RNA exosome-regulated long non-coding RNA transcription controls super-enhancer activity.

We have ablated the cellular RNA degradation machinery in differentiated B cells and pluripotent embryonic stem cells (ESCs) by conditional mutagenesis of core (Exosc3) and nuclear RNase (Exosc10) components of RNA exosome and identified a vast number of long non-coding RNAs (lncRNAs) and enhancer RNAs (eRNAs) with emergent functionality. Unexpectedly, eRNA-expressing regions accumulate R-loop structures upon RNA exosome ablation, thus demonstrating the role of RNA exosome in resolving deleterious DNA/RNA hybrids arising from active enhancers. We have uncovered a distal divergent eRNA-expressing element (lncRNA-CSR) engaged in long-range DNA interactions and regulating IgH 3' regulatory region super-enhancer function. CRISPR-Cas9-mediated ablation of lncRNA-CSR transcription decreases its chromosomal looping-mediated association with the IgH 3' regulatory region super-enhancer and leads to decreased class switch recombination efficiency. We propose that the RNA exosome protects divergently transcribed lncRNA expressing enhancers by resolving deleterious transcription-coupled secondary DNA structures, while also regulating long-range super-enhancer chromosomal interactions important for cellular function.

Exome sequencing and characterization of 49,960 individuals in the UK Biobank.

The UK Biobank is a prospective study of 502,543 individuals, combining extensive phenotypic and genotypic data with streamlined access for researchers around the world1. Here we describe the release of exome-sequence data for the first 49,960 study participants, revealing approximately 4 million coding variants (of which around 98.6% have a frequency of less than 1%). The data include 198,269 autosomal predicted loss-of-function (LOF) variants, a more than 14-fold increase compared to the imputed sequence. Nearly all genes (more than 97%) had at least one carrier with a LOF variant, and most genes (more than 69%) had at least ten carriers with a LOF variant. We illustrate the power of characterizing LOF variants in this population through association analyses across 1,730 phenotypes. In addition to replicating established associations, we found novel LOF variants with large effects on disease traits, including PIEZO1 on varicose veins, COL6A1 on corneal resistance, MEPE on bone density, and IQGAP2 and GMPR on blood cell traits. We further demonstrate the value of exome sequencing by surveying the prevalence of pathogenic variants of clinical importance, and show that 2% of this population has a medically actionable variant. Furthermore, we characterize the penetrance of cancer in carriers of pathogenic BRCA1 and BRCA2 variants. Exome sequences from the first 49,960 participants highlight the promise of genome sequencing in large population-based studies and are now accessible to the scientific community.

Activin E-ACVR1C cross talk controls energy storage via suppression of adipose lipolysis in mice.

Body fat distribution is a heritable risk factor for cardiovascular and metabolic disease. In humans, rare Inhibin beta E (INHBE, activin E) loss-of-function variants are associated with a lower waist-to-hip ratio and protection from type 2 diabetes. Hepatic fatty acid sensing promotes INHBE expression during fasting and in obese individuals, yet it is unclear how the hepatokine activin E governs body shape and energy metabolism. Here, we uncover activin E as a regulator of adipose energy storage. By suppressing ß-agonist-induced lipolysis, activin E promotes fat accumulation and adipocyte hypertrophy and contributes to adipose dysfunction in mice. Mechanistically, we demonstrate that activin E elicits its effect on adipose tissue through ACVR1C, activating SMAD2/3 signaling and suppressing PPARG target genes. Conversely, loss of activin E or ACVR1C in mice increases fat utilization, lowers adiposity, and drives PPARG-regulated gene signatures indicative of healthy adipose function. Our studies identify activin E-ACVR1C as a metabolic rheostat promoting liver-adipose cross talk to restrain excessive fat breakdown and preserve fat mass during prolonged fasting, a mechanism that is maladaptive in obese individuals.

Germline Mutations in CIDEB and Protection against Liver Disease.

