Publications by authors named "Mary E Dickinson"

112 Publications

The NIH Somatic Cell Genome Editing program.

Nature 2021 Apr 7;592(7853):195-204. Epub 2021 Apr 7.

McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA, USA.

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.
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http://dx.doi.org/10.1038/s41586-021-03191-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8026397PMC
April 2021

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

PLoS Genet 2020 12 28;16(12):e1009190. Epub 2020 Dec 28.

Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States of America.

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.
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http://dx.doi.org/10.1371/journal.pgen.1009190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822523PMC
December 2020

CreLite: An optogenetically controlled Cre/loxP system using red light.

Dev Dyn 2020 11 31;249(11):1394-1403. Epub 2020 Aug 31.

Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA.

Background: Precise manipulation of gene expression with temporal and spatial control is essential for functional analysis and determining cell lineage relationships in complex biological systems. The cyclic recombinase (Cre)-loxP system is commonly used for gene manipulation at desired times and places. However, specificity is dependent on the availability of tissue- or cell-specific regulatory elements used in combination with Cre. Here, we present CreLite, an optogenetically controlled Cre system using red light in developing zebrafish embryos.

Results: Cre activity is disabled by splitting Cre and fusing with the Arabidopsis thaliana red light-inducible binding partners, PhyB and PIF6. Upon red light illumination, the PhyB-CreC and PIF6-CreN fusion proteins come together in the presence of the cofactor phycocyanobilin (PCB) to restore Cre activity. Red light exposure of zebrafish embryos harboring a Cre-dependent multicolor fluorescent protein reporter injected with CreLite mRNAs and PCB resulted in Cre activity as measured by the generation of multispectral cell labeling in several different tissues.

Conclusions: Our data show that CreLite can be used for gene manipulations in whole embryos or small groups of cells at different developmental stages, and suggests CreLite may also be useful for temporal and spatial control of gene expression in cell culture, ex vivo organ culture, and other animal models.
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http://dx.doi.org/10.1002/dvdy.232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7931845PMC
November 2020

A global Slc7a7 knockout mouse model demonstrates characteristic phenotypes of human lysinuric protein intolerance.

Hum Mol Genet 2020 08;29(13):2171-2184

Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.

Lysinuric protein intolerance (LPI) is an inborn error of cationic amino acid (arginine, lysine, ornithine) transport caused by biallelic pathogenic variants in SLC7A7, which encodes the light subunit of the y+LAT1 transporter. Treatments for the complications of LPI, including growth failure, renal disease, pulmonary alveolar proteinosis, autoimmune disorders and osteoporosis, are limited. Given the early lethality of the only published global Slc7a7 knockout mouse model, a viable animal model to investigate global SLC7A7 deficiency is needed. Hence, we generated two mouse models with global Slc7a7 deficiency (Slc7a7em1Lbu/em1Lbu; Slc7a7Lbu/Lbu and Slc7a7em1(IMPC)Bay/em1(IMPC)Bay; Slc7a7Bay/Bay) using CRISPR/Cas9 technology by introducing a deletion of exons 3 and 4. Perinatal lethality was observed in Slc7a7Lbu/Lbu and Slc7a7Bay/Bay mice on the C57BL/6 and C57BL/6NJ inbred genetic backgrounds, respectively. We noted improved survival of Slc7a7Lbu/Lbu mice on the 129 Sv/Ev × C57BL/6 F2 background, but postnatal growth failure occurred. Consistent with human LPI, these Slc7a7Lbu/Lbu mice exhibited reduced plasma and increased urinary concentrations of the cationic amino acids. Histopathological assessment revealed loss of brush border and lipid vacuolation in the renal cortex of Slc7a7Lbu/Lbu mice, which combined with aminoaciduria suggests proximal tubular dysfunction. Micro-computed tomography of L4 vertebrae and skeletal radiographs showed delayed skeletal development and suggested decreased mineralization in Slc7a7Lbu/Lbu mice, respectively. In addition to delayed skeletal development and delayed development in the kidneys, the lungs and liver were observed based on histopathological assessment. Overall, our Slc7a7Lbu/Lbu mouse model on the F2 mixed background recapitulates multiple human LPI phenotypes and may be useful for future studies of LPI pathology.
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http://dx.doi.org/10.1093/hmg/ddaa107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7399531PMC
August 2020

Human and mouse essentiality screens as a resource for disease gene discovery.

Nat Commun 2020 01 31;11(1):655. Epub 2020 Jan 31.

