Lens Regeneration in the Newt, Triturus

Lens Regenerationin the Newt, Triturus viridescens:A Teacher-Student Research Project

* Richard J. Brady, East Leyden High School, Franklin Park, Illinois

A teacher research project is described here which has been used as a vehicle to encourage student research efforts. The embryology of the eye is the basis of the project.

The purpose of this article is two-fold. of Reyer (1954). The more recent review First, acquainting the reader with the process by Stone (1959), which is a summary of of lens regeneration. The ability of animals his own work for the most part, deals with to regenerate missing parts such as limbs and lens regeneration in adult animals and does tails has received considerable attention, and not give much consideration to larval forms Downloaded from http://online.ucpress.edu/abt/article-pdf/27/3/185/21190/4440885.pdf by guest on 02 October 2021 I am sure most biology teachers have read as does the review by Reyer. articles dealing with regenerating planaria and The process of lens regeneration from the salamander limbs and tails. The process of pupillary margin of the dorsal iris is called lens regeneration, on the other hand, is usually "Wolffian" lens regeneration after Gustav mentioned only in passing as an example of Wolff who was among the first investigators another tissue which can regenerate. Although of this process in 1894. Thirteen stages of the capacity to regenerate a missing lens in an lens regeneration have been designated by adult animal is a unique attribute of only a Sato (1940). These, with slight modification, single family of Urodele amphibians, the Sala- can be applied to the process as it occurs in mandridae, the experimental procedures which both larvae and adults. The stages can be have been employed to elucidate the factors divided into four groups and are summarized involved in this process are so straightforward as follows: that the subject could very effectively be in- I. Latent Period: Stages 1 and 2 cluded in a school biology course as il- high Thickening of the iris along pupillary lustrative material in a number of places. If margin with a formation of a cleft be- the teacher is looking for another example of tween the inner and outer laminae the "scientific method" at work, for instance, (Fig. 1, A and B). the experiments described herein will provide material which even the most well-read among II. Initial Period: Stages 3 to 6 the students will find new and interesting. Appearance of depigmented cells The second purpose of this article is to along pupillary margin followed by describe the development of this teacher-stu- formation of a vesicle. The cells of dent project at East Leyden High School. the inner wall of this vesicle become elongate to form the primary lens Part I. Lens Regeneration in the Newt, fibers (Fig. 1 C, D, E, and F). Triturus V. Viridescens III. Period of Lens Fiber Differentiation: Stages 7 to 11 The capacity of an animal to regenerate an Completion of the primary lens fiber lens has been known for many extirpated nucleus which fills the lumen of the and been the of intensive years has subject vesicle and the symmetrical enclosure There are two study by many investigators. of this nucleus by secondary lens especially ac- men, however, who have beeii fibers (Fig. 1, G). tive in this area: Dr. Leon S. Stone, Yale 12 13 Medical School, and Dr. Randall W. Reyer, IV. Period of Growth: Stages and University of West Virginia. For a more Disappearance of nuclei in primary complete development of the ideas presented lens fibers and secondary lens fibers in this paper, the reader is referred to several except in the outermost portions (Fig. excellent reviews by these investigators (Re- 1, H). yer, 1954; Stone, 1953, 1959). The most In adult Triturus v. viridescens the entire comprehensive review of the topic is that process takes approximately five weeks at or-

185 186 THE AMERICAN BIOLOGY TEACHER

that there is a stimulus of a chemical nature involved which he calls the "neural retina factor." In a series of experiments, simple to com- prehend but difficult to carry out, Stone (1953) has clearly shown the relationship of the dorsal iris to the process of lens regeneration (Figs. 2, 3 and 4). From these experimental results and others which have been reported by Dr. Stone, the following conclusions concerning the dorsal iris can be drawn:

