skeletal structures, but there is currently limited information on their role in the complex morphogenetic events of craniofacial development. Fundamental aspects of skull formation and general skeletal development are conserved from zebrafish to mammals. Using a cathepsinK reporter, we documented osteoclast location in the developing zebrafish skull over several weeks, from 5.18 mm to 9.6 mm standard length (approximately 15 to 34 days post fertilization). While broad distribution of osteoclasts is consistent across individuals, they are sparse and the exact locations vary among fish and across developmental time points. Interestingly, we observed osteoclasts concentrating at areas associated with neuromasts and their associated nerves, in particular the hyomandibular foramina and around the supraorbital lateral line. These are areas of active remodeling. In contrast, other areas of rapid bone growth, such as the osteogenic fronts of the frontal and parietal bones, show no particular concentration of osteoclasts, suggesting that they play a special role in shaping bone near neuromasts and nerves. In csf1ra mutants lacking functional osteoclasts, the morphology of the cranial bone was disrupted in both areas. The hyomandibular foramen is present in the initial cartilage template, but after the initiation of ossification, the diameter of the canal is significantly smaller in the absence of osteoclasts. The diameter of the supraorbital lateral line canals was also reduced in the mutants, as was the number of pores associated with neuromasts, which allow for the passage of associated nerves through the bone. Our findings define important and previously unappreciated roles for osteoclast activity in shaping craniofacial skeletal structures with a particular role in bone modeling around peripheral cranial nerves, providing a scaffold for wiring the sensioneural system during craniofacial development. This has important implications for the formation of the evolutionarily diverse lateral line system, as well understanding the mechanism of neurologic sequelae of congenital osteoclast dysfunction in human craniofacial development.

FaceBase 3: analytical tools and FAIR resources for craniofacial and dental research.

The FaceBase Consortium was established by the National Institute of Dental and Craniofacial Research in 2009 as a 'big data' resource for the craniofacial research community. Over the past decade, researchers have deposited hundreds of annotated and curated datasets on both normal and disordered craniofacial development in FaceBase, all freely available to the research community on the FaceBase Hub website. The Hub has developed numerous visualization and analysis tools designed to promote integration of multidisciplinary data while remaining dedicated to the FAIR principles of data management (findability, accessibility, interoperability and reusability) and providing a faceted search infrastructure for locating desired data efficiently. Summaries of the datasets generated by the FaceBase projects from 2014 to 2019 are provided here. FaceBase 3 now welcomes contributions of data on craniofacial and dental development in humans, model organisms and cell lines. Collectively, the FaceBase Consortium, along with other NIH-supported data resources, provide a continuously growing, dynamic and current resource for the scientific community while improving data reproducibility and fulfilling data sharing requirements.

Initiation and early growth of the skull vault in zebrafish.

The zebrafish offers powerful advantages as a model system for examining the growth of the skull vault and the formation of cranial sutures. The zebrafish is well suited for large-scale genetic screens, available in large numbers, and continual advances in genetic engineering facilitate precise modeling of human genetic disorders. Most importantly, zebrafish are continuously accessible for imaging during critical periods of skull formation when both mouse and chick are physically inaccessible. To establish a foundation of information on the dynamics of skull formation, we performed a longitudinal study based on confocal microscopy of individual live transgenic zebrafish. Discrete events occur at stereotyped stages in overall growth, with little variation in timing among individuals. The frontal and parietal bones initiate as small clusters of cells closely associated with cartilage around the perimeter of the skull, prior to metamorphosis and the transition to juvenile fish. Over a period of ~30 days, the frontal and parietal bones grow towards the apex of the skull and meet to begin suture formation. To aid in visualization, we have generated interactive three-dimensional models based on the imaging data, with annotated cartilage and bone elements. We propose a framework to conceptualize development of bones of the skull vault in three phases: initiation in close association with cartilage; rapid planar growth towards the apex of the skull; and finally overlapping to form sutures. Our data provide an important framework for comparing the stages and timing of skull development across model organisms, and also a baseline for the examination of zebrafish mutants affecting skull development. To facilitate these comparative analyses, the raw imaging data and the models are available as an online atlas through the FaceBase consortium (facebase.org).

