A single-cell time-lapse of mouse prenatal development from gastrula to birth.

The house mouse (Mus musculus) is an exceptional model system, combining genetic tractability with close evolutionary affinity to humans1,2. Mouse gestation lasts only 3 weeks, during which the genome orchestrates the astonishing transformation of a single-cell zygote into a free-living pup composed of more than 500 million cells. Here, to establish a global framework for exploring mammalian development, we applied optimized single-cell combinatorial indexing3 to profile the transcriptional states of 12.4 million nuclei from 83 embryos, precisely staged at 2- to 6-hour intervals spanning late gastrulation (embryonic day 8) to birth (postnatal day 0). From these data, we annotate hundreds of cell types and explore the ontogenesis of the posterior embryo during somitogenesis and of kidney, mesenchyme, retina and early neurons. We leverage the temporal resolution and sampling depth of these whole-embryo snapshots, together with published data4-8 from earlier timepoints, to construct a rooted tree of cell-type relationships that spans the entirety of prenatal development, from zygote to birth. Throughout this tree, we systematically nominate genes encoding transcription factors and other proteins as candidate drivers of the in vivo differentiation of hundreds of cell types. Remarkably, the most marked temporal shifts in cell states are observed within one hour of birth and presumably underlie the massive physiological adaptations that must accompany the successful transition of a mammalian fetus to life outside the womb.

The NIH Somatic Cell Genome Editing program.

The move from reading to writing the human genome offers new opportunities to improve human health. The United States National Institutes of Health (NIH) Somatic Cell Genome Editing (SCGE) Consortium aims to accelerate the development of safer and more-effective methods to edit the genomes of disease-relevant somatic cells in patients, even in tissues that are difficult to reach. Here we discuss the consortium's plans to develop and benchmark approaches to induce and measure genome modifications, and to define downstream functional consequences of genome editing within human cells. Central to this effort is a rigorous and innovative approach that requires validation of the technology through third-party testing in small and large animals. New genome editors, delivery technologies and methods for tracking edited cells in vivo, as well as newly developed animal models and human biological systems, will be assembled-along with validated datasets-into an SCGE Toolkit, which will be disseminated widely to the biomedical research community. We visualize this toolkit-and the knowledge generated by its applications-as a means to accelerate the clinical development of new therapies for a wide range of conditions.

Identification of a heterogeneous and dynamic ciliome during embryonic development and cell differentiation.

Primary cilia are nearly ubiquitous organelles that transduce molecular and mechanical signals. Although the basic structure of the cilium and the cadre of genes that contribute to ciliary formation and function (the ciliome) are believed to be evolutionarily conserved, the presentation of ciliopathies with narrow, tissue-specific phenotypes and distinct molecular readouts suggests that an unappreciated heterogeneity exists within this organelle. Here, we provide a searchable transcriptomic resource for a curated primary ciliome, detailing various subgroups of differentially expressed genes within the ciliome that display tissue and temporal specificity. Genes within the differentially expressed ciliome exhibited a lower level of functional constraint across species, suggesting organism and cell-specific function adaptation. The biological relevance of ciliary heterogeneity was functionally validated by using Cas9 gene-editing to disrupt ciliary genes that displayed dynamic gene expression profiles during osteogenic differentiation of multipotent neural crest cells. Collectively, this novel primary cilia-focused resource will allow researchers to explore longstanding questions related to how tissue and cell-type specific functions and ciliary heterogeneity may contribute to the range of phenotypes associated with ciliopathies.

Amphiphilic shuttle peptide delivers base editor ribonucleoprotein to correct the CFTR R553X mutation in well-differentiated airway epithelial cells.

