PLAE Web App Enables Powerful Searching and Multiple Visualizations Across One Million Unified Single-Cell Ocular Transcriptomes.

Purpose: To create a high-performance reactive web application to query single-cell gene expression data across cell type, species, study, and other factors. Methods: We updated the content and structure of the underlying data (single cell Eye in a Disk [scEiaD]) and wrote the web application PLAE (https://plae.nei.nih.gov) to visualize and explore the data. Results: The new portal provides quick visualization of over a million individual cells from vertebrate eye and body transcriptomes encompassing four species, 60 cell types, six ocular tissues, and 23 body tissues across 35 publications. To demonstrate the value of this unified pan-eye dataset, we replicated known neurogenic and cone macula markers in addition to proposing six new cone human region markers. Conclusions: The PLAE web application offers the eye community a powerful and quick means to test hypotheses related to gene expression across a highly diverse, community-derived database. Translational Relevance: The PLAE resource enables any researcher or clinician to study and research gene expression patterning across a wide variety of curated ocular cell types with a responsive web app.

Transcriptional mapping of the macaque retina and RPE-choroid reveals conserved inter-tissue transcription drivers and signaling pathways.

The macula and fovea comprise a highly sensitive visual detection tissue that is susceptible to common disease processes like age-related macular degeneration (AMD). Our understanding of the molecular determinants of high acuity vision remains unclear, as few model organisms possess a human-like fovea. We explore transcription factor networks and receptor-ligand interactions to elucidate tissue interactions in the macula and peripheral retina and concomitant changes in the underlying retinal pigment epithelium (RPE)/choroid. Poly-A selected, 100 bp paired-end RNA-sequencing (RNA-seq) was performed across the macular/foveal, perimacular, and temporal peripheral regions of the neural retina and RPE/choroid tissues of four adult Rhesus macaque eyes to characterize region- and tissue-specific gene expression. RNA-seq reads were mapped to both the macaque and human genomes for maximum alignment and analyzed for differential expression and Gene Ontology (GO) enrichment. Comparison of the neural retina and RPE/choroid tissues indicated distinct, contiguously changing gene expression profiles from fovea through perimacula to periphery. Top GO enrichment of differentially expressed genes in the RPE/choroid included cell junction organization and epithelial cell development. Expression of transcriptional regulators and various disease-associated genes show distinct location-specific preference and retina-RPE/choroid tissue-tissue interactions. Regional gene expression changes in the macaque retina and RPE/choroid is greater than that found in previously published transcriptome analysis of the human retina and RPE/choroid. Further, conservation of human macula-specific transcription factor profiles and gene expression in macaque tissues suggest a conservation of programs required for retina and RPE/choroid function and disease susceptibility.

Zfp503/Nlz2 Is Required for RPE Differentiation and Optic Fissure Closure.

Purpose: Uveal coloboma is a congenital eye malformation caused by failure of the optic fissure to close in early human development. Despite significant progress in identifying genes whose regulation is important for executing this closure, mutations are detected in a minority of cases using known gene panels, implying additional genetic complexity. We have previously shown knockdown of znf503 (the ortholog of mouse Zfp503) in zebrafish causes coloboma. Here we characterize Zfp503 knockout (KO) mice and evaluate transcriptomic profiling of mutant versus wild-type (WT) retinal pigment epithelium (RPE)/choroid. Methods: Zfp503 KO mice were generated by gene targeting using homologous recombination. Embryos were characterized grossly and histologically. Patterns and level of developmentally relevant proteins/genes were examined with immunostaining/in situ hybridization. The transcriptomic profile of E11.5 KO RPE/choroid was compared to that of WT. Results: Zfp503 is dynamically expressed in developing mouse eyes, and loss of its expression results in uveal coloboma. KO embryos exhibit altered mRNA levels and expression patterns of several key transcription factors involved in eye development, including Otx2, Mitf, Pax6, Pax2, Vax1, and Vax2, resulting in a failure to maintain the presumptive RPE, as evidenced by reduced melanin pigmentation and its differentiation into a neural retina-like lineage. Comparison of RNA sequencing data from WT and KO E11.5 embryos demonstrated reduced expression of melanin-related genes and significant overlap with genes known to be dynamically regulated at the optic fissure. Conclusions: These results demonstrate a critical role of Zfp503 in maintaining RPE fate and optic fissure closure.

