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10 Freese, E., in Brookhaven Symposia in Biology, No. 12 (1959), p. 63; Freese, E. B., these PROCEEDINGS, 47, 540 (1961). 11 Muller, H. J., in The Control of Hunman Heredity and Evolution, ed. T. M. Sonneborn (New York: Macmillan, 1965), p. 100.

EFFECTS OF RNA ON THE DEVELOPMENTAL POTENTIALITY OF THE POSTERIOR OF THE CHICK BLASTODERM* BY SOMES SANYALt AND M. C. Niu

DEPARTMENT OF BIOLOGY, TEMPLE UNIVERSITY, PHILADELPHIA Communicated by Victor C. Twitty, February 7, 1966 During early embryogenesis of the chick, the cells of the primitive streak play a key role in the development of the primary embryonic structures. However, the developmental potentiality of the streak at the different levels along its antero- posterior axis is not uniform. This is most striking when comparing the anterior regions including Hensen's node with the posterior portions under a variety of ex- perimental conditions.' Hensen's node has long been recognized as the center for the primary organization of the chick . The regions more than 0.4-0.5 mm behind the node do not participate in the formation of central nervous tissue.2 When posterior portions of the primitive streak are transplanted to other , we have noted that they do not differentiate into any histologically identifiable structure and in the majority of cases the implants become completely absorbed into the extraembryonic tissues of the host. If, however, they are combined with such tissue as Hensen's node, they are induced to undergo differentiation.' In view of the finding that RNA induces the formation of specific structure4 or specific protein,5 it was decided to treat the posterior segment of primitive streak with RNA and subsequently to study the effect of-the exogenous RNA on its developmental potency. The RNA used was extracted from brain, liver, kidney, heart, and com- mercial yeast. The purpose of this paper is to present data showing that freshly isolated RNA from the various tissues indeed affects the developmental potentiality of the posterior streak. The effect is specific and varied in accord with the kind of RNA employed. Material and Methods.-Material: All experiments reported in this study were performed on the definitive streak stage of chick embryos (Hamburger and Hamilton, stage 4).6 Fertilized eggs of the white Leghorn variety were obtained from Shaw's hatchery, W. Chester, Pa., and incubated in a forced-draft incubator for 16-18 hr at 380C. Experimental: The donor embryos were first explanted from the eggs. The portion of the primi- tive streak constituting one fourth of the total length of the streak, extending posteriorly from its middle, was dissected out under a binocular microscope. Since the length of the streak was about 1.4-1.8 mm, the anterior margin of the piece used was 0.7-0.9 mm posterior to the primitive pit. The RNA's used were separately dissolved in Pannet-Compton's saline at a concentration of 40-80 OD (optical density at 260 mu) per ml. Each OD is approximately equivalent to 50 jug of RNA. The isolates of posterior streak were transferred to the saline with and without RNA. Both were kept at 2-4oC for 6-8 hr before being transplanted to the host embryos. Embryos to serve as hosts were grown in vitro according to New's techniques The intact blastoderm after 16-18 hr of incubation was excised together with a large portion of the vitelline Downloaded by guest on October 1, 2021 744 ZOOLOGY: SANYAL AND NIU PROC. N. A. S.