Zebrafish type I collagen mutants faithfully recapitulate human type I collagenopathies.

The type I collagenopathies are a group of heterogeneous connective tissue disorders, that are caused by mutations in the genes encoding type I collagen and include specific forms of osteogenesis imperfecta (OI) and the Ehlers-Danlos syndrome (EDS). These disorders present with a broad disease spectrum and large clinical variability of which the underlying genetic basis is still poorly understood. In this study, we systematically analyzed skeletal phenotypes in a large set of zebrafish, with diverse mutations in the genes encoding type I collagen, representing different genetic forms of human OI, and a zebrafish model resembling human EDS, which harbors a number of soft connective tissues defects, typical of EDS. Furthermore, we provide insight into how zebrafish and human type I collagen are compositionally and functionally related, which is relevant in the interpretation of human type I collagen-related disease models. Our studies reveal a high degree of intergenotype variability in phenotypic expressivity that closely correlates with associated OI severity. Furthermore, we demonstrate the potential for select mutations to give rise to phenotypic variability, mirroring the clinical variability associated with human disease pathology. Therefore, our work suggests the future potential for zebrafish to aid in identifying unknown genetic modifiers and mechanisms underlying the phenotypic variability in OI and related disorders. This will improve diagnostic strategies and enable the discovery of new targetable pathways for pharmacological intervention.

The chaperone activity of 4PBA ameliorates the skeletal phenotype of Chihuahua, a zebrafish model for dominant osteogenesis imperfecta.

Classical osteogenesis imperfecta (OI) is a bone disease caused by type I collagen mutations and characterized by bone fragility, frequent fractures in absence of trauma and growth deficiency. No definitive cure is available for OI and to develop novel drug therapies, taking advantage of a repositioning strategy, the small teleost zebrafish (Danio rerio) is a particularly appealing model. Its small size, high proliferative rate, embryo transparency and small amount of drug required make zebrafish the model of choice for drug screening studies, when a valid disease model is available. We performed a deep characterization of the zebrafish mutant Chihuahua, that carries a G574D (p.G736D) substitution in the α1 chain of type I collagen. We successfully validated it as a model for classical OI. Growth of mutants was delayed compared with WT. X-ray, µCT, alizarin red/alcian blue and calcein staining revealed severe skeletal deformity, presence of fractures and delayed mineralization. Type I collagen extracted from different tissues showed abnormal electrophoretic migration and low melting temperature. The presence of endoplasmic reticulum (ER) enlargement due to mutant collagen retention in osteoblasts and fibroblasts of mutant fish was shown by electron and confocal microscopy. Two chemical chaperones, 4PBA and TUDCA, were used to ameliorate the cellular stress and indeed 4PBA ameliorated bone mineralization in larvae and skeletal deformities in adult, mainly acting on reducing ER cisternae size and favoring collagen secretion. In conclusion, our data demonstrated that ER stress is a novel target to ameliorate OI phenotype; chemical chaperones such as 4PBA may be, alone or in combination, a new class of molecules to be further investigated for OI treatment.

Evolution of the hypoxia-sensitive cells involved in amniote respiratory reflexes.

The evolutionary origins of the hypoxia-sensitive cells that trigger amniote respiratory reflexes - carotid body glomus cells, and 'pulmonary neuroendocrine cells' (PNECs) - are obscure. Homology has been proposed between glomus cells, which are neural crest-derived, and the hypoxia-sensitive 'neuroepithelial cells' (NECs) of fish gills, whose embryonic origin is unknown. NECs have also been likened to PNECs, which differentiate in situ within lung airway epithelia. Using genetic lineage-tracing and neural crest-deficient mutants in zebrafish, and physical fate-mapping in frog and lamprey, we find that NECs are not neural crest-derived, but endoderm-derived, like PNECs, whose endodermal origin we confirm. We discover neural crest-derived catecholaminergic cells associated with zebrafish pharyngeal arch blood vessels, and propose a new model for amniote hypoxia-sensitive cell evolution: endoderm-derived NECs were retained as PNECs, while the carotid body evolved via the aggregation of neural crest-derived catecholaminergic (chromaffin) cells already associated with blood vessels in anamniote pharyngeal arches.