BACKGROUND: Exome sequencing in hundreds of thousands of persons may enable the identification of rare protein-coding genetic variants associated with protection from human diseases like liver cirrhosis, providing a strategy for the discovery of new therapeutic targets. METHODS: We performed a multistage exome sequencing and genetic association analysis to identify genes in which rare protein-coding variants were associated with liver phenotypes. We conducted in vitro experiments to further characterize associations. RESULTS: The multistage analysis involved 542,904 persons with available data on liver aminotransferase levels, 24,944 patients with various types of liver disease, and 490,636 controls without liver disease. We found that rare coding variants in APOB, ABCB4, SLC30A10, and TM6SF2 were associated with increased aminotransferase levels and an increased risk of liver disease. We also found that variants in CIDEB, which encodes a structural protein found in hepatic lipid droplets, had a protective effect. The burden of rare predicted loss-of-function variants plus missense variants in CIDEB (combined carrier frequency, 0.7%) was associated with decreased alanine aminotransferase levels (beta per allele, -1.24 U per liter; 95% confidence interval [CI], -1.66 to -0.83; P = 4.8×10-9) and with 33% lower odds of liver disease of any cause (odds ratio per allele, 0.67; 95% CI, 0.57 to 0.79; P = 9.9×10-7). Rare coding variants in CIDEB were associated with a decreased risk of liver disease across different underlying causes and different degrees of severity, including cirrhosis of any cause (odds ratio per allele, 0.50; 95% CI, 0.36 to 0.70). Among 3599 patients who had undergone bariatric surgery, rare coding variants in CIDEB were associated with a decreased nonalcoholic fatty liver disease activity score (beta per allele in score units, -0.98; 95% CI, -1.54 to -0.41 [scores range from 0 to 8, with higher scores indicating more severe disease]). In human hepatoma cell lines challenged with oleate, CIDEB small interfering RNA knockdown prevented the buildup of large lipid droplets. CONCLUSIONS: Rare germline mutations in CIDEB conferred substantial protection from liver disease. (Funded by Regeneron Pharmaceuticals.).

Author Correction: The bone marrow microenvironment at single-cell resolution.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

The bone marrow microenvironment at single-cell resolution.

The bone marrow microenvironment has a key role in regulating haematopoiesis, but its molecular complexity and response to stress are incompletely understood. Here we map the transcriptional landscape of mouse bone marrow vascular, perivascular and osteoblast cell populations at single-cell resolution, both at homeostasis and under conditions of stress-induced haematopoiesis. This analysis revealed previously unappreciated levels of cellular heterogeneity within the bone marrow niche and resolved cellular sources of pro-haematopoietic growth factors, chemokines and membrane-bound ligands. Our studies demonstrate a considerable transcriptional remodelling of niche elements under stress conditions, including an adipocytic skewing of perivascular cells. Among the stress-induced changes, we observed that vascular Notch delta-like ligands (encoded by Dll1 and Dll4) were downregulated. In the absence of vascular Dll4, haematopoietic stem cells prematurely induced a myeloid transcriptional program. These findings refine our understanding of the cellular architecture of the bone marrow niche, reveal a dynamic and heterogeneous molecular landscape that is highly sensitive to stress and illustrate the utility of single-cell transcriptomic data in evaluating the regulation of haematopoiesis by discrete niche populations.

NMIHBA results from hypomorphic PRUNE1 variants that lack short-chain exopolyphosphatase activity.

Neurodevelopmental disorder with microcephaly, hypotonia and variable brain anomalies (NMIHBA) is an autosomal recessive neurodevelopmental and neurodegenerative disorder characterized by global developmental delay and severe intellectual disability. Microcephaly, progressive cortical atrophy, cerebellar hypoplasia and delayed myelination are neurological hallmarks in affected individuals. NMIHBA is caused by biallelic variants in PRUNE1 encoding prune exopolyphosphatase 1. We provide in-depth clinical description of two affected siblings harboring compound heterozygous variant alleles, c.383G > A (p.Arg128Gln), c.520G > T (p.Gly174*) in PRUNE1. To gain insights into disease biology, we biochemically characterized missense variants within the conserved N-terminal aspartic acid-histidine-histidine (DHH) motif and provide evidence that they result in the destabilization of protein structure and/or loss of exopolyphosphatase activity. Genetic ablation of Prune1 results in midgestational lethality in mice, associated with perturbations to embryonic growth and vascular development. Our findings suggest that NMIHBA results from hypomorphic variant alleles in humans and underscore the potential key role of PRUNE1 exopolyphoshatase activity in neurodevelopment.

