Book - Manual of Experimental Embryology

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Hamburger V. Manual of Experimental Embryology. (1942)

Online Editor 
Mark Hill.jpg
This historic 1942 embryology textbook by Hamburger. Currently only the an early version of the edited text has been made available online, figures will be added at a later date. My thanks to the Internet Archive for making the original scanned book available.

Viktor Hamburger (1900 - 2001) was a founding researcher in the field of developmental neuroscience, establishing the role of neurotrophic factors for neuronal survival during development.

  • University of Freiburg with Hans Spemann.
  • 1933 - United States, first in Chicago and then at Washington University.
  • 1988 - published "The Foundations of Experimental Embryology: Hans Spemann and the Organizer".
  • The Viktor Hamburger Centenary Celebration PMID 11241837 Dev Dyn.
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Pages where the terms "Historic Textbook" and "Historic Embryology" appear on this site, and sections within pages where this disclaimer appears, indicate that the content and scientific understanding are specific to the time of publication. This means that while some scientific descriptions are still accurate, the terminology and interpretation of the developmental mechanisms reflect the understanding at the time of original publication and those of the preceding periods, these terms and interpretations may not reflect our current scientific understanding.     (More? Embryology History | Historic Embryology Papers)
Viktor Hamburger (1900 - 2001)

A Manual of Experimental Embryology

The University of Chtoado Press, Ghicaoo 37 Cambridge University Pm, Loodoa, N W 1 En^Und The University Toronto Pit», Toronto 5 Canada

Ca^jniil W2 hj Tht Vmawj tf CSa tf , All rt^kij Pukulttd SepUmia 1942. fWlt Impnsam 1936. Qmfmt4 mUpmttJfyTnx UmvcaiTTY or Chxmoo Faiai,

JUmtis USA

Introduction

Experimental embryology baa achieved a pro min ent position m modern biology its classical experiments and concepts are, bj now an integral part of biological thinking Yet few students of biology have an opportunity to obtain a firsthand acquaintance with its methods and materials This is due partly to the difficulties of providing the embryomc material and partly to the technical difficulties of experimentation on living em bryos However, a large number of classiral experiments do not require exceptional rrmniml ftlcill and are suitable as classroom experiments Most of the ciqicnments described m the following pages have been made by advanced undergraduate and graduate students m a one semester course which has been offered at Washington University 1935 One of the main assets of such a course is to bring the student into intimate contact with the living developmg organism and the enthusiastic response of the students mdlcates cicarij the demand for such an approach to biology

This manual emphasizes the intrinsic factors of morphogenesis that is origin of fonn and of organs. It includes regeneration but gii'es httle con sideiation to histogenesis and growth In the selecticm of the experiments we were guided by practical considerations. Oiilj those elemental ex penments were chosen which do not require a high degree of manual skill and which can be done In the limited time of a three-hour laboratory period- We excluded all experiments which require expensi\T apparatus such as a micromanipulator Special attention was paid to the de^Tlopment of simple and inexpensive Instruments for operations. Onl> such living matenaJ as can be coDected in the field or purchased at reIatiiTl> small expense Is recommended All experiments were devised In such a wa> that sectioning of the material is not essential for the 8tud> of the results For instance regenerated lenses in amphibian Iar\’ae can be stud led by making them opaque and thus visible bj fixation m formaldehj’de chorio-allantoic grafts of limbs can be cleared and stained tn tolo with methjlene bloc etc.

Another consideration which detenmned the selection of the open ments was their analjdical \'alue that is, their expedieniy In illustrating

In Urt Appendk tlw CTpcrfmentj ire imnted in rtwp* icconCn* to the tcdudcil dif ficulila In ohed


Important principles of morphogenesis. The theoretical significance of the expenments has been strongl> emphasued I believe that a course In an experimental branch of biology not only should acquaint the student with new facts but should strengthen his power of reasoning and his logical acuitj as well He should be aware of what an experiment proves and of what It does not prove. Each openment or group of experiments is preceded by a brief outline of its theoretical implications. These general re marks Integrate the different problems handled in the Manual, but they are not to be considered as a substitute for lectures or for textbooks. On the contrary it is hoped that they will stimulate collateral reading The bibliographies serve the same purpose. They are not complete but gi\x references only to those articles which are directly related to the experl ment under consideration and to pertinent review articles. A special chapter on gastrulation In amphibians was added because familiarity with this phase of development is indispensable for experimental work on early embryos

The expenmenti arc organised according to a logical plan This is not necessarily the sequence in which they should be taken up m the labora tor> The planning of the course work wQl depend largely on the avt 3 abQity of living material To facffitate a flexible schedule each experiment has been treated as a separate unit The technical procedures are de senbed for each expenmeot separately they are not based on previous cjqpcnence with other experiments. It is immaterial whether one starts with amphibians, with regeneration m plananans or with chick expen raents The selection and arrangement of the exercises is left to the discretion of the instructor A tentative schedule for a one-scraester course wni be found m the Appendix

The instructions for technical manipulations arc gi>en m great detail I hope that they will be useful not only for students but also for research workers in biologj expenmenlal medicine, and related fields who may find one or another of the technfqaes applicable to their own problems The technical procedures have worked satiafactoril> In our course which does not mean that they cannot be further improved I hope that the students wdD feel encouraged to dci-dcp their own initially e and resource fulness m trjmg out new cipenmenU and m impro\^ng the techniques and instruments The highly spedaiiied technique of tissue culture has been omitted The role of endoennes in moiphogcncsis is adequately presented in A E Adams Studies tn Esperimenial Zadlogy (1941) which contains all In/or mation neccssar> for expenmcnlation in this field. Experiments on ma nne onimaJ eggs arc dealt with in Just (1939)


I am Indebted to many friends and coIIeagTiea Manj techniques described in the chapter on amphibians ha\T been worked out m the labors tory of Dr H. Spemann (F rtlburg, Germanj ) with whom I was assoa ated for many j'cars. Dr B H- W'dlicrand Dr Mar> Rawles generous!} made available thar experiences with operations on the chick embrvo I am much obliged to Drs L G Barth G Frankhauser T S Hall J A Moore and C Pannentcr for personal communication of technical proce dures. Grateful acknowledgment is made to all students and assistants who helped materially to improve the teclmiqucs and to re^’lse the out lines Dr K- Ga}'cr kindl} read the manuscnpt and made man} helpful suggestions.

I am grateful to Dr R. G Hamson for his kind permission to publish sketches of his unpublished stage senes of Ambysioma maculclum These sketches, as wcD as all other ongmal drawings were made b} Miss S E. Schweidi Acknowledgment is made to the Wlstar Institute Edwards Brothers, Incorporated Akadcmische Vciiags-GeseDschaft and Sprmg er 8 Verlag for permission to reproduce illustrations


Part I Equipment Axd Instruments

A. OPTICAL OUTFIT

A low power binocular microscope Is mdispcnsable for experimental embryological and regeneration studies Any standard model is accept able The V base with substage mirror is not necessary for most experiments on the contrary, it is preferable to have the glass stage for the operation dish directly on the workmg table so that the arms of the operator have optimal support Magnifications ranging between X6 and X24 are suffiaent for most purposes For illumination anj lamp which IS mounted on an adjustable support, which has a strong light source and which gives an evenly illuminated field may be used. Precautions must be taken against heating of the operation duh In our laboratoiv makeshift lamps are m use these consist of a 75 watt bulb mounted made a tin can and a 500-cc. Pjtcx boQing flask filled with water which serves as a condenser and cooler both items are clamped on an ordinary iron support A Beebe bmocular magnifying glass which is worn HLe a pair of spectacles has been found to be extreme!) useful on man> occasions for instance, m preparing glass needles selecting amphibian cmbrj’os, or m hypophysectom) of frogs,

B GLASS INSTRUMENT?

(Instruments 3-8 were designed b) Spemann)

Malerud

glass tubing 6-7 mm. m outer diameter glass tubing 9-10 mm in outer diameter rod 5-6 mm, in outer diaineter rubber tubing 11-12 mm m outer diameter rubber tubing 8-9 mm, m outer diameter rubber caps for p^tettes cover glass Bunsen burner iron support file

dansond penal*

U#e iotl glass t hroog hont do not ase Pyrei,

Id orfogtliedlamood penal, »aat£h eeJrhJfwnj troaiid Ua rod crtnbesnd tlien brtni U* ptecei spirt by gentle prtisart. Do not nmk arorol tte entire aiannference.


3


I Pipfites — U*e both 6-7 nun and ^lo-mm glass tubing Cut pieces of about 8 inches (for 2 pipettes) Heat the middle of the piece over the Bunsen bonier roll the piece constantly between your fingers to avoid one-sided melting When the glass is softened pull slowly, holding your hands horizontally until the desired diameter Is reached Wide-mouthed and gradually tapenng pipettes may best be made over a burner with wing top which gives a broad flame. Cut the pipettes to the desired length with the diamond pencil Hold both openings in the flame to smooth the edges Fit rubber caps (from medicine droppers) over the wide end Prepare pipettes of different widths nmging between 2 and 5 mm Prepare sev'eral of each kind

j Capillary plpeiles — To make capillary pipettes use the foDowing procedure Pullout a pipette with as narrow an opcmng as possible Cut it off a considerable distance from the tapenng region and bend the narrow end mto a hook bj holding It in the flame Hook the pipette over a ring on an iron support placed near the edge of the table with the pqiette suspended o\*er the edge so it will not drop on the table when heated (Fig i, h) Heat an area near the tapering part very gently and cautiously with a low Bunsen burner flame or a microbumer When properly heated, the tube will be drawn out mto a very fine capDlary tube of almost microscopic dimension by the weight of the lower wide part If too much heat 15 applied the tube will be pulled apart and drop Even so, It can be saved by placing a container with a layer of cotton on the bottom at a pomt where the pipette will hit the floor Cut off the fine end with a diamond pendL

3 Mtcroburner —The microburocr is a small gas burner used for the preparation of \’ciy fine glass needles and other instruments. It is best made of an ordinary mjection needle whose pointed end is cut off and whose wide end is fastened m rubber tubmg connecting with the gas jet It IS mounted m a honrontai position b> clampmg it on a support or otherwise The optimal length of the flame is s mm or less. The size of the flame may be controlled by a clamp on the rubber tubmg or dmcctly bj the gas jet,

Microbumer* of glass ma> be easily made of 6-7-mm tubmg Prepare a capillar} tube accordmg to section 2 and cut off the narrowest part with the diamond peadL If the opcmng is too small, the flame will seal it off b> raeltmg the edge The flame must be of blue color a \-clJow flame indicates the melting of the ed^ Control the gas supply with a clomp

4 iftavpiP<Uc(,afUrSpcmann ) — ^Thisls a ralcroprpcttc with a lateral hole (Fig I m) which is used in transplantation ezpcnmenls for trans

4



femng small objects. Pull out a fine pipette, about i-a mm in nanwest diameter using 7-S-mra glass tubing Make the handle at least 10-12 cm. long To blow a hole in the handle proceed as follows seal the capfl larj end of the pipette by holdmg it m the flame Attach a piece of rubber tubing to the wide end of the pipette. Bring one side of the wall of the handle near the pomt where it tapers very near the flime of a Bunsen bumerandheatitthoroughlyuntilitxssoft. Takcltoutof theflameand, while It IS hot blow air through the rubber tubmg The air cannot escape through the sealed capillary and will blow out the soft glass m the form of a large sausage-shaped, thin waDed bubble. The bubble usually ex plodes instantly, or it can be easily removed Scratch the arcumferrace of the hole with a diamond pencil and break off carefully all particles up to this mark Smooth the edge of the hole over the flame Cut off the closed capillary be sure to make a stratgJd cut It is practical to have the capDlaiy opening slightly bent Hold it honxontal]> over the flame of the rmcrobumer in such a way that the hole faces you and the capillary end points to junr left Gently heat the lower wall of the capillary near its opening The end piece will then bend b} its own weight With a forceps bend it farther, until it is at an angle of about i2(f to the straight part Next cover the hole with a rubber membrane Cut a piece of rubber tubing which wili co\'er the lateral bole, moisten the p^tte and the rubber piece and slip the latter over the hole. It must fit very tightly Fit a rubber cap over the wide end

Use of Ike mtcroptpeile — Place your right thumb'On the rubber mem branc over the bole. Draw m water using the distal cap until the narrow end is filled almost to the level of the hole ^VhiIe the opening is under water, squirt out a small amount of water by gentle pressure on the mem branc over the hole Retain the pressure and under the bmocular microscope place the openmg of the pipette o\’cr the object Then release the pressure slowly The object will be drawn into the capDlsiy portion and can be transferred. A slight pressure on the membrane suffices to press it out again

A good ralcropipctte must hold water in the capDlaiy end I e. no water must dnp out when ft is held with the narrow opening pointing downward

S Giass needles — These needles (Ftg t o) are the universal cutting in struments for extirpation and transplantation experiments on amphibian and chick embrjos Thej are best prepared in two parts First, make 6-10 handles using a glass rod 5-6 mm in duuneter Cut pieces about 20 cm long (for 2 handles) Draw them out In the flame of a Bunsen burner break the a handles apart, and pull each a second time on the microburner to obtain a fine pomt near the tapenng end of the handle Next pull out a piece of glass rod to an extreincl> fine elastic thread much thinner than a hair To do this heat a pomt and pull the enda apart very quickly Prepare a number of such threads of slightly different thick ness Break off a short piece of thread and hold it with youi left hand Take the handle m your right hand and bring one end of the thread to the pointed end of the handle, making contact at an angle of about 120® In this position move them slowly toward the microbumer (Fig r, n) At the moment when they are heated and fused either pull them apart with a sudden jerk or mo\'e them apart slowly The thread will thus be spun out to an exceedingly fine hair Under the bmocular microscope dip the end portion near the handle with a watchmaker forceps It should have a pomted end and should be quite elastic, A Beebe bmocular magnifier IS of great service m preparing needles^

Prepare 6-10 needles to have a supply m case one breaks during an operation. They must be tned out on the object. Most difficulties m operating result from madequate instruments needles ma> be too thick ortoothm too long or too short too clastic or not elastic enough. Differ ent stages or different types of eggs require slightly different needles,

6 Ear loop — ^The hair loop (Fig i c) is used for handling hving em brj’os or embryonic tissue Prepare a number of fine capfilaiy tubes as m section 2 Cut their ends off with the diamond penciL These are the handles They should be about 12 cm long Obtam very fine soft human hair That of babies is best suited the hair of most adults is too coarse. Cut pieces about 3 cm long Hold the handle m >our left hand and pick up a piece of hair with a watchmaker forceps which is held m jour right hand Under the binocular microscope work first one end of the hair and then the other into the capffiaiy opening of the handle This opening should not be much wider than the diameter of the two hair ends. Push one end deeper Into the capUlar} until the loop has the desired length 3-4 mm The hair must then be scaled into the capillary hIcU a small piece of paraffin on a glass plate (slide) and dip the hair loop bto it A small amount of liquid paraffin will be sucked mto the end of the tube b> capiUarj force and will harden instant]} Soraetunes a film of paraffin wUl remain m the hair loop To remo>c it warm a piece of filter paper on a slide and touch the hair to the wann paper a\ oid melting the pamflin in the handle Prepare a stock of hair loops of different sires

7 Ghss rods vnlh ball tips —Glass rods with ball tips (Fig i b) are used for making gnxnes in the Permoplast or paraffin bottom of opera tion dishes. Pull out a glass rod 5-6 mm in diameter break its slender part near the handle and hold the point downward in the flame of a


7


microbnraer (sec 3) It will melt and form a ball The ball should not ha^ c a narrow neck because it will break easQy Prepare balls of different sixes ^

8 Glass bridges — Glass bridges (Fig 1, d,f) tut used in transpUnta tions of amphibian embryos to hold the transplant m position until it is healed in With diamond pencil and ruler cut a cover glass into wnnT1 5tnp< 3-4 mm wide. Cut each str^mto small rectangular pieces lo-i* mm long Take up each piece with a watchmaker forceps and pull its four edges slowly through the microbumer so that all sharp and rough projections disappear All edges must be absolutely smooth. Gra^ e ach glass piece on one narrow end and hold it m a slanted position over the microbumer m such a way that an area a short distance from the other end Is heated from below The end opposite to the forceps will then bend down by its own weight The glass bridge, when finished, must stand firmly on its narrow edges

The sue and angle of the glass bndge must be adapted to the material The glass bridge can be easfl) cleaned and sterilixed b> pulling it quickly through a Bunsen burner Prepare 6-8 bndges of slightly different angles and lengths

C METAL INSTRUMENTS

1 Scalpds, scissors, and forceps — Scalpels sossors and forceps of dif ferent sizes will be needed for many manipulations Stamleavstccl mstru meats are preferable Chromium plated mstruments are not recom mended because particles of the plating arc liable to chip off when one tries to resharpen the Instruments.

2 ]} akhmaier forceps — ^Thesc forceps (Fig i 1 k) arc universal tools and mdispensable for work on embo'o** They must have very fine points and must work \ cry smoothly that is they must respond to the slightest pressure Instruments made of high grade steel (stainless if possible) should be selected Two tj'pcs of twc ei ers arc on the market — one with graduall} tapenng ends and one with shoulders The latter usually ba\'e finer points. Howe\*er anj tweezers must be sharpened before use and the tips ground to the finest pomts Twe eze rs ma> be purchased from an> wholesale I'-itchmakcr or jeweler tool company or through Clay Adams Compan> (44 East Twentj third Street New "iork Cit>) The

  • Clan Bffdlc*, rodi * t in trp*, ami hair may be bat mocnttdflP i gpoJf

a pifce of ioIkI t» ood, about «iX7X3cin «iUi l»o ro« â€¢ of bota ilithUjr laizrr than ibe diamrtcrt of tbe baodlcs and bored a( a dotaoceofsboat 3 aa frocn ooe aaotber (Fie- 1 left oppCT comtT)


best mstniments are mipK>rted from Switxerland (Dumont fils Arrow Roj'al) Thej are supenor to the mutation mates howev'cr the latter are entirely satisfactory for classroom experiments Skarptning is best done on an oilstone or most efficientl> with a high speed hand tool with replaceable cmeij wheels Rust is best removed with a paper towel and kitchen deanser

3 indtdemy scissors — ^Thesc scissors the finest on the market, are used bj manj invcstigatora for operations on amphibian and chick cm br} os. For certam types of operations thej are preferable to glass needles — they are more durable and the> cut m dean straight Imes, However their high price prohibits their use as instruments for the classroom and all operations described m this Manual were de\'ised for the glass-needle techmquc Three types of mdcctomj scissors are on the market m the De We^cr sossora and the McClure sassors the blades are dosed by pressure on both handles in the third t>'pe (Fig i g) one blade is a direct contmuation of the long handle and is fixed m position the cutting bemg done by pressure on the veij short handle of the other (mo\’abIe) blade All three t)*pe3 ma> be obtamed through Cla> Adams Compau) and are illustrated m Its catalogue Tbc> are equally usable as fax as our ex penence goes

4. Ins knrts — These knn'es are the finest steel kni\ts that are on the market They are required for delicate operations b which the smallest ofdmarj scalpels pto\*e to be too coarse A number of ins and cataract knives of different tjpes arc m use m ophthalmolog> We prefer the single-edged straight Elnapp ms needle (Fig i /) with a half spear pomt. For use in the dassroom we advise preparing fine knives b} grmdmg and sharpening ordinary dissecting needles or sewing needles The latter can be easily mounted m a piece of glass tubmg by heating one end of the tnhmg and mtrodudng mto it the cjt end of the needle

5 The Planana knife (after R. Stlber) — The ‘planana knife (Fig i e) has been found to be >'615 useful m cutting experiments on plananans It consists of a piece of raror blade mounted on a handle It is prepared m the following way break a double-edged razor blade in two and break off small pieces of the sharp edge Clean them thoroughly and re move all grease with acetone or alcohoL Cut pieces of 7 mm, glass tubing 10—13 cm long heat one end until it is very soft and press it against a metal plate at an angle of 45® Qcan this oblique surface carefully with acetone Glue a chip of razor blade on this surface with a high grade bquid waterproof glue (e g DuPont waterproof transparent cement) Allow the sharp edge to project a few nuUrmeters bevond the handle


9


elaborate precautions. We suggest the foUowmg simple device for their sterilization Slip a penefl dip over the handle of the hair loop or glass needle and fasten It on the edge of a bcaher or wide mouthed specimen bottle, with the needle or loop dipping into 70 per cent alcohol

BIBUOGRAPHY (A-E)

^lAKCOin 0 1919. EotnicUungsmedonlkdcrTIeTe. InT PfrEKn, Metbodik dcr wmcttschaftlicbeo Btologie, II 679 Berim Spriojer RuQa,R. 1941 Ezpenmental embo'oloey (a maimiJ of tfclmlqiKs and pmceduits) hew York New York tJmrmlly Bookitore.

SoTOTTf, 0 1930 Tnss{^uUtk>im'erfQchc liber die Deteimhution der Orpm An lagen von Anarenkeimen. Arch f Entw^medi., las 179 SiLBEK, R. H., and HAUBuaoeK, Vrcroiu 1939. The production of dupUcitaa cruoata and muUIpIe beads by rtfcnertiioa In EMpUiwia fiinna FbyaioL Z06L, la 185 SmtAJTj H. 1933 hflcrodiirurBische OpOTtionstechnik. In E. AannoiALDiH Handbuch der bioL Arbeiumetboden Part V No 3A.

SruLTZ A. 1935 Devices for experhnenti on ainpbibian embryos. Anat Rec, Snppl, 64:43


Part II Experiments on Amphibian Embryos

A. LnrtNG MATERIAL


1 AilPHIBIANS COilMOXLY USED FOR EXPERIMENTAL â– ^ORR AND THEIR BREEDING HABITS

Most expenmental work on amphibian cmbrj*os is being done on a few common iiati\’C forms namelj, 3 spcacs of salamanders {Amirysloma) 3 newts {Tnturui one of them Tr pyrrkogaster is imported from Japan) and 4 frog speaea Only these forms will be bneflj described m the fol lowing paragraphs. Other more rare forms which ina> be abundant locally, would, m all probabihty 8cr\'e manj erpenmental purposes as well However one can by no means suppose a pnon that two even closelj related forms will behave alike In a given experiment. The differences which were found between related anurans with respect to lens mduction (p 107) and between different urodeles with respect to Wolffian lens re generation (p 176) ma> sen'e as a warning To avoid expenmental fail ures due to the choice of unfit material we have mdicated for each ex penment which species are recommended Those listed are without ex cation the forms used either bj theo^gmalm^•estJgatoro^b} the author in classroom expenments Most of the data for the eastern forms are taken from the monographs of Bishop (1941) for Uredde and of Wnght (1914) for Anuta those for the western forms from Storer (1925) and Twitt> (1935) These publica tions also contain extensive blbbographies Students mterested m the hfe histories and habits of their expenmental animals should consult these books and also Wnght and W right {1933) and Noble (1931) For questioas of taxonomy see Bishop (1941) Cope (1889) Noble (1931) Ste}'neger and Barbour (1933) Storer (1925) Kc>-s for the identification of eggs end lar^’ae of eastern urodeles maj be found in Bishop (1941) Ke^-s for anuran eggs arc to be found m Wnght and Wnght (1924) for tadpoles in ^^nght (1929) Kej-s for the eggs Ur\*ac and adults of western «pecics ere gi\Tn in Storer (1925)

oaoEK Urodfla

AVBASTOUIDU:

I Ambyiloma maculaium (Shaw) frequent^ referred to os A puncta Spotted salamander A^'e^agc sue 170 mm Color deep bluish

TbcfpelliQj Ainb)->tonia’’bisb«n«kipt«dmUia J/onro/ (oIlomxr>jlt«nilrtof rotnrn claturt Mott rqinimrau] onbr^oIotrtU u«« tb bofcil f^tlLr^ Tbrrt


black, with two irregular rows of rounded j*ellow spots on the back Vcn tral side lighter The most common Widel> used for ejpen

mcnts

Breeding pjacts Woodland ponds and alowI> running streams,

Bncdtng season Vanes considerablj with latitude from January (in southern regions) to Maj (in northern regions) The breedmg season m a given locaht} lasts for se\xral weeks The animals spend the rest of the j-ear m burrows or under stones and logs on land

Egg masses 100-150 eggs, each m its own jellj membrane are held together in a common jelly envelope, which is firm and globular In shapeThey arc attached to sticks or fioat freely, within 8-10 inches from the surface,

Range ‘Nova Scotia w to Wisconsin and s. to Georgia, and Texas" (Bishop 1941 p 130)

2 A mhysloma tignnum (Green) Tiger salamander This is the largest native Amhysloma, averaging 200 mm Deep-brown to black on dorsal side ohve-}’cllow on \’entral aide Markings dorsall) and laterally are Ir regular oUve brown to brownish jtUow blotches much duller and more irregular than m A maailaium

Breeding pieces Permanent or temporary ponds

Breeding season In all regions shghUy earlier than .4 maculatum Dur ing the rest of the >‘ear the animals are on land, hidden under stones, logs etc

Egg masses Contam only 30-50 eggs the common jelly cn>’clope is softer less firm than in A maculalum They ere usually fastened to twigs and branches 12 inches or more under the surface

Range ‘ From Long Island s to m Flonda w to Mississippi and Ar kansasandn to Minnesota and Ontnno (Bishop 1941 p 173)

3 Ambysioma opacum (Gra\’cnhotst) Marbled salamander The smallest of the three about 100 mm Color black with light markings on the back and on the aides Their color is dull graj in the 9 , and bright In the d* very ^'a^able in size and shape.

Breeding piaees This form docs not lay its eggs in the water but hides them in shallow groo\'C3 under lca\-cs or under logs in woods. The breed Ing places arc on the margin of temporary ponds or swnmp\ places which arc dry at the breeding season but which will be submerged later in the


cmn be no doubt lint tl>e fatter b liDctifatlraDy comrt, loeaturf blunted moult,"

Ihe eoe vhkb eo»oj» pnorilj U raotunclm tod u prolnWy t typoftrapiwal error I tSTW toJIjr imh Dr lUmroo t charteteruabon of thU ritalkB (m Joor Erper ZoOJ 4>tJ5r )


t6


year Many females migrate to the same place so that one can nsuallj collect from man} nests m the same locationBrteding season September and October

Egg masses The eggs are laid singl} , not held together b) a common en\-elope- Particles usually stick to the surface of the eggs wherebj the eggs are well concealed- The membranes will swell and the larvae will hatch when the eggs are placed m water

Range Trom Massachusetts to Georgia w to Louisiana and Texas ilississippiBasmn-toArkansas Missoon Indiana and mmols’ (Bishop 1941 p 154)


TAAItLT SUjUlAM>lirtAZ

4- Tnlurus vtndescens Rafinesque Common newt A\Trage size S5 mm Color m the aquatic form dorsal aide ob\'C-green \Tntral side light to bnght ydlow Small black spots scattered O’er both sides A senes of black ^>ots on either side of the dorsal midline The land form called eft is bright red- During the breeding season the males can be easil} recognized by their broad, wa^y tail fins and by the presence of black bars on the ventral surface of the hmd legs Breeding places Ponds lakes and slowl} moviDg waters Breeding season \ actable with latitude April to June and more ex tended than m frogs and Amhystoma Eggs are deposited singl> on leaves of VaOisnersa Elodea and other aquatic plants. The female grasps a leaf with her hmd legs deposits the egg on It and then folds the leaf so that the egg is almost entircli con cealed The outermost egg capsule is sticky milk} white.

Females will la} eggs readfl} m the laborator} Thq should be kept m large aquana, and plent} of fresh Elodea or VaUisnena or narrow ahpa of paper should be pro\ided To insure fertilization a number of males should be added The} will court and clasp the females and de pKJBt their q^ennatophores m the aqusnum.

Range V Ontario s to Georgia w to Alabama n- to northern Hh nois and Wisconsin (Bishop 1941 p 76)

5 Tnlurvs torosus (Rathke) Pacific Coast newt A\-erBge size 200 mm andmore Dark brown on the upper side orange (oryeDow) on thei'entral side Surface rough in terrestrial iodn'iduals smoother m aquatic mdi viduals Common

Breeding places Creeks and ponds

Breeding season \ anes with latitude Januarj and Februaiy in low altitudes until carl} summer m high altitudes

Egg masses 10-25 are laid sunultaneousi} the} stick together


17


and form a firm dump but have no common envelope They are attached to water plants

Ran^e All California, from San Diego to Alaska

Twitty has discovered two new species of Tnturm in California Tr stmulans and Tr rrvulcns Both have been used for expenments (Twitty

1935. >936)

oaoEs Anura

rAim.TftAxmA£

6 Rana fnptens Schreber Leopard frog Average length 80-90 mm Dorsal side with rounded or oval dark spots rather irregularly ^ced Smaller spots on the sides Ventral side whitish or yellowish The males can be easily recognised by the swellings at the thumbs The commonest and most widdy used frog

Breeding places In ponds and swampy marshlands.

Breeding season Early qDrmg (March and April) The adults spend the summer on land and Mbemalc in the water, hidden beneath stones or logs

Bgg masics Contain approximately $ 000 eggs. The jeDy membranes of the mdividual eggs stick together but they are not inclo^ in a com mon envelope. They ore laid near the surface usually a considerable num ber of masses are deposited at the same place

Range Most parts of the United States, except the Pacific Coast.

7 Rana paluilns LeConte The pickerel frog Average siae 70 mm smaller than i? pipiens Upper aide pale brownish with dark spots these are brger and more reguUrly arrang^ than those of R pipiens namcl> , m two distinct rows They are oblong or square Undeitide j-ellowish white R paJuslns can be casfly distinguished from R. pipiens bj the bright yellow color of the underside of the thighs Males have thickened thumbs.

Breeding places Cold springs and streams, ravines ponds

Breeding season Late April and Maj The adults spend the summer in marth> places ra\'ines moss bogs etc. and tibemate In water co\eied b} logs and stones

Egg masses Contain about 3 000 eggs Thej arc laid m shallow water and are usuallj attached to sticks The masses are much like those of R pipiens but the eggs can be easfly recognized b} the brown animal pole and the >*eIlow \Tgetal pole m contrast to the black and white of i? ^ piens

Range N to Canada w to Great Plains s to Louisiana and Florida

8 Rana syi'^tica LeContc. Wood frog The smallest of the common

18


frogs, 65 mm Upper side, gray to brownish with dark streaks on both sides of the head and a few scattered dark spots. Lower side whitish Males have swollen thumbs

Breeding places Still waters ponds, transient pools m woods. Breeding season Slightly earlier than the other common frogs, late March and April as soon as the ice has left The frogs spend the summer in the woods and hibernate on land, in woods under co\’er

Egg masses Contam about 2 000 eggs. Thej are similar to those of it piptens but more globular and mdividual eggs less crowded 1 e , outer jeDy membranes thicker Most frequently attached to water plants twigs etc.

Range N to IMaine s to North Carolina w to Missouri 9 Rana caleshetana Shaw Bullfrog The largest of all native frogs up to 17-20 cm Upper aide brown ventral ade, wMte. hlalcs have thick ened thumbs

Breeding places Lakes ponds brooks marshy swamps, streams. The adults stay m the water throughout the year The tadpoles spend two j'cara in the larv'al stage

Breeding season Later than most other frogs June and July m north cm parts somewhat earlier In southern parts Egg masses Contam more eggs than those of other frogs 10 000 and more bemg reported They are laid as a surface film The jelly is loose and gclatmous and less compact than that of the other common frogs. The egg masses are deposited among brush at the edge of the ponds Range Eastern stales to the Rocky Mountains BIBLlOCRArm

Buiiop S C 1941 The nUmsoden of New lort New \oTi. Sute ilta, BoIL 3J4 AHani Um\mity of the State of New "yoTk 1943 Handbook of laUmandeis Ithaca, N \ Conotock Pnb Oj

Core E D 1SS9 Tbc of borth Amena U,S Nat, Aim. BuD S 4

Nobu:,G K 1931 TbebWogroftbe/fBi^AxAM NewAork McGraw HOL SiLi'SLcn L and Basbovs, T 1933 A check Oat of horth American amphibians and rcplilo. 3d ed Carabndge Harv-ard Unis-mity Treo.

Sioan T I 1915 A sjTWpsls of the amphlbu of California Calif Unlv Pub in Zoul *7 1

TMtTY \ c 1933 Two new ipeoes of Truant from California. Copela No i JqI> 16

1936 Correlated and etnbiyologicalexpenmenti on Tri/anu I Jour

Eiper Tool 741339

Waicirr A H 1914 Life hutonesof thevlanraof lihaca NewAork. Pub Caraejfte Inst U a. hmyilon \o 197

1939 and desmpiKm of North American Udpoles, Proc. U.S Nat

Mus 74 1


Uricht a. IL, and WtiOHT A- A- 1924. Akey totheeggjofthe^a/ifli/iaeutofthe SrUimJppi Rhtr Amcr Ivat 58 375,

1933 Handbook of frogj and toads. Ithaca, NY Comstock,

2 STAGE SERIES RATES OF DEVELOPMENT AND OTHER DATA Forpractical purposes It 13 desirable to breakup the continuous process of de%*cIopmcnt into discrete “stages ’ and to agree on standardised “stage senes ' for con\'enient reference in descriptive and experimental work. The stages should be characterued by cas3j identifiable external features In cold blooded animals stage senations in terms of age are useless be cause the rate of development vanes with temperature It should be un

TABLE I

TmrTABlXrOftA moadatMm (At Room Tetopentaie, Approx, to C)


Suou


D

D

B

B

fl

D

n

n ,

B

m

fl

n j

TImi 1

B

B

B

B

g


B

1 ..|

1 3 1

fl

g


la iMcn (r«ai n c«a,bd«7i


$Tua


mjRy

m

D

D

B

fl


m


EH


Time

M •

9

1 ’’ 1

fl


liS-jpi

Ll^


0

..-Mj

>s


derstood that, in the stage senes listed below the time intervTils between any two successive stages ore not constant but differ from stage to stage The senes for different forms have been worked out b> different authors, and their initials will be added to the stage numbers. This practice will be follors-ed throughout this Afanua/

ORDER Urodria

I Ambyitoma macidatum — Harrison s excellent senes for this widely used salamander is the most complete and most perfect stage scries desnsed so far and should be used as a model for any other urodele scries. It is not published, but it is widcl> cumulated through the courtes) of the author and general!} adopted bj experimental efflbr>*ologi3ts. It co^*cr3 with 46 stages the penod from the uncleaNxd egg to the stage m which the lar\-a begins to feed The stages are depleted in Figure 45


30






Rate of dcoelopment —This rate vanes with temperature and other ei temal factors Table i is compded tor practical purposes The data are combmed from Dempster (1933) J iloore (1939) and mj own notes all three of which are in close agreement

( r r n rs DATA OX XOtiiAL DEIXLOPVZST

Rupture of nldline membrane Late ncnnila stage Ftnl maoemenli Stages H33-H34 (for details see p 124)

Ftnt heartbeat £[34.

Hatching — 15-29 days

Beginning of feeding H45-H46 — 25 days

Metamorphons Vanes considerably with feeding etc from 70 daja (at TnflTTTrml feeding Twitty and Schwind 1931) to 120 daj's or more Grtncih curves May be foimd m Hamson (1929) Stone (1930) Twittj and Schwind (1931) Dempster (1933) Moore (1939) At metamorphosis the nnimnls have attained a length of 48-55 mm

Volumetric and dry-vceight measurements See m Dempwter (1933) Density measurements See in Brown Hamburger and Schmitt (1941) Temperature tolerance From 3 5® to 23 C (Moore 19406)

2 Amhysioma tignnum — No stage senes for this form has been worked out The H-stages for A maculaSum can be applied roughl> to this form if one uses gill and tail development for identihcation but disregards limb development. The forelimb buds which appear in A moculatum m Stages H36-H37 appear m A tignnum \try belated!) namelj m the feeding stage which corresponds to H46 for A macvlatum Another profound difference between the two forms is found in their grauth rates Adults of A tignnum are of appromnately double the sue of A moculatum and this difference is reflected in the higher growth rale (merement m length per time umt) of A tignnum from eari) stages on Growth cun-es for A are to be found m Hamson (1929) Stone (1930) Twitt) and

Schwind (1931) Moore (1939) As a result of the higher growth rate mnnmall) fed larvae of .d tignnum are about loo-i ir» mm longatmeta morphosis as compared to 48-55 mm for A moculatum Both forms if fed manmaflj metamorphose at about the same time (appronmatel) 76 daj’s after fertiliiation according to Twitt) and Schwind 1931) Under less fa\’orable conditions the time of metamoiphosis of A tignnum is ei tremelj \’anable It ma> be dcla>'ed up to 17 months (Hamson 1929) Stone (1930) gi\-es 130 daj's as an a\-erage The rapid rate of de\*cIopment of A itgnnum is expressed m the

Da STuoe tad Uutchhmo (1941 p *47) lodiale that m aH probabditj saattfc ddlc mcnm dc%ckipmaiU] rata eiirt brt»e«n eaatera and middle matm racta.


21


greater speed with which a given H stage is reached, as compared to A maculalum According to Moore (1939) and ray own limited data, the rates of development compare approximately as shown m Table 2

Uaichng tales place m approximatel> the same stage as in maculalum, which 13 the equivalent of H40-H43 (13-14 days) However the fore limbs, at that stage are barely visible buds they are comparable to those oia^TioxA maculalum

3 Ambytloma opacun — No stage senes has been worked out Growth procccdsmoreslowly even than in maculatum Growth curves m Twit t> and Elliott (1934 p 284) and Moore (1939)

Density measurements Sec in Brown Hamburger, and Schmitt (1941) and Brown (1942)


TABLE 2

Da\s Required To Reach a Given H Stage (At Approx, >0 C )


StJtoa


HI

D

m

11

^59

Ttit

A llfriam

â– 1

—



15 16

A 0 »aeidstMm

t

H



»5


4 Tnlurus —No stage senes have been published for the Amcncan ^icaes A stage series of Tr pyrrhogasler was published by C)yama (1930) several stages are described in Yamada (1939) Concemmg European newts the senes of GUlsncr (1925) for Tr vulgaris (laenialus) is not satis* factor> because the stages arc too for apart and the schematic drawings often make a diagnosis difficult Sato (1933) gives a dearly defined and well illustrated scriation of the tail bud stages of Tr vulgaris, which are numbered In conformity with Harrison s stages (1121-1132) GlQcksohn (1931) has worked out complete scnations for the larval penodsof Tr vnl garis and Tr enstalus the most commonly used European newts Her scriation begins with a stage corre^ndmg to H36 and ends with mela morphosis (Stage 62 for both forms) As entena for identification the development of fordlmbs and hmd limbs and in particular the relative sires of toes arc used The numbering b adapted to the H scries A sena tion for another widely used European newt Tr alpeslns was worked out b) Knight (1938)


IS




osDCE Anura


5 Rana piptens — stage senation has been published by Shumwa^ (1940) (see Fig 43) It covtn the phase from fertilization to the j oung tadpole in 'which the gills are just overgrown bj the operculum and the animal has not fed yet Twenty five stages are distinguished- They are convemetitl> tabulated, together with average bodj length and age (at 18® C ) This scnation is alrcad> wideJj adopted and will be referred to in the text as Shi-Shas

RaU of df?tlopmcHt —The studies of Atlas (1935) and of hloore (1939) gi\'e full information on this point (see also Shumwaj 1940) In iloore s paper the rate of de^'elopment (in hours) in terms of Sh-stages is gi\*en for four difiercnt temperatures (15 3® 18 ^ 2^ C ) The iohow

mg table includes Moore s figures for room temperature 19 8® C


TABLE i

RATnof DEVixorMtKTroRFoca ^rciis cr (Time In Hotm liter Fiat Cle»Tige at ig S* CX, from Moore 1939)


emnt D^TA 031 itosuAL DotLOTjoarr First meremenU Shi 8 First keariiKat Ship

ffalchng Shi8 (Moore 1940) Shao (Shum'wa> 1940)

Spontaneous swimmiMg Shai Feeding Sha5

ifeiatnorphoits in the field Julj weeks after egg la\'mg

(U right 1914)

Temperature tolaance C (Moore 1939 1942)

Density measurements See m Brown Hamburger and Schmitt (1941) 6 Rana syl-^tica —In the stage ^nes for this form b> Polhster and ^^oo^e (1937) the period from the unclca\-ed egg to the begmnlng of the


  • 3


overgroirth of the operculum is divided into 23 stages comparable to Shi-Sh23 for R. ptpierts ^ They wOl be designated as PM1-PM23 See Figure 44

RaU of devciopment — It 15 considerably fester than that of R^ ptptau Sec Table 3

Onm DATA ON NOUIAL DnXLOnfCTT

Ftrsl moUliiy PM 1 8 First kearibeai PM19 Hatching PM20-PM2 1 Spantanemis runmmtng PM23

Mdamorphosu tn ike field Early in July that 13 14-16 weeks after egg lajmg (Wnght 1914)

Temperature tolerance The eggs of this frog which is the earliest breed er (middle of March) can tolerate a lower temperature than others but thej ore less resistant to higher temperatures The range of tolerance is from 2 5° to 24® C (^foorc, 1939 1942)

7 Rana palusiris — No stage senes is available Rate of development — ^At 19.8® C sec Table 3 For rates for other ternperatures see Moore (1939) The development is somewhat slower than that of R piptens Hatching Stage PM17-PM18

Uetamorpkosu in the field In August^ that Is 14-17 weeks after egg laying (Wright 1914)

Temperature tolerance Frora7®-3cf*C (Moore 1939,1942)

8 Rana caJesbaana — No stage senes is available- The eggs ore laW late m the season and metamorphosis takes place two years later

Rate of de^lopmenl See Tabic 3

Hatching Stages PM17-PM18

Temperature tolerance 15®-32®C (Moore, 1942)

BIBLIOGRAPHY

Attas II 1Q3S The dTect of tanperature on the dntlopricnt of Rana pipieus Pb^aiol 2^ool 8 si/x

Brsiiop S C 1941 The nlim«t>der» of New York- hen York SUte Jin*- BuH 3J4 Albinj Umvenity of Ibe Stale of hew York Dkonr kl G 1941 An tdtplatioQ In ifeiSyt/mo 0;tecim embr>os to de%'elopnxiit

on land Ainer 76 jjj

RtonK M G ILunnnoc* \ and Scmnrr F O 1941 Denilt) itadles on am phibuQ embrj o* m itb fpcdal reference to the raechanbni of orginlxer actloa- Jour rjcpcr Zool 88 353

Sbummtjr't ftarr* «crc adapted to tboae of PoDuter and Moore «hkh acre poWabed

lint


U


DzatPSTDt, W T 1933 Gnrath b ^wWjnfofna during the embryonic and

enly Urvnl pencxL Jonr Eiper ZooL, 64 495 DuShaot, G P and Htrraiixsox C iw* Tbc effect of temperature on the de vdopcDcnt of form and bciia\Tor m amphibian embryo*. Jour Eiper Zool 87 14 S Gi-iSNER^L. xgas Normcntafcl ror EnlmcUung des gememcn W aoermolcbe*. Kef bel » Isonnentafeln, No 14

GltJcksohn S 193: Aunere EntwicUung der Extremitaten and Stadlcneinteflung dcr I-arvenpcnode ^•on Triton tamaitu Leyd and Tnion cnsiciiu Laur Arcb f Entw’mcch 115 J 4 i

TT*»PTvr>f R, G 1929 Gjrrelatiom m the deitlopment and grtnrtb of the eye Jtud led by meam of beteiofiuttc tiansplantatloii. Arcb f Enta meeb, lao i KiaaHT F C E. 1938- DleEntatcUungiaarri/fjio/^eiimbeii'encluedcDenTcm pertturen, mit NormentafeL Arch, f Entw*mcdi.^ 137 461 Moon, J A. 1939. Tnnpjcriturc tolemnce and ratea o^ development m the egg» of Amphibia. Ecnlogj 20 459 '

19400- Adaptive differtrtcea in tho egg membranei of frogs. Amer Nat 74

89

1940^ Stenothermj and emythermy of animals b relation to habatat Ibtd

p 18S

• ■ ■ ■ - i^a The role of temperature b ipedation of fro^ BloL Symposia, 6 1S9 OvAMA J 1930 Normentafel lur Entwickhing dea japanischen ifofches (rnfantr ^kogasigr) Zool Mag Tokyo 49 465 (fa Japanese )

PounTEi, A. and Moobx, J A 1937 Tables for the normal devdopment of Rana ijfta/uaj Anat Rcc 6S 489.