Institute of Developmental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health GmbH, 85764, Neuherberg, Germany.

The identification of causal variants in sequencing studies remains a considerable challenge that can be partially addressed by new gene-specific knowledge. Here, we integrate measures of how essential a gene is to supporting life, as inferred from viability and phenotyping screens performed on knockout mice by the International Mouse Phenotyping Consortium and essentiality screens carried out on human cell lines. We propose a cross-species gene classification across the Full Spectrum of Intolerance to Loss-of-function (FUSIL) and demonstrate that genes in five mutually exclusive FUSIL categories have differing biological properties. Most notably, Mendelian disease genes, particularly those associated with developmental disorders, are highly overrepresented among genes non-essential for cell survival but required for organism development. After screening developmental disorder cases from three independent disease sequencing consortia, we identify potentially pathogenic variants in genes not previously associated with rare diseases. We therefore propose FUSIL as an efficient approach for disease gene discovery.
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http://dx.doi.org/10.1038/s41467-020-14284-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994715PMC
January 2020

Soft windowing application to improve analysis of high-throughput phenotyping data.

Bioinformatics 2020 03;36(5):1492-1500

Korea Mouse Phenotyping Center (KMPC), Korea.

Motivation: High-throughput phenomic projects generate complex data from small treatment and large control groups that increase the power of the analyses but introduce variation over time. A method is needed to utlize a set of temporally local controls that maximizes analytic power while minimizing noise from unspecified environmental factors.

Results: Here we introduce 'soft windowing', a methodological approach that selects a window of time that includes the most appropriate controls for analysis. Using phenotype data from the International Mouse Phenotyping Consortium (IMPC), adaptive windows were applied such that control data collected proximally to mutants were assigned the maximal weight, while data collected earlier or later had less weight. We applied this method to IMPC data and compared the results with those obtained from a standard non-windowed approach. Validation was performed using a resampling approach in which we demonstrate a 10% reduction of false positives from 2.5 million analyses. We applied the method to our production analysis pipeline that establishes genotype-phenotype associations by comparing mutant versus control data. We report an increase of 30% in significant P-values, as well as linkage to 106 versus 99 disease models via phenotype overlap with the soft-windowed and non-windowed approaches, respectively, from a set of 2082 mutant mouse lines. Our method is generalizable and can benefit large-scale human phenomic projects such as the UK Biobank and the All of Us resources.

Availability And Implementation: The method is freely available in the R package SmoothWin, available on CRAN http://CRAN.R-project.org/package=SmoothWin.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btz744DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7115897PMC
March 2020

High Resolution Imaging of Mouse Embryos and Neonates with X-Ray Micro-Computed Tomography.

Curr Protoc Mouse Biol 2019 Jun 13;9(2):e63. Epub 2019 Jun 13.

Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas.

Iodine-contrast micro-computed tomography (microCT) 3D imaging provides a non-destructive and high-throughput platform for studying mouse embryo and neonate development. Here we provide protocols on preparing mouse embryos and neonates between embryonic day 8.5 (E8.5) to postnatal day 4 (P4) for iodine-contrast microCT imaging. With the implementation of the STABILITY method to create a polymer-tissue hybrid structure, we have demonstrated that not only is soft tissue shrinkage minimized but also the minimum required time for soft tissue staining with iodine is decreased, especially for E18.5 to P4 samples. In addition, we also provide a protocol on using commercially available X-CLARITY hydrogel solution to create the similar polymer-tissue hybrid structure on delicate early post-implantation stage (E8.5 to E14.5) embryos. With its simple sample staining and mounting processes, this protocol is easy to adopt and implement for most of the commercially available, stand-alone microCT systems in order to study mouse development between early post-implantation to early postnatal stages. © 2019 by John Wiley & Sons, Inc.
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http://dx.doi.org/10.1002/cpmo.63DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6594408PMC
June 2019

Bi-allelic Variants in TONSL Cause SPONASTRIME Dysplasia and a Spectrum of Skeletal Dysplasia Phenotypes.

Am J Hum Genet 2019 03 14;104(3):422-438. Epub 2019 Feb 14.

Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.