Base editing could correct nonsense mutations that cause cystic fibrosis (CF), but clinical development is limited by the lack of delivery methods that efficiently breach the barriers presented by airway epithelia. Here, we present a novel amphiphilic shuttle peptide based on the previously reported S10 peptide that substantially improved base editor ribonucleoprotein (RNP) delivery. Studies of the S10 secondary structure revealed that the alpha-helix formed by the endosomal leakage domain (ELD), but not the cell penetrating peptide (CPP), was functionally important for delivery. By isolating and extending the ELD, we created a novel shuttle peptide, termed S237. While S237 achieved lower delivery of green fluorescent protein, it outperformed S10 at Cas9 RNP delivery to cultured human airway epithelial cells and to pig airway epithelia in vivo, possibly due to its lower net charge. In well-differentiated primary human airway epithelial cell cultures, S237 achieved a 4.6-fold increase in base editor RNP delivery, correcting up to 9.4% of the cystic fibrosis transmembrane conductance regulator (CFTR) R553X allele and restoring CFTR channel function close to non-CF levels. These findings deepen the understanding of peptide-mediated delivery and offer a translational approach for base editor RNP delivery for CF airway disease.

Focused ultrasound-mediated brain genome editing.

Gene editing in the brain has been challenging because of the restricted transport imposed by the blood-brain barrier (BBB). Current approaches mainly rely on local injection to bypass the BBB. However, such administration is highly invasive and not amenable to treating certain delicate regions of the brain. We demonstrate a safe and effective gene editing technique by using focused ultrasound (FUS) to transiently open the BBB for the transport of intravenously delivered CRISPR/Cas9 machinery to the brain.

TMEM161B modulates radial glial scaffolding in neocortical development.

TMEM161B encodes an evolutionarily conserved widely expressed novel 8-pass transmembrane protein of unknown function in human. Here we identify TMEM161B homozygous hypomorphic missense variants in our recessive polymicrogyria (PMG) cohort. Patients carrying TMEM161B mutations exhibit striking neocortical PMG and intellectual disability. Tmem161b knockout mice fail to develop midline hemispheric cleavage, whereas knock-in of patient mutations and patient-derived brain organoids show defects in apical cell polarity and radial glial scaffolding. We found that TMEM161B modulates actin filopodia, functioning upstream of the Rho-GTPase CDC42. Our data link TMEM161B with human PMG, likely regulating radial glia apical polarity during neocortical development.

A graph theoretical approach to experimental prioritization in genome-scale investigations.

The goal of systems biology is to gain a network level understanding of how gene interactions influence biological states, and ultimately inform upon human disease. Given the scale and scope of systems biology studies, resource constraints often limit researchers when validating genome-wide phenomena and potentially lead to an incomplete understanding of the underlying mechanisms. Further, prioritization strategies are often biased towards known entities (e.g. previously studied genes/proteins with commercially available reagents), and other technical issues that limit experimental breadth. Here, heterogeneous biological information is modeled as an association graph to which a high-performance minimum dominating set solver is applied to maximize coverage across the graph, and thus increase the breadth of experimentation. First, we tested our model on retrieval of existing gene functional annotations and demonstrated that minimum dominating set returns more diverse terms when compared to other computational methods. Next, we utilized our heterogenous network and minimum dominating set solver to assist in the process of identifying understudied genes to be interrogated by the International Mouse Phenotyping Consortium. Using an unbiased algorithmic strategy, poorly studied genes are prioritized from the remaining thousands of genes yet to be characterized. This method is tunable and extensible with the potential to incorporate additional user-defined prioritizing information. The minimum dominating set approach can be applied to any biological network in order to identify a tractable subset of features to test experimentally or to assist in prioritizing candidate genes associated with human disease.

Lethal phenotypes in Mendelian disorders.

