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• W2022666276 abstract "The compartmentalization of somites along their anterior-posterior axis is crucial to the segmental organization of the vertebral column. Anterior-posterior somite polarity is generated in the anterior presomitic mesoderm by Mesp2 and Delta/Notch signaling and is further maintained by two transcriptional regulators, Uncx4.1 and Tbx18, acting in the posterior and anterior somite compartment, respectively. Here, we report that the paired box transcription factor Pax3 cooperates with the T-box protein Tbx18 in maintaining anterior somite half identity. Our findings that both genes are co-expressed in the anterior presomitic mesoderm and in early somites, that Pax3 and Tbx18 proteins physically interact, and that the loss of Pax3 gene function enhances the vertebral defects (i.e. the gain of vertebral elements derived from posterior somite halves in Tbx18 mutant mice) suggests that the two proteins cooperatively regulate the gene expression program necessary for maintaining anterior-posterior somite polarity. Genetic interaction of Pax3 with Tbx18 and the closely related T-box gene Tbx15 was also observed in the development of the scapula blade, indicating an additional cooperative function for these genes in the paraxial mesoderm. The compartmentalization of somites along their anterior-posterior axis is crucial to the segmental organization of the vertebral column. Anterior-posterior somite polarity is generated in the anterior presomitic mesoderm by Mesp2 and Delta/Notch signaling and is further maintained by two transcriptional regulators, Uncx4.1 and Tbx18, acting in the posterior and anterior somite compartment, respectively. Here, we report that the paired box transcription factor Pax3 cooperates with the T-box protein Tbx18 in maintaining anterior somite half identity. Our findings that both genes are co-expressed in the anterior presomitic mesoderm and in early somites, that Pax3 and Tbx18 proteins physically interact, and that the loss of Pax3 gene function enhances the vertebral defects (i.e. the gain of vertebral elements derived from posterior somite halves in Tbx18 mutant mice) suggests that the two proteins cooperatively regulate the gene expression program necessary for maintaining anterior-posterior somite polarity. Genetic interaction of Pax3 with Tbx18 and the closely related T-box gene Tbx15 was also observed in the development of the scapula blade, indicating an additional cooperative function for these genes in the paraxial mesoderm. The metameric organization of the vertebral column derives from the somites, segmentally repeated units in the paraxial mesoderm. Somites form in a highly periodic and synchronized fashion by condensation and subsequent epithelialization of groups of mesenchymal cells at the anterior end of the presomitic mesoderm (PSM) 3The abbreviations used are: PSMpresomitic mesodermaaamino acidsAPanterior-posteriorEnembryonic day nNLSnuclear localization sequenceGSTglutathione S-transferaseHAhemagglutinin. on both sides of the neural tube. Under the influence of signals from surrounding tissues, somites start to differentiate along their dorso-ventral axis. The ventral part undergoes an epithelial-mesenchymal transition to form the sclerotome, which contains precursors of the vertebral column and parts of the ribs. The dorsal part remains epithelial and generates the dermomyotome, from which skeletal muscles and the dermis of the skin will develop. In addition to differentiation along the dorso-ventral axis, somites become subdivided into distinct anterior and posterior compartments. Anterior-posterior (AP) polarization of somites underlies the segmental arrangement of the peripheral nervous system, since trajectories of neural crest and spinal nerves are confined to anterior somite halves. On the level of the sclerotome, the differential contribution of either compartment to the forming vertebra affects the structure of the axial skeleton. Vertebral bodies, laminae with the spinal processes, the rib heads, and the distal ribs derive from both somite halves, whereas pedicles with their transverse processes and proximal ribs derive from posterior somite halves only (1Goldstein R.S. Kalcheim C. Development. 