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TRAF4 Deficiency Leads to Tracheal Malformation with Resulting Alterations in Air Flow to the Lungs

2000; Elsevier BV; Volume: 157; Issue: 2 Linguagem: Inglês

10.1016/s0002-9440(10)64578-6

1525-2191

Helena Shiels, Xiantang Li, Paul T. Schumacker, Emin Maltepe, Philip Padrid, Anne I. Sperling, Craig B. Thompson, Tullia Lindsten,

Amoebic Infections and Treatments

TRAF4 is one of six identified members of the family of TNFR-associated factors. While the other family members have been found to play important roles in the development and maintenance of a normal immune system, the importance of TRAF4 has remained unclear. To address this issue, we have generated TRAF4-deficient mice. Despite widespread expression of TRAF4 in the developing embryo, as well as in the adult, lack of TRAF4 expression results in a localized, developmental defect of the upper respiratory tract. TRAF4-deficient mice are born with a constricted upper trachea at the site of the tracheal junction with the larynx. This narrowing of the proximal end of the trachea results in respiratory air flow abnormalities and increases rates of pulmonary inflammation. These data demonstrate that TRAF4 is required to regulate the anastomosis of the upper and lower respiratory systems during development. TRAF4 is one of six identified members of the family of TNFR-associated factors. While the other family members have been found to play important roles in the development and maintenance of a normal immune system, the importance of TRAF4 has remained unclear. To address this issue, we have generated TRAF4-deficient mice. Despite widespread expression of TRAF4 in the developing embryo, as well as in the adult, lack of TRAF4 expression results in a localized, developmental defect of the upper respiratory tract. TRAF4-deficient mice are born with a constricted upper trachea at the site of the tracheal junction with the larynx. This narrowing of the proximal end of the trachea results in respiratory air flow abnormalities and increases rates of pulmonary inflammation. These data demonstrate that TRAF4 is required to regulate the anastomosis of the upper and lower respiratory systems during development. The tumor necrosis factor receptor (TNFR) family is a still expanding family of receptors that are involved in signal transduction pathways primarily during immune and inflammatory responses.1Baker SJ Reddy EP Transducers of life and death: TNF receptor superfamily and associated proteins.Oncogene. 1996; 12: 1-9PubMed Google Scholar Each receptor binds to a ligand from the TNF family, which results in multimerization of receptor monomers and in signaling responses ranging from proliferation and differentiation to apoptosis. The various family members have considerable homology in their extracellular domains due to the presence of cysteine-rich motifs required for ligand binding. With the exception of a certain subset of receptor family members that contain the so-called death domain (DD) motif in their cytoplasmic domain, the cytoplasmic tails of the proteins have a much more limited homology.Recently, a group of proteins that bind to the cytoplasmic tail of several TNFR family members has been identified. These proteins are named TRAFs (for TNFR-associated factors), and to date six family members have been described. All TRAFs share a ∼150-amino acid C-terminal TRAF domain that mediates the interaction between the TRAF and its receptor(s)2Cheng G Cleary AM Ye ZS Hong DI Lederman S Baltimore D Involvement of CRAF1, a relative of TRAF, in CD40 signaling.Science. 1995; 267: 1494-1498Crossref PubMed Scopus (442) Google Scholar, 3Hu HM O'Rourke K Boguski MS Dixit VM A novel RING finger protein interacts with the cytoplasmic domain of CD40.J Biol Chem. 1994; 269: 30069-30072Abstract Full Text PDF PubMed Google Scholar, 4Rothe M Wong SC Henzel WJ Goeddel DV A novel family of putative signal transducers associated with the cytoplasmic domain of the 75 kDa tumor necrosis factor receptor.Cell. 1994; 78: 681-692Abstract Full Text PDF PubMed Scopus (927) Google Scholar or other proteins, as well as a more loosely conserved region of zinc finger repeats. A similar domain can also be found in the meprin family of mammalian zinc