Kato, Kenichi

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RIGHT:URL:CITATION:AUTHOR(S):ISSUE DATE:TITLE:Studies on the DifferentiatingPotencies of the Dorsal Part of theBlastoporal Lip in Triturus-Gastrula(II) : On the Differentiation ofNotochordKato, KenichiKato, Kenichi. Studies on the Differentiating Potencies of the Dorsal Part of theBlastoporal Lip in Triturus-Gastrula (II) : On the Differentiation of Notochord. Memoirs ofthe College of Science, University of Kyoto. Series B 1959, 26(1): 1-71959-07-25http://hdl.handle.net/2433/258549MEMOIRS oF THE COLLEGE OF SCIENCE, UNIVERS!TY OF KYOTO, SERIES B,             Vol. XXVI, No. 1, Article 2 (Biology), 1959Studies on the Differentiating PoteRcies of the ]])orsal   Part of the BIasteporal Lip in Triturus-Gastrula          II. Ok the Differentiation of Notochord                                    By                             Ken-ichi KATo'         Biological Laboratory, Osaka Dental Col!ege, Hirakata, Osaka-Prefecture                           (Received june 17, 1959)    In our previous paper (KATo and OKADA, '56) it was reported that the dorsal                                                          .blastoporal lip of the earliest Triturus-gastrula, consisting mainly of the presump-tive rnaterials of the foregut and prechordal plate, is able to differentiate 2ntonotochord and muscle when explanted within the envelope of the competent pre-sumptive ectoderm isolated frorn the early gastrula, but is unable when explantedwithin the eRvelope removed from the incompetent belly ectoderm of the neurula.The difference is attributed to the presence or absence of the neural tissue derivedfrom the enveloping ectoderm. In other words, there is a clese correlation betweenthe differentiation of neural tissue and the mesodermal differeRtiation. The samesituation occurred also in another experiment, in which the different parts in thedorsal blastoporal area of the early gastrula were tested for their developmentalpotency by explantation within the ectodermal vesicle taken out of the early gastrula(KATo, '58). WM the difference of the envelope be the cause for the differentresult in the differentiation of the presumptive notochorda} area of the ear}y gastrula ?    This paper deals with the closer study of such neuro-mesodermal relationshipin the differentiation of the mesodermal tissue from the dorsal blastoporal part inthe course of gastrulation.    The author wishes to express his cordial thanks to Prof. Dr. M. IcHiKAwA ofthe University of Kyoto for his supervision and encouragement during the courseof this study. His thanks are also deeply due to Dr. T. S. OKADA for the criticalreading of the manuscript and Mr. N. IKusi-iiMA for his va}uable advices. Further,it is his pleasure to record here his sincere gratitude to Prof. Dr. T. SiN-J.KE ofOsaka Dental College, who rendered aid to him in the publication of this paper.                           Material and Method    The explantation of three different regions of the dorsal part of the blastoporewas carried out, using the gastrulae of Triturus Pyrr}zogaster. The closer account2 Xen-ichi KA'roof the location and shift of tke material to be tested inwas already given in the autltor's previous paper ('58),in the following Table l.       Table 1. Sequence oÅí shifting of the blastoporal areasthe course of gastrulationand it can be summarizedduring gastru!ation."• .Stages of testl   "'-. areaslAreas". I St•to be tested'X'x-xlll l st. 12b l• St. 13c , Presumptive fatell               i       au I PrgXlllg5g9.the invaginated invaginated fOr.ehg.ltE.alnpdl.Ptr.e"       bu I MebgLa,i,.`pO.,t.he PrgXglg5g9.`he invaginated l notochord       ,u ldigtmao,gps me,gsgx,o.sse prgiggzgbgg,theinoBz2?ogS,zn.g.,g     The region tQ be tested, immediately after removal or after 4 hours precultur- ing in saline, was wrapped with the incompetent ectodermal piece taken from the presumptive be}}y region of the early neurula (stage 15 of OKADA and IcHiKAwA's standard table), and the explants thus made were kept in HoLTFRETER's solutioR for 12-25 days.                                    