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De novo heterozygous variants in the brain-specific transcription factor Neuronal Differentiation Factor 2 (NEUROD2) have been recently associated with early-onset epileptic encephalopathy and developmental delay. Here, we report an adolescent with developmental delay without seizures who was found to have a novel de novo heterozygous NEUROD2 missense variant, p.(Leu163Pro). Functional testing using an in vivo assay of neuronal differentiation in Xenopus laevis tadpoles demonstrated that the patient variant of NEUROD2 displays minimal protein activity, strongly suggesting a loss of function effect. In contrast, a second rare NEUROD2 variant, p.(Ala235Thr), identified in an adolescent with developmental delay but lacking parental studies for inheritance, showed normal in vivo NEUROD2 activity. We thus provide clinical, genetic, and functional evidence that NEUROD2 variants can lead to developmental delay without accompanying early-onset seizures, and demonstrate how functional testing can complement genetic data when determining variant pathogenicity.

Recessive variants in the GLDN gene, which encodes the gliomedin protein and is involved in nervous system development, have recently been associated with Arthrogryposis Multiplex Congenita (AMC), a heterogenous condition characterized by congenital contractures of more than one joint. Two cohorts of patients with GLDN-associated AMC have previously been described, evolving the understanding of the condition from lethal to survivable with the provision of significant neonatal support. Here, we describe one additional patient currently living with the syndrome, having one novel variant, p.Leu365Phe, for which we provide functional data supporting its pathogenicity. We additionally provide experimental data for four other previously reported variants lacking functional evidence, including p.Arg393Lys, the second variant present in our patient. We discuss unique and defining clinical features, adding calcium-related findings which appear to be recurrent in the GLDN cohort. Finally, we compare all previously reported patients and draw new conclusions about scope of illness, with emphasis on the finding of pulmonary hypoplasia, suggesting that AMC secondary to GLDN variants may be best fitted under the umbrella of fetal akinesia deformation sequence (FADS).

BackgroundCilia are dynamic cellular extensions that generate and sense signals to orchestrate proper development and tissue homeostasis. They rely on the underlying polarisation of cells to participate in signalling. Cilia dysfunction is a well-known cause of several diseases that affect multiple organ systems including the kidneys, brain, heart, respiratory tract, skeleton and retina.MethodsAmong individuals from four unrelated families, we identified variants in discs large 5 (DLG5) that manifested in a variety of pathologies. In our proband, we also examined patient tissues. We depleted dlg5 in Xenopus tropicalis frog embryos to generate a loss-of-function model. Finally, we tested the pathogenicity of DLG5 patient variants through rescue experiments in the frog model.ResultsPatients with variants of DLG5 were found to have a variety of phenotypes including cystic kidneys, nephrotic syndrome, hydrocephalus, limb abnormalities, congenital heart disease and craniofacial malformations. We also observed a loss of cilia in cystic kidney tissue of our proband. Knockdown of dlg5 in Xenopus embryos recapitulated many of these phenotypes and resulted in a loss of cilia in multiple tissues. Unlike introduction of wildtype DLG5 in frog embryos depleted of dlg5, introduction of DLG5 patient variants was largely ineffective in restoring proper ciliation and tissue morphology in the kidney and brain suggesting that the variants were indeed detrimental to function.ConclusionThese findings in both patient tissues and Xenopus shed light on how mutations in DLG5 may lead to tissue-specific manifestations of disease. DLG5 is essential for cilia and many of the patient phenotypes are in the ciliopathy spectrum.

Multipass membrane proteins have a myriad of functions, including transduction of cell-cell signals, ion transport, and photoreception. Insertion of these proteins into the membrane depends on the endoplasmic reticulum (ER) membrane protein complex (EMC). Recently, birth defects have been observed in patients with variants in the gene encoding a member of this complex, EMC1. Patient phenotypes include congenital heart disease, craniofacial malformations, and neurodevelopmental disease. However, a molecular connection between EMC1 and these birth defects is lacking. Using Xenopus, we identified defects in neural crest cells (NCCs) upon emc1 depletion. We then used unbiased proteomics and discovered a critical role for emc1 in WNT signaling. Consistent with this, readouts of WNT signaling and Frizzled (Fzd) levels were reduced in emc1-depleted embryos, while NCC defects could be rescued with β-catenin. Interestingly, other transmembrane proteins were mislocalized upon emc1 depletion, providing insight into additional patient phenotypes. To translate our findings back to humans, we found that EMC1 was necessary for human NCC development in vitro. Finally, we tested patient variants in our Xenopus model and found the majority to be loss-of-function alleles. Our findings define molecular mechanisms whereby EMC1 dysfunction causes disease phenotypes through dysfunctional multipass membrane protein topogenesis.