metalloendopeptidases.5Uren AG Vaux DL TRAF proteins and meprins share a conserved domain.Trends Biochem Sci. 1996; 21: 244-245Abstract Full Text PDF PubMed Google Scholar Among some of the TNF receptors, a PXQX(T/S) motif has been shown to be a common TRAF domain binding site.6Hsu H Solovyev I Colombero A Elliott R Kelley M Boyle WJ ATAR, a novel tumor necrosis factor receptor family member, signals through TRAF2 and TRAF5.J Biol Chem. 1997; 272: 13471-13474Crossref PubMed Scopus (154) Google Scholar, 7Miller WE Cheshire JL Raab-Traub N Interaction of tumor necrosis factor receptor-associated factor signaling proteins with the latent membrane protein 1 PXQXT motif is essential for induction of epidermal growth factor receptor expression.Mol Cell Biol. 1998; 18: 2835-2844Crossref PubMed Scopus (76) Google Scholar, 8Mizushima S Fujita M Ishida T Azuma S Kato K Hirai M Otsuka M Yamamoto T Inoue J Cloning and characterization of a cDNA encoding the human homolog of tumor necrosis factor receptor-associated factor 5 (TRAF5).Gene. 1998; 207: 135-140Crossref PubMed Scopus (13) Google Scholar With the exception of TRAF1, all TRAFs contain an N-terminal RING finger, which seems to be required for their downstream signaling potential. Dominant negative (DN) versions of the TRAFs can be generated by removal of the RING finger domain.9Lee SY Kandala G Liou ML Liou HC Choi Y CD30/TNF receptor-associated factor interaction: NF-kappa B activation and binding specificity.Proc Natl Acad Sci USA. 1996; 93: 9699-9703Crossref PubMed Scopus (160) Google Scholar, 10Takeuchi M Rothe M Goeddel DV Anatomy of TRAF2. Distinct domains for nuclear factor-kappaB activation and association with tumor necrosis factor signaling proteins.J Biol Chem. 1996; 271: 19935-199942Crossref PubMed Scopus (280) Google Scholar TRAF2, 5, and 6 share an ability to mediate NF-κB and JNK activation through their N-terminal domains.10Takeuchi M Rothe M Goeddel DV Anatomy of TRAF2. Distinct domains for nuclear factor-kappaB activation and association with tumor necrosis factor signaling proteins.J Biol Chem. 1996; 271: 19935-199942Crossref PubMed Scopus (280) Google Scholar, 11Ishida T Mizushima S Azuma S Kobayashi N Tojo T Suzuki K Aizawa S Watanabe T Mosialos G Kieff E Yamamoto T Inoue J Identification of TRAF6, a novel tumor necrosis factor receptor-associated factor protein that mediates signaling from amino-terminal domain of the CD40 cytoplasmic region.J Biol Chem. 1996; 271: 28745-28748Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar, 12Nakano H Oshima H Chung W Williams-Abbott L Ware CF Yagita H Okumura K TRAF5, an activator of NF-kappaB and putative signal transducer for the lymphotoxin-beta receptor.J Biol Chem. 1996; 271: 14661-14664Crossref PubMed Scopus (308) Google Scholar TRAFs are also able to bind to non-TNFR family members, as exemplified by TRAF6's ability to associate with the interleukin-1 (IL-1) receptor.13Cao Z Xiong J Takeuchi M Kurama T Goeddel DV TRAF6 is a signal transducer for interleukin-1.Nature. 1996; 383: 443-446Crossref PubMed Scopus (1111) Google Scholar To date, published reports have been generated on the phenotype of mice that lack TRAF2,14Yeh WC Shahinian A Speiser D Kraunus J Billia F Wakeham A de la Pompa JL Ferrick D Hum B Iscove N Ohashi P Rothe M Goeddel DV Mak TW Early lethality, functional NF-kappaB activation, and increased sensitivity to TNF-induced cell death in TRAF2-deficient mice.Immunity. 1997; 7: 715-725Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar TRAF3,15Xu Y Cheng G Baltimore D Targeted disruption of TRAF3 leads to postnatal lethality and defective T-dependent immune responses.Immunity. 1996; 5: 407-415Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar TRAF5,16Nakano H Sakon S Koseki H Takemori T Tada K Matsumoto M Munechika E Sakai T Shirasawa T Akiba H Kobata T Santee SM Ware CF Rennert PD Taniguchi M Yagita H Okumura K Targeted disruption of Traf5 gene causes defects in CD40- and CD27-mediated lymphocyte activation.Proc Natl Acad Sci USA. 1999; 17: 9803-9808Crossref Scopus (165) Google Scholar or TRAF617Lomaga MA Yeh WC Sarosi I Duncan GS Furlonger C Ho A Morony S Capparelli C Van G Kaufman S van der Heiden A Itie A Wakeham A Khoo W Sasaki T Cao Z Penninger JM Paige CJ Lacey DL Dunstan CR Boyle WJ Goeddel DV Mak TW TRAF6 deficiency results in osteopetrosis and defective interleukin-1, CD40, and LPS signaling.Genes Dev. 1999; 13: 1015-1024Crossref PubMed Scopus (1068) Google Scholar or that overexpress TRAF118Speiser DE Lee SY Wong B Arron J Santana A Kong YY Ohashi PS Choi Y A regulatory role for TRAF1 in antigen-induced apoptosis of T cells.J Exp Med. 1997; 185: 1777-1783Crossref PubMed Scopus (114) Google Scholar or the DN form of TRAF2.19Lee SY Reichlin A Santana A Sokol KA Nussenzweig MC Choi Y TRAF2 is essential for JNK but not NF-kappaB activation and regulates lymphocyte proliferation and survival.Immunity. 