PURPOSE: Existing resources that characterize the essentiality status of genes are based on either proliferation assessment in human cell lines, viability evaluation in mouse knockouts, or constraint metrics derived from human population sequencing studies. Several repositories document phenotypic annotations for rare disorders; however, there is a lack of comprehensive reporting on lethal phenotypes. METHODS: We queried Online Mendelian Inheritance in Man for terms related to lethality and classified all Mendelian genes according to the earliest age of death recorded for the associated disorders, from prenatal death to no reports of premature death. We characterized the genes across these lethality categories, examined the evidence on viability from mouse models and explored how this information could be used for novel gene discovery. RESULTS: We developed the Lethal Phenotypes Portal to showcase this curated catalog of human essential genes. Differences in the mode of inheritance, physiological systems affected, and disease class were found for genes in different lethality categories, as well as discrepancies between the lethal phenotypes observed in mouse and human. CONCLUSION: We anticipate that this resource will aid clinicians in the diagnosis of early lethal conditions and assist researchers in investigating the properties that make these genes essential for human development.

Snx3 is important for mammalian neural tube closure via its role in canonical and non-canonical WNT signaling.

Disruptions in neural tube (NT) closure result in neural tube defects (NTDs). To understand the molecular processes required for mammalian NT closure, we investigated the role of Snx3, a sorting nexin gene. Snx3-/- mutant mouse embryos display a fully-penetrant cranial NTD. In vivo, we observed decreased canonical WNT target gene expression in the cranial neural epithelium of the Snx3-/- embryos and a defect in convergent extension of the neural epithelium. Snx3-/- cells show decreased WNT secretion, and live cell imaging reveals aberrant recycling of the WNT ligand-binding protein WLS and mis-trafficking to the lysosome for degradation. The importance of SNX3 in WNT signaling regulation is demonstrated by rescue of NT closure in Snx3-/- embryos with a WNT agonist. The potential for SNX3 to function in human neurulation is revealed by a point mutation identified in an NTD-affected individual that results in functionally impaired SNX3 that does not colocalize with WLS and the degradation of WLS in the lysosome. These data indicate that Snx3 is crucial for NT closure via its role in recycling WLS in order to control levels of WNT signaling.

The spontaneous mouse mutant low set ears (Lse) is caused by tandem duplication of Fgf3 and Fgf4.

The external ear develops from an organized convergence of ventrally migrating neural crest cells into the first and second branchial arches. Defects in external ear position are often symptomatic of complex syndromes such as Apert, Treacher-Collins, and Crouzon Syndrome. The low set ears (Lse) spontaneous mouse mutant is characterized by the dominant inheritance of a ventrally shifted external ear position and an abnormal external auditory meatus (EAM). We identified the causative mutation as a 148 Kb tandem duplication on Chromosome 7, which includes the entire coding sequences of Fgf3 and Fgf4. Duplications of FGF3 and FGF4 occur in 11q duplication syndrome in humans and are associated with craniofacial anomalies, among other features. Intercrosses of Lse-affected mice revealed perinatal lethality in homozygotes, and Lse/Lse embryos display additional phenotypes including polydactyly, abnormal eye morphology, and cleft secondary palate. The duplication results in increased Fgf3 and Fgf4 expression in the branchial arches and additional discrete domains in the developing embryo. This ectopic overexpression resulted in functional FGF signaling, demonstrated by increased Spry2 and Etv5 expression in overlapping domains of the developing arches. Finally, a genetic interaction between Fgf3/4 overexpression and Twist1, a regulator of skull suture development, resulted in perinatal lethality, cleft palate, and polydactyly in compound heterozygotes. These data indicate a role for Fgf3 and Fgf4 in external ear and palate development and provide a novel mouse model for further interrogation of the biological consequences of human FGF3/4 duplication.

Comprehensive ECG reference intervals in C57BL/6N substrains provide a generalizable guide for cardiac electrophysiology studies in mice.