Sato T 1933 tlbei die DetennlnaUon dca (etalen Angenspalts bei TnSon iamoiia Arch, f Enta meeb 128 342

Shpuway W 1940 Stages in the normal derelopment of Rana fnfnau I External form. Anat Rcc. 78 139

Stowe, L. S 1930 Heteroplastic transpUntatlon of eyes betneen the lanic of tan spedes of Amiiysima Jour Eiper Zool 53 193 TiinTT V and Elliott H A. 1934 The reUthe gro» th of the amphibian eye studied by means of tra£t«pUntitkm Jour Eiper Zool 68 247 Tarm \ C and SGffxnxD J L. 1931 The gronth of ejea and hmbs transplanted beteropiastKally betaeen tao species of AwtHyjioma Jour Eiper Zohl 59 6: WBJCitT A H 1914. Life histones of the Anura of Ithaca, New \ork. Pub Came gie Inst Washington, No. 197

1929 SynopSB and description of North Aroerlcan tadpolo. Proc. U.S Nat

Mas Pub No 3756

\AUAnA, T 1939 Uber den FJnfl uia von WTrtsalter auf die Differemiening von verpflanitem Unegment Material des Moldi Embryot. Jip Jour Zool 8 365

3 CULTURE MEDIA FOR OPERATED EifBR^OS

Most experiments on amphibian embryos require the rcmot'al of all jell) membranes previous to the operatioiu The decapsulated cari) em br)*os arc \Tr> sensiUve and must be kq>t in a sterile salt solution of appropriate osmoUc pressure pH etc. From late Uil-bud and cari> swim mmg stages on embryos are much more resistant and maj be kept b tap

as


water or qarmg or pond water Amphibian Ringer solution has long been Lnown to be strongly hj’pcrtonic. Holtfrcter (1931) recommended a dilate Ringer solution which is now generally adopted and known as “Holt freter solution Its composition is shown m Table 4,

In preparing the sterile Holtfrelcr solution use distilled water The solution IS prepared without NaHCOj and autoclaved NaHCOj is sterilized dry and then added to avoid its precipitation Holtfretcr solution Is still hypertonic, and amphibian gastrulac will exogastrulate if kept in this medium after removal of the viteDine mem brane (Holtfrcter, 1933) A o 6 per cent Holtfretcr solution has approii matel> the correct osmotic pressure. In general a more dilute Holtfretcr solution IS m use Empirical data show that f or 4 - or Holtfrcter solu tions arc satisfactory we use iV throughout with good success. However

TABLE 4

Couposmov OF BcLimm and Aaiphibiah R omEn SoumoN

(la Gffl /liter)


Sohitton

K«a

xa

1

CO,

K nco,

Ilolt/reter

m

e oj

e I

e *

AwiptlMfi Rieger

0 14

e i>

e z


full-strength Holtfrcter solution facilitates healing per pnmciH in early embryos and it is a generally adopted practice to do all operations in con centrated Holtfrcter solution and keep the embr>'n* in this solution until the wound Is healed or the transplant is healed m Then they arc transferred to 4 or iV Holtfrcter solution Richards (1940) has compared the properties of the Holtfrcter solution with those of the capsular fluid, i.c , the fluid contained m the ^»ce between the vitelline membrane and the inner fell> capsule of the egg of A maculafum He pomts out that the Holtfretcr solution differs from the capsular fluid not only m Its osmotic pressure but also in the following points it lacks protein and probably as a result of this deficiency its \’iscosity is much lower He suggests addmg muon to the medium Howe^’cr no expenences with this or any other substitute ha\'e been recorded Summary Use concenlraUd UoUfreter soiulum for operatwns and 1 or A UollfreleT solutwn for the rearing of decapsidated embryos up to the snm mtng stege


s6






bibliography

Hoittmtxh J 1931 t^berdieAufmditfaonerterTeaedciAinpUbleiikeimes. Aich £ Enlw'mtcL 114 404,

iQjj Die tot^ EiogutTuUboo, out Sdbrtiblosim* da Ektodenm vcun

Entomcsodenn EntwKthmg und fnnktioiielk* Veriitltcn pervtnlow rr Orgine. Ibtd^ lap 669.

RiCHAJi*, 0 W 3940 Tbe c^MuU^ fltdd of Awdlysiowui fwicivm eggs compared with Holtfreter'i tod Rlnger'i lohitions. J<mr Eiper ZoOf 83 40:

4 REARING AND FEEDING OF LARVAE REARING OF NORUAL LARVAE

IndmduiJ spccnnens are beat kept in lily cups, Petn dishes etc- For larger cultures large finger bowls or crystallizing dishes of adequate size art recommended (see p 10) Overcrowding should be cartfoUj e\oided All should be provided with water plants- Water should be changed whenever necessary, but not too often ADow water to stand for several hours before larvae are placed m it. Tap water m manj localities is satisfactory for rearing of older larvae {dc<»psulated young embryos up to swimming stage should under all circumstances be reared m an artlficjaJ medium see p 25) If tap water proves to contain tone agents, then pond or spring water must be used

Preccuttonj at rrutamarphons — Metamorphosis is a critical stage for both anuran and urodele larvae The animals do not feed dimng this period and become weak unless they ha\T been fed well before the onset of metamorphosis. Fadlities for crawlmg on land must be pro\^ded early enough to prevent them from drowning It is best to prepare special tanks for metamorphosis and to transfer mto these all old lan.’ae which show signs of metamorphosis (color changes large hind limbs in Urodda emer gence of one forchmb and tall resorption m Anura) In these fwnlK the water should be onI> 1-3 mches deep and an mdine which rises above the water le\’el should be built either of pebbles or b} tilting a glass pbtc or a glass dish J Moore (personal communication) recommends placing a piece of filter paper on the bottom of a finger bowl tilting the finger bowl and adding a small amount of water

FEEDING OF DRODELE LAR\AE

Ambyziomc and Tnturus larv’ae begin to feed in stages corresponding to II46 The> ore camii-oroas and ha\'e to be fed In-e materml which mo\-es m front of their cj-es and thus attracts their attenUon Older larvae rcl> more on their sense of smell end will take small bits of meat Small ostnicods, daphnia and other crustaceans arc an excellent food for vtT}


  • 7


small lan’ac Eiich>'tnie ("white worms — Enchylracus albiiuj, an oltgochaete) are equally suitable and available at all times They maj be obtained from fish dealers or pet shops The> are best kept m moist humus m a dark, cool room (not warmer than 30 C ) and can be cultured easily m the following waj Fill a large earthen pot or culture dish with moist humus about 3 Inches deep Add a worm culture of at least 2 quarts. Cover it with a lid Feed the worms with white bread cereals or bofled potatoes soaked m milk. Scatter the food about or place it where the worms are cong re g a ted and cov-er it with about an mch of humus. Rerao\-e all food particles which begin to dcca> replace them by fresh food In such cultures worms of all sizes wiD be found (further details m Blount, > 937 )

Special attention should be paid to the feeding of \Try y'oong lar\'ac when they first begin to cat The very smallest worms of a culture should be selected in the following way Spread humus crumbs and food particles from the worm culture in a Petn dish under water the worms will begm to wnggle \Tgorously and the smallest ones can be picked out (under the bbocular microscope or with the Beebe loupe) At first many larvae are slow and reluctant to take food ket It is easentjal to "condition them early to feeding Particularly \*a]uable qjecimens should be fed individually for a few times. It may be ncceasaiy to mo\'e a worm slowly In front of the ey*es to eliat a snappmg reaction As the size increases, larger worms and greater quantities should be fed about e\-cry other day Lan'ac which approach metamorphosis can be fed on small bits of raw h\Tr or beef or pieces of ramworm Precaution must be taken that hu^ae do not mjure one another They wfll snap at any object that mo\'cs and will bite off one anotber^s gills or limbs. Feed the animals abundantly Valuable specimens should be kept in isolation

JEEDINC OF AXimAN TAPPOLES

The feeding habits of anuran tadpoles are entirely different Ibe tad poles are equipped with bomy teeth and are oranlTOrous they rasp off algae from the walls of the aquarium or from water plants, they feed on dead animals, meat etc. For rearing of tadpoles Adams (15141) rrcom mends the foDowing beef and wheat mixture

Dusohe one a-oi (56 68 gm ) tube of BactO'becf exUxet "Difco” lUDdirdued (Difouve Ferment Co Detroit, 3 ‘ficfa)te«ppTijiimitdj 160 cc. of wtter Jfixwbcet flour With Lbii m proportxm 1 i or 3 1 Spread paste thinly on glass pdate and dry Grind dned paste to po«der miib mortar and peiifc. Store ponder In stoppered bottle


38


Approximately a s gm of this mixture should be gnTn to each group of lo animals every daj or e\'ery other day This food is particularlj recom mended for controlled feeding (e thyroid feedmg) experiments A good substitute for this nurture is Pablum Ordmaril} algae boiled spmach^ boiled meat (finely minced liver beef or frog muscle etc ) the jolt of hard boDedeggs oranj combmation of these will give satisfactory results Tadpoles are \oraaous eaters and thar growth and metamorphosis can be speeded up considerably by hea>y feeding Carefully remove aH food debns


SOUE COMMON DISEASES

Operated animals suffer occasionally from edtma particularly m tail bud stages- Edematous blebs appear on the flank or on the \'entral side Such wniTTml^ usually die after se\'eral days and should be fixed m early stages of edema if they are valuable matenaL Sometimes puncturing of the Noside with a fine glass needle sa\es the animal (narcotize the animal if necessary)

Fungus infections {SaproU^nia) arc not infrequent m urodde cultures and are difficult to get nd of They appear as fine sticky threads first on gills and legs and eventually all over the body and are usually fatal Detwiler and McKennon (1929) recommend a bath of merairochrome (di brom-oey mercun fluoreson) Animals are kept for se%*cral days m a concentration of i 500 000-1 i 000 000 The molds usually slough off after 2-3 days. Effecti^T concentrations and lengths of exposure should be tried out for each instance

\ oxmg urodclc lar\'ac which are not properly fed or which arc slow m catching food sometimes swallow cir btibblcj Occasionally they fill their stomach with air and float on the surface TTiis condition is of course cntical and the laiwae will die of stan-ation unless the air is remo%*ed One can accomplish this either by puncturing the stomach with a glass needle or a fine steel knife or by squeezmg out the air through the mouth holding the animal cautiously with one pair of watchmaker forceps and pmching behind the air bubble with another forceps.

BIBLIOGRArm

Aruiu A E. IJ41 Studio in eipenmenUl xoologj Ami Arbor itich Edmtrdj Bn»

Blount R, T 1937 Culttvatlon ot Eackytnnts albidus In P S Gujsoit (ed.)

Culture raethoda for m\frtcbr»le inimali Ithaca, N ^ Conutock.

DcTwnxi, S R-, and McKesnox C E ioio* ifercurochrDme (<E-broin-OTj mcr cun fluorexan) as a fungiddal a^t in the growth of ampUblan emhr>os Anat, Rec 41 ao5


B EXPERIMENTS


1 SOifE TECHNICAL PROCEDURES

a) EXPERIMENTAL OVULATION BY ITYPOPHVSIB INJECTION AND ARTIFICIAL INSEMINATION IN ANURANS

It IS now possible to obtain eggs at almost anj time of the }*ear owing to our mcreascd knowledge of the control of reproduction b> the antenor lobe of the hypophj'sis, 0 M Wolf (1929) first reported expenmcntallj induced ovulation in the frog b> injection of anterior lobes of the hj*pophy sis R Rugh (1954-41) has worked out the standard tedmique on which the following directions are largely based

JnAnura Insemination Is external Male and female go into amplcrus. The eggs arc inseminated extemoUj immediately after they ha\x passed through the cloaca The routine procedure of obtaining fertile eggs is to induce ovulation stnp the eggs into a sperm suspension and submerge them in water 5 or :o minutes later to atow the membranes to swell

The following points are of unportance for obtaining optimal results.

1 Source of anlortor lobe substance — So for fresh frog pltuitarj gland is the onl> rcluible and adequatclj standardbed source for the gonadotropic prmaplc effective In frogs. Implants and extracts of mammalian anterior lobe were unsuccessful (Creascr and Gorbman, 1935, 1939)

2 Si^ of frogs {Rana pifiens) — Both donors and rcapients must be fully mature Do not use females under 75 mm or moles under 70 mm in bodj length (Rugh 1937&)

3 Condition of frogs — It is important to use only specimens (for donors and recipients) which are In an excellent condition and which have been recently caught The pituitancs of frogs which arc held In room tempera turc for anj length of time stan*cd or olhcrwac kept under Inadequate conditions lose their potency

4 Temperature — It is advisable to keep the frogs m a cold room (15 “ 20 C ) before treatment.

5 Dosage — The dosage has been standardized for R piptens implants into R pipiens (Rugh 1935a b 1941 Moore and Barth impublished)

I)r> John A Moort and L. 0 Birtb Trry Undly made ivillabic tnpobbihed daU on hjTvjphjui tn^cnn ind frrtlluaUon [n K na tiffns I »£ii to ciprm my th i n li to tbo •uthon for pcrmmjon (o todade (bnr data in the folVmins *ccounL

30


The number of pituitanes to be injected vanes with the season Ragh found that the pituitanes of mature females are twice as potent as those of mature males. The figures In Table 5 combmed from Rugh (1541) and Moore and Barth (unpublished) refer to 9 glands If cf glands are used the doses have to be doubled. Injections during the months im mediately following the natural breeding season (April Ma>) are usuallj unsuccessfuL

The size and pl^’siological potency of different hj'pophj’ses as well as the re^ionaiveness of Individual recipients var> considerabl> so that the abo\'e figures gi\’e onl} approximate values It is therefore advisable to mject two or more females for each e^iernncnt or demonstration The effect of injections is armulatn*e end it is usoaDj possible to bring a re fractory* female to ovulation bj administering a second mjection 2 daj’S after a first unsuccessful Injection.


TABLE 5

EfTtenvE Dons roa Oxtjlatiox


ScptrtBifccr N*T*tnS«r JnatuT 1

1


n«CEnb«r

Ptbruj7

1 AfnI

No- «f 9 pjraluik* re^olred

6-9

y-6

3-5


MaUrml

hj'podcnmc syringe 2 cc. hj'podcrmic needle Iso 20

dissecting instruments including strong and fine scissors and watch raaler forceps

section dishes and watch glasses finger bowls or Petn dishes ether

batterj jars or aquanum jars with Uds or wire coi-ermgs to keep the reapicnts

k Holtfreter solution or spring or pond water for dilution of sperm fluid

A Beebe binocular magnifjnng glass is verj helpful m the dissection of the bj-poph^-sis Procedure (for R pipjcnf)

I Prepare as man> battery or glass jan as there are females to be m

la R R cla^Ons tod R eralilwn hu b«n obtaiocd by Jnjectioo

of ptuuuriei. Tb« donfc fa casci Lu aol b«n lUndaniiard (Rnch,

31


jecte<L Cover the bottom with water about i mch deep Cover the jars with heavy glass lids or wire covering

2 Select two or more females as recipients Hold them ready m a small cage

3 Narcosis Is not necessarj, but it facilitates the mampulation of In jection Give a light ether narcosis. Place the frog in a tightly covered glass jar attach to the lid a cotton wad moistened with ether Take precautions that the frog does not touch the liquid ether with its sUn

4 Dissedion of the pituitary glands from the donors {J^\g 2) — Insert one blade of a strong pair of scissors into the mouth at the angle of the jaws and decapitate the frog by a transverse cut behmd the tympanic mem brane Be sure not to cut more anteriorly Pith the spinal cord and discard the body Wash the head and remove all blood Turn the head upside down Dissect and dean away the akin of the oral cavity and thus expose the base of the skull Locate the 1 shaped parasphenoid bone. Make two cuts through the floor of the skull from the cranml cavit> toward the c^xs (ab and cd m Fig 2a), by inserting the pointed blade of a fine pair of sassors into the foramen magnum or the iqiuial canal Do not injure the brain tissue With a pair of forceps carefully deflect the tn angular piece of bone, thus exposing the >*entral aq>ect of the bram (Fig 2&) Locate the hindbram the infundibulum and the optic cbiaszna The anterior lobe of the hypoph^ais can now be recogniicd by its pinkish color It is either m its normal position (attached to the mfundibulum postenor to the optic chiasma) or quite frcquentlj it will adhere to the deflected bone. Gra^ the gland with a pair of fine forceps and place it in

Holtfrctcr solution m a watch glass. (Make your first dissections under the bmocular microscope or with a loupe ) The anterior lobe is pinkish and bean shaped Attached to its anterior straight edge is usuallj a slen der whitish body, the pars mtcrmedia and piars nervosa (Fig 2c) Ranow this tissue with two pairs of watchmaker forceps Dissect as manj glands as arc required according to Tabic 5 (p 31) collect them all m the watch glass

5 Injection — The glands arc injected entire (maceration results m the loss of some actux substance) m i or 2 injections Draw the glands into the barrel of the sjTinge Hold the female which is to be injected in j our left hand and insert the needle through the flank skin into the bodj ca\nt> Push the needle forward under the skin and not mcdmllj to a\oid injuQ to the \Tsccra Inject and then withdraw the needle cau Ijouslj while }ou \s^thdmw pinch the skin at the needle entrance to a\*oid outflow of hj’pophj'sls material To insure that no material is lost



by adhering to the needle, squirt water through the syringe and Inject all material which is recovered m this way

6 Place the female m a jar X^bel the jar and indicate date and dose. Preferably heep it in a cool place not higher than 20° C

7 Tesiof crulaiu)nby*‘stnpptng — ^At room temperature most of the eggs should have ovulated in 24-48 hours after the mjection if the abmT doses arc applied To test ovulation squeeze the female m the following way Bend the legs forward and hold the frog m your right hand. Press gently m the direction of the cloaca In this way eggs will be forced out without mjur> to the frog If a string of eggs appears, release the pressure at once place the female back m the container, and prepare the sperm fluid. If only liquid or jelly ooze out, then ovulation has not yet taken place, and another test should be made 12 or 24 hours later If no eggs are obtained, a second injection should be made.

8 Preparation of the sperm fitad — ^Thc males of 22. usually con

tarn functional q)enn throughout the year so that it is not necessary to gi\ c them hypophysis injections. Prepare a diidi with 20 cc of 20 per cent Holtfrcter solution or o r per cent amphibian Ringer solution or pond or ^nng water Do not use tap water or distiUed water A quantity of 10 cc, per pair of testes is usually recommended for q^erm suspensjons, but even a tenfold dflution of this suspension sUH gives optimal results (Moore and Barth unpublished)

Decapitate and pith 2 large males not under 70 mim m length. Dissect out both pairs of testes (j-ellow, oval bodies located near the anterior borders of the kidneys) Qean them of adhering blood and tissue and macerate them thoroughly with forceps and scissors, until a milky suspension IS obtamed. Allow it to stand for 10-15 minutes, during which time the spermatozoa will become active. Observe under the microscope if active motile sperms are present.

9 Stripping and insemination — Divide the qjcnn suspension among 2-3 finger bowls or large Petn dishes so that the bottom of the dish Is just co\‘ered Hold the female as In section 7 and strip the eggs directly mto the sperm flmd by slow continued pressure The eggs will ooze out m a string Line it up m rows or in a spiral, so that all eggs arc exposed to the sperm and are not clustcrctL Shake the dish gently to insure com picte feitilitation After 5-10 mmutes flood the dish with the same medium that was used for sperm suspension nnse and wash the eggs, and let them stand submerged in dean water The jelly membranes will swell slowly A successful mscminatlon is usually indicated after about I hour by the rotation of the eggs as a result of which all dark aru mal poles nio\-c upward The first dea\’8ge Is to be expected s\ hours


34


after msemmation If egg* are clustered the} should be separated with a scalpel Do not Leap more than 30-40 eggs in a finger bowL

10 ‘ FrachcnaUd’ sinpptng — Eggs will remain viable m the oviduct for some tune (see below) if the females ore kept at a low temperature (io®-i fC) It IS therefore possible to obtam eggs from the same female over a period of several days Stnppmg Is annply mtcrrupled when the desired number of eggs is recovered, and the female is returned to the cold room

NOABILITT or ECOS, SFElil, AWD PirUItAET OLUTOS (it. pipiois)

Eggs — Eggs will remam fully viable and fcrtihzable and will give an optimal percentage of normal development for 3-4 days after the onset of ovulation if the females are kept at lo^'-is® C From then on the j>er centage of fertiliration and of normal development decreases steadily (Zimmerman and Rugh 1941 Moore and Barth, unpublished) Eggs should not be used later than 3 days after ovulatioiL

Sperm — ^According to Moore and Barth (impubliahed), a sperm suspension of 10 pairs of testes m 150CC. of o i per cent amphibian Rmgcr at 1$ C retained its fertflizability for 20 hours However it is always advlUble to use fresh suspenaons

PUutiary glands — According to Rugh (1937a) dissected glands retam their potency for a long penod If kqit m absolute alcohol m the refng crater Dilute the alcohol to 35 per cent before injection

6) EXPERntBNTAL OVUIATION AND AaTmClAI. iNSEinNATiON IN Ufvdeia

In the Urodda the male deposits a spcnnatophorc after a prolonged courtship the female takes it up Into a gland of the doaca (spennotheca.) where the spermatozoa remain functional for a long penod The eggs are inseminated mdividually while they pass through the doaca The general practice for obtaining fertile eggs outside of the breedmg season is to mject only the females and to rely for fertilization on the presence of func Uonal spermatozoa m the spermotheca The eggs laid by Tnturus vtridcscens torosus and pyrrhogaxta- females following h>'pcipLysi5 mjection were found to be fertilized in most instances but one occasionally en counters females which lay unfertilized eggs A. E Adams (1930) was the first mvestigator to obtain experimental ovulation m a urodele Tr vtndcs urn Tnlurus nndescens as well as J? ptptem hypophj’ses were used the latter arc preferable because they are larger and easier to dissect. Tnlurus females also respond to mammalian pituitar} extracts phyone (a growth stimulation fraction) and hebm (a gonadotropic fraction Adams 1934)


35


However the dosage has not been standardiicd We recommend the use of fresh R. pifncns glands for Tr vindticcm (Kaylor, 1937 Griffiths, 1941 Fankhauser, personal communication*) The females are kept m the refrigerator at about 10" C between the day of collecticm and the first implantation This keeps the ovanes in good condition for at least 2 months At room temperature ovanes deteriorate m about 2 weeks. The procedure of injection is the same as in frogs, with shght modifica tions ^Vhe^cas m the frog a large number of eggs are ovulated almost simultaneously the eggs of the newts are laid smgly over a period of sev eral weeks Therefore m order to obtain a contmuous egg prodnction, it IS advisable to Inject several doses with an mterval of i or 2 days between the injections. The standard procedure m Dr Fankhauser s laboratory is to mject a single R pipuns hypophysis on the first day and another single hypophysis on the third day Two hypophyses were found sufficient to stimulate ovulation at any time between October and May The first eggs are usually laid on the third to sixth day following the first implantation, and the egg laying period lasts between 6 and 14 days on the average. It is advisable to Inject a considerable number of females for each espenment, since the number of females which do not respond to the injections or which lay unfertihied eggs is rather high (30-40 per cent) The hy pophyses are usually implanted under the Ain of the lower jaws. In jected females should be placed m jars or tanks in which they can swim around comfortably and iiould be amply provided with fresh Ehdta or Vall^snena The ^gs are deposited on the leaves of these water plants (sec p 17) To collect them it is best to take out all water plants each day and to inspect each leaf Remove the eggs carefully with a watchmaker forceps,

Tnlunu pyrrliogaslcr may be treated in the same way (Streett, 1940) In class experiments we obtamed from 15 to 150 eggs per female after 2 mjections of 2 R. piptats glands each on 2 successive days or with a i-day interval between the 2 injections

AKZmClAL DlfEailKATIOX

Certain experiments for instance hybridization require artificial In semination The eggs cannot be stripped but must be recovered from the o\'iduct Prepare a number of Petri dishes laid out with moist filter paper ( moist chambers ) and dean microscope ahdes Decapitate and pith sc\xml males and females Dissect the testes and the (usually pigmented) vai cffcrcntla and macerate them thoroughly in 10 cc of pond or ipnng

• I am bxkbted to Dr Gcrbard Fankhsofcr for nuUnf tvalUble to roe hi* rtconi* d I®* iectioos lod for the commtmkttloo d cerUio tediakal deUib.

3fi


water or I Holtfreter solution Dissect out the oviducts place them on glass plates and vcr> carefully aht them or cut than open and set the eggs free The> are soft and dehcate and must be handled with utmost caution Jilount them smgly on the slides do not moisten them With a fine pipette drop a few drops of the spenn suq)ension ov’cr each egg so that it 13 well coated Place the slides m the moist chambers for 5-10 mmutes then submerge them m water After the membranes are swollen nnsc off the sperm suspension and remove the eggs from the shdes with a scalpel If artificial hj bndiaation is planned discard all eggs found m the cloaca because they may be fertiliml

BIBUOGRAPHY (a-b)

Ad^ A E igjo. The lEuluctlQii o( egg Uymg in Tn/ana wi/aenu bj heteropUi be pitmttrj gland grafts. Amt Rec 45 ijo

1934- The gomd- aod thyrold-stinmh bog potcftaes of ph>T 3 iie and hebm.

Jhtd^ 69 J49

CaEAtta, C. W and GoanitAN A. 1935 Appartnt ipedfiaty of the Induced ovula twn reaction m amphibia Amer Joor Phyilol 113 33

— 1939 Speaca ipccLfiaty of the gonadotropic factors m vertcbratei. Qoart

Rev Bull 14 311

GairTTTHa, R. B 1941 Trrplojdy (and hapWdy) in the newt, TriJitnu nruiesctns induced by refngeratioa of fertilized eggs Genebo 36 69 Katloi C T 1937 Eiperiments on andiogeoesa in the neat, Tn/una nnd^zeev Jour Eiper Zool 7d 375.

Rugh, R. 1934- Induced orulibou and artlfioal fertOlabon m the frog. Biol BolL, 66 33

19350 Ovulation m the frog L PUuItary rdatiotB In induced ovulatioTL

Joor Exper Zool 71 149 1935^ Pitmtary-induced aezml reactiom m the Anun BhjL Bull 68 74

1937*- Oxxilation roduced out of season- Sdence 85 588

1937^ A quantitabN'e analysis of the pituitary-ovulabon reUtiou in the frog

{Rana ftpims) Fhydol Zool so 84.

1939 Relation of the intact pitulUiy gland to artifinally induced ovulabon.

Ptoc Soc Eiper Bid and Med 40 133

1941 Expenmental embryology (a mipuil of techniques and pnxedorei)

Aea "Vork hew \oA. UnKersity Book Store.

STBErTT J C 1940 Experiments on theorfanmbon of the nrw^>g TTn»n ff 4 jp/ Imna pyrrkagasUr Jour Exper Zool 85 383

WoLi O M 1919 Effect of daily tran^lanls o( antmoi lobe of pituitary on rqwoducbon of frog Proc Soc- Eiper Bbl and iled a6 692 ZbomauAK 1 ^ and Rctch, R. 1941 Effect of age on the developtnent of the egg of the leopard frog, Aano Jour Morph 68 3 29.

c) THE REUOl AL OF EGG MESIBaAKES All amphibian eggs arc mclosed in a xutelline membrane and m a num ber of gelatinous envelopes Their number and cQn£istenc> differ in dif


37


fercnt speaes. Descriptions, measurements, and Illustrations may be found in Bishop (1941 Piersol (1929 A puiaAa/um oiidjeJ'er

tcmtcruj), Wnght and Wnght (1924, Anura)

Ambysiona — The eggs of the three species ^4 maculaium, A itgrinum, and A opccum are approximately equal m site, ranging from 2! to 3 mm The vitelline membrane 13 closely applied to the egg A second rather tough and perfectly transparent membrane forms a capsule, about 5^ 6 mm In diameter The space between the vitelline membrane and thu capsule IS filled with the capsular fluid in which the egg moves frecl> The capsule is surrounded by another tough and less transparent mem brane, which consists at least m A maetdaium, of several thm layers. In A tigrinum an additional membrane is found between the two In all


j — RsDOTtl of the }cll/ membnses of u 4 m^ jiUm a cfg (lee text)


mftyj/timc ^)eaes a thin sticky layer forms the outermost covering In A maculaium and ttgnnum the eggs arc imbedded m a common iclly» which probably originates by coalescence of individual soft jelly layers. The eggs of A opacum ore laid singly under leaves, etc. (sec p i6) Their outermost sticky membrane Is usually co\*cred with mud particles.

Two pairs of watchmaker forceps with carefully ahaipened points arc needed for the rcmo%’al of the membranes. The eggs of A macvhitum and A hgnnum are first taken out of their common jell> mass those of A o/flcwm are placed m water and allowed to swell to capaaty The egg* are transferred into a Petri HkH or other glass dish with no Permoplast or agar ground Perform all further manipulations under the low power bh nocular dissection microsco p e The two outermost ^3x13 — the stick) thin membrane and the outer capsule — can be casDy removed together N ext follows the mner capsule which is under the pressure of the capsular fluid Reino\x it in the following waj (Fig 3) Set the forceps in )*our

38


nght hand firmly on the glass bottom with extended prongs and push the egg against it Pierce the capsule with one prong of the left forceps (Fig 3 a) Carefully avoid mjury to the egg No fluid will escape because the forceps plug the hole Next close the left forceps and hold a firm gnp on the capsular membrane Next insert one pomt of the right forceps mto the hole alongside the left forceps (Fig 3 6) and rupture the membrane with a quick jerk of both pairs of forceps m opposite directions The capsular fluid will then escape and the egg will pop out or it can be shaken out. The removal of the vitelline membrane is rather difficult m yoimg stages For this purpose the forceps must be sharpened to the finest pomts possible The removal is usually unsuccessful in cleavage stages. In blastula and gastnila stages the vitellme membrane is still vcr> closely apphed to the egg but can be removed m the foltowing way Grasp the vitelline membrane with the left forceps at the animal pole or over the blastopore and tear a large hole m it with the right forceps If j'ou do not succeed then puncture the egg at two points on the animal pjole with a glass needle or the pomt of the forceps The pemntelhne fluid will escape and the egg win cohapae slightly wnnklea will appear on the egg surface and make it possible to grasp and rupture the membrane After removal of all membranes the egg will collapse It is \ary delicate and must be handled with extreme care If used for operations it should be washed m several changes of sterile Holtfreter solution and placed m a dish with agar bottom m iV Holtfreter solution If the egg was punctured place it in full Holtfreter solutwa for a while to fadlitate healing Use stenhxed pipettes and dishes end keep all dishes co^â– e^ed

Tnlurus — In the eggs of Tniurm the outer capsule mcluding the out ermost sticky membrane, can be remo\*ed with httle difficulty Howc\*er the pressure of the capsular fluid is much greater than in Ambystoma eggs and the remoi’al of the inner capsule is therefore more difficult. To re co\‘er the embryo without inpiry it is necessarj to tear a large hole m the capsule at the first attempt PuU the forceps apart with a rapid jerk. The tension of the capsular fluid is low for a abort penod Immediatel) after fertilization and workers who use Tnturm eggs m clea\’age and gastnila stages sometimes decapsulate them shortly after la^ung During clea\’age and up to tail bud stages it is ^'crJ difficult to ^emo^T the capsule successfully For this reason Tnlurus eggs are not recommended for use in classroom experiments m stages earlier than tail bud stages

Frogs — Frog eggs are imbedded m a common jcll> mass and mdi\ndu all> surrounded by loose jelly layers which howe\Tr form no tough elastic capsules The jell> is not difficult to rcmo\-c One maj cut it oS with forceps or roll the eggs on filter paper It is difficult to rcmo\-e the


39


>itelliiie membrane In early stages up to ncurulne Besides decapsulated anuran eggs arc ^XIy soft and extremely delicate. They collapse c\*cn more than Triturus eggs Frog gastrulac are therefore not suitable for classroom experiments but, m embr>TJs from late neurula stages on, all membranes, Including the viteibne membrane can be easily remov'ed BIBUOGRAPHY

Bifiirop S C. 1041 The Hdamtnderi of New York. New lork Sute ilo*. BoD 334. Albany Umvenity of the SUte of New \ork.

PmsoL, H- 1930- PathologKil p<J>'ipenny b esj» of (Green) Trans. Roy Canad. Inrt 17 57

Ricnrr A. Ht and W bight A. A. *934. A ley to the egga of the Sdieniia cast of the Mississippi Rh'cr Amer NaL, 58 375

d) NARCOSIS

Amphibians in taQ bud stages arc dhated and rotate wi thin the cap sular fluid The ciliary beat is strong enough to keep the animal m slow motion when It is taken out of the membranes This may be an impcdi ment m operations The only way to hold the embryo tight Is to bury it in a Permoplast groo\'c or under glass bndgts The first muscular motflitj begins In stages corresponding to H31 or 3a for A mcculaium (see p 175) From these stages on, the embrjw must be narcotized for operations and for protocoling Two excellent nar coUcs are at our disposal neither of which has a detrimental effect on the embryo If appUed In proper dosage.

1 CMortlone {acdone chloroform) — will dissolve rather sparingly in anj culture medium Shake thoroughly Keqj bottle* tightly stoppered and dishes co\Tred In most cases a concentration of i 3 000 will be satisfactory Old lar^Tie may require a stronger concentration It is ad\Tsable to try out the effcctl^*cness of the concentration before valuable matcnal IS narcotized Elrabryos and larvae become immobile withm a few minutes and rccowr within 5-10 minutes The heart beat should be watched Its stoppage IS a sign of too high concentration embryos can be sa^ ed if the) are transferred immediately to anonnal medium Embryos may be kept under light narcotization for several days (sec p 127 also Matthews and Detwiler 1936)

2 AFS 323 * — This IS a methan sulfonate of meta ambo-benzoic-aod ethyl-ester an isomer of anesthesm (Rothlin 1932) It is soluble m water and e\’cn less tone than chlorctonc The animals reco\tr more rapidly than from chlorctonc Copcnha\er (1939) finds that the heart beat b only thghlly affected Concentrations of i 2 000 or i 3 000 arc rccom

MS 3JJ may b< obtamed frocn Saodoi Cbcmiol Worki, Inc, 6S-“o ChaiUon SC,

\otk Gly


40


mended Scliott6 and Butler (1941) report that ‘a stock solution of I 1000 can be sterilized m the aatoclavT without apparent loss of nar cobc properties and with no increase in toxiaty Agam, a normal heart beat Is the best indicator for a proper dosls Embrj’os can be kept under light MS anesthesia for several days

BIBLIOGRAPHY

CopENHA\'Ei, ^\ 1939 IiutittioQ of be»t and mtrmalc contrtctlon rales m the

different part* of the AwMyri&ma heeit. Jour Exper Zool 80 19a MatthtuSjS a, end Detwilee, S R. iga6 Tht ntOiom oi AmlJyriewta exahrym following prolonged treitment nith chloretone Jour Exper ZooL,45 379 Rothuv E. 193a ilS aaa (losCches Anaatbesm) cm ?\triotiknm fur Kalthluter Schweii. med. Wchnvrhr^ 45 104a

ScHorri, 0 E and BurtER, E. G Morphological effects of denervation and

amputatKcn of Umbi m urodde larvae. Jour Etper ZooL, 87 379.

e) STAirDAMJ EQUIPMENT TOR OPERATIONS ON AMPHEBIAN EMBRYOS*

I EQcrmEKT roa eao rruDEsrr

Duka

several 4 inch finger bowls or No 4Lily cups to keep eggs or egg masses before operation

several Petn dishes or Syracuse dishes for decapsulation (p 37) several Syracuse dishes with Pennoplast ground (operation dishes)

6-12 SjTacuse dishes section dishes or small paraffined LDj cups for raismg of operated embryos

1 glass jar laid out with cotton for alcohol sterihzatMin of metal instru ments

Insirvmenls

binocular low power dissecting microscope microscope lamp

2 pairs of watchmaker forceps

1 pair of ordinaij small forc^a se\Tral glass needles

8e\-cral hair loops

2-3 glass rods with ball tips of different sizes sc\’tral glass bndges of different sizes

2 medicine droppers

2 wide mouthed pipettes (about 5 mm in diameter to transfer whole CEg*)

wooden bolder for glass instruments

S« Part I (p 3) for ah dctalU.


41


a CQOmCEKT FOR THE CtASS

Dishes

several oystallizuig dishes or inch finger bowls for egg masses Olher equipmcni

materials for preparing of glass instruments (see p 3)

Pcrmoplost

oil stone for sharpening of watchmaker forceps Solutions

full strength Holtfrcter solution (for operations)

] or Holtfrcter solution (for rearing of cmbrj'os)

MS 2sa, I 3 000 or chlorctonc i 3,000 (narcotics)

70 per cent alcohol for sterilization of metal mstniments Note Holtfrcter solutions must be sterilized for operations on early stages (gostrulae)

f) SOltE CEKEJLAL RULES FOR OPERATIONS

Prepare a sufficient number of glass Instruments, particular!) glass needles, to have them on hand if on instrument should break during the operatKin

Use only the best hving matcnol for operations. Discard oU embryos which do not gastrulate property, which lose individual cells on the sur face which show signs of edema, which show irregular pigmentation or other signs of on unhealthy condition

Sterilize and dean all instruments carefully Keep the operating table dean Wash the embryos after removal of the jelly membranes in sterile Holtfrcter solution Keep all dishes covered Remove dead animals at once hlark all pipettes and instruments used for fixation with a labd noxr use them for living material and keep them off the operating table Choose the proper optical magnification Operate under medium rather than highest power Be sure to hn\x on optimal illumination of the \’isual field

Operate In full strength Holtfrcter solution After healing Is well under wa> transfer the embryos to H or iV Holtfrcter solution

Always make scNTral operations of the same type The mortallt) in operations on cmbiyos is alwaj-s high despite skill and dcanllncss Keep operated cmbiytM in n cool place to hold back bacterial growth etc Carcfull) shield off direct sunlight

Fix operated cmbiyos when the) show signs of disintegration This process once started goes on rapldl) and the cmbiyo may be complctcl)

disintegrated a few hours later



Consider protocols and observations equally as important as the opera twns. Get a cle ar understanding of the theoretical implications of every experiment which you perform and try to evaluate m your own words your results in conjunction with those of the class

2. THE PROSPECmT: SIGNIFICANCE OF EMBRYONIC AREAS (\TrAL STAINING)

a) A DESCRIPnON OF CASTEULATION IN’ Ufodda

An mtimate knowledge of the process of gastrulatlon m amphibians is mdispensable for an understanding of the experimental work done on early embryos to be described m the foflowing pages During gastnilation the germ laj'ers are formed b> extensive cell movements. Moreover, transplantation explantation and other experiments have shown that, durmg and shortly after gastnilation, some of the mam organ pnmordia become more or leas ngidly ^determined. In other words profound changes m the visible and mvisible organization of the embryo take place during th i^ penod The morphogenetic cell movements are now clearly imderstood thanks to the admirable vital staimng eapenments of Walther Vogt (1925 1929) and his collaborators The following presentation is largely based on their work.

