Publications by authors named "David R Beier"

75 Publications

Reelin Mediates Hippocampal Cajal-Retzius Cell Positioning and Infrapyramidal Blade Morphogenesis.

J Dev Biol 2020 Sep 18;8(3). Epub 2020 Sep 18.

Center for Developmental Biology and Regenerative Medicine, Seattle Children's Research Institute, Seattle, WA 98101, USA.

We have previously described hypomorphic () mutant mice, , in which the morphology of the dentate gyrus is distinct from that seen in mice. In the mutant, the infrapyramidal blade of the dentate gyrus fails to extend, while the suprapyramidal blade forms with a relatively compact granule neuron layer. Underlying this defect, we now report several developmental anomalies in the dentate gyrus. Most strikingly, the distribution of Cajal-Retzius cells was aberrant; Cajal-Retzius neurons were increased in the suprapyramidal blade, but were greatly reduced along the subpial surface of the prospective infrapyramidal blade. We also observed multiple abnormalities of the fimbriodentate junction. Firstly, progenitor cells were distributed abnormally; the "neurogenic cluster" at the fimbriodentate junction was absent, lacking the normal accumulation of Tbr2-positive intermediate progenitors. However, the number of dividing cells in the dentate gyrus was not generally decreased. Secondly, a defect of secondary glial scaffold formation, limited to the infrapyramidal blade, was observed. The densely radiating glial fibers characteristic of the normal fimbriodentate junction were absent in mutants. These fibers might be required for migration of progenitors, which may account for the failure of neurogenic cluster formation. These findings suggest the importance of the secondary scaffold and neurogenic cluster of the fimbriodentate junction in morphogenesis of the mammalian dentate gyrus. Our study provides direct genetic evidence showing that normal RELN function is required for Cajal-Retzius cell positioning in the dentate gyrus, and for formation of the fimbriodentate junction to promote infrapyramidal blade extension.
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http://dx.doi.org/10.3390/jdb8030020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7558149PMC
September 2020

Differential requirements of tubulin genes in mammalian forebrain development.

PLoS Genet 2019 08 6;15(8):e1008243. Epub 2019 Aug 6.

Division of Human Genetics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States of America.

Tubulin genes encode a series of homologous proteins used to construct microtubules which are essential for multiple cellular processes. Neural development is particularly reliant on functional microtubule structures. Tubulin genes comprise a large family of genes with very high sequence similarity between multiple family members. Human genetics has demonstrated that a large spectrum of cortical malformations are associated with de novo heterozygous mutations in tubulin genes. However, the absolute requirement for many of these genes in development and disease has not been previously tested in genetic loss of function models. Here we directly test the requirement for Tuba1a, Tubb2a and Tubb2b in the mouse by deleting each gene individually using CRISPR-Cas9 genome editing. We show that loss of Tubb2a or Tubb2b does not impair survival but does lead to relatively mild cortical malformation phenotypes. In contrast, loss of Tuba1a is perinatal lethal and leads to significant forebrain dysmorphology. We also present a novel mouse ENU allele of Tuba1a with phenotypes similar to the null allele. This demonstrates the requirements for each of the tubulin genes and levels of functional redundancy are quite different throughout the gene family. The ability of the mouse to survive in the absence of some tubulin genes known to cause disease in humans suggests future intervention strategies for these devastating tubulinopathy diseases.
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http://dx.doi.org/10.1371/journal.pgen.1008243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6697361PMC
August 2019

Loss of function of disrupts collagen post-translational modification and causes musculoskeletal defects.

Dis Model Mech 2019 06 17;12(6). Epub 2019 Jun 17.

Center for Developmental Biology and Regenerative Medicine, Seattle Children's Research Institute, Seattle,  WA 98105, USA

In a screen for organogenesis defects in N-ethyl-N-nitrosourea (ENU)-induced mutant mice, we discovered a line carrying a mutation in [collagen beta(1-O)galactosyltransferase type 1], which is required for proper galactosylation of hydroxylysine residues in a number of collagens. mutant embryos have not been previously characterized; here, we show that they exhibit skeletal and muscular defects. Analysis of mutant-derived embryonic fibroblasts reveals that COLGALT1 acts on collagen IV and VI, and, while collagen VI appears stable and its secretion is not affected, collagen IV accumulates inside of cells and within the extracellular matrix, possibly due to instability and increased degradation. We also generated mutant zebrafish that do not express the duplicated orthologs of mammalian The double-homozygote mutants have muscle defects; they are viable through the larvae stage but do not survive to 10 days post-fertilization. We hypothesize that the mutant could serve as a model of a human connective tissue disorder and/or congenital muscular dystrophy or myopathy.
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http://dx.doi.org/10.1242/dmm.037176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6602307PMC
June 2019

Applicability of the Mutation-Selection Balance Model to Population Genetics of Heterozygous Protein-Truncating Variants in Humans.

Mol Biol Evol 2019 08;36(8):1701-1710

Division of Genetics, Brigham and Women's Hospital, Harvard Medical School, Boston, MA.

