Rasa3 regulates stage-specific cell cycle progression in murine erythropoiesis.

Inherited bone marrow failure syndromes (IBMFS) are heterogeneous disorders characterized by dysregulated hematopoiesis in various lineages, developmental anomalies, and predisposition to malignancy. The scat (severe combined anemia and thrombocytopenia) mouse model is a model of IBMFS with a phenotype of pancytopenia cycling through crises and remission. Scat carries an autosomal recessive missense mutation in Rasa3 that results in RASA3 mislocalization and loss of function. RASA3 functions as a Ras-GTPase activating protein (GAP), and its loss of function in scat results in increased erythroid RAS activity and reactive oxygen species (ROS) and altered erythroid cell cycle progression, culminating in delayed terminal erythroid differentiation. Here we sought to further resolve the erythroid cell cycle defect in scat through ex vivo flow cytometric analyses. These studies revealed a specific G0/G1 accumulation in scat bone marrow (BM) polychromatophilic erythroblasts and scat BM Ter119-/c-KIT+/CD71lo/med progenitors, with no changes evident in equivalent scat spleen populations. Systematic analyses of RNAseq data from megakaryocyte-erythroid progenitors (MEPs) in scat crisis vs. scat partial remission reveal altered expression of genes involved in the G1-S checkpoint. Together, these data indicate a precise, biphasic role for RASA3 in regulating the cell cycle during erythropoiesis with relevance to hematopoietic disease progression.

Differential effects of RASA3 mutations on hematopoiesis are profoundly influenced by genetic background and molecular variant.

Studies of the severely pancytopenic scat mouse model first demonstrated the crucial role of RASA3, a dual RAS and RAP GTPase activating protein (GAP), in hematopoiesis. RASA3 is required for survival in utero; germline deletion is lethal at E12.5-13.5 due to severe hemorrhage. Here, conditional deletion in hematopoietic stem and progenitor cells (HSPCs) using Vav-iCre recapitulates the null phenotype demonstrating that RASA3 is required at the stem and progenitor level to maintain blood vessel development and integrity and effective blood production. In adults, bone marrow blood cell production and spleen stress erythropoiesis are suppressed significantly upon induction of RASA3 deficiency, leading to pancytopenia and death within two weeks. Notably, RASA3 missense mutations in two mouse models, scat (G125V) and hlb381 (H794L), show dramatically different hematopoietic consequences specific to both genetic background and molecular variant. The mutation effect is mediated at least in part by differential effects on RAS and RAP activation. In addition, we show that the role of RASA3 is conserved during human terminal erythropoiesis, highlighting a potential function for the RASA3-RAS axis in disordered erythropoiesis in humans. Finally, global transcriptomic studies in scat suggest potential targets to ameliorate disease progression.

Genetic mapping in Diversity Outbred mice identifies a Trpa1 variant influencing late-phase formalin response.

Identification of genetic variants that influence susceptibility to pain is key to identifying molecular mechanisms and targets for effective and safe therapeutic alternatives to opioids. To identify genes and variants associated with persistent pain, we measured late-phase response to formalin injection in 275 male and female Diversity Outbred mice genotyped for over 70,000 single nucleotide polymorphisms. One quantitative trait locus reached genome-wide significance on chromosome 1 with a support interval of 3.1 Mb. This locus, Nociq4 (nociceptive sensitivity quantitative trait locus 4; MGI: 5661503), harbors the well-known pain gene Trpa1 (transient receptor potential cation channel, subfamily A, member 1). Trpa1 is a cation channel known to play an important role in acute and chronic pain in both humans and mice. Analysis of Diversity Outbred founder strain allele effects revealed a significant effect of the CAST/EiJ allele at Trpa1, with CAST/EiJ carrier mice showing an early, but not late, response to formalin relative to carriers of the 7 other inbred founder alleles (A/J, C57BL/6J, 129S1/SvImJ, NOD/ShiLtJ, NZO/HlLtJ, PWK/PhJ, and WSB/EiJ). We characterized possible functional consequences of sequence variants in Trpa1 by assessing channel conductance, TRPA1-TRPV1 interactions, and isoform expression. The phenotypic differences observed in CAST/EiJ relative to C57BL/6J carriers were best explained by Trpa1 isoform expression differences, implicating a splice junction variant as the causal functional variant. This study demonstrates the utility of advanced, high-precision genetic mapping populations in resolving specific molecular mechanisms of variation in pain sensitivity.