1. Injury to the dorsal iris neither stimu- Downloaded from http://online.ucpress.edu/abt/article-pdf/27/3/185/21190/4440885.pdf by guest on 02 October 2021 lates nor inhibits lens regeneration. 2. Maximum capacity for lens regeneration resides in the dorsal iris at the 12- o'clock position and decreases com- pletely as the 9 and 3-o'clock positions are reached. 3. This polarization of the iris occurs during the formation of the optic cup. Experiments by Stone and Steinitz (1957) Fig. 1. Regeneration of the Lens from the Dorsal Iris. Darkly pigmented normal iris (A); depigmentation and involving hundreds of adult newts (Trititrus thickening of iris cells (B); cell proliferation resulting v. viridescens), demonstrated the relationship in the formation of a vesicle (C, D, and E); primary the pigment cells of the retina, the lens fiber formation at the inner pole of the vesicle between (F); secondary lens fiber formation (G); detachment of neural retina, and the capacity of the dorsal lens regenerate from dorsal iris and continuation of iris to regenerate an extirpated lens. They secondary lens fiber development (H). (Redrawn from Stone, 1953) furnish conclusive evidence that the pigment cells of the retina are capable of differentiat- ing into not only neural retina cells, but also dinary room temperature, whereas in other into iris and (subsequently) into lens cells as species it may take twice that long. well. For example, in a series of experiments The exact nature of the mechanism which in which the Entire eyes of adult newts were enables these phenomena to occur has been transplanted from one animal to another, the subject of much investigation. Stone there followed a complete breakdown of the (1953), in relating the embryological de- neural retina along with the destruction of the velopment of the lens to the process of lens lens by cataract formation and subsequent de- regeneration stated that: generation. Shortly following this destruction "At first the mechanism for lens formation period, the retinal pigment cells differentiated expresses itself in the nature of some stimulus, into a new neural retina layer and a lens re- perhaps of a chemical nature, that passes from generated from the dorsal iris. the embryonic eye to the responding surface Of all the experiments involving the rela- ectoderm with which it makes contact. As the tionship of the neural retina and lens re- eye develops, the mechanism apparently be- generation, there is one kind of experiment in comes embodied within the cells of the dorsal particular which will give an insight into this iris when the latter forms. The lens-forming interesting relationship (See Fig. 5, A, B, potentiality of the cells is then restrained from and C). expressing itself by the presence of living lens Other experiments have shown that the in- fibers or by some substance produced by troduction of wax spheres or small pieces of them." tissue (other than lens tissue) does not inhibit Since making this statement eleven years the formation of a new lens in a lentectomized ago, Dr. Stone has performed a great number eye (Stone, 1945), but the replacement of an of ingenious experiments to elucidate the intact lens from the same species of sala- nature of this mechanism. It seems probably mander or from another species of salaman- LENSREGENERATION IN THENEWT 187

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Fig. 2. Dorsal iris from an intact newt eye transplanated under the ventral iris of a lensectomized eye results in the Downloaded from http://online.ucpress.edu/abt/article-pdf/27/3/185/21190/4440885.pdf by guest on 02 October 2021 development of lenses from both the normal and transplanted dorsal iris. (Redrawn from Stone, 1953)

Fig. 3. Ventral iris from an intact newt eye transplanted under the ventral iris of a lensectomized eye results in the development of a lens from only the dorsal iris. (Redrawn from Stone, 1953)

Fig. 4. Ventral iris transplanted to the 12-o'clock position in a lensectomized eye results in the development of lenses on each side of the transplant. (Redrawn from Stone, 1953) der which may Not even possess the capacity It seems logical to conclude that there is to regenerate an extirpated lens will inhibit present a factor in the lens which inhibits the lens regeneration if the transplanted lens does development of additional lens tissue. We may not degenerate. Removal of the loosely at- also conclude that there is present in the tached neural retina will also inhibit regenera- normal eye a "neural retina factor" which tion as will the daily injection of aqueous stimulates the formation of a new lens in the humor from intact eyes into lentectomized absence of the inhibiting lens factor. Experi- eyes, for many weeks in some experiments. ments by Dr. Stone (1958), using circular Following cessation of the injections, lens re- discs of plastic membranes of various poros- generation commences without delay (Stone, ities have shown that the neural retina factor 1953). will not pass through membranes which are 188 THEAMERICAN BIOLOGY TEACHER ER PLIOFILMDISCS CHOROID

PIGMEN TED RETINA

LEN3 A NEURP&LRETINA Downloaded from http://online.ucpress.edu/abt/article-pdf/27/3/185/21190/4440885.pdf by guest on 02 October 2021

