The ERBB3 gene is essential for the proper development of the neural crest (NC) and its derivative populations such as Schwann cells. As with all cell fate decisions, transcriptional regulatory control plays a significant role in the progressive restriction and specification of NC derived lineages during development. However, little is known about the sequences mediating transcriptional regulation of ERBB3 or the factors that bind them.

Innervation of the gut is segmentally lost in Hirschsprung disease (HSCR), a consequence of cell-autonomous and non-autonomous defects in enteric neuronal cell differentiation, proliferation, migration, or survival. Rare, high-penetrance coding variants and common, low-penetrance non-coding variants in 13 genes are known to underlie HSCR risk, with the most frequent variants in the ret proto-oncogene (RET). We used a genome-wide association (220 trios) and replication (429 trios) study to reveal a second non-coding variant distal to RET and a non-coding allele on chromosome 7 within the class 3 Semaphorin gene cluster. Analysis in Ret wild-type and Ret-null mice demonstrates specific expression of Sema3a, Sema3c, and Sema3d in the enteric nervous system (ENS). In zebrafish embryos, sema3 knockdowns show reduction of migratory ENS precursors with complete ablation under conjoint ret loss of function. Seven candidate receptors of Sema3 proteins are also expressed within the mouse ENS and their expression is also lost in the ENS of Ret-null embryos. Sequencing of SEMA3A, SEMA3C, and SEMA3D in 254 HSCR-affected subjects followed by in silico protein structure modeling and functional analyses identified five disease-associated alleles with loss-of-function defects in semaphorin dimerization and binding to their cognate neuropilin and plexin receptors. Thus, semaphorin 3C/3D signaling is an evolutionarily conserved regulator of ENS development whose dys-regulation is a cause of enteric aganglionosis.

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.

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.

Previously, we showed that the evolution of high acuity vision in fishes was directly associated with their unique pH-sensitive hemoglobins that allow O2 to be delivered to the retina at PO2s more than ten-fold that of arterial blood (Damsgaard et al., 2019). Here, we show strong evidence that vacuolar-type H+-ATPase and plasma-accessible carbonic anhydrase in the vascular structure supplying the retina act together to acidify the red blood cell leading to O2 secretion. In vivo data indicate that this pathway primarily affects the oxygenation of the inner retina involved in signal processing and transduction, and that the evolution of this pathway was tightly associated with the morphological expansion of the inner retina. We conclude that this mechanism for retinal oxygenation played a vital role in the adaptive evolution of vision in teleost fishes.

The retina has a very high energy demand but lacks an internal blood supply in most vertebrates. Here we explore the hypothesis that oxygen diffusion limited the evolution of retinal morphology by reconstructing the evolution of retinal thickness and the various mechanisms for retinal oxygen supply, including capillarization and acid-induced haemoglobin oxygen unloading. We show that a common ancestor of bony fishes likely had a thin retina without additional retinal oxygen supply mechanisms and that three different types of retinal capillaries were gained and lost independently multiple times during the radiation of vertebrates, and that these were invariably associated with parallel changes in retinal thickness. Since retinal thickness confers multiple advantages to vision, we propose that insufficient retinal oxygen supply constrained the functional evolution of the eye in early vertebrates, and that recurrent origins of additional retinal oxygen supply mechanisms facilitated the phenotypic evolution of improved functional eye morphology.

The speciose mammalian order Eulipotyphla (moles, shrews, hedgehogs, solenodons) combines an unusual diversity of semi-aquatic, semi-fossorial, and fossorial forms that arose from terrestrial forbearers. However, our understanding of the ecomorphological pathways leading to these lifestyles has been confounded by a fragmentary fossil record, unresolved phylogenetic relationships, and potential morphological convergence, calling for novel approaches. The net surface charge of the oxygen-storing muscle protein myoglobin (ZMb), which can be readily determined from its primary structure, provides an objective target to address this question due to mechanistic linkages with myoglobin concentration. Here, we generate a comprehensive 71 species molecular phylogeny that resolves previously intractable intra-family relationships and then ancestrally reconstruct ZMb evolution to identify ancient lifestyle transitions based on protein sequence alone. Our phylogenetically informed analyses confidently resolve fossorial habits having evolved twice in talpid moles and reveal five independent secondary aquatic transitions in the order housing the world's smallest endothermic divers.