Regulation of sensorimotor gating via Disc1/Huntingtin-mediated Bdnf transport in the cortico-striatal circuit.

Sensorimotor information processing underlies normal cognitive and behavioral traits and has classically been evaluated through prepulse inhibition (PPI) of a startle reflex. PPI is a behavioral dimension deregulated in several neurological and psychiatric disorders, yet the mechanisms underlying the cross-diagnostic nature of PPI deficits across these conditions remain to be understood. To identify circuitry mechanisms for PPI, we performed circuitry recording over the prefrontal cortex and striatum, two brain regions previously implicated in PPI, using wild-type (WT) mice compared to Disc1-locus-impairment (LI) mice, a model representing neuropsychiatric conditions. We demonstrated that the corticostriatal projection regulates neurophysiological responses during the PPI testing in WT, whereas these circuitry responses were disrupted in Disc1-LI mice. Because our biochemical analyses revealed attenuated brain-derived neurotrophic factor (Bdnf) transport along the corticostriatal circuit in Disc1-LI mice, we investigated the potential role of Bdnf in this circuitry for regulation of PPI. Virus-mediated delivery of Bdnf into the striatum rescued PPI deficits in Disc1-LI mice. Pharmacologically augmenting Bdnf transport by chronic lithium administration, partly via phosphorylation of Huntingtin (Htt) serine-421 and its integration into the motor machinery, restored striatal Bdnf levels and rescued PPI deficits in Disc1-LI mice. Furthermore, reducing the cortical Bdnf expression negated this rescuing effect of lithium, confirming the key role of Bdnf in lithium-mediated PPI rescuing. Collectively, the data suggest that striatal Bdnf supply, collaboratively regulated by Htt and Disc1 along the corticostriatal circuit, is involved in sensorimotor gating, highlighting the utility of dimensional approach in investigating pathophysiological mechanisms across neuropsychiatric disorders.

Cell type-selective targeted delivery of a recombinant lysosomal enzyme for enzyme therapies.

Lysosomal diseases are a class of genetic disorders predominantly caused by loss of lysosomal hydrolases, leading to lysosomal and cellular dysfunction. Enzyme replacement therapy (ERT), where recombinant enzyme is given intravenously, internalized by cells, and trafficked to the lysosome, has been applied to treat several lysosomal diseases. However, current ERT regimens do not correct disease phenotypes in all affected organs because the biodistribution of enzyme uptake does not match that of the affected cells that require the enzyme. We present here targeted ERT, an approach that utilizes antibody-enzyme fusion proteins to target the enzyme to specific cell types. The antibody moiety recognizes transmembrane proteins involved in lysosomal trafficking and that are also preferentially expressed in those cells most affected in disease. Using Pompe disease (PD) as an example, we show that targeted ERT is superior to ERT in treating the skeletal muscle phenotypes of PD mice both as a protein replacement therapeutic and as a gene therapy.

Structural characterization of an activin class ternary receptor complex reveals a third paradigm for receptor specificity.