1992; 116: 441-445Crossref PubMed Google Scholar, 2Aoyama H. Asamoto K. Mech. Dev. 2000; 99: 71-82Crossref PubMed Scopus (70) Google Scholar, 3Christ B. Huang R. Scaal M. Dev. Dyn. 2007; 236: 2382-2396Crossref PubMed Scopus (114) Google Scholar). presomitic mesoderm amino acids anterior-posterior embryonic day n nuclear localization sequence glutathione S-transferase hemagglutinin. Establishment of somitic AP polarity is closely coupled to the segmentation process. Work from a variety of vertebrate model systems has shown that somite formation is governed by an oscillator known as the segmentation clock that operates in the PSM (4Aulehla A. Herrmann B.G. Genes Dev. 2004; 18: 2060-2067Crossref PubMed Scopus (175) Google Scholar, 5Saga Y. Dev. Dyn. 2007; 236: 1450-1455Crossref PubMed Scopus (30) Google Scholar). It is now believed that synchronized oscillations of a number of signaling pathways, including Wnt, fibroblast growth factor, and Notch signaling, are involved in the mechanism of the segmentation clock. Gradients of secreted signaling molecules cooperatively define the segmentation border within the anterior PSM. In this region, Notch oscillation is stabilized to a narrow domain, in which cells with a high Notch pathway activity will constitute the posterior half of a newly forming somite. In an adjacent stripe of cells, Notch signaling is suppressed by the action of the basic helix-loop-helix transcription factor Mesp2. The expression domain of Mesp2 thereby defines the anterior somite half, and its anterior limit demarcates the next segmental border to be formed (6Morimoto M. Takahashi Y. Endo M. Saga Y. Nature. 2005; 435: 354-359Crossref PubMed Scopus (195) Google Scholar). Correspondingly, loss of Mesp2 activity leads to posteriorization of somites, whereas loss of Delta-like1 (Dll1) gene function and Notch signaling results in somites that bear only features of anterior halves (7Barrantes I.B. Elia A.J. Wunsch K. Hrabe de Angelis M.H. Mak T.W. Rossant J. Conlon R.A. Gossler A. de la Pompa J.L. Curr. Biol. 1999; 9: 470-480Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). Molecular players required for the further maintenance of somitic AP polarity have recently surfaced. Genetic evidence from both loss- and gain-of-function studies in the mouse suggest that this process is controlled by the combined action of a pair of transcription factors, the T-box (Tbx) protein Tbx18 and the paired type homeobox protein Uncx4.1, which are expressed in anterior and posterior somite halves, respectively (8Kraus F. Haenig B. Kispert A. Mech. Dev. 2001; 100: 83-86Crossref PubMed Scopus (144) Google Scholar, 9Neidhardt L.M. Kispert A. Herrmann B.G. Dev. Genes Evol. 1997; 207: 330-339Crossref PubMed Scopus (54) Google Scholar). Uncx4.1 is specifically required for the development of pedicles and proximal ribs (10Leitges M. Neidhardt L. Haenig B. Herrmann B.G. Kispert A. Development. 2000; 127: 2259-2267Crossref PubMed Google Scholar, 11Mansouri A. Voss A.K. Thomas T. Yokota Y. Gruss P. Development. 