To overcome the ethical and technical limitations of in vivo human disease models, the broader scientific community frequently employs model organism-derived cell lines to investigate of disease mechanisms, pathways, and therapeutic strategies. Despite the widespread use of certain in vitro models, many still lack contemporary genomic analysis supporting their use as a proxy for the affected human cells and tissues. Consequently, it is imperative to determine how accurately and effectively any proposed biological surrogate may reflect the biological processes it is assumed to model. One such cellular surrogate of human disease is the established mouse neural precursor cell line, SN4741, which has been used to elucidate mechanisms of neurotoxicity in Parkinson disease for over 25 years. Here, we are using a combination of classic and contemporary genomic techniques - karyotyping, RT-qPCR, single cell RNA-seq, bulk RNA-seq, and ATAC-seq - to characterize the transcriptional landscape, chromatin landscape, and genomic architecture of this cell line, and evaluate its suitability as a proxy for midbrain dopaminergic neurons in the study of Parkinson disease. We find that SN4741 cells possess an unstable triploidy and consistently exhibits low expression of dopaminergic neuron markers across assays, even when the cell line is shifted to the non-permissive temperature that drives differentiation. The transcriptional signatures of SN4741 cells suggest that they are maintained in an undifferentiated state at the permissive temperature and differentiate into immature neurons at the non-permissive temperature; however, they may not be dopaminergic neuron precursors, as previously suggested. Additionally, the chromatin landscapes of SN4741 cells, in both the differentiated and undifferentiated states, are not concordant with the open chromatin profiles of ex vivo , mouse E15.5 forebrain- or midbrain-derived dopaminergic neurons. Overall, our data suggest that SN4741 cells may reflect early aspects of neuronal differentiation but are likely not a suitable a proxy for dopaminergic neurons as previously thought. The implications of this study extend broadly, illuminating the need for robust biological and genomic rationale underpinning the use of in vitro models of molecular processes.

Several parameters are important when choosing the most appropriate animal to model human obstetrics, including gestation period, number of fetuses per gestation and placental structure. The domesticated long-tailed chinchilla (Chinchilla lanigera) is a well-suited and appropriate animal model of pregnancy that often will carry only one offspring and has a long gestation period of 105-115 days. Furthermore, the chinchilla placenta is of the haemomonochorial labyrinthine type and is therefore comparable to the human villous haemomonochorial placenta. This proof-of-concept study demonstrated the feasibility in laboratory settings, and demonstrated the potential of the pregnant chinchilla as an animal model for obstetric research and its potential usefulness for non-invasive measurements in the placenta. We demonstrate measurements of the placental and fetal metabolism (demonstrated in vivo by hyperpolarized MRI and in vitro by qPCR analyses), placental vessels (demonstrated ex vivo by contrast-enhanced CT angiography) and overall anatomy (demonstrated in vivo by whole-body CT).

