Transgenic Complementation Research Articles

A soybean cyst nematode resistance gene points to a new mechanism of plant resistance to pathogens

Soybean (Glycine max (L.) Merr.) is an important crop that provides a sustainable source of protein and oil worldwide. Soybean cyst nematode (Heterodera glycines Ichinohe) is a microscopic roundworm that feeds on the roots of soybean and is a major constraint to soybean production. This nematode causes more than US$1 billion in yield losses annually in the United States alone, making it the most economically important pathogen on soybean. Although planting of resistant cultivars forms the core management strategy for this pathogen, nothing is known about the nature of resistance. Moreover, the increase in virulent populations of this parasite on most known resistance sources necessitates the development of novel approaches for control. Here we report the map-based cloning of a gene at the Rhg4 (for resistance to Heterodera glycines 4) locus, a major quantitative trait locus contributing to resistance to this pathogen. Mutation analysis, gene silencing and transgenic complementation confirm that the gene confers resistance. The gene encodes a serine hydroxymethyltransferase, an enzyme that is ubiquitous in nature and structurally conserved across kingdoms. The enzyme is responsible for interconversion of serine and glycine and is essential for cellular one-carbon metabolism. Alleles of Rhg4 conferring resistance or susceptibility differ by two genetic polymorphisms that alter a key regulatory property of the enzyme. Our discovery reveals an unprecedented plant resistance mechanism against a pathogen. The mechanistic knowledge of the resistance gene can be readily exploited to improve nematode resistance of soybean, an increasingly important global crop.

A WUSCHEL-like homeobox gene, OsWOX3B responses to NUDA/GL-1 locus in rice.

BackgroundMost of the rice varieties are pubescent. However, the presence of trichomes is an undesirable characteristic in rice production because trichomes can cause atmospheric pollution. The use of glabrous rice varieties represents a solution to this problem. Yunnan Nuda Rice, a glabrous cultivar that constitutes approximately 20% of rice germplasms in Yunnan can provide important recourse for breeding of glabrous rice varieties.ResultsThe “Nuda” phenotype in Yunnan Nuda Rice was found to be controlled by a single recessive allelic gene within the well-characterized GL-1 locus. A high-resolution genetic and physical map was constructed using 1,192 Nuda individuals from the F2 population that was delivered from the cross between the Yunnan Nuda variety HMK and the pubescent TN1 variety. The NUDA/GL-1 gene was mapped to a 28.5 kb region containing six annotated genes based on the Nipponbare genomic sequence. By comparing the sequences and expression patterns of different pubescent and glabrous varieties, LOC_Os05g02730, a WUSCHEL-like homeobox gene (OsWOX3B) was identified as the candidate gene. This hypothesis was confirmed by RNA interference (RNAi) and transgenic complementation. Trichome deficiency in RNAi lines was associated with increased efficiency of grain packaging but did not affect the main agronomic traits.ConclusionNUDA/GL-1 locus encodes OsWOX3B gene.Electronic supplementary materialThe online version of this article (doi:10.1186/1939-8433-5-30) contains supplementary material, which is available to authorized users.

Genome-Wide Association Studies Identify Heavy Metal ATPase3 as the Primary Determinant of Natural Variation in Leaf Cadmium in Arabidopsis thaliana

Understanding the mechanism of cadmium (Cd) accumulation in plants is important to help reduce its potential toxicity to both plants and humans through dietary and environmental exposure. Here, we report on a study to uncover the genetic basis underlying natural variation in Cd accumulation in a world-wide collection of 349 wild collected Arabidopsis thaliana accessions. We identified a 4-fold variation (0.5–2 µg Cd g−1 dry weight) in leaf Cd accumulation when these accessions were grown in a controlled common garden. By combining genome-wide association mapping, linkage mapping in an experimental F2 population, and transgenic complementation, we reveal that HMA3 is the sole major locus responsible for the variation in leaf Cd accumulation we observe in this diverse population of A. thaliana accessions. Analysis of the predicted amino acid sequence of HMA3 from 149 A. thaliana accessions reveals the existence of 10 major natural protein haplotypes. Association of these haplotypes with leaf Cd accumulation and genetics complementation experiments indicate that 5 of these haplotypes are active and 5 are inactive, and that elevated leaf Cd accumulation is associated with the reduced function of HMA3 caused by a nonsense mutation and polymorphisms that change two specific amino acids.