The FaceBase Consortium: a comprehensive resource for craniofacial researchers.

The FaceBase Consortium, funded by the National Institute of Dental and Craniofacial Research, National Institutes of Health, is designed to accelerate understanding of craniofacial developmental biology by generating comprehensive data resources to empower the research community, exploring high-throughput technology, fostering new scientific collaborations among researchers and human/computer interactions, facilitating hypothesis-driven research and translating science into improved health care to benefit patients. The resources generated by the FaceBase projects include a number of dynamic imaging modalities, genome-wide association studies, software tools for analyzing human facial abnormalities, detailed phenotyping, anatomical and molecular atlases, global and specific gene expression patterns, and transcriptional profiling over the course of embryonic and postnatal development in animal models and humans. The integrated data visualization tools, faceted search infrastructure, and curation provided by the FaceBase Hub offer flexible and intuitive ways to interact with these multidisciplinary data. In parallel, the datasets also offer unique opportunities for new collaborations and training for researchers coming into the field of craniofacial studies. Here, we highlight the focus of each spoke project and the integration of datasets contributed by the spokes to facilitate craniofacial research.

Osterix/Sp7 limits cranial bone initiation sites and is required for formation of sutures.

During growth, individual skull bones overlap at sutures, where osteoblast differentiation and bone deposition occur. Mutations causing skull malformations have revealed some required genes, but many aspects of suture regulation remain poorly understood. We describe a zebrafish mutation in osterix/sp7, which causes a generalized delay in osteoblast maturation. While most of the skeleton is patterned normally, mutants have specific defects in the anterior skull and upper jaw, and the top of the skull comprises a random mosaic of bones derived from individual initiation sites. Osteoblasts at the edges of the bones are highly proliferative and fail to differentiate, consistent with global changes in gene expression. We propose that signals from the bone itself are required for orderly recruitment of precursor cells and growth along the edges. The delay in bone maturation caused by loss of Sp7 leads to unregulated bone formation, revealing a new mechanism for patterning the skull and sutures.

BMPs regulate msx gene expression in the dorsal neuroectoderm of Drosophila and vertebrates by distinct mechanisms.

In a broad variety of bilaterian species the trunk central nervous system (CNS) derives from three primary rows of neuroblasts. The fates of these neural progenitor cells are determined in part by three conserved transcription factors: vnd/nkx2.2, ind/gsh and msh/msx in Drosophila melanogaster/vertebrates, which are expressed in corresponding non-overlapping patterns along the dorsal-ventral axis. While this conserved suite of "neural identity" gene expression strongly suggests a common ancestral origin for the patterning systems, it is unclear whether the original regulatory mechanisms establishing these patterns have been similarly conserved during evolution. In Drosophila, genetic evidence suggests that Bone Morphogenetic Proteins (BMPs) act in a dosage-dependent fashion to repress expression of neural identity genes. BMPs also play a dose-dependent role in patterning the dorsal and lateral regions of the vertebrate CNS, however, the mechanism by which they achieve such patterning has not yet been clearly established. In this report, we examine the mechanisms by which BMPs act on cis-regulatory modules (CRMs) that control localized expression of the Drosophila msh and zebrafish (Danio rerio) msxB in the dorsal central nervous system (CNS). Our analysis suggests that BMPs act differently in these organisms to regulate similar patterns of gene expression in the neuroectoderm: repressing msh expression in Drosophila, while activating msxB expression in the zebrafish. These findings suggest that the mechanisms by which the BMP gradient patterns the dorsal neuroectoderm have reversed since the divergence of these two ancient lineages.

Distinct enhancers of ptf1a mediate specification and expansion of ventral pancreas in zebrafish.

