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BACKGROUND: In humans, vitamin B-12 (cobalamin) transport involves 3 paralogous proteins: transcobalamin, haptocorrin, and intrinsic factor. Zebrafish (Danio rerio) express 3 genes that encode proteins homologous to known B-12 carrier proteins: tcn2 (a transcobalamin ortholog) and 2 atypical β-domain-only homologs, tcnba and tcnbb. OBJECTIVES: Given the orthologous relation between zebrafish Tcn2 and human transcobalamin, we hypothesized that zebrafish carrying null mutations of tcn2 would exhibit phenotypes consistent with vitamin B-12 deficiency. METHODS: First-generation and second-generation tcn2(–/–) zebrafish were characterized using phenotypic assessments, metabolic analyses, viability studies, and transcriptomics. RESULTS: Homozygous tcn2(–/–) fish produced from a heterozygous cross are viable and fertile but exhibit reduced growth, which persists into adulthood. When first-generation female tcn2(–/–) fish are bred, their offspring exhibit gross developmental and metabolic defects. These phenotypes are observed in all offspring from a tcn2(–)(/)(–) female regardless of the genotype of the male mating partner, suggesting a maternal effect, and can be rescued with vitamin B-12 supplementation. Transcriptome analyses indicate that offspring from a tcn2(–)(/)(–) female exhibit expression profiles distinct from those of offspring from a tcn2(+/+) female, which demonstrate dysregulation of visual perception, fatty acid metabolism, and neurotransmitter signaling pathways. CONCLUSIONS: Our findings suggest that the deposition of vitamin B-12 in the yolk by tcn2(–)(/)(–) females may be insufficient to support the early development of their offspring. These data present a compelling model to study the effects of vitamin B-12 deficiency on early development, with a particular emphasis on transgenerational effects and gene–environment interactions.

Purpose North Carolina macular dystrophy (NCMD) is an autosomal dominant, congenital disorder affecting the central retina. Here, we report clinical and genetic findings in three families segregating NCMD and use epigenomic datasets from human tissues to gain insights into the effect of NCMD-implicated variants. Methods Clinical assessment and genetic testing were performed. Publicly available transcriptomic and epigenomic datasets were analyzed and the activity-by-contact method for scoring enhancer elements and linking them to target genes was used. Results A previously described, heterozygous, noncoding variant upstream of the PRDM13 gene was detected in all six affected study participants (chr6:100,040,987G>C [GRCh37/hg19]). Interfamilial and intrafamilial variability were observed; the visual acuity ranged from 0.0 to 1.6 LogMAR and fundoscopic findings ranged from visually insignificant, confluent, drusen-like macular deposits to coloboma-like macular lesions. Variable degrees of peripheral retinal spots (which were easily detected on widefield retinal imaging) were observed in all study subjects. Notably, a 6-year-old patient developed choroidal neovascularization and required treatment with intravitreal bevacizumab injections. Computational analysis of the five single nucleotide variants that have been implicated in NCMD revealed that these noncoding changes lie within two putative enhancer elements; these elements are predicted to interact with PRDM13 in the developing human retina. PRDM13 was found to be expressed in the fetal retina, with greatest expression in the amacrine precursor cell population. Conclusions We provide further evidence supporting the role of PRDM13 dysregulation in the pathogenesis of NCMD and highlight the usefulness of widefield retinal imaging in individuals suspected to have this condition.

PurposeNorth Carolina macular dystrophy (NCMD) is an autosomal dominant, congenital disorder affecting the central retina. Here, we report clinical and genetic findings in three families segregating NCMD and use epigenomic datasets from human tissues to gain insights into the effect of NCMD-implicated variants.MethodsClinical assessment and genetic testing were performed. Publicly-available transcriptomic and epigenomic datasets were analyzed and the ‘Activity-by-Contact’ (ABC) method for scoring enhancer elements and linking them to target genes was used.ResultsA previously-described, heterozygous, non-coding variant upstream of the PRDM13 gene was detected in all six affected study participants (chr6:100,040,987G>C [GRCh37/hg19]). Inter- and intra-familial variability were observed; the visual acuity ranged from 0.0 to 1.6 LogMAR and fundoscopic findings ranged from visually insignificant, confluent, drusen-like macular deposits to coloboma-like macular lesions. Variable degrees of peripheral retinal spots (which were easily detected on widefield retinal imaging) were observed in all study subjects. Notably, a 6-year-old patient developed choroidal neovascularization and required treatment with intravitreal bevacizumab injections. Computational analysis of the five single nucleotide variants that have been implicated in NCMD revealed that these non-coding changes lie within two putative enhancer elements; these elements are predicted to interact with PRDM13 in the developing human retina. PRDM13 was found to be expressed in the fetal retina, with highest expression in the amacrine precursor cell population.ConclusionsWe provide further evidence supporting the role of PRDM13 dysregulation in the pathogenesis of NCMD and highlight the utility of widefield retinal imaging in individuals suspected to have this condition.

