Pbx3 Deficiency Results in Central Hypoventilation
2004; Elsevier BV; Volume: 165; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)63392-5
ISSN1525-2191
AutoresJoon Whan Rhee, Akiko Arata, Licia Selleri, Yakop Jacobs, Satoru Arata, Hiroshi Onimaru, Michael L. Cleary,
Tópico(s)Congenital Diaphragmatic Hernia Studies
ResumoPbx proteins comprise a family of TALE (three amino acid loop extension) class homeodomain transcription factors that are implicated in developmental gene expression through their abilities to form hetero-oligomeric DNA-binding complexes and function as transcriptional regulators in numerous cell types. We demonstrate here that one member of this family, Pbx3, is expressed at high levels predominantly in the developing central nervous system, including a region of the medulla oblongata that is implicated in the control of respiration. Pbx3-deficient mice develop to term but die within a few hours of birth from central respiratory failure due to abnormal activity of inspiratory neurons in the medulla. This partially phenocopies the defect in mice deficient for Rnx, a metaHox homeodomain transcription factor, that we demonstrate here is capable of forming a DNA-binding complex with Pbx3. Rnx expression is unperturbed in Pbx3-deficient mice, but its ability to enhance transcription in vitro as a complex with TALE proteins is compromised in the absence of Pbx3. Thus, Pbx3 is essential for respiration and, like its DNA-binding partner Rnx, is critical for proper development of medullary respiratory control mechanisms. Pbx3-deficient mice provide a model for congenital central hypoventilation syndrome and suggest that Pbx3 mutations may promote the pathogenesis of this disorder. Pbx proteins comprise a family of TALE (three amino acid loop extension) class homeodomain transcription factors that are implicated in developmental gene expression through their abilities to form hetero-oligomeric DNA-binding complexes and function as transcriptional regulators in numerous cell types. We demonstrate here that one member of this family, Pbx3, is expressed at high levels predominantly in the developing central nervous system, including a region of the medulla oblongata that is implicated in the control of respiration. Pbx3-deficient mice develop to term but die within a few hours of birth from central respiratory failure due to abnormal activity of inspiratory neurons in the medulla. This partially phenocopies the defect in mice deficient for Rnx, a metaHox homeodomain transcription factor, that we demonstrate here is capable of forming a DNA-binding complex with Pbx3. Rnx expression is unperturbed in Pbx3-deficient mice, but its ability to enhance transcription in vitro as a complex with TALE proteins is compromised in the absence of Pbx3. Thus, Pbx3 is essential for respiration and, like its DNA-binding partner Rnx, is critical for proper development of medullary respiratory control mechanisms. Pbx3-deficient mice provide a model for congenital central hypoventilation syndrome and suggest that Pbx3 mutations may promote the pathogenesis of this disorder. Pbx proteins comprise a set of four TALE (three amino acid loop extension) class homeodomain transcription factors1Nourse J Mellentin JD Galili N Wilkinson J Stanbridge E Smith SD Cleary ML Chromosomal translocation t(1;19) results in synthesis of a homeobox fusion mRNA that codes for a potential chimeric transcription factor.Cell. 1990; 60: 535-545Abstract Full Text PDF PubMed Scopus (567) Google Scholar, 2Kamps MP Murre CM Sun X-H Baltimore D A new homeobox gene contributes the DNA-binding domain of the t(1;19) translocation protein in pre-B ALL.Cell. 1990; 60: 547-555Abstract Full Text PDF PubMed Scopus (580) Google Scholar, 3Monica K Galili N Nourse J Saltman