Diverse roles of strigolactone signaling in maize architecture and the uncoupling of a branching-specific subnetwork.

Strigolactones (SLs) control lateral branching in diverse species by regulating transcription factors orthologous to Teosinte branched1 (Tb1). In maize (Zea mays), however, selection for a strong central stalk during domestication is attributed primarily to the Tb1 locus, leaving the architectural roles of SLs unclear. To determine how this signaling network is altered in maize, we first examined effects of a knockout mutation in an essential SL biosynthetic gene that encodes CAROTENOID CLEAVAGE DIOXYGENASE8 (CCD8), then tested interactions between SL signaling and Tb1. Comparative genome analysis revealed that maize depends on a single CCD8 gene (ZmCCD8), unlike other panicoid grasses that have multiple CCD8 paralogs. Function of ZmCCD8 was confirmed by transgenic complementation of Arabidopsis (Arabidopsis thaliana) max4 (ccd8) and by phenotypic rescue of the maize mutant (zmccd8::Ds) using a synthetic SL (GR24). Analysis of the zmccd8 mutant revealed a modest increase in branching that contrasted with prominent pleiotropic changes that include (1) marked reduction in stem diameter, (2) reduced elongation of internodes (independent of carbon supply), and (3) a pronounced delay in development of the centrally important, nodal system of adventitious roots. Analysis of the tb1 zmccd8 double mutant revealed that Tb1 functions in an SL-independent subnetwork that is not required for the other diverse roles of SL in development. Our findings indicate that in maize, uncoupling of the Tb1 subnetwork from SL signaling has profoundly altered the balance between conserved roles of SLs in branching and diverse aspects of plant architecture.

The sensitive to freezing3 mutation of Arabidopsis thaliana is a cold-sensitive allele of homomeric acetyl-CoA carboxylase that results in cold-induced cuticle deficiencies

The sfr3 mutation causes freezing sensitivity in Arabidopsis thaliana. Mapping, sequencing, and transgenic complementation showed sfr3 to be a missense mutation in ACC1, an essential gene encoding homomeric (multifunctional) acetyl-CoA carboxylase. Cuticle permeability was compromised in the sfr3 mutant when plants were grown in the cold but not in the warm. Wax deposition on the inflorescence stem of cold-grown sfr3 plants was inhibited and the long-chain components of their leaf cuticular wax were reduced compared with wild-type plants. Thus, freezing sensitivity of sfr3 appears, from these results, to be due to cuticular deficiencies that develop during cold acclimation. These observations demonstrated the essential role of the cuticle in tolerance to freezing and drought.

Validation of the multiple sensor mechanism of the Keap1-Nrf2 system

The Keap1-Nrf2 system plays a critical role in cellular defense against electrophiles and reactive oxygen species. Keap1 possesses a number of cysteine residues, some of which are highly reactive and serves as sensors for these insults. Indeed, point mutation of Cys151 abrogates the response to certain electrophiles. However, this mutation does not affect the other set of electrophiles, suggesting that multiple sensor systems reside within the cysteine residues of Keap1. The precise contribution of each reactive cysteine to the sensor function of Keap1 remains to be clarified. To elucidate the contribution of Cys151 in vivo, in this study we adopted transgenic complementation rescue assays. Embryonic fibroblasts and primary peritoneal macrophages were prepared from mice expressing the Keap1-C151S mutant. These cells were challenged with various Nrf2 inducers. We found that some of the inducers triggered only marginal responses in Keap1-C151S-expressing cells, while others evoked responses in a comparable magnitude to those observed in the wild-type cells. We found that tert-butyl hydroquinone, diethylmaleate, sulforaphane, and dimethylfumarate were Cys151 preferable, whereas 15-deoxy-Δ12,14-prostaglandin J2 (15d-PG-J2), 2-cyano-3,12 dioxooleana-1,9 diene-28-imidazolide (CDDO-Im), ebselen, nitro-oleic acid, and cadmium chloride were Cys151 independent. Experiments with embryonic fibroblasts and primary macrophages yielded consistent results. Experiments testing protective effects against the cytotoxicity of 1-chloro-2,4-dinitrobenzene of sulforaphane and 15d-PG-J2 in Keap1-C151S-expressing macrophages revealed that the former inducer was effective, while the latter was not. These results thus indicate that there exists distinct utilization of Keap1 cysteine residues by different chemicals that trigger the response of the Keap1-Nrf2 system, and further substantiate the notion that there are multiple sensing mechanisms within Keap1 cysteine residues.