I THE iCtUdUU or TEE UXOCOX EnSttO BETOia AKD AfTEJl OASTStTLATltm

The blastula is sphencal and dearly polarized The ammal hemi sphere is charactenred b> several layers of smnll cells which form the thin roof of the ^lastocoele. They contam bltle j*oIk and are usuallj pigmented The ccHs of the ‘V^tal hemisphere are hca\’ily laden with yolk and are unpigmented

At the end of gastnilation 1 c shortlj before the meduUaij plate makes Its appearance the embryo (Figs 9 lo) is still sphencal m shape but It has acquned a bilateral 5 >Tnmetrj and its walls are formed b> three sheets the germ la> en The blastocoele is almost entirely replaced b> another central cavit>— the pnmitn-e gut or archenteron The archen teron is actuallj closed and plugged by the yolk plug op to carlj neurula stages It opens to the outside bj withdrawal of the plug (Brown 1941) The ectoderm forms a complete outer covering continuous with the mesoderm around the blastopore It is usuallj pigmented throughout the unpigmented cells ha\’e been shifted inside The mesoderm forms a mantle subjacent to the ectoderm Howe\-cr the anterior \*entnil part of


43


the embryo remains free of mesodenn, and the mesoderm mantle ends with a free edge, which extends in an oblique direction from dor»aI-tntenor to ventral postenor (pte m Fig 5) This- sharp edge fades out near the mid-dorsal line, where the antenor-dorsal part of the mesoderm mantle merges with the antenor-dorsal part of the entoderm The antenor part of the archenteron is the foregut, which is di^roportionately wide at this stage The entoderm behind the foregut forms a troughlike structure part ly inside of and aivered by, the mesoderm mantle. Its massive 6oor is formed by the large yolk laden cells Its anterior and lateral walls are thinner and rise steeply from the floor The walls do not meet m the mid dorsal Ime (at least not In the stage under consideration) but appear as two parallel lines, lateral to the median plane (e, m Figs. 7-10) Thus the archenteron has a dorsal gap and DO entodermal roof However the dorsal part of the medosenn mantle becomes mtimately applied to the free edges of the cntodermal trough and thus forms temporarily a lid over the ar chentcron for this reason the dorsal part of the mesoderm mantle at this stage has been given the misleading name "archenteron roof In the neurula stage the fret entoderm edges will converge and eventually, fuse underneath the mesodermal mantle thus giving the archenteron Its permanent entoderm al roof The temporary contact of archenteron roof and entoderm is so intimate that cross'sections may simulate an actual fusion Such pictures were taken as evidence in favor of the entoderroal origin of the mesoderm and particularly of the notochord The invcsti gations of Vogt and of others before him leave no doubt that this conception is erroneous, both germ layers originate at the blastopore and remain separate umts, despite their temporary amtact This latter statement requires e qualification There is, Indeed, true contmuity of mesoderm and entoderm at two places In a narrow sickle shaped area ventral to the blastopore and as mentioned before in the roof of the beadgut. The situation in this latter area is difBcult to visual lac and deserves further comment Ck In Figure 9 marks the antenor end of the prospecti\x notochord At this pomt the mesoderm does not end abruptly as will the notochord In later stages, but continues mto the socalled ' prechordal plate In its cellular texture the prechordal plate appears os a true transitional rone between ento- and mesodermal struc turcs A sagittal section exactly through the median plane shows therefore a continuous archenteron roof partly of entodennal and partly of mesodermal origin Such sections figure prominently in most textbooksk et they arc liable to gi\x a wrong conception of the entoderm mesodenu relation unless they are presented in conjunction with transixrse sec tions


44


7 DCTTfiXAL TtATTEEa OT CASHIULATION

The transformaUon of the blastula into the gastrula is accomplished b> a sequence of integrated cell movements The greater part of the \’egetal hcm^hcre iHN^aginatcs into the mtenor around the blastopore The am mal hemisphere spreads and o\‘crgrows the \'cgetal hemisphere and c\en tually forms the entire surface of the ncunila Dunng this process the blastopore changes its shape continuous!} These changes \’ary in differ ent forms and will be described for A rnacvJaJum In Hamson s stage senes only 3 gastnilation stages (H10-H12) are distinguished This proved to be msuffiaent for cipcnmental worker* Lehmann (1926) and Bocll and Needham {1939) ha\’c inserted 8c\Tral intermediate stages We ha\c adopted the senation of the latter authors and have added another intermediate stage (Hi2a) We distinguish the following stages (Fig 45)

suet

H9 bhsiuh

Hio earl> blastoport

ticUe-tbaped blutopoft

HtoJ itfmdnmUr blaatoport (! moon)

Hii hoi*esboe-<haped blaitopoTt (i moon)

II nj large yoik-pluf stage

Ills small yolk plug stage

Htsl slltHhaped blastopore

II13 oeunl grooNt stage

The inapient blastopore appears as an irregular line between the equator and the \Tgetal pole (stage Hio) It assumes the shape of a sickle (stage HiOj) and acquires a marked bilateral B>Tnmctry Its plane of 8>'mme lr> which coincides ^^th that of the future embrj-o onl} now becomes apparent although it is determined much earUcr ‘ The region of the animal pole Is the future antenor end of the embrj’O the blastopore Itself marks the posterior end The line connecting the animal pole with the blastopore Is the future mid-dorsal line The area abo\‘e the blastopore Is the so-eallcd upper or dorsal Iip of the blastopore Next the blastopore begins to elongate and to enarcic the }*olk field U hen it Is semi arcular (*;tagc Hioi) the areas lateral to it begin to m\Tiginatc around the lateral lips The blastopore then assumes the shape of a horseshoe (stage IIii) L%Tnluall} the lateral in\'aginalion grooNcs complete the cncudcmcnt of the >*olk field ^^hlch graduall} dirappears to the inside (formation of a NTmtral lip) The exposed part of the j oU. in the stage of the circular blastopore is called j-olk plug (stage Hii J) The rapid in

I vw-c cnyctTvrcnl »fin {crtjUialwm In ibe tttkm rf ibt

fulortoppcT p f'b'bta'tf'p'^iOfUupUneoftynuDttryfcbridyma Uthatodhicrabr^-o


45


ward movement of the yolk continues, the yoDc plug becomes smiD (stage Hia) and cvcntuallj disappears entirely The blastopore has nor assumed the shape of a short silt (stage Hi 2}), which extends m longitudi nal direction (Le , perpendicular to the early blastopore) and marts the future anus This stage is conventionally considered the end of gastnilt tiom Shortly afterward the medollaiy plate becomes visible (stage H13) From this stage on up to that of closed neural tube, the embryo is called a neurula

3 W VOOr*8 HETSOD OT XOCAUZED VITAL SIADfUtO AKD ITS OEKEIAL aaSULTS

Gastrulation 15 primarily a phenomenon of cell movements and not of cell division and proliferation Many attempts have been made to study the cell movements, c g , by inserting a fine glass needle mto the blastuU wall and following its shift This and rimnwr methods are inadequate for several reasons The most senous objection is that such mechamcal dc vices may mterfere with normal development Decisive progress wu made when W Vogt applied a method of localised vital staining ’ The procedure Is briefly, as foDows A small particle of agar stained with NUe blue sulphate or neutral red is press^ against the surface of the blastuU for a short penod Such marlu remain distmct and well circumscribed for se\*eral days According to Vogt diffusion of the stain into neighboring cell areas is neghgibic and the marks do not interfere with normal development In thi< way it is possible to follow the movements of the marks t^ugbout gastrulation by continuous obscrv’ation Particulariy instructive were those expemnents m which blastulae or early g*stnilac were marked by a senes of alternating red and blue marks (as many as fourteen on one embryTi) and their shifts relative to each other obscr^Td In an exhaustive analysis Vogt obtained almost complete records of the gastrulation movements of all parts of the surface area, and even tually he was in a position to outline a coherent picture of the mechanics of germ layer formation The same experiments solved another problem

of no less importance Smee the maria persisted over a considerable time

it possible to establish their ultimate locations in the organ primordu by microdissecLion of early tail bud stages- This in turn, enabled Vogt

to project the pattern of the organs back onto the surface of the blastula

or of the early gastrula. His maps of the organ forming areas of blastulae and early gastnilac arc well known. They arc an invaluable help not only for a better understanding of gastrulation but as guides m transplantation and other expenments

  • S rmbr mrtbods hid bern derord pcttlooBly by Coodatc (ic**) Dttrfcr (HH?)

46


The limitation of Vogt 8 method should be cleariy understood The designationa of the different areas on the maps mdicate merely their ac tual fate ("prospective significance Dnesch) in normal, undisturbed de vebpment The> do not imply that the early gastrula is built up of discrete mosaic stones which differ actually from one another m structure or otherwise Such a view would mismterpret entirely the methodological rank of the vital stauung technique It is a tool for refined observation of normal development ue, a descnptive method not an analytical method It does not reveal intrinsic properties or potcnacs of the stamed areas Only potency testa like transplantation or isolation experiments are suit able for such an analysis It is correct to refer to the areas on the map as prospective notochord etc but not as notochord or notochord pnmordium


4 KAW or THE KASLT CaODElZ OASTXtTlA

Maps of the early gastrula are reproduced in Figure 4 Similar maps for the urodele blastula and for the anuran gastrula may be found m Vogt (1929) and Pasteels (1943) The map requires little comment Its out standing landmark is the line (*/) which separates the mvaginating ma tenal (prospective entoderm and mesoderm) from the nomnvaginating prospective ectoderm The prospective mesoderm forms a nng or girdle around the yolk field It is broadest on the dorsal side and narrowest on the ventral side In the German Uteratuie it is known as the J?nndcon«  ( marginal tone ) The most peculiar feature of the map is that the areas which will form the axial organs have their greatest extent in a di rection perpendicular to the median plane ue perpendicular to their ulti mate position Thus the prospective medullary plate area forms a transverse band with pomted lateral ends the prospective notochord is a sickle shaped area above the blastopore the somites are lined up in two transverse rows etc. It requires a considerable wheeling to maneuver all prospective areas into their ultunate positions

Vogt 8 map which was largely based on experiments on the European Tnlurus speaes has been revised by Nakamura (1938) using the Japa nesc newt, Tr pyrrhegasUr and by Pasteels (1942) using the axolotl {A mewenupt) The maps of Pasteels are reproduced in Figure 4 Cand D the> apply probably to other speaes as wen Both authors

are In virtual agreement with each other Their maps are m all essential points identical with those of Vogt but differ from the latter m several details as follows

The shape of the prospective notochord area is different In particular its lateral horns are less pomted and do not extend as far lateral as the>


47


doinVogtsmap AccordingtoPaiteels^theventralmajgmalzoneislazge 1> prospecti\c lateral platematcnal(/), whereas Vogt ccnsidersthegreatcr part of this area as prospective trunk and tafl sonutc material (/) Accord mg to Pasteels the latter matcnal extends to the median dorsal line and


Fio 4 — Mip of prD«pectl%e Areas of aroddecmlnyDf at Ux begittBlDK o/fMtniUtkm^

Utcral new B donal rfew of Triignts (ftoin Qilld »ft«r ^05t) C lateral vle« ^ doml lew of axolotl (after Pasteels, ipss) Denser brolces lines, netiral plate' less do* broken Imes, seaeral ectoderm coarse stippGiif ootoebord fine slippDng mesoderm • tral Lp of blastopore <k notochord g fill area f bejliining of mrajflnatloQ 0 Umlto/lft* vaxuuUoQ / lateral mesoderm / segetalpole // prooephroi and forelimb wo ApoiP^ ti\c tail report i-io sonutes t-io


forms a narrow strip between the prospective notochord and mcdulUiy plate Furthermore Pasteels has made detailed studies of the origin of the different parts of the somites which on his map form \cr} long and narrow stnps As was to be expected that part of the prospecti^ somite region which is adjacent to the prospccti\x notochord matcnal rqsrcseflls


48


Fm j — SecoDstiQctioo al Ut£ mo^ ci D CD ti <4 tbe moodena ™«ntL» , protected on a mtddle putmli lU^ (uTodcte) Tbe dolled lioca indicate tiu antcnor edn of w mantle In foor different staces awaantenor mesodenn border at tbe end of faatnuatlon (Ft^ o) Tbearca antcnor to aw a tbe “nuaodenu free field * TbesmratliKficate tbe dfrectroia of mmements (after \ Oft, 19*9)

Flos 6-10 — GutinlatKn hi orodelea (reconttroction after diafranii and KCtlooi, In \ oft, i9j<5) FIf 6 begnuuDf cfpatmbtion. FTf 7 larft jntk-ploc atafe FIf ^ imil] yolk-phif atafe FIct 9~io early niedsllaxy-pUtettaf^c Fhi- 6^ med^ aectlona Fir le. tnum ciae aectHD cot m plane z of Fig 9 poiterxir lalf of nenrola. a^arcboitcron w > UaiUKoele f/»blaatoport ri ■ notochord r-opper edge of tbe entoderm tioafb, ref-ertodmn, rafentodenn at^medonary plate matenu t^vcntial racaodmn, t/^toIF pbir.



lie* 11-16 — YlUl-cUiniu expcHmcnU oo orodde kiMtuIm (Fig* ii “*4 i9>9 Fif* IS 16 titer Coertlier 1915)

Fio ti —Eleven miik* (t-ri) pUced b the nwdiuj lice of a lil« bUttalx. Lateral ««  Tbc opper arrov iodicatc* UK borw o{ toTaKlaatlon ♦/■•pofail of od*io of blaitoport. i»» dotted Lon lodicate Uk border* betaeoi Uk main protpecUve area* (lec Fif 4 <d)

Fio ti — Tbe«arrKembnrolne»rlf Ufl*bod*t*je,»ilhpojlllooofinark*/ // < 4 *"*®“*'

<i«*hcad ■^oearaltabe /a^^phaiyox /«taIL .

Ikj ij —JI artin* of pn»p«t]»e meaodenn and entoderm Id the caHr

view TbedottedUoeiiridicatelbebordenbetaeenprofpectlveafeaifieeliK 4.0; >i" i

(proapectivc Dolocbord) and 10 (medtaa entoderm) a* In FI* lij r-mark «i

entoderm field "mark on tmtr^teral part of (uai^inalione (proapeebre lateral njemoenn;

f mark on anterior aoTTute material .

Fio 14 — The otne embnro a* In FI* ij la middle *aitnila fta*t hoU the chan<r» ihape and in povUon of Uk marka. *nK*olwarroaaCodt£ateUKdgccUooofp**ti w^ t°

on ibe turface Ibe dotted arroni Indicate the movement* after InTuttnalion UcHto*

lio 15 — Jlarkini In Ibe proTKClIre meduHaij plate area (dotted Qoei) iraitraU itaee m H»markfia Uk median Iinc'a*btew marks /"mark on tbeammaipn* Tbe arrovt indicate the dirretloci of movrmenti


fio ti —Tbe mine embryo a* in FI*- 1 j (n meduHary plate stace.


50


the inner median edges of the fntnrc somites and the part adjacent to the prospecti\T lateral plate area (i) represents the ^Tnt^al edges of the somites The outer lateral borders of the somites cannot be stained by superficial marts and must therefore be located m the deeper laj'ers of the marginal lone (sec p 53)

5 THE rOUCATIOH OF THE MZSODBUX

The mesoderm mantle is formed by an invagination of the mesoderm girdle or marginal lone around the blastopore /«ra^ma/;<7nis one of the four basic gastrulation mo\’ement3 as distingiushcd bj Vogt, The fact that the blastopore makes its first appearance m a dorsal position and gradually encircles the j’olL mdicates a definite sequence in time of the invagination of different mesoderm areas. The dorsal mesoderm (prospcc th’c notochord) m^’aglnate3 first somite material foiloHS around the lat cral lips c\TntualIy the ^Tnt^al part of the marginal rone Is tucked in As a general rule areas which are located nearest to the blastopore will m \*aginate first and their final position inside will be farthest away from the blastopore material which m\’ag{natcs late will settle near the blastopore

The nuip shows that the blastopore originates enUrel> withm the yolL field Therefore prospective entoderm will be the first material to m vaginate it will be earned mto the head region and will form there the anterior bhnd end of the archenteroD that is the antenor parts of floor walls and roof of the future pharjiix- Accordingl>, the gill sbts are mapped out on the carlj gastrula at a short distance from the eari> blastopore TTie dorsal entoderm is hnracdlnlclj followed b) the prechordal ma tenal which m turn is followed by the antenor end of the pro 5 pecti\*e notochord matenal This then is the first roesodermal area to be lucked under In order to \'isualizc dcarlj the fasbioD m which the notochord tnatcnal is brought into its final position let us follow two marks placed on prospcctii-c notochord areas Mark 5 (Figs 11 13) is located in the median plane and has a central position m the notochord area It will mom ton-ani the blastopore Inmginatc around its dorsal hp and disappear VTiHe it is still outside it will change its shape it nTll elongate and nt the tame time become slightly narrower this eipanswn in the longitudinal direction will continue after Its mmginalion E\-cntualK the formcrlj circular mark nill stain a surpnsinglj long strip of the narrow notochord ^lark 5 (Fig 13) illustrates well the enormous degree of cion gallon which the pro'pcctli e notochordal material undergoes Almost all parts of the gastrula undergo var^nng degrees of expansion during gastrulation KJon^alton or exf^artston Is the second basic gastrulation mo\e ment


placed m the median Ime immediately above and below the blastopore fl lustrate the situation The lumen of the head gut swells rapidly and exten sivel^ in the first phases of gastrulation whereby the lumen of the blaitococle becomes obhterated Whfle the notochord and somite materials follow the head-gut material around the dorsal and lateral lips in true in lagination movements^ the ventral ghdes mto the mtenor under neath the arch of the sickle and horseshoe shaped blastopore as a contlnu ous stream without actual invagination around a groove, a ventral hp is nonexistent during these phases of gastrulation Vogt once compared this shift with the rctractiOD of a stretched-out tongue. A mark pla^ m the middle of the yolk field (lo b Figs. 13 and 14) Dlustratcs this movement The mark elongates while it approaches the blastopore and disappears un dcr the blastoporal groove ^ving arrived inside it moves forward and will be found eventually as a broad patch m the middle of the floor of the mtestinc(ng la) It can be shown that all proq>ectlvc entoderm material which was located In the median line before invagination will form the median floor of the intestme Obviously lateral parts of the yoDc field will form the lateral walls of the archentcron trough. Mark e m Figure 13 likewise moves toward the blastopore and elongates In a direction almost parallel to mark 10 In its progresion inside, It spreads farther and at the same time, nmves upward and converges tow^ the median plane. It will be found eventually in the upper edge of the left wall of the archeoteron. A mark between 10 and e would stain an area m the middle of the lateral wall of the archenteron Again, regions near the blastopore will form antenor parts of the intestine, and regions at a distance from the blastopore will bvagmatc later and form posterior mtestbe. The entoderm formation is completed with the disappearance of the yolk plug (Fig 9) end no “late m\'agination of entoderm occurs A new problem arises when we visualize mesodermal marginal zone and cntodcrmal yolk field as being conUnuous on the surface of the blastula but entire!} separate structures at the end of gastrulation (except in the pharyngeal and m the blastoporal region) Even their du^wns of move ment inside are dnTrgent The mesodenn mantle spreads forward and downward the walls of the entoderm move upward Their separation must occur sometime during gastrulation According to an earlier ■\'icw, wbch was widel> accepted for a long time this would happen by Iniagi natron of a uniform archenteron and subsequent dclarobatfon of the mesoderm from the entoderm The mesoderm would be a dcn\ativc of the entoderm i c of gaitrul origin Accorduig to an altcmaliw inter pretation the sqjaratxm takes place before or during iniaglnatlon and the two germ laj'ers m\aginatc as autonomous units peristomial on


54


gin of the mesoderm It is one of the outstanding contributions of Vogt to a theory of gastrulation to ha\’C established for Urodda the correctness of the second altcmativt- If one considers for a moment maiis e and f (Fig 13) as one single mark^ then one finds that this mark is cut In two at the moment when It arrives at the blastoporal groove (Fig 14) From then on the two parts take entirely different courses (see arrows) and cventuaBj are widelj separated — one m the lateral plate the other at the upper edge of the cntodermal trough Marks which are partly on cntodermal and partlj on mesodermal temtory were actually studied and the realitj of the rupture was demonstrated bcjTjnd doubt Accord mgl> the horseshoe shaped hue on the map (hea\y in Fig 4, B and stippled m Fig 13) designates more than the border line between entoderm and mesoderm it demarcates the line of rupture and its absence (on the map) between pro5pectl^T gills and fint somites merely expresses the fact that entoderm and mesoderm will remain continuous m the pharyn geal region

7 THE GASranuiTio^ uo^'El^E3^^3 or the raosrecTTvr rcrongm The gastrulation movements of the prospectivT ectoderm (Figs 15 and 16) were studied bj Vogts collaborator K, Gocrttler (19^5) and b> S<iechtman (1932 for Tr tensvt) Since the embrj-o retains its 5i« and its sphenctl shape throughout gastruIatioD the animal hemisphere (prospective ectoderm) must be expected to compensate for the in\Tiginating \entral hemisphere b> ertensTsx expansion and thinning This is demon strated bj c%'cr> mark placed on the prospecti\*e ectoderm except on the animal pole The extent end direction of the mo^Tment5 of different parts of the ectoderm will be discussed separately for prospccti\-c epidermis and prospcctl^T medullary plate

Frosfvclre medullary pWe — If the animal pole is stained (Fig 1 5 P) then the mark will be found first, m the anterror traim-crse part of the medullary fold and bter on m the floor of the forebrain Its shape is al most unaltered The ammal pole then is the only area of the gastruJa which remains stationary Marks placed in the median line will stay In the midlmc and elongate m the direction toward the blastopore The nearer to the blastopore i^ the nearer to the future postenor end the more will a mark elongate during gastnilatron and neurulation (compare m end n) The median marks wfll be found m the floor of the spinal cord It IS important to notice that eli material which Is located m the median line of the early gastrula remains there and thus makes true concrescence (ic grou mg together ) of lateral areas impossible The lateral parU coni-crgc toward the median line but ne\-cr concrescc The same was fUlod before for the notochord (True concrescence takes pbcc when SS


the neural folds fuse or m heart development, but nowhere m gastrula bon ) Lateral marts show deariy the convergence of the lateral parts of the prospecU\*e medullar> material Mark o in Figure r 5 is parbculariy suitable to Illustrate the “wheehng movement {Sckwcnhtng, Goerttler) toward the median line the fixed point being the median end of the mart near the animal pole The parts of the mark which are farthest away from the mldlmc traverse the longest distance This wheeling roo\'cmcnt takes place largely during the first part of gastrulation it is followed, dunng the later phases of gastrulabon by elongation

The prospecb\'e epidermis occupies the ventral sector of theanimal hem isphere Its mo\’ements are in confonmty with those of the prospccb\*c medullary material They are charactenied by a very considerable ex pansion in a fanllke fashion In ventral view this expansion appears as a ‘‘di\*ergcnce The old term epiboly* (“growing over* ) may well be applied to this maneuver, smee this spreading is at the same time, a process of growmg o\Tr the m\’aginatmg mesoderm and entodenm 8. suiauar

The proq)ecbve medullary area and the prospccbve notochord ha\*e several featura in common Their longest diameter Is In a transverse direcbon before gastrulabon and m longitudinal direction afterward The gastrulation mo\’ements of their median as well as of their lateral parts are almost identical although the one mvaginates and the other does not Their movements are perfectly mtegrated with each other, since they ha\*e a long border in common along which they remain conhnuoas throughout gastulation A similar comparison may be drawn between the duTTgence of \’entral end ventrolateral ectoderm and that of ventral and \*entrobteral mesoderm All these obsm-abons taken together illustrate cmphabcally the mtegrabon of all gastrulation movements the uni formity of the process as a whole, whose basic trends — elongation con

\*ergencc dhTTgence etc — transcend the border lines of Invaginating and

nonm^*agmati□g areas and of the prospccti\‘e germ la>Trs i) PREPARATION OP DYED AOAR

Xne blue sulphate and neutral red arc generally used as \ital (non toxic) djTS Prepare a i-s per cent solution of agar (cp , powder or shreds) m distilled water Boil brieflj Pour thin films of the solution while It IS stDl warm on carefully cleaned microscope slides or on laifer glass plates Allow them to dry thoroughly (i or more day’s) Place the agar plates in a large \-olume of i per cent Kile blue sulphate or i per cent neutral red and let them stand for i or more day's ash off thecrcessu’e dye and allow the agar to dry again The plate can be kept indefinitely

56


m a dustproof wrapping Before use, moisten a small area with a drop of water After the agar is swollen (1-2 nun ) scrape off narrow strips of the agar film with a scaIpcL Dyed agar plates may also be kept m 70 per cent alcohol, which must be nnsed off very carefully before use

c) VITAI. STAININO EXPERIIIENTS ON THE EAilY GASTRTJLA OF UrtxJela

Read carefully section aa consult Vogts and Pasteels maia of the prospective regions of the urodele cmbiyo (Fig 4)

Matendfor EspenmenU J-4

AinhysUima opaaim funcidum or tignnum Tniunu pyrrhogasUr or Tr torostis stage Hio or HioJ (p 45) agar stained with ncntral red standard cqmpment (p 41)

ExPEHxuEifT X StACnxo or THE Ufpe» Lip or the Blastopoee (PK cerpEcnvE Notooioid)

ProetduTt

Select a number 0! healthj gastnilae Removx the outer jell> mem branes but leave the vitelline membrane intact Wash the embo’os m sterile iV Holtfreter solution and transfer them to an operation dish inth the glass ball make a depression in the Permoplast into which a gastrula will fit tightly Transfer a glass bndge of suitable sue and a piece of red or blue agar to the operation dish Under the bmocular microscope cut out a square piece of agar which will co\Tr the median one third of the upper Up area Place the embr^'o m the depression The embrj-o will usually rotate into a position m which the blastopore is not visible Some time* one succeeds in moving the embryo back with the hair loop If this docs not work apply one of the following techniques Turn the blastopore upward and whfle the embryx) rotates slowly back to Its former position mark the posltwn of the blastopore on the Permoplast at the edge of the groo\*c After the embryo has come to rest, push a piece of stained agar between the Permoplast and the embryo at the marked pomt Press the embryo against the agar by gentle pressure with a glass bndge Another tnck Is to puncture the \ntcninc membrane (and the embryo) m se\“eral places with a v-eiy fine glass needle Thu will partly release the mner pressure and the embryx) wfll remain in a posibon with the blastopore upward The fine holes will heal at once Place the agar on the median part of the upper bp (5 in Fig 13) end press it against the embryx) with a glass bndge Stain for 15-45 minutes (dependmg on bow deeply stained the agar 15) Remove the glass bndge and the agar very cautiously with a h ffir loop and transfer the embryo to a dish with iV Holtfrctcr solution, Prvtocols — Protocols are of prime importance Label the embryo and make a sketch indicatmg the position of the mark. Observe the embryo once or twice dafly and make a senes of sketches Obsmo and record m the sketches the changes in position and m shape of the mark- Note its invagination and elongation After invagination a deep stain may be visible through the epidermis

Dissidion — Dissect the embryo after it has reached the tail-bod stage, either alive or immediately after a abort fetation m 10 per cent formaldc hyde (the dye will fade out slowly in formaldehyde) Mount it in a Permoplast depression with its dorsal side upward Very cautiously remove the dorsal epidermis, the brain, and spinal cord using the glass needle and hair loop thus exposing the notochord and somites hlake a sketch, mdicating precisely the position and extent of the stamed area In the pmtocol state your results dearly

Exmnforr j Vetax. SrAnriMO or the Peospective Sojotes Stam the left or right one third of the upper blastopore area (5 m Fig 13) Proceed m all details as before Observe the invagination and dissect the embry*o m &n early tail bud stage

ExpEanoarr 3 \ nAL Stadoho of the Pho sp ect ive MedulijUit Piaie Ag^ mark on the Pennoplast the po5itx>n of the blastopore or pane tore the embryo Place a mark between the blastopore and the animal pole nearer to the latter During the following da>'s note the dongation of the stained material If the maik was not exactly in the median plane note the conveigcnce of the mark toward the medum plane, m additxin to its elongation (see Figs 15-16)

ExFrancEirr 4 VrrAE Stasomo or the Axdcal Pole N ote that the mark scaredy changes its position and shape It wiH be found in the anterior part of the head {p In Figs 15 and 16)

d) VITAL STAININC 07 AREAS OF THE NEURULA OF

Matenal for Exptnmcnfs 5-7

Ambysiama any speoes stage* H14 and Hi 5 agar stained with neutral red standard equipment (p 41)

58


ExpiamErr 5 VrtAi STAUffwo at the Piospective Eye roxuwc Aeea (Mandkot 1Q29)

Remove aH mcmbranea mdudisg the vitelliiie membrane Place a mark, on the antenor median part of the meduUarj plate The mark should cover the median one-third or one fourth of the plate Its anterior border should cover the slope of the tninsvciac medullary fold, m stage

Hi 5 (Fig 17, a) AUowtheembryolodeveloptostageHaborstageHap

£W.

No*.

BoL L G.

Ot


a


St m.



Fm. 17 — \ it«l q{ bead stractnre* (alter CaipcsUr 1917} fi —border betmrai

bead aod trucik, Bai -balaoccr £ — cTe G ~s^Sa, L.— kna, Vaz.— oaaa] placode CV.— otocytt, Stmm — tTfrmodenm.

The stain on the eyes will be visible from the outside Fix the embryo m 10 per cent formaldch} de Shortly after fixation caxefuBy dissect the bead m an operation dish with Pennoplast ground Remove the epidermis with a glass needle and slit the brain open at the dorsal side. Betentune the cx tent of the stained area in the bram and the ej-ea If the ongmal mark extended too far posterior then the floor of the forebrain and midbram may be found stained It is surprising to find that the area in the medul laiy plate from which both eyes originate is one uniform, median region not separated by a piece of prospectnx brain During neurulation the mart will gradually expand to t^ sides and become dumbbell shaped The lateral parts will be folded up and come to he in the lateral walls of


59


the forebram from where thty will be evaguuted as optic vesicles The narrow median part of the mark will persist m the optic stalks. The part of the bmm which separates the eyes In later stages is den\Td from nu tenal which was located posterior to the eye area m the medullary plate and which has moved forward during ncunilation These findings have been of great importance m the interpretation of the origin of Cydopla, a malformation in which one single median eye, Instead of two eyes, fa found (sec Adelmann, 1936)

ExpEiDtEKT 6 Vital Stadiwo op th* PaosppcmT hAsAL, Baiakcxi, Lots, Gnx, Eax EertmEau

(Carpenter 1937)

Procedure

Remove the jeDy membranes but not the vitelline membrane Mount the neurula in a depression m an operation dish, so that the prospective head region points upward Press Pennoplast from the edge of the groora gently against the embryo to hold It tightly m position Stain one of the areas listed above using Figure 17 for your orientation Make several experiments FoUow the shifting of the marks during neimilation and make sketches of transitional stages and of the position of the mark to stages H29-H3S

ExpEanoofT 7 Vieal Staikino op mr Boann MTwrni Head AimTamra

(Manchot, 1929)

Procedure

Place a mark in the middle of the medullary plate at the point where the folds come closest together or place the maik at the same level on the left or right prospective epidermis outside of the medullary folds {B to Fig 17 c) Allow the embryo to develop to at least stage H24 Identify the position of the mark, dissect if necessary' Note the number of the soimtcs m front of the mark Visuallre that about two-thirds of the medullary plate m the early neurula is prospective head and only one third Is prospective trunk and taD The latter part stretches enormously in tail bud stages the taxi originates largely by growth from the tail bud

e) VITAL STADm^O OF LATEEAL-UKE PLACODES

(After L S Stone)

Aquatic vertebrates, including amphibian larvae, possess a special iyT< of sense organs — the lateral line organs which arc receptors for water pressure and aid the animal In its orientation In flowing water They are cop

60


shaped stmctuies composed of sensoij and supporting cells and are ei posed to the surface. The} are arrange m lines which fonn specific pat terns on the head trunk and tail Those of the head are innen-ated b} a special branch of the nervtis faaaha those of the tninl. and tafi by a branch of the nemis vagus

Their mode of ongin is nmqut in several The three trunk end

tail lines charactenstic for urodele larvae onginate from ectodermal thick

eningSjOr placodes irhich are part of the vagus system and are located

immediately behind the otocyst The deeper cells of these placodes be come detached and migrate a.udad m a body Thej glide along the inner surface of the epidermis In three distinct columns forming one dorsal


a


Pxo s — ^Mul tUinlos of the UtenJ hne pbcodfs (£roDi Sume igu) Set text

one middle and one I’cntral line On their 'wa> they deposit at regular Intervals clusters of cells which differentiate Into the cup-shaped sense organs The latter push through the ectoderm and are thus exposed to the surface- All placode material is used up when the migrating pnmordia ha\'e reached the caudal end of the taiL The pnmordium of the sense or gans IS accompanied by the lateral line branch of the \agu5 nerve which originated at about the same time and in a fashion Kimilwr to that of the sense organs le from a %'agus placode which, bowe\*er was supplemented bj neural-crest material Side branches of this nerve innei^*ate each in dindual sense organ The lateral Ime organs of the head and their ner\xs originate In a simDar fashion from placodes belonging to the facialis s\'stem

The mode of origin of the lateral line sense organs Is of piartlcular in tercst because it demonstrates long range directional migration of cell

6i


groups along speafic patlis This process can be observed on the hving embryo with the aid of vital staining The intriguing problem of the de termination of the lateral line pathways has instigated the rlnwira l a perimentof Homson (1904) in which parts of darily pigmented embryos of R sybattca were combined with parts of the lighter R. paJusiru embryo and the deposition of dark sense organs on the light epidermis was observed This was one of the first instanixs m which the method of heteroplastic transplantation (previously worked out by G Bom) was applied in an analytical experiment. Stone has ointinued this analysis using the methods of transplantation of placode pnmordia and of vital staining His papers (1922, and particularly 1933) should be consulted

ExpaancxNT 8

AfaknuJ

Ambyst&ma — anyspeaes stages H28-H30 operation dish

agar stained with neutral red glass bridges lily cups

Procedure

1 Remove all membranes and plan the embryos In an operabon groove, ngbt side up

2 Locate the otocyst above the second visceral arch Place a piece of red agar on the epidermis covering thcoto<yst and the region immediatd) behind it (Fig rS, 0) Hold the agarin position with a glass bndge Press tightly

3 Stam for 15-25 minutes untfl the epidermis is stained deeply red

4. Remove the glass bndge and transfer the embrjo to a Lily cup

5 Make a sketch of the head indicating the stained area

6 Observe the embryo twice a daj for several days under high power of the binocular Watch the middle line grow out boriEontaDy across the middle of the somites It is an elongated, club-shaped structure which mo\Ts backward and lea\’e5 behind on Its path darklj stained spots, the lateral Ime sense organs. A similar though smaller red mass— the pn mordium of the dorsal line — grows out somewhat later It turns dorsad and follows the upper border of the somites (sec Fig 18 b and Stone, 1922 Figs 1-12 also Stone, 1933 Figs, 1-3) Make careful sketches


7 Students who wish to observe the finer details of the differtntiattoa furno should construct an obscrv’ation chamber (Stone 1933 PP


winch allows observations on the narcotittd animal under the compound microscope

8 The de\Tlopment of the lateral lines of the head may be studied m a similar way by staining the prcauditor facialis placode


Adeliiank H B 1936 The problem of Cjxlopia- Quart. Rev BloL, iz 161 384 Boell, E. Jt and Needham, J 1939 Morphogenesis and metabolism studie* with the dner oltramkromanametcr Ell. Respiratory rate of the regions of

the amphibdm gastnila Proc Rzrv Soc London, B 1*7 363 Baows M G xo 4 i CoUapte of the arcbenteron In embryos of and Raiw

Jonr Eiper Zool 8 S 95

CAapEsmat, E. 1937 The bead pattern studied by ntal staining and

transplantation methods. Jour Exper ZooL, 75 roj Drrwmai, R. S 1917 On the use of Nile bhie sulfate In embrj-onic tissue transplantatioa. AoaL Rec. 13 493

GOEXTTLEa, K, 1935 Die Formbddang der MeduHaianlagt bo Urodden. Arch, f Entw’nwdL, 106 503

Goodalx, H, D 1911 The early desxlopment of Spderpa bUxnealtu Amer Jour Anat za 173

Hauisox R, G 1904 Experimentdle Untersochangen Uber die EntwfcUung der StnneforgiXM der SeitenBnie bei AmphJbten. Arch. mUx Anat 63 35 LzJQiASfS F E. 1936 Ents^IeUungjstonmgen to d« MeduBaianlagt s-on Tnicn eneugt dorefa 0 QteTUgmmgs-Defel.te. Arch, f Entw^mech zo8 143 Maxoiot E Z939 Abgrenxnng des Augenmatoiala und anderer TeJbearie in der Medullarplatto- Arch, f Etotvmech iz6 689.

ISAXAMtrtA 0 193&. Tall forraatwa In the urodeie. ZooL hlag ToLyo 50 44a PastxeU J 194a bea obvrvatfoQs coBcemlng the maps of prwimiptr.*e areas of the young amphibian gastrula (/f «A/yi/<»aio and Dweflejtuj) Jour Exper ZoOl^ 89 355

SamarrMAX A if Z933 ifostment aod kicalization of the presumptive epidermis in TnJitrtis tarcrus (RalhLe) Umx of Cahf Pub m Zool., 36 335 Sro t, L S 1931 Eipenments on the dc\*ek»pmeiit of the cranial pngli.t md the lateral line sense organs In A mNysiema pumdolam Jour Exper ZoQL, 35 43Z

1933 The doelopment of literal 4 ine seme orgam In amphibians ob*er>‘td

in b\ing and >^la^ltalned prepariUoai. Jour Comp NruroL, 57 307 \ OCT W 1935. GestaliangMni])-se am AiDphitIenl.eun mit ortlIcbCT Mtalfarbuag I Metbodik. Arch, f Entm mech., zo6 543

1939 Geilaltungsanalyse am ArophlWeiiEeim mit ertDcher \lUlfarbung II

Gt trulalKm und MesodennbOdang ba Urodeleo and Anurea. Ibid ise 384


3 SOME ANALYTICAL EXPERIMENTS IN PREGASTRULATION STAGES

a) ARnnClAL PARTITEXOGENXSIS in TTTE frog PRICKINC

(After Parroeotcr)

Frrtilt^ljem that is the union of the two gametes has two Important consequences B> the fusion of egg and sperm nuclei the diploid chromo63


some number is restored, and two different sets of nuclear hereditary fac tors arc combined Furthermore, fertilisation activates the first cieavage and thus initiates development

The occurrence of parikcnogenuis (i^ , development of the egg without insemination) m a number of animals — for instance, in rotifers, aphidi, Cladocera and the honeybee — abows that neither the fusion of two celb nor the fusion of two nuclei is a prerequisite for the Initiation of de\-ck)fh ment In 1901 Jacfjues Loeb made the discovery that the sea urchin egg can be stimulated to develop into a normal larva without fcrtllizatwn, by placmg it In hypertonic sea water ('artificial parthenogenesis ) The con* elusions drawn from normally parthcnogenetic eggs can thus be extended to eggs which normally require the sperm for activation Many different chemical and physical agents are now known to activate eggs— for in* stance surface active substances like fatty aads which have a iligblly cytolyzmg effect, hypertonic and hypotonic salt solutions, temperatare changes, irradiation and even pneUng with a fine needle In addition to the sea urchin and starfish eggs those of several annelids, of molluaki of the frog and others, have responded to such treatments Students who arc interested in the theories which have been advanced to account for the activating role of the on the basis of artificial parthenogenesis a penments are referred to J Loeb (1913), \Mlson (19^5), Spek Just (1939), Tyler (1941)

Aritjxcial perthenegenetu tn the frog s egg — The frog s egg is rather rt fractoiy to chemical agents, but m 1910 BataiUon discovered that artlfi* cial parthenogenesis may be obtained by puncturing the egg with a fine needle of glass or platinum However this treatment is successful onl> If the needle is dipped Into frog s blood and a small amount of blood Is In troduced into the egg The role which the blood plays has not been ex plained satisfactorily but its mdispcnsabUlty has been confirmed by all subsequent observers A small percentage of porthenogcnctic frogs develop into tadpoles and Loeb, Parmenter and others have succeeded In rabmg a number of these through metamorphosis to sexual maturity (see photographs m Loeb 1931)

The chromosome situation In these specimens is of particular interest Parmenter (1933 1940) found a number of young tadpoles to be baploW but oil those which had metamorphosed were dfplofd According to Par mentor the regulation of the chromosome number may occur cren cleavage starts It will be remembered that in the frog the egg Is in stage of the second maturation spindle when insemination takes pl^ce In cases of experimental parthenogenesis the spindle may be withdrawn into the egg and the formation of the second polar body suppressed to


64


that the egg starts with a diploid number of chromosomes (all den\*ed from the female pronudeus) Or both polar bodies may be formed but the first nudear ivisioo may not be followed b) a cy'toplasmic di\*iaiOD


ExrntiuENTo

2 oMilating females olILpt pterts Petn dishes or finger bowls

or other frog speaes i S>Tacusc dish

I nonovulatmg female as a 3-4 >'ei> fine glass needles

source of blood paper towels

10-12 dean slides spring or pond water

2-3 pipettes

StcrUuatton —In this experiment it is imperative that contaminatMn with sperm be av-oided SterfUae glassware, etc. m the autoclave and wash yoMT hands with 70 per cent alcohol wash the frogs under ru n n in g water Have no male frogs m the laboratory It is also advnsable to autodave all water to be used in this expennsent.