Reference ranges provide a powerful tool for diagnostic decision-making in clinical medicine and are enormously valuable for understanding normality in pre-clinical scientific research that uses in vivo models. As yet, there are no published reference ranges for electrocardiography (ECG) in the laboratory mouse. The first mouse-specific reference ranges for the assessment of electrical conduction are reported herein generated from an ECG dataset of unprecedented scale. International Mouse Phenotyping Consortium data from over 26,000 conscious or anesthetized C57BL/6N wildtype control mice were stratified by sex and age to develop robust ECG reference ranges. Interesting findings include that heart rate and key elements from the ECG waveform (RR-, PR-, ST-, QT-interval, QT corrected, and QRS complex) demonstrate minimal sexual dimorphism. As expected, anesthesia induces a decrease in heart rate and was shown for both inhalation (isoflurane) and injectable (tribromoethanol) anesthesia. In the absence of pharmacological, environmental, or genetic challenges, we did not observe major age-related ECG changes in C57BL/6N-inbred mice as the differences in the reference ranges of 12-week-old compared to 62-week-old mice were negligible. The generalizability of the C57BL/6N substrain reference ranges was demonstrated by comparison with ECG data from a wide range of non-IMPC studies. The close overlap in data from a wide range of mouse strains suggests that the C57BL/6N-based reference ranges can be used as a robust and comprehensive indicator of normality. We report a unique ECG reference resource of fundamental importance for any experimental study of cardiac function in mice.

Overlapping roles for PLK1 and Aurora A during meiotic centrosome biogenesis in mouse spermatocytes.

The establishment of bipolar spindles during meiotic divisions ensures faithful chromosome segregation to prevent gamete aneuploidy. We analyzed centriole duplication, as well as centrosome maturation and separation during meiosis I and II using mouse spermatocytes. The first round of centriole duplication occurs during early prophase I, and then, centrosomes mature and begin to separate by the end of prophase I to prime formation of bipolar metaphase I spindles. The second round of centriole duplication occurs at late anaphase I, and subsequently, centrosome separation coordinates bipolar segregation of sister chromatids during meiosis II. Using a germ cell-specific conditional knockout strategy, we show that Polo-like kinase 1 and Aurora A kinase are required for centrosome maturation and separation prior to metaphase I, leading to the formation of bipolar metaphase I spindles. Furthermore, we show that PLK1 is required to block the second round of centriole duplication and maturation until anaphase I. Our findings emphasize the importance of maintaining strict spatiotemporal control of cell cycle kinases during meiosis to ensure proficient centrosome biogenesis and, thus, accurate chromosome segregation during spermatogenesis.

Mouse mutant phenotyping at scale reveals novel genes controlling bone mineral density.

The genetic landscape of diseases associated with changes in bone mineral density (BMD), such as osteoporosis, is only partially understood. Here, we explored data from 3,823 mutant mouse strains for BMD, a measure that is frequently altered in a range of bone pathologies, including osteoporosis. A total of 200 genes were found to significantly affect BMD. This pool of BMD genes comprised 141 genes with previously unknown functions in bone biology and was complementary to pools derived from recent human studies. Nineteen of the 141 genes also caused skeletal abnormalities. Examination of the BMD genes in osteoclasts and osteoblasts underscored BMD pathways, including vesicle transport, in these cells and together with in silico bone turnover studies resulted in the prioritization of candidate genes for further investigation. Overall, the results add novel pathophysiological and molecular insight into bone health and disease.

Large-scale discovery of mouse transgenic integration sites reveals frequent structural variation and insertional mutagenesis.

Transgenesis has been a mainstay of mouse genetics for over 30 yr, providing numerous models of human disease and critical genetic tools in widespread use today. Generated through the random integration of DNA fragments into the host genome, transgenesis can lead to insertional mutagenesis if a coding gene or an essential element is disrupted, and there is evidence that larger scale structural variation can accompany the integration. The insertion sites of only a tiny fraction of the thousands of transgenic lines in existence have been discovered and reported, due in part to limitations in the discovery tools. Targeted locus amplification (TLA) provides a robust and efficient means to identify both the insertion site and content of transgenes through deep sequencing of genomic loci linked to specific known transgene cassettes. Here, we report the first large-scale analysis of transgene insertion sites from 40 highly used transgenic mouse lines. We show that the transgenes disrupt the coding sequence of endogenous genes in half of the lines, frequently involving large deletions and/or structural variations at the insertion site. Furthermore, we identify a number of unexpected sequences in some of the transgenes, including undocumented cassettes and contaminating DNA fragments. We demonstrate that these transgene insertions can have phenotypic consequences, which could confound certain experiments, emphasizing the need for careful attention to control strategies. Together, these data show that transgenic alleles display a high rate of potentially confounding genetic events and highlight the need for careful characterization of each line to assure interpretable and reproducible experiments.

