Publications by authors named "Ophir D Klein"

146 Publications

Refining nosology by modelling variation among facial phenotypes: the RASopathies.

J Med Genet 2022 Jul 20. Epub 2022 Jul 20.

Department of Human Genetics, KU Leuven, Leuven, Flemish Brabant, Belgium

Background: In clinical genetics, establishing an accurate nosology requires analysis of variations in both aetiology and the resulting phenotypes. At the phenotypic level, recognising typical facial gestalts has long supported clinical and molecular diagnosis; however, the objective analysis of facial phenotypic variation remains underdeveloped. In this work, we propose exploratory strategies for assessing facial phenotypic variation within and among clinical and molecular disease entities and deploy these techniques on cross-sectional samples of four RASopathies: Costello syndrome (CS), Noonan syndrome (NS), cardiofaciocutaneous syndrome (CFC) and neurofibromatosis type 1 (NF1).

Methods: From three-dimensional dense surface scans, we model the typical phenotypes of the four RASopathies as average 'facial signatures' and assess individual variation in terms of direction (what parts of the face are affected and in what ways) and severity of the facial effects. We also derive a metric of phenotypic agreement between the syndromes and a metric of differences in severity along similar phenotypes.

Results: CFC shows a relatively consistent facial phenotype in terms of both direction and severity that is similar to CS and NS, consistent with the known difficulty in discriminating CFC from NS based on the face. CS shows a consistent directional phenotype that varies in severity. Although NF1 is highly variable, on average, it shows a similar phenotype to CS.

Conclusions: We established an approach that can be used in the future to quantify variations in facial phenotypes between and within clinical and molecular diagnoses to objectively define and support clinical nosologies.
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http://dx.doi.org/10.1136/jmedgenet-2021-108366DOI Listing
July 2022

MusMorph, a database of standardized mouse morphology data for morphometric meta-analyses.

Sci Data 2022 05 25;9(1):230. Epub 2022 May 25.

Alberta Children's Hospital Research Institute, University of Calgary, 28 Oki Dr NW, Calgary, AB, T3B 6A8, Canada.

Complex morphological traits are the product of many genes with transient or lasting developmental effects that interact in anatomical context. Mouse models are a key resource for disentangling such effects, because they offer myriad tools for manipulating the genome in a controlled environment. Unfortunately, phenotypic data are often obtained using laboratory-specific protocols, resulting in self-contained datasets that are difficult to relate to one another for larger scale analyses. To enable meta-analyses of morphological variation, particularly in the craniofacial complex and brain, we created MusMorph, a database of standardized mouse morphology data spanning numerous genotypes and developmental stages, including E10.5, E11.5, E14.5, E15.5, E18.5, and adulthood. To standardize data collection, we implemented an atlas-based phenotyping pipeline that combines techniques from image registration, deep learning, and morphometrics. Alongside stage-specific atlases, we provide aligned micro-computed tomography images, dense anatomical landmarks, and segmentations (if available) for each specimen (N = 10,056). Our workflow is open-source to encourage transparency and reproducible data collection. The MusMorph data and scripts are available on FaceBase ( www.facebase.org , https://doi.org/10.25550/3-HXMC ) and GitHub ( https://github.com/jaydevine/MusMorph ).
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http://dx.doi.org/10.1038/s41597-022-01338-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9133120PMC
May 2022

Parallels in signaling between development and regeneration in ectodermal organs.

Curr Top Dev Biol 2022 26;149:373-419. Epub 2022 Mar 26.

Program in Craniofacial Biology and Department of Orofacial Sciences, University of California, San Francisco, CA, United States; Department of Pediatrics and Institute for Human Genetics, University of California, San Francisco, CA, United States. Electronic address:

Ectodermal organs originate from the outermost germ layer of the developing embryo and include the skin, hair, tooth, nails, and exocrine glands. These organs develop through tightly regulated, sequential and reciprocal epithelial-mesenchymal crosstalk, and they eventually assume various morphologies and functions while retaining the ability to regenerate. As with many other tissues in the body, the development and morphogenesis of these organs are regulated by a set of common signaling pathways, such as Shh, Wnt, Bmp, Notch, Tgf-β, and Eda. However, subtle differences in the temporal activation, the multiple possible combinations of ligand-receptor activation, the various cofactors, as well as the underlying epigenetic modulation determine how each organ develops into its adult form. Although each organ has been studied separately in considerable detail, the mechanisms underlying the parallels and differences in signaling that regulate their development have rarely been investigated. First, we will use the tooth, the hair follicle, and the mammary gland as representative ectodermal organs to explore how the development of signaling centers and establishment of stem cell populations influence overall growth and morphogenesis. Then we will compare how some of the major signaling pathways (Shh, Wnt, Notch and Yap/Taz) differentially regulate developmental events. Finally, we will discuss how signaling regulates regenerative processes in all three.
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http://dx.doi.org/10.1016/bs.ctdb.2022.02.006DOI Listing
March 2022

CNPY4 inhibits the Hedgehog pathway by modulating membrane sterol lipids.