1997; 7: 703-713Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar The TRAF2-, TRAF3-, or TRAF6-deficient mice that are born appear normal at birth but die at a very young age as a result of severe runting. TRAF5-deficient mice appear outwardly normal.The immune system of TRAF2-deficient mice is affected, with depletion of lymphocytes in thymus and spleen and an absence of secondary germinal centers, perhaps due to a combination of hypersensitivity to TNF and increased serum TNF levels. B-cell development is impaired, but T cells develop normally. TNF-mediated NF-κB responses are normal, but JNK activation is deficient. Interestingly, in TRAF2 DN mice with the transgene being expressed only in lymphocytes, there is an increase in the number of B cells, splenomegaly, and lymphadenopathy. JNK activation, but not NF-κB responses, is affected just as in TRAF2-deficient mice.In mice lacking TRAF3, despite hypotrophy of spleen and thymus similar to what is seen in TRAF2-deficient animals, the immune system is defective when it comes to T-cell-dependent responses to antigen but otherwise is fairly normal.TRAF5-deficient mice show defects in CD40- and CD27-mediated signaling. TRAF6-deficient mice have enlarged spleens, despite normal T-cell function and reduced B-cell proliferation in response to CD40 or lipopolysaccharide stimulation. IL-1, CD40, or lipopolysaccharide-mediated NF-κB activation is also disrupted, as is IL-1-mediated JNK activation. In addition, these mice are osteopetrotic, which is most likely due to the recently described association between TRAF6 and the receptor activator of NF-κB (RANK),20Galibert L Tometsko ME Anderson DM Cosman D Dougall WC The involvement of multiple tumor necrosis factor receptor (TNFR)-associated factors in the signaling mechanisms of receptor activator of NF-kappaB, a member of the TNFR superfamily.J Biol Chem. 1998; 273: 34120-34127Crossref PubMed Scopus (279) Google Scholar which is required for osteoclast differentiation and lymph node formation.21Dougall WC Glaccum M Charrier K Rohrbach K Brasel K DeSmedt T Daro E Smith J Tometsko ME Maliszewski CR Armstrong A Shen V Bain S Cosman D Anderson D Morrissey PJ Peschon JJ Schuh J RANK is essential for osteoclast and lymph node development.Genes Dev. 1999; 13: 2412-2424Crossref PubMed Scopus (1188) Google ScholarTRAF4 was cloned in a differential expression screen, using mRNA from metastatic breast cancer samples.22Tomasetto C Regnier C Moog-Lutz C Mattei MG Chenard MP Lidereau R Basset P Rio MC Identification of four novel human genes amplified and overexpressed in breast carcinoma and localized to the q11–q21.3 region of chromosome 17.Genomics. 1995; 28: 367-376Crossref PubMed Scopus (226) Google Scholar, 23Regnier CH Tomasetto C Moog-Lutz C Chenard MP Wendling C Basset P Rio MC Presence of a new conserved domain in CART1, a novel member of the tumor necrosis factor receptor-associated protein family, which is expressed in breast carcinoma.J Biol Chem. 1995; 270: 25715-25721Crossref PubMed Scopus (201) Google Scholar The 2-kb gene is located on human chromosome 17, q11-q12,22Tomasetto C Regnier C Moog-Lutz C Mattei MG Chenard MP Lidereau R Basset P Rio MC Identification of four novel human genes amplified and overexpressed in breast carcinoma and localized to the q11–q21.3 region of chromosome 17.Genomics. 