The fate of alleles in the human population is believed to be highly affected by the stochastic force of genetic drift. Estimation of the strength of natural selection in humans generally necessitates a careful modeling of drift including complex effects of the population history and structure. Protein-truncating variants (PTVs) are expected to evolve under strong purifying selection and to have a relatively high per-gene mutation rate. Thus, it is appealing to model the population genetics of PTVs under a simple deterministic mutation-selection balance, as has been proposed earlier (Cassa et al. 2017). Here, we investigated the limits of this approximation using both computer simulations and data-driven approaches. Our simulations rely on a model of demographic history estimated from 33,370 individual exomes of the Non-Finnish European subset of the ExAC data set (Lek et al. 2016). Additionally, we compared the African and European subset of the ExAC study and analyzed de novo PTVs. We show that the mutation-selection balance model is applicable to the majority of human genes, but not to genes under the weakest selection.
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http://dx.doi.org/10.1093/molbev/msz092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6738481PMC
August 2019

Reply to 'Selective effects of heterozygous protein-truncating variants'.

Nat Genet 2019 01;51(1):3-4

Division of Genetics, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.

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http://dx.doi.org/10.1038/s41588-018-0301-yDOI Listing
January 2019

Optimizing Genomic Methods for Mapping and Identification of Candidate Variants in ENU Mutagenesis Screens Using Inbred Mice.

G3 (Bethesda) 2018 02 2;8(2):401-409. Epub 2018 Feb 2.

Center for Developmental Biology and Regenerative Medicine, Seattle Children's Research Institute, Seattle, Washington 98101

Positional cloning of ENU-induced mutations has traditionally relied on analysis of polymorphic variation between two strains. In contrast, the application of whole-genome sequencing (WGS) has enabled gene discovery in mutant lines maintained on an inbred genetic background. This approach utilizes genetic variation derived from ENU-induced variants for mapping and reduces the likelihood of phenotypic variation, making it an ideal method for genetic modifier screening. Here, we describe the results of such a screen, wherein we determined the minimal number of mutant genomic DNA samples to include in our analyses and improved the sensitivity of our screen by individually barcoding each genomic DNA library. We present several unique cases to illustrate this approach's efficacy, including the discovery of two distinct mutations that generate essentially identical mutant phenotypes, the ascertainment of a non-ENU-induced candidate variant through homozygosity mapping, and an approach for the identification of putative dominant genetic modifiers.
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http://dx.doi.org/10.1534/g3.117.300292DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5919724PMC
February 2018

Generation of a multipurpose mouse allele by targeted gene trapping.

Dis Model Mech 2017 07 19;10(7):909-922. Epub 2017 Apr 19.

Department of Biochemistry, Chicago College of Osteopathic Medicine, Midwestern University, Downers Grove, IL 60515, USA

Gene trap mutagenesis is a powerful tool to create loss-of-function mutations in mice and other model organisms. Modifications of traditional gene trap cassettes, including addition of conditional features in the form of Flip-excision (FlEx) arrays to enable directional gene trap cassette inversions by Cre and Flpe site-specific recombinases, greatly enhanced their experimental potential. By taking advantage of these conditional gene trap cassettes, we developed a generic strategy for generating conditional mutations and validated this strategy in mice carrying a multipurpose allele of the transcription factor gene. We demonstrate that the gene trap insertion creates a null mutation replicating the Pierre Robin sequence-type cleft palate phenotype of other mutant mice. Consecutive breeding to and deleter mice spatially restricted loss to regions of the forebrain expressing the homeobox gene , demonstrating the utility of the technology for the analysis of tissue-specific gene functions.
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http://dx.doi.org/10.1242/dmm.029561DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5536910PMC
July 2017

Estimating the selective effects of heterozygous protein-truncating variants from human exome data.

Nat Genet 2017 May 3;49(5):806-810. Epub 2017 Apr 3.

Division of Genetics, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.

The evolutionary cost of gene loss is a central question in genetics and has been investigated in model organisms and human cell lines. In humans, tolerance of the loss of one or both functional copies of a gene is related to the gene's causal role in disease. However, estimates of the selection and dominance coefficients in humans have been elusive. Here we analyze exome sequence data from 60,706 individuals to make genome-wide estimates of selection against heterozygous loss of gene function. Using this distribution of selection coefficients for heterozygous protein-truncating variants (PTVs), we provide corresponding Bayesian estimates for individual genes. We find that genes under the strongest selection are enriched in embryonic lethal mouse knockouts, Mendelian disease-associated genes, and regulators of transcription. Screening by essentiality, we find a large set of genes under strong selection that are likely to have crucial functions but have not yet been thoroughly characterized.
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http://dx.doi.org/10.1038/ng.3831DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5618255PMC
May 2017

C-Terminal Region Truncation of RELN Disrupts an Interaction with VLDLR, Causing Abnormal Development of the Cerebral Cortex and Hippocampus.

J Neurosci 2017 01;37(4):960-971

Center for Developmental Biology and Regenerative Medicine and

We discovered a hypomorphic reelin (Reln) mutant with abnormal cortical lamination and no cerebellar hypoplasia. This mutant, Reln, carries a chemically induced splice-site mutation that truncates the C-terminal region (CTR) domain of RELN protein and displays remarkably distinct phenotypes from reeler The mutant does not have an inverted cortex, but cortical neurons overmigrate and invade the marginal zone, which are characteristics similar to a phenotype seen in the cerebral cortex of Vldlr mice. The dentate gyrus shows a novel phenotype: the infrapyramidal blade is absent, while the suprapyramidal blade is present and laminated. Genetic epistasis analysis showed that Reln/Apoer2 double homozygotes have phenotypes akin to those of reeler mutants, while Reln/Vldlr mice do not. Given that the receptor double knock-out mice resemble reeler mutants, we infer that Reln/Apoer2 double homozygotes have both receptor pathways disrupted. This suggests that CTR-truncation disrupts an interaction with VLDLR (very low-density lipoprotein receptor), while the APOER2 signaling pathway remains active, which accounts for the hypomorphic phenotype in Reln mice. A RELN-binding assay confirms that CTR truncation significantly decreases RELN binding to VLDLR, but not to APOER2. Together, the in vitro and in vivo results demonstrate that the CTR domain confers receptor-binding specificity of RELN.