membrane and immediately transferred to a watch glass with the facing up. A glass ring about an inch in diameter was so placed on the that the blastoderm was located in the center, with the periphery of the membrane wrapping around the ring. Fluid albumen from the same eggs was added to the outside of the ring as nutrient. Both RNA-treated and untreated isolates were rinsed quickly in Pannet-Compton's saline and grafted onto the blastoderm (one in each host embryo) at the level of Hensen's node near the margin of the area pellucida. This was accomplished by making a very small slit on the hypoblast through which the isolate could be pushed into the space between the and the hypoblast. The microsurgery was performed with a pair of stainless steel needles. Sterile precautions were observed throughout the operation. After operation at room temperature (22-240C) the embryos were immediately returned to the incubator and allowed to develop for 24-26 hr. They were fixed in Bouin's fluid, sectioned at 8 ,u, and stained with hematoxylin and eosin. Preparation of RNA: RNA was extracted from chick brain, chick liver, calf brain, calf liver, calf kidney, and calf heart. Brains and livers of 10-12-day chick embryos were pooled and im- mediately frozen on dry ice. The calf tissues were brought from the slaughterhouse in ice-cold sucrose solution (0.25 M) and used immediately for RNA isolation or stored frozen in dry ice no more than 10 days before use. The RNA's were prepared by a modifications of Kirby's8 procedure. The tissue homogenates were deproteinized twice with water-saturated phenol. Polysaccharides and other particles were removed by centrifugation at 20C (Spinco-30,000 rpm for 45 min). The supernatant was precipitated by alcohol and redissolved in saline. This was repeated three times. The last alcohol precipitate was dissolved in Pannet-Compton's saline. The yeast RNA, pur- chased from Schwarz BioResearch, Inc., was reprecipitated by 2 vol of alcohol. All of our RNA preparations gave a positive orcinol reaction and a maximum UV absorption at 258-260 mM and a minimum at 230 my. The ratio between 230 and 260 was over 2 and that of 260 and 280 was above 0.45 but less than 0.5. It should be emphasized that the RNA was either freshly prepared or quickly frozen in acetone-dry ice mixture and subsequently stored in a deep freezer for 3-5 days. In experiments with ribonuclease, the liver RNA and crystallized ribonuclease (Worthington Biochemical Co.) were dissolved separately in Pannet-Compton's saline and mixed to make up the final concentration of 80 OD units of RNA and 1 mg of ribonuclease per ml of the RNA solution. This mixture was incubated at 37CC, pH 7, for 1 hr and cooled to 20C before use. RNase activity was inactivated by the rabbit antiserum against RNase. However, there was practically no difference observed in experiments with and without the presence of added RNase. Results.-Development of untreated fragments of the primitive streak: A total of 39 cases were studied in which the posterior isolates of the definitive streak were kept in the saline solution for 6-8 hr. After grafting to the host embryos, 24-26 hr were allowed for further development. Of the 39 grafts, 28 showed no trace of the implanted cells in serial sections of the host embryos. Thickened re- sembling a neural plate developed in one case. Persistent grafts were found in ten embryos. A typical example is shown in Figure 1, where it can be seen that the ec- toderm is slightly thicker than the adjoining extraembryonic ectoderm overlying a mass of very loosely scattered mesenchymal cells. No definite structure or pattern was noted in any of these ten grafts. Thus, the posterior portions of the primi- tive streak under the experimental condition employed here can develop at best into mesenchyme cells.

FIG. 1.-Cross section of a trans- _ ___plant from the posterior primitive AT streak of chick blastoderm. (control). ~~~~~~Itwas allowed to develop in the ex- traembryonic area for 24 hr. Note layer of ectoderm cells overlying a mass of mesodermal cells and absence of any definite histological structure. X 50. Downloaded by guest on October 1, 2021 VOL. 55, 1966 ZOOLOGY: SANYAL AND NIU 745

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FIG. 2.-Cross section of two transplants treated with chick brain RNA. Note the development of neuralAtissue which*~~~~~~~~~~~~~~~~~~~Aroverlies a mass of loose mesodermal cells. X 50.

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FIG. 3.-Cross section of two transplants treated with liver RNA. (A) Calf liver RNA. Note the presence of neural tissue and the enhanced development of mesodermal cells (arrow). X100. (B) Chick liver RNA. X50.

Effects of RNA isolated from different tissueson development of the posterior primi- tive streak: Pretreatment with various RNA's evoked a distinct shift in the de- velopmental potentiality of the posterior portion of the primitive streak. Table 1 shows the frequency of occurrence of neural structures, nonneural tissues, and ab- sorption in the various series. Brain RNA: Isolates of the posterior portion of the streak were exposed to RNA Downloaded by guest on October 1, 2021 746 ZOOLOGY: SANYAL AND NIU PROC. N. A. S.

TABLE 1 NUMBERS AND PERCENTAGES* OF GRAFTS SHOWING DIFFERENTIATION IN CONTROL AND 1ANA-T'REATED SERIES No. of No. of No. of neural nonneural No. of cases RNA source cases tissue tissue absorbed Control 39 1(2.6) 10(25.6) 28(71.8) Chick brain 29 10(34.5) 9(31.0) 10(34.5) Chick liver 28 10(35.7) 10(35.7) 8(28.6) Calf liver 35 18(51.4) 6(31.4) 11(31.4) Calf kidney 35 2(5.7) 12(34.3) 21(60.0) Calf heart 30 1(3.4) 14(46.6) 15(50.0) Commercial yeast 22 0(0) 3(13.6) 19(86.4) Calf liver RNA plus RNase 26 4(15.3) 8(30.9) 14(53.8) * Percentages in parentheses.