2000; 127: 2251-2258Crossref PubMed Google Scholar), elements exclusively derived from the posterior lateral sclerotome. In contrast, loss of Tbx18 function results in expansion of pedicles and proximal ribs in the cervical and thoracic region of the axial skeleton (12Bussen M. Petry M. Schuster-Gossler K. Leitges M. Gossler A. Kispert A. Genes Dev. 2004; 18: 1209-1221Crossref PubMed Scopus (138) Google Scholar). Notably, the forced misexpression of Tbx18 in posterior somite halves results in reduction of pedicles and proximal ribs (12Bussen M. Petry M. Schuster-Gossler K. Leitges M. Gossler A. Kispert A. Genes Dev. 2004; 18: 1209-1221Crossref PubMed Scopus (138) Google Scholar), suggesting that Tbx18 is sufficient to specify anterior versus posterior somite fates. Opposing phenotypic consequences of loss of either factor are based on molecular cross-regulation. In Uncx4.1 mutants, Tbx18 expression is derepressed in posterior somite halves, whereas in Tbx18 mutants, expression of Uncx4.1 progressively expands in anterior somite halves (12Bussen M. Petry M. Schuster-Gossler K. Leitges M. Gossler A. Kispert A. Genes Dev. 2004; 18: 1209-1221Crossref PubMed Scopus (138) Google Scholar). On the molecular level, Uncx4.1 may therefore act as transcriptional repressor of Tbx18, whereas Tbx18 may regulate Uncx4.1 indirectly by controlling expression of the Notch ligand Dll1 (13Farin H.F. Bussen M. Schmidt M.K. Singh M.K. Schuster-Gossler K. Kispert A. J. Biol. Chem. 2007; 282: 25748-25759Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). To get further insight into the molecular function of Tbx18, thus into the control of AP-somite compartmentalization, we sought to identify and characterize protein binding partners of Tbx18. This may also help to define transcriptional targets of Tbx18 and their molecular regulation. Here, we report on the identification of the paired box (Pax) transcription factor Pax3 as a protein binding partner of Tbx18. We characterize this interaction on the biochemical level and define genetically that both transcription factors synergize in the development of the paraxial mesoderm, including anterior-posterior somite compartmentalization and scapula development. Expression Constructs—Bacterial expression constructs were generated as N-terminal glutathione S-transferase (GST)-fusions in pGEX-4T3 (GE Healthcare). Generation of GST-Tbx18 fusion proteins has been described (13Farin H.F. Bussen M. Schmidt M.K. Singh M.K. Schuster-Gossler K. Kispert A. J. Biol. Chem. 2007; 282: 25748-25759Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), and constructs covering the T-box region of mouse Tbx15 (aa 110–313), human TBX22 (aa 96–291), and mouse Brachyury (aa 41–225) were PCR-amplified from the cDNAs NM_009323, NM_016954, and NM_009309, respectively. For in vitro expression of proteins, cDNA fragments were cloned with C-terminal Myc or HA tags in the vector pSP64 (Promega) that was modified to contain a 5′-β-globin leader anda3′-β-globin trailer. Fragments encoding Pax3 partial (Fig. 1D) and full-length (aa 1–479) proteins were amplified from the mouse cDNA NM_008781. Expression plasmids of full-length Pax1 (aa 1–361), Pax7 (aa 1–503), and Pax9 (aa 1–342) were amplified from mouse cDNAs NM_008780, NM_011039, and NM_011041, respectively. For cytomegalovirus promoter/enhancer-driven expression in cells, the globin leader/cDNA/globin trailer cassette was shuttled into EcoRI and HindIII sites of pcDNA3 (Invitrogen). The expression vector for Tbx18ΔNLS has been described (13Farin H.F. Bussen M. Schmidt M.K. Singh M.K. Schuster-Gossler K. Kispert A. J. Biol. Chem. 2007; 282: 25748-25759Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). All plasmids were sequenced; details on cloning strategies and primer sequences are available upon request. Yeast Two-hybrid Screen—The construct for the generation of a fusion protein between the DNA binding domain of GAL4 and Tbx18 (aa 1–345) was cloned into pGBKT7 (Clontech). This bait vector was transformed into the yeast strain AH109 (Clontech), that was