TGFß family ligands, which include the TGFßs, BMPs, and activins, signal by forming a ternary complex with type I and type II receptors. For TGFßs and BMPs, structures of ternary complexes have revealed differences in receptor assembly. However, structural information for how activins assemble a ternary receptor complex is lacking. We report the structure of an activin class member, GDF11, in complex with the type II receptor ActRIIB and the type I receptor Alk5. The structure reveals that receptor positioning is similar to the BMP class, with no interreceptor contacts; however, the type I receptor interactions are shifted toward the ligand fingertips and away from the dimer interface. Mutational analysis shows that ligand type I specificity is derived from differences in the fingertips of the ligands that interact with an extended loop specific to Alk4 and Alk5. The study also reveals differences for how TGFß and GDF11 bind to the same type I receptor, Alk5. For GDF11, additional contacts at the fingertip region substitute for the interreceptor interactions that are seen for TGFß, indicating that Alk5 binding to GDF11 is more dependent on direct contacts. In support, we show that a single residue of Alk5 (Phe84), when mutated, abolishes GDF11 signaling, but has little impact on TGFß signaling. The structure of GDF11/ActRIIB/Alk5 shows that, across the TGFß family, different mechanisms regulate type I receptor binding and specificity, providing a molecular explanation for how the activin class accommodates low-affinity type I interactions without the requirement of cooperative receptor interactions.

Effects of BMP7 produced by group 2 innate lymphoid cells on adipogenesis.

Group 2 innate lymphoid cells (ILC2s) are type 2 cytokine-producing cells that have important roles in helminth infection and allergic inflammation. ILC2s are tissue-resident cells, and their phenotypes and roles are regulated by tissue-specific environmental factors. While the role of ILC2s in the lung, intestine and bone marrow has been elucidated in many studies, their role in adipose tissues is still unclear. Here, we report on the role of ILC2-derived bone morphogenetic protein 7 (BMP7) in adipocyte differentiation and lipid accumulation. Co-culture of fat-derived ILC2s with pluripotent mesenchymal C3H10T1/2 cells and committed white preadipocyte 3T3-L1 cells resulted in their differentiation to adipocytes and induced lipid accumulation. Co-culture experiments using BMP7-deficient ILC2s revealed that BMP7, produced by ILC2s, induces differentiation into brown adipocytes. Our results demonstrate that BMP7, produced by ILC2s, affects adipocyte differentiation, particularly in brown adipocytes.

Context-dependent functions of angiopoietin 2 are determined by the endothelial phosphatase VEPTP.

The angiopoietin (ANGPT)-TIE2/TEK signaling pathway is essential for blood and lymphatic vascular homeostasis. ANGPT1 is a potent TIE2 activator, whereas ANGPT2 functions as a context-dependent agonist/antagonist. In disease, ANGPT2-mediated inhibition of TIE2 in blood vessels is linked to vascular leak, inflammation, and metastasis. Using conditional knockout studies in mice, we show TIE2 is predominantly activated by ANGPT1 in the cardiovascular system and by ANGPT2 in the lymphatic vasculature. Mechanisms underlying opposing actions of ANGPT2 in blood vs. lymphatic endothelium are poorly understood. Here we show the endothelial-specific phosphatase VEPTP (vascular endothelial protein tyrosine phosphatase) determines TIE2 response to ANGPT2. VEPTP is absent from lymphatic endothelium in mouse in vivo, permitting ANGPT2/TIE2-mediated lymphangiogenesis. Inhibition of VEPTP converts ANGPT2 into a potent TIE2 activator in blood endothelium. Our data support a model whereby VEPTP functions as a rheostat to modulate ANGPT2 ligand effect on TIE2.

Monoallelic BMP2 Variants Predicted to Result in Haploinsufficiency Cause Craniofacial, Skeletal, and Cardiac Features Overlapping Those of 20p12 Deletions.

Bone morphogenetic protein 2 (BMP2) in chromosomal region 20p12 belongs to a gene superfamily encoding TGF-ß-signaling proteins involved in bone and cartilage biology. Monoallelic deletions of 20p12 are variably associated with cleft palate, short stature, and developmental delay. Here, we report a cranioskeletal phenotype due to monoallelic truncating and frameshift BMP2 variants and deletions in 12 individuals from eight unrelated families that share features of short stature, a recognizable craniofacial gestalt, skeletal anomalies, and congenital heart disease. De novo occurrence and autosomal-dominant inheritance of variants, including paternal mosaicism in two affected sisters who inherited a BMP2 splice-altering variant, were observed across all reported families. Additionally, we observed similarity to the human phenotype of short stature and skeletal anomalies in a heterozygous Bmp2-knockout mouse model, suggesting that haploinsufficiency of BMP2 could be the primary phenotypic determinant in individuals with predicted truncating variants and deletions encompassing BMP2. These findings demonstrate the important role of BMP2 in human craniofacial, skeletal, and cardiac development and confirm that individuals heterozygous for BMP2 truncating sequence variants or deletions display a consistent distinct phenotype characterized by short stature and skeletal and cardiac anomalies without neurological deficits.