Prwdun

1 Prepare the blood of the nonov’ulating female as follows ash the female and pith it Open the abdomen and expose the heart Cut ofi the tip of the ventnde and allow the blood to accmnulale m the pwiordial cant) or in the coelom Close the abdominal skin flaps until you arc ready to use the blood

2 Strip eggs on sterile slides (p 34) Stnp 2 rows of eggs onto each slide. Prepare 6-10 slides m this wa>

3 Smear eggs with blood. Dip a piece of musde into the blood pool of the frog prev'iousl) prepared and smear all eggs with blood. Be careful not to exert any pressure on the eggs

4 Pnehng — Under appropriate illamination pnek each egg with the glass needle somewhere withm the annual hemisphere The germinal vesicle IS usuallj under the animal pole. It should not be injured. PniioL, 3 539

ilotoAW T H I9»7 Experimental embryology chap xrffl. New\ork Cotnnha Uaivenlty Pim.

PAuaarmc CL. 1933 HapWd dfploM tnploW ami tetnpWd chroniosooie im»ben and their ori^n In partbeo<^aietJCs^ developed larvae and frogs cf Eau pipiev and Rana pclmsiru Jour Eiper Zbbl 66 409.

1940 Chromosome numbers in RoMe fusca parthenogeneticaHy dertloped

from eggs mith knoim pxikr body and deavage histones. Jour hfosplu, 66:34* Spar, J 1931 Allgeincine Phyilologle der Entwlddung und Fonnbfldung. In £• Ga.Lnoajf Lehrbuch der allgemeuien Pb^iiologie. Ldptzig Thicme.

Tvlet, A 1941 Artificial parthenogenesis. BioL Rev 16 S91 \\ilso:<E,B 1935 TbeceDIndevdopmcntandberedity cbap.v Newlork Ms^ mUlan.


b) ALTERATION OF Tin: CLEAVAGE PLANE DY PRESSURE

(After G Bom)

Some of the earliest experiments m embryology were inspired b> Wclsmann 8 theory of devdopment and heredity (see Wcismann, 189a) Thi 5 theory was the first to attribute a decisive role in organ detcnnlnation to factors located in the chromosomes These factors which are equivalent to our genes were called detenmnants by Wdsmaim The cytoplasm he considered merdy as building material He construed an mgeaiou* ichcmc bj which the determinants would be distributed o\‘cr the dif ferent areas of the doxloplng cmbryTi According to his hypothesis o quahtatnx nudear dmslon the two daughter cells of a di\'iding cell would obtain qualitatively different assortments of determinants and in thk way the germ plasm would be broken up mto its units by successive mitotic divisions. For instance m cases where the first cleavage plane comcides with the median plane of the future organism one blastomerc would obtam all determinants for left organs and the other all detenni nants for “right organs In a later step the determinants for neural structures would be segregated from those for epidermis and so forth^ un til cell would be left with one determinant, which would then be instrumental in its structural differentiation Welsmann s theory is thus the prototype of a prefonnlstic theor}?^ — more spe cifical ly of a nuclear prefonnatiOE It is now abandoned as a theory of embryomc differentia bon, because we have ample evidence that in mitotic nuclear divisions both daughter rrlin receive quanbtatrvely and qualitatively equal chromosome matenals However, parts of Wosmann 8 theory are mcorporated m the present gene theory of heredity Moreover his theory of differentia bon challenged the most outstanding embryologists of the turn of the century and uu^iired several dasrfcal eaqicnments which arc now comer stones of experimental embryology — for instance, Dnesch s eiqpeiuncnt of separating the blastomeres of the sea-urcMn egg Roux's famous pricking experiment on the frog's egg and Spemann s constnction experiment (s«p 69)

The experiment of ‘ deavage under presarurc was devised by Dnesch (1892) to test the validity of this theorj In the sea urchin egg and m the frog s egg the first two deavage planes arc meridional, but the third is equatonaL If the eggs are mounted on a glass plate animal pole upward, and then slightly compressed by placmg another glass plate on top of them, the third clea\'age plane will also be mendwnaL If the pressure Is released at the S-celi stage the fourth deavage plane will be honrontal This procedure does not change the arrangement of the cytoplasmic structure, but it results In a complete reshuffling of the nudei (see Weiss 1939 Fig 33 p aoo) Some nuclei whidi in normal development would be located m dorsal organs now find themselves m a ventral position and should accordmg to Weismaun detcraunc dorsal structures at the wrong place Generally speaking a completely disorganued patchwork of struc tures should result. If the hj’pothcsis of unequal nudear division were cor rcct Instead normal embryos developed which proves that the blastomcrc nudei cannot be quahUtivdy different The orderly pattern of difTcrcntiation must be brought about by other mechanisms Dnesch experimented on the sea-urchin egg The experiment was repeated successfully on the frogs egg by G Bom (1893) and O Hertwig (*S93)

EXPEBIMENT lO


Material

fertilized eggs of JL ptpiens or other ^>ecie 3 (artificiall> msen mated

[p 30D

scalpel finger bowls

10 ilides Lfl> dishes

Pcnnoplast

Procedure

I Obtain fertilized eggs b} artificial insemination (p 30) a Qean and di^ 10 microscope shdes thoroughl) Place narrow stnps of Pcnnoplast near both ends of 5 shdes and parallel to the short edges. The strips should not be much higher than the diameter of the eggs and the membranes,

3 Ahnnt t hour ii rt-ifirmnn^fnina ttnn when the jclly membranes are swollen cut out 30-40 mdi\ndua] eggs, using knife (scalpel) and for ceps. Be sure to pick out fertilized eggs. Eggs whose \TgctaI (light) poles face lateral or upward are usually not fertilized On each shde with Permoplast stnps pbee 4-6 eggs scpanitcl> , each In a small drop of water Allow tune for the eggs to assume their nonnal position, animal pede upward Set 10-15 eggs aside, as controls

4 Place a second slide oN-cr the eggs so that the slides arc held together

by Permoplast, Press the ends bIow1> but fiimlj with j-our thumbs until the eggs are flattened Control this procedure under the binocular micro* scope

5 Watch the appearance of the first c 3 ca^‘age, 2-2 J boun after fertilization The second cleavage will follow I hour later (The tunes for other amphibians are different.)

6 Observe the appearance and the plane of the third clca\Tige whidi IS perpendicular instead of parallel to the plane of the slides hfihe sketches of se>'eral such S-ccU stages and compare with Dormal 8-ceB stages from the same batch of eggs

7 ^^^len all or most of the ^gs on a shde have reached the 8-ccll release the pressure cautiously lift the upper slide by pushing two point Instruments through the Permoplast stnps on each end Discard the shnormal eggs and place all others in a finger bowl or a Lily cup Ascertain that no horuontal clca^’age plane is present by turning the egg* sideww

8 Label the dishes and protocol os foIloi\*s

nzssTTu Ecrauveer llirch jjd t 00 PJC. fertilized

a 00 TM. 30 ej*s prened


6S


3 00 PJC firtt dcavige la *8 eggs

3? p P If third pliae mcfidiotttl in 3^ tboonnal, 5

etc.

9 One half to i hour later observ’c the appearance of the first hon xonlal clea>'age planes.

10 On the following dai^ maLc observatjons on the further de\*eloi>ment of compressed eggs Those which dc\'elop normally are of course of special importance for the problem under discussion AfaLe protocols sketches formulate m j’our own words the imphcalwn and the results of the experiment

BIBUOGRArJn

Boait G iS93 Gber Drudcvmudit »a Ftojch-Eieiii. AaaL Aai 8 6o^

DuEsen II iSgJ ExpertmenteDe \ craodmiog des Tj-pus dcr Furchung und ttire Fol|;ea. Zeattdir f â– nns.Zool 5511

IlraTWiOjO 1&93 tlberdenttertdermtenFurchunpxrilenhirdieOrjanbildaiig des Em'bO ‘0 f miVx Antt 4x166)

Hmacr J S., and De Bra G R- 1934. The tlmcau of eipahncntal embiytdojj pf) 838 Jseir\ork MacmlHaa.

MotcAK T H 1917 Expemnental embriolosy pp 473 3 Newkotk Colombia Unlverdtj Prws

tSeisiLurs A. 1891 D« KeimpUama «id* Tbaonc der \ c erb une Jen* G Fischer

1893 The eerm pU«m EajJish timas New ^ ork Scribner’s.

^\Eas,P 1939 rnnapla ol devdoptnenl, pp 19SS Newport. HoIl

c) THE PEODUenOV OF TWD, EitDtt\03 A^^) OE DUT»UCAnON*S IN Urodda by coNSTEJcnoN (After Spemann)

Few experiments ha\*c influenced the course of experimental embrjt)! og> moTC deeply than Dnesch % expenment on the sea urchin egg m which cmbr>*08 were produced out of 7 egg b> isolating the firat blaatomcres This expenment demonstrated an unexpected rcgulaln e property in carlj dc\tlopmcntal stages of an organism which shov-s little regenera tl\*c power in adult life This result was at once interpreted os a strong argument ogalnst anj mosaic thcor> of dcxTlopmcnt and pai*ed the wa} for our modem epigenetic concept of dcxtlopmcnt Because of its funda menial importance it was repeated on the eggs of man> other iniTrte brates and xtrlcbratc^ in manj instances with the same result In the urodclc \.v,m embrvos and duplications can be obtained best b} constricting the a-ctU stage m the plane of the first clcav-age using a child s hair (Spemann 1901 190a 1903 and others) Spemann 5 papers giNT an ediaustut analpis of the results If the J blastomcres

  • 59


were separated completely by a deep constnctwn, then the following results were obtained In a certam percentage of cases i whole embrjtu, that IS ‘identical twins, developed They were small m me bat other wise normal In a larger percentage, only i blistomcrc gave a noniul embryo whereas the other half remained an unorganixed, though vulic, sphencal structure. The explanation is as follows In the first case ttc first cleavage plane (plane of constnction) comaded with the fntnre median plane of the embryo In the second instance the first dettagt plane separated future dorsal from future ventral structures, end only the dorsal half gave a normal embryo The unorganixcd sphere of ccUi was therefore called *T>eIly piece {BaHcksiUci) This implies that, in the sah mander the first cleavage plane and the median plane of the embryo have no constant relation TTiat this explanation is correct can be demonatnted by examining constricted eggs dunng gastnilation Some eggs will be found in which both isolated blastomcrcs develop blastopores and domJ bps and these mvorlably give identical twins. In the majority of cisa only I embryo has a dorsal hp and undergoes complete gastnilation tod It Is this half which develops mto the complete embryo Spemann concluded that as early as the a-ccU stage the dorsal half differs quabtathrely from the ventral half In that it contains “something*' which enables it to undergo typical, proportionate differentiation, l^is “dorsal qnilityi which IS lacUng m the \entral half was Identified later as the “oigtnijer” and the constriction experiment may thus be considered as the first step In Its discovery

When the two blastomcrcs arc not separated entirely but constricted slightly so that they form a dumbbcD shaped structure, anterior doph® tion duplidtas anterior may result (Spemann, 1903), resembling tirt two-headed monsters found o rra^inniill y m mwn y n^prf^hrfttea Thcricgit^ of duplication depends on the d^ree of constriction alight constdctioB results in slight dupbcation of the head and such cases may even bi'*« * median ej'c in common deep constnction almost to the point of separs turn results in embryos which are fused together only at their tall ends. Strangely enough dupliatas postenor (monsters with one bead sod two posterior ends) never occurred m this experimenL Spemann explains this fact on the basis of the mechanics of gastnilation in constricted efiJ* (see Spemann 1938 p 159) Thus the constnction experiment contr^ utes materially to an undemanding of the origin of identical twins sad anterior duplications

In some bat not all cases of complete and incomplete twinning the symmetry rdationsof the internal organs (curvalureof the heart and of the

intestine posiUonofm-er gallbladder etc.) of one twin were found to be


70


inverted This condition is known as “situs in\*eTSTis ^'iscc^lm ct cordis” and IS again identical with the same abnormalit} found occasional!} m vertebrates In this abnormality is vcr> rare even m identical twins but it is the rule m laterall) conjomed human twins (Newman it^o) It IS significant that in the constriction experiments aH left embr>-os showed normal situs and right cmbrj’os showed the minor imagmg The ci penment of constnction thus broaches another important problem — that of the origin of bilateral a 5 }’mmeti> m vertebrates


Exnantnrr ii


JUaUnal

Tnturus nndercenr* or Tr pyrrhogasitr a watchmaker forceps


child a hair hair loop

glass without Permoplast


Procedure

I Prepare Ike hair for ccmstnction — Select a fine hair hold it between the thumh and forefinger of your left hand so that it forms a loop as mdicated m Figure 19 a Use the natural bend if it a curly Under the bi nocular microscope or loupe tie the upper end through the loop twice using the watchmaker for c e ps Then puD on both ends until the diameter of the loop is slightly larger than the smaller diameter of the oval capsule (a-ai mm m the case of Tr vtndescens) Cot both ends at some datance from the loop and submerge the loop in the operation d?<h (without Permoplast or paraffin bottom)

a Prepare ike emiryo — TrUurusTtrtdacensfcmjiia caught m the field during March and April will la} their eggs m captrvit} The eggs are fold ed between the lea>’e5 of £Z<xi«x The first dca\-age occurs 8-10 hours after fertiliralion Eggs should be collected twice a day and watched at short intervals unless the laying has been observed The outer sticky membrane B milL} In some eggs it can be peeled off easily and the egg is then visible inside the tran^iarent capsule If one finds it difficult to re move the outer membrane, one may leaiT it mtact.

3 Consindum — \\ ait until the a-cefl stage is wdl under way Hold the hair loop upright directl} m the center of the visual field so that it appears as one streak. Hold it in place with one forceps and push the egg into the loop with the other forceps (Fig 19 6) V.'hen the loop a appromnatel} around the middle of the egg capsule pull gentl} at its

• TrUns tiridmtnj t» prtfoaHe beextne in Tr fT^hetisUr tht Titelhne mi^hr a pf £rt quently bants noder tie inner prenore of tbe constricted capsale, reniltm* tmnlly In the loss of one half (see Strettt,i«o) inTr »ir«etcn« both twins nirTtv etna hI*bperanU*e of free ends so that it fits loosely but docs not yet constnet- Shift the loop until It IS CCTctly in the middle of the capsule The slightest a^Tniaetry will result m a conspicuous sixe difference of the egg halves after constre tion, which will obscure the results Constnet the capsule slightly lo thit the egg is still free to move m the capsular fluid Again control the ijm metry of the half -capsules, start anew if the hal\xs are unequal Tilt the egg from one side to the other until the first cleavage plane is exactly under the loop Then pull slo^y and evenly on both ends of the hair until the desired degree of constnction is reached (Fig 19, c)



Flo xg — CcPditrklloa o( ft xrrodcl« egg (?-cd! sU^) «iUi A b&lr «-prepftr»li® ^ hair loop t^pltcing of the egj In the loop c—cooMUkOca,


In order to obtain compute hdns it Is usually not necessarj to constnet until the two halves ore entirely separated Such a deep constrictwn uw ally results in the bursting of the vitelline membrane and the loss of one or both halt’d. If the bridge between the halves is narrow, it will breti apart by itself after a few hours Another way of avoiding rupture of the ntellinc membrane is to complete the constnction in several steps 10-30 minutes apart.

In order to obtain conjoined antenor duplications constrict only slfeht

1 >

A ole The same results may be obtained by constricting In one of the planes of the 4-cdl lUgc


4 After constnction is finished cut off the free ends of the loop near the egg Make a ^tch indicating the degree of constnction (diameUr of “handle in proporbon to diameter of the lateral halves)

5 Place the egg in a clean dish.

6 Observe the gastnilatiom It is indicative of the future result Twins wQl be obtained If the blastopore and upper Up are shared by both sides If

you find gastrulaUon in one half onl> then you may conclude that the plane of constnction was frontal The half which docs not gastrulate will form a bcHy piece,

7 Observe further development and make aketches In the late tail bud stages the twins will be crowded In the narrow capsule and thus cn dangered Try to grasp the hair loop with your sharpest forceps chp it^ and remove it- In later stages, when the cajsule has lost its turgor It may be rcmo\Td easily However it is dangerous to do this In early tail bud stages because the embryos arc liable to be squeexed to pieces when the highly turgid capsule is punctured If wbole mounts of the constricted egg with the hau loop intact are desired fix the egg within the capsules m formaldehyde

8 Try to raise twins or dupbcations to stage H40 or older Observe the atus viscenim of the twins (sec p 70)

r emma suooEsnon uzaooorr

In 1914 Spemann discovered that salamander eggs can be constricted shortly after fertilization before cleavage starts. 'Qrodelea are known to exhibit ‘ph>'fl»ok)gical polyspermy that is se^'cral spermatozoa enter the egg In nonnal development all but one disintegrate In constricted undtaved eggs one half contidns the egg nucleus and will subsequently develop with the diploid zj’gote nudeus The other half contains accessory fpermatoioa and m the absence of the rj*gotc nudeus, one of them will become activated In a few favorable cases a haploid embrj’o will de \Tlop out of this combinatwn of maternal cytoplasm and paternal nu deus Embr>-os developing from egg fragments are called *meTOgoni those which dc\-elop with the sperm nudeus only are called 'andro^merogoni Fankhauscr and his associates ha^•e used this method for an analj'zls of cjlological problems connected with poljTpenny fertilization and haploid) (sec Fankhauser 1937(1 and & and Fankhauscr and iloore

Baltxcr and his associates (in Bern Switzerland) ha\*e seized upon this unique opportunity of building up lndi\'iduals of maternal cv'toplosm and patcmal nudeus and haNt inseminated such nonnudealed egg fragments With sperms of different spcdei These expenments ha\‘c gi\‘cn ^•cr> important dues as to the role of nudeus and cytoplasm in heredity and de vdopment (cf reviewB m Baltzer 1933 1940, Hadora, 1937) Students who were successful in the preceding eipeninent should try to obtim dro-merogons by constriction

Ezpkxiuxnt iia

i/o/erw/

According to Fankhauscr (1932), ondro merogons of Tr tindertwib not develop beyond gastrulation, whereas those of Tr pynhoiasiaxsxj^ velop mto older embryos (Fankhauser, 1937ft Streett, 1940) Horoer the number of successful cases will be small Proetdure

If possible use eggs whose deposition was observed Notice an onpgraented area at the animal pole with a small black spot In its center lie latter Is the second polar spmdlc Constnet, as before, through animd and \'egetal pole For mterprctatlon of your results consult Fankhaaor (19376) and Streett (1940)

BIBUOGRAPHY

Baltzei F 193^ tlber die Entwtddung von Tritoo Biitaidcn ohne EHhil d. »oL Godbdl p 119,

1940 tlber abilche, leuJe Entiricklong und Auitaosciitaikrit artw**^

dener kerne ba Btsurden. Natnrwus., a8 177 196.

FxjiciACsra G 193: C>toplaamicIocalkalJonintheun3«gnientedeucrf U*

Tniarnt nndactm Anat Rec, SappL, 54 73 ,1 ju

' 10378 The developroent of fragment! of the fertfli*ed TrU**

egg nudeus alone (gyno-merogony) Jour Exper 2^ooL, 75 413 . ■ " 1937ft The prodartton flf wV^ nTfT*t ‘‘‘f ■elim andcfbrTte. J

IIcre(L, s8 s .

Fakthausei G u»daiooxn,C. 1041 CjiologkdandeiperimcntilstQdkJofp*

•penny In the newt, Tnlunu nridcfcens h U Jour Moiph Mi347

lUiwax K. 1937 DieentwicklangspbjwiologiKhe AuswlrkungderdiibinBOO®^

Kem-ria nn i Kombinatioa bdtn Baatnrd Merogon TrUait ^

tw Arch, ! Entw medi 1361400.

kEWjUK IL H 1940. The question of mirror Imaging In human oce^ Hunan BioL, la ji j

SrenAJo* IT 1901 Entwickiungspb^ilologucbe Studlcn am Tritt® El !■

Bnl» mech la ^

' 190* Eniwicklungspbjaiologischc StudJen am Triton*Ei H ||

1903 ^twicUungspbysiologisdio Studien am Triton El HL / gf.

“ I 9 t 4 - tlber versogerte K em T Ci iorgung von Kemteileiu Verb,

•rilich, p J16

— ~ *' 535 - Embryonic de\Tlopmenl and Induction, New Haven

Prm. ^

J ^ 1940 Experimenta on the organisation of the

Tritunu Jour Exper ZooL 85 383


74


d) SCHXTLTZE S EXPEilllENT PRODUCnO^ OF DUPUCATIO^S BY imrERSIOV

(After Schultzc and Pennere and Schleip)

In 1895 0 Schultzc studied the role of gravity in frog devTlopment bj compressing eggs m the a-cell stage between glass plates and then turning them upside down Thej were left m this position for several hours and then released from pressure If this is done part of the heavy ymlh of the vegetal pole sinks down lighter parts of the egg cell move upward^ and a complete rearrangement of the mner egg substances takes place whereas the cortical layer seems to remain rather stable O Schultzc made the unexpected discovery that a high percentage of doublt monsters resulted

Hi


Fta to — DcpbdUi CTodiU m the ffog, pi od occ J by lavmlop rf the fgg 0**eajlnila iU£c (••uvednllarT' fold fUs« ca-uH bod â– tage' U— blutopon IT H t«o heads 7 **, n»the t«o UlU (modiSed alter ScUerp, 1979)

from this procedure Penners and Schleip (19280 and 6) and Pasteels (1938) repeated the eipenraeot on a large scale and obtamed the same rc suits These duplicatwns undoubtedlj owe their origin to the shift of yo\k. and other materials which m turn causes great disturbances of the gastrulation process As a result m many instances two upper Ups in stead of one are formed which give nsc to ti\o separate axial-organ systems, One type of duplication is of particular interest the so-called duplidtas cruciata It dcnv’cs its name from the crosslike appearance of the duplicated structures In this strange monstrositj two heads are opposite each other and so arc the two trunks and tails but the plane of s>Tnmctry of the heads is perpendicular to that of the trunks and tails (Fig 20 fc c) This duplication onginatcs apparently from an elongated blastoporal groove {bl m Fig 20 o) the edges of which behave as upper hps OnU a few embryw will be found to represent duplications of the diagrammatic dearness of Figure jo Most of them arc asymmetneal and distorted Houever all the possible modifications of this and other types



75


have been catalogued and analysed by Pcnner* and S<^eip (i5h 8) Hie student should consult the Illustrations of their papers to Identify hh ms tenaL It is Impossible to discuss here the far reaching theoretical implfca tions of this experiment The student Is referred to the papers quoted above and to Schleip (1929) and Daicq (1938) The experiment is prtsented here because It demonstrates by means of a very simple tedmicsl procedure a fundamental fact of experimental embryology that more than one embryo can develop from one egg ExpaaoaifT is

MaUnaJ

fertilised, undeaved eggs of Petn dlsbes or finger bowls JL pipiens (sec p 30) Pcrmoplast

microscope slides Procedure

1-3 As in Experiment 10 (alteration of dcavage plane, p 68)

4 Place the second slide over the eggs and press gently sothatthejdlf membranes are comptcaed but the eggs remain movable

5 Wlien the first dcavage is weD under way i-e , a-j hours after Icr tlhzabon (in R. fiptent) press the ends of the upper slide firmly with your thumbs untfl the eggs are flattened Control this under the binocular microscope turning the slides edge up

6 Turn oU slides upside down while the eggs are in the a-cell stage. Do not place them in water The eggs should be pressed so tightly that the white poles remain uppermost Eggs which rotate back or those whose axes arc not exactly perpendicular to the ahdes must be discarded later

7 After 10 minutes submerge ell double slides m water In the in\*ertcd position use Petn dishes or finger bowls

8 During the following hours watch the flow of light and dark materials Make sketches

9 After s-34 hours remove the eggs from the slides and place those embryos which arc In good condition In a finger bowl or a Lily dish.

10 Study the gastrulatlon and subsequent development, isolate the best cases and protocol them individually Make sketches The late ncu rula stage with raised neural folds Is the most Interesting stage, because it shows the general structure (tymmetry relations etc.) of duplications more dcari> than later stages do

BIBLIOGRAPin

Daico A- M mjS 1 onti lod cauunt> io earl/ Oevtlopmenl *cepp.5pff Cxni

tndse Endarx! Cambnd^ Unlierut} Prest.

PAfTtru, J lojS. Rcchcrthe* lar les facteun WtUoi de la morphog^fl^v cbe* le*

76


tnpUbKna tDOores. L R£salt>ts de 1 exp£ne&ce d£ Scholtze et leor Interprftatioa. Arch, 49 629.

PrjiKtis, and Scmzu Vi 19280 Dh> Entuldionf dcr Scholtxeschen Doppd bQdimgcn ita den El von Xana fksta, I-I\ Zdtschr f wiss ZooL 130 306

1928^ pTit-wi rVTung dw SfbnltwAi^ TWTpp rihildnng m am dem Ei von

Ranafusca \-\X Ibid 131 i

ScsLEiP W 19^9. Ehe Detennuatioa der Pnmitiv Entwicklung Akad. ^ erL Ge •eUsdi. (fee pp 696-715 for a brW rcvtcir)

S on j L T r E, 0 189s Die ktmitEdie Errcafiing von DoppdbDdunxen bd Fiosdhlar Ten. Arch, f Entw'mech, z 269,

«) CXNTRIFUCING

(After Jenkinson)

Centnfagiiig of eggs has been one of the standard methods of expen mental embryologj smee the beginning of this century Centrifuging with sofikiently high force results m a redistribution of the formed inclusions of the egg cytoplasm For instance m the centrifuged frog’s egg three distinct layers can be seen a yellow cap of oil and fat gbbules at the centnp>etal pole a colorless middle la)^- composed of proteins water salts etc. and a thurd Layer containing )’oIk and pigment granules at the centnf ugal pole It is of considerable interest to find out whether or not the re arrangement of these egg substances affects the differentiation of the em br>*o Many mvertebrate and \ertebrate eggs ha\e been centrifuged at different stages and with different forces (comprehensi\-e re\ncw m Mor gan[i937] chap iiu) The essence of all these experiments is that high speed centrifuging will result m abnormal devTlopmcnt but for most egg* a moderate rate of rotation can be found which will result In an abnormal stratification of the egg mdosions jtt will not mterfere with nor mal dc\*elopment For Instance, fat globules or pigment granules may be accumulated in abnormal concentration m the nervous sj'stem of a tad pole and be missmg in other organs where they arc normallj found. An unportant condusion can be drawn from these experiments these formed cj'toplasmic indusions cannot be considered as organ-determining or formati\T substances m the sense that their presence m a gi\“en organ pnmordium Is necessary for its normal differentiation However one •hould not forget that the cortical laj*cr of the egg is m all probabiht> rather stable and not disturbed b> centnfuging and that there maj exist in the cj'toplasm a micellar framework which also maj rcniam more or less unaffected b> hcav'j centrifugal forces Therefore It would be unwar ranted to condude that there exists no preformed structuration whatsoevtr In the egg All we can sa> is that its \Tsible components and their distnbutkin ha\T nothing to do with the future organuation of the em brj-o


77


In batches of centrifuged eggs there always will be found numerous monstrosities of all kinds, for instance double monsters multiple Uflj headless monsters ^ina bifida, etc. (Jenkinson, 1915, Banta and Gortner 1915 Beams King and Risley, 1934) This is not surprismg since the dislocation of the jiDlk and of other substances will, m many m sta n ces, interfere with normal gastrulatwn etc and also a dislocation of the or ganiaer material may be expected

Centrifuging is an effective way of segregating the different constituents of the egg The different layers actually can be separated and subjected to a chemical and physicochemical analysis The application of the ultra-ccntnfuge which provides a much more effiaent tool for m\’cstlgi tions along this line, has already brought a revival of Interest m centri fuging experiments

Experiment 13

Afatmal

unclcavcd eggs of Rana (any species) obtained by artificial inscnilna tion Cp 30)

any type of clectnc centrifuge finger bowls large mouthed pipettes Procedurt

1 Use eggs 3-1 hour after fcrtiliaatlon when the jelly racpihmncs art swollen Do not remo\*c the latter Fill the tubes of the centrifuge with eggs do not crowd them

2 Run scN-cral senes of expenments Vary the speed (between 1 , 5 °® and 3 000 rcN-olutions per minute) and the time of exposure ( 5 ”^° utes) Take careful protocols and keep the different batches in separate carefully labeled finger bowls. Find the optimal procedure for the partico Inr centnfuge and the spedes of frogs which j-ou use

3 Observe under the microscope the redistribution of materials (con suit Jenkinson 1915)

4 The first clca\Tige is to be expected 2] hours after fertilisation ^ R ptpiens) Calculate the percentage of normal cleavage and disct the nonclea\dng eggs

5 Observe gastnilation and j-oung tadpoles 2-5 days later Take protocols Trj to identify the malformations

BIDLIOGRAPJI\

Bant\ a ifld GorrHrs R. A 1915 AccKV)r> appendagto arKl other •b*®*' maBuei produced m imphIbUn larvae through the tcUon of cenirlfui^l Jour Liper Zool 18:433


78


Btjua,H.W Kimo R-U uwIRislet P 1934. Studies on centrifu^ frog egg». Proc Soc Eiper BwL and iIe<L, 3a 181

Ji^mirtow J W 191S- On the rekbon between the •tructure and the development of the centnfnged egg of the frog. Quart. Jour Ulcar So., 60 61 UoaOAK T H, 1937 Expenincntal embryology Ncw\ork Columbia Umvmity Prtm.

Transplantation Experiments to Demonstrate Self Differentiation

a) Introduction to Transplantation Terminology

Before starting traniplantation cipenments the student should read the chapter* on ‘ determination in one of the textbooks of experimental embryology

Experiments m which the firat a blastomeres were Isolated or mverted (pp 69 7 5) have shown that eadiblaslomere can produce a whole embij-o In the terminology of Dnesch, Its *prospective potency^ is greater than its *prospective significance (actual fate in normal deN-ebpment) This implies that the destiny of a given part of the embryo m the a-ceU stage is not yet definitely fixed or "determined it should therefore be possible to mterchange embryonic matenah during eariy stages without daturbmg the oormal development On this reasoning are based the classical transplantation cipenments of Spemann (1918) in which prospective epi dentils and prospective meduUaiy plate of the eari> gastnila of a urodele were mtcrchanged The result was that prospective epidermis formed part of the ner\’Ou* system and pro^iective medullary material formed part of the epidermis Thus it was shown that m earij gMtrula stages these embryonic areas are not yet irreversibly fixed or determined They differentiate m accordance with their new environment (ortsgerTuiss) In other words their fate is being determined during or after gastnilabon by factorswhich reside ID the tissnes outside of the transplant ( extrinsic’ factors) It was found m later expenmenU that the subjacent mesoderm detennmes the medullary plate during gastnilation.

If a similar experiment of kdcrotopic Iransplaniaiion (ue transplanta tion to an abnormal positwn) is done In stages after gastnilation many part* win differentiate according to their prospectn. e significance in regard of their new environment as if th^ had been left m their normal place For instance the prospective eye brain or limb areas of the neunila stage will form eye brain or hmb when transplanted to the fl*nV W Roux coined the term sdf-differentiation for this capaat\ Obvn oosly a change has taken place m these primordia during gastnilation They ha\-e acquired mdependence of their cn>'ironment and now contain mthm themselves all factors which are essential for their further differentiation From now on they are “determined and one may properij call them e>T bnun, or limb pnmordta although thc> are not ytt MsibI) different from one another

The terms "self-differcntiation* and “determination must be qualified precisely whenever they arc used otherwise th^ are ambiguous and misleading In applying the term “sclf-diffcrentiation to an embr^tinlc area it Is necessary to state at what stage the latter has acquired Its self differentiatmg capaatj For instance the eje forming area is self differcntiating/rww Ike neurula stage on Furthermore, self-differentiation IS alwa^-s relati\’e Absolute sclf-differentiation does not exist an organ pnmordium remains dependent on its environment m certam respects until its de\'elopment is completed It is necessary to state what specific structural features are to be tested with respect to their self differentiation or dependent differentiation because a pnmordrum may be self-differentiating in one aspect of Its development but, at the same time be dependent on extrinsic factors m other aspects of its differentia twn For instance m the carlj tail bud stage of a salamander a certain area m the flank will differentiate mto a fortlunb when transplanted heterotopicall) it is self-differentiating from that stage on as far as its general morphogenesis is concerned Howrvxr experiments of Hamsoa have shown that the same pnmordium at the same stage a not >tt irrc\‘ef*Ibl> determined with r espect to its “lateralitj this pnmordium ma> dc\'clop Into a nght or a left forclrmb depending on the site of to plantation It Is self-differentiating os a whole but slDl depends on ex trinsic factors for its syraractrj relations In Experiment 14 the following question will be raised Is the balancer pninordjum which is self differentiating as a whole from tail bud stages on dependent on, or mdc pendent of its adjacent structures with respect to its direction of out growth and the tunc of its resorption^ Likewise the central nerv’ous system Is blocked out roughly in the roedullarj plate stage It will differ cntlate Into nervous tissue and c\*en Into special parts (forebrain spinal cord etc ) when transplanted helcrotopIcaD} Howe\Tr man> structural detaQs arc not j-ct 'dclemilned at that stage but become fixed In later stages under the influence of factors cxtnnsic to the nervous 5>atcm For instance the site of the spinal ganglia and the number of neurons In the motor column are controlled b> the developing pcnphcral structures to be Innervated the extirpation of a limb pnmordium results m a sire reduc Iron of the limb-innervating ganglia and motor column In this respect the nervous s>alcra is not self-diffcrentiating but remains under extnnsh: control up to a rcmarkabl> late stage of differentiation In other words determination is not a single act but a process of gradual emancipation So from citnnsic factors ilan> other instances of the relatmt^ of sdf diflerentiation vrill be Hliistrated In the following experiments

In the analysis of the process of detenmnation the following three questions are pertinent

1 At»hit/faxec/^dfl^j««f/does*givtnembr>onIc*rtabcconiertliUvd} lelf dIfferentUring thit b {odependent of certam ertniuic factors^

3 WUch derdofmenlal pnccsm or linutittal deiails (c moipbogcnttb of the whole organ, its syinmetr> rekdon Its quanduti\*e gr ow t h) are self-differentiitxng at a given stage?

3 Wth respect to what exinnnc sirudmrts or facicn (e g adjacent flank tissue, innemtion, honnoQcs) b the primordiam under dbamiooindqscndent or dependent?

The following experiments fDostratc relati\-e seIf-<iifferentiation of whole organ pnmorduu Thc> are designed to stress these pomts (i) to make the student acquamted with the method of transplantation (2) to illustrate the fact that m the neurula and tail bud stages of amphibians many organ forming areas are self-differentiating units as far as their gross morphological differentiation is concerned and (3) to impress the student with the fact that the process of determination mvohes changes in the in vulble properties of embrj-omc materials The pnmordia to be transplanted are m no wa> distinguishable from adjacent areas yet thej be hav'e differently in transplantation experiments The balancer pnmordium was found to be the most favorable object for the first exercise In transplantation It is cas} to locate and easy to h»n die The transplant shows visible differentiation two dap after operation and no dissection or sectioning is neccssarj for its identification Further more the balancer offers an excellent opportunit> for the stud} of mter esUng side issues For instance the question of whether the durction of Its outgrowth IS determined b} mtnnsic factors or b} the surrounding host tissue ma} be anal}-zed bj varjTng the onenta lion of the transplanL Likewise the questwn of whether the tune of its resorption is determined b} mtnnsic factors or b} the host ma} be stodied" b} using hosts and donors of different ages

b) TaASSPLANTATION OF BALANCEKS Urodtla (After Hamson)

The balancers are a pair of slender rod like appendages which project from the side of the head a little behmd and below the c}'es and which serve as props to hold the head off the bottom and to prevent the Lrva from falling over on its side until the forelegs devxlop and assume that office (Harrison 19 4 p 349)

The} arecharactenicdb} a club-shaped thickening at their ends which secretes a sticky mucus This "secretion cone * and its stickiness should be used as a entenon for the identification of tran^lants as ‘Tiakncers ” Transplants which do not show it may be rudimentary balancers or mere ly epidermal outgrowths Stickiness can be tested easily with a hair loop The balancer is innervated by a fine nerve and vascularized by a small artery and a small \'em Its rigidity is maintained by the firm ancer membrane ' which is located at the base of the epithelium In A macuhttm the balancers become visible externally in stage H54 they begm to secrete mucus In stage H38 and reach their full size in stages H40 or H41 The balancers arc transitory larval organs which havT only a short life span In A maculatum, regressive charges (constriction at the base) begin at stage H4S soon afterward the entire balancer Is shed by breaking off at the base In A cpacum the balancer disappears m a different fashion It shrivels, beginning at the tip, and is largely re sorbed The remnant seems to be cast off as In other forms Balancers arc found only In certain speaes of urodcles, for instance, in most spcoes of Tniunts in A macuhtHm jeferumtanum, opcevm, and microsiomum but not in tigrinum (except for a few local strains m which rudimentary balancers were described by Nicholas 1924)

Students should study the structure development and disappearance of normal balancers before starting experimental work Hamson (1924) should be consulted for all details concerning their development and histological structure Kollros (1940) for detail* concerning their disappearance The experiment Is supposed to demonstrate the self-differentiation of the balancer pnmordium after it Is determmed but before it is visible Hamson (1924) has found that when prospective balancer ectoderm m stages H28 or younger Is transplanted to the head of another embryo it will form a balancer but that when tran^lanlcd to the flank It will not do so The same ectoderm taken from stages older than H28 will grow oat to form a balancer in any region Hamson concluded that up to stage H28 prospecti\’e balancer epidermis b not entirely self -differentiating but still dependent on the underlying head mesoderm Therefore in order to obtain balancer formation it b necessary' to transplant young epidcrmb to the head or young epklcrmb plus undcrlymg mesoderm to the flank old epidermis wiU gut balancer in any position

EjcreaiuzKT 14

Material for Exp^nmcnls 14 J4a-b

Ambystoma maeulalum or opacum {A tigrinum has no balancers) In stages H38 H32 standard equipment (p 41)

Procedure

I Select two embrj-oa of approximately the same stage remove all membranes including the \ntellme membrane (see p 37) HU the opera tion dish with fuU-strength Holtfrcter solntion

7 Transfer both embryos m a xnde mouthed pipette to the operation dish (Syracuse dish with smooth Permoplast ground) Dip the pipette im der the surface of the water before releasing the crabry-o



Frc ji — Balancei trxnfplinUOoQ a-dofior«nbf>-o ^Uu nndk (xfl uid hair knp (U) In tlie poritioo In «hicti they ire held donne tbt eitiiiuUoo repan

otocyit /XBpronq)lirot ( — hott embryo, i j j 4*bok prepared for tbe reception er{ the tmopUat (Kt text)

3 Wlth a glass rod with baU tip mahe two gToo^*c3 one beside the other of such size and shape that the embryos will fit mlo them when lynng right Side up CarcfuUy smooth the edges Place the embryos m the En>D\-e3 right side up

General rule Operate on the right side onl\ use the left side as a control

4 Prepare Ike host embryo — Choose the site of implantation either the trunk region just below and close to somites 6-S (behind pronephros not too far \*entrall\) or the head region m the 1 c\t1 of and immediately be hind the otoo-st (Hg 21 6) B\ means of the glass needle cut out a rec langular area of ectoderm approximately the size of the optic \tsic1c To do this pierce the ectoderm ^ith the point of the needle push the

nccdk gently fonrard undenieath and parallel to the ectoderm and prcree again at the upper comer of the desired hole as if you were malung a stitch in sewing (hne 1-2 in Fig 20, b) Stroke the hair loop gently against the needle until the ectoderm is cut Do the same on the other three edges first line 4-3 then line 1-4 and Ime 2-3 Lift out and discard the ectoderm and some of the underlying mesoderm

5 In order to assure the right onentation of the transplant in the host cmbrji), It IS advisable to stamp a vital stain mark on the upper right comer of the transplant Before atartmg the extirpation, place a small piece of red or blue agar on the upper posterior end of the right cj-e and adjacent regions of the donor embryo Press it against the embryo with a glass bridge lea\'e it there for 10-15 minutes

6 Exitrpate tJi 6 prospedm balancer region from the donor embryo {J>al in Fii 21, a) — ^Thls region lies postenor and slightly ventral to the eye on the mandibular arch The dorsal border Is on a level with the dorsal margm of the eye, the ventral border is slightly above the mid ventral Ime and parallel to it. The antenor limit is along the posterior margin of the eye The posterior limit is bebmd the first gill slit Cut these four edges with the glass needle and hair loop (as imder sec. 4) Remove ectoderm and mesectoderm very gently transfer the piece on the tip of the needle or with the hair loop to the host embryo and place it temporarily on the Pennoplast ground near the host embrj*© Notice the mesodermal cells attached to the mner side of ectoderm In transplanting try to retain the proper onentation of the transplant with respect to the longitudinal axis of the host embryo do not rotate It

7 Implanialwn — It ma> be nccc5sar> to enlarge the hole in the host embrjo b^ pulhng the edges apart or by remov’ing some mesenchyme cells \\nth the tip of the needle With hair loop or needle lift the transplant Into the hole Make the edges fit and place a glass bridge of proper slic o\*er the transplant $0 that it co\-er3 the transplant completely W ork as quickly os possible smCT the transplant Is likely to curl Try to Implant in undisturbed onentation If the transplant was rotated intentionally or unintentionally note this in your protocol immediately after operation Press the glass bndge Into the Pennoplast ground until it bolds the transplant firmly in place Too strong pressure may cause disintegration of the transplant

8 After 20 minutes lift the glass bndge gently rcmo’c the loose cells adhenng to the edges of the transplant using the tip of the glass needle or the hair loop If the transplant has not healed in completely place the glass bndge back for 15 2om!nutcs. Otherwise lift the embryo out of the groo'V'e using the hair loop and clean it of all adhering cells and Permoplast Transfer it very gently m a wide mouthed pipette to a section dish or a Lily cup Half filled with Holtfrcter solution Dip the pipette under the surface before releasing the embryo Place the embryo nght side up

9 Clean the donor embryo and transfer it to the same dish right side up

10 Give the embryos a serial number (c g bcl i), label the dish, and prepare a protocol Carefulness in drafting the protocol is as important as cartfulnese m the operation. Make sketches of donor and host indicating the position orientation etc , of the transplant.

