Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Biology, June 1999."May 19, 1999."Includes bibliographical references (leaves [12]-[13]).by M. Elizabeth Hick.S.M

Hick, M. Elizabeth, 1974-

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Identification and Characterization of fullback, a Novel Posteriorly-Expressed Xenopus GeneMaster's thesis submitted by M. Elizabeth HickMassachusetts Institute of TechnologyMay 19, 1999- f-r - -The auwth bebyg itto MfrprrnIsuiont I o adue ndtodtMaute putcA papr andelcwonic copis of thb the*documtent h whjoI or In partM. Elizabeth Hick, StudentHazel Sive, AdvisorTerry Orr-Weaver, Co-Chair of Graduate Committee,Department of BiologyMASSACHUSETTS INSTITUTEOF TECHNOLOGYI-- k*168Abstract:We have identified fullback, a Xenopus laevis gene which shows highestsimilarity to the low affinity nerve growth factor receptor p75. Extracellular portionsof the protein are conserved between fullback and p75, although regions of p75which have been implicated in its role in apoptosis are not especially conserved infullback. fullback is expressed in the posterior of the embryo from gastrulationonwards and is found dorsally during tailbud stages. This pattern of expressionapparently does not coincide with the incidence of apoptosis in the early Xenopusembryo and therefore indicates that fullback may have a different function duringembryogenesis.Results:The low affinity nerve growth factor receptor p75 is a member of the "deathreceptor" family of transmembrane proteins, most of which have some role inapoptosis (Liepinsh, et al, 1997). This receptor sends a pro-apoptotic signal leading toneuronal death unless it binds a neurotrophic ligand such as NGF which causes it tosend an anti-apoptotic signal instead (reviewed in Bredesen and Rabizadeh, 1997).Using a yeast invertase screen to find secreted and transmembrane proteins whichmay play a role in patterning the early embryo, we have identified fullback, aXenopus gene with highest similarity to p75 (Jacobs et al, 1997). Here we report asequence comparison of fullback to p75 in other vertebrates and a spatial andtemporal analysis of fullback expression in Xenopus.fullback encodes a 427 amino acid protein with a signal sequence peptide atthe amino terminus (von Heijne, 1986) and a single putative transmembranedomain. While the p75 genes from human, rat and chicken share between 75-85%identity with each other, fullback is only approximately 40% similar to any of themand is therefore unclear whether fullback is a Xenopus homolog of p75 (Fig 1). Thedeath receptor family of proteins is characterized by their cysteine-rich extracellulardomains (Liepinsh et al, 1997). A protein sequence alignment of fullback with thethree p75 proteins illustrates that all 25 of the extracellular cysteines in p75 are alsoconserved in this region of fullback (Fig 1, boxed). This implies that fullback mayhave extracellular structural similarity to p75. However, the "death domain", a six-helix portion of the intracellular domain present in most death receptor familymembers which is thought to be essential for apoptotic signaling (underlined, Fig 1)(Tartaglia, et al, 1993) is not well conserved in fullback, suggesting that this proteinmay have an entirely different, non-apoptotic function in the embryo.Northern analysis of fullback expression (Fig 2), shows a full length transcriptof approximately 3kb. Weak maternal expression can be detected by RT-PCR (datanot shown), but is not apparent by Northern blot. Zygotic expression of fullbackbegins at midgastrula stages, peaks during neurulation and then declines byswimming tadpole stages.In situ hybridization analysis was performed in order to determine thelocation of fullback transcripts within the embryo (Fig 3). During gastrula andneurula stages,fullback expression is posteriorly localized. At early gastrula (Fig 3A,panel a) weak expression is found in a wide rim around the blastopore. Atmidgastrula (panel b)fullback expression remains around the blastopore andincreases in intensity. A cross section through a halved embryo of this stage (panel f)shows that fullback is expressed in the superficial tissue of the blastopore lip but notin the involuted mesoderm. At neurulation (panel c)fullback reaches its highestlevels