subsequently mated to the yeast strain Y187 that was pretransformed with a prey library of poly(T)-primed mouse embryonic day 11.5 (E11.5) whole embryo cDNAs (Clontech) following the manufacturer's instructions. Clones were selected on plates lacking leucine, tryptophan, histidine, and alanine. After this selection step, prey plasmids were isolated, amplified in Escherichia coli, and sequenced. GST Pull-down, Immunofluorescence, and Co-immunoprecipitation Assays—These assays were performed as described (13Farin H.F. Bussen M. Schmidt M.K. Singh M.K. Schuster-Gossler K. Kispert A. J. Biol. Chem. 2007; 282: 25748-25759Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Mice and Genotyping—Mice carrying a null allele of Pax3 (Pax3lacZ) (14Mansouri A. Pla P. Larue L. Gruss P. Development. 2001; 128: 1995-2000Crossref PubMed Google Scholar), Tbx18 (Tbx18tm2Akis (12Bussen M. Petry M. Schuster-Gossler K. Leitges M. Gossler A. Kispert A. Genes Dev. 2004; 18: 1209-1221Crossref PubMed Scopus (138) Google Scholar) (synonym: Tbx18GFP), and Tbx15 (Tbx15tm1Akis) (15Singh M.K. Petry M. Haenig B. Lescher B. Leitges M. Kispert A. Mech. Dev. 2005; 122: 131-144Crossref PubMed Scopus (91) Google Scholar) (synonym: Tbx15lacZ) were maintained on an outbred (NMRI) background. For the generation of compound mutants, double heterozygous mice were intercrossed. Genomic DNA prepared from yolk sacs or tail biopsies was used for genotyping by PCR (details on PCR strategies are available upon request). For timed pregnancies, vaginal plugs were checked in the morning after mating, and noon was taken as E0.5. Skeletal Preparations—Skeletal preparations of E14.5 embryos and newborns were prepared essentially as previously described (12Bussen M. Petry M. Schuster-Gossler K. Leitges M. Gossler A. Kispert A. Genes Dev. 2004; 18: 1209-1221Crossref PubMed Scopus (138) Google Scholar). Embryos were fixed in 95% ethanol overnight, and cartilaginous elements were then stained for 2 days in Alcian blue solution (150 mg/liter Alcian blue 8GX in 80% ethanol, 20% acetic acid). Embryos were transferred in methanol and cleared in benzylbenzoate/benzylalcohol (2:1). In Situ Hybridization Analysis—Whole mount in situ hybridization analysis was performed with digoxigenin-labeled antisense riboprobes following a standard procedure (16Wilkinson D.G. Nieto M.A. Methods Enzymol. 1993; 225: 361-373Crossref PubMed Scopus (724) Google Scholar). Stained specimens were transferred into 80% glycerol prior to documentation on a Leica M420 microscope with a Fujix digital camera HC-300Z. Images were processed in Adobe Photoshop CS. Details about probes are available upon request. T-box and Pax Proteins Interact in Vitro—In order to identify protein interaction partners of Tbx18, we performed a yeast two-hybrid screen. We initially tested a number of fusion constructs of the GAL4-DNA-binding domain with subregions of Tbx18 protein for their quality as bait. A construct encoding a fusion protein with the N terminus and the T-domain of Tbx18, which was expressed and lacked autoactivation in yeast, was transformed into yeast, and the resulting bait strain was mated to a strain pretransformed with a mouse cDNA library from E11.5 whole embryos. One of the clones identified by the yeast two-hybrid screen harbored a partial cDNA for Pax3, a member of the gene family encoding paired box transcription factors (data not shown). To validate and further investigate the interaction between Tbx18 and Pax3, we performed a series of in vitro binding assays using bacterially expressed subregions of Tbx18 fused to GST and in vitro expressed HA-tagged Pax3 protein (Fig. 1, A and B). In GST pull-down assays, Pax3 was specifically bound to GST-Tbx18 fusion proteins harboring the N-terminal domain and the T-box region (GST-Tbx18(N+T)) and