Microinjection is an important technique used to study development in the oocyte and early embryo. In

Recessive variants in the GLDN gene, which encodes the gliomedin protein and is involved in nervous system development, have recently been associated with Arthrogryposis Multiplex Congenita (AMC), a heterogenous condition characterized by congenital contractures of more than one joint. Two cohorts of patients with GLDN-associated AMC have previously been described, evolving the understanding of the condition from lethal to survivable with the provision of significant neonatal support. Here, we describe one additional patient currently living with the syndrome, having one novel variant, p.Leu365Phe, for which we provide functional data supporting its pathogenicity. We additionally provide experimental data for four other previously reported variants lacking functional evidence, including p.Arg393Lys, the second variant present in our patient. We discuss unique and defining clinical features, adding calcium-related findings which appear to be recurrent in the GLDN cohort. Finally, we compare all previously reported patients and draw new conclusions about scope of illness, with emphasis on the finding of pulmonary hypoplasia, suggesting that AMC secondary to GLDN variants may be best fitted under the umbrella of fetal akinesia deformation sequence (FADS).

Cpf1 is a novel class of CRISPR-Cas DNA endonucleases, with a wide range of activity across different eukaryotic systems. Yet, the underlying determinants of this variability are poorly understood. Here, we demonstrate that LbCpf1, but not AsCpf1, ribonucleoprotein complexes allow efficient mutagenesis in zebrafish and Xenopus. We show that temperature modulates Cpf1 activity by controlling its ability to access genomic DNA. This effect is stronger on AsCpf1, explaining its lower efficiency in ectothermic organisms. We capitalize on this property to show that temporal control of the temperature allows post-translational modulation of Cpf1-mediated genome editing. Finally, we determine that LbCpf1 significantly increases homology-directed repair in zebrafish, improving current approaches for targeted DNA integration in the genome. Together, we provide a molecular understanding of Cpf1 activity in vivo and establish Cpf1 as an efficient and inducible genome engineering tool across ectothermic species., Cpf1 is a promising addition to the CRISPR toolkit but displays wide variability of activity in different eurkaryotes. Here the authors identify temperature as a modulator of activity and use this to efficiently edit ectothermic vertebrate species.

Microinjection is an important technique used to study development in the oocyte and early embryo. In Xenopus, substances such as DNA, mRNA, and morpholino oligonucleotides have traditionally been injected into Xenopus laevis, because of their large embryo size and the relatively long time from their fertilization to first division. In the past few decades, Xenopus tropicalis has become an important model in developmental biology; it is particularly useful in genetic studies. The advent and rapid development of CRISPR–Cas9 technology has provided an array of targeted gene manipulations for which X. tropicalis is particularly suited. The equipment and protocol for X. tropicalis microinjection is broadly transferable from X. laevis. There are important differences between the species to consider, however, including the smaller embryo size and faster embryo development time in X. tropicalis. There are a number of solutions and reagents that differ in concentration and composition as well. Here we describe a microinjection protocol specifically for studies in X. tropicalis.

Mucociliary flow is an important defense mechanism in the lung to remove inhaled pathogens and pollutants. Disruption of ciliary flow can lead to respiratory infections. Multiple factors, from drugs to disease can cause an alteration in ciliary flow. However, less attention has been given to injury of the ciliated epithelium. In this study, we show how optical coherence tomography (OCT) can be used to investigate injury to the ciliated epithelium in a multi-contrast setting. We used particle tracking velocimetry (PTV-OCT) to investigate the cilia-driven flow field and 3D speckle variance imaging to investigate size and extent of injury caused to the skin of Xenopus embryos. Two types of injuries are investigated, focal injury caused by mechanical damage and diffuse injury by a calcium chloride shock. We additionally investigate injury and regeneration of cilia to calcium chloride on ex vivo mouse trachea. This work describes how OCT can be used as a tool to investigate injury and regeneration in ciliated epithelium.

Optical coherence tomography (OCT) imaging can be used to visualize craniocardiac structures in the Xenopus model system. OCT is analogous to ultrasound, utilizing light instead of sound to create a gray-scale image from the echo time delay of infrared light reflected from the specimen. OCT is a high-speed, cross-sectional, label-free imaging modality, which can outline dynamic in vivo morphology at resolutions approaching histological detail. OCT imaging can acquire 2D and 3D data in real time to assess cardiac and facial structures. Additionally, during cardiac imaging, Doppler imaging can be used to assess the blood flow pattern in relation to the intracardiac structures. Importantly, OCT can reproducibly and efficiently provide comprehensive, nondestructive in vivo cardiac and facial phenotyping. Tadpoles do not require preprocessing and thus can be further raised or analyzed after brief immobilization during imaging. The rapid development of the Xenopus model combined with a rapid OCT imaging protocol allows the identification of specific gene/teratogen phenotype relationships in a short period of time. Loss- or gain-of-function experiments can be evaluated in 4–5 d, and OCT imaging only requires ∼5 min per tadpole. Thus, we find this pairing an efficient workflow for screening numerous candidate genes derived from human genomic studies to in-depth mechanistic studies.