Reference Trait Analysis Reveals Correlations Between Gene Expression and Quantitative Traits in Disjoint Samples.

Systems genetic analysis of complex traits involves the integrated analysis of genetic, genomic, and disease-related measures. However, these data are often collected separately across multiple study populations, rendering direct correlation of molecular features to complex traits impossible. Recent transcriptome-wide association studies (TWAS) have harnessed gene expression quantitative trait loci (eQTL) to associate unmeasured gene expression with a complex trait in genotyped individuals, but this approach relies primarily on strong eQTL. We propose a simple and powerful alternative strategy for correlating independently obtained sets of complex traits and molecular features. In contrast to TWAS, our approach gains precision by correlating complex traits through a common set of continuous phenotypes instead of genetic predictors, and can identify transcript-trait correlations for which the regulation is not genetic. In our approach, a set of multiple quantitative "reference" traits is measured across all individuals, while measures of the complex trait of interest and transcriptional profiles are obtained in disjoint subsamples. A conventional multivariate statistical method, canonical correlation analysis, is used to relate the reference traits and traits of interest to identify gene expression correlates. We evaluate power and sample size requirements of this methodology, as well as performance relative to other methods, via extensive simulation and analysis of a behavioral genetics experiment in 258 Diversity Outbred mice involving two independent sets of anxiety-related behaviors and hippocampal gene expression. After splitting the data set and hiding one set of anxiety-related traits in half the samples, we identified transcripts correlated with the hidden traits using the other set of anxiety-related traits and exploiting the highest canonical correlation (R = 0.69) between the trait data sets. We demonstrate that this approach outperforms TWAS in identifying associated transcripts. Together, these results demonstrate the validity, reliability, and power of reference trait analysis for identifying relations between complex traits and their molecular substrates.

Mutant KLF1 in Adult Anemic Nan Mice Leads to Profound Transcriptome Changes and Disordered Erythropoiesis.

Anemic Nan mice carry a mutation (E339D) in the second zinc finger of erythroid transcription factor KLF1. Nan-KLF1 fails to bind a subset of normal KLF1 targets and ectopically binds a large set of genes not normally engaged by KLF1, resulting in a corrupted fetal liver transcriptome. Here, we performed RNAseq using flow cytometric-sorted spleen erythroid precursors from adult Nan and WT littermates rendered anemic by phlebotomy to identify global transcriptome changes specific to the Nan Klf1 mutation as opposed to anemia generally. Mutant Nan-KLF1 leads to extensive and progressive transcriptome corruption in adult spleen erythroid precursors such that stress erythropoiesis is severely compromised. Terminal erythroid differentiation is defective in the bone marrow as well. Principle component analysis reveals two major patterns of differential gene expression predicting that defects in basic cellular processes including translation, cell cycle, and DNA repair could contribute to disordered erythropoiesis and anemia in Nan. Significant erythroid precursor stage specific changes were identified in some of these processes in Nan. Remarkably, however, despite expression changes in large numbers of associated genes, most basic cellular processes were intact in Nan indicating that developing red cells display significant physiological resiliency and establish new homeostatic set points in vivo.

RASA3 is a critical inhibitor of RAP1-dependent platelet activation.

The small GTPase RAP1 is critical for platelet activation and thrombus formation. RAP1 activity in platelets is controlled by the GEF CalDAG-GEFI and an unknown regulator that operates downstream of the adenosine diphosphate (ADP) receptor, P2Y12, a target of antithrombotic therapy. Here, we provide evidence that the GAP, RASA3, inhibits platelet activation and provides a link between P2Y12 and activation of the RAP1 signaling pathway. In mice, reduced expression of RASA3 led to premature platelet activation and markedly reduced the life span of circulating platelets. The increased platelet turnover and the resulting thrombocytopenia were reversed by concomitant deletion of the gene encoding CalDAG-GEFI. Rasa3 mutant platelets were hyperresponsive to agonist stimulation, both in vitro and in vivo. Moreover, activation of Rasa3 mutant platelets occurred independently of ADP feedback signaling and was insensitive to inhibitors of P2Y12 or PI3 kinase. Together, our results indicate that RASA3 ensures that circulating platelets remain quiescent by restraining CalDAG-GEFI/RAP1 signaling and suggest that P2Y12 signaling is required to inhibit RASA3 and enable sustained RAP1-dependent platelet activation and thrombus formation at sites of vascular injury. These findings provide insight into the antithrombotic effect of P2Y12 inhibitors and may lead to improved diagnosis and treatment of platelet-related disorders.