of expression and is found in a ring around the closed blastopore in theposterior of the embryo, both in the ectoderm and in the underlying mesoderm(panel g). At this stage, dorsal expression begins to extend out from the posterior ofthe embryo in a small streak along the midline (arrow, panel c). Cross sections ofneurula embryos suggest that this staining does not coincide with the notochord butmay correspond to migrating neural crest. At late neurula (panel d) expressionpersists in the posterior of the embryo and continues to extend dorsally into regionsnear the developing somites. A comparative in situ done on halved embryos withfullback and m-actin probes clearly shows non-overlapping regions of expression,implying that this dorsal fullback expression is not myotomal (panels h and j). Attailbud stages fullback is maintained in the posterior and the dorsal expressionexpands ventrally (panel e). A transverse cut through these embryos reveals that thedorsal staining overlaps with muscle actin staining in the somite, implying that atthis stage fullback RNA is present in the myotome (panels i and k).In order to assess whether fullback plays a role in modulating apoptosis, wecompared its expression with TUNEL assays which profile the temporal and spatialoccurrence of apoptosis within the Xenopus embryo (Hensey and Gautier, 1998).Using this assay, these authors found that apoptosis begins at midgastrula stages in avariable pattern including every region of the embryo except the cells immediatelysurrounding the blastopore. Later, TUNEL staining coincides with regions ofneurogenesis in the ectoderm during neurula and tailbud stages. These regions ofthe embryo do not correspond with those expressing fullback, implying that fullbackis not essential for apoptotic signaling. These data, together with the significantintracellular sequence divergence between p75, suggest that the function of fullbackis different from that of p75.Methods:The full length clone of fullback was isolated from a random-primed cDNAlibrary made from ventral mesoderm and ectoderm from mid-gastrula Xenopusembryos (stage 11.5). Embryos were staged according to Nieuwkoop and Faber,1967. Two oligos were used to form an EcoR I adapter: BIS3, 5'-AATTCCCATAGCAACAAACAGTA-3', unphosphorylated and BIS4, 5'-TACTGTTTGTTGCTATGGG-3', phosphorylated.The random-primed cDNA library was subjected to a yeast selection asdescribed previously (Jacobs et al., 1997). The Genbank accession number of fullbackis AF131890.in situ hybridization was performed as described in Kuo, et al., 1998. Most insitu panels show albino embryos, although some show wild type embryos which havebeen bleached in 2% hydrogen peroxide for 45 minutes.Northern blot analysis was performed as described in Sagerstrom et al., 1997.-MG AG AMR RAG A- -MD KRGM1 IV TT G R MD G 0!11 1!ACSM DR LR....R- -MG FV L4LI> 1K ISfs L G G/ S S G Gfullbackhp75rp75cp75fullbackhp75rp75cp75fullbackhp75rp75cp75fullbackhp75rp75cp75fullbackhp75rp75cp75fullbackhp75rp75cp75fullbackhp75rp75cp75fullbackhp75rp75cp75fullbackhp75rp75cp75fullbackhp75rp75cp75fullbackhp75rp75cp75K A F SPSLTLESNT RTEPE g SS E..... :E L - M AETOG V 1,A 18 K. I'sP X:KT'3 S V S S KS SES V F LM P s... ... 'S S E SF 3IT E - - -aS TP P E.S TP P EH TPS LA-N H L S K A K I E PQ-H T !T A S LT H T T A S .LPPN4S TQ P &PKDO"."Q R1 lis..... ... YD TW RH L AG6ELGYD TWRH A .EDE .A.?T VQ IG DNE RML RTERQL RHN: E 1V M V K- - - - i - - - G - - -mT A P S 'Q E IE A PT A S 70 E ;iE V P~E I D P ---E E'T2 P EHJPE(ED LPQHRW APW AA TSP E Q DP E 0 DF N T EL IAL V PG MAY.IND sH( A dif ~.ST; G E DS THEA CS HACCR E S AI %IDEEDTLEA Dq Gq -s sA3SK ETQDAQDA K E3239403172798071111118119110151158159150191197198189203237238226242277278266281317318306316356354345356396394385427425416387K D VP LKI PG RNI TI PG RWI PL H P WT TS lb- A. VVTS .1V WDM VTT L D VTFigure 1: Protein Sequence Alignment of fullback with p75 from Other VertebratesThe p75 proteins shown are from human (hp75), rat (rp75) and chicken (cp75).Identical amino acids are shaded. Numbers to the right of the sequences indicate thenumerical positions of the amino acids. Boxes denote cysteines conserved among allfour proteins. The six-helix death domain region is underlined and overlined.Gastrula10 11fullbackrRNAANeurula1I _q 1 r. 4 %f %w IFigure 2: Northern Analysis of fullback mRNAfullback RNA (upper panel). Lane 1 contains unfertilized egg RNA. Lane 2contains RNA from early gastrula, stage 10. Lane 3 contains RNA from midgastrula,stage 11. Lane 4 contains RNA from early neurula, stage 13. Lane 5 contains RNAfrom midneurula, stage 15. Lane 6 contains RNA from early tailbud, stage 22. Lane 7contains RNA from swimming tadpole, stage 35. 