1995; 28: 367-376Crossref PubMed Scopus (226) Google Scholar which is a region that also contains the oncogene c-erbB2, a gene known to be involved in breast cancer. The predicted molecular weight of TRAF4 is 53 kd. TRAF4 was originally found to be expressed in the nucleus of the breast cancer cells, using a polyclonal rabbit anti-serum against a TRAF-C domain synthetic peptide. In mice, using Northern blot analysis, as well as in situ hybridization,24Masson R Regnier CH Chenard MP Wendling C Mattei MG Tomasetto C Rio MC Tumor necrosis factor receptor associated factor 4 (TRAF4) expression pattern during mouse development.Mech Dev. 1998; 71: 187-191Crossref PubMed Scopus (39) Google Scholar it was shown that TRAF4 is expressed at high levels during embryogenesis, peaking at embryonic day 13.5. Specifically, transcripts are present in the developing neuroepithelium, neural crest cell condensations, and the first, second, and third branchial arches. Transcripts are also detected in the neuroepithelium of the nasal fossae, as well as in thymus, salivary glands, and intestine of embryos. In the adult, TRAF4 was among the genes induced in postmitotic and undifferentiated cells of the central nervous system.Recently, a second TRAF4-specific polyclonal antibody was used to investigate expression patterns in normal embryonic and adult human tissue, as well as in tissue from breast and prostate cancers.25Krajewska M Krajewski S Zapata JM Van Arsdale T Gascoyne RD Berern K McFadden D Shabaik A Hugh J Reynolds A Clevenger CV Reed JC TRAF4 expression in epithelial progenitor cells: Analysis in normal adult, fetal, and tumor tissues.Am J Pathol. 1998; 152: 1549-1561PubMed Google Scholar Surprisingly, by using this antibody for immunohistochemistry, TRAF4 was found to be primarily localized to the cytoplasm of the cells, and was not expressed in most breast cancer samples. Most epithelial cells in this study, including thymic epithelial cells and lymph node dendritic cells, expressed TRAF4, whereas the thymocytes and lymphocytes did not demonstrate any TRAF4 staining. In addition, Krajewska et al found evidence that TRAF4 is able to associate with the lymphotoxin-β receptor, which has an overlapping expression pattern in the scaffolding tissues of thymus and lymph nodes. A weak interaction with the p75 neural growth factor receptor was also detected. The differences in the expression patterns of TRAF4 in this report as compared to the study using in situ hybridization could be explained by posttranscriptional modification of TRAF4 transcripts.24Masson R Regnier CH Chenard MP Wendling C Mattei MG Tomasetto C Rio MC Tumor necrosis factor receptor associated factor 4 (TRAF4) expression pattern during mouse development.Mech Dev. 1998; 71: 187-191Crossref PubMed Scopus (39) Google ScholarTo better understand the role of TRAF4 in development, we have generated TRAF4-deficient mice through gene targeting. The mice are viable and appear outwardly normal. However, about 20% of TRAF4 knock-out mice present with a high-pitched wheezing noise as they breathe. In addition, there is a decrease in weight of the animals, averaging 10% for adult males and 5% for adult females. Pathological analysis demonstrates that lack of TRAF4 leads to failure to properly form the proximal end of the trachea, at the region where the trachea connects to the larynx, leading to various degrees of constriction in all knock-out mice examined. The air flow is altered, as evidenced by a marked decrease in response to a challenge with aerosolized antigen in immunized mice lacking TRAF4. TRAF4-deficient mice also develop spontaneous inflammation of the lungs, correlating with the occurrence of wheezing. The immune system of TRAF4 knock-out mice appears to respond normally to a variety of stimuli. Taken together, the data suggest that the expression of TRAF4 normally seen in branchial arches 2 and 3 during embryogenesis correlates with a required role for TRAF4 during normal laryngotracheal development.Materials and MethodsTargeting Constructs for the TRAF4 GenePolymerase chain reaction amplification of a human cDNA library, using primers derived from the published sequence of human TRAF4, was used to subclone human TRAF4 cDNA. A 5′ 0.6-kb probe, containing mostly the three zinc finger repeats, was isolated from the cDNA clone and used to screen a phage library (Lambda FIX II; Stratagene Cloning Systems, La Jolla, CA) derived from 129/SvJ mice. The resulting genomic clones were mapped by restriction digest analysis, Southern blotting, and sequencing. A 2.2-kb Kpn I-Xba I fragment located 3′ of the last exon (exon 7), in