Significance Statement: Reelin signaling is important for brain development and is associated with human type II lissencephaly. Reln mutations in mice and humans are usually associated with cerebellar hypoplasia. A new Reln mutant with a truncation of the C-terminal region (CTR) domain shows that Reln mutation can cause abnormal phenotypes in the cortex and hippocampus without cerebellar hypoplasia. Genetic analysis suggested that CTR truncation disrupts an interaction with the RELN receptor VLDLR (very low-density lipoprotein receptor); this was confirmed by a RELN-binding assay. This result provides a mechanistic explanation for the hypomorphic phenotype of the CTR-deletion mutant, and further suggests that Reln mutations may cause more subtle forms of human brain malformation than classic lissencephalies.
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http://dx.doi.org/10.1523/JNEUROSCI.1826-16.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5296787PMC
January 2017

Reduction of ciliary length through pharmacologic or genetic inhibition of CDK5 attenuates polycystic kidney disease in a model of nephronophthisis.

Hum Mol Genet 2016 06 5;25(11):2245-2255. Epub 2016 Apr 5.

Department of Rare Diseases, Sanofi-Genzyme R&D Center, 49 New York Avenue, Framingham, MA 01701, USA

Polycystic kidney diseases (PKDs) comprise a subgroup of ciliopathies characterized by the formation of fluid-filled kidney cysts and progression to end-stage renal disease. A mechanistic understanding of cystogenesis is crucial for the development of viable therapeutic options. Here, we identify CDK5, a kinase active in post mitotic cells, as a new and important mediator of PKD progression. We show that long-lasting attenuation of PKD in the juvenile cystic kidneys (jck) mouse model of nephronophthisis by pharmacological inhibition of CDK5 using either R-roscovitine or S-CR8 is accompanied by sustained shortening of cilia and a more normal epithelial phenotype, suggesting this treatment results in a reprogramming of cellular differentiation. Also, a knock down of Cdk5 in jck cells using small interfering RNA results in significant shortening of ciliary length, similar to what we observed with R-roscovitine. Finally, conditional inactivation of Cdk5 in the jck mice significantly attenuates cystic disease progression and is associated with shortening of ciliary length as well as restoration of cellular differentiation. Our results suggest that CDK5 may regulate ciliary length by affecting tubulin dynamics via its substrate collapsin response mediator protein 2. Taken together, our data support therapeutic approaches aimed at restoration of ciliogenesis and cellular differentiation as a promising strategy for the treatment of renal cystic diseases.
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http://dx.doi.org/10.1093/hmg/ddw093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5081056PMC
June 2016

Dysfunction of intraflagellar transport-A causes hyperphagia-induced obesity and metabolic syndrome.

Dis Model Mech 2016 07 11;9(7):789-98. Epub 2016 May 11.

Department of Anatomy and Cell Biology and The Kidney Institute, University of Kansas Medical Center, Kansas City, KS 66160, USA Genetics Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA

Primary cilia extend from the plasma membrane of most vertebrate cells and mediate signaling pathways. Ciliary dysfunction underlies ciliopathies, which are genetic syndromes that manifest multiple clinical features, including renal cystic disease and obesity. THM1 (also termed TTC21B or IFT139) encodes a component of the intraflagellar transport-A complex and mutations in THM1 have been identified in 5% of individuals with ciliopathies. Consistent with this, deletion of murine Thm1 during late embryonic development results in cystic kidney disease. Here, we report that deletion of murine Thm1 during adulthood results in obesity, diabetes, hypertension and fatty liver disease, with gender differences in susceptibility to weight gain and metabolic dysfunction. Pair-feeding of Thm1 conditional knock-out mice relative to control littermates prevented the obesity and related disorders, indicating that hyperphagia caused the obese phenotype. Thm1 ablation resulted in increased localization of adenylyl cyclase III in primary cilia that were shortened, with bulbous distal tips on neurons of the hypothalamic arcuate nucleus, an integrative center for signals that regulate feeding and activity. In pre-obese Thm1 conditional knock-out mice, expression of anorexogenic pro-opiomelanocortin (Pomc) was decreased by 50% in the arcuate nucleus, which likely caused the hyperphagia. Fasting of Thm1 conditional knock-out mice did not alter Pomc nor orexogenic agouti-related neuropeptide (Agrp) expression, suggesting impaired sensing of changes in peripheral signals. Together, these data indicate that the Thm1-mutant ciliary defect diminishes sensitivity to feeding signals, which alters appetite regulation and leads to hyperphagia, obesity and metabolic disease.
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http://dx.doi.org/10.1242/dmm.025791DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4958314PMC
July 2016

Improvement of ENU Mutagenesis Efficiency Using Serial Injection and Mismatch Repair Deficiency Mice.

PLoS One 2016 21;11(7):e0159377. Epub 2016 Jul 21.