Progress towards completing the mutant mouse null resource.

The generation of a comprehensive catalog of null alleles covering all protein-coding genes is the goal of the International Mouse Phenotyping Consortium. Over the past 20 years, significant progress has been made towards achieving this goal through the combined efforts of many large-scale programs that built an embryonic stem cell resource to generate knockout mice and more recently employed CRISPR/Cas9-based mutagenesis to delete critical regions predicted to result in frameshift mutations, thus, ablating gene function. The IMPC initiative builds on prior and ongoing work by individual research groups creating gene knockouts in the mouse. Here, we analyze the collective efforts focusing on the combined null allele resource resulting from strains developed by the research community and large-scale production programs. Based upon this pooled analysis, we examine the remaining fraction of protein-coding genes focusing on clearly defined mouse-human orthologs as the highest priority for completing the mutant mouse null resource. In summary, we find that there are less than 3400 mouse-human orthologs remaining in the genome without a targeted null allele that can be further prioritized to achieve our overall goal of the complete functional annotation of the protein-coding portion of a mammalian genome.

Annotated expression and activity data for murine recombinase alleles and transgenes: the CrePortal resource.

Recombinase alleles and transgenes can be used to facilitate spatio-temporal specificity of gene disruption or transgene expression. However, the versatility of this in vivo recombination system relies on having detailed and accurate characterization of recombinase expression and activity to enable selection of the appropriate allele or transgene. The CrePortal ( http://www.informatics.jax.org/home/recombinase ) leverages the informatics infrastructure of Mouse Genome Informatics to integrate data from the scientific literature, direct data submissions from the scientific community at-large, and from major projects developing new recombinase lines and characterizing recombinase expression and specificity patterns. Searching the CrePortal by recombinase activity or specific recombinase gene driver provides users with a recombinase alleles and transgenes activity tissue summary and matrix comparison of gene expression and recombinase activity with links to generation details, a recombinase activity grid, and associated phenotype annotations. Future improvements will add cell type-based activity annotations. The CrePortal provides a comprehensive presentation of recombinase allele and transgene data to assist researchers in selection of the recombinase allele or transgene based on where and when recombination is desired.

The Mafb cleft-associated variant H131Q is not required for palatogenesis in the mouse.

BACKGROUND: Orofacial clefts (OFCs) are common birth defects with complex etiology. Genome wide association studies for OFC have identified SNPs in and near MAFB. MAFB is a transcription factor critical for structural development of digits, kidneys, skin, and brain. MAFB is also expressed in the craniofacial region. Previous sequencing of MAFB in a Filipino population revealed a novel missense variant significantly associated with an increased risk for OFC. This MAFB variant, leading to the amino acid change H131Q, was knocked into the mouse Mafb, resulting in the MafbH131Q allele. The MafbH131Q construct was engineered to allow for deletion of Mafb ("Mafbdel "). RESULTS: Mafbdel/del animals died shortly after birth. Conversely, MafbH131Q/H131Q mice survived into adulthood at Mendelian ratios. Mafbdel/del and MafbH131Q/H131Q heads exhibited normal macroscopic and histological appearance at all embryonic time points evaluated. The periderm was intact based on expression of keratin 6, p63, and E-cadherin. Despite no effect on craniofacial morphogenesis, H131Q inhibited the Mafb-dependent promoter activation of Arhgap29 in palatal mesenchymal, but not ectodermal-derived epithelial cells in a luciferase assay. CONCLUSIONS: Mafb is dispensable for murine palatogenesis in vivo, and the cleft-associated variant H131Q, despite its lack of morphogenic effect, altered the expression of Arhgap29 in a cell-dependent context.