combination with either a 7-kb Sal I-Bgl II fragment containing the first exon (construct 1, Figure 1A) or a 4.1-kb Sal I-Bam HI fragment containing only sequence 5′ of the gene (construct 2, Figure 1A), was cut out from the same phage preparation (the Sal I site is derived from the phage) and subcloned into the pPNT vector.26Tybulewicz VL Crawford CE Jackson PK Bronson RT Mulligan RC Neonatal lethality and lymphopenia in mice with a homozygous disruption of the c-abl proto-oncogene.Cell. 1991; 65: 1153-1163Abstract Full Text PDF PubMed Scopus (1158) Google Scholar On linearization, the resulting targeting constructs contained the PGK-neor gene in the opposite transcriptional orientation as compared to the TRAF4 gene, between the two subcloned genomic fragments that were flanked on one side by the PGK-TK gene.Generation of TRAF4-Deficient MiceThe two linearized targeting constructs were used to electroporate R1 ES cells, derived from an F1(129/Sv × 129/SvJ) blastocyst.27Nagy A Rossant J Nagy R Abramow-Newerly W Roder JC Derivation of completely cell culture-derived mice from early-passage embryonic stem cells.Proc Natl Acad Sci USA. 1993; 90: 8424-8428Crossref PubMed Scopus (1960) Google Scholar Two × 107 ES cells were used for each electroporation at 270 V, 500 μF, using a Bio-Rad Genepulser (Bio-Rad Laboratories, Hercules, CA), and the cells were plated onto eight 10-cm dishes containing mitomycin C-treated neor mouse embryo fibroblasts.28Askew GR Doetschman T Lingrel JB Site-directed point mutations in embryonic stem cells: a gene-targeting tag-and-exchange strategy.Mol Cell Biol. 1993; 13: 4115-4124Crossref PubMed Scopus (119) Google Scholar Culture and selection of neor, TK-negative ES cells were as previously described,29Joyner AL Gene Targeting: A Practical Approach. Oxford University Press, New York1993Google Scholar with a few modifications as described below. High-glucose liquid Dulbecco's minimum essential medium with l-glutamine (GibcoBRL, Grand Island, NY) was used with the addition of penicillin-streptomycin (GibcoBRL), but without the addition of sodium pyruvate. The fetal bovine serum (HyClone, Logan, Utah) was screened for the optimal growth of undifferentiated ES cells. Selection for targeted ES cells was performed 24 hours after electroporation, using ES cell medium containing 250 μg/ml neomycin and 2 μM gancyclovir. Surviving colonies of ES cells were picked 7 days after electroporation.The resulting expanded ES cell clones were screened by Southern blot analysis of Hin dIII-digested genomic DNA isolated from each clone. The 1-kb Xba I-Hin dIII probe used to identify homologously recombined ES cell clones is shown in Figure 1A.ES cells from the selected clones were injected into C57/BL6 blastocysts, and chimeric mice with a large contribution of targeted ES cells could be identified because of their mainly agouti coat color. Largely agouti chimeras were bred to C57/BL6 females, and the resulting F1 progeny were screened by Southern blot analysis of tail DNA to determine their genotype. Mice with germline transmission of a targeted TRAF4 allele were intercrossed to generate TRAF4-deficient mice.Western and Northern Blot AnalysisWhole-cell extracts of homogenized tissues from wild-type mice and mice derived from all three of the germline transmitted ES cell lines were made using RIPA buffer (50 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 0.1% Triton X-100, 0.5% deoxycholate, 0.1 mmol/L EGTA, 0.1 mmol/L EDTA) with the addition of protease inhibitors. The protein concentration was determined by colorimetric assay (BCA Protein Assay; Pierce, Rockford, IL), and equal amounts of protein were loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel. The Western blot was probed with a polyclonal antibody raised against an N-terminal peptide from human TRAF4 (Santa Cruz Biotechnology, Santa Cruz, CA).Total RNA was purified from wild-type and knock-out mice with the use of TRIzol (GibcoBRL). The RNA was equalized as previously described,30June CH Ledbetter JA Gillespie MM Lindsten T Thompson CB T-cell proliferation involving the CD28 pathway is associated with cyclosporine-resistant interleukin 2 gene expression.Mol Cell Biol. 1987; 7: 4472-4481Crossref PubMed Scopus (473) Google Scholar and the concentration