from chick brain (40 OD units/ml) for 6-8 hr before being grafted to host embryos. Ten of the 29 cases studied gave rise to well-differentiated neural tissue (neural plate or neural fold). Figures 2A and B show that these neural structures are continuous with the host ectoderm overlying a mass of loose mesenchyme cells. The con- tinuity of the neural structure with the host ectoderm points to the host origin of the neural tissue in response to the inducing action of the grafted cells, which them- selves have not differentiated into any recognizable structure. Nine other cases have varying amounts of undifferentiated mesenchymal cells at the site of im- plantation, but no neural structures developed. No sign of organization can be found in these mesenchymal cells. In the remaining ten cases the grafts were ap- parently absorbed. Liver RNA: RNA's from both embryonic chick liver (concentration used-40 OD units/ml) and calf liver (80 OD units/ml) were used. Of 28 implants pre- treated with chick liver RNA, there were ten cases showing neural induction (Fig. 3B). Recognizable grafted tissue was present in an additional ten cases, in some of which it had differentiated into organized, but as yet unidentified, structures. It is hoped, however, that more prolonged cultivation, or possibly other experiments now in progress, will help identify these structures. The remaining eight grafts were absorbed completely. Of 35 implants treated with calf liver RNA, 18 yielded neural inductions, six persisted as unorganized grafts, and 11 underwent absorption. The neural struc- tures were similar to those obtained in the experiments using brain RNA. The RNA from calf liver also induced in some cases the formation by the grafts of or- ganized but unidentifiable structures. Both chick and calf liver RNA's promoted the development of -like structures (Fig. 3A). Kidney RNA: Two of the 35 posterior isolates pretreated with RNA from calf kidney (80 OD units/ml) induced neural structures. In 12 cases the graft de- veloped into a mass of mesodermal cells, overlaid by thin ectoderm similar to the ex- traembryonic ectoderm. Figures 4A and B show that the mesodermal mass ap- pears to have been organized into irregular tubular structures. The lumen is sur- rounded by rows of lining cells which bear some resemblance to nephrogenic tissue at early stages of development. In the remaining 21 embryos the graft tissue was completely absorbed. Heart RNA: Thirty pieces of posterior primitive streak were exposed to RNA from calf heart (80 OD units/ml). In one case the transformation of epiblast into Downloaded by guest on October 1, 2021 VOL. 55, 1966 ZOOLOGY: SANYAL AND NIU 747

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FIG. 4.-Cross section of two transplants treated with calf kidney RNA. Note the absence of neural tissue and note also that some mesodermal cells have organized into tubular structures. X50. neural plate was noted. Fourteen grafts underwent considerable growth and differ- entiation. It can be seen from Figures 5A and B that cells of the implant are or- ganized in the form of 1 or 2 large vesicles. The cells lining the vesicles are compact and form a continuous sheath. Apparently they differ from both the tubular struc- tures observed in the series treated by kidney RNA and the loose mesenchymal cells observed in the brain RNA series. Yeast RNA: Pretreatment with yeast RNA (80 GD units/ml) produced no appreciable effect on the development of the posterior streak cells. Generally speaking, the results with yeast RNA resembled those with the controls. Twenty- two grafts were made, of which 19 were completely absorbed and three yielded loose mesenchyme cells without any visible differentiation. No neural tissue was noted. Effect of ribortuclease: The activity of calf liver RNA was decreased significantly by treatment of ribonuclease. Four out of 26 cases (15.3%) showed the presence of neural tissue, compared with 51.4 per cent obtained with untreated calf liver RNA. This 15 per cent of neural tissue was probably caused by the indigestible residue of the liver RNA. Varying amounts of mesenchymal cells were also seen, but without definite structure. Discussion.-Recently, Spratt and Haas'0 have suggested that the primitive streak of the chick embryo is an elongated blastema from which all primary struc- tures of the embryonic body develop. The posterior segments on isolation do not realize their presumptive fate, but can be made to differentiate if grown in as- sociation with the inducing tissue, Hensen's node.3 This has led us to use the pos- Downloaded by guest on October 1, 2021 748 ZOOLOGY: SANYAL AND NIU PROC. N. A. S.