Resu!tsI. Differentiating Po'tency of the eecPlanted Piece.     (a) Part aU. In most cases the explanted material remained a mass or un- differentiated cells containing many yolk platelets, and only in a very few cases the deve}opment of notochord and muscle occurred. This was universal, regardless of whether the test piece was taken either before or after its invagination. Besides, a few specimens had a neural tissue.     (b) Part bU. Unlike the results of the previous series, the mesodermal tissues such as notochord and muscle were found in many cases. There existed, however, a considerabie difference in the frequency ef their appearance according to the stage at which the test piece was taken. Namely, the explants taken at stage 11, i.e., from the part medial to the blastopore, differentiated into notochord and/or muscle only in 24 per cent and 16 per cent of all cases respectively. On the ether hand, when the bU material was removed at stage 12b, i.e., from the part located immedi- ately above the biasÅíopore, the frequency was increased to 77 per cent and 43 per cent respectively. Moreover, the deveioped structures of the mesodermal tissues were irnproved inversely to the decline of the undifferentiated rnass or fragment.     There were specimens in which the neural tissue was established, and such an unexpected productien of neural tissue occurred more eften when the test piece was removed at stage 11 than when it was isolated at stage 12b. Here wili arise a dottbt as to the origin of the neural tissue. In order to ascertain this point, theDifferentiating Potencies of the          Table 2. TissuesDorsal Partdeveloped inof the Blastoporalthe explants.Lip, rl g         ÅísS "ata eq$s H-;ti B         ÅíN exqs" Sxto b).g" s.sts "Åí rc>IliliIIlbRSh"'gb"l"INl.INi{ei '"lptIIbIvlbÅéSh"'gbvl1lNumber oÅí available case$65 26 49 27 8 il42 8NotochordMuscleMesenchymel  5  5l 32ll     i( 8)i)i( 8) i(49)1(4) 2( 8)12 (46)ilI12823sgiI?;ggi(47) l24 (ss)i 875(100)( 88)( 63)EIll91020(45)(24)(48)2) i  lNeural tissue3) i        iAtypicalepidermis2(3)I/it-'Lt-ttttt'tttnvtttttt  itttttttnvtttttttttttttttttttlttmmnv       /1       Il1 ( 4) l 11 (22) I 3       II       //o I2(4)Io       //(ll) io]l ol iolle (24)lE7 (17)876(loe)( 88)( 75)oo g,n,dG,fferentiatediss (sg) I2s (7o) Is(63) 3o (72) I2(2s)    1) The number in.parenthesi$ indicates the percentage against the total number of       available cases.    2) Blood cells appeared in one specimen (cf. Fig. 2).    3) Archencephalic induction took place in one explant of the part aU at st. Z2b; in       ether cases neural tisstte appeared as a small mass of a neural tube or an amor-       phous fragment (cf. Figs. 4 and 5).test piece was taken sometimes from the embryos previously stained with Nile bluesulphate and was enveloped with the unstained ectoderm. The results revealedthat the neural tissue had been derived solely from the test piece. The xenoplasticcombination using the ectoderm of Rana-neurula as enveloping material aiso con-firmed this point. These evidences indicate that the part bU at stage 11 has apotency to differentiate even into the neural tissue under favourable cenditions.    (c) Part cU. As to the differentiation of the mesodermal tissue, the part cUbehaved quite like the previous part bU in explantation, i.e., the notochord andmuscle appeared very frequentiy in the explants coming from the part proximal tothe biastopore at stage 13c (100.0ei and 889o! respectively), while the explaRts fromthe part medial to the biastopore at stage 12b gave rise to the mesodermal tissuesin less than half of the total cases, i.e., 4596 in the notochordal and 24.0of in themuscular differentiation. The neural differentiation took place only in the latterexpiants.    