Development of the pancreas and cerebellum require Pancreas-specific transcription factor-1a (Ptf1a), which encodes a subunit of the transcription factor complex PTF1. Ptf1a is required in succession for specification of the pancreas, proper allocation of pancreatic progenitors to endocrine and exocrine fates, and the production of digestive enzymes from the exocrine acini. In several neuronal structures, including the cerebellum, hindbrain, retina and spinal cord, Ptf1a is transiently expressed and promotes inhibitory neuron fates at the expense of excitatory fates. Transcription of Ptf1a in mouse is maintained in part by PTF1 acting on an upstream autoregulatory enhancer. However, the transcription factors and enhancers that initially activate Ptf1a expression in the pancreas and in certain structures of the nervous system have not yet been identified. Here we describe a zebrafish autoregulatory element, conserved among teleosts, with activity similar to that described in mouse. In addition, we performed a comprehensive survey of all non-coding sequences in a 67kb interval encompassing zebrafish ptf1a, and identified several neuronal enhancers, and an enhancer active in the ventral pancreas prior to activation of the autoregulatory enhancer. To test the requirement for autoregulatory control during pancreatic development, we restored ptf1a function through BAC transgenesis in ptf1a morphants, either with an intact BAC or one lacking the autoregulatory enhancer. We find that ptf1a autoregulation is required for development of the exocrine pancreas and full rescue of the ptf1a morphant phenotype. Similarly, we demonstrate that a ptf1a locus lacking the early enhancer region is also capable of rescue, but only supports formation of a hypoplastic exocrine pancreas. Through our dissection of the complex regulatory control of ptf1a, we identified separate cis-regulatory elements that underlie different aspects of its expression and function, and further demonstrated the requirement of maintained ptf1a expression for normal pancreatic morphogenesis. We also identified a novel enhancer that mediates initiation of ptf1a expression in the pancreas, through which the signals that specify the ventral pancreas are expected to exert their action.

GRG5/AES interacts with T-cell factor 4 (TCF4) and downregulates Wnt signaling in human cells and zebrafish embryos.

Transcriptional control by TCF/LEF proteins is crucial in key developmental processes such as embryo polarity, tissue architecture and cell fate determination. TCFs associate with ß-catenin to activate transcription in the presence of Wnt signaling, but in its absence act as repressors together with Groucho-family proteins (GRGs). TCF4 is critical in vertebrate intestinal epithelium, where TCF4-ß-catenin complexes are necessary for the maintenance of a proliferative compartment, and their abnormal formation initiates tumorigenesis. However, the extent of TCF4-GRG complexes' roles in development and the mechanisms by which they repress transcription are not completely understood. Here we characterize the interaction between TCF4 and GRG5/AES, a Groucho family member whose functional relationship with TCFs has been controversial. We map the core GRG interaction region in TCF4 to a 111-amino acid fragment and show that, in contrast to other GRGs, GRG5/AES-binding specifically depends on a 4-amino acid motif (LVPQ) present only in TCF3 and some TCF4 isoforms. We further demonstrate that GRG5/AES represses Wnt-mediated transcription both in human cells and zebrafish embryos. Importantly, we provide the first evidence of an inherent repressive function of GRG5/AES in dorsal-ventral patterning during early zebrafish embryogenesis. These results improve our understanding of TCF-GRG interactions, have significant implications for models of transcriptional repression by TCF-GRG complexes, and lay the groundwork for in depth direct assessment of the potential role of Groucho-family proteins in both normal and abnormal development.

Skeletogenic fate of zebrafish cranial and trunk neural crest.

The neural crest (NC) is a major contributor to the vertebrate craniofacial skeleton, detailed in model organisms through embryological and genetic approaches, most notably in chick and mouse. Despite many similarities between these rather distant species, there are also distinct differences in the contribution of the NC, particularly to the calvariae of the skull. Lack of information about other vertebrate groups precludes an understanding of the evolutionary significance of these differences. Study of zebrafish craniofacial development has contributed substantially to understanding of cartilage and bone formation in teleosts, but there is currently little information on NC contribution to the zebrafish skeleton. Here, we employ a two-transgene system based on Cre recombinase to genetically label NC in the zebrafish. We demonstrate NC contribution to cells in the cranial ganglia and peripheral nervous system known to be NC-derived, as well as to a subset of myocardial cells. The indelible labeling also enables us to determine NC contribution to late-forming bones, including the calvariae. We confirm suspected NC origin of cartilage and bones of the viscerocranium, including cartilages such as the hyosymplectic and its replacement bones (hymandibula and symplectic) and membranous bones such as the opercle. The cleithrum develops at the border of NC and mesoderm, and as an ancestral component of the pectoral girdle was predicted to be a hybrid bone composed of both NC and mesoderm tissues. However, we find no evidence of a NC contribution to the cleithrum. Similarly, in the vault of the skull, the parietal bones and the caudal portion of the frontal bones show no evidence of NC contribution. We also determine a NC origin for caudal fin lepidotrichia; the presumption is that these are derived from trunk NC, demonstrating that these cells have the ability to form bone during normal vertebrate development.