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FIG. 5.-Cross section of two transplants treated with calf heart RNA. Note the for- mation of vesicular structures by mesodermal cells. X 50.

tenior primitive streak as a target tissue for the inducing actioii of RNA. The re- sults of various experiments reported in this paper provide direct evidence that the RNA-treated segments of the posterior streak acquire the capacity to undergo self-differentiation, to induce the overlying epiblast to become neuralized, or both. The type of differentiation depends on the RNA used. It can be seen from Table 1 that RNA's from brain and liver cause neural formation, while those from kidney and heart tend to induce the development of nonneural structures. Examination of the nonneural structures reveals the presence of mesenchyme cells in the control, yeast, and brain RNA-treated series, both somite-like and organized (but not identi- fiable) structures in the liver RNA-treated series, tubular structures in the kidney RNA-treated series, and vesicular structure in the heart RNA-treated series. It may be relevant to add here that tiny pieces of presumptive urodele gastrula ecto- derm developed in hanging drops of Niu-Twitty solution containing liver RNA into highly organized but unidentifiable structures."I Implanting these cells into tail-bud stage embryos resulted in their development into neural tissue, gall bladder, intes- tine, and connective tissue.'2 The correlation between RNA tissue source and the specific structures produced suggests the transfer of organ specificity or information by its RNA to the target cells. As a result, the target cells induce, or develop into, structures similar to those of the RNA donor tissues. It is possibly this mechanism bywhich homeogenetic in- duction is achieved." In normal development, however, primary induction pre- cedes homogenetic induction. Unlike the latter, primary induction may not in- volve the transfer of organ specific informational macromolecules. Otherwise, the macromolecules emanating from the invaginated chordamesoderm during gastrula- tion would induce the overlying ectoderm to develop into and/or instead of the nerve plate. This concept is suIpported by the observation that medium conditioned 7-10 days by the developing posterior medullary plate (pre- sumptive tail muscle) seldom initiates the differentiation of muscle cells, whereas medium conditioned for 10-14 days will do so frequently.'14 In the previous sec- Downloaded by guest on October 1, 2021 VOL. 55, 1966 ZOOLOGY: SANYAL AND NIU 749

tion we have also seen that the action of liver RNA on the posterior primitive streak cells varied according to its donor species. Chick liver RNA gave the same fre- quency of occurrence of neuralization as did brain RNA, whereas calf liver RNA, the concentration of which was twice that of the other RNA's, elicited neuralization in a considerably higher percentage of cases than did chick liver and chick brain RNA's. Although the liver RNA's produce neuralization, their action is not as specific as that of chick brain RNA. In the case of the liver RNA-treated streak, mesenchymal cells were also induced to form organized structures that were com- pletely absent from the brain RNA-treated samples. Notochord, somites, pronephros, and heart appear earlier than any of the endo- dermal derivatives (liver, pancreas, stomach, etc.). The present experiments were limited to 24-26 hr, and during this period none of the organs derived from the appear in the developing chick blastoderm. Accordingly the liver RNA-treated grafts would not be expected to develop into any of the endodermal derivatives, but possibly become organized, unidentifiable tissue. Unfortunately the technique used is not designed to permit cultivation of the grafts for longer pe- riods of development. In conclusion, the present results indicate that by treatment with exogenous RNA the posterior part of the primitive streak can be made active for inducing neuralization, for self-differentiation, or for both. Summary.-Posterior segments of the primitive streak of chick blastoderm were grafted between epiblast and hypoblast at the level of Hensen's node near the margin of the area pellucida. The grafts were absorbed in most cases, and seldom induced the formation of neural structure or underwent differentiation. When pretreated with RNA, the implants acquired the capacity to induce or develop into neural and nonneural tissues depending on the tissue source of RNA. Kidney and heart RNA seldom induced neural tissue but differentiated into tubular and vesicular struc- tures, respectively. RNA from chick brain resulted in the development of neural tissue. Chick and calf liver RNA caused the formation of neural structures and organized, but as yet unidentified, mesodermal tissues, and also promoted growth of mesenchymal cells.