De novo transcriptome construction from short-read RNA-seq is a common method for reconstructing mRNA transcripts within a given sample. However, the precision of this process is unclear as it is difficult to obtain a ground-truth measure of transcript expression. With advances in third generation sequencing, full length transcripts of whole transcriptomes can be accurately sequenced to generate a ground-truth transcriptome. We generated long-read PacBio and short-read Illumina RNA-seq data from a human induced pluripotent stem cell- derived retinal pigmented epithelium (iPSC-RPE) cell line. We use long-read data to identify simple metrics for assessing de novo transcriptome construction and optimize a short-read based de novo transcriptome construction pipeline. We apply this this pipeline to construct transcriptomes for 340 short-read RNA-seq samples originating from healthy adult and fetal human retina, cornea, and RPE. We identify hundreds of novel gene isoforms and examine their significance in the context of ocular development and disease.

In humans, transport of food-derived cobalamin (vitamin B12) from the digestive system into the bloodstream involves three paralogous proteins: transcobalamin (TC), haptocorrin (HC), and intrinsic factor (IF). Each of these proteins contains two domains, an α-domain and a β-domain, which together form a cleft in which cobalamin binds. Zebrafish (Danio rerio) are thought to possess only a single cobalamin transport protein, referred to as Tcn2, which is a transcobalamin homolog. Here, we used CRISPR/Cas9 mutagenesis to create null alleles of tcn2 in zebrafish. Fish homozygous for tcn2-null alleles were viable and exhibited no obvious developmentally or behaviorally abnormal phenotypes. For this reason, we hypothesized that previously unidentified cobalamin-carrier proteins encoded in the zebrafish genome may provide an additional pathway for cobalamin transport. We identified genes predicted to code for two such proteins, Tcn-beta-a (Tcnba) and Tcn-beta-b (Tcnbb), which differ from all previously characterized cobalamin transport proteins as they lack the α-domain. These β-domain–only proteins are representative of an undescribed class of cobalamin-carrier proteins that are highly conserved throughout the ray-finned fishes. We observed that the genes encoding the three cobalamin transport homologs, tcn2, tcnba, and tcnbb, are expressed in unique spatial and temporal patterns in the developing zebrafish. Moreover, exogenously expressed recombinant Tcnba and Tcnbb bound cobalamin with high affinity, comparable with binding by full-length Tcn2. Taken together, our results suggest that this noncanonical protein structure has evolved to fully function as a cobalamin-carrier protein, thereby allowing for a compensatory cobalamin transport mechanism in the tcn2−/− zebrafish.

The human eye is built from several specialized tissues which direct, capture, and pre-process information to provide vision. The gene expression of the different eye tissues has been extensively profiled with RNA-seq across numerous studies. Large consortium projects have also used RNA-seq to study gene expression patterning across many different human tissues, minus the eye. There has not been an integrated study of expression patterns from multiple eye tissues compared to other human body tissues. We have collated all publicly available healthy human eye RNA-seq datasets as well as dozens of other tissues. We use this fully integrated dataset to probe the biological processes and pan expression relationships between the cornea, retina, RPE-choroid complex, and the rest of the human tissues with differential expression, clustering, and GO term enrichment tools. We also leverage our large collection of retina and RPE-choroid tissues to build the first human weighted gene correlation networks and use them to highlight known biological pathways and eye gene disease enrichment. We also have integrated publicly available single cell RNA-seq data from mouse retina into our framework for validation and discovery. Finally, we make all these data, analyses, and visualizations available via a powerful interactive web application (https://eyeintegration.nei.nih.gov/).