SPONASTRIME dysplasia is an autosomal-recessive spondyloepimetaphyseal dysplasia characterized by spine (spondylar) abnormalities, midface hypoplasia with a depressed nasal bridge, metaphyseal striations, and disproportionate short stature. Scoliosis, coxa vara, childhood cataracts, short dental roots, and hypogammaglobulinemia have also been reported in this disorder. Although an autosomal-recessive inheritance pattern has been hypothesized, pathogenic variants in a specific gene have not been discovered in individuals with SPONASTRIME dysplasia. Here, we identified bi-allelic variants in TONSL, which encodes the Tonsoku-like DNA repair protein, in nine subjects (from eight families) with SPONASTRIME dysplasia, and four subjects (from three families) with short stature of varied severity and spondylometaphyseal dysplasia with or without immunologic and hematologic abnormalities, but no definitive metaphyseal striations at diagnosis. The finding of early embryonic lethality in a Tonsl murine model and the discovery of reduced length, spinal abnormalities, reduced numbers of neutrophils, and early lethality in a tonsl zebrafish model both support the hypomorphic nature of the identified TONSL variants. Moreover, functional studies revealed increased amounts of spontaneous replication fork stalling and chromosomal aberrations, as well as fewer camptothecin (CPT)-induced RAD51 foci in subject-derived cell lines. Importantly, these cellular defects were rescued upon re-expression of wild-type (WT) TONSL; this rescue is consistent with the hypothesis that hypomorphic TONSL variants are pathogenic. Overall, our studies in humans, mice, zebrafish, and subject-derived cell lines confirm that pathogenic variants in TONSL impair DNA replication and homologous recombination-dependent repair processes, and they lead to a spectrum of skeletal dysplasia phenotypes with numerous extra-skeletal manifestations.
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http://dx.doi.org/10.1016/j.ajhg.2019.01.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6408318PMC
March 2019

Identification of genes required for eye development by high-throughput screening of mouse knockouts.

Commun Biol 2018 21;1:236. Epub 2018 Dec 21.

Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, 77030, USA.

Despite advances in next generation sequencing technologies, determining the genetic basis of ocular disease remains a major challenge due to the limited access and prohibitive cost of human forward genetics. Thus, less than 4,000 genes currently have available phenotype information for any organ system. Here we report the ophthalmic findings from the International Mouse Phenotyping Consortium, a large-scale functional genetic screen with the goal of generating and phenotyping a null mutant for every mouse gene. Of 4364 genes evaluated, 347 were identified to influence ocular phenotypes, 75% of which are entirely novel in ocular pathology. This discovery greatly increases the current number of genes known to contribute to ophthalmic disease, and it is likely that many of the genes will subsequently prove to be important in human ocular development and disease.
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http://dx.doi.org/10.1038/s42003-018-0226-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6303268PMC
December 2018

Rapid and Integrative Discovery of Retina Regulatory Molecules.

Cell Rep 2018 08;24(9):2506-2519

Department of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA; Huffington Center on Aging, Baylor College of Medicine, Houston, TX 77030, USA; Program in Developmental Biology, Baylor College of Medicine, Houston, TX 77030, USA. Electronic address:

Retinal function relies on precisely organized neurons and synapses and a properly patterned vasculature to support them. Alterations in these features can result in vision loss. However, our understanding of retinal organization pathways remains incomplete because of a lack of methods to rapidly identify neuron and vasculature regulators in mammals. Here we developed a pipeline for the identification of neural and synaptic integrity genes by high-throughput retinal screening (INSiGHT) that analyzes candidate expression, vascular patterning, cellular organization, and synaptic arrangement. Using this system, we examined 102 mutant mouse lines and identified 16 unique retinal regulatory genes. Fifteen of these candidates are identified as novel retina regulators, and many (9 of 16) are associated with human neural diseases. These results expand the genetic landscape involved in retinal circuit organization and provide a road map for continued discovery of mammalian retinal regulators and disease-causing alleles.
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http://dx.doi.org/10.1016/j.celrep.2018.07.090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6170014PMC
August 2018

The phenotypic and functional properties of mouse yolk-sac-derived embryonic macrophages.

Dev Biol 2018 10 30;442(1):138-154. Epub 2018 Jul 30.

Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX, USA; Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA; Cardiovascular Research Institute, Baylor College of Medicine, Houston, TX, USA; Department of Bioengineering, Rice University, Houston, TX, USA. Electronic address:

Macrophages are well characterized as immune cells. However, in recent years, a multitude of non-immune functions have emerged many of which play essential roles in a variety of developmental processes (Wynn et al., 2013; DeFalco et al., 2014). In adult animals, macrophages are derived from circulating monocytes originating in the bone marrow, but much of the tissue-resident population arise from erythro-myeloid progenitors (EMPs) in the extra-embryonic yolk sac, appearing around the same time as primitive erythroblasts (Schulz et al., 2012; Kierdorf et al., 2013; McGrath et al., 2015; Gomez Perdiguero et al., 2015; Mass et al., 2016). Of particular interest to our group, macrophages have been shown to act as pro-angiogenic regulators during development (Wynn et al., 2013; DeFalco et al., 2014; Hsu et al., 2015), but there is still much to learn about these early cells. The goal of the present study was to isolate and expand progenitors of yolk-sac-derived Embryonic Macrophages (EMs) in vitro to generate a new platform for mechanistic studies of EM differentiation. To accomplish this goal, we isolated pure (>98%) EGFP populations by flow cytometry from embryonic day 9.5 (E9.5) Csf1r-EGFP mice, then evaluated the angiogenic potential of EMs relative to Bone Marrow-Derived Macrophages (BMDMs). We found that EMs expressed more pro-angiogenic and less pro-inflammatory macrophage markers than BMDMs. EMs also promoted more endothelial cell (EC) cord formation in vitro, as compared to BMDMs in a manner that required direct cell-to-cell contact. Importantly, EMs preferentially matured into microglia when co-cultured with mouse Neural Stem/Progenitor Cells (NSPCs). In conclusion, we have established a protocol to isolate and propagate EMs in vitro, have further defined specialized properties of yolk-sac-derived macrophages, and have identified EM-EC and EM-NSPC interactions as key inducers of EC tube formation and microglial cell maturation, respectively.
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http://dx.doi.org/10.1016/j.ydbio.2018.07.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6190604PMC
October 2018

The effects of reduced hemodynamic loading on morphogenesis of the mouse embryonic heart.

Dev Biol 2018 10 17;442(1):127-137. Epub 2018 Jul 17.

Department of Biology, Missouri State University, United States. Electronic address:

Development of the embryonic heart involves an intricate network of biochemical and genetic cues to ensure its proper growth and morphogenesis. However, studies from avian and teleost models reveal that biomechanical force, namely hemodynamic loading (blood pressure and shear stress), plays a significant role in regulating heart development. To study how hemodynamic loading impacts development of the mammalian embryonic heart, we utilized mouse embryo culture and manipulation techniques and performed optical projection tomography imaging followed by morphometric analysis to determine how reduced-loading affects heart volume, myocardial thickness, trabeculation and looping. Our results reveal that hemodynamic loading can regulate these features at different thresholds. Intermediate levels of hemodynamic loading are sufficient to promote proper myocardial growth and heart size, but insufficient to promote looping and trabeculation. Whereas, low levels of hemodynamic loading fails to promote proper growth of the myocardium and heart size. These results reveal that the regulation of heart development by biomechanical force is conserved across many vertebrate classes, and this study begins to elucidate how these specific forces regulate development of the mammalian heart.
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http://dx.doi.org/10.1016/j.ydbio.2018.07.007DOI Listing
October 2018

Comparative analysis of single-stranded DNA donors to generate conditional null mouse alleles.

BMC Biol 2018 06 21;16(1):69. Epub 2018 Jun 21.

Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, MS BCM225, Houston, TX, 77030, USA.

Background: The International Mouse Phenotyping Consortium is generating null allele mice for every protein-coding gene in the genome and characterizing these mice to identify gene-phenotype associations. While CRISPR/Cas9-mediated null allele production in mice is highly efficient, generation of conditional alleles has proven to be more difficult. To test the feasibility of using CRISPR/Cas9 gene editing to generate conditional knockout mice for this large-scale resource, we employed Cas9-initiated homology-driven repair (HDR) with short and long single stranded oligodeoxynucleotides (ssODNs and lssDNAs).

Results: Using pairs of single guide RNAs and short ssODNs to introduce loxP sites around a critical exon or exons, we obtained putative conditional allele founder mice, harboring both loxP sites, for 23 out of 30 targeted genes. LoxP sites integrated in cis in at least one mouse for 18 of 23 genes. However, loxP sites were mutagenized in 4 of the 18 in cis lines. HDR efficiency correlated with Cas9 cutting efficiency but was minimally influenced by ssODN homology arm symmetry. By contrast, using pairs of guides and single lssDNAs to introduce loxP-flanked exons, conditional allele founders were generated for all four genes targeted, although one founder was found to harbor undesired mutations within the lssDNA sequence interval. Importantly, when employing either ssODNs or lssDNAs, random integration events were detected.