* Supported by grants from The National Foundation (CRMS-213) and NSF (G-23644). t Present address: Institute for Cancer Research, Fox Chase, Philadelphia, Pa. 1 Butros, J. M., J. Exptl. Zool., 143, 259-282 (1960); Rudnick, D., J. Exptl. Zool., 78, 369-383 (1938); Rudnick, D., "Embryogenesis: progressive differentiation; teleosts and birds,"inAnalysis of Development, ed. Willier, Weiss, and Hamburger, pp. 297-314 (1955). 2 Butros, J. M., J. Exptl. Zool., 143, 259-282 (1960); Spratt, N. T., Jr., J. Exptl. Zool., 120, 109-130 (1952); Spratt, N. T., J. Expd. Zool., 128, 121-163 (1955). 3 Butros, J. M., J. Exptl. Zool., 149, 1-20 (1962). 4Benitez, H. H., M. R. Murray, and E. Chargaff, J. Biophys. Biochem. Cytol., 5, 25-32 (1959); Butros, J. M., J. Embryol. Exptl. Morphol., 13, 119-128 (1965); Hillman, N. W., and M. C. Niu, these PROCEEDINGS, 50, 486-493 (1963); Jones, P. C. T., Nature, 202, 1226-1227 (1964); Niu, M. C., these PROCEEDINGS, 44, 1264-1274 (1958). 6 Mansour, A. M., and M. C. Niu, these PROCEEDINGS, 53, 764-770 (1965); Niu, M. C., Develop. Biol., 7, 379-393 (1963); Niu, M. C., in Symposium on Nucleic Acid and Biological Func- tion (Lombardo Institute, Milan (1964), pp. 352-371; Niu, M. C., Science, 148, 513-516 (1965); Niu, M. C., C. C. Cordova, and L. C. Niu, these PROCEEDINGS, 47, 1689-1700 (1961); Niu, M. C., C. C. Cordova, L. C. Niu, and C. T. Radbill, these PROCEEDINGS, 48, 1964-1969 (1962). 6 Hamburger, V., and H. L. Hamilton, J. Morphol., 88, 49-92 (1951). 7New, D. A., J. Embryol. Exptl. Morphol., 3, 320-331 (1955). Downloaded by guest on October 1, 2021 750 ZOOLOGY: CALDER AND SCHMIDT-NIELSEN PROC. N. A. S.

8Kirby, K. S., Biochem. J., 64, 405-408 (1956). 9 Niu., M. C., these PROCEEDINGS, 44, 1264-1274 (1958). 10 Spratt, N. T., Jr., and H. Haas, J. Exptl. Zool., 158, 9-38 (1965). 11 Niu, M. C., Evolution in Nervous Control (London: Bailey and Swinfen, 1959), pp. 7-30. 12Niu, M. C., unpublished. 13 Butros, J. M., J. Exptl. Zool., 154,125-133 (1963); Butros, J. M., J. Embryol. Exptl. Morphol., 13, 119-128 (1965); Hillman, N. W., and M. C. Niu, these PROCEEDINGS, 50, 486-493 (1963); Mansour, A. M., and M. C. Niu, these PROCEEDINGS, $3, 764 (1965); Niu, M. C., in Cellular Mech- anisms in Differentiation and Growth, ed. Rudnick (Princeton University Press, 1956), pp. 155- 171; Niu, M. C., these PROCEEDINGS, 44, 1264-1274 (1958); Niu, M. C., Evolution in Nervous Con- trol (1959), pp. 7-30. 14 Niu, M. C., in Cellular Mechanisms in Differentiation and Growth (1956), pp. 155-171.

EVAPORATIVE COOLING AND RESPIRATORY ALKALOSIS IN THE PIGEON* BY WILLIAM A. CALDER, JR.,t AND KNUT SCHMIDT-NIELSEN DEPARTMENT OF ZOOLOGY, DUKE UNIVERSITY Communicated February 18, 1966 Some birds can withstand exposure to high air temperatures and keep their body temperature from rising to a lethal level by increasing evaporation of water. This is achieved by an increase in respiratory ventilation similar to panting in dogs. Most noticeable in birds is a change in respiratory rate which may increase more than 20-fold. An increase of this magnitude should have considerable consequences for the gas exchange in the lungs. In particular, the removal of carbon dioxide can be expected to increase, and unless this loss can somehow be prevented, the result will be a severe alkalosis. The respiratory system of birds differs from that of mammals in that the lungs communicate with large, membranous air sacs which are thought to act as bellows. The air can reach the air sacs via two different pathways, either directly through the mesobronchi, or via the numerous, smaller parabronchi which penetrate the lung parenchyma (for anatomical details, see refs. 1 and 2). It has been suggested that this arrangement could permit air to pass to and from the air sacs without passing the gas exchange surfaces of the lung.2-5 Thus, respiratory ventilation could increase without causing alkalosis due to removal of CO2 from the lungs, but experimental evidence that could clarify this point has been inadequate. We have approached this problem by examining the acid-base balance of the blood, and found that during heat regulation pigeons sustain a severe alkalosis due to excess loss of CO2. Methods.-Animals: Adult domestic pigeons (Columba livia) weighing 270 to 425 (mean = 347) gm were obtained commercially. They were kept and fed in outside aviaries, but were main- tained inside at ordinary room temperature for one week preceding any experiment. Temperature measurements; Air and body (cloacal) temperatures accurate to ±0.2CC were re- corded with copper constantan thermocouples connected to a Leeds and Northrup Speedomax G 16-channel potentiometer. Air temperatures above 500C were measured with a mercury ther- mometer. Cloacal thermocouples were inserted about 2 cm and taped to the tail feathers. All Downloaded by guest on October 1, 2021