Xenopus is a powerful model system for cell and developmental biology in part because frogs produce thousands of eggs and embryos year-round. For cell biological studies, egg extracts can mimic many processes in a cell-free system. For developmental biology, Xenopus embryos are a premier system, combining cut-and-paste embryology with modern gene manipulation tools. Xenopus tropicalis are particularly suited to genetic studies because of their diploid genome, as compared to the tetraploid genome of Xenopus laevis. When collecting eggs, there are differences in timing of steps, amounts of hormone administered, and handling of females between these species. In this protocol, X. tropicalis females are induced with a hormone that stimulates ovulation, and then eggs are collected. To administer the ovulation hormone and express eggs, it is necessary to be comfortable with handling frogs. Proficient handling of X. tropicalis requires practice, as they are relatively small, active, and slippery.

In the US and Europe, birth defects are the leading cause of infant mortality. Among birth defects, Congenital Heart Disease (CHD) occurs in approximately 8 out of 1000 live births, affects 1.3 million newborns per year worldwide, and has the highest mortality rate. While there is evidence to indicate that CHD does have a genetic basis, most of the CHD burden remains unexplained genetically. Fortunately, new genomics technologies are enabling genetic analyses of CHD patients. Whole exome sequencing of trios as well as copy number variations assayed by high-density SNP arrays can now be obtained at high efficiency and relatively low cost. These efforts are identifying a number of sequence variations in patients with CHD, but only a small percentage have second unrelated alleles to validate them as disease causing. Importantly, most of these candidate genes do not have an identified molecular mechanism implicating them in cardiac development. Therefore, there is a pressing need to develop rapid functional assays to evaluate candidate genes for a role in cardiac development, and then to investigate the underlying developmental mechanisms. Most recently, the advent of CRISPR/Cas9 genome editing technology has greatly enhanced the ability to manipulate and observe the function of the genome in model systems and cell culture. Incorporating the power of a developmental system such as Xenopus tropicalis with the CRISPR/Cas9 system and the microscale imaging modality optical coherence tomography (OCT), the analysis of thousands of different genes in cardiac development becomes possible.

BackgroundEarly infantile epileptic encephalopathies are severe disorders consisting of early-onset refractory seizures accompanied often by significant developmental delay. The increasing availability of next-generation sequencing has facilitated the recognition of single gene mutations as an underlying aetiology of some forms of early infantile epileptic encephalopathies.ObjectivesThis study was designed to identify candidate genes as a potential cause of early infantile epileptic encephalopathy, and then to provide genetic and functional evidence supporting patient variants as causative.MethodsWe used whole exome sequencing to identify candidate genes. To model the disease and assess the functional effects of patient variants on candidate protein function, we used in vivo CRISPR/Cas9-mediated genome editing and protein overexpression in frog tadpoles.ResultsWe identified novel de novo variants in neuronal differentiation factor 2 (NEUROD2) in two unrelated children with early infantile epileptic encephalopathy. Depleting neurod2 with CRISPR/Cas9-mediated genome editing induced spontaneous seizures in tadpoles, mimicking the patients’ condition. Overexpression of wild-type NEUROD2 induced ectopic neurons in tadpoles; however, patient variants were markedly less effective, suggesting that both variants are dysfunctional and likely pathogenic.ConclusionThis study provides clinical and functional support for NEUROD2 variants as a cause of early infantile epileptic encephalopathy, the first evidence of human disease caused by NEUROD2 variants.

In the US and Europe, birth defects are the leading cause of infant mortality. Among birth defects, Congenital Heart Disease (CHD) occurs in approximately 8 out of 1000 live births, affects 1.3 million newborns per year worldwide, and has the highest mortality rate. While there is evidence to indicate that CHD does have a genetic basis, most of the CHD burden remains unexplained genetically. Fortunately, new genomics technologies are enabling genetic analyses of CHD patients. Whole exome sequencing of trios as well as copy number variations assayed by high-density SNP arrays can now be obtained at high efficiency and relatively low cost. These efforts are identifying a number of sequence variations in patients with CHD, but only a small percentage have second unrelated alleles to validate them as disease causing. Importantly, most of these candidate genes do not have an identified molecular mechanism implicating them in cardiac development. Therefore, there is a pressing need to develop rapid functional assays to evaluate candidate genes for a role in cardiac development, and then to investigate the underlying developmental mechanisms. Most recently, the advent of CRISPR/Cas9 genome editing technology has greatly enhanced the ability to manipulate and observe the function of the genome in model systems and cell culture. Incorporating the power of a developmental system such as Xenopus tropicalis with the CRISPR/Cas9 system and the microscale imaging modality optical coherence tomography (OCT), the analysis of thousands of different genes in cardiac development becomes possible.