The Sg-1 Glycosyltransferase Locus Regulates Structural Diversity of Triterpenoid Saponins of Soybean

Triterpene saponins are a diverse group of biologically functional products in plants. Saponins usually are glycosylated, which gives rise to a wide diversity of structures and functions. In the group A saponins of soybean (Glycine max), differences in the terminal sugar species located on the C-22 sugar chain of an aglycone core, soyasapogenol A, were observed to be under genetic control. Further genetic analyses and mapping revealed that the structural diversity of glycosylation was determined by multiple alleles of a single locus, Sg-1, and led to identification of a UDP-sugar-dependent glycosyltransferase gene (Glyma07g38460). Although their sequences are highly similar and both glycosylate the nonacetylated saponin A0-αg, the Sg-1(a) allele encodes the xylosyltransferase UGT73F4, whereas Sg-1(b) encodes the glucosyltransferase UGT73F2. Homology models and site-directed mutagenesis analyses showed that Ser-138 in Sg-1(a) and Gly-138 in Sg-1(b) proteins are crucial residues for their respective sugar donor specificities. Transgenic complementation tests followed by recombinant enzyme assays in vitro demonstrated that sg-1(0) is a loss-of-function allele of Sg-1. Considering that the terminal sugar species in the group A saponins are responsible for the strong bitterness and astringent aftertastes of soybean seeds, our findings herein provide useful tools to improve commercial properties of soybean products.

Mature erythrocyte membrane homeostasis is compromised by loss of the GATA1-FOG1 interaction

AbstractGATA1 plays essential roles in erythroid gene expression. The N-terminal finger of GATA1 (GATA1-Nf) is important for association with FOG1. Substitution mutations in GATA1-Nf, such as GATA1V205M that diminish the GATA1-FOG1 association, have been identified in human thrombocytopenia and anemia cases. A mouse model of human thrombocytopenia has been established using a transgenic complementation rescue approach; GATA1-deficient mice were successfully rescued from embryonic lethality by excess expression of GATA1V205G, but rescued adult mice suffered from severe thrombocytopenia. In this study, we examined GATA1-deficient mice rescued with GATA1V205G at a comparable level to endogenous GATA1. Mice rescued with this level of GATA1V205G rarely survive to adulthood. Rescued newborns suffered from severe anemia and jaundice accompanied with anisocytosis and spherocytosis. Expression of Slc4a1, Spna1, and Aqp1 genes (encoding the membrane proteins band-3, α-spectrin, and aquaporin-1, respectively) were strikingly diminished, whereas expression of other canonical GATA1-target genes, such as Alas2, were little affected. Lack of these membrane proteins provoked perturbation of membrane skeleton. Importantly, the red cells exhibited increased reactive oxygen species accumulation. These results thus demonstrate that the loss of the GATA1-FOG1 interaction causes a unique combination of membrane protein deficiency and disturbs the function of GATA1 in maintaining erythroid homeostasis.