18s Ribosomal RNA is shown inthe lower panel as a loading control.- ---------------- ----Figure 3: In Situ Hybridization Analysis of fullback RNA ExpressionIn situ hybridization was performed on whole (panels a-e) or halved (panelsf-k) Xenopus embryos. Dashed lines in panels b-e denote the plane in which eachembryo was cut in half.a. Vegetal view of an early gastrula (stage 10.5). b. Posterior view of an embryo atmid-gastrula (stage 11.5). c. Dorsal view of an early neurula (stage 13). Arrowindicates the weakly perceptible stripe of staining at the dorsal midline. d. Dorsalview of a late neurula (stage 18). e. Lateral view of a tailbud (stage 25). f. Interiorview of a halved embryo at mid-gastrula (stage 11.5). g. Interior view of an earlyneurula (stage 13) which has been parasagittally cut in half. h. Interior view of a lateneurula halved transversely. The dorsal expression of fullback is shown. i. Interiorview of a late tailbud (stage 25) embryo cut transversely in half.Panels j and k: Comparative in situ hybridizations were performed using a probe formuscle actin, which is expressed in the myotome of the Xenopus somite. Panel jshows a late neurula embryo (stage 18) cut in half transversely. Panel k shows atailbud stage embryo (stage 25).In all panels, Vg= vegetal, A= anterior, P= posterior, D= dorsal, V= ventral.References:Bredesen, D. E., and Rabizadeh, S. (1997). p75NTR and apoptosis: Trk-dependent andTrk-independent effects. Trends Neurosci 20, 287-90.Hensey, C., and Gautier, J. (1998). Programmed cell death during Xenopusdevelopment: a spatio-temporal analysis. Dev Biol 203, 36-48.Jacobs, K. A., Collins-Racie, L. A., Colbert, M., Duckett, M., Golden-Fleet, M.,Kelleher, K., Kriz, R., La Vallie, E. R., Merberg, D., Spaulding, V., Stover, J.,Williamson, M. J., and McCoy, J. M. (1997). A genetic selection for isolating cDNAsencoding secreted proteins. Gene 198, 289-96.Johnson, D., Lanahan, A., Buck, C. R., Sehgal, A., Morgan, C., Mercer, E., Bothwell,M., and Chao, M. (1986). Expression and structure of the human NGF receptor. Cell47, 545-54.Kaplan, D. R., and Miller, F. D. (1997). Signal transduction by the neurotrophinreceptors. Curr Opin Cell Biol 9, 213-21.Kuo, J. S., Patel, M., Gamse, J., Merzdorf, C., Liu, X., Apekin, V., and Sive, H.L. (1998)opl: A Zinc Finger Protein that Regulates Neural Determination and Patterning inXenopus. Development 125, 2867-2882.Large, T. H., Weskamp, G., Helder, J. C., Radeke, M. J., Misko, T. P., Shooter, E. M.,and Reichardt, L. F. (1989). Structure and developmental expression of the nervegrowth factor receptor in the chicken central nervous system. Neuron 2, 1123-34.Liepinsh, E., Ilag, L. L., Otting, G., and Ibanez, C. F. (1997). NMR structure of thedeath domain of the p75 neurotrophin receptor. Embo J 16, 4999-5005.Nieuwkoop, P., and Faber, J. (1967). Normal table of Xenopus laevis (Daudin), 2ndEdition (Amsterdam: North-Holland Publishing Co.).Radeke, M. J., Misko, T. P., Hsu, C., Herzenberg, L. A., and Shooter, E. M. (1987).Gene transfer and molecular cloning of the rat nerve growth factor receptor. Nature325, 593-7.Sagerstr6m, C. and Sive, H. L. (1997). Northern hybridization analysis. RNAMethods. Krieg, P., ed. Academic Press, pp 83-104.Tartaglia, L. A., Ayres, T. M., Wong, G. H., and Goeddel, D. V. (1993). A noveldomain within the 55 kd TNF receptor signals cell death. Cell 74, 845-53.von Heijne, G. (1986). A new method for predicting signal sequence cleavage sites.Nucleic Acids Res 14, 4683-90.

Identification and characterization of fullback, a novel posteriorly-expressed Xenopus gene : master's thesis submitted

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Identification and characterization of fullback, a novel posteriorly-expressed Xenopus gene : master's thesis submitted

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