Nat Commun 2022 05 3;13(1):2407. Epub 2022 May 3.

Program in Craniofacial Biology and Department of Orofacial Sciences, University of California, San Francisco, CA, USA.

The Hedgehog (HH) pathway is critical for development and adult tissue homeostasis. Aberrant HH signaling can lead to congenital malformations and diseases including cancer. Although cholesterol and several oxysterol lipids have been shown to play crucial roles in HH activation, the molecular mechanisms governing their regulation remain unresolved. Here, we identify Canopy4 (CNPY4), a Saposin-like protein, as a regulator of the HH pathway that modulates levels of membrane sterol lipids. Cnpy4 embryos exhibit multiple defects consistent with HH signaling perturbations, most notably changes in digit number. Knockdown of Cnpy4 hyperactivates the HH pathway in vitro and elevates membrane levels of accessible sterol lipids, such as cholesterol, an endogenous ligand involved in HH activation. Our data demonstrate that CNPY4 is a negative regulator that fine-tunes HH signal transduction, revealing a previously undescribed facet of HH pathway regulation that operates through control of membrane composition.
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http://dx.doi.org/10.1038/s41467-022-30186-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9065090PMC
May 2022

A Deep Invertible 3-D Facial Shape Model for Interpretable Genetic Syndrome Diagnosis.

IEEE J Biomed Health Inform 2022 07 1;26(7):3229-3239. Epub 2022 Jul 1.

One of the primary difficulties in treating patients with genetic syndromes is diagnosing their condition. Many syndromes are associated with characteristic facial features that can be imaged and utilized by computer-assisted diagnosis systems. In this work, we develop a novel 3D facial surface modeling approach with the objective of maximizing diagnostic model interpretability within a flexible deep learning framework. Therefore, an invertible normalizing flow architecture is introduced to enable both inferential and generative tasks in a unified and efficient manner. The proposed model can be used (1) to infer syndrome diagnosis and other demographic variables given a 3D facial surface scan and (2) to explain model inferences to non-technical users via multiple interpretability mechanisms. The model was trained and evaluated on more than 4700 facial surface scans from subjects with 47 different syndromes. For the challenging task of predicting syndrome diagnosis given a new 3D facial surface scan, age, and sex of a subject, the model achieves a competitive overall top-1 accuracy of 71%, and a mean sensitivity of 43% across all syndrome classes. We believe that invertible models such as the one presented in this work can achieve competitive inferential performance while greatly increasing model interpretability in the domain of medical diagnosis.
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http://dx.doi.org/10.1109/JBHI.2022.3164848DOI Listing
July 2022

SRSF1 governs progenitor-specific alternative splicing to maintain adult epithelial tissue homeostasis and renewal.

Dev Cell 2022 03 23;57(5):624-637.e4. Epub 2022 Feb 23.

Department of Orofacial Sciences and Program in Craniofacial Biology, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Pediatrics and Institute for Human Genetics, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address:

Alternative splicing generates distinct mRNA variants and is essential for development, homeostasis, and renewal. Proteins of the serine/arginine (SR)-rich splicing factor family are major splicing regulators that are broadly required for organ development as well as cell and organism viability. However, how these proteins support adult organ function remains largely unknown. Here, we used the continuously growing mouse incisor as a model to dissect the functions of the prototypical SR family protein SRSF1 during tissue homeostasis and renewal. We identified an SRSF1-governed alternative splicing network that is specifically required for dental proliferation and survival of progenitors but dispensable for the viability of differentiated cells. We also observed a similar progenitor-specific role of SRSF1 in the small intestinal epithelium, indicating a conserved function of SRSF1 across adult epithelial tissues. Thus, our findings define a regulatory mechanism by which SRSF1 specifically controls progenitor-specific alternative splicing events to support adult tissue homeostasis and renewal.
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http://dx.doi.org/10.1016/j.devcel.2022.01.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8974236PMC
March 2022

LGL1 binds to Integrin β1 and inhibits downstream signaling to promote epithelial branching in the mammary gland.

Cell Rep 2022 02;38(7):110375

School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China. Electronic address:

Branching morphogenesis is a fundamental process by which organs in invertebrates and vertebrates form branches to expand their surface areas. The current dogma holds that directional cell migration determines where a new branch forms and thus patterns branching. Here, we asked whether mouse Lgl1, a homolog of the Drosophila tumor suppressor Lgl, regulates epithelial polarity in the mammary gland. Surprisingly, mammary glands lacking Lgl1 have normal epithelial polarity, but they form fewer branches. Moreover, we find that Lgl1 null epithelium is unable to directionally migrate, suggesting that migration is not essential for mammary epithelial branching as expected. We show that LGL1 binds to Integrin β1 and inhibits its downstream signaling, and Integrin β1 overexpression blocks epithelial migration, thus recapitulating the Lgl1 null phenotype. Altogether, we demonstrate that Lgl1 modulation of Integrin β1 signaling is essential for directional migration and that epithelial branching in invertebrates and the mammary gland is fundamentally distinct.
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http://dx.doi.org/10.1016/j.celrep.2022.110375DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9113222PMC
February 2022

Microbial signals, MyD88, and lymphotoxin drive TNF-independent intestinal epithelial tissue damage.