Center for Developmental Biology and Regenerative Medicine, Seattle Children's Research Institute, Seattle, WA, United States of America.

ENU mutagenesis is a powerful method for generating novel lines of mice that are informative with respect to both fundamental biological processes and human disease. Rapid developments in genomic technology have made the task of identifying causal mutations by positional cloning remarkably efficient. One limitation of this approach remains the mutation frequency achievable using standard treatment protocols, which currently generate approximately 1-2 sequence changes per megabase when optimized. In this study we used two strategies to attempt to increase the number of mutations induced by ENU treatment. One approach employed mice carrying a mutation in the DNA repair enzyme Msh6. The second strategy involved injection of ENU to successive generations of mice. To evaluate the number of ENU-induced mutations, single mice or pooled samples were analyzed using whole exome sequencing. The results showed that there is considerable variability in the induced mutation frequency using these approaches, but an overall increase in ENU-induced variants from one generation to another was observed. The analysis of the mice deficient for Msh6 also showed an increase in the ENU-induced variants compared to the wild-type ENU-treated mice. However, in both cases the increase in ENU-induced mutation frequency was modest.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0159377PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4956170PMC
July 2017

Mutations in Dnaaf1 and Lrrc48 Cause Hydrocephalus, Laterality Defects, and Sinusitis in Mice.

G3 (Bethesda) 2016 08 9;6(8):2479-87. Epub 2016 Aug 9.

Center for Developmental Biology and Regenerative Medicine, Seattle Children's Research Institute, Washington 98101 Division of Genetic Medicine, Department of Pediatrics, University of Washington Medical School, Seattle, Washington

We have previously described a forward genetic screen in mice for abnormalities of brain development. Characterization of two hydrocephalus mutants by whole-exome sequencing after whole-genome SNP mapping revealed novel recessive mutations in Dnaaf1 and Lrrc48 Mouse mutants of these two genes have not been previously reported. The Dnaaf1 mutant carries a mutation at the splice donor site of exon 4, which results in abnormal transcripts. The Lrrc48 mutation is a missense mutation at a highly conserved leucine residue, which is also associated with a decrease in Lrrc48 transcription. Both Dnaaf1 and Lrrc48 belong to a leucine-rich repeat-containing protein family and are components of the ciliary axoneme. Their Chlamydomonas orthologs are known to be required for normal ciliary beat frequency or flagellar waveform, respectively. Some Dnaaf1 or Lrrc48 homozygote mutants displayed laterality defects, suggesting a motile cilia defect in the embryonic node. Mucus accumulation and neutrophil infiltration in the maxillary sinuses suggested sinusitis. Dnaaf1 mutants showed postnatal lethality, and none survived to weaning age. Lrrc48 mutants survive to adulthood, but had male infertility. ARL13B immunostaining showed the presence of motile cilia in the mutants, and the distal distribution of DNAH9 in the axoneme of upper airway motile cilia appeared normal. The phenotypic abnormalities suggest that mutations in Dnaaf1 and Lrrc48 cause defects in motile cilia function.
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http://dx.doi.org/10.1534/g3.116.030791DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4978901PMC
August 2016

High-resolution genetic localization of a modifying locus affecting disease severity in the juvenile cystic kidneys (jck) mouse model of polycystic kidney disease.

Authors:
David R Beier

Mamm Genome 2016 06 25;27(5-6):191-9. Epub 2016 Apr 25.

Center for Developmental Biology and Regenerative Medicine, Seattle Children's Research Institute, Seattle, WA, USA.

We have previously demonstrated that a locus on proximal Chr 4 modifies disease severity in the juvenile cystic kidney (jck) mouse, a model of polycystic kidney disease (PKD) that carries a mutation of the Nek8 serine-threonine kinase. In this study, we used QTL analysis of independently constructed B6.D2 congenic lines to confirm this and showed that this locus has a highly significant effect. We constructed sub-congenic lines to more specifically localize the modifier and have determined it resides in a 3.2 Mb interval containing 28 genes. These include Invs and Anks6, which are both excellent candidates for the modifier as mutations in these genes result in PKD and both genes are known to genetically and physically interact with Nek8. However, examination of strain-specific DNA sequence and kidney expression did not reveal clear differences that might implicate either gene as a modifier of PKD severity. The fact that our high-resolution analysis did not yield an unambiguous result highlights the challenge of establishing the causality of strain-specific variants as genetic modifiers, and suggests that alternative strategies be considered.
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http://dx.doi.org/10.1007/s00335-016-9633-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5039125PMC
June 2016

Variant mapping and mutation discovery in inbred mice using next-generation sequencing.

BMC Genomics 2015 Nov 9;16:913. Epub 2015 Nov 9.

Department of Pediatrics, University of Washington School of Medicine, Seattle, WA, USA.

Background: The development of powerful new methods for DNA sequencing enable the discovery of sequence variants, their utilization for the mapping of mutant loci, and the identification of causal variants in a single step. We have applied this approach for the analysis of ENU-mutagenized mice maintained on an inbred background.

Results: We ascertained ENU-induced variants in four different phenotypically mutant lines. These were then used as informative markers for positional cloning of the mutated genes. We tested both whole genome (WGS) and whole exome (WES) datasets.