Cell-autonomous lipid-handling defects in Stargardt iPSC-derived retinal pigment epithelium cells.

Stargardt retinopathy is an inherited form of macular degeneration caused by mutations in gene ABCA4 and characterized by the accumulation of lipid-rich deposits in the retinal pigment epithelium (RPE), RPE atrophy, and photoreceptor cell death. Inadequate mechanistic insights into pathophysiological changes occurring in Stargardt RPE have hindered disease treatments. Here, we show that ABCA4 knockout and induced pluripotent stem cell-derived RPE (STGD1-iRPE) from patients with Stargardt differentiate normally but display intracellular lipid and ceramide deposits reminiscent of the disease phenotype. STGD1-iRPE also shows defective photoreceptor outer segment (POS) processing and reduced cathepsin B activity-indicating higher lysosomal pH. Lipid deposits in STGD1-iRPE are lowered by increasing the activity of ABCA1, a lipid transporter, and ABCA4 ortholog. Our work suggests that ABCA4 is involved in POS and lipid handling in RPE cells and provides guidance for ongoing gene therapy approaches to target both RPE and photoreceptor cells for an effective treatment.

Epithelial phenotype restoring drugs suppress macular degeneration phenotypes in an iPSC model.

Age-related Macular Degeneration (AMD), a blinding eye disease, is characterized by pathological protein- and lipid-rich drusen deposits underneath the retinal pigment epithelium (RPE) and atrophy of the RPE monolayer in advanced disease stages - leading to photoreceptor cell death and vision loss. Currently, there are no drugs that stop drusen formation or RPE atrophy in AMD. Here we provide an iPSC-RPE AMD model that recapitulates drusen and RPE atrophy. Drusen deposition is dependent on AMD-risk-allele CFH(H/H) and anaphylatoxin triggered alternate complement signaling via the activation of NF-κB and downregulation of autophagy pathways. Through high-throughput screening we identify two drugs, L-745,870, a dopamine receptor antagonist, and aminocaproic acid, a protease inhibitor that reduce drusen deposits and restore RPE epithelial phenotype in anaphylatoxin challenged iPSC-RPE with or without the CFH(H/H) genotype. This comprehensive iPSC-RPE model replicates key AMD phenotypes, provides molecular insight into the role of CFH(H/H) risk-allele in AMD, and discovers two candidate drugs to treat AMD.

Building the mega single-cell transcriptome ocular meta-atlas.

BACKGROUND: The development of highly scalable single-cell transcriptome technology has resulted in the creation of thousands of datasets, >30 in the retina alone. Analyzing the transcriptomes between different projects is highly desirable because this would allow for better assessment of which biological effects are consistent across independent studies. However it is difficult to compare and contrast data across different projects because there are substantial batch effects from computational processing, single-cell technology utilized, and the natural biological variation. While many single-cell transcriptome-specific batch correction methods purport to remove the technical noise, it is difficult to ascertain which method functions best. RESULTS: We developed a lightweight R package (scPOP, single-cell Pick Optimal Parameters) that brings in batch integration methods and uses a simple heuristic to balance batch merging and cell type/cluster purity. We use this package along with a Snakefile-based workflow system to demonstrate how to optimally merge 766,615 cells from 33 retina datsets and 3 species to create a massive ocular single-cell transcriptome meta-atlas. CONCLUSIONS: This provides a model for how to efficiently create meta-atlases for tissues and cells of interest.

North Carolina Macular Dystrophy: Phenotypic Variability and Computational Analysis of Disease-Associated Noncoding Variants.