J Clin Invest 2022 03;132(5)

Department of Medicine, UCSF, San Francisco, California, USA.

Anti-TNF antibodies are effective for treating patients with inflammatory bowel disease (IBD), but many patients fail to respond to anti-TNF therapy, highlighting the importance of TNF-independent disease. We previously demonstrated that acute deletion of 2 IBD susceptibility genes, A20 (Tnfaip3) and Abin-1 (Tnip1), in intestinal epithelial cells (IECs) sensitized mice to both TNF-dependent and TNF-independent death. Here we show that TNF-independent IEC death after A20 and Abin-1 deletion was rescued by germ-free derivation or deletion of MyD88, while deletion of Trif provided only partial protection. Combined deletion of Ripk3 and Casp8, which inhibits both apoptotic and necroptotic death, completely protected against death after acute deletion of A20 and Abin-1 in IECs. A20- and Abin-1-deficient IECs were sensitized to TNF-independent, TNFR1-mediated death in response to lymphotoxin α (LTα) homotrimers. Blockade of LTα in vivo reduced weight loss and improved survival when combined with partial deletion of MyD88. Biopsies of inflamed colon mucosa from patients with IBD exhibited increased LTA and IL1B expression, including a subset of patients with active colitis on anti-TNF therapy. These data show that microbial signals, MyD88, and LTα all contribute to TNF-independent intestinal injury.
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http://dx.doi.org/10.1172/JCI154993DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8884902PMC
March 2022

Application of full-genome analysis to diagnose rare monogenic disorders.

NPJ Genom Med 2021 Sep 23;6(1):77. Epub 2021 Sep 23.

Children's Hospital Oakland Research Institute, Benioff Children's Hospital Oakland, University of California San Francisco, Oakland, CA, USA.

Current genetic testenhancer and narrows the diagnostic intervals for rare diseases provide a diagnosis in only a modest proportion of cases. The Full-Genome Analysis method, FGA, combines long-range assembly and whole-genome sequencing to detect small variants, structural variants with breakpoint resolution, and phasing. We built a variant prioritization pipeline and tested FGA's utility for diagnosis of rare diseases in a clinical setting. FGA identified structural variants and small variants with an overall diagnostic yield of 40% (20 of 50 cases) and 35% in exome-negative cases (8 of 23 cases), 4 of these were structural variants. FGA detected and mapped structural variants that are missed by short reads, including non-coding duplication, and phased variants across long distances of more than 180 kb. With the prioritization algorithm, longer DNA technologies could replace multiple tests for monogenic disorders and expand the range of variants detected. Our study suggests that genomes produced from technologies like FGA can improve variant detection and provide higher resolution genome maps for future application.
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http://dx.doi.org/10.1038/s41525-021-00241-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8460793PMC
September 2021

MEK-inhibitor-mediated rescue of skeletal myopathy caused by activating Hras mutation in a Costello syndrome mouse model.

Dis Model Mech 2022 02 19;15(2). Epub 2021 Nov 19.

Department of Pediatrics, University of California Davis, Sacramento, CA 95817, USA.

Costello syndrome (CS) is a congenital disorder caused by heterozygous activating germline HRAS mutations in the canonical Ras/mitogen-activated protein kinase (Ras/MAPK) pathway. CS is one of the RASopathies, a large group of syndromes caused by mutations within various components of the Ras/MAPK pathway. An important part of the phenotype that greatly impacts quality of life is hypotonia. To gain a better understanding of the mechanisms underlying hypotonia in CS, a mouse model with an activating HrasG12V allele was utilized. We identified a skeletal myopathy that was due, in part, to inhibition of embryonic myogenesis and myofiber formation, resulting in a reduction in myofiber size and number that led to reduced muscle mass and strength. In addition to hyperactivation of the Ras/MAPK and PI3K/AKT pathways, there was a significant reduction in p38 signaling, as well as global transcriptional alterations consistent with the myopathic phenotype. Inhibition of Ras/MAPK pathway signaling using a MEK inhibitor rescued the HrasG12V myopathy phenotype both in vitro and in vivo, demonstrating that increased MAPK signaling is the main cause of the muscle phenotype in CS.
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http://dx.doi.org/10.1242/dmm.049166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8617311PMC
February 2022

Salivary gland: A budding genius.