Conclusions: Our studies demonstrate that Cas9-mediated HDR with pairs of ssODNs can generate conditional null alleles at many loci, but reveal inefficiencies when applied at scale. In contrast, lssDNAs are amenable to high-throughput production of conditional alleles when they can be employed. Regardless of the single-stranded donor utilized, it is essential to screen for sequence errors at sites of HDR and random insertion of donor sequences into the genome.
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http://dx.doi.org/10.1186/s12915-018-0529-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6011517PMC
June 2018

The role of FREM2 and FRAS1 in the development of congenital diaphragmatic hernia.

Hum Mol Genet 2018 06;27(12):2064-2075

Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030, USA.

Congenital diaphragmatic hernia (CDH) has been reported twice in individuals with a clinical diagnosis of Fraser syndrome, a genetic disorder that can be caused by recessive mutations affecting FREM2 and FRAS1. In the extracellular matrix, FREM2 and FRAS1 form a self-stabilizing complex with FREM1, a protein whose deficiency causes sac CDH in humans and mice. By sequencing FREM2 and FRAS1 in a CDH cohort, and searching online databases, we identified five individuals who carried recessive or double heterozygous, putatively deleterious variants in these genes which may represent susceptibility alleles. Three of these alleles were significantly enriched in our CDH cohort compared with ethnically matched controls. We subsequently demonstrated that 8% of Frem2ne/ne and 1% of Fras1Q1263*/Q1263* mice develop the same type of anterior sac CDH seen in FREM1-deficient mice. We went on to show that development of sac hernias in FREM1-deficient mice is preceded by failure of anterior mesothelial fold progression resulting in the persistence of an amuscular, poorly vascularized anterior diaphragm that is abnormally adherent to the underlying liver. Herniation occurs in the perinatal period when the expanding liver protrudes through this amuscular region of the anterior diaphragm that is juxtaposed to areas of muscular diaphragm. Based on these data, we conclude that deficiency of FREM2, and possibly FRAS1, are associated with an increased risk of developing CDH and that loss of the FREM1/FREM2/FRAS1 complex, or its function, leads to anterior sac CDH development through its effects on mesothelial fold progression.
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http://dx.doi.org/10.1093/hmg/ddy110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5985720PMC
June 2018

Comparison and combination of rotational imaging optical coherence tomography and selective plane illumination microscopy for embryonic study.

Biomed Opt Express 2017 Oct 22;8(10):4629-4639. Epub 2017 Sep 22.

Department of Biomedical Engineering, University of Houston, Houston, TX 77204, USA.

Several optical imaging techniques have been applied for high-resolution embryonic imaging using different contrast mechanisms, each with their own benefits and limitations. In this study, we imaged the same E9.5 mouse embryo with rotational imaging optical coherence tomography (RI-OCT) and selective plane illumination microscopy (SPIM). RI-OCT overcomes optical penetration limits of traditional OCT imaging that prohibit full-body imaging of mouse embryos at later stages by imaging the samples from multiple angles. SPIM enables high-resolution, 3D imaging with less phototoxicity and photobleaching than laser scanning confocal microscopy (LSCM) by illuminating the sample with a focused sheet of light. Side by side comparisons are supplemented with co-registered images. The results demonstrate that SPIM and RI-OCT are highly complementary and could provide more comprehensive tissue characterization for mouse embryonic research.
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http://dx.doi.org/10.1364/BOE.8.004629DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5654805PMC
October 2017

A dual-modality optical coherence tomography and selective plane illumination microscopy system for mouse embryonic imaging.

Annu Int Conf IEEE Eng Med Biol Soc 2017 Jul;2017:4038-4040

Both optical coherence tomography (OCT) and selective plane illumination microscopy (SPIM) are frequently used in mouse embryonic research for high-resolution three-dimensional imaging. Each of these imaging methods provide a unique and independent advantage: SPIM provides morpho-functional information through immunofluorescence and OCT provides a method for whole-embryo 3D imaging. In this study, we have combined rotational imaging OCT and SPIM into a single, dual-modality device to image E9.5 mouse embryos. The results demonstrate that the dual-modality setup is able to provide both anatomical and functional information simultaneously for more comprehensive tissue characterization.
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http://dx.doi.org/10.1109/EMBC.2017.8037742DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6322396PMC
July 2017

A large scale hearing loss screen reveals an extensive unexplored genetic landscape for auditory dysfunction.