NO LENS RF6CVA'A7S SMALLt ts RCCANCATCS Fig. 5. Pliofilm implant in lensectomized eye (A) results in complete lack of lens regeneration if the neural retina is entirely separated from the dorsal iris (B) and in reduced regeneration if a small portion of the neural retina is not separated from the dorsal iris (C). (Redrawn from Stone, 1958)

permeable to water soluble substances (Du se, such a situation is not at all surprising. Pont No. 600 cellophane), although, during Another noticeable, if not significant differ- the experiments, the membranes were cov- ence is the fact that lens regeneration does ered with a film of cells which may have not involve the formation of a blastema as in affected their permeability. tail and limb regeneration. One of the most notable differences between the regeneration of a missing lens and Part 11. The Teacher-StudentResearch Program School that of limb is the fact that, in the case of a at East Leyden High missing lens, the "stimulus of injuty" is not Purposeand Objectives: necessary to initiate the regenerative process. The purpose and objectives of this program The mere absence of the lens is sufficient. can be considered from the viewpoint of both Consistent with these findings is the fact that the student and the teacher. hypophysectomy of a newt previous to len- There is a place within the framework of tectomy will slightly retard, but not inhibit the modern, secondary school for a program the process of lens regeneration. Removal of of original, carefully selected research. It is the thyroid has the same effect (Stone, 1959). true that in many schools the brighter stu- Since the adrenal hormones play an important dents have been encouraged to engage in part in only the early stages of wound healing project work of one kind or another in an (Needhan, 1952) and such healing processes effort to stimulate their interest. However, are not a part of normal lens regeneration per these efforts cannot be compared to participa- LENSREGENERATION IN THENEWT 189 tion in a project as a member of a well-or- plementary Training for Secondary School ganized team. To borrow a quote from a Teachers). fellow teacher, "We expect the able-bodied I am especially indebted to Dr. Ronald R. boys to play football, but we don't require Novales, Department of Biological Sciences, them to organize their own football team!" Northwestern University, for the important The sooner a student begins to direct his role he played in communicating with the attention towards specific areas of vocational NSF during the long months when the project interest, the more ambitious that student is was being reviewed and for acting as Science going to be in regards to that interest and Advisor to the project for the past several others related to it. Those concerned with the years. education of teen-agers will agree that a major The project was initiated in the summer of cause of intellectual laziness on the part of 1961 at Northwestern University. A thorough high school students is lack of direction. They study of the literature and a pilot study of lens Downloaded from http://online.ucpress.edu/abt/article-pdf/27/3/185/21190/4440885.pdf by guest on 02 October 2021 don't know where they are going so they don't regeneration was started for the purpose of go anywhere! The final choice in a matter as learning the necessary skills. important as this should not have to be made The following September the project activi- until the student has an adequate concept of ties were moved to the high school and stu- his particular abilities and interests, so in dent participation was begun. Since that time some cases this decisioin can be properly about twenty-five students have taken an ac- made only after years of study. This does tive part in the project. The degree of partici- not, however, preclude the possibility of es- pation is left entirely to the student. Some tablishing broad vocational aims which could students have specialized in certain aspects serve as a source of motivation in the attain- such as sectioning, staining, photomicrog- ment of higher and more specific vocational raphy, etc. Others have undertaken individ- goals. To expose a student to research deal- ual projects on related topics such as: lens ing with lens regeneration may not result in regeneration in the mosquito fish, Gambusia his choosing to become a research biologist in affinis; the embryology of the eye of the that particular area for the rest of his life, nor Gambusia affinis; the histology of the frog's is this intended to be the case. It will, how- eye, Rana pipiens; and a study of lens regen- ever, provide him with a concrete situation eration in the largemough bass, Micropterus against which he can pit his abilities and am- salmoides. All of these "sub-projects" require bitions and thereby temper his goals in the re- basically the same type of equipment and re- fining fire of experience. agents which are needed for the main project and yet allow the students to work com- Background: pletely independently if they have the ability While I was attending an NSF Academic and the inclination. In most cases the stu- Year Institute in 1959-60 at Brown Univer- dents work on the main project for a year or sity, Dr. Leon S. Stone was a visiting lecturer. so to learn the basic techniques and then He spoke at some length and with a con- launch out on their own when they discover tagious enthusiasm about his work which is related problems in the literature. This pro- mainly concerned with the visual apparatus of cedure has resulted in a much more effective the newt. Taking advantage of a general in- utilization of time and equipment and seems vitation to visit his laboratory, several other to be a much more natural way of choosing a teachers and myself spent a day with Dr. problem. Stone in the Department of Anatomy at Yale University Medical School. Certain aspects of Materialsand Methods: his work dealing with lens regeneration seemed The following is a description of the pro- suitable for project work at the high school cedures used to study lens regeneration as it level so, with the cooperation and encourage- normally occurs. The ability to complete such ment of Dr. Stone I applied to the NSF and a study is a necessary prerequisite to any received help in terms of time (Summer Fel- work involving an experimental and con- lowship Program for Secondary School Teach- trol situation. Such a study concerning the ers - NSF 71053) and also in terms of effects of the drug Actinomycin D on lens re- financial assistance (NSF -- 022704 - Sup- generation is now being made and will be 190 THE AMERICANBIOLOGY TEACHER