PURPOSETo develop an accessible and reliable RNA-seq transcriptome database of healthy human eye tissues and a matching reactive web application to query gene expression in eye and body tissues. METHODS: We downloaded the raw sequnce data for 1375 RNA-seq samples across 54 tissues in the GTEx project as a non-eye reference set.We then queried several public repositories to find all healthy, non-perturbed, human eye-related tissue RNA-seq samples. The 916 eye and 1375 GTEx samples were sent into a Snakemake-based reproducible pipeline we wrote to quantify all known transcripts and genes, removes samples with poor sequence quality and mislabels, normalizes expression values across each tissue, performs 882 differential expression tests, calculates GO term enrichment, and outputs all as a single SQLite database file: the Eye in a Disk (EiaD) dataset. Furthermore, we rewrote the web application eyeIntegration (https://eyeIntegration.nei.nih.gov) to display EiaD.RESULTSThe new eyeIntegration portal provides quick visualization of human eye-related transcriptomes published to date by database version, gene/transcript, 19 eye tissues, and 54 body tissues. As a test of the value of this unified pan-eye dataset, we showed that fetal and organoid retina are highly similar at a pan-transcriptome level but display distinct differences in certain pathways and gene families like protocadherin and HOXB family members.CONCLUSIONThe eyeIntegration v1.0 web app serves the pan-human eye and body transcriptome dataset, EiaD. This offers the eye community a powerful and quick means to test hypotheses on human gene and transcript expression across 54 body and 19 eye tissues.

Considerable progress has been made in testing stem cell-derived retinal pigment epithelium (RPE) as a potential therapy for age-related macular degeneration (AMD). However, the recent reports of oncogenic mutations in induced pluripotent stem cells (iPSCs) underlie the need for robust manufacturing and functional validation of clinical-grade iPSC-derived RPE before transplantation. Here, we developed oncogenic mutation-free clinical-grade iPSCs from three AMD patients and differentiated them into clinical-grade iPSC-RPE patches on biodegradable scaffolds. Functional validation of clinical-grade iPSC-RPE patches revealed specific features that distinguished transplantable from nontransplantable patches. Compared to RPE cells in suspension, our biodegradable scaffold approach improved integration and functionality of RPE patches in rats and in a porcine laser-induced RPE injury model that mimics AMD-like eye conditions. Our results suggest that the in vitro and in vivo preclinical functional validation of iPSC-RPE patches developed here might ultimately be useful for evaluation and optimization of autologous iPSC-based therapies.

DNA methylation is a dynamic process through which specific chromatin modifications can be stably transmitted from parent to daughter cells. A large body of work has suggested that DNA methylation influences gene expression by silencing gene promoters. However, these conclusions were drawn from data focused mostly on promoter regions. Regarding the entire genome, it is unclear how methylation and gene transcription patterns are related during vertebrate development. To identify the genome-wide distribution of CpG methylation, we created series of high-resolution methylome maps of Danio rerio embryos during development and in mature, differentiated tissues. We found that embryonic and terminal tissues have unique methylation signatures in CpG islands and repetitive sequences. Fully differentiated tissues have increased CpG and LTR methylation and decreased SINE methylation relative to embryonic tissues. Unsupervised clustering analyses reveal that the embryonic and terminal tissues can be classified solely by their methylation patterning. Novel analyses also identify a previously undescribed genome-wide exon methylation signature. We also compared whole genome methylation with genome-wide mRNA expression levels using publicly available RNA-seq datasets. These comparisons revealed previously unrecognized relationships between gene expression, alternative splicing, and exon methylation. Surprisingly, we found that exonic methylation is a better predictor of mRNA expression level than promoter methylation. We also found that transcriptionally skipped exons have significantly less methylation than retained exons. Our integrative analyses reveal highly complex interplay between gene expression, alternative splicing, development, and methylation patterning in zebrafish.

RET, a gene causatively mutated in Hirschsprung disease and cancer, has recently been implicated in breast cancer estrogen (E2) independence and tamoxifen resistance. RET displays both E2 and retinoic acid (RA)-dependent transcriptional modulation in E2-responsive breast cancers. However, the regulatory elements through which the steroid hormone transcriptional regulation of RET is mediated are poorly defined. Recent genome-wide chromatin immunoprecipitation-based studies have identified 10 putative E2 receptor-alpha (ESR1) and RA receptor alpha-binding sites at the RET locus, of which we demonstrate only two (RET −49.8 and RET +32.8) display significant E2 regulatory response when assayed independently in MCF-7 breast cancer cells. We demonstrate that endogenous RET expression and RET −49.8 regulatory activity are cooperatively regulated by E2 and RA in breast cancer cells. We identify key sequences that are required for RET −49.8 and RET +32.8 E2 responsiveness, including motifs known to be bound by ESR1, FOXA1 and TFAP2C. We also report that both RET −49.8 regulatory activity and endogenous RET expression are completely dependent on ESR1 for their (E2)-induction and that ESR1 is sufficient to mediate the E2-induced enhancer activity of RET −49.8 and RET +32.8. Finally, using zebrafish transgenesis, we also demonstrate that RET −49.8 directs reporter expression in the central nervous system and peripheral nervous system consistent with the endogenous ret expression. Taken collectively, these data suggest that RET transcription in breast cancer cells is modulated by E2 via ESR1 acting on multiple elements collectively.