Conclusion: Both approaches were successful as a means to localize a region of homozygosity, as well as identifying mutations of candidate genes, which could be individually assessed. As expected, the WGS strategy was more reliable, since many more ENU-induced variants were ascertained.
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http://dx.doi.org/10.1186/s12864-015-2173-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4640199PMC
November 2015

Grhl2 is required in nonneural tissues for neural progenitor survival and forebrain development.

Genesis 2015 Sep 22;53(9):573-582. Epub 2015 Jul 22.

Division of Human Genetics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio.

Grainyhead-like genes are part of a highly conserved gene family that play a number of roles in ectoderm development and maintenance in mammals. Here we identify a novel allele of Grhl2, cleft-face 3 (clft3), in a mouse line recovered from an ENU mutagenesis screen for organogenesis defects. Homozygous clft3 mutants have a number of phenotypes in common with other alleles of Grhl2. We note a significant effect of genetic background on the clft3 phenotype. One of these is a reduction in size of the telencephalon where we find abnormal patterns of neural progenitor mitosis and apoptosis in mutant brains. Interestingly, Grhl2 is not expressed in the developing forebrain, suggesting this is a survival factor for neural progenitors exerting a paracrine effect on the neural tissue from the overlying ectoderm where Grhl2 is highly expressed. genesis 53:573-582, 2015. © 2015 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/dvg.22875DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4713386PMC
September 2015

Amyloid accelerates tau propagation and toxicity in a model of early Alzheimer's disease.

Acta Neuropathol Commun 2015 Mar 24;3:14. Epub 2015 Mar 24.

Introduction: In early stages of Alzheimer's disease (AD), neurofibrillary tangles (NFT) are largely restricted to the entorhinal cortex and medial temporal lobe. At later stages, when clinical symptoms generally occur, NFT involve widespread limbic and association cortices. At this point in the disease, amyloid plaques are also abundantly distributed in the cortex. This observation from human neuropathological studies led us to pose two alternative hypotheses: that amyloid in the cortex is permissive for the spread of tangles from the medial temporal lobe, or that these are co-occurring but not causally related events simply reflecting progression of AD pathology.

Results: We now directly test the hypothesis that cortical amyloid acts as an accelerant for spreading of tangles beyond the medial temporal lobe. We crossed rTgTauEC transgenic mice that demonstrate spread of tau from entorhinal cortex to other brain structures at advanced age with APP/PS1 mice, and examined mice with either NFTs, amyloid pathology, or both. We show that concurrent amyloid deposition in the cortex 1) leads to a dramatic increase in the speed of tau propagation and an extraordinary increase in the spread of tau to distal brain regions, and 2) significantly increases tau-induced neuronal loss.

Conclusions: These data strongly support the hypothesis that cortical amyloid accelerates the spread of tangles throughout the cortex and amplifies tangle-associated neural system failure in AD.
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http://dx.doi.org/10.1186/s40478-015-0199-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4371800PMC
March 2015

ANKS6 is the critical activator of NEK8 kinase in embryonic situs determination and organ patterning.

Nat Commun 2015 Jan 20;6:6023. Epub 2015 Jan 20.

1] Department of Systems Biology, Harvard Medical School, 4 Blackfan Circle, HIM 568, Boston, Massachussetts 02115, USA [2] Renal Division, Brigham and Women's Hospital, Boston, Massachussetts 02115, USA.

The ciliary kinase NEK8 plays a critical role in situs determination and cystic kidney disease, yet its exact function remains unknown. In this study, we identify ANKS6 as a target and activator of NEK8. ANKS6 requires NEK8 for localizing to the ciliary inversin compartment (IC) and activates NEK8 by binding to its kinase domain. Here we demonstrate the functional importance of this interaction through the analysis of two novel mouse mutations, Anks6(Streaker) and Nek8(Roc). Both display heterotaxy, cardiopulmonary malformations and cystic kidneys, a syndrome also characteristic of mutations in Invs and Nphp3, the other known components of the IC. The Anks6(Strkr) mutation decreases ANKS6 interaction with NEK8, precluding NEK8 activation. The Nek8(Roc) mutation inactivates NEK8 kinase function while preserving ANKS6 localization to the IC. Together, these data reveal the crucial role of NEK8 kinase activation within the IC, promoting proper left-right patterning, cardiopulmonary development and renal morphogenesis.
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http://dx.doi.org/10.1038/ncomms7023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4361001PMC
January 2015

Downregulating hedgehog signaling reduces renal cystogenic potential of mouse models.

J Am Soc Nephrol 2014 Oct 3;25(10):2201-12. Epub 2014 Apr 3.

Genetics Division, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts; Center for Developmental Biology and Regenerative Medicine, Seattle Children's Research Institute, Seattle, Washington.