D Cleary ML PBX2 and PBX3, new homeobox genes with extensive homology to the human proto-oncogene PBX1.Mol Cell Biol. 1991; 11: 6149-6157Crossref PubMed Scopus (262) Google Scholar, 4Wagner K Mincheva A Korn B Lichter P Pöpperl H Pbx4, a new Pbx family member on mouse chromosome 8, is expressed during spermatogenesis.Mech Dev. 2001; 103: 127-131Crossref PubMed Scopus (64) Google Scholar that are implicated in developmental gene expression. They form hetero-oligomeric DNA-binding complexes and function as transcriptional regulators in cells of different developmental lineages.5Mann RS Affolter M Hox proteins meet more partners.Curr Opin Genet Dev. 1998; 8: 423-429Crossref PubMed Scopus (327) Google Scholar Pbx proteins are orthologs of Drosophila extradenticle,6Rauskolb C Peiper M Weischaus E Extradenticle, a regulator of homeotic gene activity, is a homolog of the homeobox-containing human proto-oncogene, Pbx1.Cell. 1993; 74: 1101-1112Abstract Full Text PDF PubMed Scopus (216) Google Scholar which contributes to axial and limb patterning,7Mercader N Leonardo E Azpiazu N Serrano A Morata G Martinez C Torres M Conserved regulation of proximodistal limb axis development by Meis1/Hth.Nature. 1999; 402: 425-429Crossref PubMed Scopus (249) Google Scholar eye development,8Pai CY Kuo TS Jaw TJ Kurant E Chen CT Bessarab DA Salzberg A Sun YH The homothorax homeoprotein activates the nuclear localization of another homeoprotein, extradenticle, and suppresses eye development in Drosophila.Genes Dev. 1998; 12: 435-446Crossref PubMed Scopus (272) Google Scholar and patterning of the embryonic peripheral nervous system.9Kurant E Pai CY Sharf R Halachmi N Sun YH Salzberg A Dorsotonals/homothorax, the Drosophila homologue of meis1, interacts with extradenticle in patterning the embryonic PNS.Development. 1998; 125: 1037-1048Crossref PubMed Google Scholar Similarly, loss-of-function studies in knockout mice demonstrate a role for Pbx1 in patterning and morphogenesis.10Selleri L Depew MJ Jacobs Y Chanda SK Tsang KY Cheah K Rubenstein JLR O'Gorman S Cleary ML Requirement for Pbx1 in skeletal patterning and programming chondrocyte proliferation and differentiation.Development. 2001; 128: 3543-3557PubMed Google Scholar, 11DiMartino J Selleri L Traver D Firpo M Rhee JW Warnke R O'Gorman S Weissman IL Cleary ML The Hox cofactor and proto-oncogene Pbx1 is required for maintenance of definitive hematopoiesis in the fetal liver.Blood. 2001; 98: 618-626Crossref PubMed Scopus (135) Google Scholar, 12Kim SK Selleri L Lee JS Zhang AY Gu X Jacobs Y Cleary ML Pbx1 inactivation disrupts pancreas development and in Ipf1-deficient mice promotes diabetes mellitus.Nat Genet. 2002; 30: 430-435Crossref PubMed Scopus (146) Google Scholar, 13Schnabel CA Godin RE Cleary ML Pbx1 regulates nephrogenesis and ureteric branching in the developing kidney.Dev Biol. 2002; 254: 262-276Crossref Scopus (94) Google Scholar Most organs are abnormally developed in Pbx1−/− embryos and some of the observed deficiencies in organogenesis and cellular differentiation appear to result from compromise of orphan Hox proteins.12Kim SK Selleri L Lee JS Zhang AY Gu X Jacobs Y Cleary ML Pbx1 inactivation disrupts pancreas development and in Ipf1-deficient mice promotes diabetes mellitus.Nat Genet. 2002; 30: 430-435Crossref PubMed Scopus (146) Google Scholar To further define the developmental roles of Pbx proteins and their potential isotype-specific contributions, we have determined the expression pattern of Pbx3 and created Pbx3-deficient mice. We report here that lack of Pbx3 results in neonatal death due to central respiratory failure, a phenotype similar to that of mice deficient for Rnx,14Shirasawa S Arata A Onimaru H Roth KA Brown GA Horning S Arata S Okumura K Sasazuki T Korsmeyer SJ Rnx deficiency results in congenital central hypoventilation.Nat Genet. 