Plasticity of gene regulatory encryption can permit DNA sequence divergence without loss of function. Functional information is preserved through conservation of the composition of transcription factor binding sites (TFBS) in a regulatory element. We have developed a method that can accurately identify pairs of functional noncoding orthologs at evolutionarily diverged loci by searching for conserved TFBS arrangements. With an estimated 5% false-positive rate (FPR) in approximately 3000 human and zebrafish syntenic loci, we detected approximately 300 pairs of diverged elements that are likely to share common ancestry and have similar regulatory activity. By analyzing a pool of experimentally validated human enhancers, we demonstrated that 7/8 (88%) of their predicted functional orthologs retained in vivo regulatory control. Moreover, in 5/7 (71%) of assayed enhancer pairs, we observed concordant expression patterns. We argue that TFBS composition is often necessary to retain and sufficient to predict regulatory function in the absence of overt sequence conservation, revealing an entire class of functionally conserved, evolutionarily diverged regulatory elements that we term “covert.”

Zebrafish transgenesis is a powerful and increasingly common strategy to assay vertebrate transcriptional regulatory control. Several challenges remain, however, to the broader application of this technique; they include increasing the rate with which transgenes can be analyzed and maximizing the informational value of the data generated. Presently, many rely on the injection of individual constructs and the analysis of resulting reporter expression in mosaic G0 embryos. Here, we contrast these approaches, examining whether injecting pooled transgene constructs can increase the efficiency with which regulatory sequences can be assayed, restricting analysis to the offspring of germ line transmitting transgenic zebrafish in an effort to reduce potential subjectivity. We selected a 64kb interval encompassing the human ASCL1 locus as our model interval and report the analysis of 9 highly conserved putative enhancers therein. We identified 32 transgene-positive zebrafish, transmitting one or more independent constructs displaying ASCL1-like regulatory control. Through examination of embryos harboring one or more transgenes, we demonstrate that five of the nine sequences account for the observed control and describe their likely roles in ASCL1 regulation. These data demonstrate the utility of this approach and its potential for further adaptation and higher throughput application.

Numerous linkage and association studies by our group and others have implicated DPYSL2 at 8p21.2 in schizophrenia. Here we explore DPYSL2 for functional variation that underlies these associations. We sequenced all 14 exons of DPYSL2 as well as 27 conserved noncoding regions at the locus in 137 cases and 151 controls. We identified 120 variants, eight of which we genotyped in an additional 729 cases and 1542 controls. Several were significantly associated with schizophrenia, including a three single-nucleotide polymorphism (SNP) haplotype in the proximal promoter, two SNPs in intron 1, and a polymorphic dinucleotide repeat in the 5′-untranslated region that alters sequences predicted to be involved in translational regulation by mammalian target of rapamycin signaling. The 3-SNP promoter haplotype and the sequence surrounding one of the intron 1 SNPs direct tissue-specific expression in the nervous systems of Zebrafish in a pattern consistent with the two endogenous dpysl2 paralogs. In addition, two SNP haplotypes over the coding exons and 3′ end of DPYSL2 showed association with opposing sex-specific risks. These data suggest that these polymorphic, schizophrenia-associated sequences function as regulatory elements for DPYSL2 expression. In transient transfection assays, the high risk allele of the polymorphic dinucleotide repeat diminished reporter expression by 3- to 4-fold. Both the high- and low-risk alleles respond to allosteric mTOR inhibition by rapamycin until, at high drug levels, allelic differences are eliminated. Our results suggest that reduced transcription and mTOR-regulated translation of certain DPYSL2 isoforms increase the risk for schizophrenia.

Hirschsprung disease (HSCR) is a common, multigenic neurocristopathy characterized by incomplete innervation along a variable length of the gut. The pivotal gene in isolated HSCR cases, either sporadic or familial, is RET . HSCR also presents in various syndromes, including Shah–Waardenburg syndrome (WS), Down (DS), and Bardet–Biedl (BBS). Here, we report 3 families with BBS and HSCR with concomitant mutations in BBS genes and regulatory RET elements, whose functionality is tested in physiologically relevant assays. Our data suggest that BBS mutations can potentiate HSCR predisposing RET alleles, which by themselves are insufficient to cause disease. We also demonstrate that these genes interact genetically in vivo to modulate gut innervation, and that this interaction likely occurs through complementary, yet independent, pathways that converge on the same biological process.