was later determined by spectrophotometric analysis. Approximately 5 μg of total RNA was used for each sample. The samples were loaded on an agarose gel, electrophoresed, and blotted as described.30June CH Ledbetter JA Gillespie MM Lindsten T Thompson CB T-cell proliferation involving the CD28 pathway is associated with cyclosporine-resistant interleukin 2 gene expression.Mol Cell Biol. 1987; 7: 4472-4481Crossref PubMed Scopus (473) Google Scholar TRAF4 transcripts were detected using a probe specific for exons 4–7. (Similar results were obtained with the use of a probe containing the RING domain.) The relative amounts of RNA were visualized with the use of an end-labeled 5S RNA probe.Examination of Tracheal MorphologyThe mice were sacrificed with a lethal dose of ketamine. The tissues in front of the trachea were removed and the chest was opened, but the mice were otherwise left intact. The outer diameter of each cartilage ring was measured, using a dissection microscope equipped with a scale bar with 0.1-mm increments. The average width of either the first three rings below the cricoid cartilage or the last three cartilage rings before the bifurcation of the trachea was used as a measure of proximal and distal tracheal width, respectively. After the tracheal diameter had been documented, tissue containing the entire airway was removed and fixed. The fixed tissue was sectioned in the coronal plane and stained with hematoxylin-eosin. More detailed histological analysis was performed to determine the circumference, width, and density of the cartilage rings, as well as the occurrence of inflammatory cells in the airway tissue.Ovalbumin Immunization, Nebulization, and Analysis of Lavage FluidThe method used for ovalbumin (OVA) immunization and nebulization was as described by Krinzman et al,31Krinzman SJ De Sanctis GT Cernadas M Kobzik L Listman JA Christiani DC Perkins DL Finn PW T cell activation in a murine model of asthma.Am J Physiol. 1996; 271: L476-L483PubMed Google Scholar with some modifications. Specifically, TRAF4-deficient mice, or littermate controls, were left untreated or were immunized intraperitoneally with 10 μg of OVA (grade IV; Sigma, St. Louis, MO) in 20 μl of 5 mg/ml alum in phosphate-buffered saline (PBS). Each experimental group contained three mice. The immunization was repeated 1 week later. Four days after the second immunization, the mice were challenged with aerosolized 6% OVA in sterile PBS, using a nebulizer, for 3 consecutive days for 30 minutes each time. Five days later the mice were sacrificed with a lethal dose of ketamine. Day 5 was chosen to maximize the presence of eosinophils in the bronchoalveolar lavage (BAL) fluid.32Zuany-Amorim C Haile S Leduc D Dumarey C Huerre M Vargaftig BB Pretolani M Interleukin-10 inhibits antigen-induced cellular recruitment into the airways of sensitized mice.J Clin Invest. 1995; 95: 2644-2651Crossref PubMed Scopus (257) Google Scholar The airways were rinsed four times with 0.8 ml of sterile PBS, using a syringe with a blunt needle inserted halfway down the trachea, and sealed off with suture thread around the trachea. Similar volumes of BAL fluid were recovered from each mouse. Total white blood cell counts were made directly from the BAL fluid. In addition, samples were centrifuged onto microscope slides with a cytospin (Shandon, Pittsburgh, PA). The cells were stained by standard methods, and the relative contribution of eosinophils versus other white blood cell types was determined.In Situ HybridizationEmbryos from timed matings (noon of the day of vaginal plug was considered embryonal day 0.5, E0.5d) were dissected out, fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned. A 0.6-kb probe from the human TRAF4 cDNA 5′ end was first used on a Northern blot with total mouse RNA to verify specific recognition of mouse TRAF4 transcript. The probe was subcloned in pBluescript (Stratagene Cloning Systems), linearized on either side of the cloning cassette, and in vitro transcribed and α-35S-labeled with T3 (sense) or T7 (antisense) polymerase, depending on the site of linearization. The riboprobes were quantitated and used for in situ hybridization, with washing and developing conditions as previously described.33Wilkinson DG Nieto MA Detection of messenger RNA by in situ hybridization to tissue sections and whole mounts.Methods Enzymol. 