Dev Cell 2021 08;56(16):2271-2272

Program in Craniofacial Biology and Division of Craniofacial Anomalies, Department of Orofacial Sciences, University of California San Francisco, San Francisco, CA, USA; Division of Medical Genetics, Department of Pediatrics and Institute for Human Genetics, University of California San Francisco, San Francisco, CA, USA. Electronic address:

During salivary gland development, branches arise through formation of new end buds (budding) and division of existing buds (clefting). In a recent Cell study, Wang et al., (2021) report that mouse salivary gland budding is reliant on a delicate interplay between epithelial cells and the extracellular matrix surrounding them.
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http://dx.doi.org/10.1016/j.devcel.2021.08.001DOI Listing
August 2021

Early perturbation of Wnt signaling reveals patterning and invagination-evagination control points in molar tooth development.

Development 2021 07 22;148(14). Epub 2021 Jul 22.

Program in Craniofacial Biology and Department of Orofacial Sciences, University of California, San Francisco, CA 94143, USA.

Tooth formation requires complex signaling interactions both within the oral epithelium and between the epithelium and the underlying mesenchyme. Previous studies of the Wnt/β-catenin pathway have shown that tooth formation is partly inhibited in loss-of-function mutants, and gain-of-function mutants have perturbed tooth morphology. However, the stage at which Wnt signaling is first important in tooth formation remains unclear. Here, using an Fgf8-promoter-driven, and therefore early, deletion of β-catenin in mouse molar epithelium, we found that loss of Wnt/β-catenin signaling completely deletes the molar tooth, demonstrating that this pathway is central to the earliest stages of tooth formation. Early expression of a dominant-active β-catenin protein also perturbs tooth formation, producing a large domed evagination at early stages and supernumerary teeth later on. The early evaginations are associated with premature mesenchymal condensation marker, and are reduced by inhibition of condensation-associated collagen synthesis. We propose that invagination versus evagination morphogenesis is regulated by the relative timing of epithelial versus mesenchymal cell convergence regulated by canonical Wnt signaling. Together, these studies reveal new aspects of Wnt/β-catenin signaling in tooth formation and in epithelial morphogenesis more broadly.
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http://dx.doi.org/10.1242/dev.199685DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8326921PMC
July 2021

Large-scale open-source three-dimensional growth curves for clinical facial assessment and objective description of facial dysmorphism.

Sci Rep 2021 06 9;11(1):12175. Epub 2021 Jun 9.

Department of Human Genetics, KU Leuven, 3000, Leuven, Belgium.

Craniofacial dysmorphism is associated with thousands of genetic and environmental disorders. Delineation of salient facial characteristics can guide clinicians towards a correct clinical diagnosis and understanding the pathogenesis of the disorder. Abnormal facial shape might require craniofacial surgical intervention, with the restoration of normal shape an important surgical outcome. Facial anthropometric growth curves or standards of single inter-landmark measurements have traditionally supported assessments of normal and abnormal facial shape, for both clinical and research applications. However, these fail to capture the full complexity of facial shape. With the increasing availability of 3D photographs, methods of assessment that take advantage of the rich information contained in such images are needed. In this article we derive and present open-source three-dimensional (3D) growth curves of the human face. These are sequences of age and sex-specific expected 3D facial shapes and statistical models of the variation around the expected shape, derived from 5443 3D images. We demonstrate the use of these growth curves for assessing patients and show that they identify normal and abnormal facial morphology independent from age-specific facial features. 3D growth curves can facilitate use of state-of-the-art 3D facial shape assessment by the broader clinical and biomedical research community. This advance in phenotype description will support clinical diagnosis and the understanding of disease pathogenesis including genotype-phenotype relations.
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http://dx.doi.org/10.1038/s41598-021-91465-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8190313PMC
June 2021

Inflation-collapse dynamics drive patterning and morphogenesis in intestinal organoids.

Cell Stem Cell 2021 09 28;28(9):1516-1532.e14. Epub 2021 Apr 28.

Department of Systems Biology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

How stem cells self-organize to form structured tissues is an unsolved problem. Intestinal organoids offer a model of self-organization as they generate stem cell zones (SCZs) of typical size even without a spatially structured environment. Here we examine processes governing the size of SCZs. We improve the viability and homogeneity of intestinal organoid cultures to enable long-term time-lapse imaging of multiple organoids in parallel. We find that SCZs are shaped by fission events under strong control of ion channel-mediated inflation and mechanosensitive Piezo-family channels. Fission occurs through stereotyped modes of dynamic behavior that differ in their coordination of budding and differentiation. Imaging and single-cell transcriptomics show that inflation drives acute stem cell differentiation and induces a stretch-responsive cell state characterized by large transcriptional changes, including upregulation of Piezo1. Our results reveal an intrinsic capacity of the intestinal epithelium to self-organize by modulating and then responding to its mechanical state.
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http://dx.doi.org/10.1016/j.stem.2021.04.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8419000PMC
September 2021

Transit-Amplifying Cells Coordinate Changes in Intestinal Epithelial Cell-Type Composition.

Dev Cell 2021 02 22;56(3):356-365.e9. Epub 2021 Jan 22.

Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address:

Renewing tissues have the remarkable ability to continually produce both proliferative progenitor and specialized differentiated cell types. How are complex milieus of microenvironmental signals interpreted to coordinate tissue-cell-type composition? Here, we investigate the responses of intestinal epithelium to individual and paired perturbations across eight epithelial signaling pathways. Using a high-throughput approach that combines enteroid monolayers and quantitative imaging, we identified conditions that enrich for specific cell types as well as interactions between pathways. Importantly, we found that modulation of transit-amplifying cell proliferation changes the ratio of differentiated secretory to absorptive cell types. These observations highlight an underappreciated role for transit-amplifying cells in the tuning of differentiated cell-type composition.
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http://dx.doi.org/10.1016/j.devcel.2020.12.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7917018PMC
February 2021

Engineering synthetic morphogen systems that can program multicellular patterning.

Science 2020 10;370(6514):327-331

Cell Design Institute, Department of Cellular and Molecular Pharmacology, and Howard Hughes Medical Institute, University of California San Francisco, San Francisco, CA 94158, USA.

In metazoan tissues, cells decide their fates by sensing positional information provided by specialized morphogen proteins. To explore what features are sufficient for positional encoding, we asked whether arbitrary molecules (e.g., green fluorescent protein or mCherry) could be converted into synthetic morphogens. Synthetic morphogens expressed from a localized source formed a gradient when trapped by surface-anchoring proteins, and they could be sensed by synthetic receptors. Despite their simplicity, these morphogen systems yielded patterns reminiscent of those observed in vivo. Gradients could be reshaped by altering anchor density or by providing a source of competing inhibitor. Gradient interpretation could be altered by adding feedback loops or morphogen cascades to receiver cell response circuits. Orthogonal cell-cell communication systems provide insight into morphogen evolution and a platform for engineering tissues.
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http://dx.doi.org/10.1126/science.abc0033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7986291PMC
October 2020

Asymmetric Stratification-Induced Polarity Loss and Coordinated Individual Cell Movements Drive Directional Migration of Vertebrate Epithelium.

Cell Rep 2020 10;33(2):108246

School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China. Electronic address:

Collective migration is essential for development, wound repair, and cancer metastasis. For most collective systems, "leader cells" determine both the direction and the power of the migration. It has remained unclear, however, how the highly polarized vertebrate epithelium migrates directionally during branching morphogenesis. We show here that, unlike in other systems, front-rear polarity of the mammary epithelium is set up by preferential cell proliferation in the front in response to the FGF10 gradient. This leads to frontal stratification, loss of apicobasal polarity, and leader cell formation. Leader cells are a dynamic population and move faster and more directionally toward the FGF10 signal than do follower cells, partly because of their intraepithelial protrusions toward the signal. Together, our data show that directional migration of the mammary epithelium is a unique multistep process and that, despite sharing remarkable cellular and molecular similarities, vertebrate and invertebrate epithelial branching are fundamentally distinct processes.
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http://dx.doi.org/10.1016/j.celrep.2020.108246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7668195PMC
October 2020

KrasP34R and KrasT58I mutations induce distinct RASopathy phenotypes in mice.

JCI Insight 2020 11 5;5(21). Epub 2020 Nov 5.

Department of Pediatrics, University of California, San Francisco, San Francisco, California, USA.

Somatic KRAS mutations are highly prevalent in many cancers. In addition, a distinct spectrum of germline KRAS mutations causes developmental disorders called RASopathies. The mutant proteins encoded by these germline KRAS mutations are less biochemically and functionally activated than those in cancer. We generated mice harboring conditional KrasLSL-P34Rand KrasLSL-T58I knock-in alleles and characterized the consequences of each mutation in vivo. Embryonic expression of KrasT58I resulted in craniofacial abnormalities reminiscent of those seen in RASopathy disorders, and these mice exhibited hyperplastic growth of multiple organs, modest alterations in cardiac valvulogenesis, myocardial hypertrophy, and myeloproliferation. By contrast, embryonic KrasP34R expression resulted in early perinatal lethality from respiratory failure due to defective lung sacculation, which was associated with aberrant ERK activity in lung epithelial cells. Somatic Mx1-Cre-mediated activation in the hematopoietic compartment showed that KrasP34R and KrasT58I expression had distinct signaling effects, despite causing a similar spectrum of hematologic diseases. These potentially novel strains are robust models for investigating the consequences of expressing endogenous levels of hyperactive K-Ras in different developing and adult tissues, for comparing how oncogenic and germline K-Ras proteins perturb signaling networks and cell fate decisions, and for performing preclinical therapeutic trials.
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http://dx.doi.org/10.1172/jci.insight.140495DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710308PMC
November 2020

Dental cell type atlas reveals stem and differentiated cell types in mouse and human teeth.

Nat Commun 2020 09 23;11(1):4816. Epub 2020 Sep 23.