Structural Basis of Transcriptional Gene Silencing Mediated by Arabidopsis MOM1

Shifts between epigenetic states of transcriptional activity are typically correlated with changes in epigenetic marks. However, exceptions to this rule suggest the existence of additional, as yet uncharacterized, layers of epigenetic regulation. MOM1, a protein of 2,001 amino acids that acts as a transcriptional silencer, represents such an exception. Here we define the 82 amino acid domain called CMM2 (Conserved MOM1 Motif 2) as a minimal MOM1 fragment capable of transcriptional regulation. As determined by X-ray crystallography, this motif folds into an unusual hendecad-based coiled-coil. Structure-based mutagenesis followed by transgenic complementation tests in plants demonstrate that CMM2 and its dimerization are effective for transcriptional suppression at chromosomal loci co-regulated by MOM1 and the siRNA pathway but not at loci controlled by MOM1 in an siRNA–independent fashion. These results reveal a surprising separation of epigenetic activities that enable the single, large MOM1 protein to coordinate cooperating mechanisms of epigenetic regulation.

Retracted: Opaque7 Encodes an Acyl-Activating Enzyme-Like Protein That Affects Storage Protein Synthesis in Maize Endosperm

In maize, a series of seed mutants with starchy endosperm could increase the lysine content by decreased amount of zeins, the main storage proteins in endosperm. Cloning and characterization of these mutants could reveal regulatory mechanisms for zeins accumulation in maize endosperm. Opaque7 (o7) is a classic maize starchy endosperm mutant with large effects on zeins accumulation and high lysine content. In this study, the O7 gene was cloned by map-based cloning and confirmed by transgenic functional complementation and RNAi. The o7-ref allele has a 12-bp in-frame deletion. The four-amino-acid deletion caused low accumulation of o7 protein in vivo. The O7 gene encodes an acyl-activating enzyme with high similarity to AAE3. The opaque phenotype of the o7 mutant was produced by the reduction of protein body size and number caused by a decrease in the α-zeins concentrations. Analysis of amino acids and metabolites suggested that the O7 gene might affect amino acid biosynthesis by affecting α-ketoglutaric acid and oxaloacetic acid. Transgenic rice seeds containing RNAi constructs targeting the rice ortholog of maize O7 also produced lower amounts of seed proteins and displayed an opaque endosperm phenotype, indicating a conserved biological function of O7 in cereal crops. The cloning of O7 revealed a novel regulatory mechanism for storage protein synthesis and highlighted an effective target for the genetic manipulation of storage protein contents in cereal seeds.

Ubiquitin Homeostasis Is Critical for Synaptic Development and Function

The ubiquitin-proteasome system (UPS) controls protein abundance and is essential for many aspects of neuronal function. In ataxia (ax(J)) mice, profound neurological and synaptic defects result from a loss-of-function mutation in the proteasome-associated deubiquitinating enzyme Usp14, which is required for recycling ubiquitin from proteasomal substrates. Here, we show that transgenic complementation of ax(J) mice with neuronally expressed ubiquitin prevents early postnatal lethality, restores muscle mass, and corrects developmental and functional deficits resulting from the loss of Usp14, demonstrating that ubiquitin deficiency is a major cause of the neurological defects observed in the ax(J) mice. We also show that proteasome components are normally induced during the first 2 weeks of postnatal development, which coincides with dramatic alterations in polyubiquitin chain formation. These data demonstrate a critical role for ubiquitin homeostasis in synaptic development and function, and show that ubiquitin deficiency may contribute to diseases characterized by synaptic dysfunction.

Transgenic complementation of MeCP2 deficiency: phenotypic rescue of Mecp2-null mice by isoform-specific transgenes

Rett syndrome (RTT) is a disorder that affects patients' ability to communicate, move and behave. RTT patients are characterized by impaired language, stereotypic behaviors, frequent seizures, ataxia and sleep disturbances, with the onset of symptoms occurring after a period of seemingly normal development. RTT is caused by mutations in methyl-CpG binding protein 2 (MECP2), an X-chromosome gene encoding for MeCP2, a protein that regulates gene expression. MECP2 generates two alternative splice variants encoding two protein isoforms that differ only in the N-terminus. Although no functional differences have been identified for these splice variants, it has been suggested that the RTT phenotype may occur in the presence of a functional MeCP2-e2 protein. This suggests that the two isoforms might be functionally distinct. Supporting this notion, the two variants show regional and age-related differences in transcript abundance. Here, we show that transgenic expression of either the MeCP2-e1 or MeCP2-e2 splice variant results in prevention of development of RTT-like phenotypic manifestations in a mouse model lacking Mecp2. Our results indicate that the two MeCP2 splice variants can substitute for each other and fulfill the basic functions of MeCP2 in the mouse brain.