Example of protocol

boL 7 Ambytiama piauiaJitm Mirch II donor stige H30 host, fta^e H31

trpl right biltncer ectoderm (refer to ikctch) a few mesoderm cells ad here to ectoderm

impJanUd ventral to somites 7-9 (refer to sketch) In nonnal onentabOQ healed after 30 minates

saved aound be^Lniung to heal

March 13 donor stage H35 OE left bafsam just visible wound on right side healed

koO. stage H36 OK both host balasctn lost visible

trpL healed fint of outi^crrth, m typical dutcrion etc.

Note all changes In the transplant particularly Umc and direction of the outgrowth of the transplanted balancer Compare with the control b a l ancer of the donor or of normal embiyo* of the same stage as the donor

Make 3-4 operations choose different sites of implantation

iVo/ff — In rare instances the transplant wiD form a double balancer Occasionally the tissue which healed over the wound of the donor will regenerate a balancer

Expebdeest 14a

Rotaio the transplarii go or ijo* — Observe carefully (and sketch) the di rection of outgrowth of the balancer compare it with the normal balancer In order to assure the desired oneutation of the transplant it is to vital stain its upper right comer before exdsion as under section 5 la the direction of outgrowth determined exclusively by Intrinsic factors or u it influenced by the host?

ExpEKOcorr i4i

Ofifam tmbryof of dt^erent stages — ^Transplant from a young donor onto a host which is several stages older and vice versa Operate with great care m order to keep the donor ahve Observe the first appearance of the transplanted balancer and compare it with that of the left donor (con trol) balancer Likewise observ'c the time of shedding In the transplant^ the host and the donor (control) balancer Is the life-cycle of the transplant in an> way influenced by the host? (Sec KoUros, 1940 )

Bibliography

IIauuson R. G 1934. The devekpment of the balancer in itndxtdbr the method of tnosplaotatlon and lo rtlatioQ to the connective tissue problaa Jour Eipef Zobl 41 349.

Koluos J J 1940 The disappearance of the balanctT in 4 mbtpimalimt. Jour Eipcr Zook 85133

NicnoiAS J S 1934. The deveJopment of the balancer in AmbJpifma Utriitim AnaL Ret 38:317

c) TRANSPLANTATION OF FOREUIID PRIUOROIA

(After Hamson)

Study first the normal development of the forcllmbs in Amh)' 3 tema (Hamson 1918) In stage H^S and earlier notice the pronephros swcUing immediately beneath somites 3-5 and a short distance posterior to the gill swelling The prospective forchmb area is rtfpresented b> ectodermal and mesodermal material Immcdlatel> \cntral to the pronephros, including the ventral slope of the prooephne bud In stage H36 the hmb bud be comes visible as a prominence separate from the pronephros In stage H37 It IS a distinct bud which points In caudal direction It Is at first cone shaped and then flattens at its distal end In stage H41 on indentation at its distal outline marks the two digits i and 2 which grow out rapidl> In the succcedmg stages Digits 3 and 4 follow successive!) at the ulnar border At the same time the elbow joint becomes visible Note the posture of the limb At first Its palmar side faces the flank Then it rotates forward in the shoulder Joint so that the animal supports Itself with the forcllmbs when it Is at rest The balancers, which performed this function prc\'K)usly disappear at the time when the digits touch the ground Limb-bud transplantations were among the earliest embryonic transpbntalions The pioneer work was done by Braus (1904) and Hamson (1907) on anurant in connection with certain problems of ncr\c outgrowth Later on Harrison introduced embryos as an unusuoll) fa\'or

able object for limb transplantation since then wry exlcnsl\’c eipcn mental work has been done by Harrison and his students and b> man) other m\*c 3 tigator 8 using the lirab bud as an object for the rinal)'Sis of fundamental problems of determioatwn (rcvTCWs In Mangold 1929 Swcit 1937) Detwilcr (1953) has shown that the prospective bmb material is deter mined as early as in the late yolk plug stage For our purpose it is ad visable to use older stages preferably stages H2S-H31 Hamson (1918) has demonstrated that the limh-fonning potencies reside in the mesoderm and not m the epidermis of the primordlum Removal of ectoderm does not mterferc with Innb development, whereas extirpation of the entire hmb mesoderm, keeping the ectoderm intact m position usually results m the lack of a limb Also hetcrotopicaDy transplanted hmb mesoderm without ectoderm will give nse to a bmb It is therefore essential for the success of the following experiments to include the mesoderm m the graft as completely as possible

OccasKmaHy the transplant will form The dupbcation

may aficct only the digits or it maj affect the whole limb or an> mter mediate degree of duplication may occur In such cases the double limbs are mirror images of each other These dupbcations are of mterest m sev eral respects First they prove for an organ pnmordium what constnc twn and inversion expenmentB(pp 69-75) have proved for the whole egg in earlier stages that at the time of transplantation the individual skeletal muscle, and other elements were not ngidly determined in a mosaic like fashkin (see p 94) Second the mirror imaging of all duplications shows that once a hmb bud segregates mto two there must be a reversal of the lymmctry rclatwns of one of the partners under the influence of the other partner The same mirror imaging is frequently found in the viscera heart, etc, of naturally occurring and artifioallj produced double mon sters where it is known as situs mversus (see p 70) The problem of symmetT} and the causes of symmetry m\'ersion m dupbeated limbs arc discussed by Hamson (1921)

It is of Interest to raise the donor embryos of limb transplantations and to find out if the extirpated pnmordium will be regenerated or not If the donor embryo shows no bmb defect and the transplant is entirely re sorbed one ma> suspect that not the bmb primordium proper but adja cent tissue has been transplanted by mistake.

ExpotnoixT 15

ifalcnal

Ambysloma (anj speacs) orTnlurus stages H25-H31 standard equipment (p 41) o I per cent solution of Nile blue sulphate Procedure

1 Prepare at least a dozen embiyos of stages H25-H31 preferably stages H28-H30 Select ^’CTy healthy embryos, RcmoiT all membranes 87


including the vitelhne membrane, and tranafer the embryos in a sterile pipette to sterile iV Holtfrctcr solutbn Older embryos which begin to show mo\'ements most be narcotised in chloretone or MS 332 during the operation (see p 40)

3 It Is advisable to vital stain the donor embryos tn tclo by placing them for an hour in a o i per cent solution of N 3 e blue sulphate

3 Transfer 2 embryos into the operation dish and, with the glass rod with ball tip mold two grooves, side by side into which the embryos will


tm. — TnofpUnUUon of t forcUfflb primonlluza. •■"donor embrye J-borteolxTO I 1 3 4 “limbErea- #r-otocytt /»-croiKphro» Sj etc.

fit when l>Tng right side up Smooth the edges of the grooves very carefully Place the embr^xis in the grooves Have several glass bridges ready , slip them over the cnibrj’o and make sure that they have the proper ste and bend

4 Prepare the host embryo All transplants should be made to the flank at some distance postenor to the pronephros swelling of the host, and approximatel) below somites 8-10 The position of the graft should be in exactly the same lc^ cl as the host limb i e , Immediately below the somites Transplants grafted to a more \*entnil position that is into the j^olk do not take well and arc frequently rcsorbed or extruded

With the glass needle cut a hole 2-3 somites in length into the ectoderm adjacent to and just beneath somites 8-10 Follow the technique as


88


m Experiment 14 under section 4 ^ 83) Remo>*e carefu]l> a consider able amoimt of mesoderm cells with the tip of the glass needle so that a rather deep hole IS laid open (Fig 22 &)

5 Extirpate the prospective limb area of the donor (Fig 22 a) Locate the pronephros sw ellin g The limb area extends from the third to the fifth somites umnediatelj below the pronephros swelling and mcludes the ventral part of the latter 'With the glass needle maLe a stitch through ectoderm and mesodenn m the groove between gill and pronephros swell ing and stroLc the hair loop against the needle until a cut is made (Ime 1-2 m Fig 22 o) Make a second cut parallel to the first one behmd the pronephros usmg the sixth somite as a landmark (line 3-4) Then make two horizontal cuts one across the middle of the pronephne swelling (Ime 2-3 pronephne tubules may be exposed) and the other one parallel to the \Tntral midline of the embr^’o (Ime 1-4) Next with the hp of the needle lift out the entire cut area mcluding as much of the deep mesoderm as possible Be sure not to lose too many mesoderm cells during the following transfer Work as fast as possible.

h ole — Hamson and his students use the indectomy sossors for extir pations Operations with this instrument are probablj easier but m dectomy sossors are erpensrve and therefore usuallj not available for class use

6 For Implantation transfer the transplant on the tip of the needle to the hole m the Imst proceed with the greatest care and tiy not to lose the orientation of the transifiant If neccssar> drop the transplant near the host without changing its onentation and enlarge the hole Implant the graft m normal onentation press it mto the bole with the glass needle or the hair loop and cover it quicklj with a glass bndge Press the bndge finnlj against the transplant push the edges of the bndge mto the PermoplasL

7 Gi\x the embrj-o a protocol number make a sketch and take a care ful protocol mdudmg data on the amount of mesoderm transplanted and the onentation of the graft.

8 After a half hour remove the glass bndge ven, cautiouslj If the transplant has not healed m proper!) place the bndge back for another half hour The danger of damaging the cmbrj’o bj prolonged pressure is much less than the chance of losmg an unpropcrly healed transplant, which IS usually extruded

9 Transfer the embiyo m a wide mouthed pipette to a Lflj (label ft) dean the wound of the donor embrj-o of adhering cells and transfer it to the same or to another Hub

A ole — 'Make 3 or 4 operations of the same kind


Fio >3 —Limb trmtplantJboci with fanenton of n« â€¢ "ti»fl*plintjtloo of a Icfl foftLmb pfUDordjom to ibe njcM fl»nV_ » 5 ih tmcmoo ol tl* dr (Mrf»-orifnUUon) A •«tenor F-poftmor C-donal » -\enu»L Hie dotted drcle and ibe irttm tail the poi-UQo and tie* d the bort forelimb the ioBd drde arid the letter* Jo It lodictte W trtniplint and 111 tie* the trajupliat,re*iiltiiiit Jrom thUoperallu C”tf*D*plaotauM“ a left Jorehr b pnmordiotn to the njiht fiuik tJlh m\cnk)n ef the a> aiii (a^-oneBlalwjh a"trtBfpliaiaiiOT d a ryjht fortLmb pnmordjom to the ftint, after rotatwri d iScr (a/dTHmcnUbaej f" the titft*pUnt, retuHtnrfromopcTttkisieajidd (inodihed afterS etl.



10 Dunng the following weeks observe both host and donor frequent ly Narco tixe both and study them in tim narcotic. Take careful protocols and make sketches of all changes which you observe on the transplant and on the Innb region of the donor Compare the development of the transplant with that of the host Umb and with the left forelimb of the donor Watch carefully for a possible duplication of the transplant try to detect and sketch it at its inception This will help m the later mterpretation of the BjTnmetry relations (see Hamaon, iqai)

d) UUB TEANSPLAirrATIONS COMBINED WITH INVEISION OF AXES

The ongm of polarization and of symmetiy relations m an organism or m an organ is one of the central problems of experimental embryology Hamson (1921) m a dassical study analyzed this problem by transplant mg limb pnmordia in such a way that their axes wonld not comcide with those of the host embryo In a limb pnmordium Hamson distinguishes 3 axes the antenor posterior axis, the doraoventral (d-p) axis and the mediolateral (m-f) axis which comade with the corresponding axes of the cmbr}*o Left pnmordia were transplanted to the right flank (Fig 33 a (md c) or right pnmordia were rotated iSo but kept on the same aide (Fig 23 d) Transplantatwns from one side to the other can be done In two ways either by moving the tranqilant over the back, so to speak (Fig 23 0)— in which case the dp-aus is mverted with reflect to the host but the of the host and transplant eomadc (oadtMinentatKm m

Hamson s terminology) — or by ahiftlng the transplant around the tall so to speak (Fig 23 c ) — in which case the o/^-axis is reversed but the dp-ans IS not (o/^id-onentation) Rotation by 180 on the same nde results in an apdp-onentation Bnefly the results were as follows The apftns IS irreversibly fixed In all Instances a transplant m o^ncntation win grow forward Instead of caudad (Fig 33 c) It will follow its original trend umnflacnced by adjacent tissues The dr-axis however is not Ir fcversibly fixed at least not up to stage H33 (m A pimcUitum) Transplant* in dr-onentation bcha%‘e as if the> were in a dd-onentation with the fint digit growing outward It is to be concluded that the dr-axis becomes polanied by influences from ad}aceDt host tissue This explains the star tluig phenomenon that, m certain combinations a left pnmordium will pvc nse to a right hmb (for Instance In operation Fig 23 a &)oranght pnmordium can be made to form a left limb even If it stays on the right (Fig 23 d e) From stage H3S on the dr-aiis is also inrver»lbl> fixed Stages H33 and H34 are transitional stages For all details see Htnison (1921) More recent studies are re^dewed In Swett (1937) The Experiments clearij Illustrate a point which was emphasized on page 80


9 *


vir, a pnrnordium may self-diffcrcntiatc with respect to one charactcratic and at the same time, be dependent on extrinsic factors mother rcspccti. ExrouMEfT 16a. Revemal or Tire Axis

MaUnal

Ambystema not older than stage H31 standard -equipment

Procedure

In order to facilitate the onentatlon of the transplant vital-staln the anterior border of the left foreltmb pnmordium Operate as m Expen raent 15 Implant to the right flank Orient the transplant as m Figure 23 a Fit the anterior (marked) border of the transplant to the anterior border of the Implantation groove In the host. Note the direction of out growth

Experiuekt 166. Revexsai. or Tire op-bxa Procedure as before but fit the anterior (vital stained) border of the transplant to the postenor border of the implantation groove (Fig 33, c) Do you expect a result different from Experiment 160^

ExrotivzjfT 16c Rotatiov 180

Proceed as mdicated m Figure 23 d What do you expect? How will the results compare with those of the preceding experiments?

EXFOUlfEKT l6d REVEXSAL 01 UIZ ^AXIS Df OlDEt StAOZS

RcpeatExpcrlments i6(Jor i6em8tagesoldcrthanH35 Howdocsthe outcome compare with that of Experiments i6a and ibcT

BlBLIOGRAPirV (c-rf)

Braos, IL 1904. FHnlge Ergcbolsie der TimmpUntaljon von OrpuunlRgen bd Bomblnator Larven. \erh. <L axuL Geseibcb 185s

Detwilei S R- ig^ On the time of detennlnatlon of the anleropoiterlor txb of the forelimb in Jour Exper 2 ^ooL 64 405 ILuous<r« R- G 1907 ExperimenU In truxpUntIng limbs and their bearing up®® the problems of the development of nerve*. Jour Exper ZobL 4 ejg.

• ~ 1918. Elxpenmentson tbedevefepment of theforehmbof 1 »df

dlBcrentiAtinc eq\iipotenti*l s>'s(cin, Ibld^ XS 413

191J On reUboni of *>10016117 in transplanted hmb*. /Wif ja i

Ma-scold 0 rgrp. Das Dctcrminabooqirobleni- II Diepaarifen ErtitmlUlendcr

Wlrbdtkre In der EntrkidJang Etjebn d Biol 51190.

SwETT F If 1937 Detcrmmitloa of lunb-axe*. Quart. Rev BioL,ia»jJJ

e) TRAhSPLAKTATlOV or GILL PRniOamA

(After ScA'cnngbaus)

The external gills of salamander lan.’ae consist of three branches with secondary filaments They ore full> developed in stage II41 In the stage


9 *


senes (Fig 45) trace them to earlier stages In ta3 bud stages as earij as stage H35 or stage H26 they are clearly distinguishable as lateral swellings on the head slightlj ventral and posterior to the optic \Tsidc Familianie yourself with the normal development of gills

Harrison (1921) has shown that the gills of A maculaium are deter mmed as early as m stage H21 All three germ layers contribute to the formation of the giDs The respective role of each one of them m the de termination of the slxc and shape of gills has been investigated by means of hetero topic, heteroplastic and xenoplastic* transplantation of the three components separately The considerable hterature on this subject 15 discussedm Sevennghaus (1930) and Rotmann (1935) The following eipen ments will demonstrate merely that the three la yer ed giU swelling is self differentiating m its gross morphology m early tail bud stages, that is long before visible differentiation takes place


Malcnal


F.rprBrvrw T 17


Ambyit^ma any speaea stages BL26-H29 standard equipment (p 41)

Procedure

1 Select two embryos of approximately the same stage Reino\‘e all membranes Place them side by side in two giooves in the operating dkh (as m Espt 14 under secs 1-3 [p 83D right side up Operate m full strength Holtfreter solution

2 Prepare the hast embryo — With glass needle and hair loop prepare a rather large groove m the flank of the host embryo Choose one of the following sites postenor or ventral to anterior limb bud Immediately ventral to somites 9-1 2 m the place of the eye For this and the following procedure follow the technique described in Experiment 14, imdcr sections 4-8 (p 83)

3 Esitrpalion of the gUl pnmordhtm — Cut out the larger part of the gill swelling using glass needle and hair loop Cut vei> deeply so that the pharyngeal cavity is exposed Lift out the block of tissue and transfer it cautiouslj to the site of implantation using the tip of the glass needle or the hair loop

4 Enlarge the hole if necessary Fit the transplant in and bold it in position with a glass bndge for 30 mmutes or longer

5 After the tran^lant is healed in lift the glass bridge cautiously


Heterotf^^c — truuplantatxiQ to s different pasdon betcroplistk — tnmplinutkci bet* eto anbiyo* bdoogtn* to different ipecie* xencplutk — tnmplintatioei b e tw e e n embfyw beknfin^ to different fcocn, funllie*, or more duUnt Umaomic cnteforles


93


dean the edges of the wound and transfer the embryo to a dish filled with iV Holtfreter solution

6 Take a protocol, note the onentation of the tian^lant Givx the embrj-o a serial number Operate 3-5 embryos m the same wa) In some of them onent the transplant m m\’trted positioiL

7 On the following day observe and protocol all changes In the transplant Compare its development with that of the host gflls. In what onentation do the transplanted giDs grow out? Arc they of normal size and shape? Arc they vasculanred? They wiD be resorbed eventually

Furifur su^estums — Study the papers of Hamson SevTringhaus, and Rotmann and repeat some of the experiments m which ectoderm alone or mesoderm alone was transplanted or rotated

BIBUOGRAPHY

HAmso'7 R. G 1931 EiperimeDts on the devdopaent of the gOIs In the ampblb* Un embryo. B»oLBtiIL,4t 15^

Rothaxi. E. 1935 Der Antcil von loduktorund reagJertndem Gewebe an der Eat

vncUung dcr Klemen tmd {brer Cefease. Arch. L Entw meth. ijj 335. SnTajacHAua, A. E 1930. Gin devekjpment In AmUjstfma ptndaiim. Joor Ei per ZoOL $6 r

5 REGULATnT PROPERTIES OF ORGAN PRBIORDIA (MORPHOGENETIC FIELDS)

a) nnaODUCTORY REUARKS TERlfTNOLOCY In the neunila stage many regions of the amphfbun crabrj*o have ac qulred a considerable degree of adf-differentiating capaaty This boWs for the limb-formmg area, the eye forming area, for the nose, car, heart, balancer region etc These areas have \'eiy peculiar properties First of all the) show a high regulative power WTienpartofalunb eye, or heart pnraordium etc. Is tran^lanted the transplant will tend to form a whole structure as will the fragment which was left behind m the donor In this respect the organ pnraordia resemble the egg In the 2 or The) arc not composed of a mosaic pattern of smaller self-differentialuig and speciahied units On the contrar) each part must contain within Itself a full complement of all factors which arc neccssar) for the for malion of a whole Dnesch called such B)’3tems ‘ harmonloas cquipoIcnual s)'steins. If an) given part of such a ^’Stem is potentially ca pable of forming the whole organ j*ct onl) one proportionate hmb cyt or heart dc%'clop5 evtnluall) then ngid restnclions of potenaes must be imposed on the parts The) arc assigned to limited specific tasLs within the framework of a whole and mutual adjustments between the parts must take place At a tunc when the limb area as a whole is self


94


differentiating as is sbcrwn by tran^lantatlon eaqicnments (p 86) the finer structural details within thl«t area, such as individual skeletal ele menta musdes etc. are not yet determined they gradually become established in later stages by mutual mteractions of the parts and possibly through other mechanisms Harmonious eqmpotential systems il lostratc the epigenetic nature of development, as well as the relativity of the terms self-differentiation and “detcnmnat»n (see p 8o)

The organ forming areas which are thus blocked out m the rough ha\’e at first no distmct boundaries. The capaaty for hmb or eye formation may extend beyond the cell area which, m normal development, will ac tually form the hmb or the ejr This was demonstrated by experiments m which the entire prospective limb- or cyt forming area was extirpated yet a limb or an eye was formed by adjacent cells which closed the wound Finally withm each pnmordium there seems to exist a gradient of organ forming capaaty with a peak in the center of the field and a gradual de dine toward the periphery Embryomc areas which exhibit the foDowmg four charactensticB are called ‘morphogenetic fields (i) Thc> are self dtffenrUtaitng systems, as shown by hctcrotopic transplantation (a) they are rtguUUn^ systems as shown by the formation of normal organs after remcn,al or transplantation of half pnmordia or after supenmposition of two whole pnmordia (3) the specific organ forming /v/enr*ejci<enii‘ey«?nd the bertUrs of the prospecti^ organ forming areas and (4) these self differcntiatmg regulative areas are gradtmt fidds For further discussions of the field concept see the books by Huxley and Dc Beer, by Spemaim and by Wqss

In the following experiments the properties of morphogenetic fidds will be illustrated by experiments on the limb the heart, and the cj'c

b) EXTIRPAITON EXPERIMENTS ON THE T-Tim FIELD

(After Hamson 1918)

The first extensive analysis of fidd properties was made on the forclimb of Ambysiema b> Hamson (1918) In tail bud stages the hmb area is a disk hnmediatdy ventral to the pronephros extending from the antenor ^fder of the third to the posterior border of the fifth somite Its dorsal ® 03 t part covers the ventral part of the pronephros (Fig aa a) Thelunbfonaing potenaes reside in the mesodermal cells of this area,

Hamson fint made a sj*3tematic potency test of half-disks antenor dorsal, and ventral halves we re extirpated It was found that half-disk IS capable of forming a whole hmb although the percentage of normal limbs resulting from the operation vaned considcrablj Detwiler U918) supplemented these eipcnmcnts by heterotopic transplantation of


9 S


dorsal or of \’entral half-disks, both of which gave nsc to normal limbt The limb area has thus been shown to be a ^'self-differentiating haimoni ous cquipotential sj’stem as the title of Hamson s paper indicates. Next the entire limb disk was removed Normal limbs developed from cells which migrated mto the wound from the periphery 'When the diameter of the extirpated di<k was mereased from 3 to 4 and 4^ somites, normal limbs were still formed m a certain percentage of cases. WTicn the amount of extirpated tissue was further mereased, no regencraticra took place The percentage of "regenerating limbs was lower when larger disks were removed or when the wound was thoroughly cleaned of mesoderm cells Thus it is shown that the limb-forming potencies extend bcj'ond the area which actually enters into limb formation (1 e , the prospectnx limb area) and that they arc higher in the center than at the penphery

The student should consult the paper of Hamson (1918) for all dctafli. Malmalfor Exptnmenlt iS-2i

Ambysioma any stage between H24 and H30 standard equipment (p 41)

Exptnmenlcl proccdurt for Expenmettis 18-21

Operate m full strength Holtfrctcr solution Operate aJwa>'8 on the right aide of the embryo Narcotixe m chlorctonc 1 3/500 or hISaa*

1 3 000 if nccessarj Proceed as foUows

I Choose a number of healthy tail bud stages Remove all membranes. Wash id stcrUc Holtfrctcr solution

a Transfer one embryo to the operation dish Prepare a groove of adc quate size into which the embrj’O fits nght side up Locate the pronephros Bwellmg and the limb disk (Fig aa, a)

3 Ulth the glass needle (or the indectoro> sossors) cut out the desired part of the limb area make a rectangular hole if jiiu use the glass

4 Make a careful sketch prepare a protocol label the embiyo and transfer it to a Lil) dish containing Holtfrctcr solution

5 Obsenx the heahng of the wxiund In the following hours and the differentiation of the limb in the following weeks Hake sketches and tike careful protocols Docs the regenerating limb catch up with the left (control) limb’

ExrouirrxT 18

Removal of the dorsal half of the limb area — Cut out the dorsal half of the square r i 3 4 in Figure aa a Hake 3-5 operations In some clean the wound carefully of all mesoderm cells with the tip of the glass

96


needle m other oses leave some mesoderm b ehin d. Protocol m each m stance the amoimt of mesoderm left m the wound Note differences m the regulation

ExPERDfEfT {9

R£mrcl of ike anienor half of the square 1334m Figure 23 a

ExpnautECT 10

RemoTol of the entire prospectiee Umh area 3 somites m diameter (1-23-4 m Fig 22, c)

ExPERIUENT 21

Revirral of an area ^ somites in dtameier centenng \'entnil^ to the fourth somite

Note — In evaluating the results one must bear m mmd that not all quadrants of the limb di^ share equally m the fonnation of the limb Itself For instance the %'entral half contributes less than docs the dorsal half etc. The pro^ctn*e signmcance of the different sectors has been woited out by Swett (1923) usmg the vital staining method

BIBUOGRAPm

DiiviLER, S R. zgiS Expeninents 00 the derdopment of the shoulder girdle and tbeantenorCmb of pundatam Joor Eiper Zoo] 35 499 >

Hahhox R. G igi8. Eiperunents on the doelopmeat of the foreCmb of Apthty^ stoma a •eU’diferentfotlog eqaipotoatal t\stem Jour Eiper Zooh 35 413 Smr F H. 1933. The prospective eignifioioce of the ceDs contamed in the four qoadianti of the primldve limh due m AmHystoma Jour Eipcr Zool 37 207

c) FOailATION or TWO HUAiTS

The amphibian heart onginates from two separate lateral pnmordia the free ventral edges of the left and nght hj'pomcre which fuse e\-entuall) m the mid %’entral Ime AcconJmgtoCopenha\Tr<':926) fn^ macalatum stage H22 the two pnmordia are a considerable distance apart from each other they reach the mid ^■ent^^! line m stage H27 The outline of the nght heart pnmordium at this stage is mdicated m Figure 24 c Bj stage H33 the endocardial tube is formed but not j-et curved The formation of the different sub<ii\'i 5 ion 5 of the heart bj differential growth occurs m the succeeding stages Circulation begins at about stages H36-H37 the fiiTt spontaneous pulsations b^in somewhat eaiiier The student should ^e^•iew details of heart de\'ek)pmcnt m a textbooL of embryology and should read Copcnha^•c^ (1926 1939) before starting the experimental work.

The_^efd properties of the heart forming area and of the surrounding tttsuc ha^-e been demonstrated by the some potency tests which were applied to the limb


97


1 R£lcixv€ :df-dtffercnitalton — Hetcro topic transplAntation of the heart pnmordium at rtage H28 results m the formation of a fairly itfldifferentiated pulsating heart (Copenhaver, 1926) Suchheartsmayihor typical subdivisions, curvature, etc. yet they are never completely nor mal, although they may be mcorporated In the blood stream of the host As Stochr (1925, 1929) has pointed out, certam structural differentiaboiu are dependent, even in late stages on extrinsic factors (normal blood transfusion normal spatial relations to adjacent structures such as the li\Tr etc) This pomt illustrates again the relativity of the concept of “sclf-differentiation ’

2 Regidalm properties — These have been demonstrated in the foDcnr mg way If one mtcrfcrcs with the union of the two lateral pnmordu by implanting foreign tissue mid ventraDy between them, etch of the pnmordia will form a whole heart (Eiman, 1925 Copenhaver, 1916) Such double hearts usually pulsate with different rates They arc usatBy mirror images of each other, the left heart being normal, the right beirt showing an mversion of its symmetry (situs inversus cordis, sec Fig 24 ^ Exactly the same type of inversion may be found in identical twins of Tnton produced by constriction (p 70) Regulations may also be don* onstrated by exitrp<Ui<m of ports of the pnmordium (Copenhaver, 19*6) Anterior posterior, left, and right halves will regulate to form more of less typical whole structures Such hearts from fragments of the primor dium approach normal tubes with typical subdivisiODS they art dch* nitcly not half -structures This experiment ran be done successfully even at a stage when the heart tube is already formed, 1 e In stage Hjy of Amhy stoma

3 The heart field extends beyond the hmiis of ike prospedne keart-fonffH area — ^The complete extirpation of the latter in AmbyzUma is usasBy followed by the regeneratioD of cither one or two hearts Even the citir pation of an area exceeding considerably the heart formmg area proper may be followed by the formation of small single or double hearts (Copen* haver 1926)

The foDowmg experiments illustrate pomta a and 3

Expeiiwznt 13 BLocxnro oFTnr FenoM or Lxrr AKD Rioirr PawouuA

(After Ekman, 1925 and Copenhaver, 1926)

llatenaX

Rana or Ambysloma embryos (For hosts use meduUaiy-pIatc or roedJ lary fold stages for donors use late ncurulac [dosing folds] or canT tail-bud stages )

standard equipment (p 41)


98


Proctdve

I Take donor and host out of all membranes and plajx them side by side m grooves m an operating dish. Onent the donor right side up and the host ventral side up Operate m full strength Holtfreter solution,

3 With the glass needle make a longitudinal sht m the median ventral line of the head of the host- The sht should extend from the outer edge of the medullary fold backward to about one third of the entire length of the embryo Cut deeply and make a wide gap without mjunng the heart mesoderm to the left and the right (Fig 34 6)


Fla. 14. — ProdtxrtlflQ tj two batrti ect of one pnmonSam (« d fcfto Copc[Lha>er 1916) ••dmor ezubtTo the dotted hoci ladiCAle the atrip of Uairc (gUl ajra) wt^ U buplaated In the reglcQ Indkaled In i by dotted Imet 6— hostanbryo ••heart region of host ezobryo directed Cootc that the two hearts are minor images of each othes) Ci^the dght half of the prcapetliTt heart >rea (ri) In stage 17

3 The tissue which is to serve as a block must be self-differentiating or else it might be mcorporated In the host heart Therefore slightly older donors are chosen The prospective gill region was found to be smtable as a block but any other tissue — for instance somites — will serve the same purpose With a gUai needle cut out a long and narrow strip from the prospective gill area of the donor (Fig 34, c) The stnp should mdude all three g erm layers. Cut deepl> until you reach the pharyngeal cavity Implant this strip in the slit of the host m a longitudinal direction Take care that the epidermis of the tram^lant is adjommg to that of the bostI’lace a glass bndge over the transplant for 30 minutes or longer

4 Remove the glass bndge after the transplant has healed in discard the donor place the host m another dish m tV Holtfrctcr solution Take a

protocoL Rear it together with a few unoperated embryos of host age

99



iVo/e — ilfttc 3 or more operations

5 Allow the embryos to develop for 3-4 days Narcotize the host tod a control place them m grooves in an operatwn dish, \Tiitiil ade op. Obsm’e pulsations through the epidermis If the epidermis is not mfr aently transparent dissect it away carefully with a strong glass needk mdectomy sossors or watchmaker forceps Carefully a\*oid dtmige to the beating hearts Note the heart beat Are two hearts present? Dothey beat synchronously? Take notes Pmctjcx the dissection ttt toc, on t normal cmbiyo first

6 Disstdton — Fix the host and several controls in ro per cent for raaldehyde + 5 per cent aqueous solution of nitnc aad Dissect artfully with a gloss needle Dissect a control embryo first identify the parts of the heart Then dissect theoperated animals, makeakctdies,iiid take a protocol You will find partial duplications if the block did not a tend fufliaentlj far antenor or posterior If two hearts are present, stnify their symmetry relations (Fig 24, d)


d) PAJimt REMOVAL OF THE PROSPECTIVE HEART RECION EXPERIMENT 93 (After Copenhaver, 1936)

llalenal

Amlnsioma stages H35-H38 standard equipment (p 41)

Procedure

I Remove oU merabrancs. Place the embryo in a groove m the opert tion dish ^’entral side up

3 ith a glass needle remove the right half of the heart area as indl cated in Figure 24, c Apply the technique described fn Eipenmcnt 14 under sections 4-S (pp 83-84) Extirpate ectoderm and me«>denn Do not co\'er the wound Make several operations

3 Transfer the embryos to LU^ dishes (Vr Holtfrctcr solution) tnd allow them to de^’clop for several days Notice the heart beat

4 Fix and dissect the embryos from the ventral side with a strong needle as in Experiment 23 Take protocols make sketches.

c) REMOVAL OP THE ENTIRE PROSPECTIVE HEART REGION EXPERIMENT 24 (After Copenhaver 1926)

Procedure

Proceed as m Eipcnmcnt *3 Remow area rk (Fig 24 responding area on the left side


xoo


BIBUOGRAPH\ (c-#)

CoFEraAV-n^ T\ M 1936. Eipenments on the development erf the hoirt of Am puMciaiim Jonr Eiper Zool 45 331

1939* Initoition of bc»t «nd mtnnslc contnictioa ntes In the (Cffeient puts

of the AmiJysfoma hart. Ibui 80 193

EeiaX G 1935 Tlr p enmentene Beitrage mrHetTentm ci.lnn gder Amphi hien, Ardu f Entw^mech 106:330

1939 Expenmentdle UntersQchimgai uber (he fnihate HerzentwidJimg bd

Jfirtj/wca. Ihd 116 337

Sioim^ P 1935 Entitehong der Herzfonn. Arch f Entv’medu, 106 409.

1959- Zur cmbr\ onakn Herxtfmnsplantatlon. Ihd p.300.

/) EXTIRPATION OF PASTS OF THE EYE FOSIIINC IIATESIAL IN THE NEUSHIA STAGE

(After Adelmann)

The prospective eye forming area has been localized m the medullary plate stage by Manebot (1929) using the method of vital stammg (see p 59 and Fig 17 a) The materials for the trro c>Ts are located close together near the median region of the antenor meduDarj plate Thc> are connected by a narrow median strip which repre sents the pTOspecti\'e ghiacnfl region Dunng the process of neumlation these pnmordia mo\*e apart, m a lateral direction and material which was onginally located postcnorl> mo\es forward and forms that part of the brain floor which e\*entuallj separates the c>c3 The antenor part of the meduUarj plate repre sents an eye field m the sense defined abo\-e (P 9S) It is self-differentiating and at the same time a harmonious equipotenbal sj-stem It shows a gradation of eye forming potenaes and it extends bej-ond the boundaries of the actual prospccti\*e eye forming areas These conclusions wc deduced primarily from the extensive senes of transplantation and t^irpation experiments on the antenor medullary plate bj H B Adel rnann (19290 1929^1 1930) The student is referred to Adelmann s reTplew (1936) and to Mangold (1931)

If a median strip of the medullarj platc(F1g 25 a-6-(/-e)i5 transplanted to the fla nk of another cmbr)*o the donor ma> dc\’clop two complete c^•ca M regulatwn and the transplant ma> also form either i or 2 well formed Thus the C) e field ma) giN-e nsc to 3 or 4 e> es Theremo\'alofalater •i area b-c-d maj likewise be followed bj complete regulation In order


FlO. 35 — Extirpations In tbc e>e 6dd of the medolhiy-fold staee (tee text)


to suppress the fonnatioii of i eye entirely, it is necessary to mate a defect which extends to the median line and m posterior directwn as far as the level of the broadest portion of the neural plate This shows that the e>T forming potenaes extend beyond the prpspective eye for ming area A racdiolateral gradient of eye forming potenaes was established by comparing the percentages of eye formation from lateral and from raediari strips It was as high as 70 per cent for median rtnps and only 1 1 per cent for lateral stnps under otherwise identical conditions If the “peak ’ of eye forming capaaty is in the center of the medullary plate wh> are 3 lateral eyes instead of i single median eye formed m normal development? Adelraann (1930) has shown that the underlying entomesoderm a rt sponsible for this effect It creates bilaterality by “rcinforang ’ the eye potenaes m lateral regions WTien median stnps of the medullary plate were transplanted with and without the entomeaodcnnal substrate (pit chordal mesoderm) those with substrate formed 2 eyes m almost 50 per cent of the cases, while those without substrate never formed 3 whole eyes and at best only a single eye The explanation of the origin of i-ejxd monsters (cyclopia) is along these lines The presence or absence of the substrate wiD have to be given speoal consideration b the followbg ex periments

llaicnal Jot ExpenmonU is~29

Ambytioma, tny stages H15-H18

standard equipment (p 41)

ExptaiuofT 15 Rntcn AL or THT Medlui Oin: rani) or iHz lIxDUUAir

PiATE wira SuamAiE (9-b-d~t Fla #5)

Procedure

I Select a number of healthy embryos Remow all membranes

3 In an operation dish prepare a groove of proper sue and place an embryo m it Operate In full strength Holtfretcr solution

3 \\lth the glass needle and hair loop cut out a block of tissue along the lines a~b~d-t Follow the technique described in Experiment 14 under section 4 (p 83) Cut deeply until the archenteron is exposed Remove the medullary material and its substrate

4 Make a protocol sketch Allow the wound to heal b full-strength Holtfretcr solution for 1-2 hours

5 Make sc>’cnil operations of this Place them all b a dish in Vf Holtfretcr solution

6 After 24 hours take a protocol of the condition of the wound Discard all disintegratbg cmbrj'os

7 During the following daj’S observe the development of the c^'cs


ro2


8 When the embryos have reached stages H36-H40 fix the entire ma tcnal m 10 per cent formaldehyde. Dissect the head skin away Compare the Mp* of normal and regulated eyes.

ExraiMEirr a6 Reuoval or tht. Mdjian Stbip wnncroT SoHSiaxTr {a-b-d-e Fia *5)

Proceed as m Experiment 25 but carefully awid mjury to the pre chordal entomesoderm. Scrape off the ectoderm with the tip of the glass needle and the hair loop

ExPEKMurr 27 REum al or the Latoai Ows thud with Substrate { b - c-d Fio. 35)

Proceed as in Experiment 25 Make first cut h—d, then b—c then c-d

ExTEinnjfr aS Revovai. or the Laizeai. Oie thted wiihout Substrate { h - c-d Fio 35)

Proceed as m Experiments 25 and 27 Carefully separate medullary plate from substrate tiding hair loop and glass needle

ExTOTviirr 79 RzumAL or TaO'TBUDS or the ilniuuAST Piaze

wnHODT SuBStEAia {o-^-d-t Fia 25)

Proceed as before. Make the cuts in the following sequence o-e a-c,

e~t

Make several operations of each type Compare j-our results with those of Adclmann


BIBLIOGRAPHY

Adeluanm H. B 1929a ExperimentEl itadies on the development erf the eye. I The effect of the remortl of medbiD Utenl areu of the anterior end of the Prodrbn neonl plate oa the devefopment of the ctcs {Trilon latniaius and Am ^itndai$tm) Jour Exper Zool 54 149.

Experimentil itadief on the development of the eye. IL The eyefciUuIug potencies of the median portioos of the urodefan neoial plate (Tnifin and Amilytioma fvndaiUM') Ibid 54 *91 1930. Expenmental studiet on the development of the eye. HL The effect of the labstrate (Uniaiaiernn/) on the heterotopic development of median and lateral strips of the anterior end of the neural plate erf AmUyti^wta Ibid^ S 7 t »»3 ■ 1936 The problem of CytJopia. Quart. Rev Biol ir itfr *84,

â–  1937 Experimental studies on the devdopment of the eye. I\ The effect erf the partoal and complete exoflon of the prechordal substrate oa the devdopment of the eyes of Jour Exper Zool 751199.