2000; 24: 287-290Crossref PubMed Scopus (141) Google Scholar a metaHox protein that is also expressed in the medulla and capable of associating with Pbx3 in a higher-order DNA-binding transcriptional complex. Our studies implicate Pbx3 as being critical for development of central control of respiration in vertebrates and suggest the broader hypothesis that mutations of Pbx3, its cofactors, or its regulators may promote the pathogenesis of congenital central hypoventilation syndrome. The Pbx3 gene was mutated by deletion of a HindIII-BamH1 fragment spanning exon 3 and replacement with a PGK neo cassette (from the pNT vector) in opposite transcriptional orientation. The Pbx3 deletion creates an early frame-shift mutation that causes termination of the Pbx3 reading frame upstream of its dimerization and DNA-binding domains. A 12-kb XbaI-EcoR1 fragment of genomic DNA spanning the disrupted Pbx3 exon 3 was then cloned into the targeting vector. Homologous recombinant clones were identified by Southern blot analyses. Out of 132 informative clones, 8 were identified as homologous recombinants. Euploid clones were microinjected into C57BL/6 host blastocysts, which were then implanted in pseudo-pregnant females. Chimeric male mice were backcrossed with wt C57BL/6 females to obtain germline transmission of the targeted allele. Phenotypes were analyzed in neonates derived from third backcross generations on a C57BL/6 background. Monoclonal antibodies were raised against maltose-binding protein (MBP) fusion proteins containing the 80 or 30 carboxy-terminal amino acids of human Pbx3a or Pbx3b, respectively. Specificity for Pbx3 proteins was established by Western blot analysis of in vitro translated Pbx family proteins (Pbx1, 2, and 3). Anti-Rnx rabbit antisera were raised against an affinity-purified MBP-Rnx fusion protein containing the 75 C-terminal amino acids of mouse Rnx. Immune sera were purified by Protein A Sepharose affinity chromatography (Amersham-Pharmacia Biotech, Piscataway, NJ) and their specificity determined by Western blot analysis of in vitro translated Hox11 family proteins. Embryos were fixed in formalin and embedded in paraffin using standard procedures. Sectioned tissues were stained with anti-Pbx3a (0.008 mg/ml), anti-Pbx3b (0.04 mg/ml), or anti-Meis15Jacobs Y Schanbel CA Cleary ML Trimeric association of Hox and TALE homeodomain proteins mediates HoxB2 hindbrain enhancer activity.Mol Cell Biol. 1999; 19: 5134-5142Crossref PubMed Scopus (215) Google Scholar (0.043 mg/ml) monoclonal antibodies followed by biotinylated rabbit anti-mouse IgG secondary antibody (1:150). Immune complexes were visualized using horseradish peroxidase-conjugated streptavidin (Jackson Immunoresearch, West Grove, PA) and DAB chromagen (Sigma, St. Louis, MO). For Rnx/Pbx3 co-localization, paraffin sections were stained with anti-Rnx (1:25) and Pbx3a (0.16 mg/ml) antibodies. Secondary antibodies (1:150) consisted of Texas red-conjugated goat anti-rabbit IgG (Accurate Antibodies, Westbury, NY) and FITC-conjugated goat anti-mouse IgG (Accurate Antibodies). Stained slides were mounted in DAPI-containing slide mount medium with antiquench (Vector Labs, Burlingame, CA) and examined by fluorescence microscopy. Whole body plethysmography was performed on P0 mice less than 1 day of age.16Kuwaki T Cao W Kurihara Y Kurihara H Ling G Onodera M Ju K Yazaki Y Kumada M Impaired ventilatory responses to hypoxia and hypercapnea in mutant mice deficient in endothelin-1.Am J Physiol. 