As mentioned above, the neural tissue was occasionally developed in spite of thebelly ectoderm of neurula having been used as envelope. The production of thistissue occurred more frequently in the explants of the distal part than in those of4 Ken-ichi KA'rothe proximal part. Conseq"ently, the correlation of the neural tissue to the differ-entiation of the notochord was surmised in the former case, in which alrnost all ofthe explants with notochord had the neural tissue, too. Contrary to this, the explantsfrom the payt proximal to the blastopore produced often the notochord independentof the productioR oÅí the neural tissue. These situations are summarized in Table 3.On the other hand, as is seen in the table, the muscular differentiation had nothingto do with the neural tissue, as was pointed out by MucKMoRE ('51).         Tab}e 3. Differentiation of the notochord and musc}e in relation to the            presence or absence of the neural tissue in the explants from the            presumptive notochordal areas (parts bU and cU).Å~ PieÅëes to be    x.      x tested i      XIDiffs,gg:ti"x. I                   x.II Part apart from thel blastoporeiIl1V"'-'-'-r--T---_-.------"nvww.lmr1Part immediate}y above    the blastopore. Z;llNHNta'  "q"'  :6g       gsbbsnvFÅíI"qI b.I s-;IigIgI,lÅívosby '-.k cato PgProduction ofneural tissue                   i                   {{  with notochord                   'i                   ''                   'withottt notoehord                   '{  with musc}e                   i  without muscle                   ill921101091372218 (86) 3 (14) 4 (59)17 (81);Ii33e3oooooo33 (100)o3 (100)o                                                 i  Non-production of I  neural tissue 38 32 70 i 24 8 32   Swith notochord I 3 10 13 (19) 18 8 26 (81)   twithout notochord 35 22 57 (81) 6 e 6(i9)                                                                   16 (50)14 (2e)   {                 9       7with muscle 7 7                     'l                     i                          56 (80)                           16 (50)without muscle             31            25    25        1                     l       The number in parenthesis indicates the percentage against the total number       of explants with or without the neural tissue.II. Differe"tiating Pote•ncy of tke Part aPart from tke btastoPore aftev 4-hour    ceslturing.    The results of the experiments so far described indicate that the notochordaldifferentiation from the parts bU and cU occurred with high frequency, only whenthese parts come to occupy the area irnmediately above the blastopore. As to thecause which enhances this potency during the course of gastrulation, we may surmisetwo possibilities; one is the mere aging of the tissue and the other is the influencefrom the surrounding tissues. In order to examine the first possibility, the partbU at st. 11 Iocated media} to the blastopore had been previously cultured inHoLTFRETER's soiution for about 4 hours. i)uring this period this part would shiftto the area immediately above the blastopore, if it would be left in the intact em-bryo. Then, the pre-cultured rnateyial was wrapped with an incompetent ectodermalDifferentiating Potencies of the Dorsal Part of the Blastoporal Lip, II 5Table 4.   and Differentiationpre-cultured in from the partXOLTFRETER'SbU removed atsolution fer 4 st. 11hours.   No. ofavailable cases, Notochord l Muscle I Mesenchyrne Undiffmtraesnstiated27lI 3 (18e/.)t/'   2 (12o%) 9 (530/a) I 17 (20eo/.)piece. The results are shown in Table 4. A comparisen ef the results in Tabie4with those in Table 2 indicates that the pre-culturing does not enhance the meso-dermal differentiation, but suppresses it slightly. Consequently, it seems unlikelythat a mere aging of the part is an active cause for the enhancement of thenotochordal differentiation. The secoBd possibility of the surrounding influencewould tentatively be considered as the cause in question, though the direct evidencefor it is lacking.                                 