Multiple enhancers associated with ACAN suggest highly redundant transcriptional regulation in cartilage.

The chondroitin sulfate proteoglycan core protein aggrecan is the major protein constituent of cartilage aside from collagen, and is largely responsible for its distinctive mechanical properties. Aggrecan is required both for proper cartilage formation in development and maintenance of mature cartilage. Prominent ACAN transcription is a conserved feature of vertebrate cartilage, although little is known about its specific transcriptional regulation. We examined the genomic interval containing human ACAN for transcriptional enhancers directing expression to cartilage, using a functional assay in transgenic zebrafish. We tested 24 conserved non-coding sequences, representing ~6% of the total sequence in the interval, and identified eleven independently capable of regulating reporter gene expression in cartilage. These enhancers were widely spaced, from >100kb upstream of the gene to within the first intron. While the majority displayed broad cartilage expression in zebrafish larvae, several were restricted to a subset of cartilage cells in the craniofacial skeleton. In older fish, the enhancers displayed differential activity; some maintained expression, either in all cartilage or preferentially in articular cartilage at the joints, while others were not active. This remarkable degree of overlapping regulatory control has been highly conserved; we identified clear orthologues of six enhancers at the chicken ACAN locus, arranged in the same order relative to the gene. These were also functional in directing expression to cartilage in transgenic zebrafish. Several enhancers contain potential binding sites for Sox9, consistent with its described role as an upstream regulator of ACAN expression. However, others lacked Sox9 consensus binding sites, implicating additional pathways and transcription factors as regulators of ACAN expression in cartilage, either in development or adult tissue. Our identification of these enhancer sequences is the necessary first step in detailed examination of the upstream regulators of ACAN expression.

Evolution of the bone gene regulatory network.

Current fossil, embryological and genetic data shed light on the evolution of the gene regulatory network (GRN) governing bone formation. The key proteins and genes involved in skeletogenesis are well accepted. We discuss when these essential components of the GRN evolved and propose that the Runx genes, master regulators of skeletogenesis, functioned in early cartilages well before they were co-opted to function in the making of bone. Two rounds of whole genome duplication, together with additional tandem gene duplications, created a genetic substrate for segregation of one GRN into several networks regulating the related tissues of cartilage, bone, enamel, and dentin. During this segregation, Runx2 assumed its position at the top of the bone GRN, and Sox9 was excluded from bone, retaining its ancient role in cartilage.

Identification of RNA binding motif proteins essential for cardiovascular development.

BACKGROUND: We recently identified Rbm24 as a novel gene expressed during mouse cardiac development. Due to its tightly restricted and persistent expression from formation of the cardiac crescent onwards and later in forming vasculature we posited it to be a key player in cardiogenesis with additional roles in vasculogenesis and angiogenesis. RESULTS: To determine the role of this gene in cardiac development, we have identified its zebrafish orthologs (rbm24a and rbm24b), and functionally evaluated them during zebrafish embryogenesis. Consistent with our underlying hypothesis, reduction in expression of either ortholog through injection of morpholino antisense oligonucleotides results in cardiogenic defects including cardiac looping and reduced circulation, leading to increasing pericardial edema over time. Additionally, morphant embryos for either ortholog display incompletely overlapping defects in the forming vasculature of the dorsal aorta (DA), posterior caudal vein (PCV) and caudal vein (CV) which are the first blood vessels to form in the embryo. Vasculogenesis and early angiogenesis in the trunk were similarly compromised in rbm24 morphant embryos at 48 hours post fertilization (hpf). Subsequent vascular maintenance was impaired in both rbm24 morphants with substantial vessel degradation noted at 72 hpf. CONCLUSION: Taken collectively, our functional data support the hypothesis that rbm24a and rbm24b are key developmental cardiac genes with unequal roles in cardiovascular formation.