1996; 270: 1279-1286PubMed Google Scholar Respiratory frequency, tidal volume, and minute volume were calculated from 50 to 70 respiratory cycles during quiet breathing. Medulla and spinal cords for in vitro preparations were isolated from P0 mice under deep ether anesthesia as described previously.14Shirasawa S Arata A Onimaru H Roth KA Brown GA Horning S Arata S Okumura K Sasazuki T Korsmeyer SJ Rnx deficiency results in congenital central hypoventilation.Nat Genet. 2000; 24: 287-290Crossref PubMed Scopus (141) Google Scholar, 17Suzue T Respiratory rhythm generation in the in vitro brain stem-spinal cord preparation of the neonatal rat.J Physiol. 1984; 354: 173-183PubMed Google Scholar, 18Onimaru H Arata A Homma I Primary respiratory rhythm generator in the medulla of brainstem-spinal cord preparation from newborn rat.Brain Res. 1988; 445: 314-324Crossref PubMed Scopus (140) Google Scholar Respiratory-like activity corresponding to the inspiration rhythm was monitored at the C4/C5 ventral root through a glass capillary suction electrode and a high-pass filter with a 0.3 seconds time constant. Values are presented as mean ± SD. Statistical significance was determined by analysis of variance (analysis of variance) for all three genotypes. Significant differences between groups were identified using post-hoc Bonferroni tests. Membrane potentials of inspiratory neurons in the ventrolateral medulla19Arata A Onimaru H Homma I Respiration-related neurons in the ventral medulla of newborn rats in vitro.Brain Res Bull. 1990; 24: 599-604Crossref PubMed Scopus (89) Google Scholar were recorded using conventional whole-cell patch-clamp methods.20Onimaru H Homma I Whole cell recordings from respiratory neurons in the medulla of brainstem-spinal cord preparations isolated from newborn rats.Plugers Arch. 1992; 420: 399-406Crossref PubMed Scopus (116) Google Scholar The membrane potentials were recorded with a single-electrode voltage-clamp amplifier (CEZ-3100, Nihon Kohden, Tokyo, Japan) after compensation of the series resistance (25 to 50 mol/LΩ) and capacitance. Proteins used in DNA-binding assays were produced in vitro from SP6 expression plasmids using a coupled rabbit reticulocyte lysate system (Promega, Madison, WI). The DNA probe consisted of a gel-purified, end-labeled, double-stranded oligonucleotide encoding a modified HoxB2 r4 enhancer,15Jacobs Y Schanbel CA Cleary ML Trimeric association of Hox and TALE homeodomain proteins mediates HoxB2 hindbrain enhancer activity.Mol Cell Biol. 1999; 19: 5134-5142Crossref PubMed Scopus (215) Google Scholar containing consensus Pbx and Meis sites and a Hox site that was altered to favor binding by Rnx.21Shen WF Chang CP Rozenfeld S Sauvageau G Humphries RK Lu M Lawrence HJ Cleary ML Largman C Hox homeodomain proteins exhibit selective complex stabilities with Pbx and DNA.Nucleic Acids Res. 1996; 24: 898-906Crossref PubMed Scopus (102) Google Scholar Transient transfection assays were performed as described22Di Rocco G Mavilio G Zappavigna V Functional dissection of a transcriptionally active, target-specific Hox-Pbx complex.EMBO J. 1997; 16: 3644-3654Crossref PubMed Scopus (99) Google Scholar using a pGL3 luciferase reporter (driven by SV40 early promoter containing one copy of the modified HoxB2 r4 enhancer) and internal control (pCMV-1 β-gal) plasmids. Luciferase activity was measured in light units using a Monolight 2010 luminometer; β-gal activity was used to normalize luciferase activity to account for differences in transfection efficiency. Pbx3 expression was studied using immunohistochemical staining methods using highly specific monoclonal antibodies directed against two different isoforms (Pbx3a and Pbx3b) that arise from differential splicing of the Pbx3 RNA.3Monica K Galili N Nourse J Saltman D Cleary ML PBX2 and PBX3, new homeobox genes with extensive homology to the human proto-oncogene PBX1.Mol Cell Biol. 1991; 11: 6149-6157Crossref PubMed Scopus (262) Google