the T-box region alone (GST-Tbx18(T)), respectively. Binding was observed neither with GST nor with GST-Tbx18(N) or GST-Tbx18(C), indicating that the T-domain of Tbx18 mediated the binding to Pax3 (Fig. 1C). We next generated a series of deletion mutants of the Pax3 cDNA for expression in vitro as HA-tagged peptides to determine which region of Pax3 confers interaction with Tbx18 (Fig. 1, D and E). Pax proteins are characterized by the presence of a conserved N-terminal DNA-binding region, the paired domain. Some of the eight murine family members, including Pax3, additionally contain a homeodomain as a second DNA-binding region. Pax3 peptides containing the paired domain were efficiently bound to GST-Tbx18(N+T), whereas peptides containing the homeodomain only and/or the C terminus of Pax3 were not efficiently retained (Fig. 1E). In summary, our in vitro binding assays showed that binding of Tbx18 and Pax3 is mediated by the two conserved DNA binding regions, the T-domain and the paired domain. The interaction between Tbx18 and Pax3 was additionally validated in a mammalian cell system using a nuclear recruitment assay. Transfection of an expression construct for HA-tagged Pax3 revealed constitutive nuclear localization of Pax3. In contrast, Myc-tagged Tbx18 protein lacking the nuclear localization signal (Tbx18ΔNLS) (13Farin H.F. Bussen M. Schmidt M.K. Singh M.K. Schuster-Gossler K. Kispert A. J. Biol. Chem. 2007; 282: 25748-25759Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) was excluded from the nucleus and localized to the cytoplasm. Upon co-transfection of constructs encoding HA-tagged Pax3 protein and Myc-tagged Tbx18ΔNLS protein, nuclear localization of Tbx18 was regained (Fig. 1F), suggesting that Tbx18ΔNLS in complex with Pax3 is shuttled to the nuclear environment. Furthermore, co-immunoprecipitation assays were performed in HEK293 cells transfected with full-length constructs for Myc-tagged Tbx18 alone or in the presence of HA-tagged Pax3. In immunoprecipitates obtained with the HA antibody, an enrichment of Myc-tagged Tbx18 protein was detected only upon co-transfection of the Pax3 expression construct (Fig. 1G, left). Conversely, Pax3.HA protein was specifically coimmunoprecipitated with the anti-Myc antibody when Tbx18.Myc protein was present (Fig. 1G, right), providing further proof for complex formation of Tbx18 and Pax3 in a cellular system. T-box and Paired Box Interaction Is Promiscuous—We next investigated whether binding of Tbx18 to Pax3 is unique among T-box and Pax proteins or whether Tbx18 and Pax3 interact with additional members of the other family as well. In a GST pull-down assay, we found that, similar to Pax3, the closely related Pax7 and the more divergent proteins Pax1 and Pax9 exhibited binding to the T-domain of Tbx18 (Fig. 2A). This interaction was confirmed in the nuclear recruitment assay, where Tbx18ΔNLS was shuttled to the nucleus upon co-expression of HA-tagged Pax1 and Pax9 but not with unrelated nuclear proteins (data not shown). Conversely, we analyzed if Pax3 is able to bind to other members of the T-box protein family. Therefore, GST fusions of the T-box region of the closely related Tbx15, Tbx18, and Tbx22 proteins and the distant family member Brachyury (Fig. 2B) were expressed in bacteria, purified (Fig. 2C), and incubated with in vitro expressed Pax3 protein. Binding of Pax3 protein was detected to all T-box proteins analyzed; however, binding of Pax3 to Tbx18 was the strongest (Fig. 2D). Together, these findings suggest promiscuity of binding between T-box and paired box regions, but binding affinities between individual family members might differ substantially. Comparative Expression Analysis of Tbx15, Tbx18, Tbx22, and Pax3—The facts that we only detected Pax3 but not other