Role of SLAM in NKT Cell Development Revealed by Transgenic Complementation in NOD Mice

Allelic variation of SLAM expression on CD4(+)CD8(+) thymocytes has been proposed to play a major role in NKT cell development. In this article, this hypothesis is tested by the production of subcongenic mouse strains and Slamf1 transgenic lines. The long isoform of the C57BL/6 allele of Slamf1 was transgenically expressed on CD4(+)CD8(+) thymocytes under control of an hCD2 minigene. NOD.Nkrp1b.Tg(Slamf1)1 mice, which had a 2-fold increase in SLAM protein expression on CD4(+)CD8(+) thymocytes, had a 2-fold increase in numbers of thymic NKT cells. The additional thymic NKT cells in NOD.Nkrp1b.Tg(Slamf1)1 mice were relatively immature, with a similar subset distribution to those of congenic NOD.Nkrp1b.Nkt1 and NOD.Nkrp1b.Slamf1 mice, which also express increased levels of SLAM on CD4(+)CD8(+) thymocytes and produce larger numbers of NKT cells. Transgenic enhancement of SLAM expression also increased IL-4 and IL-17 production in response to TCR-mediated stimulation. Paradoxically, NOD.Nkrp1b.Tg(Slamf1)2 mice, which had a 7-fold increase in SLAM expression, showed no significant increase in NKT cells numbers; on the contrary, at high transgene copy number, SLAM expression levels correlated inversely with NKT cell numbers, consistent with a contribution to negative selection. These data confirm a role for SLAM in controlling NKT cell development and are consistent with a role in both positive and negative thymic selection of NKT cells.

Molecular Determinants for Small Maf Protein Control of Platelet Production

MafG and p45 possess basic region-leucine zipper (bZip) domains and form a heterodimer called NF-E2, a key regulator of megakaryopoiesis. NF-E2 binds to the Maf recognition element (MARE) and activates transcription of many platelet genes. Since the bZip domain, which mediates DNA binding and heterodimerization, is the only functional domain established for MafG, it has been assumed that MafG is required only for p45 binding to MARE and to facilitate p45-mediated transcriptional activation. Analysis of the C-terminal region of MafG, which is distinct from the bZip domain, revealed that this region contains a nuclear matrix-targeting signal. We used a transgenic complementation rescue assay to delineate the function of the MafG C terminus in vivo. Transgenic mice expressing a mutant MafG protein lacking the C terminus (MafGΔC) were crossed into a MafG-null background. The compound mutant mice displayed severe thrombocytopenia and splenomegaly, which phenocopied p45-null mice. The MafG C terminus is essential for proplatelet formation and platelet gene activation but not for p45 binding to MARE. These results demonstrate that the MafG C terminus is required for NF-E2 function and suggest that efficient targeting of NF-E2 to a specific nuclear scaffold is important to achieve high-level activity.

Transgenic Targeting of Recombinant Rabies Virus Reveals Monosynaptic Connectivity of Specific Neurons

Understanding how neural circuits work requires a detailed knowledge of cellular-level connectivity. Our current understanding of neural circuitry is limited by the constraints of existing tools for transsynaptic tracing. Some of the most intractable problems are a lack of cellular specificity of uptake, transport across multiple synaptic steps conflating direct and indirect inputs, and poor labeling of minor inputs. We used a novel combination of transgenic mouse technology and a recently developed tracing system based on rabies virus (Wickersham et al., 2007a,b) to overcome all three constraints. Because the virus requires transgene expression for both initial infection and subsequent retrograde transsynaptic infection, we created several lines of mice that express these genes in defined cell types using the tetracycline-dependent transactivator system (Mansuy and Bujard, 2000). Fluorescent labeling from viral replication is thereby restricted to defined neuronal cell types and their direct monosynaptic inputs. Because viral replication does not depend on transgene expression, it provides robust amplification of signal in presynaptic neurons regardless of input strength. We injected virus into transgenic crosses expressing the viral transgenes in specific cell types of the hippocampus formation to demonstrate cell-specific infection and monosynaptic retrograde transport of virus, which strongly labels even minor inputs. Such neuron-specific transgenic complementation of recombinant rabies virus holds great promise for obtaining cellular-resolution wiring diagrams of the mammalian CNS.