Scholar Pbx3a was present throughout the mesenchyme at early gestational days, but was progressively more restricted to tissues of the developing central nervous system (CNS) by E11.5 (data not shown). At E15, nuclear Pbx3a (and to a much lesser extent Pbx3b) staining was observed in the dorsal thalamus, cerebellum, pons, medulla, dorsal root ganglia, and neurons in both the ventral and dorsal columns of the spinal cord (Figure 1A to D). Staining was most intense throughout the medulla (Figure 1, A and D) and was present in regions that overlap with those involved in central control of respiration.23Bianchi AL Denavit-Saubie M Champagnat J Central control of breathing in mammals: neuronal circuitry, membrane properties, and neurotransmitters.Am Phys Soc. 1995; 75: 1-45Google Scholar Pbx3a was also found at high levels in myenteric neurons and at lower levels in smooth muscle myocytes of the intestine (Figure 1, E and F). Even at late gestational days, Pbx3a was not exclusively confined to the nervous system, as lower levels were also observed in chondrocytes throughout the axial and appendicular skeleton (Figure 1C). To assess the functional role of Pbx3, gene targeting was used to generate Pbx3-deficient (Pbx3−/−) mice (Figure 2A). Chimeric male mice carrying the Pbx3 null allele were bred with wild-type C57BL/6 mice until the allele was transmitted through the germline as determined by Southern blot analysis (Figure 2B). Mice heterozygous for the targeted Pbx3 gene were viable and fertile. They developed normally and showed no histopathological abnormalities. Intercross matings of Pbx3+/− mice, however, did not yield viable Pbx3−/− mice at 3 weeks of age (0 of 155 pups, P < 0.001). A high number of dead pups were observed at P0, and most were found to be null for Pbx3. Analysis of all neonates showed a Mendelian distribution of genotypes (wild-type, 23 of 93, 25%; Pbx3+/−, 49 of 93, 53%; Pbx3−/−, 21 of 93, 23%) indicating that the absence of Pbx3 did not result in embryonic lethality. Western blot analysis confirmed the lack of Pbx3 protein in the brains of late gestation Pbx3−/− embryos (Figure 2C). Close observation showed that Pbx3−/− pups were viable at birth with intact heart rate but died within a few hours. They appeared apneic and developed marked cyanosis shortly after birth (Figure 2D). Gross anatomical examination of Pbx3−/− mice showed that all internal organs were present, properly developed, and of appropriate size; however the lungs were not inflated indicating an absence of appropriate ventilation. Neurofilament staining of E10.5 embryos showed no gross abnormalities of cranial nerve ganglia, indicating correct specification of hindbrain rhombomeres and associated cranial nerve nuclei (data not shown). The brain and spinal cord appeared grossly normal in Pbx3−/− mice at P0 and no histological abnormalities of the cranial nerves, ganglia, or brainstem nuclei were observed. No apparent neuronal loss or gliosis was observed in the brainstem, including areas of the ventral medulla implicated in respiratory control and associated with high-level Pbx3 expression. The physiological abnormality in Pbx3−/− mice was examined by monitoring spontaneous inspiration, using whole body plethysmography.16Kuwaki T Cao W Kurihara Y Kurihara H Ling G Onodera M Ju K Yazaki Y Kumada M Impaired ventilatory responses to hypoxia and hypercapnea in mutant mice deficient in endothelin-1.Am J Physiol. 