Renal cystic diseases are a leading cause of renal failure. Mutations associated with renal cystic diseases reside in genes encoding proteins that localize to primary cilia. These cystoproteins can disrupt ciliary structure or cilia-mediated signaling, although molecular mechanisms connecting cilia function to renal cystogenesis remain unclear. The ciliary gene, Thm1(Ttc21b), negatively regulates Hedgehog signaling and is most commonly mutated in ciliopathies. We report that loss of murine Thm1 causes cystic kidney disease, with persistent proliferation of renal cells, elevated cAMP levels, and enhanced expression of Hedgehog signaling genes. Notably, the cAMP-mediated cystogenic potential of Thm1-null kidney explants was reduced by genetically deleting Gli2, a major transcriptional activator of the Hedgehog pathway, or by culturing with small molecule Hedgehog inhibitors. These Hedgehog inhibitors acted independently of protein kinase A and Wnt inhibitors. Furthermore, simultaneous deletion of Gli2 attenuated the renal cystic disease associated with deletion of Thm1. Finally, transcripts of Hedgehog target genes increased in cystic kidneys of two other orthologous mouse mutants, jck and Pkd1, and Hedgehog inhibitors reduced cystogenesis in jck and Pkd1 cultured kidneys. Thus, enhanced Hedgehog activity may have a general role in renal cystogenesis and thereby present a novel therapeutic target.
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http://dx.doi.org/10.1681/ASN.2013070735DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4178433PMC
October 2014

NEK8 links the ATR-regulated replication stress response and S phase CDK activity to renal ciliopathies.

Mol Cell 2013 Aug;51(4):423-39

Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA 94025, USA.

Renal ciliopathies are a leading cause of kidney failure, but their exact etiology is poorly understood. NEK8/NPHP9 is a ciliary kinase associated with two renal ciliopathies in humans and mice, nephronophthisis (NPHP) and polycystic kidney disease. Here, we identify NEK8 as a key effector of the ATR-mediated replication stress response. Cells lacking NEK8 form spontaneous DNA double-strand breaks (DSBs) that further accumulate when replication forks stall, and they exhibit reduced fork rates, unscheduled origin firing, and increased replication fork collapse. NEK8 suppresses DSB formation by limiting cyclin A-associated CDK activity. Strikingly, a mutation in NEK8 that is associated with renal ciliopathies affects its genome maintenance functions. Moreover, kidneys of NEK8 mutant mice accumulate DNA damage, and loss of NEK8 or replication stress similarly disrupts renal cell architecture in a 3D-culture system. Thus, NEK8 is a critical component of the DNA damage response that links replication stress with cystic kidney disorders.
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http://dx.doi.org/10.1016/j.molcel.2013.08.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3790667PMC
August 2013

A forward genetic screen in mice identifies mutants with abnormal cortical patterning.

Cereb Cortex 2015 Jan 22;25(1):167-79. Epub 2013 Aug 22.

Genetics Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA Center for Developmental Biology and Regenerative Medicine, Seattle Children's Research Institute, University of Washington School of Medicine, Seattle, WA 98101, USA.

Formation of a 6-layered cortical plate and axon tract patterning are key features of cerebral cortex development. Abnormalities of these processes may be the underlying cause for a range of functional disabilities seen in human neurodevelopmental disorders. To identify mouse mutants with defects in cortical lamination or corticofugal axon guidance, N-ethyl-N-nitrosourea (ENU) mutagenesis was performed using mice expressing LacZ reporter genes in layers II/III and V of the cortex (Rgs4-lacZ) or in corticofugal axons (TAG1-tau-lacZ). Four lines with abnormal cortical lamination have been identified. One of these was a splice site mutation in reelin (Reln) that results in a premature stop codon and the truncation of the C-terminal region (CTR) domain of reelin. Interestingly, this novel allele of Reln did not display cerebellar malformation or ataxia, and this is the first report of a Reln mutant without a cerebellar defect. Four lines with abnormal cortical axon development were also identified, one of which was found by whole-genome resequencing to carry a mutation in Lrp2. These findings demonstrated that the application of ENU mutagenesis to mice carrying transgenic reporters marking cortical anatomy is a sensitive and specific method to identify mutations that disrupt patterning of the developing brain.
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http://dx.doi.org/10.1093/cercor/bht209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4259276PMC
January 2015

Loss of the ciliary kinase Nek8 causes left-right asymmetry defects.

J Am Soc Nephrol 2013 Jan;24(1):100-12

Division of Genetics, Brigham and Women's Hospital, Boston, MA 02115, USA.

A missense mutation in mouse Nek8, which encodes a ciliary kinase, produces the juvenile cystic kidneys (jck) model of polycystic kidney disease, but the functions of Nek8 are incompletely understood. Here, we generated a Nek8-null allele and found that homozygous mutant mice die at birth and exhibit randomization of left-right asymmetry, cardiac anomalies, and glomerular kidney cysts. The requirement for Nek8 in left-right patterning is conserved, as knockdown of the zebrafish ortholog caused randomized heart looping. Ciliogenesis was intact in Nek8-deficient embryos and cells, but we observed misexpression of left-sided marker genes early in development, suggesting that nodal ciliary signaling was perturbed. We also generated jck/Nek8 compound heterozygotes; these mutants developed less severe cystic disease than jck homozygotes and provided genetic evidence that the jck allele may encode a gain-of-function protein. Notably, NEK8 and polycystin-2 (PC2) proteins interact, and we found that Nek8(-/-) and Pkd2(-/-) embryonic phenotypes are strikingly similar. Nek8-deficient embryos and cells did express PC2 normally, which localized properly to the cilia. However, similar to cells lacking PC2, NEK8-depleted inner medullary collecting duct cells exhibited a defective response to fluid shear, suggesting that NEK8 may play a role in mediating PC2-dependent signaling.
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http://dx.doi.org/10.1681/ASN.2012050490DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3537214PMC
January 2013

A spontaneous Fatp4/Scl27a4 splice site mutation in a new murine model for congenital ichthyosis.

PLoS One 2012 30;7(11):e50634. Epub 2012 Nov 30.

Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, United States of America.

Congenital ichthyoses are life-threatening conditions in humans. We describe here the identification and molecular characterization of a novel recessive mutation in mice that results in newborn lethality with severe congenital lamellar ichthyosis. Mutant newborns have a taut, shiny, non-expandable epidermis that resembles cornified manifestations of autosomal-recessive congenital ichthyosis in humans. The skin is stretched so tightly that the newborn mice are immobilized. The genetic defect was mapped to a region near the proximal end of chromosome 2 by SNP analysis, suggesting Fatp4/Slc27a4 as a candidate gene. FATP4 mutations in humans cause ichthyosis prematurity syndrome (IPS), and mutations of Fatp4 in mice have previously been found to cause a phenotype that resembles human congenital ichthyoses. Characterization of the Fatp4 cDNA revealed a fusion of exon 8 to exon 10, with deletion of exon 9. Genomic sequencing identified an A to T mutation in the splice donor sequence at the 3'-end of exon 9. Loss of exon 9 results in a frame shift mutation upstream from the conserved very long-chain acyl-CoA synthase (VLACS) domain. Histological studies revealed that the mutant mice have defects in keratinocyte differentiation, along with hyperproliferation of the stratum basale of the epidermis, a hyperkeratotic stratum corneum, and reduced numbers of secondary hair follicles. Since Fatp4 protein is present primarily at the stratum granulosum and the stratum spinosum, the hyperproliferation and the alterations in hair follicle induction suggest that very long chain fatty acids, in addition to being required for normal cornification, may influence signals from the stratum corneum to the basal cells that help to orchestrate normal skin differentiation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0050634PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3511458PMC
May 2013

Mutation of the BiP/GRP78 gene causes axon outgrowth and fasciculation defects in the thalamocortical connections of the mammalian forebrain.

J Comp Neurol 2013 Feb;521(3):677-96

Department of Cell Biology, University of Virginia, Charlottesville, Virginia 22908, USA.

Proper development of axonal connections is essential for brain function. A forward genetic screen for mice with defects in thalamocortical development previously isolated a mutant called baffled. Here we describe the axonal defects of baffled in further detail and identify a point mutation in the Hspa5 gene, encoding the endoplasmic reticulum chaperone BiP/GRP78. This hypomorphic mutation of BiP disrupts proper development of the thalamocortical axon projection and other forebrain axon tracts, as well as cortical lamination. In baffled mutant brains, a reduced number of thalamic axons innervate the cortex by the time of birth. Thalamocortical and corticothalamic axons are delayed, overfasciculated, and disorganized along their pathway through the ventral telencephalon. Furthermore, dissociated mutant neurons show reduced axon extension in vitro. Together, these findings demonstrate a sensitive requirement for the endoplasmic reticulum chaperone BiP/GRP78 during axon outgrowth and pathfinding in the developing mammalian brain.
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http://dx.doi.org/10.1002/cne.23199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3515720PMC
February 2013

Mutation mapping and identification by whole-genome sequencing.

Genome Res 2012 Aug 3;22(8):1541-8. Epub 2012 May 3.

Genetics Division, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.

Genetic mapping of mutations in model systems has facilitated the identification of genes contributing to fundamental biological processes including human diseases. However, this approach has historically required the prior characterization of informative markers. Here we report a fast and cost-effective method for genetic mapping using next-generation sequencing that combines single nucleotide polymorphism discovery, mutation localization, and potential identification of causal sequence variants. In contrast to prior approaches, we have developed a hidden Markov model to narrowly define the mutation area by inferring recombination breakpoints of chromosomes in the mutant pool. In addition, we created an interactive online software resource to facilitate automated analysis of sequencing data and demonstrate its utility in the zebrafish and mouse models. Our novel methodology and online tools will make next-generation sequencing an easily applicable resource for mutation mapping in all model systems.
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http://dx.doi.org/10.1101/gr.135541.111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3409267PMC
August 2012

Production of a natural antibody to the mouse polyoma virus is a multigenic trait.

G3 (Bethesda) 2012 Mar 1;2(3):353-5. Epub 2012 Mar 1.

MA/MyJ mice express a natural antibody to the highly oncogenic polyoma virus. C57BR/cdJ mice lack this antibody but mount an adaptive T-cell response to the virus. Analysis of F2 progeny of a cross between these strains reveals a pattern of inheritance of expression of the natural antibody involving two genes in an epistatic relationship.
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http://dx.doi.org/10.1534/g3.111.001701DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3291505PMC
March 2012

ENU mutagenesis in mice identifies candidate genes for hypogonadism.

Mamm Genome 2012 Jun 19;23(5-6):346-55. Epub 2012 Jan 19.

Division of Endocrinology, Department of Medicine, Northwestern University Feinberg School of Medicine, 303 E. Superior Street, Chicago, IL 60611, USA.