Trisomy for human chromosome 21 (Hsa21) results in Down syndrome (DS), one of the most genetically complex conditions compatible with human survival. Assessment of the physiological consequences of dosage-driven overexpression of individual Hsa21 genes during early embryogenesis and the resulting contributions to DS pathology in mammals are not tractable in a systematic way. A recent study looked at loss-of-function of a subset of Caenorhabditis elegans orthologs of Hsa21 genes and identified ten candidates with behavioral phenotypes, but the equivalent over-expression experiment has not been done. We turned to zebrafish as a developmental model and, using a number of surrogate phenotypes, we screened Hsa21 genes for effects on early embyrogenesis. We prepared a library of 164 cDNAs of conserved protein coding genes, injected mRNA into early embryos and evaluated up to 5 days post-fertilization (dpf). Twenty-four genes produced a gross morphological phenotype, 11 of which could be reproduced reliably. Seven of these gave a phenotype consistent with down regulation of the sonic hedgehog (Shh) pathway; two showed defects indicative of defective neural crest migration; one resulted consistently in pericardial edema; and one was embryonic lethal. Combinatorial injections of multiple Hsa21 genes revealed both additive and compensatory effects, supporting the notion that complex genetic relationships underlie end phenotypes of trisomy that produce DS. Together, our data suggest that this system is useful in the genetic dissection of dosage-sensitive gene effects on early development and can inform the contribution of both individual loci and their combinatorial effects to phenotypes relevant to the etiopathology of DS.

In this study, we sought to establish a novel method to prospectively and dynamically identify live human oligodendrocyte precursor cells (OPCs) and oligodendrocyte lineage cells from brain dissociates and pluripotent stem cell culture. We selected a highly conserved enhancer element of the Sox10 gene, known as MCS5, which directs reporter expression to oligodendrocyte lineage cells in mouse and zebrafish. We demonstrate that lentiviral Sox10-MCS5 induced expression of GFP at high levels in a subpopulation of human CD140a/PDGFαR-sorted OPCs as well as their immature oligodendrocyte progeny. Furthermore, we show that almost all Sox10-MCS5:GFP(high) cells expressed OPC antigen CD140a and human OPCs expressing SOX10, OLIG2, and PDGFRA mRNAs could be prospectively identified using GFP based fluorescence activated cells sorting alone. Additionally, we established a human induced pluripotent cell (iPSC) line transduced with the Sox10-MCS5:GFP reporter using a Rex-Neo cassette. Similar to human primary cells, GFP expression was restricted to embryoid bodies containing both oligodendrocyte progenitor and oligodendrocyte cells and co-localized with NG2 and O4-positive cells respectively. As such, we have developed a novel reporter system that can track oligodendrocyte commitment in human cells, establishing a valuable tool to improve our understanding and efficiency of human oligodendrocyte derivation.

We take a comprehensive approach to the study of regulatory control of gene expression in melanocytes that proceeds from large-scale enhancer discovery facilitated by ChIP-seq; to rigorous validation in silico, in vitro, and in vivo; and finally to the use of machine learning to elucidate a regulatory vocabulary with genome-wide predictive power. We identify 2489 putative melanocyte enhancer loci in the mouse genome by ChIP-seq for EP300 and H3K4me1. We demonstrate that these putative enhancers are evolutionarily constrained, enriched for sequence motifs predicted to bind key melanocyte transcription factors, located near genes relevant to melanocyte biology, and capable of driving reporter gene expression in melanocytes in culture (86%; 43/50) and in transgenic zebrafish (70%; 7/10). Next, using the sequences of these putative enhancers as a training set for a supervised machine learning algorithm, we develop a vocabulary of 6-mers predictive of melanocyte enhancer function. Lastly, we demonstrate that this vocabulary has genome-wide predictive power in both the mouse and human genomes. This study provides deep insight into the regulation of gene expression in melanocytes and demonstrates a powerful approach to the investigation of regulatory sequences that can be applied to other cell types.

Elevated transforming growth factor (TGF)-β signaling has been implicated in the pathogenesis of syndromic presentations of aortic aneurysm, including Marfan syndrome (MFS) and Loeys-Dietz syndrome (LDS). However, the location and character of many of the causal mutations in LDS intuitively imply diminished TGF-β signaling. Taken together, these data have engendered controversy regarding the specific role of TGF-β in disease pathogenesis. Shprintzen-Goldberg syndrome (SGS) has considerable phenotypic overlap with MFS and LDS, including aortic aneurysm. We identified causative variation in ten individuals with SGS in the proto-oncogene SKI, a known repressor of TGF-β activity. Cultured dermal fibroblasts from affected individuals showed enhanced activation of TGF-β signaling cascades and higher expression of TGF-β-responsive genes relative to control cells. Morpholino-induced silencing of SKI paralogs in zebrafish recapitulated abnormalities seen in humans with SGS. These data support the conclusions that increased TGF-β signaling is the mechanism underlying SGS and that high signaling contributes to multiple syndromic presentations of aortic aneurysm.