E. 1939. Abgrtnioiig des Aogenmaterials ond anderer Teilbeilrke in der lledaHaiplatte. Arch, f Entw’mech,, 116 689 ^Aiccou) 0 1931 Das Detennlnatlon^rroblem. HL Das W ubdUer-Aage. Eigebn.

iBloL,T 193


roj


g) PARTIAL AND TOTAL EXTIRPATION OF THE OPTIC VESICLE

In the early tail bud stages the optic pnmordium has proceeded to fona the optic vesicle The prospective significance of the different parts of the \*cside — retina pigment epithelium, staDc — has been mapped b> Petersen (1933, see Fig 26, h)

The optic vesicle is still capable of regulation, both in urodcles and In anurons Even small fragments of the vesicle may form normal, though smaller eyes (review in Mangold 1931) The prospective retina maj re place the prospective pigment epithelium if the latter is removed and Viet versa (Dragomirow, 1932, 1933, 1935) The optic vesicle still has field properties These findings Qlustmte dearly a pomt of theoretical ini portonce (p 80) The optic primordlum is self -differentiating as a whole but its parts (retina etc,) are not yet self-differentiatmg units, c\*cn in relatively late stages Determination Is a process which continues ow a considerable period, dunng which first the general and then the detailed characters of an organ pnmordium become Irreversibly fixed The optic vcsldc differs from other morphogenetic fidds in one respect eye-fonnlng properties do not transcend the boundaries of the prospccti\*e eye area No eye regeneration occurs w hen the optic vealdc is entirely extirpated This result again Illustrates a general prlnaple to be emphasiicd further in the chapter on “RegeneraUon relation and regeneration ore properties of fields and not of the organism as a whole A fragment of a roor phogcnetic fidd Is capable of restoring the lost parts, but the organism Is not capable of restonng a field once it is lost entirely

ExFEantEKT JO RivoAt or tor Distal Reojoh or tot Optic Vehcle

}[aUrtaJ

Rana sylrcttca, paluslns, stages PM16-PM17 (R pipieru embiyos are 8tich> In these stages and therefore leas desirable They may be used if no other material is available )

standard equipment (p 41)

Procedure

The student is advdsed to dissect the e>*es of normal cmbiytis (fresh or fixed In 10 per cent formaldehj’dc) before the ojjcrationi arc started

1 Select a number of healthy embryos remote all membranes Place one embrjT) in a grooN’e m the operation dish, right side up Operate In full strcngthHoltfreteraolution

2 Locate the right optic \*c5idc It forms a slight bulge on the surface With the glass needle and hair loop cut out a square piece of epidermis


  • 04


over the (/ e inFig 26 a) Remove the skm The window should be 10 large that the optic vesicle {ox ) is clearly exposed

3 Remove the outer (distal) half of the optic •v'eside with the glass needle (cut a-h m Fig 26 h) Make sure under the high power that the cavity of the \Tsicle is exposed Leave the embryo m full strength Holt freter solution until wound heahng is well under wa> — for 1-2 hours (It IS not necessary to cover the wound with foreign epidermis Adjacent epidermis will grow over )

4. Transfer the embryo to a dish with tV Holtfreter solution Take a protocol


Fd >6 — Ejc cxUrpttloQ* In the Uil bod tUst «-tbe optic \eskle caposed {-{jllre CUD It •■leoi epUbetsm Coptic >«dcle l•tQckm prapect i yc um of tbe

cptc^esicle (CnxD Klufold, ipji tfurretenea) /oiiiiBeat tplU^om r^ntnu {f~op tKttilk.

5 ^\Tien the cornea o\‘er the eyes has beoime transparent (i week or longer) fix the embryo in 10 per cent fonnaldch} dc and dissect the skin 0er the eye Note the degree of regeneration the sire, etc, of the opiated eye m comparison with the left (control) eye Make sketches ilake several operations

ExrancENT 31 Totai ExriRPAinrf or the Otttc \ esicie Material and procedure as in Experiment 30 except that the entire '^IcleisrciiKrvTd bj a cut near thebrain (c-dln Fig 36 6) Make certain oader the high power that only the narrow opening of the stalk into the train rather than the wide lumen of the optic \-c3icIc remams Fix and dissect the einbrj*o after 1 week Notice the complete absence of the ey c

BIDUOGRAPH\

bsicoMiow \t 1933 Uber Entricklung \on tugenbecbeni *U3 tianspUnlJerteo Stttckdien del emboonalea Tapetum Arch f EntVmecb 126636 ” tlber die KoordloatjoQ der TedproRue in der cnbrvoniten Motpbo da Aojenbeefaen. Ibtd 129 533


DiAOOuxow W I9J5. Detennlcitkxi des Aagttifcnmts bd AnpbitrfetL Aad. id dTJknlDe tnv Inst lOo] et bioL 8:25.

XtAfTOOLD 0 1931 Dis DctmnlMttomprobkm. in Das IVTrbdtiertnge. Erxeia. cL BfoL 7 1Q3.

PrmiSQf H 1933 Sencfate tiber Entwicklungsraedmlk I E i g d cu <L Aut n. Entw gesdi^ 14 337


6, EMBRYONIC INDUCTION

a) INTEODUCTORY EEMAiZS 'TERMINOLOGY

The experimenta to be discussed next center around the following problems ^Vhat factors are instrumental in the process of determination? How is the determination of the medullary plate of the eye, of the bal anccr, accomplished? There is no one answer to these questions, A mold tude of different agents are at work in each instance and each pnmordnra requires a ^leoal analysis

Of the few general mechanisms so far discovered, that of "embrjunlc induction is of considerable importance It may be defined as a process m which one embryonic area, the snduc/or, calls forth a spcofic diffcrcn tiation m an adjacent embryomc tissue by contact The two best ina lysed and most widelj known cases arc the lens InductioD by the opbc vesicle and the induction of the medullary plate by the anderi>in| mesoderm

The term * induction includes apparently, quite heterogeneous types of mteractioas ranging from mere “trigger actions to highly specific Interactions Induction undoubtedly docs not comprise a phj’siologlcalJy uniform group of phenomena. If one bears this in mind then the use of this conv'cnient term as defined above will do no harm

Embryonic mduction can be demonstrated In two ways by extirpa tlon and b> transplantation If, after the extirpation of an embryonic area an adjacent organ foils to differentiate, then a causal interaction of the inductor tj’pe suggests itself However, crudal evndcncc for Induction can be obtained only from transplantation experiments because they a posIU\’e rather than a negative Indication Transplantations as proofs for induction arc alwaj’S of the following tj’pc the structure which o suspected of being an mductor is combined with relatively indifferent (that U not sclf-differcnUating) tissue from a remote part of the same or of an other embryo or ei'en from an embryo of another speacs For instance the optic \Tiiclc Is combined with belly ectodenn or the archenteron roo of one speaes is made to Interact with ventral gostrula ectoderm of an other speacs If under these circumstances a lens or a neural tube rt Bpecti\*clj appears in itruclurcs which would nevTr haw formed these 106


structures by themselves then the mductor capaaties of ej'c or of ar chentcron roof are positively established- Both of these examples will be treated m the foUowmg experiments. The student is referred to the chapters on mduebon m the books of Huxley and Dc Beer, of Spemann and of Weiss

6) TAiLini: or lens toeuation ajtee extirpation of thf eye

PEmOEDrUM IN THE MEDTJILAIY PLATE

(After Spemann)

ITie optic cup and the lens originate from two different sources the former as an evagmation of the forebrain, the latter as an mvaginatron of the head ectoderm The fact that the lens is formed at the pomt where the optic veside makes contact with the owrlying ectoderm is suggesti\'e of a causal relation between the two Spemann was the first to test this asumption experimentally He extirpated the pnmordimn of the optic \Tside and found that no lens would differentiate although the prospec ti\’e lens area was left undisturbed It was conduded that the latter re quires an inductive stunulus from the optic veside for lens formation His first experiments were made on the European grass frog ILtanporana When another speaes R escuUrUa was used a lens though small and m complete was formed even In the absence of the optic veside These ex penmenta were extended to other ^leacs and repeated b> other m\‘estigators. Spemann s conduswns were confirmed first the optic cup is Instrumental in the formation of the lens second the dependence of the lens epithelium on the optic cup diffcia m different speaes. The lens ^ since become one of the classical objects for the analysis of embiyomc induction (renews m all textbooks sec also Spemann 1938 and Mangold

1931)

Most of the Amencan urodeles and anurans m which the extirpation of the optic pnmordium has been performed so far show a complete lack of lens differentiation in the absence of the optic \’eside This holds for R- tyitctica R, paJusiru (Lewis 1904 1907) R caiesbaana (PaSqumi ^933) and X maculalum (HsLmson 1910)

The extirpation of the cyt pnmordium can be done cither m the mcdul ^ plate stage or In the carl> tail bud stage before a contact between ®Ptic iTside and epidermis is established Experimentation on the earlier •tage has the advantage that the lens-forming area need not be disturbed ^ ^ whereas m the tail bud stage the lens epidermis must be lifted to access to the eye and then healed back ogam


107


Expewmektji


Mafmal

Rana syltaltcn (stage PM14) or R pipuns (stage Shi4) or CDirespoud mg stages of R palustris or catesbetana or Amhystoma stage H15 or stage H16

standard equipment (p 41)

Procedure

The ami of this experiment is to remove the eye pnraordium (part of antenor raedullarj plate) without disturbing the lens-fonning area, which IS located just outside the medullary plate or fold (sec Fig 17, L) The experiment is Identical with Experiment 28 (p 103), except that a median cut should be made mstcad of b-d (Fig 25), In order to prevent eye re generation Again, first male the median cut then the cut in direction fr-c and finally the cut m direction c-d Carefully avoid any damage to the mesodermal substrate and to the pro5pccti\’c lens area. Follow the tcdmique described for Expenmenta 25 and 26 (pp 102-3) Operate In full strength Holtfreter solution and allow wound healing m this solution for se\*eral hours Tate protocols of the operation

Operate on several embryos

Fix the larvae In 10 per cent formaldehyde when the swunmlng stage is reached and the cornea over the left cy^ is dear Note the opaque left lens and the absence of a transparent cornea on the right side Carefoll> dissect the stin of the right tide of the head Find out if the right e>*e is completely absent. The presence of a small eye may be doe to an mcom plete extirpation -and a regulation of tbc fragment. Note If 1 small, v-hitish opaque lens is attached to such a regenerated eye The complete absence of aU rudiments of a lens can be definitely established onl> bj sectioning

c) FAILUaE or LENB rORlIATION AJTEB EXTIWATION OF THE OPTIC \TSICLE IN EARL'V TAIL BUD STAGES

EXPEEDIOfT JJ

Matmal

Rana xyl-'otica stage PM16 or stage rMi7 or R pipiens stage Shi6 or correspondmg stages of f? paJuslris {R syivcticaa.ndp<dHSlnstre preferable )

standard equipment (p 41)

Procedure

Note the projecting optic ^*esfdcs inspect the head from all sides

I Rcmo\-c all membranes

loS


a Operate m full strength Holtfrctcr solution Place the cmbi^'o m a Pcrmoplast groo>T, right side up With the glass needle cut out a square flap of epidermis over the cj-e, cuttmg on three sides onlj (Fig 26 a) Foflow the technique described in Erpenment 14 (p 82) Peel the epidermis oS the optic reside carefully In early stages there is no close contact as yet between the two Reflect the flap of ectoderm Cut out the optic \’eside at its base using the glass needle as m Eipcnmentyi (p 105) Turn the flap bacL again flatten it out cautiously with the hair loop or glass needle and place a glass bridge o%’eriL If It fits well it win be healed m 20-30 minutes Control the healing carefully if the flap shn\'el 3 then neighboring epidermis will grow o\Tr the ^^ound The lens will be absent m either case but only the instance m which pro^jective lens epithelium proper fails to form a lens is condusnT for the present problertu

3 Prepare a protocol and transfer the embry*© to a dish with Holt freter solution.

Operate scvTral embryxis In the same way

4 Fix the swimming lar\ae se\'eral day's later in 10 per cent formalde hyde Proceed as m Experiment 32

d) LENS INDUCTION AFTER REUOt A1 OF THE PROSPECmX LENS EPITHELIUII

Once a causal relation is established between optic ^‘eslde and lens for niation a number of questions ansc concerning the nature of lens mduc tioQ For instance is the optic \*csidc capable of inducing a lens out of foreign epidermis? This can be tested cither by transplanting foreign cpi dermis m the place of the prospectna lens epidermis or simply by remo\ ing the lens epidermis and allowing adjacent bead epidcnnis to grow o\ er the eye

ExyrKnnzcT >4

ilalmaj

05 in Experiment 33

Procedure

1 Rcmo^*e all membranes

3 Operate in full strength Holtfrctcr solution Place the embryo m a I’crmoplast groo\*c nght side up With the glass needle remo\-c a square piece of epidermis which co\crs the optic xTsicle f/ e in Fig 26 c) Care lufly a\*oid any injury to the optic \*c 5 iclc Lea^c the embryo in the full »trength Holtfrctcr solution for o 40 minutes Obsenx the growth of ‘djacent head epidermis o\xr the wound X09


3 Take a protocol and transfer the embryo to a Lfly dUh contaliung iV Holtfrctcr solution

4. After the embryo has reached the swimming stage fix It m 10 per cent formaldehyde After a few mmutes the lenses will become visible as opaque white structures Note that the epidermis which has grown over the nght tyt has become transparent and forms a normal cornea. Dissect the cornea away from both eyes and study the size and shape of the nght lens which has been mduced from head epidermis Further suggestion — ^Transplant the optic vesicle to the flank of an other embrj-o Make a deep groove ventral to the middle somites and fit the optic vesicle into it. Heal a flap of epidermis over the transplant or place a glass bridge over the eye to keep It in position and allow adjacent epidermis to grow o\Tr Again find the lens by dissection after fixation in 10 per cent formaldehyde

BlBUOGRAPm ( 4 -d)

JIauusov IL G zgio. Experiments on the lens In Awthiysioma Proc Soc Exper BfoL and lied., 17 igg.

Lewis U H 1904. ExpenmeoUlstQdiesontbede\’elopinento{thee7tInAinphn]U. L On the on^m of the Ic« Rntia pdusUis Aner Joar AnxL, $1505

1907 ExpeiimeaUl stwhes 00 IhedcveJoptDOStof theeyt Id Aiophlbia. m

On the origio and dif’erentutlon of the lens. Ibii, 61473 llAKCOtD 0 1931 Du DetenalmtioaspiDblaD. m. Du UlrbdtienDge b der

Entwkklong uod Re^eratioo. Ersebo. d. DIoL, 7 193

PASQonia P 1931 Sulla dctennln&ztooe e sul diflerenxkiaento dd crhtalliDO In Aud

caiahdena (Shaw) Joor Exper Zoti 61:45 SrotAJCf H. 193S. Embrj'oidc devdopenent and Induction- bewIUvtn kale Uni %Trsit> Preu.

e) THE OBOANIZEn EXPERIUEKT TRANSPUtNTATlON OF THE UPPER LIP OF THE BLASTOPORE

(After Spemann and H Mangold, 19*4)

A ole — Before startmg this experiment read the chapter on organizers in an> textbook Read the section on gastrulalion (p 43)

The organizer experiment (Spemann and H Mangold 1924) represents the classical case of a complex mductlon The authors disco^tred that the upper lip of the blastopore of a urodele gastmla when transplanted to the flank or the ^'ent^ll region of another crabrj'o of the same stage would sclf-differcntiatc into mcsodennal structures such as notochord and somites and also would induce adjacent host tissue to form additional mesodermal and ectodermal structures. Host and transplant structures supplemented each other in the formation of a whole new or ganisra This capaatj for integrated inductions has earned for the upper


xio


lip tte designation "organizer The further analj’sis vraa greatl} en hanced by the appbcation of ‘heteroplastic transplantation i c cx change between embryos belonging to two different speaes making it possible to distmgmsh between self-ffiffcrcntiating donor tissue and mdneed host tissue

In the organizer action the following omponents can be distinguished

I The transplanted upper bp mvaginates. It possesses autonomous gastnilatjon tendenaes

a It self-<liffercntiates mto notochord and somites or other mesodermal tissue

3 The transplant mduces adjacent host mesoderm to form mesodermal structures such as notochord, somites pronephros lateral plate etc Host and transplant structures aapplcment one another to form a com plete set of mesodermal axial and panuDal organs The share of the transfdant m this secondary set is NTinable and depends partlj on the mitial size of the tran^lant.

4 The transplant induces overlymg host ectoderm to form a neural tube which is frequentlj subdivided into brain with optic vesicles spinal cord etc

5 Occasionally a secondary gut is induced In host entoderm

In the following a few points will be discussed which arc of bnportance in planning the ex pe ri mental work Bautxmann (19:6) has shown that the regwn of the early gastrula which possesses organizer capaaty corre sponds to the chorda mesoderm area (see map Fig 4) Therefore it is not necessary to use strictly median parts of the upper bp The results of the expenment will differ when upper blastopore bps from different stages of gastrulation are used It will be remembered that the upper bp of the early gastrula is composed of material which mvaginates first and will come to be head mesoderm The upper bp of late gastrulae is prospectrv’e trunk mesoderm Spemann (1931) has shown that the two differ m thdr inductive capaaties the former has a tendency to induce head structures ^nd Is therefore called Tiead organizer and the latter the trunk or ganirer tends to Induce trunk structures It is therefore necessary to chedc and to protocol carcfuUj the developmental stage of the donor Furthermore, the host level to which the transplantation is made inffu cnees the result Spemann (i 931) has found that the hcad-organlzer tend cnacs arc strong enough to induce a head in any host level Howc^'e^ the trunk organizer will mducc a trunk only in the trunk le% el whereas it will induce bead structures m the head 1 c\tI In the latter instance the host influence o\-cmdes the inherent tendency of the transplant. Since many of the inductions obtamed by the student will be partial rather than com


III


plete embr^'os these findings may help to interpret the results Finafly the anal orientation of the induced embrj'o will be found to vary considerably the axes of the host and of the mduced embryo may be at any angle with each other Again Spenttann (1931) has found that the transplant has its intrinsic polanty but that the more powerful gastruUtjon rao%ements of the host will cause those of the transplant to deviate, to that the final onentatwn of the mduced embryo will be either parallel to the host axis or the resultant between the two tendenaes

Altogether the most complete secondary embryos can be obtained when large median pieces of the upper lip of early gastrulae are implanted in the ventral hp opposite to the upper lip of the host (Fig 27 i) In this instance the directions of invagination of the host and tran^Jant win be parallel and host and donor structures will be in corresponding levels In this way the host will Interfere little with the formation of the second ary embrj'o

ExrauMEiirr

Ifaienal

gastrulae of Amiyj^omo, any speaes stages Hio-Hii Standard equipment (p 41)

hoU — Use dishes with agar bottom for operations and for raising (p xi) Steriltze all instruments carefully Procedure

Note — Since the color differences between donor and host are usually not very striking the transplant will soon be lost sight of unless it b \ntal stained Therefore vital staining of the donor cmbiyos in tolo m o i per cent Nile blue sulphate is advisable. Remember that early gastrulae ere very delicate when taken out of their membranes and that your success will depend on careful handling and working under sterile conditions

1 Select 8-1 2 healthy gastrulae In the stage of the sickle-shaped blastopore (preferabl> stage HioJ or stage Hiof)

2 Renure all membranes — Stnp off the outer jelly membranes with two pairs of watchmaker forceps with not too sharp points. Wash the embiyos B<r\-eral times m Holt/rctcr solution Transfer these to a dish containing sterile Vo- Iloltfreter solution Remove the vitelline membrane (follow the technique described on p 39) Transfer the embiyos to full strength Holtfrcter solution Keep the dish tightly closed avcild an> shaldng of the dish

3 Prqjorc the operation dish fill it with full strength Holtfrcter solu tlon and prepare 5c^'cral glass bndges Make a smooth flat wide groo\*e in the agar it should be larger than the embrj'o If the bole b too small


1X3


il M iliroiU to rcrm\T ihc cmSrvo uninjuiT«i Tran'^rr enlnwcfthc u—f fta-’c into thr <!j h Lvr a tcrPr p priic t\li ch not l<Tn u ntl l>c'o c for anoth r pjrpov: Cb'V' r a a Iki t an c^\ m» I'hich ha' httlc or no injjnr' from thr rcrooN-al of t> '‘Midi rrmljrarc With the hair loop >ho\T It into the nmoxc fnith \cf> pmtle r~o\T~nt'^ I bee it tjndc I’oirn v> that the upper hp facc< j-ou (I i:: 3^ f'

4 Pref'jrr th A V »n // / f — With the tip of a am 1 nc pb ' needle rjt ojl a j^uarc area of the <urfacc U\rroppo itc to the I b'lnpi'T ap


ll t ~<>r /r* I r- - f « Â». J ;

' n iJ- r* ^ *1 'l ** - j

t I 1 » i' *--• I f r - I T- I 1 ^ « f ^ 1 I t f -I rf T cf I » - f ,

I I —at U n tl rr-j f» rl r il \r tr^l 1 j » ' 1 *| ,»‘ar birr Tl

^ ' 1 1 ml !<â–  ir*» larrr rt '<t| It I 1 r— \rafrwo‘t!

h ' V «r ’

lartl rv, '(»: llatt*

^1 I tana'fr-i\ (t ta rrj

‘ ' b tl an t' I I IJ 1 Mai t» rr i » I j 1 t}

' 1 p It ' ax 1} ( t‘i r • I j I - xr I ^ r- ' I* I t 1 t ^ t* t’ I , ‘ I* 1 b r I r

‘t’ If , t t ^ } r * Il 1

  • â–  I la rr l‘ I j a l » 1*1 1*

■^ * l‘ 1 a r I 1“ I J t V 1» i t i' I \ t 1

tt



plant 15 opposite that of the host (Fig 27, b) Press the transplant gently into position, fit it in tightly, and enlarge the hole if necessary Pi^ the glass bndge over the transplant (with 2 hair loops) and press it down so that the transplant fits well and is in the desired oncntation. Discard the donor embryo Cover the operation dish at once and sbo\T it gcntl} aside, do not lift it up Healing tales hour Control the progress of healing but disturb the embryo as httic as poasible. Readjust the glass bndge if necessary

7 After 45-60 minutes, cautiously remove the glass bndge Transfer the embryo in a clean stenle pipette to another dish with agar bottom filled with iV Holtfrcter solution Dip the mouth of the pipette under the surface near the bottom before y'ou release the embryo Place the embryo with both blastopores directed upward otherwise gastrulation will be Impeded Cover the dish at once

8 Take a careful protocol, make sketches Indicate stage sire, shape, onentation of transplant iVote — Make 3-4 operations

9 During the following days disturb the embryo as little as possible handle it with utmost care After gastrulaUon is completed tunl It r^t side up to allow neural folds to de\’elop normally Watch for a secondary neural plate Make sketches

10 Tlie mduced embryo IS usuaflyat its best m stages during and after the closure of the neural folds (Fig 27, d) and in earliest tail bud stages. Study and draw these stages carefully The early tail bud stage is a enti cal stage, from then on mortality is high andonlyafewcmbrjoswillsar vive ^ m stage H20 or stageHai If you wish to rear an embryo longer watch It every few hours for the first signs of disintegration or edema and fix at once Disintegration proceeds very rapidly once it has started

/) tUPLANTATION OF INDITCTOBS IN HIE BLASTOCOELE

(After Spemann and 0 Mangold)

Spemann and O hlangold found that a piece of the organiser when placed m the blastocoele of a blastula will be shifted into the region of the h\'er primordium b> the gastrulation movements of the host and will Induce a secondary embr^x) in this position( £trtsteci method) In some m stances it will be found m other locations Its final location cannot be c tcrmincd exactly b} the cxpcninentcr This dlsad\'antagc, bowner i* outweighed b) the advantages of this technique. It is a much simp er and faster experiment than the transplantation Furthermore it it possible to test the inductive capacity of structures which cannot transplanted to the surface of the gastrula— for instance adult tissues


114


killed tissues or even foreign bodies or extracts or chemical substances which one can adsorb to agar and implant m this way The rapid prog ress in the organizer analj’sis and the discov'crj that medullary mductwn IS mediated by a chemical substance were gTcatl> enhanced b) this tech meal ad\Tince

ExpaaDmNT

Maknal

Ambysiama anj speaes stages Hio-Hii as donors stage H9 as hosts

standard equipment (p 41)

operation dish with agar bottom Procedure

1-3 As m Experiment 35 Place one donor and one host m the opera twn HlsH. Operate m full strength Holtfreter solution.

4 Prepare the host — Place the host m a wide groova with the animal pole upward ^Mth the point of the glass needle make a aht m the roof of the blastula near the animal pole (Fig a; c) Destroj as few cdls as possible

5 Cut oul the Iraruplant a ptece oj the upper Up (Fi; 27 a) — ^Use the hair loop and glass needle. Spemann has frequently used the hair loop as a cutting instrument. It p\*es an oval transplant with sharp edges.

6 Tmplanialton — Place the transplant near the sht m the host and work It through the slit with the tip of the glass needle (Fig 27 e) Push It deep down and try to bring the edges of the sht together Place a glass badge o\-cr the wound and apply sbght pressure TATien the edges of the wound fit well together it is ad\T5able not to use a glass badge at all

7 The host has a tendency to extrude the transplant, unless it is pushed mto a deep positwo. \^ atch the operated embryos closely and re implant if necessary Do not shake the operation dish

8 After healing is completed transfer the embryo) to a dish with -jV Holtfreter solution Take protocob

Make a number of operations The mortality is high Keep all dishes bghtly dosed Use sterile pipettes and hair loops for inspection

Sections 9 and 10 as in Experiment 35

ExpraniEfT 360

Study the papers of Holtfreter (193411 and 6) and try to obtam mdoc tbns with living or dead tissue of adult salamanders (brain, rctma) or With pieces of medullary plate or ectoderm of A mbysloma which have been tilled by heat or In alcohol (wash carefully before implanting) There b


15


a strong tendency for such implants td be extruded Healing has to be watched closely Ho not try these c^jenments until you have had success with Expenment 36

BIBLIOGRAPHY {t-f)

Bautzuaxn H. ipjA. EipenmentcBeOntmodnuiseniurAbjreimmgdaOnswin tkaiMcntrumi bd THltw Itfflriotitf mil anem Anhang ubcrlndokbondurdiBliitulamateriaL Arcb f EctiPmedi 108 3S3 HotTnETrRjJ 19343 Der Elnflots ihmnlsdier roedttimeher end ciietirf»db«r Eingriffc tuf die lodtmerfihlgkeit voo Triton Kecnteflen. Arcb f Entw^mecli-, ip t»6

1934^ tlbcr die Verbrritong iodozieresder Subitameo und Hire I/GiUmjen

im Tntop-Kcm. p 308.

SrzxAKN H 1931 tlber den Anted voo Implintat and WlrUkeun to derOricDber ung and BescfatfTcphrit der Induserten Embryontlanltce. Arch f Eotw'medu, 133 390 1938 EmbOTJnicdevtJopmenttnd indnctloo. New Htvtn Ytle Urdvenity

Press.

SraUMS, H, and ilAwooto H, 1914 Uberlndottko von Embryomlsnla^dttfcfa ImpkntaUon artfronder Or^misatoren. Arch / mir Aiut a. Entw'mech. root S99 7 PARABIOSIS

(After R- K- Bums, Jr 19J5)

This method consists of fusing together s whole embryos aide b> side or in other positions and allowing them to develop as conjoined twins. This can be accomplished readily by creating wound surfaces on the td jacent and pressing them together until healing is completed Such twins have been reared through metamorphosis.

The method has been found useful for several purposes Hamson (19®® 1924) in an experiment in which the fate of the neural crest was studied, healed together 2 frog larvae from which the dorsal halves of the spinal cord were removed to prevent their regeneration Dctwiler (1926) in hb analysis of the influence of peripheral fields on the dcvclopracnt oI the central nen’ous sjatcra used parabiotic twins to obtain a substantial slan loss without a proportionate muscle loss The most fruitful application of the method has been in the field of physiology of sex detenmoition Conyimcd twins share a common blood circulation and their sex honuMCS are distributed to both partner* Since 50 per cent of all parabiotic t are expected to be male female combinations valuable rnatcnal can be obtained for the study of the interaction between genetic ^

monal sex detennmers Bums and Ultschi (reviews In 1934 applied this technique cxtenshTly for mvcstigations of this pm eio Ultschi has occasionallj used an end to-end fusion of embrj-os (tclo tosi* 116


ErPEKMTNT 37


Maitnal

urodeJe larvae, stages Hsa-HaS

anuran larvae stages Pili6 or PM17 (embrj’os m later stages arc motile and more difficult to handle)

standard equipment (p 41)

Procedure

I With the glass rod with ball tip mate a deep groove in the Pennoplast wide enough to bold both embryos ^en pressed together and when in upside-down position- Smooth the edges Fill the dish with full strength Holtfreter solution


Fra. iS. — Ptnlkaiii experiment (modified after Adim*, i^x) e-embryn la openUon riece enw-betebed^ktio nee to be reouned freon left t«u embryo (lee text) &-tbetikO enbiyoi b the opermtloo groore,

2 Take a considerable number of embryos of identical stages out of all membranes

3 ^Vith the glass needle cut out a orcular area of ectoderm of consider able extent on the left flank of one embryo and on the right flank of an other embryo (Fig a8 o) Remove either the skm co\’enng the gill swell ing or the skin behind the limb and pronephros swelling

4 Place both embo'os m the groove m npside-down position and with the wound surfaces adjacent to each other Partiallj imbed the cmbr^xis in Pennoplast by building it up on the sides (Hg aS b) Place a glass tnidge on top of the embr >’05 to bold them m position Otherwise they will move by ciliary motion which 13 rather strong in these stages Keep the cmbrj’os In the operation groo'V'e for i hour or longer

5 After 1-3 hours transfer the ctnbrj’os to a glass dish or LOj cup m tV Holtfreter solution

6 Obsovx the de\'elopment of the twins their heart beat swimming


17


reactions etc. 1/ tune permit!, raise the twin! to stages in which the gonads arc differentiated Dissect the gonads and compare them irith normal gonad! Sectiomng i! necessary for detailed studies Consult Wt schi (1934 1939)


BIBLIOCRAPHl

Bcas‘S,R-K.,Ja- 1913. The ser ot paiablotic tirau In AmpliJbii. Jour. Eiper, ZofiJ 31

DcnviLa S R- 1526 Tlie effect of redocuoo oJsUn and of mmdeoa Utederdop* ment of ipinaJ cansTca. Jour Eiper 2 o 6 L, 45 J99.

Hauscct R. G 190S. Embr^Ttmc tnospluiUtloa mod desTlopmeat of tbc oenoos fvftaiL Amt- Rec, Ji 385 19*4- J^eoroblxit vrous ilteath ctfl fn lie devekipmciit of pcrfpBcnl nerva,

JooT Cocrp 'NeurDL,37

\\ iTscni, £. 1927 Sex revemi fo psrmyotjc tvios of lie Anerfcu wood fro^ BW. BuD. 52 137

1934- Gena tad Inductor* of aci djffejtnliatjon In â– w plifWtni. BIoL Ret,,

9 460.

— 1039. ilodifictbon of tie devefopment of »ei In knrer rtiiebralo tod la

mt m mt U. lo Aixcf? Set tod Infenud tearuoax BiflfniOTe \\lIStBa

LWmJos.


1 EJCTEWhAL factors in DE\ ELOPilEST

a) TSE PROoccnov or cicxom and oror* abnouiauties b^ TREATMENT WITH UTHTDll CHLORIDE

(After T S HaU)

Embr5X)s will tolerate moderate changes in their cntironmcnts but ci cessive changes in crtemal factors auch a* temperature chemical substrate etc wQl result i/t abnormal de\elopraent Since ezteniti tgents permit a quantitati>T approach thej are a valuable tool In dcvcJopraental mechanics

Of the large bod> of material which is a\’aflable onJ^ two expenments were selected Thej concem the effects of changes in the chemical com position of the culture medium on the de%Tlopment of amphibian cm brj-os. Thej Illustrate certam general aspects of the role of external agents in dexxJopincnt- ExperimenU on the temperature effect on de^■e^opmcnl can be set up easH} and require no specuU treatment "Manj other ex penmen ts concerning the role of light, of oxj'gcn tenawn and other chem icai properties of the mflieu, of grasifj etc cad he planned b^ the stu dent on the basis of literature itudies

The peculiar effect of lithium chlonde on csrl> dmTiopmcflt mas u* cohered bj Herbst (1893) in his ^atcmatic studi of the effects of certain

X18


ions on sea urchin development. Lithium chloride if added to the sea water m a ccrtam concentration and if applied before gastrulation re nits in eiDgastrulation that is the entoderm grows to disproportion ately large size and instead of mvagmating evaginates. This mteresting phenomenon of entoderrmaation m the sea urchin cmbry’o has been thoronghly analyzed b} Lmdahl (1940) Child (1940) and others These Btndics give an insight mto the physiological mechanism of hthimn action and should therefore be consulted m connection with the amphibian cipenments to be discussed here

The hthium effect on amphibians has been studied by Adelmann (1934 1936) Lehmann (1937, 1938) T S Hall (1942) and others Ifearl> gastnilac are kept m a high concentration of hthiiun chlondc for se\*eral hours, eiogastrulation results. The msTigmation of mesoderm and entoderm Is inhibited (see p 122) If alowcra)ncentrationisapphedfor6-24 hours then a variety of abnormahties results Most common among them are complex defiaenaes of the head In the least affected cases the head is disproportionately small (tmcrocephaly) Its bilateral structures (nose eyes forebram hemispheres sucLen or balancers) may be found m slide grees of appronmabon Extreme cases of this tj^jc maj show an un paired single nose (monorhyny) a single median cyt (c>x]opia) or e\tn a complete suppression of an> or all of these structures These monsters are strikingly similar to monsters found occastonaB) in human and mam malian fetuses Microscopic studies show that mtemaJ head structures — for instance the mandibular arch and the pbarjTu— are also affected Obviously the median part of the head is more severely affected than the lateral parts and the head as a whole is more sevxrclj affected than posterior r^ions. Different structures show a differential susceptibil it> to the lithium action Adelmann Lehmann and Hall each ha\*e demonstrated that the pnraary effect is on the median strip of the mesoderm (organizer) and that the effects on the ectodermal structures arc secondary effects caused by faul^ inductions. Lehmann applied the Iith linn treatment at different stages of gastrulation and showed that dif ferent regions of the organizer arc diffcrentiall> susceptible at different times For instance in earl> gastrulation the posterior head and anterior trunk lc^•cl are the most highlj affected regions Microcephaly and cy clopia were more frequent after treatment of the middle gastrula Vana tionsof the concentration and of the time of exposure will likewise modify the effecu (Han 1942)

Obviously the same agent can produce a number of different end cf feels depending on its concentration on the duration of its application oa the phase of de\’elopmcnt etc (see Stockard 1921) The ‘pwafiaty


of the end effecta appoura to be due to diaturbancca of the dehcately bah anced Intrinalc developmental pattern rather than to the chemical prop, crtiea of the lithium chloride Thia point la emphaaized further by the fact that the same abnormahtiea (microcephaly, cyclopia etc ) can be produced by a vanety of other agenta aa well (Bellamy, 1919, 1922), for matance, by hypertonic sodium chloride solution, by magneahim chloride, etc. or by high temperature (Hoadley 1938)

However it would be misleading to make a sharp distinction between “nonspecific ’ external agents and a specific ' mtnnsic developoienUl pattern. If an eitcmal agent affects a fundamental physiological activity of all cells then its cffecta arc widespread, and the end effect depend! Jargelyon the differential susceptibility of the organ pnmordia— the agent will give the impresaion of a “nonspecific * entity If by virtue of Hj chemical properties, it affecta selectively a local process, then it will appear as a ‘specific agent In the last analysis differentiation is brought about by a complex mterplay of mtnnsic and extrinsic conditions, and the tcra “speafic Is dispensable

ExpotntEifT $8

MaUrtd

beginning gastnilae of R, ptpiens or R tyhdtca or domiitms or R caksbcpma or A trujoiUUum or A tignttum glass Jars or Lfly cups with tightly fitting lids pipettes

lithium chlonde c-p , i 9 gm. m r,ooo mL distilled water lithium chloride, c.p , i 9 gm m ^50 icL distilled water Note — Concentratrons and time of exposure which arc necessary to produce microcephaly vary shghtly with the species used The foDoriof data apply to R ptpifns Considerable vanations will be found, ertn d the material is treated uniformly If you find eirogastrulac, consult tec

tion 6 (p 122) If possible keep the temperature constant (optimal temperatures between 18® and 22 C)

Procedure^

1 Remove all membranes except the vitelline membrane, fmm loo*

200 eggs .

2 Place 30-50 embryos (shortly after appearance of the blastopore into each of the following solutions

a) I 9 gm LiC] c-p in I 000 mL distilled water

b) I 9 gm LiCl cp in 250 ml distilled water I un rmUy iodebted to Dr T S

Ulbof ihaerperijBmt preAiou 5 to tbdrpoblla


120


c) iV Holtfrcter solution (control)

Do not crowd the eggs Co\’er the dishes

3 After 24 hours transfer the embrj’os from solution a to tV Holtfreter solution After 2-7 hours transfer part of the embrj’os from solution b to Holtfreter solution lea^’C the rest of the embr}’os m solution b for 24 hours Take careful protocols of the time of exposure etc. Label all dishes carefully

4. Observe the gastrulation and following stages Note and sketch the first delations from normal devxlopment 5 After the swimming stage Is reached (e>TS pigmented) fix all em bryos in 10 per cent fonnaldehj*de Dissect the most mtcrestmg cases with glass needle and watchmaker forceps Compare with normal em bryos Notice the great variations m the effect even within the same group of cmbry’os Arrange all cases m a graded senes Record }*our re suits in a tabulated form (consult Hall 1942)

BIBUOGRAPBn

AamiAiK H- B 1934- A study of axiopi* In AmAijrtima finjvtjiajrt with special reference to the mesoderm. Jour Eiper Zotd 67 31

1936 The problem of cj-dopa. Quart. Rm BwU ii i6j 2S4.

Beuakt 1919 DiffereotialfaKepdbihty o a bacii for modihaDoo and COD' trdof earl} derdopmest In the fros Btol Boll 37313 ~~ — 193] Dileratlal suscepdbiht> as a basis for modihcatiaD and cootrol of eidy derdopmeat in the frog, CL T}'pe3 of modification seen in liter derdopmeu til stages. Amer Jour Anat., 30 473

C hitp C. hL 1940. Liihlom and cdunodenn ctogastralatioQ with a re%new of the phynoiogicatgTadiait concept PhNicoi Zool 13 4Haii, T S 1943 The mode of icbon of bthram sails m amphibian de^dop^lellt jesrt. Eiper ZoOL, 89 i

Hnasr C, 1893 t\otere3 uber die morphologische WiiLong der Lithlumsilze u.

QuetheoretucheBedeutmig Jlitt lool Stat Neapd, jj 136 Hqaoixt L. 1938 The effect of sapixmaiiniinn tempeiatures on the dcveloptnent of piftfns Growth, a 35

Limtuor F E. 1937 lleiodenmscning des prasumpti\cn Chorda matenals doich ^owiitnnj Ton LlthiumchJond aof die Gastrola vxm Tnlon alptiirts Arch, f Entw’mech., 136 nj

1938 Regioca]c\ enchledcnhelci desOrganoataii \on Tet/OB insbesoodere in der vouloen and hinteren Kopfregwo, nachgewiesen doich phasenspenfische ErteuximgTmT.bhTTTTnJ MHUnf tfn nnd operatiyhenAten Regionaldcfct.ten. Ibtd tj* 106.