1993; 225: 361-373Crossref PubMed Scopus (721) Google Scholar The sections were dipped in NTB2 emulsion (Kodak, Rochester, NY), and autoradiography was allowed to proceed for 1 week before the slides were developed and the cell nuclei were counterstained using Hoechst 33258 (Boehringer Mannheim, Mannheim, Germany). Adjacent sections from the same embryo probed with either sense or antisense riboprobe were photographed, using the same exposure time, with a Zeiss Axiophot microscope for epifluorescence and dark-field microscopy.ResultsGene Targeting and Generation of TRAF4-Deficient MiceThe TRAF4 gene was disrupted by homologous gene targeting. Three independent ES cell lines derived from two different constructs (Figure 1A) were used to generate TRAF4-deficient mice. Construct I eliminated all but part of the RING finger (exon 1), whereas construct II eliminated the entire gene from the targeted chromosome. Either of the two constructs resulted in loss of both detectable TRAF4 protein (Figure 1B) and TRAF4 mRNA (Figure 1C), and all three lines of the TRAF4−/− mice have the same phenotype, verifying the successful targeting of TRAF4 in these mice.TRAF4-deficient mice were born at the expected Mendelian ratio. The mice bred as well as their wild-type littermates. On gross examination of the knock-out mice, two findings were evident. First, the mice were smaller overall than their littermates. Figure 2 shows the weight at 7 weeks of 66 knock-out mice and 89 wild-type mice grouped by sex. There is a statistically significant reduction in the average weight of mice lacking TRAF4. Males have undergone a 10% decrease in weight and females a 5% decrease at 7 weeks of age (Figure 2). The weight reduction is not due to loss of any specific tissue, inasmuch as overall gross and histopathological examination revealed no significant differences between TRAF4 wild-type and knock-out mice. Second, a certain percentage (approximately 20%) of the mice at any one time displayed inspiratory stridor, as evidenced by a high-pitched wheezing noise. Mice as young as 2 weeks of age could be heard making the noise. Affected mice could have severe stridor at one time, but within weeks could be asymptomatic and make no or very little noise.Figure 2Average total body weight of TRAF4 wild-type and knock-out mice. The mice were placed on a Boston trip balance. Weight was recorded to within an accuracy of 0.1 g. The weights of male and female mice at 7 weeks of age are shown. The mean and SEM can be seen, as well as the calculated P values for each data set. The number of animals in each group is denoted by n.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Histological Examination of the Airways of TRAF4-Deficient MiceTo understand the underlying defect(s) of TRAF4-deficient mice, a more detailed analysis of the airways was performed. A total of nine TRAF4-deficient mice and six littermate controls were subjected to analysis. Tissue samples were delivered to the pathologist in a blinded fashion to prevent introduction of investigator bias. On studying the trachea, it became clear that all mice lacking TRAF4 had a narrowing of the proximal part of the trachea, involving the first few cartilage rings immediately below the larynx. When the external diameter of the upper trachea was measured, an average 18% reduction was found in TRAF4-deficient mice as compared to littermate control mice, resulting in an estimated decrease of the cross-sectional area of about 32%. There was no difference in the width of the cartilage rings themselves, and the U-shaped form of the rings remained intact. However, a ventrally pointed appearance was often evident. Lack of TRAF4 expression had no effect on the width of the trachea below this region. The results are summarized in Figure 3A. The location of the tracheal constriction just below the larynx in TRAF4 knock-out mice correlates with the observed inspiratory stridor, because extrathoracic tracheal stenosis would be expected to give rise to inspiratory stridor, whereas intrathoracic tracheal stenosis would result in expiratory stridor.Figure 3Morphology of the airways in TRAF4 wild-type or knock-out mice, showing the deformed upper trachea of TRAF4-defic