Background Transcriptional regulatory elements are central to development and interspecific phenotypic variation. Current regulatory element prediction tools rely heavily upon conservation for prediction of putative elements. Recent in vitro observations from the ENCODE project combined with in vivo analyses at the zebrafish phox2b locus suggests that a significant fraction of regulatory elements may fall below commonly applied metrics of conservation. We propose to explore these observations in vivo at the human PHOX2B locus, and also evaluate the potential evidence for genome-wide applicability of these observations through a novel analysis of extant data. Results Transposon-based transgenic analysis utilizing a tiling path proximal to human PHOX2B in zebrafish recapitulates the observations at the zebrafish phox2b locus of both conserved and non-conserved regulatory elements. Analysis of human sequences conserved with previously identified zebrafish phox2b regulatory elements demonstrates that the orthologous sequences exhibit overlapping regulatory control. Additionally, analysis of non-conserved sequences scattered over 135 kb 5' to PHOX2B, provides evidence of non-conserved regulatory elements positively biased with close proximity to the gene. Furthermore, we provide a novel analysis of data from the ENCODE project, finding a non-uniform distribution of regulatory elements consistent with our in vivo observations at PHOX2B. These observations remain largely unchanged when one accounts for the sequence repeat content of the assayed intervals, when the intervals are sub-classified by biological role (developmental versus non-developmental), or by gene density (gene desert versus non-gene desert). Conclusion While regulatory elements frequently display evidence of evolutionary conservation, a fraction appears to be undetected by current metrics of conservation. In vivo observations at the PHOX2B locus, supported by our analyses of in vitro data from the ENCODE project, suggest that the risk of excluding non-conserved sequences in a search for regulatory elements may decrease as distance from the gene increases. Our data combined with the ENCODE data suggests that this may represent a genome wide trend.

Despite its recognized utility, the extent to which evolutionary sequence conservation-based approaches may systematically overlook functional noncoding sequences remains unclear. We have tiled across sequence encompassing the zebrafish phox2b gene, ultimately evaluating 48 amplicons corresponding to all noncoding sequences therein for enhancer activity in zebrafish. Post hoc analyses of this interval utilizing five commonly used measures of evolutionary constraint (AVID, MLAGAN, SLAGAN, phastCons, WebMCS) demonstrate that each systematically overlooks regulatory sequences. These established algorithms detected only 29%–61% of our identified regulatory elements, consistent with the suggestion that many regulatory sequences may not be readily detected by metrics of sequence constraint. However, we were able to discriminate functional from nonfunctional sequences based upon GC composition and identified position weight matrices (PWM), demonstrating that, in at least one case, deleting sequences containing a subset of these PWMs from one identified regulatory element abrogated its regulatory function. Collectively, these data demonstrate that the noncoding functional component of vertebrate genomes may far exceed estimates predicated on evolutionary constraint.

Inflammatory skin disorders result in significant epidermal changes, including keratinocyte hyperproliferation, incomplete differentiation, and impaired barrier. Here we test whether, conversely, an impaired epidermal barrier can promote an inflammatory response. Mice lacking the transcription factor Kruppel-like factor 4 (Klf4) have a severe defect in epidermal barrier acquisition. Transcription profiling of Klf4(-/-) newborn skin revealed similar changes in gene expression to involved psoriatic plaques, including a significant upregulation of the gap junction protein connexin 26 (Cx26). Ectopic expression of Cx26 from the epidermis-specific involucrin (INV) promoter (INV-Cx26) demonstrated that downregulation of Cx26 is required for barrier acquisition during development. In juvenile and adult mice, persistent Cx26 expression kept wounded epidermis in a hyperproliferative state, blocked the transition to remodeling, and led to an infiltration of immune cells. Mechanistically, ectopic expression of Cx26 in keratinocytes resulted in increased ATP release, which delayed epidermal barrier recovery and promoted an inflammatory response in resident immune cells. These results provide a molecular link between barrier acquisition in utero and epidermal remodeling after wounding. More generally, these studies suggest that the most effective treatments for inflammatory skin disorders might concomitantly suppress the immune response and enhance epidermal differentiation to restore the barrier.

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.