Sox10 is a dynamically regulated transcription factor gene that is essential for the development of neural crest-derived and oligodendroglial populations. Developmental genes often require multiple regulatory sequences that integrate discrete and overlapping functions to coordinate their expression. To identify Sox10 cis-regulatory elements, we integrated multiple model systems, including cell-based screens and transposon-mediated transgensis in zebrafish, to scrutinize mammalian conserved, noncoding genomic segments at the mouse Sox10 locus. We demonstrate that eight of 11 Sox10 genomic elements direct reporter gene expression in transgenic zebrafish similar to patterns observed in transgenic mice, despite an absence of observable sequence conservation between mice and zebrafish. Multiple segments direct expression in overlapping populations of neural crest derivatives and glial cells, ranging from pan-Sox10 and pan-neural crest regulatory control to the modulation of expression in subpopulations of Sox10-expressing cells, including developing melanocytes and Schwann cells. Several sequences demonstrate overlapping spatial control, yet direct expression in incompletely overlapping developmental intervals. We were able to partially explain neural crest expression patterns by the presence of head to head SoxE family binding sites within two of the elements. Moreover, we were able to use this transcription factor binding site signature to identify the corresponding zebrafish enhancers in the absence of overall sequence homology. We demonstrate the utility of zebrafish transgenesis as a high-fidelity surrogate in the dissection of mammalian gene regulation, especially those with dynamically controlled developmental expression.

Haploid cell lines are a valuable research tool with broad applicability for genetic assays. As such the fully haploid human cell line, eHAP1, has been used in a wide array of studies. However, the absence of a corresponding reference genome sequence for this cell line has limited the potential for more widespread applications to experiments dependent on available sequence, like capture-clone methodologies. We generated ~15× coverage Nanopore long reads from ten GridION flowcells and utilized this data to assemble a de novo draft genome using minimap and miniasm and subsequently polished using Racon. This assembly was further polished using previously generated, low-coverage, Illumina short reads with Pilon and ntEdit. This resulted in a hybrid eHAP1 assembly with >90% complete BUSCO scores. We further assessed the eHAP1 long read data for structural variants using Sniffles and identify a variety of rearrangements, including a previously established Philadelphia translocation. Finally, we demonstrate how some of these variants overlap open chromatin regions, potentially impacting regulatory regions. By integrating both long and short reads, we generated a high-quality reference assembly for eHAP1 cells. The union of long and short reads demonstrates the utility in combining sequencing platforms to generate a high-quality reference genome de novo solely from low coverage data. We expect the resulting eHAP1 genome assembly to provide a useful resource to enable novel experimental applications in this important model cell line.

Clinically pertinent electrocardiogram (ECG) data from model systems, such as zebrafish, are crucial for illuminating factors contributing to human cardiac electrophysiological abnormalities and disease. Current zebrafish ECG collection strategies have not adequately addressed the consistent acquisition of high-quality traces or sources of phenotypic variation that could obscure data interpretation. Thus, we developed a novel platform to ensure high-quality recording of in vivo subdermal adult zebrafish ECGs and zebrafish ECG reading GUI (zERG), a program to acquire measurements from traces that commercial software cannot examine owing to erroneous peak calling. We evaluate normal ECG trait variation, revealing highly reproducible intervals and wave amplitude variation largely driven by recording artifacts, and identify sex and body size as potential confounders to PR, QRS and QT intervals. With this framework, we characterize the effect of the class I anti-arrhythmic drug flecainide acetate on adults, provide support for the impact of a Long QT syndrome model, and establish power calculations for this and other studies. These results highlight our pipeline as a robust approach to evaluate zebrafish models of human cardiac electrophysiological phenotypes.