Multifunctional Alleles: A novel method for the generation of "All-In-One" null and conditional alleles.

The engineering of conditional alleles has evolved from simple floxing of regions of genes to more elaborate methods. Previously, we developed Conditional by Inversion (COIN), an allele design that utilizes an exon-splitting intron and an invertible genetrap-like module (COIN module) to create null alleles upon Cre-mediated inversion. Here we build upon COINs by generating a new Multifunctional Allele (MFA), that utilizes a single gene-targeting step and three site-specific recombination systems, to generate four allelic states: 1. The initial MFA (generated upon targeting) functions as a null with reporter (plus drug selection cassette) allele, wherein the gene of interest is inactivated by both inversion of a critical region of its coding sequence and simultaneous insertion of a reporter gene. MFAs can also be used as 'reverse-conditional' alleles as they are functionally wild type when they are converted to COIN alleles. 2. Null with reporter (minus drug selection cassette), wherein the selection cassette, the inverted critical region, and the COIN module are removed. 3. COIN-based conditional-null via removal of the selection cassette and reporter and simultaneous re-inversion of the critical region of the target. 4. Inverted COIN allele, wherein the COIN allele in turn is reconverted to a null allele by taking advantage of the COIN module's gene trap while simultaneously deleting the critical region.

Noncoding RNA transcription targets AID to divergently transcribed loci in B cells.

The vast majority of the mammalian genome has the potential to express noncoding RNA (ncRNA). The 11-subunit RNA exosome complex is the main source of cellular 3'-5' exoribonucleolytic activity and potentially regulates the mammalian noncoding transcriptome. Here we generated a mouse model in which the essential subunit Exosc3 of the RNA exosome complex can be conditionally deleted. Exosc3-deficient B cells lack the ability to undergo normal levels of class switch recombination and somatic hypermutation, two mutagenic DNA processes used to generate antibody diversity via the B-cell mutator protein activation-induced cytidine deaminase (AID). The transcriptome of Exosc3-deficient B cells has revealed the presence of many novel RNA exosome substrate ncRNAs. RNA exosome substrate RNAs include xTSS-RNAs, transcription start site (TSS)-associated antisense transcripts that can exceed 500 base pairs in length and are transcribed divergently from cognate coding gene transcripts. xTSS-RNAs are most strongly expressed at genes that accumulate AID-mediated somatic mutations and/or are frequent translocation partners of DNA double-strand breaks generated at Igh in B cells. Strikingly, translocations near TSSs or within gene bodies occur over regions of RNA exosome substrate ncRNA expression. These RNA exosome-regulated, antisense-transcribed regions of the B-cell genome recruit AID and accumulate single-strand DNA structures containing RNA-DNA hybrids. We propose that RNA exosome regulation of ncRNA recruits AID to single-strand DNA-forming sites of antisense and divergent transcription in the B-cell genome, thereby creating a link between ncRNA transcription and overall maintenance of B-cell genomic integrity.

Induction of the Hajdu-Cheney Syndrome Mutation in CD19 B Cells in Mice Alters B-Cell Allocation but Not Skeletal Homeostasis.