Machine learning-based automated phenotyping of inflammatory nocifensive behavior in mice.

The discovery and development of new and potentially nonaddictive pain therapeutics requires rapid, yet clinically relevant assays of nociception in preclinical models. A reliable and scalable automated scoring system for nocifensive behavior of mice in the formalin assay would dramatically lower the time and labor costs associated with experiments and reduce experimental variability. Here, we present a method that exploits machine learning techniques for video recordings that consists of three components: key point detection, per frame feature extraction using these key points, and classification of behavior using the GentleBoost algorithm. This approach to automation is flexible as different model classifiers or key points can be used with only small losses in accuracy. The adopted system identified the behavior of licking/biting of the hind paw with an accuracy that was comparable to a human observer (98% agreement) over 111 different short videos (total 284 min) at a resolution of 1 s. To test the system over longer experimental conditions, the responses of two inbred strains, C57BL/6NJ and C57BL/6J, were recorded over 90 min post formalin challenge. The automated system easily scored over 80 h of video and revealed strain differences in both response timing and amplitude. This machine learning scoring system provides the required accuracy, consistency, and ease of use that could make the formalin assay a feasible choice for large-scale genetic studies.

Mutations in SMG9, Encoding an Essential Component of Nonsense-Mediated Decay Machinery, Cause a Multiple Congenital Anomaly Syndrome in Humans and Mice.

Nonsense-mediated decay (NMD) is an important process that is best known for degrading transcripts that contain premature stop codons (PTCs) to mitigate their potentially harmful consequences, although its regulatory role encompasses other classes of transcripts as well. Despite the critical role of NMD at the cellular level, our knowledge about the consequences of deficiency of its components at the organismal level is largely limited to model organisms. In this study, we report two consanguineous families in which a similar pattern of congenital anomalies was found to be most likely caused by homozygous loss-of-function mutations in SMG9, encoding an essential component of the SURF complex that generates phospho-UPF1, the single most important step in NMD. By knocking out Smg9 in mice via CRISPR/Cas9, we were able to recapitulate the major features of the SMG9-related multiple congenital anomaly syndrome we observed in humans. Surprisingly, human cells devoid of SMG9 do not appear to have reduction of PTC-containing transcripts but do display global transcriptional dysregulation. We conclude that SMG9 is required for normal human and murine development, most likely through a transcriptional regulatory role, the precise nature of which remains to be determined.

APOBEC1 complementation factor (A1CF) is dispensable for C-to-U RNA editing in vivo.

Editing of the human and murine ApoB mRNA by APOBEC1, the catalytic enzyme of the protein complex that catalyzes C-to-U RNA editing, creates an internal stop codon within the APOB coding sequence, generating two protein isoforms. It has been long held that APOBEC1-mediated editing activity is dependent on the RNA binding protein A1CF. The function of A1CF in adult tissues has not been reported because a previously reported null allele displays embryonic lethality. This work aimed to address the function of A1CF in adult mouse tissues using a conditional A1cf allele. Unexpectedly, A1cf-null mice were viable and fertile with modest defects in hematopoietic, immune, and metabolic parameters. C-to-U RNA editing was quantified for multiple targets, including ApoB, in the small intestine and liver. In all cases, no changes in RNA editing efficiency were observed. Blood plasma analysis demonstrated a male-specific increase in solute concentration and increased cellularity in the glomeruli of male A1cf-null mice. Urine analysis showed a reduction in solute concentration, suggesting abnormal water homeostasis and possible kidney abnormalities exclusive to the male. Computational identification of kidney C-to-U editing sites from polyadenylated RNA-sequencing identified a number of editing sites exclusive to the kidney. However, molecular analysis of kidney C-to-U editing showed no changes in editing efficiency with A1CF loss. Taken together, these observations demonstrate that A1CF does not act as the APOBEC1 complementation factor in vivo under normal physiological conditions and suggests new roles for A1CF, specifically within the male adult kidney.