1996; 270: 1279-1286PubMed Google Scholar The respiratory pattern in Pbx3−/− mice exhibited a marked irregularity of the tidal volume in each respiratory cycle (Figure 3, A and B). In addition, the average respiratory frequency of 81 ± 35 per minute in Pbx3−/− mice was significantly lower than that observed in Pbx3+/− and wild-type (wt) mice (Table 1). As a consequence, the mean respiratory minute volume of 0.72 ± 0.29 ml/min in Pbx3−/− mice was significantly lower than that of wt (1.57 ± 0.62 ml/min; P < 0.01) and Pbx3+/− mice, suggesting that hypoventilation was a likely cause of death within several hours after birth.Table 1In Vivo and in Vitro Respiratory Activity of Pbx3-Deficient MiceIn vivo respiratory activity measured by plethysmographyGenotypeFrequency (per min)Tidal volume (μl)Minute volume (ml/min)+/+ (n = 7)124 ± 2412.6 ± 4.51.57 ± 0.62+/− (n = 9)124 ± 3811.8 ± 4.91.47 ± 0.91−/− (n = 8)81 ± 35*, p < 0.01 compared to +/+ and +/−.9.0 ± 2.50.72 ± 0.29†, p < 0.05 compared to +/+ and +/−.C4 inspiratory activity recorded in vitroGenotypeFrequency (per min)Duration (ms)Amplitude (μV)+/+ (n = 8)6.0 ± 0.7934 ± 371246 ± 73+/− (n = 15)8.1 ± 1.4958 ± 275‡, p < 0.05 compared to +/+.159 ± 52§, p < 0.01 compared to +/+.−/− (n = 11)9.0 ± 2.1*, p < 0.01 compared to +/+ and +/−.713 ± 239†, p < 0.05 compared to +/+ and +/−.65 ± 35*, p < 0.01 compared to +/+ and +/−.* , p < 0.01 compared to +/+ and +/−.† , p < 0.05 compared to +/+ and +/−.‡ , p < 0.05 compared to +/+.§ , p < 0.01 compared to +/+. Open table in a new tab To localize the level of the defect in Pbx3−/− mice, C4 ventral nerve root activity was recorded in medulla-spinal cord preparations in vitro as a measure of respiratory output. Ventral C4/C5 activity has been shown to be synchronous with phrenic nerve discharge and contraction of inspiratory intercostal muscles.17Suzue T Respiratory rhythm generation in the in vitro brain stem-spinal cord preparation of the neonatal rat.J Physiol. 1984; 354: 173-183PubMed Google Scholar The C4 ventral root recordings from Pbx3−/− mice showed a characteristic pattern of motoneuronal outputs, consisting of small bursts at a frequency of 6 to 13 per minute, interspersed with large bursts appearing at a frequency of 0.2 to 0.5 per minute (Figure 3C). The latter bursts were usually followed by apnea (up to 30 seconds), or by bursts with minimum activity whose amplitude gradually recovered with time. Mean values of parameters of C4 inspiratory activity revealed a significantly higher frequency but lower amplitude of the bursts in the medulla-spinal cord preparations of Pbx3−/− mice (Table 1). This reduced amplitude likely produces insufficient pressure changes by ventilatory movement in some respiratory cycles leading to a significant decrease of minute respiratory volume in Pbx3−/− mice. To investigate whether the altered inspiratory pattern of Pbx3−/− mice may result from abnormal function of respiratory centers in the medulla, we assessed the membrane potentials of inspiratory neurons in this region.19Arata A Onimaru H Homma I Respiration-related neurons in the ventral medulla of newborn rats in vitro.Brain Res Bull. 1990; 24: 599-604Crossref PubMed Scopus (89) Google Scholar, 24Onimaru H Studies of the respiratory center using isolated brainstem-spinal cord preparations.Neurosci Res. 1995; 21: 183-190Crossref PubMed Scopus (48) Google Scholar, 25Rickling JC Champagnat J Denavit-Saubie M Electroresponsive properties and membrane potential trajectories of three types of inspiratory neurons in the newborn mouse brain stem in vitro.J Neurophysiol. 1996; 75: 795-810PubMed Google Scholar The membrane potential trajectory of inspiratory neurons (n = 6) in Pbx3−/− mice correlated with the pattern of C4 motoneuronal outputs (Figure 3D). The coordinate patterns indicate that an underlying basis for respiratory failure in Pbx3−/− mice is, at least in part, dysfunction of central respiratory networks in the medulla. Neonatal death due to a central hypoventilation syndrome has been reported in Rnx-deficient mice.14Shirasawa S Arata A Onimaru H Roth KA Brown GA Horning S Arata S Okumura K Sasazuki T Korsmeyer SJ Rnx deficiency results in congenital central hypoventilation.Nat Genet. 