Pax family member in our yeast two-hybrid screen and the high affinity binding of Tbx18 with Pax3 in the in vitro assays prompted us to analyze whether this interaction is functionally relevant in vivo. To determine in which tissues such a molecular interaction may occur, we compared the expression patterns of Pax3 and Tbx18 and the two closely related Tbx15 and Tbx22 genes using in situ hybridization analysis of E9.5 wild-type mouse embryos (Fig. 3). At this stage, Tbx15 expression was confined to the mesenchyme of the forelimb buds (Fig. 3A, arrow). Tbx18 was co-expressed with Tbx15 in this tissue (Fig. 3B, arrow) but showed additional expression domains in the sinus venosus, the proepicardial organ (Fig. 3B, white arrowhead), and the head mesenchyme (Fig. 3B, black arrowhead). In derivatives of the paraxial mesenchyme, Tbx18 expression was observed in the anterior halves of epithelial somites and additionally in two stripes representing the anterior halves of somites that were about to form (S0 and S-1) (Fig. 3E). With differentiation of somites, Tbx18 expression in anterior somite halves became restricted to the lateral sclerotome (Fig. 3E, arrow). Tbx15 expression was absent during somite development (Fig. 3A). However, the closely related Tbx22 gene was co-expressed with Tbx18 in anterior halves of somitomeres and early somites (S-1 to S1) (Fig. 3, C and F). Expression of Tbx22 in anterior somite halves was then rapidly down-regulated, but expression was reinitiated in forming myotomes (Fig. 3F, arrow). Pax3 was strongly expressed in the dorsal neural tube (Fig. 3D, arrow). Furthermore, Pax3 expression was found in the anterior PSM and in epithelial somites (Fig. 3, D and G). Expression was maintained in the dermomyotomal compartment (Fig. 3G, arrow) and in migrating precursors of the limb musculature (Fig. 3D, arrowhead) (17Schubert F.R. Tremblay P. Mansouri A. Faisst A.M. Kammandel B. Lumsden A. Gruss P. Dietrich S. Dev. Dyn. 2001; 222: 506-521Crossref PubMed Scopus (60) Google Scholar). Hence, Tbx18, Tbx22, and Pax3 are co-expressed in the PSM and undifferentiated somites, but expression domains segregate during the differentiation of the sclerotome, myotome, and dermomyotome. Tbx18 and Pax3 Cooperate in the Development of the Axial Skeleton—The observed physical interaction and the co-expression of Tbx18 (and Tbx22) with Pax3 during somitogenesis suggested that these factors also interact genetically during this process. We analyzed this possibility by generating embryos compound mutant for null alleles of Tbx18 (Tbx18GFP) and Pax3 (Pax3lacZ). On the outbred background on which we maintained these alleles, Pax3-/- embryos were viable at E14.5. This is in contrast to studies where lethality of Pax3-/- embryos was observed between E13.5 and E14.5 when the mutant allele was kept on an inbred background, such as a mix of C3H/101 and BA/Ca or C57Bl6 (17Schubert F.R. Tremblay P. Mansouri A. Faisst A.M. Kammandel B. Lumsden A. Gruss P. Dietrich S. Dev. Dyn. 2001; 222: 506-521Crossref PubMed Scopus (60) Google Scholar, 18Henderson D.J. Conway S.J. Copp A.J. Dev. Biol. 1999; 209: 143-158Crossref PubMed Scopus (67) Google Scholar). Tbx18-/- embryos died shortly after birth as reported before (12Bussen M. Petry M. Schuster-Gossler K. Leitges M. Gossler A. Kispert A. Genes Dev. 2004; 18: 1209-1221Crossref PubMed Scopus (138) Google Scholar). Mice double heterozygous for Tbx18GFP and Pax3lacZ mutant alleles were viable and fertile and were intercrossed to obtain all possible allelic combinations. We harvested embryos at E14.5 and analyzed the skeletons as a read-out of defects of somite patterning and differentiation. We noted that embryos double homozygous for Pax3 and Tbx18 null alleles were severely underrepresented at this stage. Of a total