Department of Molecular Neuroscience, Center for Brain Research, Medical University of Vienna, Vienna, Austria.

Understanding cell types and mechanisms of dental growth is essential for reconstruction and engineering of teeth. Therefore, we investigated cellular composition of growing and non-growing mouse and human teeth. As a result, we report an unappreciated cellular complexity of the continuously-growing mouse incisor, which suggests a coherent model of cell dynamics enabling unarrested growth. This model relies on spatially-restricted stem, progenitor and differentiated populations in the epithelial and mesenchymal compartments underlying the coordinated expansion of two major branches of pulpal cells and diverse epithelial subtypes. Further comparisons of human and mouse teeth yield both parallelisms and differences in tissue heterogeneity and highlight the specifics behind growing and non-growing modes. Despite being similar at a coarse level, mouse and human teeth reveal molecular differences and species-specific cell subtypes suggesting possible evolutionary divergence. Overall, here we provide an atlas of human and mouse teeth with a focus on growth and differentiation.
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http://dx.doi.org/10.1038/s41467-020-18512-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7511944PMC
September 2020

FaceBase 3: analytical tools and FAIR resources for craniofacial and dental research.

Development 2020 09 21;147(18). Epub 2020 Sep 21.

Human Medical Genetics and Genomics Program, School of Medicine, University of Colorado, Aurora, CO 80045, USA.

The FaceBase Consortium was established by the National Institute of Dental and Craniofacial Research in 2009 as a 'big data' resource for the craniofacial research community. Over the past decade, researchers have deposited hundreds of annotated and curated datasets on both normal and disordered craniofacial development in FaceBase, all freely available to the research community on the FaceBase Hub website. The Hub has developed numerous visualization and analysis tools designed to promote integration of multidisciplinary data while remaining dedicated to the FAIR principles of data management (findability, accessibility, interoperability and reusability) and providing a faceted search infrastructure for locating desired data efficiently. Summaries of the datasets generated by the FaceBase projects from 2014 to 2019 are provided here. FaceBase 3 now welcomes contributions of data on craniofacial and dental development in humans, model organisms and cell lines. Collectively, the FaceBase Consortium, along with other NIH-supported data resources, provide a continuously growing, dynamic and current resource for the scientific community while improving data reproducibility and fulfilling data sharing requirements.
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http://dx.doi.org/10.1242/dev.191213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7522026PMC
September 2020

Generation of Knockout Gene-Edited Human Intestinal Organoids.

Methods Mol Biol 2020 ;2171:215-230

Department of Pediatrics, University of California, San Francisco, San Francisco, CA, USA.

We discuss a methodology to generate and study knockout gene-edited human intestinal organoids. We describe the generation of knockout human embryonic stem cell lines that we then differentiate into mature human intestinal organoid tissue in Matrigel using several growth factors. We also discuss a pair of assays that can be used to study the integrity of the intestinal epithelial barrier of the human intestinal organoids under inflammatory stress conditions.
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http://dx.doi.org/10.1007/978-1-0716-0747-3_13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8086508PMC
March 2021

LGR5 in breast cancer and ductal carcinoma in situ: a diagnostic and prognostic biomarker and a therapeutic target.

BMC Cancer 2020 Jun 10;20(1):542. Epub 2020 Jun 10.

Department of Anatomy and the Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA, 94143-0452, USA.

Background: Novel biomarkers are required to discern between breast tumors that should be targeted for treatment from those that would never become clinically apparent and/or life threatening for patients. Moreover, therapeutics that specifically target breast cancer (BC) cells with tumor-initiating capacity to prevent recurrence are an unmet need. We investigated the clinical importance of LGR5 in BC and ductal carcinoma in situ (DCIS) to explore LGR5 as a biomarker and a therapeutic target.

Methods: We stained BC (n = 401) and DCIS (n = 119) tissue microarrays with an antibody against LGR5. We examined an LGR5 knockdown ER cell line that was orthotopically transplanted and used for in vitro colony assays. We also determined the tumor-initiating role of Lgr5 in lineage-tracing experiments. Lastly, we transplanted ER patient-derived xenografts into mice that were subsequently treated with a LGR5 antibody drug conjugate (anti-LGR5-ADC).

Results: LGR5 expression correlated with small tumor size, lower grade, lymph node negativity, and ER-positivity. ER patients with LGR5 tumors rarely had recurrence, while high-grade ER patients with LGR5 expression recurred and died due to BC more often. Intriguingly, all the DCIS patients who later died of BC had LGR5-positive tumors. Colony assays and xenograft experiments substantiated a role for LGR5 in ER tumor initiation and subsequent growth, which was further validated by lineage-tracing experiments in ER /triple-negative BC mouse models. Importantly, by utilizing LGR5 patient-derived xenografts, we showed that anti-LGR5-ADC should be considered as a therapeutic for high-grade ER BC.