Nat Commun 2017 10 12;8(1):886. Epub 2017 Oct 12.

RIKEN BioResource Center, Tsukuba, Ibaraki, 305-0074, Japan.

The developmental and physiological complexity of the auditory system is likely reflected in the underlying set of genes involved in auditory function. In humans, over 150 non-syndromic loci have been identified, and there are more than 400 human genetic syndromes with a hearing loss component. Over 100 non-syndromic hearing loss genes have been identified in mouse and human, but we remain ignorant of the full extent of the genetic landscape involved in auditory dysfunction. As part of the International Mouse Phenotyping Consortium, we undertook a hearing loss screen in a cohort of 3006 mouse knockout strains. In total, we identify 67 candidate hearing loss genes. We detect known hearing loss genes, but the vast majority, 52, of the candidate genes were novel. Our analysis reveals a large and unexplored genetic landscape involved with auditory function.The full extent of the genetic basis for hearing impairment is unknown. Here, as part of the International Mouse Phenotyping Consortium, the authors perform a hearing loss screen in 3006 mouse knockout strains and identify 52 new candidate genes for genetic hearing loss.
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http://dx.doi.org/10.1038/s41467-017-00595-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5638796PMC
October 2017

Loss of Apela Peptide in Mice Causes Low Penetrance Embryonic Lethality and Defects in Early Mesodermal Derivatives.

Cell Rep 2017 Aug;20(9):2116-2130

Developmental Biology Program, Sloan Kettering Institute, New York, NY 10065, USA. Electronic address:

Apela (also known as Elabela, Ende, and Toddler) is a small signaling peptide that activates the G-protein-coupled receptor Aplnr to stimulate cell migration during zebrafish gastrulation. Here, using CRISPR/Cas9 to generate a null, reporter-expressing allele, we study the role of Apela in the developing mouse embryo. We found that loss of Apela results in low-penetrance cardiovascular defects that manifest after the onset of circulation. Three-dimensional micro-computed tomography revealed a higher penetrance of vascular remodeling defects, from which some mutants recover, and identified extraembryonic anomalies as the earliest morphological distinction in Apela mutant embryos. Transcriptomics at late gastrulation identified aberrant upregulation of erythroid and myeloid markers in mutant embryos prior to the appearance of physical malformations. Double-mutant analyses showed that loss of Apela signaling impacts early Aplnr-expressing mesodermal populations independently of the alternative ligand Apelin, leading to lethal cardiac defects in some Apela null embryos.
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http://dx.doi.org/10.1016/j.celrep.2017.08.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5580402PMC
August 2017

Prevalence of sexual dimorphism in mammalian phenotypic traits.

Nat Commun 2017 06 26;8:15475. Epub 2017 Jun 26.

Mouse Genetics Project, The Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK.

The role of sex in biomedical studies has often been overlooked, despite evidence of sexually dimorphic effects in some biological studies. Here, we used high-throughput phenotype data from 14,250 wildtype and 40,192 mutant mice (representing 2,186 knockout lines), analysed for up to 234 traits, and found a large proportion of mammalian traits both in wildtype and mutants are influenced by sex. This result has implications for interpreting disease phenotypes in animal models and humans.
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http://dx.doi.org/10.1038/ncomms15475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5490203PMC
June 2017

Disease model discovery from 3,328 gene knockouts by The International Mouse Phenotyping Consortium.

Nat Genet 2017 Aug 26;49(8):1231-1238. Epub 2017 Jun 26.

CELPHEDIA, PHENOMIN, Institut Clinique de la Souris (ICS), Illkirch-Graffenstaden, France.

Although next-generation sequencing has revolutionized the ability to associate variants with human diseases, diagnostic rates and development of new therapies are still limited by a lack of knowledge of the functions and pathobiological mechanisms of most genes. To address this challenge, the International Mouse Phenotyping Consortium is creating a genome- and phenome-wide catalog of gene function by characterizing new knockout-mouse strains across diverse biological systems through a broad set of standardized phenotyping tests. All mice will be readily available to the biomedical community. Analyzing the first 3,328 genes identified models for 360 diseases, including the first models, to our knowledge, for type C Bernard-Soulier, Bardet-Biedl-5 and Gordon Holmes syndromes. 90% of our phenotype annotations were novel, providing functional evidence for 1,092 genes and candidates in genetically uncharacterized diseases including arrhythmogenic right ventricular dysplasia 3. Finally, we describe our role in variant functional validation with The 100,000 Genomes Project and others.
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http://dx.doi.org/10.1038/ng.3901DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5546242PMC
August 2017

Biallelic Variants in OTUD6B Cause an Intellectual Disability Syndrome Associated with Seizures and Dysmorphic Features.