Mice harboring Notch2 mutations replicating Hajdu-Cheney syndrome (Notch2tm1.1ECan) have osteopenia and exhibit an increase in splenic marginal zone B cells with a decrease in follicular B cells. Whether the altered B-cell allocation is responsible for the osteopenia of Notch2tm1.1ECan mutants is unknown. To determine the effect of NOTCH2 activation in B cells on splenic B-cell allocation and skeletal phenotype, a conditional-by-inversion (COIN) Hajdu-Cheney syndrome allele of Notch2 (Notch2[ΔPEST]COIN) was used. Cre recombination generates a permanent Notch2ΔPEST allele expressing a transcript for which sequences coding for the proline, glutamic acid, serine, and threonine-rich (PEST) domain are replaced by a stop codon. CD19-Cre drivers were backcrossed into Notch2[ΔPEST]COIN/[ΔPEST]COIN to generate CD19-specific Notch2ΔPEST/ΔPEST mutants and control Notch2[ΔPEST]COIN/[ΔPEST]COIN littermates. There was an increase in marginal zone B cells and a decrease in follicular B cells in the spleen of CD19Cre/WT;Notch2ΔPEST/ΔPEST mice, recapitulating the splenic phenotype of Notch2tm1.1ECan mice. The effect was reproduced when the NOTCH1 intracellular domain was induced in CD19-expressing cells (CD19Cre/WT;RosaNotch1/WT mice). However, neither CD19Cre/WT;Notch2ΔPEST/ΔPEST nor CD19Cre/WT;RosaNotch1/WT mice had a skeletal phenotype. Moreover, splenectomies in Notch2tm1.1ECan mice did not reverse their osteopenic phenotype. In conclusion, Notch2 activation in CD19-expressing cells determines B-cell allocation in the spleen but has no skeletal consequences.

MAPPIN: a method for annotating, predicting pathogenicity and mode of inheritance for nonsynonymous variants.

Nonsynonymous single nucleotide variants (nsSNVs) constitute about 50% of known disease-causing mutations and understanding their functional impact is an area of active research. Existing algorithms predict pathogenicity of nsSNVs; however, they are unable to differentiate heterozygous, dominant disease-causing variants from heterozygous carrier variants that lead to disease only in the homozygous state. Here, we present MAPPIN (Method for Annotating, Predicting Pathogenicity, and mode of Inheritance for Nonsynonymous variants), a prediction method which utilizes a random forest algorithm to distinguish between nsSNVs with dominant, recessive, and benign effects. We apply MAPPIN to a set of Mendelian disease-causing mutations and accurately predict pathogenicity for all mutations. Furthermore, MAPPIN predicts mode of inheritance correctly for 70.3% of nsSNVs. MAPPIN also correctly predicts pathogenicity for 87.3% of mutations from the Deciphering Developmental Disorders Study with a 78.5% accuracy for mode of inheritance. When tested on a larger collection of mutations from the Human Gene Mutation Database, MAPPIN is able to significantly discriminate between mutations in known dominant and recessive genes. Finally, we demonstrate that MAPPIN outperforms CADD and Eigen in predicting disease inheritance modes for all validation datasets. To our knowledge, MAPPIN is the first nsSNV pathogenicity prediction algorithm that provides mode of inheritance predictions, adding another layer of information for variant prioritization.

Sustained Notch2 signaling in osteoblasts, but not in osteoclasts, is linked to osteopenia in a mouse model of Hajdu-Cheney syndrome.