and their circulation is checked. When it be- comes noticeably depressed anesthesia is adequate. Incidentally, using the newt to demon- strate the circulation of the blood is much simpler than the use of either the tail of the goldfish or the webbed foot of the frog and the results are excellent . . . it would be worth having several newts on hand just for this demonstration. The newts are then weighed to the nearest 10 mg and measured to facilitate identifica- tion. Each animal is assigned a number and Downloaded from http://online.ucpress.edu/abt/article-pdf/27/3/185/21190/4440885.pdf by guest on 02 October 2021 this number is entered in a permanent log along with a description of the animal. The lens is removed from the eye in the fol- "'I~~~~o lowing manner: 1. The anesthetized animal is placed in a shallow petri dish filled with amphibian Ringer's solution. A piece of cotton is placed in the mouth to avoid excessive movement of the eye during the operation. Small glass rods (I"), bent at a slight angle are placed over the newt in Fig. 6. (35 x) An intact right eye of the newt, Triturus one or two places to hold him in posi- viridescens. The lower jaw of the animal has been tion. removed. The darkly stained horseshoe shaped structure is the neural retina with several distinct layers clearly 2. While observing the animal under ap- visible. proximately 25x magnification (a stereo- reported soon. The animals used in the project are adult pond newts, Tr-iturus viridescens. They are purchased from a local aquarium supply house. The stock animals are kept in a 50 gallon aquarium and the experimentals are kept in half-gallon plastic containers with perforated lids, usually one to each container. At first, small bits of calves' liver were fed to the newts, but at present they are being fed mainly tubifex worms, also obtained from a local supply house. Because the worms are living they can be left in the water for long periods of time without fouling the water. If liver is fed, the water must be changed a few hours after feeding. They are fed at least three times each week. The water in the small containers is changed daily. Before undergoing the lentectomy operation the animals are anesthetized by placing them in a 0.1% solution of MS 222 (Sandoz Pharm.) for a period of 15 to 20 minutes. The blood vessels in the iris and subepidermal layers are easily visible under a low power (20-30x) dissecting microscope. The newts Fig. 7. (100x) An early stage af lens regeneration showing the formation of a vesicle and the depigmentation of are removed from the anesthetic periodically the cells along the dorsal border of the pupillary margin. LENSREGENERATION IN THENEWT 191

zoom dissecting microscope is ideal for this purpose), a small slit is made in the cornea over the pupil using a micro- scalpel or a sharpened needle. t~~~~4 . 3. The slit is then extended to the nasal and temporal borders of the cornea using iridectomy scissors. 4. While spreading this slit with one pair of fine forceps, a second pair is used to grasp the elastic capsule of the lens and pull it through the pupil opening and the opening in the cornea. It is pos- Downloaded from http://online.ucpress.edu/abt/article-pdf/27/3/185/21190/4440885.pdf by guest on 02 October 2021 sible to do this entire procedure without any bleeding or damage to the iris. 4% If the operation is not properly carried out the neural retina may become de- tached and appear as a bluish material protruding from the pupil. If this hap- pens lens regeneration will be consider- ably delayed until this layer has first regenerated to some extent. 5. The lens may be preserved in 6%'/o formalin where it will maintain crystal- line appearance. By placing the lens Fig. 9. (lOOx) Beginning of secondary lens fiber formation on a piece of newsprint in a watch glass which will eventually enclose primary lens fiber nucleus. filled with water the students can see then gently brought together and the the lens act as a magnifying glass. animal is placed in a cool place on 6. The edges of the corneal opening are moist paper towels for 24 hours to allow for wound closure. 7. Each newt can then be transferred to its labelled aquarium where it is kUpt in fresh water for further observations. Approximately every three days for forty 4,. days several animals are again anesthetized and then decapitated. The several heads are placed in 5% acetic Zenker's fixative for 24 hours and then washed for 24 hours in run- ning tap water. Decalcification with 6% nitric acid in 80% alcohol is then followed by final dehydration, clearing with methyl benzoate