B-function expression in the flower center underlies the homeotic phenotype of Lacandonia schismatica (Triuridaceae).

Spontaneous homeotic transformations have been described in natural populations of both plants and animals, but little is known about the molecular-genetic mechanisms underlying these processes in plants. In the ABC model of floral organ identity in Arabidopsis thaliana, the B- and C-functions are necessary for stamen morphogenesis, and C alone is required for carpel identity. We provide ABC model-based molecular-genetic evidence that explains the unique inside-out homeotic floral organ arrangement of the monocotyledonous mycoheterotroph species Lacandonia schismatica (Triuridaceae) from Mexico. Whereas a quarter million flowering plant species bear central carpels surrounded by stamens, L. schismatica stamens occur in the center of the flower and are surrounded by carpels. The simplest explanation for this is that the B-function is displaced toward the flower center. Our analyses of the spatio-temporal pattern of B- and C-function gene expression are consistent with this hypothesis. The hypothesis is further supported by conservation between the B-function genes of L. schismatica and Arabidopsis, as the former are able to rescue stamens in Arabidopsis transgenic complementation lines, and Ls-AP3 and Ls-PI are able to interact with each other and with the corresponding Arabidopsis B-function proteins in yeast. Thus, relatively simple molecular modifications may underlie important morphological shifts in natural populations of extant plant taxa.

Natural Variation in Arabidopsis Leads to the Identification of REME1, a Pentatricopeptide Repeat-DYW Protein Controlling the Editing of Mitochondrial Transcripts

In vascular plants, organelle RNAs are edited by C-to-U base modification. Hundreds of mitochondrial C residues are targeted for editing in flowering plants. In this study, we exploited naturally occurring variation in editing extent to identify Required for Efficiency of Mitochondrial Editing1 (REME1), an Arabidopsis (Arabidopsis thaliana) pentatricopeptide repeat protein-encoding gene belonging to the DYW subclass that promotes editing of at least two C residues on different mitochondrial transcripts. Positional cloning identified REME1 unambiguously as the gene controlling editing of nad2-558. Virus-induced gene silencing of REME1 confirmed its role in editing of nad2-558 and allowed us to identify orfX-552 as a second C whose editing is positively controlled by REME1. An unexpected outcome of REME1 silencing was the finding of a number of mitochondrial C targets whose editing extent exhibits a significant and reproducible increase in silenced tissues. That increase was shown to be partly due to the virus inoculation and partly to REME1-specific silencing. Analysis of an insertional T-DNA mutant within the REME1 coding sequence confirmed the findings of the virus-induced gene silencing experiments: decrease in editing extent of nad2-558 and orfX-552 and increase in editing extent of two sites, matR-1771 and rpl5-92. Transgenic complementation of the low-edited accession (Landsberg erecta) restored the editing of nad2-558 and orfX-552 to high-edited accession (Columbia)-type levels or to even higher levels than Columbia. There was no effect of the transgene on editing extent of matR-1771 and rpl5-92. The strategy and tools used in this report can be applied to identify additional genes that affect editing extent in plant mitochondria.