TMEM14C is required for erythroid mitochondrial heme metabolism.

The transport and intracellular trafficking of heme biosynthesis intermediates are crucial for hemoglobin production, which is a critical process in developing red cells. Here, we profiled gene expression in terminally differentiating murine fetal liver-derived erythroid cells to identify regulators of heme metabolism. We determined that TMEM14C, an inner mitochondrial membrane protein that is enriched in vertebrate hematopoietic tissues, is essential for erythropoiesis and heme synthesis in vivo and in cultured erythroid cells. In mice, TMEM14C deficiency resulted in porphyrin accumulation in the fetal liver, erythroid maturation arrest, and embryonic lethality due to profound anemia. Protoporphyrin IX synthesis in TMEM14C-deficient erythroid cells was blocked, leading to an accumulation of porphyrin precursors. The heme synthesis defect in TMEM14C-deficient cells was ameliorated with a protoporphyrin IX analog, indicating that TMEM14C primarily functions in the terminal steps of the heme synthesis pathway. Together, our data demonstrate that TMEM14C facilitates the import of protoporphyrinogen IX into the mitochondrial matrix for heme synthesis and subsequent hemoglobin production. Furthermore, the identification of TMEM14C as a protoporphyrinogen IX importer provides a genetic tool for further exploring erythropoiesis and congenital anemias.

Precise genetic mapping and integrative bioinformatics in Diversity Outbred mice reveals Hydin as a novel pain gene.

Mouse genetics is a powerful approach for discovering genes and other genome features influencing human pain sensitivity. Genetic mapping studies have historically been limited by low mapping resolution of conventional mouse crosses, resulting in pain-related quantitative trait loci (QTL) spanning several megabases and containing hundreds of candidate genes. The recently developed Diversity Outbred (DO) population is derived from the same eight inbred founder strains as the Collaborative Cross, including three wild-derived strains. DO mice offer increased genetic heterozygosity and allelic diversity compared to crosses involving standard mouse strains. The high rate of recombinatorial precision afforded by DO mice makes them an ideal resource for high-resolution genetic mapping, allowing the circumvention of costly fine-mapping studies. We utilized a cohort of ~300 DO mice to map a 3.8 Mbp QTL on chromosome 8 associated with acute thermal pain sensitivity, which we have tentatively named Tpnr6. We used haplotype block partitioning to narrow Tpnr6 to a width of ~230 Kbp, reducing the number of putative candidate genes from 44 to 3. The plausibility of each candidate gene's role in pain response was assessed using an integrative bioinformatics approach, combining data related to protein domain, biological annotation, gene expression pattern, and protein functional interaction. Our results reveal a novel, putative role for the protein-coding gene, Hydin, in thermal pain response, possibly through the gene's role in ciliary motility in the choroid plexus-cerebrospinal fluid system of the brain. Real-time quantitative-PCR analysis showed no expression differences in Hydin transcript levels between pain-sensitive and pain-resistant mice, suggesting that Hydin may influence hot-plate behavior through other biological mechanisms.

Strain-specific hyperkyphosis and megaesophagus in Add1 null mice.

The three adducin proteins (α, ß, and γ) share extensive sequence, structural, and functional homology. Heterodimers of α- and ß-adducin are vital components of the red cell membrane skeleton, which is required to maintain red cell elasticity and structural integrity. In addition to anemia, targeted deletion of the α-adducin gene (Add1) reveals unexpected, strain-dependent non-erythroid phenotypes. On an inbred 129 genetic background, Add1 null mice show abnormal inward curvature of the cervicothoracic spine with complete penetrance. More surprisingly, a subset of 129-Add1 null mice develop severe megaesophagus, while examination of peripheral nerves reveals a reduced number of axons in 129-Add1 null mice at four months of age. These unforeseen phenotypes, described here, reveal new functions for adducin and provide new models of mammalian disease.