of 123 embryos harvested, we only obtained two double mutants (1.6%) instead of the expected eight (1 of 16; 6.3%). Similarly, the observed number of nine Tbx18-/-,Pax3+/- embryos (7.3%) displayed a reduction from the expected value (15 embryos; 1 of 8; 12.5%), suggesting that the removal of one or two copies of one wild-type allele in the mutant background of the other gene dramatically enhanced the severity of the embryonic defects. All other genotypes were found in the expected Mendelian frequencies (data not shown). In wild-type embryos of E14.5, the cartilagenous preskeleton was invested with a segmental array of orderly spaced ribs and vertebra (Fig. 4A). At the thoracic level, ribs were connected to vertebral pedicles (Fig. 4G, black arrowhead). Strikingly, in 12 of 30 embryos (40%) double heterozygous for both Tbx18 and Pax3 null alleles, we detected isolated expansions of proximal ribs (Fig. 4, B and H, white arrowhead), whereas these malformations were never observed in single heterozygous embryos. In Tbx18-/- embryos, pedicles and proximal ribs were expanded and formed contiguous cartilagenous bands in the vertebral column at the cervical and thoracic levels and the rib cage, respectively (Fig. 4, C (brackets) and I (arrowheads)) (12Bussen M. Petry M. Schuster-Gossler K. Leitges M. Gossler A. Kispert A. Genes Dev. 2004; 18: 1209-1221Crossref PubMed Scopus (138) Google Scholar). In all Tbx18-/-,Pax3+/- embryos analyzed (n = 9), expansions of proximal ribs were increased in frequency and extended more caudally (Fig. 4D, brackets) and medially (Fig. 4J, white arrowheads) compared with Tbx18-/- embryos. A further expansion of pedicles was not observed (Fig. 4J, black arrowhead). In mutants double homozygous for Tbx18 and Pax3 null alleles, the body axis was dramatically shortened (n = 2; Fig. 4E). The severity of the skeletal defects in cervical vertebrae was unchanged compared with Tbx18 mutant embryos. In contrast, the lateral parts of the vertebrae, neural arches and pedicles, were largely expanded at the thoracic and lumbar level (Fig. 4E, black arrowhead) and frequently misconnected to the vertebral bodies that were often split (Fig. 4E, asterisks and white arrow, respectively). Proximal parts of ribs constituted large contiguous plates of cartilage on both sides of the vertebral column (Fig. 4K, white arrowheads). In Pax3-/- embryos, defects of the axial skeleton, including fusions of neural arches of adjacent vertebrae, occurred mainly in the lumbosacral region (Fig. 4F, arrowhead), and rib fusions and bifurcations were apparent (Fig. 4F, arrow). However, skeletal defects were generally less severe than the ones described for Pax3 alleles maintained on inbred genetic backgrounds (17Schubert F.R. Tremblay P. Mansouri A. Faisst A.M. Kammandel B. Lumsden A. Gruss P. Dietrich S. Dev. Dyn. 2001; 222: 506-521Crossref PubMed Scopus (60) Google Scholar, 18Henderson D.J. Conway S.J. Copp A.J. Dev. Biol. 1999; 209: 143-158Crossref PubMed Scopus (67) Google Scholar). Notably, and in contrast to Tbx18 mutants, the proximal ribs were unaffected, and the pedicles were spaced regularly (Fig. 4L). Together, our results demonstrate genetic interaction of Pax3 and Tbx18 in the formation of the axial skeleton. Removal of Pax3 function enhances the phenotypic changes associated with the loss of Tbx18, namely the expansion of derivatives of the posterior lateral sclerotome, pedicles, and proximal ribs. Tbx18 and Pax3 Cooperate in the Maintenance of Anterior Somite Halves—Co-expression of Tbx18 and Pax3 in undifferentiated somites suggests that not only the lateral sclerotome but also other somitic compartments could be affected by the combined loss of Pax3 and Tbx18 functions. To determine patterning and differentiation of the somitic mesoderm into