ΦC31 integrase mediates efficient cassette exchange in the zebrafish germline.

Site-specific recombinases (SSRs) are powerful tools for genome manipulation, used in diverse organisms including Drosophila melanogaster, mouse, Arabidopsis, zebrafish, and human cultured cells. The integrase from the bacteriophage ΦC31 belongs to the large serine family of integrases, and in contrast to other widely used SSRs such as Cre and Flp, recombination is directional and therefore irreversible. We have developed a vector system for recombinase-mediated cassette exchange (RMCE) in the zebrafish, allowing swapping of the coding sequence in an integrated transgene. Utilizing codon-optimized ΦC31 integrase RNA bearing the 3'UTR from the nanos1 gene, we replaced the egfp coding sequence of an integrated reporter transgene with mCherry coding sequence. Recombination was achieved at high efficiency in both somatic cells and in the germline. We demonstrate an effective approach to RMCE, increasing the repertoire of tools available to manipulate the zebrafish genome.

Bone regenerates via dedifferentiation of osteoblasts in the zebrafish fin.

While mammals have a limited capacity to repair bone defects, zebrafish can completely regenerate amputated bony structures of their fins. Fin regeneration is dependent on formation of a blastema, a progenitor cell pool accumulating at the amputation plane. It is unclear which cells the blastema is derived from, whether it forms by dedifferentiation of mature cells, and whether blastema cells are multipotent. We show that mature osteoblasts dedifferentiate and form part of the blastema. Osteoblasts downregulate expression of intermediate and late bone differentiation markers and induce genes expressed by bone progenitors. Dedifferentiated osteoblasts proliferate in a FGF-dependent manner and migrate to form part of the blastema. Genetic fate mapping shows that osteoblasts only give rise to osteoblasts in the regenerate, indicating that dedifferentiation is not associated with the attainment of multipotency. Thus, bone can regenerate from mature osteoblasts via dedifferentiation, a finding with potential implications for human bone repair.

Mosaic zebrafish transgenesis for evaluating enhancer sequences.

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non coding sequences. One prominent function carried out by the non coding fraction of the genome is to regulate gene transcription; however, there are no effective methods to broadly predict cis-regulatory elements from primary DNA sequence. We have developed an efficient protocol to functionally evaluate potential cis-regulatory elements through zebrafish transgenesis. Our approach offers significant advantages over cell-culture based techniques for developmentally important genes, since it provides information on spatial and temporal gene regulation. Conversely, it is faster and less expensive than similar experiments in transgenic mice, and we routinely apply it to sequences isolated from the human genome. Here we demonstrate our approach to selecting elements for testing based on sequence conservation and our protocol for cloning sequences and microinjecting them into zebrafish embryos.

Functionally conserved cis-regulatory elements of COL18A1 identified through zebrafish transgenesis.

Type XVIII collagen is a component of basement membranes, and expressed prominently in the eye, blood vessels, liver, and the central nervous system. Homozygous mutations in COL18A1 lead to Knobloch Syndrome, characterized by ocular defects and occipital encephalocele. However, relatively little has been described on the role of type XVIII collagen in development, and nothing is known about the regulation of its tissue-specific expression pattern. We have used zebrafish transgenesis to identify and characterize cis-regulatory sequences controlling expression of the human gene. Candidate enhancers were selected from non-coding sequence associated with COL18A1 based on sequence conservation among mammals. Although these displayed no overt conservation with orthologous zebrafish sequences, four regions nonetheless acted as tissue-specific transcriptional enhancers in the zebrafish embryo, and together recapitulated the major aspects of col18a1 expression. Additional post-hoc computational analysis on positive enhancer sequences revealed alignments between mammalian and teleost sequences, which we hypothesize predict the corresponding zebrafish enhancers; for one of these, we demonstrate functional overlap with the orthologous human enhancer sequence. Our results provide important insight into the biological function and regulation of COL18A1, and point to additional sequences that may contribute to complex diseases involving COL18A1. More generally, we show that combining functional data with targeted analyses for phylogenetic conservation can reveal conserved cis-regulatory elements in the large number of cases where computational alignment alone falls short.