Comparative proteomics reveals deficiency of SLC9A1 (sodium/hydrogen exchanger NHE1) in ß-adducin null red cells.

Spherocytosis is one of the most common inherited disorders, yet presents with a wide range of clinical severity. While several genes have been found mutated in patients with spherocytosis, the molecular basis for the variability in severity of haemolytic anaemia is not entirely understood. To identify candidate proteins involved in haemolytic anaemia pathophysiology, we utilized a label-free comparative proteomic approach to detect differences in red blood cells (RBCs) from normal and ß-adducin (Add2) knock-out mice. We detected seven proteins that were decreased and 48 proteins that were increased in ß-adducin null RBC ghosts. Since haemolytic anaemias are characterized by reticulocytosis, we compared reticulocyte-enriched samples from phenylhydrazine-treated mice with mature RBCs from untreated mice. Among the 48 proteins increased in Add2 knockout RBCs, only 11 were also increased in reticulocytes. Of the proteins decreased in Add2 knockout RBCs, α-adducin showed the greatest intensity difference, followed by SLC9A1, the sodium-hydrogen exchanger previously termed NHE1. We verified these mass spectrometry results by immunoblot. This is the first example of SLC9A1deficiency in haemolytic anaemia and suggests new insights into the mechanisms leading to fragile RBCs.

Comparative proteomics reveals deficiency of NHE-1 (Slc9a1) in RBCs from the beta-adducin knockout mouse model of hemolytic anemia.

Hemolytic anemia is one of the most common inherited disorders. To identify candidate proteins involved in hemolytic anemia pathophysiology, we utilized a label-free comparative proteomic approach to detect differences in RBCs from normal and beta-adducin (Add2) knock-out mice. We detected 7 proteins that were decreased and 48 proteins that were increased in the beta-adducin knock-out RBC ghost. Since hemolytic anemias are characterized by reticulocytosis, we compared reticulocyte-enriched samples from phenylhydrazine-treated mice with mature RBCs from untreated mice. Label-free analysis identified 47 proteins that were increased in the reticulocyte-enriched samples and 21 proteins that were decreased. Among the proteins increased in Add2 knockout RBCs, only 11 were also found increased in reticulocytes. Among the proteins decreased in Add2 knockout RBCs, beta- and alpha-adducin showed the greatest intensity difference, followed by NHE-1 (Slc9a1), the sodium-hydrogen exchanger. We verified these mass spectrometry results by immunoblot. This is the first example of a deficiency of NHE-1 in hemolytic anemia and suggests new insights into the mechanisms leading to fragile RBCs. Our use of label-free comparative proteomics to make this discovery demonstrates the usefulness of this approach as opposed to metabolic or chemical isotopic labeling of mice.

Targeted deletion of betaIII spectrin impairs synaptogenesis and generates ataxic and seizure phenotypes.

The spectrin membrane skeleton controls the disposition of selected membrane channels, receptors, and transporters. In the brain betaIII spectrin binds directly to the excitatory amino acid transporter (EAAT4), the glutamate receptor delta, and other proteins. Mutations in betaIII spectrin link strongly to human spinocerebellar ataxia type 5 (SCA5), correlating with alterations in EAAT4. We have explored the mechanistic basis of this phenotype by targeted gene disruption of Spnb3. Mice lacking intact betaIII spectrin develop normally. By 6 months they display a mild nonprogressive ataxia. By 1 year most Spnb3(-/-) animals develop a myoclonic seizure disorder with significant reductions of EAAT4, EAAT1, GluRdelta, IP3R, and NCAM140. Other synaptic proteins are normal. The cerebellum displays increased dark Purkinje cells (PC), a thin molecular layer, fewer synapses, a loss of dendritic spines, and a 2-fold expansion of the PC dendrite diameter. Membrane and expanded Golgi profiles fill the PC dendrite and soma, and both regions accumulate EAAT4. Correlating with the seizure disorder are enhanced hippocampal levels of neuropeptide Y and EAAT3 and increased calpain proteolysis of alphaII spectrin. It appears that betaIII spectrin disruption impairs synaptogenesis by disturbing the intracellular pathways selectively regulating protein trafficking to the synapse. The mislocalization of these proteins secondarily disrupts glutamate transport dynamics, leading to seizures, neuronal damage, and compensatory changes in EAAT3 and neuropeptide Y.