P E. 1940. Iseue Batrige sur ph>siok5(pschen Gnmdlage der tegetathdoctuiig des dnreh T.itliinmtnnpn Aich f Entw’mcch 140 16S

SrociAiD Ch. R. 1931 Dcvdopmentsl rate and itmctural erprcMon an open f^tal study of twins, double monsters »nH single defonnibes and the mteraction GnbtjocJc organs during their origin and deveiopm cut Amer Joor Anat

a* IIS


121


fc) EX0CASTK17LATI0N A^'D SPINA BIPIDA PBODUCED BY TREATMENT WITH HYPEBTONIC SALT SOLUTION (After Holtfrcter)

If amphibian embryos are exposed to a h>'pcrtomc saJt solatron before the onset of gastnilation the entoderm and mesoderm fdl to im-igi nate. Instead they cvaginatc and a constnction appear* between the ectoderm and the partly or fully evagmated mner germ layer* (Fig 29 a)



Fic. * 9 . — Tot*l &od pullft] exocvtroIaUQii (Inaa HoUfrrter XQjjf) • trolatios #»Ia cutroU J-Ia Ufl^pod K/»*Ktodem »o4-#rl"fT»ffea*

ao o d ma-itotodtna t rf“parti»l 5^»jroCkpln5%ljklitiB»lolii>»rto*^ wfpiiift bi£di (open cptoal cord tnd xatebru)


Eiogastrulation is one of the most common disturbances of cari> a® phibian development and can be produced m man> wa>’» such as b> treatment with hthmm chloride or with magnesium chloride etc. An eihausU^t analj’sis of erogastrulation m amphibians was gi\'en b> Holt

freler (1933b) who applied a dBute Ringer solution (o 35 per cent

the so^aHed ‘^Holtfrcter solution”) JCs paper contains a discusswn a^ btbltogniphj of earlier wort. His experiments arc rc\^cwcd cjlcnsii*e ) in the Appendix of Huxic) and He Beer (1934)


131


Holtfrcter obtained a graded senes of exogaslnilac Total eiogastnda Uon (Fig 39 j 6) m which the ectoderm is separated almost completel\ from the entoderm and mesoderm is rare. In partial cxogastnilation the anterior head organizer and head entoderm fail to ini’aginate \s a re suit parts of the head are not diffcrcnUated In the least affected cases (Fig 39 c) the large jx)!): plug merelj fails to be absorbed The fusion of the medullar} folds which differentiate at its circumference is bloded In later stages this defiaenc} appears as a sht in the spinal cord (it is known as * spina bifida [Fig 39 J]) Holtfrcter followed the c\agina twn moi-cments b} means of Mtal stain marks and showed that the mor pbogcnetic mo\'ements described b} \ogl(pp 51 ff ) proceed m a t}*pical fashwn though In re\*crscd direction This indicates a ^■cr} carl} deter mlnation of the gastrulation mo%'emcnts- Holtfrcter succeeded m nusmg complete ciogastnilae to ad\-anccd stages In all instances the entomesodcrmal part proceeded surpnsingl} far m its differentiation m spite of the absence of the ectodermal co\‘ermg and of all ner%-ous structures and in spite of the complete In\xrsK)n of all structures (entoderm outside mesoderm inside) The ectodermal part however failed to undergo an} differ entiatwn (Fig 39 6) This strongl} supports the contention that indue tive stimuli from the mesoderm arc neccssai} for neural differentiation It IS \*cr} difficult to raise exogastnilae be}*ond the gaslrulation stages therefore the following obscr\-aUons wiU be concerned most!} with a study of the at}'pical gastrulation itself

ExrauursTjo

ifaUnai

Ambjsloma an} speaes stage H7 or stage H8 (The cjpenment is un successful if carl} gastrube are used ) glass dishes or LH} cups with lids full slrengih Holtfrcter v>lulKjn pipette* i‘, HoUfrtler solution

Procedure

I Renlo^•c all membranes from 30 30 eggs Holtfrcter rcmo\Td the 'itcHine membrane also HowriTr this is difficult to do without injur} to the crabr}t) It is therefore recommended that it be left Intact 3 Place two-lhirds of the embrvos m full strength Holtfrcter solution and the other* in iV Holtfrcter solution as controls Lea\c them in this Solution until the controls arc in the ncurula stage Obvn c the process of ^gastrulation A considerable \*anaiion of the effect wilJ be noticed I'olatc and draw the most interesting cases Tabulate }‘our results Rai e cmbr}-osuhich show slight degreesofexoga trulaiion <rig 0 cltoswun itag« and obvmt spma bifida (Fig 9 d)


BrBLIOGRAFH\


Hourmiat,/ ipijc Chjim£3enm^tnfe3nxchretJciiitIerKotnbm»ttaiTciaEnlO' ZBcwderm mit EttodemL SioL ZatzmlbL 53.404.

ipijj Djc tot*Ie Erosostmlitioo, one SeDwUhlSsnng da Utodcna ran

EntnnMsodeniL Entincihing end fnnltlcmeOa \eriitltai nerrsilcHer Orpsc. ArdiL L Eotw’xnech., tag 669.

1933c Elmgeig gwdin die ^nnhtlrfnng rq im TJrhtgpfwrrr Am rHhii»n<» T T« i mente. Sttanpb Ges. iforph. o- PhyjioL, Mflncten, 43 1.

Hcxltt J S^tndDrBEn^G R. 1934. TbedonefltsofapmnienUlanbocJoiy Creibndfe, En^nd. Ounhnd^ UmrenJlj* Pjtsi.

9 THE DE\'ELOPiIENT OF BEHA\70R PATIERXS a) TEE OaiCIN OF EAILY HXTIXXES {Alter Cbghni)

BehaMorpattenu like organs have an ontogencUc developmcnL The development of behavior is studied best in the snnple reflexes of knrer vertebrate embr^vis. The classical work of G E CogHU on the carij re flexes of salamander larvae has led him to condusjoas of great importance for bwlogy and ps}*choJogE Someofhisobser^'aUonson Awiyhcwlar w can easily be repeated and are therefore introduced here. Ha senes of lectures Ancifimy and ike problem of beba-wvr (1939) should be consulted In connection enth this exercise.

The gy imming reflex is the first integrated actixtly of an amp hib ia n larra. Its origin was studied bj Cbghill with the mteresl focused on the problem Is this reflex the result of beaming or u it cntirel) theresolt of the maturation of the nerrous system with do contributxm from * ex penence” and external sumnli?

Swimming is preceded by spontaneous wngglmg motions within the membranes long before hafrhmg CoghiD studied in detail these earhest reflexes and classified them m a number of behaiTor stages (p 125) senes shows dearth the progressjon in the complexity of the behavior pattern. A slight bending of the head is the first perceptible moirmcnL It IS followed bj a more intense bend, or coll^ extending from the bead U3ward. The additjon of a second cofl m the opposite directwn before the first has reached the tail results m an S-like wriggle therfnmimnge“°E^ from thi< double flexure as a senes of continuous and more powerful S-rc actions CoghiD succeeded in accounting for each step m this beha^W development in terms of a stepwise increase in the complexity of the nervous system- 'Ulth each stage a new type of ncurtros, or connedioit^ IS added. The following expcnmenti and observations obtain significance only m the light of these neurological data. The stu cut


134


Bbould study the diagrams of the organization of the nervous system in different stages of behavior (in Coghfll 1929)

The general conclusions at which CoghiU am\Td have influenced and modi^ed considerably our concepts concerning the ongin of beha\’ior and of reflexes. In contradiction to the views held by many psj chobgists and biologists he demonstrated at least for his object that complex behavior patterns do not ongmate by assembling separate simple reflexes and m tegratmg them secondarily but that the re\Trse is true all activities are integrated first and each step emerges from the preceding one as an mte grated umt Local reflexes arc secondary emanapalions from an mte grated total pattern The origin of the swimming reflex illustrates this principle. The ongm of mdependent limb movtments is another example To quote Coghill

The fint movoaent of the fort hmb ts addactioa and abduction, WTien thi< movement of the limb fa fint performed it occura only mith trunk movemoit When the trunk acU vlgorousb as hi fnmming the fore hinba art drawn doee against the body

A dav or tao ordiaarfl> ekp«ei betwneo the time when the arm begins to mmT ▼fth the action of the trunk before it acquiits the ability to respond to a local stinmlus without the perceptible action of the trunk. It Is obvious, therefore, that the fint kmh movement is an Integral part of the total reaction of the ammal, and that it B only later that the Umb acquires an indlvidoal]t> of its own in behaviour [1939 PP. iSfl.

The same bolds for jaw movements in feeding gill movements etc Altogether complex activities such as swimming or feeding are not the result of expcnence or learmng but the result of an orderlj sequence of devdopmental steps of the neural mechanisms

BCHAVIOR STAGES

These stages were worked out by Coghill The following dcfimtions arc taken verbatim from DuShane and Hutchinson (1941 pp 250-51) with a few additions

A-R, PranolUt siuge — Iso response to repeated touch and deep pressure on the myotomes

The nonmoUle or myotomte respome — This occurs m the absence of and carher than the ‘ early flexure It is charactensticall> a slow contraction towTird the side stimulated followed by a slow r el a xa tion It with a bending of the head This Is regarded as a direct nonnervous response of the myotomes

^ The early flexure response — ^This is a rather rapid reflex response of the animal to gentle touch The bendmg of the body beginning at the bead fa always away from the side stimulated (contralateral) The reac


125


tion IS bnef m duratwn, the relaiation being abrupt ^Ith further de%-c] cement of the m^utonjes and the ncnuus system, the contracted phase tends to be held for a longer pcnod of time and the tail is brought progresBivcl> nearer the head There is no sharp natural distinction between the more sd\*anced flexures and the next stage.

Coil — ^The coD reaction is aptly named It is the culmination of the carl> flexure and is attained wben the tail touches or passes the bead at the height of the response It is again awa> from the stimulated side (Le, contralateral) Some cmbrj’os show the coil reaction in tjpical fashion followed b> acoflmtheoppositedircctionwithoutadditional external stmnUaboiL

S-recdum — This is a reaction superimposed upon the coil reaction It results when a wa>’e of contraction passes down the stimulated side before the original contralateral contraction has relaxed The cmbr^ti fs transf torily in the form of the letter S Occasionally m response to a single touch the reaction may be repeated scxtral time* sucrcssi\’ely but it does not jet result m locomotion

£ 5 * The eariy-mmrmng response — Repeated S-reaction* become *o or ganized and strengthened that the anbr>*o makes some forward progress, Embr^’os which show any progression not more than tpproxnnatcl) 3 bod\ lengths fall mto this categor>

SS Strong itnmmerr— Embryos which swim for more than 3 bod> lengths to less than 10 bod} lengths

LS Lcte ssnmmers — ‘Embr^us winch swim 10 or more lengths

ExreRUtEjrr+o OasnvA-noxscrcTHEEAEiJEfrRcrLCXEior Awibyzl^ma IjUtiAZ

Matenal

cmbi}-os of an} speoes of Ambysioma in all stages from H31 to H46

dishes

a hair loop or a fine hair or bristle mounted m a glass handle Procedure

Choose crabr}!?* of diflerent stages. Place one after another in a dish. Make all observations under the binocular microscope I\ 1 th the hair loop stroke the embr}*o gentl} akmg the row of the nght ra}‘Otomes Observe the reaction as cai^ulI} as possible "Make a record of the reaction m terms of the bchavTor stages listed aboxT and of the Ham»on stages Repeat these observations on a considerable number of embr}*os and find reprcsentaln*c specimens for each Coghfll stage Try to correlate the be havTor stages with the Hamson stages kou will observT that there is a considerable variation of behaMor reactions In embr}^ of the same Har


n5on stage- For instance embr^-os in stage H35 ma> exhibit an> of the following reflex responses NAf EF CoO S Such \'an3tK)ns ma> be found even m matenal from the same lot of eggs, DuShane and Hotchm son (1941) have de\'Dtcd a special m\TStigation to this vanabilit) Thcj ha\T given precise data for the range of \'anation of beha\'ior stages m terms of Hamson stages for two different temperatures using A macula lum The results of the class should be compiled and compared with Table i and Figure i of the paper quoted abo\*e

J) THE DEVELOpaiENT OF REFLEX ACTHTn IN NARCOTZZZD EJCBRi OS

(After Matthews and Detwiler 1926)

It IS possible to give experimental e\idcn<^ for the contention that ex pcriencc plays no role m the formation of the svnmmmg reflex of sala minders In connection with another 8tud> Hamson (1904) placed frog embrj-os of early tail bud stages — 1 e prc\’iou 5 to the first mo\Tmcnts — m chlorctonc and kept them in a narcotized condition for as long as 7 flag’s They were returned to normal water at a stage when the controls were swimming lanme After a short period of rccover> they began to swim normally Obviously the nervous system and also the musculature had dmxloped normallv in complete absence of functional octiMt> Later ex penments along similar lines ha\T shown that we arc dealing here with a general prmaple of de%clopment which bolds for organs and structures as sell as for the ontogeny of bcha\Tor Most structures such as the eje or ihekidnej are differentiated first and begin to function later The narcotization experiments of Hamson were repeated on A mbystoma embryos by Matthews and Detwiler (1926) The following expenment is based on this paper which should be consulted for details

Expeeiment 41

AlalcnaJ

Ambysloma an> spieaes stage H28 or stage H29 Tetri dishes or section dishes with tightl> fitting bds chloretone i 3 000 or MS 222 i 6000 id ,V Holtfrctcr solution hair loop

— ^Thc mortalitj in narcotics is high Different batches of eggs require different concentrations The appropnatc concentration

  • bould be tested In ad\'ance

Pfocedurt

  • Select 15 healthy embiyos in astage preceding the onset of muscular

“^'emenli (H28 or H29) RcTno\'C all membranes Place 10 *pcamcns


in A dish with one of the narcotics mentioned above, keep 5 embr)x« in iV Holtfretcr solution as controls Cover both dishes

2 During the following week diange the narcotic daily keep the dlsbcs tightl} covered to prc\'ent evaporation, remove all dead animals. By gently stroking the narcotized animals with the hair loop, check each day to sec if they are completely immobDlked If not, transfer them to t stronger concentration

3 When the embryos have reached stages H38-H40 and the controli are swimming larvae, transfer the narcotized embryos to iV Holtfretcr solution Watch their recovery Stimulate with the hair loop Take notes.

BIBUOGRAPHY (a-A)

CocHiLL, G. £. 1939. Anatomy and the prohlen of behavfoor Cambrfctge, Bnthad Camhrklge University Press.

DoSHAirz:, G P and HoTtEnnot C 1941 Tbe effect of tsnpentiire oo the de vdopment of form and befaavwr In amphlblaD gnhr>'oa. Jour Expcf ZoCl.,B7 >45 Hamikm R. G 1904. An experimeatal stedy of the rdadon 0/ the oervoas lyttan tothedevekipliif musaiUtarelatheembryoof tbelio(. Amer Jour Anit,jt }97 ilATTimn,S A~, and Derwrera S.R. ipj 6 T^ttteikmoS Am 6 ipl*maaiiinyc$ fdkndog prokm^ treatment with chlorttooe. Jour Exper Zod 45 ^


PART m

EXPERIMENTS ON THE CHICK EMBRYO



A MATERIAL AND TECHNICAL PROCEDURES 1 Lime ilATERIAL INCUBATIOV The cluck embryo rank* second to the amphibian embr>*o as a material for the experimental analysis of embryoruc dc\’elopment- During the past decades the classical methods of extirpation transplantation explantation and vital staining have been applied successfully to the chick In addi tion the method of cbono-allantoic grafting has gn^n \-aIuable informs tion concerning the potenaes of carl> pnmordia The a\Tulabilitj of eggs at slmott any tunc of the j^ear and the short duration of earl> development are great ach’antages m experimental work.

Data on different breeds on the pnnaples and practice of meubatwn on factors influencing hatchabilitj etc., are easDj accessible and will not be presented here Full information roaj be found m JuU (1938) and Lippmeott and Card (1939) The following remarks will be limited to a few essential details

The most unportant prerequisite for successful operations is a suppl> of fint rate stnctly fresh eggs with a high percentage of fertility and a low percentage of abnormal de\’eIopment The quaht> of the eggs should be tested rigorously before operations are started on a large scale There B apparently no difference m quabtj between the different breeds Storage — ^Eggs should not be stored longer than 6 da>*s the> detenorate when thej arc older They should be stored m a cool place The optimal temperature for storage is 55” F

Incubaiors — ^Two t^pes are on the market models without forced air draft in which the warm air diffuses downward onto the egg and models with forced au circulation Smaller units are usually of the first They arc cntirelj satisfactory for laboratory use but require more atten tion than the latter Incubators are best installed in a room with even temperature and with good \’cntiIation They should not be exposed to direct sunlight It is ad\'isable to follow closelj the directions of the man ufseturer concerning temperature humidity etc Tempcralure — In the itiU-air type the temperature ^*a^cs consider •hly in different le\‘cls of the incubator space The temperatures 3-3 niches abo\*e the eggs (readings on hanging thermomclcrs) arc i®-2* ^•Bher than temperatures on top of the eggs The optimal temperature for bcubators without forced air draft is 100* F throughout the meuba


tion penod (readings on thermometers which are placed on top of the

eggs)

Humidity — ^Humidity is a vwy essential factor in successful incubi

tjon RefiUingof the water pans or of other devices for evaporation ihoold be carefully attended to In general a relative humidity of 6o per cent was found to be optimal The humidity in the Incubator is of course, closely related to the humidity in the room and to the vcntilatjon within the b cubator and in the room

Tumtns —Eggs should be turned twice a day

Fertility, katekabilUy, fxartaltty~^The natural breeding season is February to June, and the best material can be obtained during this season but acceptable material can be obtained at almost any time except dunng hot summer months Very cold weather reduces the percentage of fer tility Fertility and hatcfaabfllty arc not correlated with etch other A hatchabOity of 8o per cent, which imphes an even higher percentage of fertility, is considered satisfactory Two peahs of mortality occur durbg incubation, one around the third or fourth day and another around the eighteenth to twentieth day (see Landauer, ip 4 i)

Testing by candling —Candies can be obtained from farm supply houses, or they can be easily prepared in the following way Make a or cular hole approximately a inches m diameter in the bottom of a tin can. Mount a loo-watt bulb on a wooden base and invert the tin can over it Place the egg over the hole. Candle in a darkened room. The yolk lac arculatwn becomes visible at days of bcubation as a network of blood vessels radiating from an indistinct dark spot which is the embryo In the following days the rodong movements of the embiyo can be recog nlrcd in candling and the expanding vitelline circulation as well as the begimung chono-allantoic di^Iabon can be seen. From the third to about the seventh day dead cmbiyos can be rccognixcd by the "blood ring — blood settles at the periphery of the area vasculosa. From the seventh day on the chono-aHantoIc circulation can be seen in live cm bryos as an irregular network closely applied to the shclL From the thlr tcenth day on living embryos appear increasingly dark and the line of demarcation against the air chaniber is very sharp and distinct In ero bryos which die during these days this line is indistinct and hasy


BrBUOCRATHy


jenx, it A. 1938 Poultry husbsDdry New York itcCnw IDIL

LAWUca W 1941 TbeirtUitMityotdilcienegptsinflaeactd byeovlrc*^


and hereditj Storr* Aaric. Eipcr SUt BoD. J36.

Limvcorr U A. * 11(1 Caw L.E. 1939- Poultry pnxIgctk«L CtlieA PtflKWpni* Lea Si Fcbigcr


2, LUIB BUD STAGES


Iso satisfactory stage series of the chid, exists thatof Kclbdand Abra ham (1900) IS not \*cry useful Many textbooks particularly thatofLflbc (1919) contain illustrations of different stages In most of the follow mg expenmenU the limb buds of embryos of 4S-72 hours of mcubation are used for transplantation etc. To facilitate the quid identification of such embryos we distinguish the following stages which are based on somite number* and on the shape of the wing buds (Hamburger 1939) Slcie 1 — A slight thidenmg of the somitopleurc appears in the wing 1c\t1 (adjacent to somites 14-16) 24-27

lomites Incubation period 45-55 hours Stage 2 — The Wolffian ndge a thid emng along the lateral border of the somites IS \nsible in the wing le\*cl 27-28 somites Incubation period 5156 hours

5 /(jge 3 {?\g JO <j) — Wing and leg pnmordla ore marked off as slight twcUmgs, 29-32 somites Incubation penod 52-64 hours Stage4{Ftg jo b) — \Wings are small buds length width - 5 i 30-36 sormtes Incubation period 65-69 hours Stage s (Fig jo e) — ^\'ing* are median buds length width ■* 4 1 36 38 somites Incubation penod 6S-72 hours Stage 6 (Fig JO J) — \\ mgs are brge buds length width -251 39-42 somites Incubation penod 70-72 hours

Stages i and 2 arc difficult to handle os donors stage 6 Is too old for donors but may be Uocd os hosts

BIBLIOCRAPin

lUifiuicn \ yhe dr\-dopment and inncrviUon of trin^plintcd bmb pri

“ordii of chick embrjo^. Jour Erper Zool 80 347 KnstL, r,, lod Abkaiuu K ipoo. NormenUfd lur Eni»IcUunp'pr*chIchie do Hohacs (Go/fuj dMciItTKi) Jena C FUcher

f" R X919, The dcsTlopmcQl of ibc chick, -d ed m Nem\orL HolL

^ STVVD^RD EQUIPMENT FOR OPEILtTIONS ON CHICK >AIUK\OS â– Ifflffnaf/ijr joinf use of ikf class

meubator wiih turning trays and with haUhing tri\-s ('Tiirc trays) It 1' ad\n able to u‘<r tv-Ti incubators one Hilh wire traN'i m which the eggs on their ne^ts can l>c placed temporanU dunng the opera twn and one mth turning trais in which the operateil egg? arc


O h c d


Fio JO — Sta^ of wiO£ buds of chSck em b ry o s (from Ilambnrxw

iftj9) fl-itace i 4 « sta^ S 6 (se« test)


133


placed for further incobabon The latter incubator should be opened as little aa possible I autoclave

I or more heating plates for wanning the egg* dunng the opcxttJOQ I or more mounted candles (p 132) several containers with melted paraffin and a briuh cotton *ne3ts (Place a padding of cotton on a watch glass. Mold the cotton into a groove in which the egg will fit Eggs are placed on these "nests dunng the operatioD ) agar plates stained with Nfle blue sulphate and neutral red for vital staining (for preparation of the plates see p 56)

•several large 5 liter flaafcs with NaCl o p per cent*

•paper towels

several buckets for discards


Each student needs


watch glasses 3 with hds small scalpel hack saw blade pain of watchmaker forceps mi knifeor finclancet (sharpened steel needle, Fig i,/}


I pair of hne scissors 1 pair of large sdssors 6 square cover gla.ssca, 12-15 I micropipctte (p 4)

•i pipette with very wide mouth •several medicine droppers


1 water glass or Jar with cotton on the bottom and half filled with 70 per cent alcohol for steriUiation of fine steel Instruments


4 THE LIWDVAIX TEaiNIQUE OF CARTILAGE STAINING W#

(After LundvaD, Anat Ana. 25 1904 ^7 1906)

Ufually It is not feasible in courses of experimental cmbrj-ology to sec tion the transplants Therefore U is suggested that limb pnraordia be used for chorio-allantolc, cocloraic and flank grafts Carliltginous limb skeletons of transplants, 9-13 days old can easily be stained tn Mo mthm

less than a week using the simple method described below For older, ossified skeletons the alkann red method is recommended It is suggested that normal (host) limbs be stained together with the transplants for com panson

AD ittnxi Darted with an aiterijk ( J most t* »ulocb'«t.


134


I-UND\ALL TECHKIQUE


I Fix m Bourn s fluid

ptcnc »dd, saL aq lot 75 parts

40 per cent farzaa](Ieh>'de >5

giacial tcttic aad 5

or m fonnaldeh^'de 1 10 for 1-2 da>'S.

3 W ash m 70 per cent alcohol After Bouin fixation add a few drops of bthium carbonate (saturated solutioa In 70 per cent alcohol) Change the fluid until the jtUow color has complete)} disappeared

3 Remo\T sljn with feathers adhering iiscera and fat masses Use a watchmaker forCTps

4 Stain in meth^dene blue (o 25 gm per 100 cc. of 70 per cent alcohol mtih 3 per cent of HQ b} \’olume) or m toluidin blue (same solutwn) for 3-3 da)-s

5 Destain in 70 per cent alcohol for at least 4S hours (change sc^Tral times) in 95 per cent alcohol for 3-4 hours (change) and m absolute alcohol for 13 hours (change) In absolute alcohol the soft tissues should be cotQfJetdj dcstained, but the Wuc cartflage can be seen faintJ> through the other tissues

6 Qear harden and store m 3 parts of oil of wintergreen plus 1 part beaz)*! benioate.


  • 35


B EXPERIMENTS


1 THE PROSPECm’E SICNDICANCE OF EilBRIOMC AREAS

(iTrALOTAimNG experiments;

O) PKOSHECTIVE OKGAN TORIUNG ASEAS AND UOIPHOGEKETIC UOVEUENTS IN THT. CHICK

Before starting the following cipenments the student should scqtuunt himself thoroughly with the no rmal dcvelopincnt of the duck embryo, from the pnimtxve streak stage to the early somite stage (between 12 and 36 hours of incubation)

The expenments suggested below do not deal with the two earliest phases of gastrulabon, namely, the fonnatiOD of the entodenn and the fonnatiOD of the primitive streak itself Both phases, tbough of great in* terest, are not fawrable for dassroom eapenments. Our startlng-pomt is the stage of the definitive primitive streak (about 16-18 hours of incaba twn) The pnimtn-e streak itsdf is a thickening of the upper layer, ei tending anteriorly from near the posterror margm of the oval area pch luoda for about two-thuds of its length (Fig 33 a) The anterior end of the primitive streak is a knoblike thickening, called “Hensen s node.” The streak is the r^ion of mesodcim m\’aginatjon It is formed of dense

ly packed embryonic cells whldi continue laterally as organized epitheha

both in the ectodermal and m the mesodermal laj-er The underi^mg entodenn apphes itself doscly to the streak, particularly in the node Ic\el yet it remains a separate unit The streak and the adjacent regions con tain the material for all structures of the embryo proper with the excep' tion of the forebraln portion of the head wfalci originates from mitenal Ijing m front of Hensen t node The peripheral regions of the area pci lodda and the entire area opaca form extra -enabrj’onlc structures Shortly after the dcfiniti\T streak stage is reached the differentiation of the em biyo begins with the formation of the head process, which is an anterior extension m front of Hensen s node (Fig Sections show that the

head process is the 5rst snsiblj differentiated structure it is composed 0 the nntenor end of the notochord and the crvTrlying medullary plate From then on organ formation proceeds rapKfl> m anterior posterwr

rection Hensen s node recedes whereb> the primitn*e streak shortens

at the same time the differentiated organized antenorpartof tbccrobOTJ

136




  • 37


lengthens (Fig 33, c-c) The antenor end of the neural tube doses, while the neural plate m more posterior levels begins to form One somite after another is laid down the notochord lengthens Hensen’s node alwap marks the boundai> between the antenor organised and the posterior unorganized part of the embr>*o

The localization of the prospective organ forming areas m the pnmi tive streak stage has been mapped out by Wcttel (1929) and Pasteds (1937), usmg the vital-staming technique I ha\‘c adopted as the basts for the following experiments the map of Pasteds, whose entiosms of Wet zel 8 map I consider as justified. The map of the definitive primitnt streak stage (Fig 31 e d) can best be understood if one goes back to earlier stages The pattern of the different areas In a stage preceding the formation of the prmutn'C streak is shown in Figure 31 a This stage cor rc^nds to an amphibian blastola (except for the fact that, in the cluck, entoderm formation is already completed the similanty of the maps for both forms IS striking cf Fig 4) The morphogenetic mov'ementsdunng the subsequent phase of pmmtive-stieak fonnatx>n also have many fca tures m common with those of the amphibian gastnilation, we find again the three basic movements coowrgence elongation and bvagmaBoo All areas swing toward the midlme (convergence) , thej elongate consider abl^ and mesodenn invaginates through the pnmitive streak (the left half of Fig 31 c-e, is a surface view and the right half shows the mesoderm after removal of the ectoderm) The lateral and ventral mesodenn (/jn ) is the first material to invaginate around the growing pnmitive streak (^ j in Fig 3 1 A) In the phase between stages c and d the prospec tive notochord material (nx ) invaginates around Hensen s node the prospective somite material (j(«w ) invaginates around tie antenorhal/of tic

primitive streak and the lateral mesoderm continues to invaginate around

the posterior half of the primitive streak. The pr 05 pccti\T medullary ma tcrial (med ) con\'ergc3 toward the median line in conjunction with the adjacent mesodenn It amv'es near the median hne when stage d a reached However it is likely that left and nght haJ^Ts of the medoJlaij plate do not fuse in the midlinc that Is no concrescence takes place. The median strip of the meduDfliy plate (future floor of the neural tube) i* probablj formed by material which, in the stage under discussion Is lo* cated m Hensen a node abovT the notochord and which m subsequent stages moiTs backward and elongates cnonnousij Both WcUcI and Fas teds found that when Hensen s node was vital sUmed dcep]> so as to stain both its ectodermal and its mesodermal component then the en notochord as well as the entire length of the fkxir of the neural tube were

138


stained m the embryo This nnphes that Hensen s node is the actual ma tenal for the notochord and for the floor of the neiiral tube and that from stage d on Hensen s node mora badeward and leavTs m its wake the notochord and the floor of the neural tube. It dimimshcs m size while it recedes The regression of Hensen s node is part of a very conspicuous dongation m posterior direction of the posterior area pelludda^ whereby the latter changes from a circular or oval to a pear shaped elongate form These movements are mdicated by the arrows m Figure 3 2 Theprocesses of convergence and of mvagmabon are probably completed m stage d before the extensive elongation begms and the latter would then be a separate final phase of the morphogenetic gastrulation mo\‘cment3 A peculiar feature of this spreading is that the median part (Hensen s node material) moves and elongates more rapidly than the adjacent lateral materials As a result the somites and the overlymg medul liry material which are at first staggered at the anterior part of the primitive streak unfold and spread m a fanlikc fashion

31 *)

According to this account, a vital stain mark placed on Hensen s node will result m a itain of the entire length of the notochord ind of the floor of the neural tube. A mark I^ced on the anterior primitive streak (Fig d) win stam somites lateral walls and roof of the neural tube The material for the ^trrior part of the head in pnrmti\'c-streak is located m front of Hensen s node In order to stam the eye one must place a ®ark on the preuodal area slightly lateral to the median Ime It should be dearly understood that \uta] s tainin g ^^Pciments concern only the prospective significance of organ form ^ areas The mherent potenaes of the diflercnt regions are much Pcatcr as 15 shown by chono-allantoic grafts and explantation open Gicnti (see p 143)


Fio 3» — Head proceti itaft The airowt hidJcatc the mmemenU Hesxnt Dode aod adjacent matoiaL Bju** Henjcn a oode i t “ bead proc ta i fj "primitive itreab (after Paiteeb, ipj?)


"n* otaemUom oi Jacobaoo ( gjS) 00 acctiooed natenaj are in part, at raruiice with 'tawBL JacofaaoQ pocnla oat a pcncible lource of errora in iia] aiauunc erperimeBli on ‘«cbck Intbchttertbe vital djCB taken op b> the>oIk and fat ffobuln and not bj- pi* paonlea a* b the tmphiiaanj He foand that m pnatni* matemh feicrpt notochord)

    • ‘t®! lipid content Aj a mnlt the d>e maj diffiae into other ctlh and Ona gne an or

picture of the arloal cdl mm emcnli Hoaever lha wnrot of error doe* not affect the bterpreUUon of the three eipenn»enta *a*ge*ted below


139


d) GENERAL PBOCEDUKE OT VITAL STAININC (EXPERIMENTS 41-44) (After Wetzel and Pastcels)

Wetzel was the first to apply Vogt's technique of vital staining to the chick embryo The results are not so satisfactory as in amphlbums. In the chick the stain is taken up by yoDc and fat globules, which break down m cell metabolism As a result the marks fade out more rapidly than b amphibians, in which the pigment granules are the chief camerB of the Etam It IS therefore necessary to apply a deep stain. On the other hand, overstammg must be avoided, because it may result in a disint^ration of the stamed cells and In death of the embryo Vital stained embryos should be inspected not later than 24 hours after the experiment

MakHcl Jot Expnhnmis 4^44

6-8 eggs per student standard equipment (p 133)

Procedure

1 Incubate the ^gs for 16-24 hours depending on the temperature and moisture of the incubator It is adviaahle to make preliminary checks of the rate of devekipment under the particular conditions of the incubator used

2 Several hours before the laboratory perKHll>^ins,autocIa\’e the nil

tenals marked with on asterisk ^ 134)

3 Oiher preparatiofu for the whole dcu — B^in the heating of the warming plate and of the paraffin j boor before operations.

4 Each student should tnohe the following preparations — Place forceps, scalpel, hack saw blade and scissors m 70 per cent alcohol r hour before operations start Prepare 2-3 nests by placing a padding of cotton on a watch glass and mold it mto a depression into which the egg win fit Fill 2 watch glasses with sterile saline and cover them Keep a third watch glass dry and uncovered Before the operations start, cover the table with sterile towels Wipe off all alcohol from the steel instruments and place them in a fold of a sterile towel, to the right of the binocular microscope. Moisten a small strip of the red and of the blue agar piste with saline solu

tion After 1-2 minutes scrape off small strips of the swollen agar rla« 

them in one of the watch glasses in saline solution and cut theni into small square pieces.

5 Saw the window in the shell Candling is not necessary In early stages the blastoderm will always float on the top of the 3‘olk P ce the egg on the cotton nest with the blunt end to your left, shake it gen > to loosen the blastoderm In case it should stick to the membrane w



Fn u — apcrimenU b the primltj^Mtrtak itt^ (alter Wetzel, r{iJ9) «,t>T]til«ubbg d a rccloQ b Iroot d Heit*eD»Dode (protpectjve njht fortbrab and rlfht •"Imiaedlateljr alter itabbs the aame embryo a4bonnlater f-f "vital rtabltn Heaiea anode f "bnncdiately alter atauunj* boura later r-14 boon later / x" •tebl^ of the anterior part ol the pnmltiNC ateak /— buocdutely alter atabbx later i a. " Hcnaen a node i / "head proem ax "Dotoebord «r-opUc /j "prlmltiTe itrtak 5 r"fint aocnlu.


141


a square window, about 10-15 nun square in the uppennost part of the shell, using the hack saw blade. Carefully avoid any injury to the shell membrane. Lift the window out with the scalpel and watchmaker forceps.

6 Moisten the shell membrane thoroughly with saline solution , rupture and remove the shell membrane with the watchmaker forceps, CarefuDj avoid any Injury of the blastoderm Remove excessive egg albumen with the pipette, if necessary Use sterile pipettes,

7 Usually the primitive streak does not stand out clearly To make it distinctly visible, place a rather large piece of blue agar on the central area of the blastoderm Do not remove the vitelline membrane, Thestam diffuses rapidly through It Remove the agar as soon as the streak becomes visible awid deep staining Identily Hensen s node Select embryos in definitive pnmltive-streak stages

8 With the watchmaker forceps place a small piece of red agar, slightly larger than Hensen s node, m the desired position Stain deeply for sev era! minutes (the time depends on the concentration of the dye in the agar) Moisten the blastoderm before you remo\’e the red agar, then pick it up with the watchmaker forceps

9 Make a sketch of the embryo and of the mark It is necessary to have an exact drawing of the position of the mark, In order to be able to interpret the results,

10 Seal a cover glass over the window In the following way with a small bru^ apply hot melted paraffin to the edges of the window Place the cover glass over the window and press it gently Add paraffin if necessary

11 After 14 hours Tcmove the cover glass enlarge the window, cut out the entire blastoderm with a fine for c eps transfer it with an extra wide pipette to a watch glaa with aalme Study and draw the extent of the mark Its shape etc. Handle the embryo with watchmaker forceps. Dissect the skin away if necessary Use a very strong light source.

Do 1-3 experiments of each type

c) VITAL STAININC OF HENSEN S NODE (nO 33 C~t)

ExrrsiMJjrT 43

Procedure

Stain Hensen s node following the procedure given on page 140 After 24 hours remove the blastodcmi from the egg Slit the neural tube In the midline with a glass needle hold the embryo with a forceps or turn the

Some loTwtitaloT* rccorameod ptadne a BnaH rtanBle 0/ DeuliaJ n 6 ponder on the to be lUloed. CooUo] antfuIlT* ormUlnlof


14a


blastodenn upside down Locate the stain m the notochord and \-entral t^ject of the neural tube Use high power and ^*e^J bright fllummation d) VITAL STAINDiC OF PAiT OF THE AKTEEIOE HALF OF THE

ntntrm'E stseah (no 33 f-f)

ExyaaiMETr 43

Procedure

Vitil-stiin a small area of the primitive streak a short distance behind Hensen s node Make an exact sketch of the position and extent of the mark- After 24 hours, rcco«r and dissect the embrj-o as before- Beter mine the number of sormtes which axe g tamed Tr> to find the stain m the neural tube- Use a strong light sourcc «) VTEAL STAIKn.C OF A PEENODAL AEEA (tIG $3 a b) ExrajMxzTT 44

Procedure

Place the mart m front of Hensen s node, near it and to the right of the median plane. Make a precise sketch. After 24 hours dissect the embrjo find the stain Notice which parts of the brain are stamed

BIBUOGRAPHk (®-e)

JiCOBsox W 1938. The eatdy de v 'e bpai mt of the grim eiibt> a IL Moodennfor miOon and the distriimtion ol presomptire tsnbxyvcac aatuiaL Joor Moeph^ 445.

Pasieeli, J 1936. Analyte de» mouv e m cats moTphotfaitniues de jajtndatroa dttz fcs ooeani. Aad. roy de Bdgiqae, BoD- da we d. to. lir., 23 737

â–  1937 Etndei inr la gartmliban dea vert fl gfa miroblastMiaea. HL O iwgT .

Arch, de Hd., 48 3S3

^>^nEL,R, 15*^ Unlenadmngen am Hflhncfcenu DieEntwidthmgde* Keinairtlir ad der enten hdden Bnrttagc, Ardi. L Entw^medL, 119 i8S 1931 Unmmd wml Prfmitrvjtreifen. Ercebn. d. AnaL n. Entw'feadi-, ap i

2. CHORIO-ALLANTOIC GRAFTS

a) GEh~EEAL

^^^view the formation and structure of the cbono-allantoic membrane Fig 34, a 6)

The mam experimental methods m chick embryos are as m amphibians isolation extirpaUon and transplantation of embrjomc areas Complete bolahon can be accomplished b> in rtl/o culture on a watch glass or a slip using embrjomc extract as a nutnent substrate The in rtiro IS too difficult to be used in Ha. w T nom experiments Those who ^ mterested in it will find techmcal details in W addington (1932) and Spratt (1940)



The tediiuqat of cborio-allaDtoJc grafting is wide]) adopted aa an laola tion methocL It was worked out m the present form in Dr F R. LHhe s laboratory at the Umverslt) of Chicago by Hoadlc) (1924) and Willicr (1924) It makrfl use of the highly vascularized chono-allantoic membrane of avian embryos which becomes closcl) apphed to the shell membrane and is thus withm easy reach of the cjpenmenter (Fig 34) The mem brine of Sj-io-day chid: embryos is suffiaently developed to servT as a substrate The structures whose potenaes are to be tested are placed on the membrane through a window in the shell which is sealed up after the operatioiL The grafts become vascularized and can be reared for 9-10 days. The) have to be rccos'ered before the chono-allantoic membrane breaks down on the nmeteenth day of mcubation The transplants are complctel) isolated from the structures of the host embryo proper, so that inductive effects etc. cannot obscure the results. On the other hand, the transplants being meorporated mto the blood or culition of the host, are exposed to all substances earned m the blood stream for instance the hormones In this respect the isolation is incom plete and the possible effects of these agents on the differentiation of the transplant must be taken into consideration This aspect has been made use of m the analysis of the mffuence of sex hormones on mdifferent gonad pnmordia (see Wilher 1939) Another limitation of the method lies m the inadequate space which tbe transplant frnds on the membrane It flattens and is buned m membrane cells Therefore one ne\ar obtains normally shaped eyes or limbs In evaluating the results one must be caatious not to attribute such failures to the lack of inherent potenaes of the tran^lanted pnmordia

A promising new field was opened when it was found that mammalian ti»oe can be grown successful!) on the chick membrane (see Nicholas and Rodnick 1933) Recentl) the membrane was found to be a suitable sub»tnte for a large number of \irusc5 and of bacteria mcludmg forms pathogenic for man and forms which arc difficult to culture otherwise (rc\iew m Goodpasture 1938)

b) CHORIO-ALLAKTOIC CRAFTS OF LIUB PRBJORDIA

The eipcnment demonstrates that limb structures are capable of self differentutwn when isolated from the bod) at 2-3 da)*» of incubation Thu cipenment was made first b) Murra) and Hiudc) (1935) and b)

Mumj (1926) This author grafted whole hmb buds and proximal dis^ or longitudinal hal^•es from 3-5'da) erobr)-os and found a rather fijid mosaic de\-elopment of fragments For instance the proximal part


of a leg bud from a s-day donor formed femur, and the distal part formed the tibiotaraus the fibuJa, and foot The cartilages are usually abnormal in shape and the sVcletona incomplete owing to unfavorable condJtionj on the membrane The differentiation of the limb musculature on the membrane was studied b> E A Hunt {1933) The percentage of weO developed transplants is usually smalL Figure 34, c, showi an exceptionally well-differentiated graft (from Hunt, 1933) It is advisable to make 4-6 operations at a time


Maiemi


ExratnoNT 45

10-12 eggs per atudent standard equipment (p. 133)


Procedure

1 Incubation of the hosts — ^Start the incubatfon of the hosts (5-d per student) 9 or 10 days before the operation day Mark the date of incuba tion, or an U, on each egg Turn the eggs once or twice daily

2 Incuhalton of the donors — Start the meubatton of the donors (j~6 per student) hours before the laboratory penod Mark the date of meubatiod, or a i? on each egg Roll the eggs once or twice a da>

3 Autoclave the material rnariced with an astenak (p ija) screraJ hours before the beginning of the laboratory

4 Other preparations to be made tn advance — Begin the heating of the warming plate and of the paraffin i hour before operations, Ha\T the candles ready Place donors and hosts in separate trays mark all trays clearly Have empty wire trays (hatching trap’s) ready for the temporar} placement of hosts in the nest and have empty turning trays retd> for the hosts after operation

5 Preparations to be made by each student — ^Placc forceps scalpels, scosors end hack-saw blade in 70 per cent alcohol, i hour before the opera tlons start Prepare two nests by plaong a padding of cotton on watch glasses. Mold the cotton into a shallow groove into which the egg will fit Fill 3 watch glasses with sterfle saline solution and coitt them with a lid Keep the third watch glass dry and covered Immedlatclj before operation cover the operation table with sterile towels Dry all stnmienta carefully Place them and all pipettes in a fold of a sterile towel and place the towel to the right of the binocular microscope

>’Our hands

Ao/e— FoUow two rules Work as fast as you can but not bn3tU> Keep mTiy tool as sterile as possible


146


HXPAZATICet Of TgT HOST ElIBETO

6 Candle host eggs Discard those which are sterile and those with dead embo"®* * Living embo’os show clcarl> the chono-alkntoic \'es$els and mdistinctlj the embr5*o as a dark area. Its rocking movements maj be seen in candling

7 Select and mark a suitable site for miplantabon. CHtt the candle locate the mam blood vessels of the chono-allantoic membrane Choose a point of junction of two strong %'essds at some distance from the embrj-o Mart this pomt with a pencil on the shelL

8 Saw the wmdow It should be small to reduce the chance of mfec twtt. Place the egg on a nest marked side up Saw a rectangular hole about i-i cm Saw onlj 3 sides The fourth side will break when the window is lifted up Saw slowl> and steadil> without jerks Be sure to awid sawing through the shell membrane The chono-aDantoic mem brane is closely apphed to the latter and its mjurj results in a hemor rfiagc Before you remoix the window draw a pencil line across one edge of the wmdow so that j*ou can fit it m agam m the right onentation Lift the window out with the scalpel and place it m the di> watch glass Cow the latter with a Ud

9 Moisten the shell membrane thoroughly with saline solutwn usmg a small pipette. This is absolutely necessary because otherwise the shell membrane cannot be removed without mjury to the chono aflantoic mem brane. Under the bw power of the bmocular microscope pick up the shell membrane cautiously usmg the watchmaker forceps rupture and rtmo\-e it or sever it on three sides only The junction of the blood vessels should now be visible,

10 The host IS now prepared. Place the shell piece back m the wmdow Mirk the ^ with y-our mitials and place it on its nest on a wire tray m the meubator until the transplant is prepared.