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.

Loss of the Vitamin B-12 Transport Protein Tcn2 Results in Maternally Inherited Growth and Developmental Defects in Zebrafish.

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 ß-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.

Eye in a Disk: eyeIntegration Human Pan-Eye and Body Transcriptome Database Version 1.0.

Purpose: We develop an accessible and reliable RNA sequencing (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 sequence data for 1375 RNA-seq samples across 54 tissues in the Genotype-Tissue Expression (GTEx) project as a noneye reference set. We then queried several public repositories to find all healthy, nonperturbed, 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, perform 882 differential expression tests, calculate GO term enrichment, and output all as a single SQLite database file: the Eye in a Disk (EiaD) dataset. Furthermore, we rewrote the web application eyeIntegration (available in the public domain at https://eyeIntegration.nei.nih.gov) to display EiaD. Results: The 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, such as protocadherin and HOXB family members. Conclusions: The 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.

Clinical-grade stem cell-derived retinal pigment epithelium patch rescues retinal degeneration in rodents and pigs.

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.

Functional and phylogenetic characterization of noncanonical vitamin B12-binding proteins in zebrafish suggests involvement in cobalamin transport.

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 ß-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 ß-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.

Identifying core biological processes distinguishing human eye tissues with precise systems-level gene expression analyses and weighted correlation networks.

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 with 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, retinal pigment epithelium (RPE)-choroid complex, and the rest of the human tissues with differential expression, clustering and gene ontology 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/).

A Common Polymorphism in HIBCH Influences Methylmalonic Acid Concentrations in Blood Independently of Cobalamin.

Methylmalonic acid (MMA) is a by-product of propionic acid metabolism through the vitamin B12 (cobalamin)-dependent enzyme methylmalonyl CoA mutase. Elevated MMA concentrations are a hallmark of several inborn errors of metabolism and indicators of cobalamin deficiency in older persons. In a genome-wide analysis of 2,210 healthy young Irish adults (median age 22 years) we identified a strong association of plasma MMA with SNPs in 3-hydroxyisobutyryl-CoA hydrolase (HIBCH, p = 8.42 × 10(-89)) and acyl-CoA synthetase family member 3 (ACSF3, p = 3.48 × 10(-19)). These loci accounted for 12% of the variance in MMA concentration. The most strongly associated SNP (HIBCH rs291466; c:2T>C) causes a missense change of the initiator methionine codon (minor-allele frequency = 0.43) to threonine. Surprisingly, the resulting variant, p.Met1?, is associated with increased expression of HIBCH mRNA and encoded protein. These homozygotes had, on average, 46% higher MMA concentrations than methionine-encoding homozygotes in young adults with generally low MMA concentrations (0.17 [0.14-0.21] µmol/L; median [25(th)-75(th) quartile]). The association between MMA levels and HIBCH rs291466 was highly significant in a replication cohort of 1,481 older individuals (median age 79 years) with elevated plasma MMA concentrations (0.34 [0.24-0.51] µmol/L; p = 4.0 × 10(-26)). In a longitudinal study of 185 pregnant women and their newborns, the association of this SNP remained significant across the gestational trimesters and in newborns. HIBCH is unique to valine catabolism. Studies evaluating flux through the valine catabolic pathway in humans should account for these variants. Furthermore, this SNP could help resolve equivocal clinical tests where plasma MMA values have been used to diagnose cobalamin deficiency.

Functional variants in DPYSL2 sequence increase risk of schizophrenia and suggest a link to mTOR signaling.

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.

Genomics of CpG methylation in developing and developed zebrafish.

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.

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.

Steroid hormone modulation of RET through two estrogen responsive enhancers in breast cancer.

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.

Genome-wide identification of conserved regulatory function in diverged sequences.

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."

Efficient discovery of ASCL1 regulatory sequences through transgene pooling.

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.

Epistasis between RET and BBS mutations modulates enteric innervation and causes syndromic Hirschsprung disease.

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.