Analysis of novel sph (spherocytosis) alleles in mice reveals allele-specific loss of band 3 and adducin in alpha-spectrin-deficient red cells.

Five spontaneous, allelic mutations in the alpha-spectrin gene, Spna1, have been identified in mice (spherocytosis [sph], sph(1J), sph(2J), sph(2BC), sph(Dem)). All cause severe hemolytic anemia. Here, analysis of 3 new alleles reveals previously unknown consequences of red blood cell (RBC) spectrin deficiency. In sph(3J), a missense mutation (H2012Y) in repeat 19 introduces a cryptic splice site resulting in premature termination of translation. In sph(Ihj), a premature stop codon occurs (Q1853Stop) in repeat 18. Both mutations result in markedly reduced RBC membrane spectrin content, decreased band 3, and absent beta-adducin. Reevaluation of available, previously described sph alleles reveals band 3 and adducin deficiency as well. In sph(4J), a missense mutation occurs in the C-terminal EF hand domain (C2384Y). Notably, an equally severe hemolytic anemia occurs despite minimally decreased membrane spectrin with normal band 3 levels and present, although reduced, beta-adducin. The severity of anemia in sph(4J) indicates that the highly conserved cysteine residue at the C-terminus of alpha-spectrin participates in interactions critical to membrane stability. The data reinforce the notion that a membrane bridge in addition to the classic protein 4.1-p55-glycophorin C linkage exists at the RBC junctional complex that involves interactions between spectrin, adducin, and band 3.

Targeted deletion of alpha-adducin results in absent beta- and gamma-adducin, compensated hemolytic anemia, and lethal hydrocephalus in mice.

In the red blood cell (RBC), adducin is present primarily as tetramers of alpha- and beta-subunits at spectrin-actin junctions, or junctional complexes. Mouse RBCs also contain small amounts of gamma-adducin. Platelets contain alpha- and gamma-adducin only. Adducin functions as a barbed-end actin capping protein to regulate actin filament length and recruits spectrin to the ends of actin filaments. To further define adducin's role in vivo, we generated alpha-adducin knockout mice. alpha-Adducin is absent in all tissues examined in homozygous null mice. In RBCs, beta- and gamma-adducin are also absent, indicating that alpha-adducin is the limiting subunit in tetramer formation at the spectrin-actin junction. Similarly, gamma-adducin is absent in alpha-null platelets. alpha-Adducin-null mice display compensated hemolytic anemia with features characteristic of RBCs in hereditary spherocytosis (HS), including spherocytes with significant loss of surface area, decreased mean corpuscular volume (MCV), cell dehydration, and increased osmotic fragility. Platelets maintain their normal discoid shape, and bleeding times are normal. alpha-Adducin-null mice show growth retardation at birth and throughout adulthood. Approximately 50% develop lethal communicating hydrocephalus with striking dilation of the lateral, third, and fourth ventricles. These data indicate that adducin plays a role in RBC membrane stability and in cerebrospinal fluid homeostasis.

Targeting the MEK1 cascade in lung epithelium inhibits proliferation and fibrogenesis by asbestos.

The extracellular signal-regulated kinases 1 and 2 (ERK1/2) are phosphorylated after inhalation of asbestos. The effect of blocking this signaling pathway in lung epithelium is unclear. Asbestos-exposed transgenic mice expressing a dominant-negative mitogen-activated protein kinase kinase-1 (dnMEK1) (i.e., the upstream kinase necessary for phosphorylation of ERK1/2) targeted to lung epithelium exhibited morphologic and molecular changes in lung. Transgene-positive (Tg+) (i.e., dnMEK1) and transgene-negative (Tg-) littermates were exposed to crocidolite asbestos for 2, 4, 9, and 32 days or maintained in clean air (sham controls). Distal bronchiolar epithelium was isolated using laser capture microdissection and mRNA analyzed for molecular markers of proliferation and Clara cell secretory protein (CCSP). Lungs and bronchoalveolar lavage fluids were analyzed for inflammatory and proliferative changes and molecular markers of fibrogenesis. Distal bronchiolar epithelium of asbestos-exposed wild-type mice showed increased expression of c-fos at 2 days. Elevated mRNA levels of histone H3 and numbers of Ki-67-labeled proliferating bronchiolar epithelial cells were decreased at 4 days in asbestos-exposed Tg+ mice. At 32 days, distal bronchioles normally composed of Clara cells in asbestos-exposed Tg+ mouse lungs exhibited nonreplicating ciliated and mucin-secreting cells as well as decreased mRNA levels of CCSP. Gene expression (procollagen 3-a-1, procollagen 1-a-1, and IL-6) linked to fibrogenesis was also increased in lung homogenates of asbestos-exposed Tg- mice, but reduced in asbestos-exposed Tg+ mice. These results suggest a critical role of MEK1 signaling in epithelial cell proliferation and lung remodeling after toxic injury.