A New Kv1.2 Channelopathy Underlying Cerebellar Ataxia

A forward genetic screen of mice treated with the mutagen ENU identified a mutant mouse with chronic motor incoordination. This mutant, named Pingu (Pgu), carries a missense mutation, an I402T substitution in the S6 segment of the voltage-gated potassium channel Kcna2. The gene Kcna2 encodes the voltage-gated potassium channel α-subunit Kv1.2, which is abundantly expressed in the large axon terminals of basket cells that make powerful axo-somatic synapses onto Purkinje cells. Patch clamp recordings from cerebellar slices revealed an increased frequency and amplitude of spontaneous GABAergic inhibitory postsynaptic currents and reduced action potential firing frequency in Purkinje cells, suggesting that an increase in GABA release from basket cells is involved in the motor incoordination in Pgu mice. In line with immunochemical analyses showing a significant reduction in the expression of Kv1 channels in the basket cell terminals of Pgu mice, expression of homomeric and heteromeric channels containing the Kv1.2(I402T) α-subunit in cultured CHO cells revealed subtle changes in biophysical properties but a dramatic decrease in the amount of functional Kv1 channels. Pharmacological treatment with acetazolamide or transgenic complementation with wild-type Kcna2 cDNA partially rescued the motor incoordination in Pgu mice. These results suggest that independent of known mutations in Kcna1 encoding Kv1.1, Kcna2 mutations may be important molecular correlates underlying human cerebellar ataxic disease.

An intrinsic gut leptin-melanocortin pathway modulates intestinal microsomal triglyceride transfer protein and lipid absorption

Fat is delivered to tissues by apoB-containing lipoproteins synthesized in the liver and intestine with the help of an intracellular chaperone, microsomal triglyceride transfer protein (MTP). Leptin, a hormone secreted by adipose tissue, acts in the brain and on peripheral tissues to regulate fat storage and metabolism. Our aim was to identify the role of leptin signaling in MTP regulation and lipid absorption using several mouse models deficient in leptin receptor (LEPR) signaling and downstream effectors. Mice with spontaneous LEPR B mutations or targeted ablation of LEPR B in proopiomelanocortin (POMC) or agouti gene related peptide (AGRP) expressing cells had increased triglyceride in plasma, liver, and intestine. Furthermore, melanocortin 4 receptor (MC4R) knockout mice expressed a similar triglyceride phenotype, suggesting that leptin might regulate intestinal MTP expression through the melanocortin pathway. Mechanistic studies revealed that the accumulation of triglyceride in the intestine might be secondary to decreased expression of MTP and lipid absorption in these mice. Surgical and chemical blockade of vagal efferent outflow to the intestine in wild-type mice failed to alter the triglyceride phenotype, demonstrating that central neural control mechanisms were likely not involved in the observed regulation of intestinal MTP. Instead, we found that enterocytes express LEPR, POMC, AGRP, and MC4R. We propose that a peripheral, local gut signaling mechanism involving LEPR B and MC4R regulates intestinal MTP and controls intestinal lipid absorption.

Modulation of rumpshaker phenotype with wild‐type PLP/DM20 suggests several pathogenic mechanisms

The rumpshaker mutation of the murine myelin proteolipid protein 1 (Plp1) gene generates misfolded PLP/DM20 protein, resulting in dysmyelination, increased oligodendrocyte apoptosis, and death prior to P40 when expressed on the C57 BL/6 background. In this study, we used transgenic complementation to normalize the levels of PLP/DM20 in myelin with wild-type protein to determine whether loss of normal PLP function or gain of toxic function is responsible for dysmyelination in the rumpshaker. Restoring myelin PLP/DM20 levels extended the survival time to at least P60, significantly reduced the density of apoptotic cells, increased myelin volume, and restored normal periodicity of myelin. Biochemical analysis found that several myelin proteins that are reduced in rumpshaker, including MAG, CNP, and SirT2, are markedly elevated at peak myelination (P20) in the rumpshaker transgenic mouse. Myelin basic protein, however, remained low at peak myelination but was restored at P60 when myelin had matured and entered into a maintenance phase. Markers of the unfolded protein response (UPR), BiP and XBP1, remained activated with the introduction of wild-type PLP. These data demonstrate that restoring wild-type PLP/DM20 levels in rumpshaker improves the phenotype and the integrity of myelin, but hypomyelination persists and stress pathways remain activated. This suggests that both gain- and loss-of-function mechanisms are involved in the pathogenesis of the rumpshaker.