Am J Hum Genet 2017 Apr 23;100(4):676-688. Epub 2017 Mar 23.

Laboratory for Pediatric Brain Disease, Howard Hughes Medical Institute, Department of Neurosciences, University of California, San Diego, CA 92093, USA.

Ubiquitination is a posttranslational modification that regulates many cellular processes including protein degradation, intracellular trafficking, cell signaling, and protein-protein interactions. Deubiquitinating enzymes (DUBs), which reverse the process of ubiquitination, are important regulators of the ubiquitin system. OTUD6B encodes a member of the ovarian tumor domain (OTU)-containing subfamily of deubiquitinating enzymes. Herein, we report biallelic pathogenic variants in OTUD6B in 12 individuals from 6 independent families with an intellectual disability syndrome associated with seizures and dysmorphic features. In subjects with predicted loss-of-function alleles, additional features include global developmental delay, microcephaly, absent speech, hypotonia, growth retardation with prenatal onset, feeding difficulties, structural brain abnormalities, congenital malformations including congenital heart disease, and musculoskeletal features. Homozygous Otud6b knockout mice were subviable, smaller in size, and had congenital heart defects, consistent with the severity of loss-of-function variants in humans. Analysis of peripheral blood mononuclear cells from an affected subject showed reduced incorporation of 19S subunits into 26S proteasomes, decreased chymotrypsin-like activity, and accumulation of ubiquitin-protein conjugates. Our findings suggest a role for OTUD6B in proteasome function, establish that defective OTUD6B function underlies a multisystemic human disorder, and provide additional evidence for the emerging relationship between the ubiquitin system and human disease.
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http://dx.doi.org/10.1016/j.ajhg.2017.03.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5384096PMC
April 2017

Three-dimensional microCT imaging of mouse development from early post-implantation to early postnatal stages.

Dev Biol 2016 11 23;419(2):229-236. Epub 2016 Sep 23.

Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030, USA; Optical Imaging and Vital Microscopy Core, Baylor College of Medicine, Houston, TX 77030, USA; Cardiovascular Research Institute, Baylor College of Medicine, Houston, TX 77030, USA. Electronic address:

In this work, we report the use of iodine-contrast microCT to perform high-throughput 3D morphological analysis of mouse embryos and neonates between embryonic day 8.5 to postnatal day 3, with high spatial resolution up to 3µm/voxel. We show that mouse embryos at early stages can be imaged either within extra embryonic tissues such as the yolk sac or the decidua without physically disturbing the embryos. This method enables a full, undisturbed analysis of embryo turning, allantois development, vitelline vessels remodeling, yolk sac and early placenta development, which provides increased insights into early embryonic lethality in mutant lines. Moreover, these methods are inexpensive, simple to learn and do not require substantial processing time, making them ideal for high throughput analysis of mouse mutants with embryonic and early postnatal lethality.
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http://dx.doi.org/10.1016/j.ydbio.2016.09.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5405732PMC
November 2016

Lethal lung hypoplasia and vascular defects in mice with conditional Foxf1 overexpression.

Biol Open 2016 Nov 15;5(11):1595-1606. Epub 2016 Nov 15.

Department of Molecular & Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA

FOXF1 heterozygous point mutations and genomic deletions have been reported in newborns with the neonatally lethal lung developmental disorder, alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). However, no gain-of-function mutations in FOXF1 have been identified yet in any human disease conditions. To study the effects of FOXF1 overexpression in lung development, we generated a Foxf1 overexpression mouse model by knocking-in a Cre-inducible Foxf1 allele into the ROSA26 (R26) locus. The mice were phenotyped using micro-computed tomography (micro-CT), head-out plethysmography, ChIP-seq and transcriptome analyses, immunohistochemistry, and lung histopathology. Thirty-five percent of heterozygous R26-Lox-Stop-Lox (LSL)-Foxf1 embryonic day (E)15.5 embryos exhibit subcutaneous edema, hemorrhages and die perinatally when bred to Tie2-cre mice, which targets Foxf1 overexpression to endothelial and hematopoietic cells. Histopathological and micro-CT evaluations revealed that R26Foxf1; Tie2-cre embryos have immature lungs with a diminished vascular network. Neonates exhibited respiratory deficits verified by detailed plethysmography studies. ChIP-seq and transcriptome analyses in E18.5 lungs identified Sox11, Ghr, Ednrb, and Slit2 as potential downstream targets of FOXF1. Our study shows that overexpression of the highly dosage-sensitive Foxf1 impairs lung development and causes vascular abnormalities. This has important clinical implications when considering potential gene therapy approaches to treat disorders of FOXF1 abnormal dosage, such as ACDMPV.
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http://dx.doi.org/10.1242/bio.019208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5155529PMC
November 2016

High-throughput discovery of novel developmental phenotypes.