Conclusion: LGR5 has distinct roles in ER vs. ER BC with potential clinical applicability as a biomarker to identify patients in need of therapy and could serve as a therapeutic target for high-grade ER BC.
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http://dx.doi.org/10.1186/s12885-020-06986-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7285764PMC
June 2020

Fully Automatic Landmarking of Syndromic 3D Facial Surface Scans Using 2D Images.

Sensors (Basel) 2020 Jun 3;20(11). Epub 2020 Jun 3.

Department of Radiology, Alberta Children's Hospital Research Institute and Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Alberta, AB T2N 4N1, Canada.

3D facial landmarks are known to be diagnostically relevant biometrics for many genetic syndromes. The objective of this study was to extend a state-of-the-art image-based 2D facial landmarking algorithm for the challenging task of 3D landmark identification on subjects with genetic syndromes, who often have moderate to severe facial dysmorphia. The automatic 3D facial landmarking algorithm presented here uses 2D image-based facial detection and landmarking models to identify 12 landmarks on 3D facial surface scans. The landmarking algorithm was evaluated using a test set of 444 facial scans with ground truth landmarks identified by two different human observers. Three hundred and sixty nine of the subjects in the test set had a genetic syndrome that is associated with facial dysmorphology. For comparison purposes, the manual landmarks were also used to initialize a non-linear surface-based registration of a non-syndromic atlas to each subject scan. Compared to the average intra- and inter-observer landmark distances of 1.1 mm and 1.5 mm respectively, the average distance between the manual landmark positions and those produced by the automatic image-based landmarking algorithm was 2.5 mm. The average error of the registration-based approach was 3.1 mm. Comparing the distributions of Procrustes distances from the mean for each landmarking approach showed that the surface registration algorithm produces a systemic bias towards the atlas shape. In summary, the image-based automatic landmarking approach performed well on this challenging test set, outperforming a semi-automatic surface registration approach, and producing landmark errors that are comparable to state-of-the-art 3D geometry-based facial landmarking algorithms evaluated on non-syndromic subjects.
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http://dx.doi.org/10.3390/s20113171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7309125PMC
June 2020

Automated syndrome diagnosis by three-dimensional facial imaging.

Genet Med 2020 10 1;22(10):1682-1693. Epub 2020 Jun 1.

Department of Pediatrics, University of Colorado School of Medicine, Aurora, CO, USA.

Purpose: Deep phenotyping is an emerging trend in precision medicine for genetic disease. The shape of the face is affected in 30-40% of known genetic syndromes. Here, we determine whether syndromes can be diagnosed from 3D images of human faces.

Methods: We analyzed variation in three-dimensional (3D) facial images of 7057 subjects: 3327 with 396 different syndromes, 727 of their relatives, and 3003 unrelated, unaffected subjects. We developed and tested machine learning and parametric approaches to automated syndrome diagnosis using 3D facial images.

Results: Unrelated, unaffected subjects were correctly classified with 96% accuracy. Considering both syndromic and unrelated, unaffected subjects together, balanced accuracy was 73% and mean sensitivity 49%. Excluding unrelated, unaffected subjects substantially improved both balanced accuracy (78.1%) and sensitivity (56.9%) of syndrome diagnosis. The best predictors of classification accuracy were phenotypic severity and facial distinctiveness of syndromes. Surprisingly, unaffected relatives of syndromic subjects were frequently classified as syndromic, often to the syndrome of their affected relative.

Conclusion: Deep phenotyping by quantitative 3D facial imaging has considerable potential to facilitate syndrome diagnosis. Furthermore, 3D facial imaging of "unaffected" relatives may identify unrecognized cases or may reveal novel examples of semidominant inheritance.
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http://dx.doi.org/10.1038/s41436-020-0845-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7521994PMC
October 2020

Tools and Concepts for Interrogating and Defining Cellular Identity.

Cell Stem Cell 2020 05;26(5):632-656

Department of Orofacial Sciences, University of California, San Francisco, San Francisco, CA, USA; Program in Craniofacial Biology, University of California, San Francisco, San Francisco, CA, USA; Department of Pediatrics, University of California, San Francisco, San Francisco, CA, USA; Institute for Human Genetics, University of California, San Francisco, San Francisco, CA, USA. Electronic address:

Defining the mechanisms that generate specialized cell types and coordinate their functions is critical for understanding organ development and renewal. New tools and discoveries are challenging and refining our definitions of a cell type. A rapidly growing toolkit for single-cell analyses has expanded the number of markers that can be assigned to a cell simultaneously, revealing heterogeneity within cell types that were previously regarded as homogeneous populations. Additionally, cell types defined by specific molecular markers can exhibit distinct, context-dependent functions; for example, between tissues in homeostasis and those responding to damage. Here we review the current technologies used to identify and characterize cells, and we discuss how experimental and pathological perturbations are adding increasing complexity to our definitions of cell identity.
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http://dx.doi.org/10.1016/j.stem.2020.03.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7250495PMC
May 2020

Lgr5+ telocytes are a signaling source at the intestinal villus tip.