myotome and sclerotome more carefully, we analyzed expression of molecular markers at E10.5. Within the collected embryos at this stage (n = 221), all genotypes were found in the expected frequencies, indicating that lethality of Tbx18/Pax3 double mutant embryos occurred between E10.5 and E14.5. In the E10.5 wild-type embryo, Myogenin was expressed in the myotomes in a repeating metameric pattern (Fig. 5A) (19Edmondson D.G. Olson E.N. Genes Dev. 1989; 3: 628-640Crossref PubMed Scopus (602) Google Scholar). In Pax3-/- embryos, Myogenin was segmentally expressed in myotomes, but its hypaxial domain appeared truncated (Fig. 5C, arrowhead). This is in agreement with the known role of Pax3 as a regulator of migration and survival of myotomal cells (20Williams B.A. Ordahl C.P. Development. 1994; 120: 785-796Crossref PubMed Google Scholar, 21Bober E. Franz T. Arnold H.H. Gruss P. Tremblay P. Development. 1994; 120: 603-612Crossref PubMed Google Scholar, 22Daston G. Lamar E. Olivier M. Goulding M. Development. 1996; 122: 1017-1027Crossref PubMed Google Scholar, 23Borycki A.G. Li J. Jin F. Emerson C.P. Epstein J.A. Development. 1999; 126: 1665-1674Crossref PubMed Google Scholar). In Tbx18 mutant embryos, Myogenin expression was unchanged (Fig. 5B), and no increase of the Pax3-/- phenotype was observed in double mutants (Fig. 5D, arrowhead), indicating that both genes do not cooperate in the myogenic program. Next we analyzed Pax9 that is expressed in the ventro-lateral sclerotome compartment with a strong up-regulation in the posterior somite halves in wild-type embryos (Fig. 5E) (24Neubüser A. Koseki H. Balling R. Dev. Biol. 1995; 170: 701-716Crossref PubMed Scopus (241) Google Scholar). In Pax3-/- embryos, polarized expression of Pax9 was maintained, whereas in Tbx18-/- mutant embryos, Pax9 expression became progressively homogenous with somite maturation (Fig. 5F). In Tbx18-/-,Pax3-/- embryos (n = 3), Pax9 expression was homogeneously strong in somites along the entire axial extension (Fig. 5H), suggesting that Pax3 cooperates with Tbx18 in AP-somite polarization. To further analyze AP-somite patterning in Tbx18/Pax3 compound mutant embryos, we used Uncx4.1 as a marker of the posterior somite half and the caudo-lateral sclerotome (Fig. 5I) (9Neidhardt L.M. Kispert A. Herrmann B.G. Dev. Genes Evol. 1997; 207: 330-339Crossref PubMed Scopus (54) Google Scholar). In Tbx18 mutant embryos, Uncx4.1 expression was progressively expanded, demonstrating the gain of posterior and the loss of anterior somite fates (Fig. 5J) (12Bussen M. Petry M. Schuster-Gossler K. Leitges M. Gossler A. Kispert A. Genes Dev. 2004; 18: 1209-1221Crossref PubMed Scopus (138) Google Scholar). In Pax3 mutant embryos (n = 2), the domain of Uncx4.1 was reduced in its dorso-ventral extension. However, AP polarization of expression was largely unaffected (Fig. 5K). In Pax3-/-,Tbx18-/- embryos (n = 2), up-regulation of Uncx4.1 expression was even enhanced compared with Tbx18-/- embryos, demonstrating a further expansion of posterior somitic identity (Fig. 5L). In embryos heterozygous mutant for Pax3 or Tbx18, Uncx4.1 expression was normal. In contrast, in 3 of 10 double heterozygous embryos, expansions of Uncx4.1 expression into anterior halves of differentiated somites were detect" @default.
• W2022666276 created "2016-06-24" @default.
• W2022666276 creator A5040494071 @default.
• W2022666276 creator A5040922232 @default.
• W2022666276 creator A5075279504 @default.
• W2022666276 creator A5084217132 @default.
• W2022666276 date "2008-09-01" @default.
• W2022666276 modified "2023-09-27" @default.
• W2022666276 title "T-box Protein Tbx18 Interacts with the Paired Box Protein Pax3 in the Development of the Paraxial Mesoderm" @default.
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