The mouse as a model for human biology: a resource guide for complex trait analysis.

The mouse has been a powerful force in elucidating the genetic basis of human physiology and pathophysiology. From its beginnings as the model organism for cancer research and transplantation biology to the present, when dissection of the genetic basis of complex disease is at the forefront of genomics research, an enormous and remarkable mouse resource infrastructure has accumulated. This review summarizes those resources and provides practical guidelines for their use, particularly in the analysis of quantitative traits.

Dlx5 and Dlx6 homeobox genes are required for specification of the mammalian vestibular apparatus.

The mammalian inner ear is a complex organ that develops from a surface ectoderm into distinct auditory and vestibular components. Congenital malformation of these two components resulting from single or multiple gene defects is a common clinical occurrence and is observed in patients with split hand/split foot malformation, a malformation which is phenocopied by Dlx5/6 null mice. Analysis of mice lacking Dlx5 and Dlx6 homeobox genes identified their restricted and combined expression in the otic epithelium as a crucial regulator of vestibular cell fates. Otic induction initiates without incident in Dlx5/6(-/-) embryos, but dorsal otic derivatives including the semicircular ducts, utricle, saccule, and endolymphatic duct fail to form. Dlx5 and Dlx6 seem to influence vestibular cell fates by restricting Pax2 and activating Gbx2 and Bmp4 expression domains. Given their proximity to the disease locus and the observed phenotype in Dlx5/6 null mice, Dlx5/6 are likely candidates to mediate the inner ear defects observed in patients with split hand/split foot malformation.

The murine fork head gene Foxn2 is expressed in craniofacial, limb, CNS and somitic tissues during embryogenesis.

The fork head domain-containing gene family (Fox) comprises over 20 members in mammals and is defined by a conserved 110 amino-acid motif containing a winged helix structure DNA-binding domain. The members of this gene family have been implicated as key regulators of embryogenesis, cell cycling, cell lineage restriction and cancer. The Foxn2 gene (Ches1) is expressed in postgastrulation embryos in multiple tissues that serve as important signaling centers as well as end-stage-differentiated cell types that arise from different germ layers of the developing embryo. The dynamic and specific expression of Foxn2 during embryonic development suggest multiple independent roles for Foxn2 function during gestation.

The Dlx5 and Dlx6 homeobox genes are essential for craniofacial, axial, and appendicular skeletal development.

Dlx homeobox genes are mammalian homologs of the Drosophila Distal-less (Dll) gene. The Dlx/Dll gene family is of ancient origin and appears to play a role in appendage development in essentially all species in which it has been identified. In Drosophila, Dll is expressed in the distal portion of the developing appendages and is critical for the development of distal structures. In addition, human Dlx5 and Dlx6 homeobox genes have been identified as possible candidate genes for the autosomal dominant form of the split-hand/split-foot malformation (SHFM), a heterogeneous limb disorder characterized by missing central digits and claw-like distal extremities. Targeted inactivation of Dlx5 and Dlx6 genes in mice results in severe craniofacial, axial, and appendicular skeletal abnormalities, leading to perinatal lethality. For the first time, Dlx/Dll gene products are shown to be critical regulators of mammalian limb development, as combined loss-of-function mutations phenocopy SHFM. Furthermore, spatiotemporal-specific transgenic overexpression of Dlx5, in the apical ectodermal ridge of Dlx5/6 null mice can fully rescue Dlx/Dll function in limb outgrowth.