CRISPR-Cas9 technology provides a powerful system for genome engineering. However, variable activity across different single guide RNAs (sgRNAs) remains a significant limitation. We analyzed the molecular features that influence sgRNA stability, activity and loading into Cas9 in vivo. We observed that guanine enrichment and adenine depletion increased sgRNA stability and activity, whereas differential sgRNA loading, nucleosome positioning and Cas9 off-target binding were not major determinants. We also identified sgRNAs truncated by one or two nucleotides and containing 5' mismatches as efficient alternatives to canonical sgRNAs. On the basis of these results, we created a predictive sgRNA-scoring algorithm, CRISPRscan, that effectively captures the sequence features affecting the activity of CRISPR-Cas9 in vivo. Finally, we show that targeting Cas9 to the germ line using a Cas9-nanos 3' UTR led to the generation of maternal-zygotic mutants, as well as increased viability and decreased somatic mutations. These results identify determinants that influence Cas9 activity and provide a framework for the design of highly efficient sgRNAs for genome targeting in vivo.

Premature infants are at a high risk for respiratory diseases owing to an underdeveloped respiratory system that is very susceptible to infection and inflammation. One aspect of respiratory health is the state of the ciliated respiratory epithelium which lines the trachea and bronchi. The ciliated epithelium is responsible for trapping and removing pathogens and pollutants from the lungs and an impairment of ciliary functionality can lead to recurring respiratory infections and subsequent lung damage. Mechanisms of cilia-driven fluid flow itself but also factors influenced by development like ciliary density and flow generation are incompletely understood. Furthermore, medical interventions like intubation and accidental aspiration can lead to focal or diffuse loss of cilia and disruption of flow. In this study we use two animal models, Xenopus embryo and ex vivo mouse trachea, to analyze flow defects in the injured ciliated epithelium. Injury is generated either mechanically with a scalpel or chemically by calcium chloride (CaCl2) shock, which efficiently but reversibly deciliates the embryo skin. In this study we used optical coherence tomography (OCT) and particle tracking velocimetry (PTV) to quantify cilia driven fluid flow over the surface of the Xenopus embryo. We additionally visualized damage to the ciliated epithelium by capturing 3D speckle variance images that highlight beating cilia. Mechanical injury disrupted cilia-driven fluid flow over the injured site, which led to a reduction in cilia-driven fluid flow over the whole surface of the embryo (n=7). The calcium chloride shock protocol proved to be highly effective in deciliating embryos (n=6). 3D speckle variance images visualized a loss of cilia and cilia-driven flow was halted immediately after application. We also applied CaCl2-shock to cultured ex vivo mouse trachea (n=8) and found, similarly to effects in Xenopus embryo, an extensive loss of cilia with resulting cessation of flow. We investigated the regeneration of the ciliated epithelium after an 8 day incubation period, and found that cilia had regrown and flow was completely restored. In conclusion, OCT is a valuable tool to visualize injury of the ciliated epithelium and to quantify reduction of generated flow. This method allows for systematic investigation of focal and diffuse injury of the ciliated epithelium and the assessment of mechanisms to compensate for loss of flow.

We developed ultra-high-speed, phase-sensitive, full-field reflection interferometric confocal microscopy (FFICM) for the quantitative characterization of in vivo microscale biological motions and flows. We demonstrated 2D frame rates in excess of 1 kHz and pixel throughput rates up to 125 MHz. These fast FFICM frame rates were enabled by the use of a low spatial coherence, high-power laser source. Specifically, we used a dense vertical cavity surface emitting laser (VCSEL) array that synthesized low spatial coherence light through a large number of narrowband, mutually-incoherent emitters. Off-axis interferometry enabled single-shot acquisition of the complex-valued interferometric signal. We characterized the system performance (~2 μm lateral resolution, ~8 μm axial gating depth) with a well-known target. We also demonstrated the use of this highly parallelized confocal microscopy platform for visualization and quantification of cilia-driven surface flows and cilia beat frequency in an important animal model (Xenopus embryos) with >1 kHz frame rate. Such frame rates are needed to see large changes in local flow velocity over small distance (high shear flow), in this case, local flow around a single ciliated cell. More generally, our results are an important demonstration of low-spatial coherence, high-power lasers in high-performance, quantitative biomedical imaging.