Bicuspid aortic valve (BAV) is a common congenital heart defect (population incidence, 1-2%)1-3 that frequently presents with ascending aortic aneurysm (AscAA)4. BAV/AscAA shows autosomal dominant inheritance with incomplete penetrance and male predominance. Causative gene mutations (for example, NOTCH1, SMAD6) are known for ≤1% of nonsyndromic BAV cases with and without AscAA5-8, impeding mechanistic insight and development of therapeutic strategies. Here, we report the identification of variants in ROBO4 (which encodes a factor known to contribute to endothelial performance) that segregate with disease in two families. Targeted sequencing of ROBO4 showed enrichment for rare variants in BAV/AscAA probands compared with controls. Targeted silencing of ROBO4 or mutant ROBO4 expression in endothelial cell lines results in impaired barrier function and a synthetic repertoire suggestive of endothelial-to-mesenchymal transition. This is consistent with BAV/AscAA-associated findings in patients and in animal models deficient for ROBO4. These data identify a novel endothelial etiology for this common human disease phenotype.

Comparative anatomy and physiology are disciplines related to structures and mechanisms in three-dimensional (3D) space. For the past centuries, scientific reports in these fields have relied on written descriptions and two-dimensional (2D) illustrations, but in recent years 3D virtual modelling has entered the scene. However, comprehending complex anatomical structures is hampered by reproduction on flat inherently 2D screens. One way to circumvent this problem is in the production of 3D-printed scale models. We have applied computed tomography and magnetic resonance imaging to produce digital models of animal anatomy well suited to be printed on low-cost 3D printers. In this communication, we report how to apply such technology in comparative anatomy and physiology to aid discovery, description, comprehension and communication, and we seek to inspire fellow researchers in these fields to embrace this emerging technology.

Vertebrate sensory organs originate from both cranial neural crest cells (CNCCs) and placodes. Previously, we have shown that the olfactory placode (OP) forms from a large field of cells extending caudally to the premigratory neural crest domain, and that OPs form through cell movements and not cell division. Concurrent with OP formation, CNCCs migrate rostrally to populate the frontal mass. However, little is known about the interactions between CNCCs and the placodes that form the olfactory sensory system. Previous reports suggest that the OP can generate cell types more typical of neural crest lineages such as neuroendocrine cells and glia, thus marking the OP as an unusual sensory placode. One possible explanation for this exception is that the neural crest origin of glia and neurons has been overlooked due to the intimate association of these two fields during migration. Using molecular markers and live imaging, we followed the development of OP precursors and of dorsally migrating CNCCs in zebrafish embryos. We generated a six4b:mCherry line (OP precursors) that, with a sox10:EGFP line (CNCCs), was used to follow cell migration. Our analyses showed that CNCCs associate with and eventually surround the forming OP with limited cell mixing occurring during this process.

Genetic variation modulating risk of sporadic Parkinson disease (PD) has been primarily explored through genome-wide association studies (GWASs). However, like many other common genetic diseases, the impacted genes remain largely unknown. Here, we used single-cell RNA-seq to characterize dopaminergic (DA) neuron populations in the mouse brain at embryonic and early postnatal time points. These data facilitated unbiased identification of DA neuron subpopulations through their unique transcriptional profiles, including a postnatal neuroblast population and substantia nigra (SN) DA neurons. We use these population-specific data to develop a scoring system to prioritize candidate genes in all 49 GWAS intervals implicated in PD risk, including genes with known PD associations and many with extensive supporting literature. As proof of principle, we confirm that the nigrostriatal pathway is compromised in Cplx1-null mice. Ultimately, this systematic approach establishes biologically pertinent candidates and testable hypotheses for sporadic PD, informing a new era of PD genetic research.