o- d:EE.i.i:- - ...... s .....we . ... and cedar oil, and embedding in "Tissuemat." The tissues are then sectioned at 7 microns 4 ~ ~ ~~~4 and stained with Ehrlich's hematoxylin and erythrosin for study. Resultsand Conclusions: A complete set of permanent slides has been produced which show all of the important phases of lens regeneration. The ac- companying photomicrographs (Figs. 6-10) clearly show distinct stages in the regenerative process. Fig. 8. (100x) Vesicle formation with the lumen being filled These pictures are much more significant with primary lens fibers from the inner wall (the right side) of the vesicle. than that, however, because they reflect to 192 THEAMERICAN BIOLOGY TEACHER

us have experiencedthis same type of feeling, for example, while being conducted through laboratorieswhere important research is in progress.It is an intangiblefeeling of awe and respect which extends even beyond the per- sons involved to the equipment being used. I have found this change in student attitude to be present among the vast majorityof biology studentsin spite of the fact that only a

. o . ! ? + comparativelysmall number of students are {~~~~~~~~~~~~...... 4 actuallyinvolved in the project and the topic is broughtup in class only very brieflyin re- .. Downloaded from http://online.ucpress.edu/abt/article-pdf/27/3/185/21190/4440885.pdf by guest on 02 October 2021 Fig 10; (lOO) Detachmet of t s...... c l f sponse to students'questions at varioustimes. dorsal iris Len--s will gradually .e lr .ut .l it rece The concept of teacher-orientedresearch at the high school level is analagousto the situation which obtains in most institutions of higher learning. If the dual role of teacher- ~~,.z.;,s-*-'':vsi...... | I .. researcheris not only desirable at the uni- I w ...' ~~~~~~~~~~~~~~.:...... f... k. versity level, it is a criterionof academicex- it rginaWFsize in abu on year Seodr len fibe cellence, it would be reasonable to assume that some of the benefitsof such a role might also be obtained at the high school level. I am not advocatingthe adoptionof a research Fig. 10. (100x) Detachment of the lens capsule from the project for the purpose of self-aggrandize- dorsal iris. Lens will gradually enlarge until it reaches ment. I am simply sayingthat such a project, its original size in about one year. Secondary lens fiber im- nuclei are visible within the lens. if diligentlypursued, will often resultin an proved academicclimate which will make it- some degree the success of the project thus self felt in the daily classroomsituations. far. From the time these animals were len- The recent BSCS publication, "Research tectomizeduntil their tissues were processed Problemsin Biology"(Brandwein, et al, 1963) and photographedand the picturesprinted in is invaluablefor the teacherwho is consider- the dark room, every phase of the operation ing establishing a research project in his was carried out by the students. Some in- school on a permanentteacher-student basis dividualstudents have progressedto the point and is searchingfor a suitabletopic. where they could carry out the entire procedure by themselves. Such a studentis cer- Acknowledgments: tainly well preparedto engage in significant I am deeply indebtedto Dr. Leon S. Stone individualresearch in any relatedarea. for the help and encouragementwhich he has Competencein an area of original investi- given since the beginningof the project and gation cannot help but have a stimulating for his continuedinterest in its progress. effect on the professionaloutlook and growth Sincereappreciation is also extendedto Dr. of the teacher. This is bound to carry over Ronald R. Novales, Departmentof Biological into the daily classroomroutine. The dichot- Sciences,Northwestern University, for the im- omy which exists in the minds of many stu- portantrole which he has played in obtaining dents in regards to school and "real life" the supportof the NSF and for acting as Ad- plays a great part in the lackadaisicalattitude visor for. the project during the past three which they exhibit toward their school work. years. I am convinced that the existence of the re- This project was supported by National search project here at East Leyden has Science FoundationGrant G22704 to North- engenderedin the minds of the studentsthe westernUniversity in supportof a programof feeling that "somethingreal" is going on in SupplementaryTraining for Science Teachers the departmentand that what we are teaching at East LeydenHigh School, District212, and in class must, therefore, be meaningfuland by the Administrationof East Leyden High importanteven in terms of "real life," All of School. LENSREGENERATION IN THENEWT 193