2000; 24: 287-290Crossref PubMed Scopus (141) Google Scholar Rnx is a member of the Hox11 orphan homeobox gene family,26Hatano M Roberts CWM Minden M Crist WM Korsmeyer SJ Deregulation of a homeobox gene, HOX11, by the t(10;14) in T-cell leukemia.Science. 1991; 253: 79-82Crossref PubMed Scopus (375) Google Scholar, 27Kennedy MA Gonzalez-Sarmiento R Kees UR Lampert F Dear N Boehm T Rabbitts TH HOX11, a homeobox-containing T-cell oncogene on human chromosome 10q24.Proc Natl Acad Sci USA. 1991; 88: 8900-8904Crossref PubMed Scopus (244) Google Scholar whose representatives contain DNA-binding homeodomains that differ in their recognition helix from those encoded by clustered Hox genes,28Dear TN Sanchez-Garcia I Rabbitts TH The HOX11 gene encodes a DNA-binding nuclear transcription factor belonging to a distinct family of homeobox genes.Proc Natl Acad Sci USA. 1993; 90: 4431-4435Crossref PubMed Scopus (120) Google Scholar but contain conserved motifs (hexapeptide) implicated in Pbx dimerization.29Chang CP Shen WF Rozenfeld S Lawrence HJ Largman C Cleary ML Pbx proteins display hexapeptide-dependent cooperative DNA binding with a subset of Hox proteins.Genes Dev. 1995; 9: 663-674Crossref PubMed Scopus (353) Google Scholar Immunofluorescence analysis demonstrated that both Rnx and Pbx3a proteins were present in the nuclei of neurons of the caudal portion of the medulla (Figure 4A). Since the nuclear localization of Pbx proteins is dependent on dimerization with TALE class homeoproteins of the Meis/Pknox1 family,30Berthelsen J Kilstrup-Nielsen C Blasi F Mavilio F Zappavigna V The subcellular localization of PBX1 and EXD proteins depends on nuclear import and export signals and is modulated by association with PREP1 and HTH.Genes Dev. 1999; 13: 946-953Crossref PubMed Scopus (199) Google Scholar we also examined the distribution of Meis protein expression in the medulla. Meis proteins were present in the nuclei of neurons whose distribution extensively overlapped with those expressing Pbx3a (Figure 4B), consistent with a Meis-mediated mechanism for Pbx3 nuclear import.31Toresson H Parmar M Campbell K Expression of Meis and Pbx genes and their protein products in the developing telencephalon: implications for regional differentiation.Mech Dev. 2000; 94: 183-187Crossref PubMed Scopus (89) Google Scholar Furthermore, the nuclear co-localization of Rnx and Meis proteins was unaltered in Pbx3−/− mice (Figure 4C), demonstrating that neither the expression nor nuclear localization of either protein was dependent on Pbx3. Expression of Rnx, Pbx3, and Meis in the medulla raised the possibility that they function together as a hetero-oligomeric complex whose transcriptional activity may be altered by the lack of Pbx3. Therefore, their ability to form a DNA-binding complex was examined on a modified endogenous enhancer that contained consensus sites for all three proteins. This genetically defined response element (Hoxb2 r4) has been shown to rely on combinatorial interactions between Hox and TALE proteins to mediate appropriate HoxB2 developmental expression in vivo.15Jacobs Y Schanbel CA Cleary ML Trimeric association of Hox and TALE homeodomain proteins mediates HoxB2 hindbrain enhancer activity.Mol Cell Biol. 1999; 19: 5134-5142Crossref PubMed Scopus (215) Google Scholar, 32Ferretti E Marshall H Popperl H Maconochie M Krumlauf R Blasi F Segmental expression of Hoxb2 in r4 requires two separate sites that integrate cooperative interactions between Prep1, Pbx, and Hox proteins.Development. 