Nat Commun 2020 04 22;11(1):1936. Epub 2020 Apr 22.

Department of Molecular Cell Biology, Weizmann Institute of Science, 7610001, Rehovot, Israel.

The intestinal epithelium is a structured organ composed of crypts harboring Lgr5+ stem cells, and villi harboring differentiated cells. Spatial transcriptomics have demonstrated profound zonation of epithelial gene expression along the villus axis, but the mechanisms shaping this spatial variability are unknown. Here, we combine laser capture micro-dissection and single cell RNA sequencing to uncover spatially zonated populations of mesenchymal cells along the crypt-villus axis. These include villus tip telocytes (VTTs) that express Lgr5, a gene previously considered a specific crypt epithelial stem cell marker. VTTs are elongated cells that line the villus tip epithelium and signal through Bmp morphogens and the non-canonical Wnt5a ligand. Their ablation is associated with perturbed zonation of enterocyte genes induced at the villus tip. Our study provides a spatially-resolved cell atlas of the small intestinal stroma and exposes Lgr5+ villus tip telocytes as regulators of the epithelial spatial expression programs along the villus axis.
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http://dx.doi.org/10.1038/s41467-020-15714-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7176679PMC
April 2020

Safety and immunogenicity of Fc-EDA, a recombinant ectodysplasin A1 replacement protein, in human subjects.

Br J Clin Pharmacol 2020 10 24;86(10):2063-2069. Epub 2020 Apr 24.

Center for Ectodermal Dysplasias, University Hospital Erlangen, Germany.

In X-linked hypohidrotic ectodermal dysplasia, the most frequent ectodermal dysplasia, an inherited deficiency of the signalling protein ectodysplasin A1 (EDA1) impairs the development of the skin and its appendages, various eccrine glands, and dentition. The severe hypohidrosis common to X-linked hypohidrotic ectodermal dysplasia patients may lead to life-threatening hyperthermia, especially during hot weather or febrile illness. Fc-EDA, an EDA1 replacement protein known to prevent the disease in newborn animals, was tested in 2 clinical trials (human adults and neonates) and additionally administered under compassionate use to 3 infants in utero. The data support the safety of Fc-EDA and efficacy if applied prenatally. Anti-drug antibodies were detected after intravenous administration in adult males and nonpregnant females, but not in pregnant women when Fc-EDA was delivered intra-amniotically. Most importantly, there was no detectable immune response to the investigational drug in neonates treated by intravenous infusions and in infants who had received Fc-EDA in utero. In conclusion, the safety profile of this drug encourages further development of prenatal EDA1 replacement therapy.
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http://dx.doi.org/10.1111/bcp.14301DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7495278PMC
October 2020

From gut to glutes: The critical role of niche signals in the maintenance and renewal of adult stem cells.

Curr Opin Cell Biol 2020 04 6;63:88-101. Epub 2020 Feb 6.

Program in Craniofacial Biology and Department of Orofacial Sciences, University of California, San Francisco, CA, USA; Department of Pediatrics and Institute for Human Genetics, University of California, San Francisco, CA, USA. Electronic address:

Stem cell behavior is tightly regulated by spatiotemporal signaling from the niche, which is a four-dimensional microenvironment that can instruct stem cells to remain quiescent, self-renew, proliferate, or differentiate. In this review, we discuss recent advances in understanding the signaling cues provided by the stem cell niche in two contrasting adult tissues, the rapidly cycling intestinal epithelium and the slowly renewing skeletal muscle. Drawing comparisons between these two systems, we discuss the effects of niche-derived growth factors and signaling molecules, metabolic cues, the extracellular matrix and biomechanical cues, and immune signals on stem cells. We also discuss the influence of the niche in defining stem cell identity and function in both normal and pathophysiologic states.
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http://dx.doi.org/10.1016/j.ceb.2020.01.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7247951PMC
April 2020

Molecular and cellular mechanisms of tooth development, homeostasis and repair.

Development 2020 01 24;147(2). Epub 2020 Jan 24.

Program in Craniofacial Biology and Department of Orofacial Sciences, University of California, San Francisco, CA 94143, USA

The tooth provides an excellent system for deciphering the molecular mechanisms of organogenesis, and has thus been of longstanding interest to developmental and stem cell biologists studying embryonic morphogenesis and adult tissue renewal. In recent years, analyses of molecular signaling networks, together with new insights into cellular heterogeneity, have greatly improved our knowledge of the dynamic epithelial-mesenchymal interactions that take place during tooth development and homeostasis. Here, we review recent progress in the field of mammalian tooth morphogenesis and also discuss the mechanisms regulating stem cell-based dental tissue homeostasis, regeneration and repair. These exciting findings help to lay a foundation that will ultimately enable the application of fundamental research discoveries toward therapies to improve oral health.
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http://dx.doi.org/10.1242/dev.184754DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6983727PMC
January 2020
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