Diseussion    The results of the present experiments have demonstrated that the presumptivearea of notochordcan differentiate into a definitive notochord in explantation, whenit occupies the area immediately above the blastopore, but that the same materialfails very often to develop into the notochordal structure, if it is taken out when itlies apart from the blastopore. Thus clearly there exists a difference in the dif-ferentiating potency of the presupaptive notochordal materia} between when it isdistal to the blastopore and when proximal to it. As meRtioned already, the causefor this dlfference can not be attributed to a mere lapse of time required for theshifting of the area from the distal location to the proximal site. The unknowninfluence exerted from the surroundings may be taken into consideration to explainthe present results. Recently, from the biochemical, histochemical and electron-microscopical studies a wealth of data has been accumulated that the dorsal partof gastrula is the most active site in proteolysis and protein synthesis (BRAcHET,'50; KARAsAKi, '58). Although no work from such an angle of study has everbeen reported about any difference between such small areas as studied in the pre-sent experiment, the data presented by RouND and FmcmNGER ('58) seem to besuggestive in relation to the present results. They found that RNA content of theresidual fraction of tlte dorsal blastoporal area ef Rana-gastrula decreases duringthe course of gastrulation. From this, it seems permissible to assume that theactivities in the protein breakdown and in the new protein synthesis differ betweenthe distal and proximal parts of the dorsal blasteporal region. These differencesrevealed in the biochemical level might refiect the difference of the notochordforming potency obtained by the present experiments.                                  Summary    (1) The developmental potencies of the different parts of the blastoporal iip6 Ken-ichi KAToisoiated from the ga$trulae at various stages were tested by explanting each withinan ectodermal envelope removed from the early neurula.    (2) At the same stage of gastrula, there were differences in the deve}opmentalpotency between the parts distal and proximal to the blastoporal lip. Occasionalproduction of the neural tissue occurred only from the distal part, while the noto-chord and muscle were frequently deveioped from the proximal part.    (3) When the differentiation of the notochord from the distal part tool< place,it was mainly accompanied with the simultaneous production of the neural tissue.    (4) The pre-culturing of the distal part in HoL'rFRETER's so}ution for 4 hourswhich is equivalent to the time of shifting of this part tG the area just above theblastopore in the course of gastrulation, could not enhance the differentiation of themesodermal tissues.                                 ReferencesBRAcffET, J., 1950. Chemical Embryology. New York, Interscience Publishers.KARAsAi<i, S., 1958. Jap. J. Exp. Mi orph., i2:21-32.I<ATo, K, & T. S. Oi<ADA, 1956. Mem. Coll. Sci. Univ. Kyoto, <B), 23:1-9.KATo, K., 1958. Ibid., 25:1-10.MucffMoRE, W. B., l951. J. Exp. Zool., 118:l37-186.OKADA, Y. K., & M. Icl{mAwA, 1947. 3ap. J. Exp. Morph., 3: 1-6.RouND, D. E., & R. A. FLici<tw'GER, 1958. J. Exp. Zool., 126: 21-32.Fig.                   Explanation of FiguresAbbreviatiens : B!, blood cells ; Ms, muscular bundle ; N, notocherd ; Nt,neural tube or tissue ; U, undifferentiated mass of cells.1. Differentiation of notochord and sma}1 muscular bundles in the explantof the part bUat stage 21.-Fig. 2. Differentiation of notochord and bloodcells in the explant of the part cUat stage 13c.-Fig. 3. Diffeventiation ofnotochord and muscie in the expiant of the part bU at stage 12b.-Fig. 4.Differentiation of notochord, small neural tube and undifferentiated mass ofcells in the explant of the part bU at stage il.-Fig. 5. Differentiatien ofnetocherd and small fragment of neural tissue in the expJant of the part cUat stage 12b.--Fig. 6. Unclifferentiated mass of cells in the explant of thepart bU at stage 11.Differentiating Potencies of the Dorsal Part of the Blastoporal Lip, IIFig.2NitNBINMsFig.4Fig.6UrxiT        •1x "'      - '"'   Fig3Fig.57

Studies on the Differentiating Potencies of the Dorsal Part of the Blastoporal Lip in Triturus-Gastrula (II) : On the Differentiation of Notochord

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Studies on the Differentiating Potencies of the Dorsal Part of the Blastoporal Lip in Triturus-Gastrula (II) : On the Differentiation of Notochord

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