2000; 127: 155-166Crossref PubMed Google Scholar For the current studies, we modified the Hox consensus site to optimize for recognition by the divergent homeodomain of Rnx.21Shen WF Chang CP Rozenfeld S Sauvageau G Humphries RK Lu M Lawrence HJ Cleary ML Largman C Hox homeodomain proteins exhibit selective complex stabilities with Pbx and DNA.Nucleic Acids Res. 1996; 24: 898-906Crossref PubMed Scopus (102) Google Scholar As expected, in vitro-produced Pbx3a and Meis1a formed a dimeric complex on this enhancer element (Figure 5A, lane 5). Addition of Rnx resulted in the formation of an additional slower migrating band consistent with formation of a trimeric complex (Figure 5A, lane 6), similar to those observed in previous studies of Hoxb1 and Hoxa9.15Jacobs Y Schanbel CA Cleary ML Trimeric association of Hox and TALE homeodomain proteins mediates HoxB2 hindbrain enhancer activity.Mol Cell Biol. 1999; 19: 5134-5142Crossref PubMed Scopus (215) Google Scholar, 33Schnabel CA Jacobs Y Cleary ML HoxA9-mediated immortalization of myeloid progenitors requires molecular interactions with TALE cofactors Pbx and Meis.Oncogene. 2000; 19: 608-616Crossref PubMed Scopus (109) Google Scholar This indicated that Rnx has the capacity to assemble into a higher order, trimeric DNA-binding complex with TALE homeodomain proteins Pbx3 and Meis1a. To assess the functional consequences of trimeric Rnx/Pbx3/Meis interactions, transient transfection assays were performed. For these studies, a reporter gene that contained the modified enhancer situated upstream of the SV40 promoter was used in human embryonic kidney cells, which are permissive for Hox transcriptional activity. Co-transfection of expression constructs Pbx3a and Meis1a displayed minimal activation above background (Figure 5B), as previously noted for comparable TALE heterodimers.15Jacobs Y Schanbel CA Cleary ML Trimeric association of Hox and TALE homeodomain proteins mediates HoxB2 hindbrain enhancer activity.Mol Cell Biol. 1999; 19: 5134-5142Crossref PubMed Scopus (215) Google Scholar, 33Schnabel CA Jacobs Y Cleary ML HoxA9-mediated immortalization of myeloid progenitors requires molecular interactions with TALE cofactors Pbx and Meis.Oncogene. 2000; 19: 608-616Crossref PubMed Scopus (109) Google Scholar Co-transfection of Rnx, Pbx3a, and Meis1a, however, resulted in a 7.5-fold activation above background levels of transcription obtained with Rnx alone. In contrast, Rnx and Meis1a in the absence of Pbx3a displayed a reduced level of activation that approximated only a third of that observed in the presence of all three homeodomain proteins (Figure 5B). These data reveal trimeric interactions between Rnx, Pbx3a, and Meis1a function to promote heightened transcriptional activation, and highlight the critical role of Pbx3a in trimeric transcriptional activity. The respiratory pattern in Pbx3−/− mice is characterized by irregular amplitude of inspiration and reduced respiratory frequency. Although a higher than normal frequency of C4 inspiratory neuronal activity is observed in the in vitro brainstem preparations of Pbx3−/− mice, the burst amplitude of the phrenic motoneurons is significantly smaller than wt. This reduced amplitude likely produces insufficient pressure changes by ventilatory movement in some respiratory cycles leading to a significant decrease of minute respiratory volume in Pbx3−/− mice, and appears insufficient to fully inflate the lungs. Importantly, the coordinate patterns of membrane potentials for inspiratory neurons in the ventrolateral medulla and C4 motoneuronal outputs indicate that an underlying basis for respiratory failure in Pbx3−/− mice is dysfunction of central respiratory networks in the medulla. We cannot exclude a possibility that abnormality of afferent inputs contributes to respiratory dysfunction in vivo. Dysfunction of central re
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