Genome-wide mutagenesis was performed in mice to identify candidate genes for male infertility, for which the predominant causes remain idiopathic. Mice were mutagenized using N-ethyl-N-nitrosourea (ENU), bred, and screened for phenotypes associated with the male urogenital system. Fifteen heritable lines were isolated and chromosomal loci were assigned using low-density genome-wide SNP arrays. Ten of the 15 lines were pursued further using higher-resolution SNP analysis to narrow the candidate gene regions. Exon sequencing of candidate genes identified mutations in mice with cystic kidneys (Bicc1), cryptorchidism (Rxfp2), restricted germ cell deficiency (Plk4), and severe germ cell deficiency (Prdm9). In two other lines with severe hypogonadism, candidate sequencing failed to identify mutations, suggesting defects in genes with previously undocumented roles in gonadal function. These genomic intervals were sequenced in their entirety and a candidate mutation was identified in SnrpE in one of the two lines. The line harboring the SnrpE variant retains substantial spermatogenesis despite small testis size, an unusual phenotype. In addition to the reproductive defects, heritable phenotypes were observed in mice with ataxia (Myo5a), tremors (Pmp22), growth retardation (unknown gene), and hydrocephalus (unknown gene). These results demonstrate that the ENU screen is an effective tool for identifying potential causes of male infertility.
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http://dx.doi.org/10.1007/s00335-011-9388-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3358541PMC
June 2012

Cholesterol metabolism is required for intracellular hedgehog signal transduction in vivo.

PLoS Genet 2011 Sep 1;7(9):e1002224. Epub 2011 Sep 1.

Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States of America.

We describe the rudolph mouse, a mutant with striking defects in both central nervous system and skeletal development. Rudolph is an allele of the cholesterol biosynthetic enzyme, hydroxysteroid (17-beta) dehydrogenase 7, which is an intriguing finding given the recent implication of oxysterols in mediating intracellular Hedgehog (Hh) signaling. We see an abnormal sterol profile and decreased Hh target gene induction in the rudolph mutant, both in vivo and in vitro. Reduced Hh signaling has been proposed to contribute to the phenotypes of congenital diseases of cholesterol metabolism. Recent in vitro and pharmacological data also indicate a requirement for intracellular cholesterol synthesis for proper regulation of Hh activity via Smoothened. The data presented here are the first in vivo genetic evidence supporting both of these hypotheses, revealing a role for embryonic cholesterol metabolism in both CNS development and normal Hh signaling.
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http://dx.doi.org/10.1371/journal.pgen.1002224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3164675PMC
September 2011

The naive airway hyperresponsiveness of the A/J mouse is Kit-mediated.

Proc Natl Acad Sci U S A 2011 Aug 18;108(31):12787-92. Epub 2011 Jul 18.

Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA.

There is a wide variation among humans and mice in airway hyperresponsiveness (AHR) in the absence of allergen sensitization, i.e., naïve AHR. Because mast cell (MC) activation is thought to mediate AHR in atopic asthmatic subjects, we asked whether MCs mediate naïve AHR in A/J mice. We generated an A/J congenic strain lacking c-Kit by introgression of the Wv mutation, which resulted in the elimination of MCs and the abrogation of naïve AHR. Imatinib, which disrupts Kit signaling, also abrogated AHR in A/J mice. Remarkably, introduction of the Vga9 Mitf mutation into the A/J background resulted in the ablation of MCs but did not ameliorate AHR. These results indicate that c-Kit is required for development of AHR in an MC-independent fashion.
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http://dx.doi.org/10.1073/pnas.1106582108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3150901PMC
August 2011

Lipoxygenases mediate the effect of essential fatty acid in skin barrier formation: a proposed role in releasing omega-hydroxyceramide for construction of the corneocyte lipid envelope.

J Biol Chem 2011 Jul 10;286(27):24046-56. Epub 2011 May 10.

Department of Pharmacology and the Vanderbilt Institute of Chemical Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.

A barrier to water loss is vital to maintaining life on dry land. Formation of the mammalian skin barrier requires both the essential fatty acid linoleate and the two lipoxygenases 12R-lipoxygenase (12R-LOX) and epidermal lipoxygenase-3 (eLOX3), although their roles are poorly understood. Linoleate occurs in O-linoleoyl-ω-hydroxyceramide, which, after hydrolysis of the linoleate moiety, is covalently attached to protein via the free ω-hydroxyl of the ceramide, forming the corneocyte lipid envelope, a scaffold between lipid and protein that helps seal the barrier. Here we show using HPLC-UV, LC-MS, GC-MS, and (1)H NMR that O-linoleoyl-ω-hydroxyceramide is oxygenated in a regio- and stereospecific fashion by the consecutive actions of 12R-LOX and eLOX3 and that these products occur naturally in pig and mouse epidermis. 12R-LOX forms 9R-hydroperoxy-linoleoyl-ω-hydroxyceramide, further converted by eLOX3 to specific epoxyalcohol (9R,10R-trans-epoxy-11E-13R-hydroxy) and 9-keto-10E,12Z esters of the ceramide; an epoxy-ketone derivative (9R,10R-trans-epoxy-11E-13-keto) is the most prominent oxidized ceramide in mouse skin. These products are absent in 12R-LOX-deficient mice, which crucially display a near total absence of protein-bound ω-hydroxyceramides and of the corneocyte lipid envelope and die shortly after birth from transepidermal water loss. We conclude that oxygenation of O-linoleoyl-ω-hydroxyceramide is required to facilitate the ester hydrolysis and allow bonding of the ω-hydroxyceramide to protein, providing a coherent explanation for the roles of multiple components in epidermal barrier function. Our study uncovers a hitherto unknown biochemical pathway in which the enzymic oxygenation of ceramides is involved in building a crucial structure of the epidermal barrier.
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http://dx.doi.org/10.1074/jbc.M111.251496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3129186PMC
July 2011