Individuals with Hajdu-Cheney syndrome (HCS) present with osteoporosis, and HCS is associated with NOTCH2 mutations causing deletions of the proline-, glutamic acid-, serine-, and threonine-rich (PEST) domain that are predicted to enhance NOTCH2 stability and cause gain-of-function. Previously, we demonstrated that mice harboring Notch2 mutations analogous to those in HCS (Notch2HCS) are severely osteopenic because of enhanced bone resorption. We attributed this phenotype to osteoclastic sensitization to the receptor activator of nuclear factor-κB ligand and increased osteoblastic tumor necrosis factor superfamily member 11 (Tnfsf11) expression. Here, to determine the individual contributions of osteoclasts and osteoblasts to HCS osteopenia, we created a conditional-by-inversion (Notch2COIN ) model in which Cre recombination generates a Notch2ΔPEST allele expressing a Notch2 mutant lacking the PEST domain. Germ line Notch2COIN inversion phenocopied the Notch2HCS mutant, validating the model. To activate Notch2 in osteoclasts or osteoblasts, Notch2COIN mice were bred with mice expressing Cre from the Lyz2 or the BGLAP promoter, respectively. These crosses created experimental mice harboring a Notch2ΔPEST allele in Cre-expressing cells and control littermates expressing a wild-type Notch2 transcript. Notch2COIN inversion in Lyz2-expressing cells had no skeletal consequences and did not affect the capacity of bone marrow macrophages to form osteoclasts in vitro In contrast, Notch2COIN inversion in osteoblasts led to generalized osteopenia associated with enhanced bone resorption in the cancellous bone compartment and with suppressed endocortical mineral apposition rate. Accordingly, Notch2 activation in osteoblast-enriched cultures from Notch2COIN mice induced Tnfsf11 expression. In conclusion, introduction of the HCS mutation in osteoblasts, but not in osteoclasts, causes osteopenia.

Canonical and noncanonical Wnts use a common mechanism to activate completely unrelated coreceptors.

Wnt ligands signal through ß-catenin and are critically involved in cell fate determination and stem/progenitor self-renewal. Wnts also signal through ß-catenin-independent or noncanonical pathways that regulate crucial events during embryonic development. The mechanism of noncanonical receptor activation and how Wnts trigger canonical as opposed to noncanonical signaling have yet to be elucidated. We demonstrate here that prototype canonical Wnt3a and noncanonical Wnt5a ligands specifically trigger completely unrelated endogenous coreceptors-LRP5/6 and Ror1/2, respectively-through a common mechanism that involves their Wnt-dependent coupling to the Frizzled (Fzd) coreceptor and recruitment of shared components, including dishevelled (Dvl), axin, and glycogen synthase kinase 3 (GSK3). We identify Ror2 Ser 864 as a critical residue phosphorylated by GSK3 and required for noncanonical receptor activation by Wnt5a, analogous to the priming phosphorylation of low-density receptor-related protein 6 (LRP6) in response to Wnt3a. Furthermore, this mechanism is independent of Ror2 receptor Tyr kinase functions. Consistent with this model of Wnt receptor activation, we provide evidence that canonical and noncanonical Wnts exert reciprocal pathway inhibition at the cell surface by competition for Fzd binding. Thus, different Wnts, through their specific coupling and phosphorylation of unrelated coreceptors, activate completely distinct signaling pathways.

Comparative Transcriptomics Identifies Novel Genes and Pathways Involved in Post-Traumatic Osteoarthritis Development and Progression.

Anterior cruciate ligament (ACL) injuries often result in post-traumatic osteoarthritis (PTOA). To better understand the molecular mechanisms behind PTOA development following ACL injury, we profiled ACL injury-induced transcriptional changes in knee joints of three mouse strains with varying susceptibility to OA: STR/ort (highly susceptible), C57BL/6J (moderately susceptible) and super-healer MRL/MpJ (not susceptible). Right knee joints of the mice were injured using a non-invasive tibial compression injury model and global gene expression was quantified before and at 1-day, 1-week, and 2-weeks post-injury using RNA-seq. Following injury, injured and uninjured joints of STR/ort and injured C57BL/6J joints displayed significant cartilage degeneration while MRL/MpJ had little cartilage damage. Gene expression analysis suggested that prolonged inflammation and elevated catabolic activity in STR/ort injured joints, compared to the other two strains may be responsible for the severe PTOA phenotype observed in this strain. MRL/MpJ had the lowest expression values for several inflammatory cytokines and catabolic enzymes activated in response to ACL injury. Furthermore, we identified several genes highly expressed in MRL/MpJ compared to the other two strains including B4galnt2 and Tpsab1 which may contribute to enhanced healing in the MRL/MpJ. Overall, this study has increased our knowledge of early molecular changes associated with PTOA development.