References 613. Stone, Leon S., 1945, "Heteroplastic Lens Grafts Brandwein, Paul F., et al, 1962, "Teaching High Related to Factors Inhibiting Lens Regenerationin School Biology: A Guide to Working with Poten- Triturus,"Proc. of the Society for Exp. Biol. and tial Biologists," BSCS Bulletin No. 2, AIBS, Med., Vol. 60, No. 10. Washington, D. C. ..., 1953, "An Experimental Analysis of Lens Re- 1963 "Research Problems in Biology: In- generation," Amer. Jour. of Ophthalmology, Vol. vestigations for Students," Doubleday and Co., 36, No. 6, Part II, 31-39. New York. ., 1958, "Lens Regeneration in Adult Newt Eyes 1 81473 Interstate Prtrs. 20593 1-20-65 LLW-6 Related to Retina Pigment Cells and the Neural Needham, A. E., 1952, "Regeneration and Wound Retina Factor," Journal of Exp. Biology, Vol. Healing," Methuen's Monographs on Biological 139, No. 1, 69-84 Subjects, John Wiley and Sons, New York. . ., 1959, "Regeneration of the Retina, Iris, and Reyer, R. W., 1954, "Regeneration of the Lens in Lens," Part I of Regeneration in the Vertebrates,

the Amphibian Eye," Quarterly Review of Bi- C. S. Thornton, ed., University of Chicago Press, Downloaded from http://online.ucpress.edu/abt/article-pdf/27/3/185/21190/4440885.pdf by guest on 02 October 2021 ology, Vol. 29, No. 1, 1-46. Chicago Sato, T., 1940, "Vergleichende Studien uber die . , and H. Steinitz, 1957, "Regeneration of Neural Geschwindigkeit der Wolffschen Linsenregenera- Retina and Lens from Retina Pigment Cell Grafts tion bei Triton taeniatus und bei Diemyctylus in Adult Newts," Jour. Exp. Zool., Vol. 135, No. pyrrhogaster,"Arch. EntwMech. Org., 140: 570- 2, 301-317.

Urban Problems Projects ranged all the way from the Stev- Suburban sprawl and industrial expansion ens Creek Park Chain in Santa Clara County, have almost buried most of the open space California, to historic Monmouth Battlefield remaining in the nation's growing metropoli- in New Jersey. Copies of the report "Pre- tan centers. More than 100,000 acres of serving Urban Open-Space Land: The First valuable urban land in 177 communities have Three Years" are available upon request been saved, however, for present and future from the Open-Space Land Division of the generations, with the aid of the Urban Re- Urban Renewal Administration, Washington, newal Administration's three-year-old Open- D. C. Space Land Program. Commenting on a special report just pub- Color Slides lished by the Urban Renewal Administration, Sportsmen and other outdoor enthusiasts Commissioner William L. Slayton pointed now have an oportunity to view wildlife on out that the $32 million in Federal grants for U. S. Bureau of Land Management areas of open-space land were primarily concentrated the west in a series of 35 mm color slides. in the large fast-growing regions of the Na- The 50-slide set depicts a wide variety of tion where problems of open space are most wild birds and animals found on the deserts, critical. The Northeast, Middle Atlantic, plains, and mountains and portrays the ex- Midwest, and West Coast regions received panding wildlife and recreation programs be- the majority of the 219 grants, with nearly ing developed by the Bureau. The slides, half of them going to metropolitan areas hav- plus a complete narrative script, can be ing populations of over one million people. obtained for showings to sportsmen's clubs, California led with 33 projects, followed by school groups and other interested organi- New York (31), New Jersey (26) and Illi- zations through any of the Bureau's 11 state nois (15). offices in the West. The Open-Space Land Program was au- thorized by Congress in the Housing Act of Cover Picture 1961. It authorizes the Federal Government "The facets of a crayfish eye suggest opaque to make grants to localities of up to 30% windows. What does the animal see through of the cost of undeveloped land for recrea- these grids? They suggest orientation based tional, scenic historic, and conservation pur- on pattern in the environment, and perhaps poses. The 101,947 acres were acquired by the use of ultraviolet light." These are the states, counties, special park districts, cities suggestions of Millard Davis, Blair Academy, and towns in 31 states at a total cost of over Blairstown, New Jersey, who sent in this $121 million. photograph.