Nature 2016 09 14;537(7621):508-514. Epub 2016 Sep 14.

Department of Molecular Physiology and Biophysics, Houston, Texas 77030, USA.

Approximately one-third of all mammalian genes are essential for life. Phenotypes resulting from knockouts of these genes in mice have provided tremendous insight into gene function and congenital disorders. As part of the International Mouse Phenotyping Consortium effort to generate and phenotypically characterize 5,000 knockout mouse lines, here we identify 410 lethal genes during the production of the first 1,751 unique gene knockouts. Using a standardized phenotyping platform that incorporates high-resolution 3D imaging, we identify phenotypes at multiple time points for previously uncharacterized genes and additional phenotypes for genes with previously reported mutant phenotypes. Unexpectedly, our analysis reveals that incomplete penetrance and variable expressivity are common even on a defined genetic background. In addition, we show that human disease genes are enriched for essential genes, thus providing a dataset that facilitates the prioritization and validation of mutations identified in clinical sequencing efforts.
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http://dx.doi.org/10.1038/nature19356DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5295821PMC
September 2016

Applicability, usability, and limitations of murine embryonic imaging with optical coherence tomography and optical projection tomography.

Biomed Opt Express 2016 Jun 19;7(6):2295-310. Epub 2016 May 19.

Department of Biomedical Engineering, University of Houston, 3605 Cullen Boulevard, Houston, 77204, USA; Molecular Physiology and Biophysics, Baylor College of Medicine, One Baylor Plaza, Houston, 77584, USA; Department of Electrical Engineering, Samara National Research University, Samara, 34 Moskovskoye sh., 443086, Russia.

We present an analysis of imaging murine embryos at various embryonic developmental stages (embryonic day 9.5, 11.5, and 13.5) by optical coherence tomography (OCT) and optical projection tomography (OPT). We demonstrate that while OCT was capable of rapid high-resolution live 3D imaging, its limited penetration depth prevented visualization of deeper structures, particularly in later stage embryos. In contrast, OPT was able to image the whole embryos, but could not be used in vivo because the embryos must be fixed and cleared. Moreover, the fixation process significantly altered the embryo morphology, which was quantified by the volume of the eye-globes before and after fixation. All of these factors should be weighed when determining which imaging modality one should use to achieve particular goals of a study.
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http://dx.doi.org/10.1364/BOE.7.002295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4918583PMC
June 2016

Optical coherence tomography for embryonic imaging: a review.

J Biomed Opt 2016 05;21(5):50902

University of Houston, Department of Biomedical Engineering, 3517 Cullen Boulevard, Room 2027, Houston, Texas 77204-5060, United StatesbBaylor College of Medicine, Department of Molecular Physiology and Biophysics, One Baylor Plaza- BCM335, Houston, Texas.

Embryogenesis is a highly complex and dynamic process, and its visualization is crucial for understanding basic physiological processes during development and for identifying and assessing possible defects, malformations, and diseases. While traditional imaging modalities, such as ultrasound biomicroscopy, micro-magnetic resonance imaging, and micro-computed tomography, have long been adapted for embryonic imaging, these techniques generally have limitations in their speed, spatial resolution, and contrast to capture processes such as cardiodynamics during embryogenesis. Optical coherence tomography (OCT) is a noninvasive imaging modality with micrometer-scale spatial resolution and imaging depth up to a few millimeters in tissue. OCT has bridged the gap between ultrahigh resolution imaging techniques with limited imaging depth like confocal microscopy and modalities, such as ultrasound sonography, which have deeper penetration but poorer spatial resolution. Moreover, the noninvasive nature of OCT has enabled live imaging of embryos without any external contrast agents. We review how OCT has been utilized to study developing embryos and also discuss advances in techniques used in conjunction with OCT to understand embryonic development.
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http://dx.doi.org/10.1117/1.JBO.21.5.050902DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4881290PMC
May 2016