PtrPAlATIO'J or THE TEAKSTLAr^


II Candle the donor embryo and mark ita position Place it on a cot ton nest and saw a large square wmdow wound the marked region. No Precautions are necessary the shell may crack and the vitcUme %'cs$els


rupture

12 Mobten the shell membrane ovtr the embryo and remove it with toe watchmaker forceps The embryo will now be exposed

13 Mlth a pair of fine scissors cut out the blastoderm a short dis t ance toim the embryo Transfer it qmckly to a watch glass containmg warm

wlutioa, usmg the wide-mouthed pipette or pick it up with a fine

DBomitheegg

U bert done by the Imtfoctor before th *ibor»lor> perwd


147


14 The embryo is covered with amnion and cbonon Rupture and re rao>T these very carefully with 3 watchmaker forceps. Use one to hold the cmbiyo and the other to remove the membranes Work under the low power of the binocular microscope against a dark background

15 Xocatethe wingand thcJegbuds Before isolating them, first make two trarmrersc cuts through the entire embryo, one through the neck one between wing and leg buds (x, y in Hg 35, a) Use a forceps for hold mg the embryo and an ms knife (Fig i,y) for the cutting Next, dissect out one bud by making four cuts m the sequence i, 2 3, 4 or 5 678 (Fig 35, 0) Do not cut too close to the bud rather include parts of the somites Remove fragments of yolk and blastoderm adhering to the bud \Vhen the transplant is ready for grafting cover the watch glass and put It on a heating plate


TaAMSPIANTAITON

16 Take the host out of the incubator and rcmo\'e the shell piece from the window, placing it in a covered sterilised watch glass.

17 Suck the transplant mto the micropipette with a small amount of saline Introduce the tip of the mlcropipctte into the window (Fig 34 J) and drop the transplant onto the chorio-allantolc membrane by applying gentle pressure on the rubber membrane of the pipette. Operate under the bmocular miscroscope and try to place the graft near the blood vessel. Observe carefuU> to see if the transplant Is actually on the membrane. It sometunes sticks to the pipette U this happens then pipette it back into the watch glass with strong squirts and repeat the procedure.

18 Fit the piece of shell back and seal it in with hot paraffin \Mththe paraffin brush go over all four edges. Gi\*e the egg a protocol number Return the host to the incubator, with the window facing downward

19 Take a careful protocol indicate the exact age of donor and host. Note whether it was a wing or leg bud etc.

Repeat the experiment using both wing and leg buds If the matenaJ is scarce scieral buds from the same donor should be taken It a also possible but not advisable to implant 2 grafts in the same host


acccn-EiT or tub caArr


llaicrxals

5c\‘cral finger bowls sciTial liters of warm saline (NaCl o 9 per cent ) a pair of strong sossors and a pair of fine scissors watchmaker forceps


Bourn B fixative

small nals with cork stoppers

labels

a jar with cthcrixcd cotton /of discarded host embrjw


148


20 The graft should be taken out when the host cmbrj’o is 18-19 da}*3 old Le. 9-10 days after operation- If the graft has taken it will be found under the window Make a circular cut through shell and chonotllantox membrane at a radius of about 1-2 cm from the window ^^ash this piece of shell in the finger bowl and inspect it from the inner side The chono-allantoic membrane covers the inside of the shelly and the graft will be seen under the wmdow as a pink nodule of ■varying siae sometimes with fluid filled ■ntsIcIcs attached to iL Lrmb structures are rarely recognizable the tran^lant must be cleared and stained to make them visible Cut the graft out very carefuDj using fine scissors and transfer it to a vial half filled with Bourn s fixing fluid Label the \'ial with your mitialK and the case number Enter jour findmgw m the protocol Place the host embryo m the jar with etherized cotton and discard it later on 21 Staining and cUanng of the graft — ^The Lund\’all technique of stam mg cartilage is recommended for a quick preparation of the skeleton of the grafts for further study (see p 135 and Fig 34 c) Students who are Interested m the differentiation of the musculature etc. should section and stam their grafts

22 Ifaie protocol of the siatnedgrafli also of the best specimens

obtained by other members of the class. Compart with the figures in Murray (1926) and E A Hunt (1932)

e) CHORIO-ALLAJJTOIC CRATTS OF ETX PJmiORHIA Exfzbuizkt 46

The optic vesicle of a 33 hour embry'o will undergo considerable differ entiation when isolated and grown on the chono-allantoic membrane Retina, pigment epithelium and lens may be distinguished m the graft However there is a high degree of variation m the shape of the transplants most of them will be small solid nodules with obnornial arrange taent of the different layers and only a few will be \e8icular structures approachmg a normal eye (Hoadley 1924 Alexander 1937) These abnormalities arc due to anfa>’orablc conditions on the membrane since primordia of the same stage proceed much further toward normalcy when Shafted to the flank or into the coelom

the same as m Eixpenment 45

Procedure

The method is the same as in Expenment 45 except for the following changes


149


2 (p 146) Incubate the donors for about 33 hours to obtain cmbrjtn of about p-i2 somites

15 (p 148) Amputate the head in the hind bratn region Cut out the right optic veside and forcbrain follow the dotted lines in Figure 36 b Remove the underlying blastoderm The forcbrain and epidermis with lens epithelium arc induded In order to make the graft bulkier

21 (p 149) Clearing m oil of wintergrccn or xylol will bring out the main structures, such as pigment epithelium and lens. Stamuig is not necessary The grafts must be sectioned if liislological studies arc desired.

BlBLIOGRAPin (a~c)

Alexakder, Ih E 1937 An ejpmmaiUJitudy of the role of optic cup tad o\Trl>hic ectodenn in lent fonnalion in lie eWefc embrvo Jour Exper ZobL 75141 GoocMSTTOa, E. tv 1938 Some uses of the ctidc embryo for tbe »tudy of infectkio and inimmut> Anier Jour H>g. *8 nr

HoAPLar L. tgn Tbe iodepezKlent ddrcreottatioo of Uolnted chick ptimonlli In eborio-AUanterfe crtfti. fifol BuIL 46 281 Hukt £. A. 1933 The dl/TereatUtion of chick limb buds in chorio-allutcfc ptSlt with spead reference to tbe musdes. Jour Exper Zod. 6x157

hlCKRAV PDF 1936 An experimcnUl study of tbe devtJ{^eBt of the Embi of

the chick. Proc r.fnrwxn Soc, 51 180

1938 Chorioallantoic grafts of frw^menU 0/ the two-day cbick, »Ith ipeciil

reference to the derelopmat of the limbs intestine, tod ikln. AustraHta Jour Exper Btol tnd hfed Sa., s 337

1936 Bocei. Ctfflbndge, Digit od Ctmbndge Univmjty Prets,

hiinuuY PDF tndlfDXLrr J S 1935 Srif-diUcrenthitloo In tbe grafted limbbod of tbe chick. Jour Anit, 59 379.

Motulay r D F^aod Selby D 1930 Inlnnsic and ertnnsiefseton in the primary derelopmcnt of the ikektott. Ardu f Entw^mech isa 639.

NrenoLAJ J S and Rtnufrcx, D 1933 Tbe dcvthjpmeot of embivonlc rtt lluocs upon the duck chorio allantoii. Jour Eiper ZooL 661193 SniATT N T 1940. An /• tilro anabab of the ori^UatJoo of the cj'e-fomilog area In tbe earlv chick blastoderm. Jour Exper Zoo! 85 i7r

\VAl^I)£^OTO'^ C II 1933 Experiments on the doTlopment of chick and duck cm

brjtis cultiv'ated rf/ro Phil Trans. Ro> Soc London B 321:179 \\ iLim D II 1934. Tbe endocrine glands and the devdojuneol of the chick- I The efiecls of th>Toid grafts. Amer Joor AnaL, 33 67

1939 The embfj oolc development of sre In Alle-y D (ed ) Sex and to temal secreLlons. ad ed. Baltimore WTUiams & WTIkins.

3 DsTRA EMBR^ OVIC TRANSPLANTATIONS a) COELOJt CRAFTS OF LDIB PjaMORWA

(After Hamburger 1938 1939)

The coelomic of the chia embrj-o « an cjpcdall^ foTOrahle site

for the transplantation of primordia It has certain ad\Tintagc3 t c

chono-allantoic membrane It allows for undlsturlicd expansion an nor

150


mal morphogenesis of such pnmordia as limb bads and optic -vTsides Farthennore it gives the tran^lant a longer life span since 3-daj em br)*os are being used as hosts The transplants are slipped through a hole in the somatopleore and attach thenisel\'es to the coelomic walls mesen tenes or to parts of the umbflical cord and receiw blood supply from the host Since transplants which de\‘elop at a distance from the central nen ous system have httle chance to rcccu'c a ncr>T supplj it is possible in this way to obtain noninnervated organs for instance completelj nei^x less limbs which are paralyzed from the start The> were found to be re martably normal, particularly with respect to their skeleton and their jomts (Hamburger 1939 Hamburger and Waugh 1940) E\Tn the musculature will undergo normal differentiation and cross-stnation but innervation is required for its mamtenance and without this it degener ates shortly after its initial differentiation These cases demonstrate that innervation and functional activity during development play only a min or role as causal factors in Umb morphogenesis Other pnmordia haNT been reared successfully m the coelomic ca\it> for instance optic vesicles Qoy 1939) spinal cord (Bueter unpublished) and mouse tissue (Rawles 1^0 Gluecksohn Schoenheimer 1941)

Expeehiest 47

ISaUmi

3-4 donors and 3-4 hosts per student standard equipment (p 133)

Procedure

1 Start the mcubation of both donors and hosts about ai 3 daj’S be fore the operation For your particular incubator find out the tunc re Tured to tcada stages 3-5 (39-3S somites see p 133)

2 Autoclave all material mart:cd with an asterisk (*) on page 134

  • ^*rting se\’cral hours before the operations

3 Make other preparations as m sections 4 and 5 on page 146

4 ^ Cut out small pieces of red agar for \ntal staining and place them m sterile saline solution Their size can best be determined when the first oiihryo u stained It should be large enough to co^xr the nght half of the ®ibryo posterwr to the tenth somite Prepare half of a dozen agar pieces or more They must be replaced by fresh pieces when they begin to fade

MTAl STAININ-C OF THE HOST ElCBZlO

5 Candle the egg If the embrj’o is stuck on the side it is often pos•OjIc to bring it mto the desired position on top b> gentle shaking or roll ^ With pencil mark a square of about cm edge on the shell aboix embryo


6 Place the egg on a nest (cotton pad on watch glass), marked side op, and saw a square window as marked Very carefully avoid sawmg through the shell membrane and thus injuring the cmbi>*o HcnjojThiges arc usuallj fatal Saw slowly and steadfly Saw only 3 sides and br^ the fourth tide when the window is lifted up with the scalpel If the iheD piece IS to be scaled back place it in a sterile, dry dish Avoid the loosening of small shell partidea on the edges of the T?indow




Fic J5 —limb-bod truupUflUtkn la tbe fMct emtoyo. c^doDor tmwyo »“bo« cm biyo (both J4 jomites, 7» boon of locuhadoo) i>*»Iejbod j-riJt for tie reception cd tb* tftntpbnt 5 r/ 5 yo«>amfta 15 jo 6- — Irwujlaat, bod "MoUEtn rWir’

for other letters *»d namberi see text

7 Moisten the shell membrane thoroughly with saline solution other wise it cannot be rcmo\'ed without hcmoirbages Under the binocular dissecting nucroscope remow it s’ery cautiously in the area of the window using a pair of watchmaker forceps The embryo should now be ctposed If it sticks on one side roll or shake the egg gently and try to mort it to the top Add suffiaent sterile salt solution to keep it moist The cm bryo should settle rather deep under the shell

8 For Nntal stainmg place a piece of red agar o\"cr the right flank co^^ ing the right wing bud and the rcgwn posterior to it Co^tt the win


with the shell piece or with a cover glass Mark the egg with j-our imtmN and with a serial number and return it to the mcubator for 5-10 minutes Note — If the agar piece is deep red and rather thick then it is best to stain through the vitelline membrane The latter stains first but will soon give off all dye to the adjacent tissues We prefer thin agar films Rupture the vitelline membrane with the watchmaker forceps before staining then place the agar directlj on the embryo and thus obtam a deep stam within a short time.


hlepaiahos or the TaAwspLwr

9 Candle another egg marking the embryo as before Place the egg on a nest and break or saw a large bole considerably wider thwn that m the host so that the embryo and the adjacent area %*asailosa are laid open. With a pair of (steriUred) scissors cut out the entire area vasculosa with the embryo in its center AVlth the watchmaker forceps or with a wide-mouthed pipette transfer it to a dish with saline solution

10 Under the binocular microscope turn the blastoderm right side up flatten it and hold it with the left forceps Use a daik background Locate wing and leg buds Count the somites determine the stage (p 133) ud protocol these data Amputate the head with a Knapp ins knife (level * m Fig 35 j) and discard it Hold the embryo with the left for ceps and cut out the right wing or leg bud with the ins knife Make four cats the first longitudinal and median to the bud dose to the somites the second and third perpendicular to this m front of and behind the bud snd the fourth lateral and not too dose to the bud (1 234 or 5678 ® lug 35 a) The entoderm may be peeled off usmg 2 forceps but this IS a rather dehcate procedure and is not necessary ^Mien the transplant i* isolated cover the di?di with a lid and place it on a heating plate (not warmer than 39® C )

ElEPAiATIOJl OT THE SITE OJ IMPLANTATION IN THE ITOST ElrB*\0

Reopen the host embryo add salme solution and under the binoc °la^mlc^o5cope^cmo^*c the agarwithapairof watchmaker forceps Shake die embryo gently if the blastoderm adheres to the edge of the window

12 If the ammon and chonon co\-cr the operation region it is neccsto slit them open This is done with a glass needle Hold the egg dicD with j-our left hand insert the needle into the amniotic ca\ntj ^ the mldline where the raphe (suture) is ^^3Iblc and with a jerLj upffioiTment of the needle rupture the membranes Continue this un dl at least the posterior half of the nght wing bud is exposed The mem require no further care Tbej will heal back owr the embryo


  • 53


13 The transplant is to be implanted through a hole posterior to the mng bud (j m Fig 35, i) Locate wing and leg buds and vitelliae artcnci In the following steps it is absolutely necessary to avoid heraorrfaigei. Be sure not to get too dose to the lateral edges of the somites, to avoid puncturing of the posterior cardinal win, which runs underneath the lat cral edges of the somites Do not push the needle so decpl> as to In/ure the splanchnic layer whidi is highly vasculanxcd Under high power of the binocular microscope push the glass needle through the somatoplcurc at a pomt between the wing and the 1^ bud and a short distance htcml to the outer edges of the somites whidi stand out dearly m red From this hole vrork forward and backward and make a longitudinal slit m the soraatopleure parallel to the mom axis and large enough to allow the passage of a limb bud Add saline to keep the embryo moist

TlUlflPIAWTATTOH

14 Under the bmocular microscope suck the prepared limb bud Into the distal part of a micropipctlc B> gentle pressure on the rubber mem brane which covers the lateral hole drop the transplant onto the host near the slit This should be done under the bmocular microscope (lowest power) to avoid the loss of the transplant. Hold the egg shell with j'our left hand and manipulate the transplant through the slit into the codom using the tip of the gloss needle (Fig 35 b) The transplant may be ori ented dunng or after the implantation If one wishes to have the transplant adhere to the umbDical cord, it has to be pushed into a lateral pori tjon at a considerable distance from the somites Otherwise lea\T it near the somites above the root of the vitcllmc artery in longitudinal orienla tion Add a small amount of talme solution Take a complete protocol note if wing or 1^ has been transplanted orientation stage of host etc.

1 5 Seal the window Place cither the ongmaJ piece of shell which was sawed out or a square or circular cover glass over the window Wth a brush apply warm paraffin around the edges Be sure that the window b sealed completely on all sides Return the egg to the incubator Place It on a stnp of cotton the window faang upward

aacemaT or mz raANsn-ufT

16 Allow the host to dc\xIop for 7-9 days the best stage for fixation is lo-i 3 days of incubation Do not roll the eggs during this period Pre pare a pan or finger bowl wdth warm saline solution Remora the window and widen the hole Carefully dissect away the cbono-allantoic mem brane Carefully sever the umbilical cord and lift the cTnbrjTi into t e dtsh of laJmc If no transplant is suslblc from the outside it roa> be cn Urcl> hidden Inside of the coclomlc easily or it ma> ha\T been rcsorbed


154


CircfuUy sht open the ventral body will sJightlj to the left (apparent nght) of the median line and Inspect the bside of the body cavi^ Tate careful protocols Fix the host embryo together with the tran^lant m Bouin and stain them with methylene blue (p 135) to make the host and the transplant skeleton visible

6) PLANE GRAPTS OF LDIB PRUrOUHA

(After Hamburger 1938 1939)

The method of implantation into the coelomic cavity has the Himd vantage that the transplants cannot alwaj’S be fixed m a gn’en position^ If a definite orientation is desired the pnmordia are best implanted m the outer body wall

£xFEaDirKr4S

IfaUrtaJ

a-3 donors and 3-4 hosts per student standard equipment (p 133)

Procedurt

As in Eiperrment 47 with the following modifications 10 (p 153) Preparation of the transpilant In cuttmgout thehrabbud leave small strips of tissue (somites or adjacent mesodennl attached to the antenor and posterior ends of the base of the bud these will be tucked into the slit The other Umb buds maj be used for further transplan ta bons

^3 (p 154) Preparation of the slit m the host embryo Make the sht between wing and leg bud as dose to the somites as possible and not bxt long Rather lengthen it while >*00 implant 14 (p 154) Implantation With the tip of the glass needle first tuck the antenor then the posterior end of the hmb bud into the sht (see Fig 35 i 4 ) If the hole is slightly shorter than the bud the transplant will be held in position by the tension of the tissues Take care that the major F*rtof the transplant is exposed and that it does not slip mto the coelom No other precaution is necessary to keep the transplant m position ^5 (p 154) In handling the egg after the operabon be exceedingly cautious avoid all sudden or jerl^ movements Place it on cotton m ^ incubator and do not disturb it for a da> or two

c) FLANK GBAFTS OF KYE PBniOKDIA

(After Gaj'er 1942)

The first flank grafts of optic vesides were made b> Alexander (1937) ™ ®nnection with the problem of lens mduction Gajxr (194 ) obtained


155


well formed eyts by the same method, using optic vcsidei of lo-ap. somite stages Ihe shape and general structures of the transplants were normal This demonstrates the high degree of iclf-differcntlatmg capac ity of the optic vesicle with respect to its surrounding structures. How ever, practically all flank grafts showed a deficiency in the closure of the choroid fissure of the type which is occasionally found as a congenital abnormality m human eyes and which Is known In Ophthalmol^ as ‘*coIoboma,'


Fia, j6 — TnuupIuUtian ot ao oplk reifdo [o Ibe ciikk (tJla Gtyw iw*) tffflbiyo (jo fOQiItei, 6o boun of IscobiUan) vfth iHt for tbt laiplutatioD ot tbe optic rwfcfc >-baidoi tbedooofgnbT7o(mamItn,36boanQfbcub«ti<«) tb* dotted Hd** lb« 

caU by videb ibe nsht oplk rejieJe and (be adfacost brain part are iCTtrcd (â– â– trwuplanf /â– CTOP- a ectlon tbrooffa a, In Itrei A-A abowias tbc traniplaat tn po ri tke.


Exrai>asfT49

i/a/ma/

a-3 donors 3-4 hosts per student standard equipment (p 133)

Procedure

As in Experiment 47 with the following modifications I (p 151) Start the Incubation of the host embrjirt j}-3 d”}"* ‘J’'

incubation of the donors 36-41 hours before operation At operation t e donor should have 10-15 somites The host should be In stages 3 5 fP

  • 33)


8 (p 153) Vital stain the nght wing le\'cl rather than more posterior parts of the host 10^ 153) Cut out the nght optic \'e3iclc together with the right half of the forebrain. Firsts mate a transverae cut behind the nght optic vcside through the head to the midlme Second mate a median cut through the anterior part of the head Tie left eye nmy be used for an other transplantation (Fig 36 b)

13 (p 154) Make the aUt at the base of the wing at about the level of the twentieth somite (Fig 36 a)

14 (p 154) Transfer the optic vesicle with adhering brain tissue onto the host blastoderm, using the micropipettc. Drop it near the sht With the tip of the glass needle tuck the bram portion Into the slit, thus lea\’mg the optic vcside expxjsed on the surface (Fig 36 d)

16 (p 154) Incubate the host for not more than g-io] days (total age) Le. 7-8 days after operation at which stage all essential c>'c structures are differentiated In order to make visible such details as lens retina ins choroid fissure etc without sectioning the transplant the embryo may be fijoKi in Boom dehjtlrated and deared in ofl of winte i green (p 135)

S) TSAKBPLAHTATION OF tJEUlAL CH£ST JJtOU DASX TO WHITE BREEDS TO DEilONSTRATE THE ORIGIN AND UIGRATION or UELAKOPHOEES

(After WUhcT and Rawlcs 1938 1940)

The daik pigment of vertebrates is contained m granular form in pig Dttnt cells which are called ‘melanophores They ongmate m the neural creaL This has been convmcingly demonstrated for amphibians and birds by extirpation transplantatioa, and tissue-culture experiments (rtrviews mHamson 1938 Wllher and Rawlcs 1940) The potential melanophore like other neural-crest arc wandering cells They migrate from

the dorul part of the neural tube to their final locations where thc> prohferate and form complex color patterns for example in the skm of am phibons or m the feathera of buds. In the latter mitance they migrate “ito the developing feather germs and deposit pigment granules m the barb pnmordia. The migration of melanophores can be demonstrated by the foDowmg experiment devised by Wilher and Rawles (1938 1940)

A inull piece of embryomc head epidermis from a dark breed of fowl hiaDdmg some prospective melanophores was transplanted to the base ^ the Wing bud of a 2-'3-day embryo of a white breed The transE^ted epidermis was not Incorporated in the epidermis of the host, but P^^®pectrve mdanophorca adhermg to it migrated mto host territory


  • 57


The^ settled down m the developing feather germa of the host tnng and there deposited their pigment As a result the fully de^-eloped vring was partly or cntirel> covered with blacl. down feathers, all other fealhcn being uncolored In some cases the pigmented area extended as far is to the ventral midhne of the body The melanophores thus exhibited not only an extensive migration but an eitTaordjnar> prolifcrati\*c capaat; they ail originated from the few neural-cxcst rgHs which adhered to the epidermis transplant

Willier and Rawles and their associates earned the eipenment farther The host* were allowed to hatch In due tunc the down feather* were re placed by the adult plumage WTutc I^hom wings which earned a transplant of Barred Ro<i. rnelanopborcs ejdubited a typical barred pattern in their feathers These experiinents rcx^al the unexpected fact that the color pattern is largely determined by the genetic cooititutlon of the melanophores rather than b> that of the feathers themsclm However the boat feather germs modif> and control the acti\i^ of the melanophores to a certain extent. The role of both partners In the detennination of the final pattern was analyzed by means of extenn\'e senes of redproctl traa^lantatlons between many different breeds of fowl (review in WTlUer I94J)

Bxpcanresr so

ifaleriol

donors any dark breed c g , Brown L^boms a*-3 eggs per student

hosts 3-4 eggs, UTutc Leghorn

standard equipment (p 133)

Procedure

r Start the incubation of the donors and hosts 3§^3 days before opera tiom HowoTr younger donors of 30*40 hours of incubation ( 5 ~*® ^ mites) may be used (Dorris, 1939) Ihc hosts should not be older than stage 4 (p 133)

2-7 As In Experiment 47 (p 151)

8 Prepare the slit in the host. Rupture chorion and amnion over the right wing bud open up a large hole The membranes wili heal ov'cr again With the glass needle make a small but rather deep hole at the base of the wing Vital staimng Is not necessary Return the host to the incubator

9 Vital stain the head skin on the donor Place a piece of red agar on

the head in front of the otocyit. Stain for 5*10 mmutes.

10 Mcanwhae take the host out of the meubator, place it withm easy reach When the »tm of the donor Is sufTidcntly itoincd cut out the

15S


transplant as /oIIottb With the glass needle or vratchmaher forceps stnp t piece of flkm from the dorsal and dorsolateral surface of the head m front of the otocj’sts This piece contains neural crest cells II Transfer this piece directlj onto the host using the micropipette 13 Implant the graft m the prepared sUt burj it deeply Be sure that the transplant sticLs to the sht

13 Seal the window and return the egg to the meubator the window facmg upward hlark the egg with j'ourimtiah. and a protocol number

14 Recover the host about 2 weets after the operation Pigment has formed at that stage Stud} the color of the wing the extent of the pig mented area etc, (sec papers quoted abo^x)

BffiUOGRAPm (a -<0

AuxutDEt L. E, 1937 An etpmmenUl *lud) of lie njle of opijc cup and o\TrI\ ing ectoderm xn leta fonnttion in tie chick embr>‘o Jour Eiper ZooL 75 41 Doms F 1939, The production of pigment cbici neural emt In grafts to the 3-d*> limb bad Jour Eipcr Zool 80 315.

G\TEa, H. 1943 A itud> of cokiboma and other abnonnaiiUea m transpUnU of ejepruaardufttnnoormalandCrecperehickembriCb Joar Eiper Zool 89 103

Girtoxoax ScBOE?ranaEB S 194: Tbe derdopmeot of eari\ mouse embow m the eitneabr} onic coelom of the cbici Saence, 93 HAUBCTOEa, ^ 1938 hforphogeneuc and ami seU-did^erotiaUoo of trui*phnted Imib pomorda of a-da> ^ck embryos Jour Eiper Zool 77 i 9

1939. The development and LoDervation of transplanted limb pnmordia of

dud cmbrjoi. Ilnd go 347

lUsanca \ ^ and \\ acok, ML 1940 Tbepnmar) de%e]opmeiit of the lideton In amdeaiand poorij mnervated lintb Iran plants 0/ cb»ci embnos Pb>sfol Zool

  • 3 367

fitaaijox R G 1938 Die Neundloste ErganruDg-hefl i anat Vnx 85 3 Jot E a, 1939. Intra-coelomic grafts of the eje pninordium of the chici. Jour Eiper ZooL, 74 461

Eawlo M E 1940 Thede^'clopmento^meUnophons.fromembr>on^cmoa»etiswe* grown m the coelom of chici crabn 01 Proc Nall \cad Sa 36673 ^ruin B II i^r Ananalvsuoffcathercolorpattcrnproducedbi grafungmclan

opborei during ernbr^tjnic devdopoient \iner Nat 75 *3®

B IE and Rawixs, M E 1038 Feather charactcruauon as tcdied in ^*0*1 graft CDinbinationi betneen cbici embr'O' of different breeds Proc NatL Ami Sd X4 446

■ • »94o The control of feather color pattern b mdacophore* grafted from one

erabrjo to another of a different breed of foul IhMiolZool 1317 '\aura B II Ratuxi M E andHuxas C 103? Sim tran<pbnU between eubrios of different breeds of fowl Proc NaU \cad ba 33 541


  • 59



PART IV

REGENERATION (RECONSTITUTION)



A GE>rERAL REMARKS


The regeneratn'e potcnaes \TLr> greatly among anminU E\dra and other Coelenterates the plananans among the Platjhelimnthes and the Urodda among the «rtebrates ore Lnovrn for their extraordinan. rcgener atn-e power A survty of the rcgenerativt properties of the different am mal groups maj be found m Korschclt (1927) see also Morgan (1901) and Child (1941)

It has long been recognized that the repair of lost parts can be accora plished In two waj** bj internal reorganizabon and transformation of old tissues without addition of new growth or b> ontgrowtb of new tissue from the cut surface m the form of a regeneration bud or 'blastema Head and tail regeneration in Planona Innb and tall regeneration In Urodda arc examples of the latter t>'pe The change m shape as well as the formation of a new phorj-ux b> the old tissue in the irgenerotion of Planarta is an example of the former t>pe. In man) instances both tj^cs tre oombmed m the same form and there ma> be no fundamental differ ence between them but for practical purposes it is desirable to designate them with different technical terms Morgan (1901) distinguishes be bron ‘ eplraorphosis (proliferation of new tissue) and morphalbxis (changes wiihm the old tissue) and mcludcs both under the general head log regeneration Child (1941 p 30) uses the term reconstitution to Include all tjpes and defines regeneration in the narrower sense as reconstitution b^ outgrowth and reorganization as reconstitution b} Internal changes

Sc\‘eral experiments described below illustrate the fact that prcci5<I\ those parts which w ere remoN-ed ore regenerated This is to be expected from the teleological point of %new but the underhuig causal factors which tnusl be different at different Icrvels of the regenerating organ are not clear Jy understood Cases m which more or less tissue is regenerated than was r^rno\*ed and cases in which the regenerated structure is different from the lost part (heteromorphosis) axe of particular mterest in this connee ^3 These points will be illustraled bdow

The >'oung regeneration blastema with its undcrlnng stump ha\T all ^UiractCTuticsof a morphogenetic field (p 94) The organ forming prop0/ the blastema arc at first fabflclj determined andgradcaJI^ become Thlswassbownbj tnmspbntationeTpcnmcnt»(«ccSchottf 1939 163


Weiss, 1939) The bUstema passes through a stage in which it is deter mmed as a whole but not as a mosaic of details Duplications ansmg from single blastemas bear out this poinL Regeneration fields,’ like cm bryonic fields extend beyond the boundaries of the regenerating organs. For instance, if a urodele limb is extirpated with its girdle, tissue adjacent to It will regenerate a complete limb However, the limb-regeneration field, as well as other regeneration fields do have definite limits, and no re generation occurs if the entire field Is erthpated In this sense regenera tion is not a prop>erty of the Ti^le organism but of a local field P Weiis (1925, etc.) and Guytfnot (1927, Guy£not and Ponsc, 1930) ha\‘c de veloped the concept of r^cneratlon fields

BIBLIOGRAPHY

Crtnn C. 1941 Patterns and problems of devtlopmcot Chka^ University of Chicap) Preas.

GoyfiroT E. 1937 La Perte da pouvotr r<gfa£rateur dea anourcs <tncii£c par U m^thode des U ootkio de tcrdtoiita. Rev sulase de sooL, J4!i

Gtnrfjioi E., and PavsE, K. 193a Territoirts de r^gfafratkm et tranapUntJtkws. Bull Biol 64 35s

Koasemeu E. 1937 RejenentloQ and Tran^jlanUiUoD \oLi BerllD Borolrie8«T

MotOAK T H. 1901 Reseoeradoa. New York MtcmlQan.

Senorrf, 0 E. 1939. The origiQ and morphogeaetJe potcades of rtjeocrates. Grovtl] SuppL 1939. p S9*

Weiss, P 1915 Upabha^gkdt der Estremltatenitp^o efa tloo voto SkdeU (t*!

hit cnsiclus) Arch, f mikr Arut q Ent* medu, io4i3S9»

1939. Prindples of dereloproent Nnr York Holt


164


B REGENERATION I^ PI^ARU 1 LIVn^G ZIATERUL CULTURE METUODS

Two nath’C apeaea are commonly used for expenniejits Vugcsta /t gnna Girard (83m Planana macuJaia Euplanana Ifgnna Lcidj), and D dcroiocephda Girard (5301 PI dorotoctphcla \\ oodworth E dorotoccpk eh Woodworth) They can be distinguished readil} as follows DugesPi dorctocepkala is larger than itgnna (length up to 25 mm ) it B uniformly dark (brown or black) its auricles are elongated and pointed (Fig 37 a) it lives in springs and spring fed streams and can be collected b3 baiting It is found in middle western states Dugexia itgnna is, at best 15-18 mm long it is variable m color usuall3 it shows a spotted color pattern (white Irregular spots on a brown ish or bla(±Ish background) or a hght mid-dorsal

  • tnpe. Its aundes are broader and blunter than

tho« of D derolacepliala (Fig 37 i) It Irves m ponds, lakes and alow flowing streams on the ander surface of stones and lca\-es and can be collected b3 tummg these over Its distribution is eastern and middle western states wrst to the iliisissippi south to the Carolmas

Culiure of plananans — Plananans collected m the held or bought from

  • dealer raa) be kept in large glass containers or dark enamel dishpans

Fhe3 should be co\'cred and kept in a dork cool place *^pnng or well ^ter IS preferable to tap water Plananans should be fed twice a week

  • >th itnps of calf or beef h\Tr Before feeding lower the water to a depth

of t few inches Distribute the stnpsof meat and rcmo\-c them after 2 3 l»un of feedmg Thereafter nnsc the dish or pan thorough^ and fill it 'ilh fresh water The animals arc ver3 susceptible to fouling of water (ilost of these data arc taken from H>Tnan 193/ where more details ^3 be found )

2, GENERAL E\PERIME.VTAL PROCEDl RE Since the eighteenth centur} Planana has been one of the fa\*onte Eternals for the 8tud> of regeneration A s3Stcmatic anaKsis was begun

Ccrctiulat itw pTkdt> of ll>e rmta came Kt Hmqu (iftjti)

165


a b


Fio j7 — <“bc*d of Dw[oia {Pla aric) ctf^eh • bead of P {.Fl (from

Ifrmafi i9Jt)


by Morgan (1898, 1900) Later on, Child and his assoaates used Plcnana extcnsi\Tly for reconstitutwn eipcnmeats m connection with the gradient theory The volmmnous hterature on the subject is reviewed m Morgan (1901), Korschelt (19*7), and ChDd (1941, and previous books) A large number of cutting experiments can be done as rlw experiments In the following only a few experiments were selected Others may be taken from the bterature, Expenments which ha\T a special bearing on the gradient theory will be found on page 189

Mctmal for Expcnmenls 51-53

Dugesia doroiocepkeda or D Itgnna^ about 1 2 specimens per student and per experiment (Speaes differences exist with respect to regener atjve power, time of regeneration, etc- Since thesp^ of regenera tron vanes with temperature. It is desirable to run all expenments at constant temperatures )

finger bowls or Petn dishes small brush to transfer planarians

microscope sbdes pipettes

Planaria Lmfe (p 9)

Gtnercl procedure for ExpcnmtnU 51-53

Noie — Do not feed expenmental animals for a week before operations For each experiment select 8 or 10 speomens of as uniform nxe as potnble. ifake all operations under the low power of the binocular microscope

I Prepare and label finger bowls or Petri dishes one for 8-10 sped mens Prepare a dish for discarded pieces

i Wth a brush place a specimen in a drop of water on a dean slide Allow the animal to expand manmally Then make a cut with the Pla nana knife in the desired plane. Theknlfcmustcutdowninapcrpcnd^cu lar direction not obbquelj The cut surface must be sharp and dean Practice on a few anirriiil^ before >*00 perform the protocoled experiments

3 Take a protocol and make a sketch

4 Transfer the piece which is to regenerate into the labeled dish and discard the rest of the animal at once

Note — Do not feed regenerating animals. Keep them in a cool dark place or cowr the dishes containing regenerating animals with an in>'trtcd cardboard box.

5 Inspect the regenerating pieces cv«t> second or third day depend mg on the t>*pc of eipenmcnt Discard ail dead pieces Indicate

on sketches the border line of old and new tissue ChTr a long period the regenerating tissue can be distinguished \-cry clearl> from the old tissue x66


by its lact of pigmentation Pay speaal attention to the first appearance of regenerating eye spots and pharynx Under ordinary conditions regen cratiOD will be complete in about 8-14 daj'S

3 REGENERATION AfTER TRANSl'ERSE LONGITUDINAL AND OBUQUE CUTTING Experiment 5: Rxoeneiahos op TRANsvntsE Pieces Follow the directwns on piage 166 First decapitate the nmmnl by cattmg m plane a (Fig 38 a) Next make section c which is at one third of the length of the decapitated animal Remo\’e the pbarjiii if it protrudes Place each piece m a separate dish label the dish Repeat the apenment on 6 animals Place all identical pieces m the same dish Make careful observations and sketches of ^epTesentatl^'C cases Payspe 0*1 attention to the followmg points wound healing the appearance of the unpigmented regeneration blastemas The head which is character lied by the eye spots is the first differentiated structure to appear m posterior pieces It is alwa >-3 formed by blastema cells Ob 5 cn*e the first appearance of the pharynx It appears near the postenor cut surface m the anterior pieces and near the anterior cut surface in the posterior pieces usually being formed bj old tissue Note the time required for completion of the regeneration The end result is in each instance a smaller but proportionate mdiNudual The original polant> is maintained in all fragment*. Anj le\cl is capable of forming a head

Exteument sa Reocneration or Short Tka.v 5 VE 25 E Pieces Sectwn behind the head (Fig 38 a le\’el a) and bisect the bodj (.level d) Cut the anterior half in 3 (one sixth) or 4 (one-eighth) pieces of equal lengths and keep all pieces of the same Ic%el m one dish Label the dishes Operate 6 animals In this experiment stud> mainl} the at Imminent of the typical proportions bj the formation of blastemas as well w bj morphalkns AD short pieces are at the beginning much too wide w compared to their lengths The beads (old or regenerated) will at first be disproportionatelj large Note the gradual adjustments of the proportions b> changes m shape Note agam the regeneration of a new phaiyni, usually m the old tissue The head malformations which frequently m short transverse pieces are discussed on page 187 (swFig 43)

SecU« ( !• rtcomnjcnded bewue tin* cut «ill serve si s cnntrol for Eipetunent it IS ^ operiment b not bknued, then sectMU d (uutesd of c) »ludi cuts the deenpUsted ‘aifflalhhalf.


Fro ^ — RcfCfierttiooiDpUturitBJC/fnKDlIiiZcr / ( aittx St^bo tad lUjahcrta

<939) «~diffcreDt lerelf of tft aim ' j e irctloQs fk>"p\aryT\x h — iaedixn (i) tod obbqitf tecUxtO ot ) c d^prDducUaaofdDpQdUsutaiirtyaectioainglnlmtKaod* f-product^OQ of trfpotar fonns bj re^ceenUioQ of F-fdcca / /■■prodactiao of dopbdtu cru^U by trctiont */ axid r Tbe dotted are*s tad t aad g ire old tiwue tiic Cgbt arcu to CbcK firrure* are rftrumted ti»ae.


ExKsaujiEfT 5j Lauxal REOEtfiiAiiay

Sectwn several plananans m the median plane (A Fig 38 b) Section others m a paramedian plane Discard all pieces of the old pharynx. Observe lateral regeneration

F.T-r rpniTN T 54 RnoEirnuTtON noi£ Oblique Suxtaceb

Make an obhque cut and discard the anterior piece. In another series rnakp two parallel obhque cuts in the prephaiyngeal region (/ and m Fig 38 b) discard head and posterior end Note that the head regenerat mg at the antcrwr surface makes its appearance not m the middle of the ent surface but at its most anterior pomt It is asymmetneal at first. This asymmetry is clearly expressed m the earher appearance of the left (antenor) eye The tail blastema is likewise asvTnmetncal The regener ates fllustrate another pomt of general interest the main direction of outgrowth both of head and of tail blastemas is at first perpendicular to the cut surface and not m the mAin ana of the old piece Barfurth (1891) was the first to observe this on tail regenerates from obhque scctioiis m amphibian tadpoles and regeneration perpendicular to the cut surface is occasionally referred to as Barfurth a rule In later stages the heads and taila straighten out Rukn (1936) has inUrpreted these results on the base of Child s gradient theory


4. PRODUCTION OF TTtO-HEADED PLANARIANS (DUPLICITAS ANTERIOR)

Expeeoiewt 55

Remove the head behmd the cyts (o Fig 38 c) and spht the antenor tro-thirds of the animal in the median line («) The separated parts ha\-e a strong tendency to heal together and if necessary the sht must be re ®P«ied several times withm the next 12-24 hours Note the appearance oi new eyes and new pharynges The experiment shows that the regener ate of a Planana behaves like a 2-cclI stage of a salamander or of a sea orchlii. Each half tends to reconstitute a whole organism (Fig 38 d)


5 PRODUCTION OF TWO-TAILED PLANARIANS (DUPLICITAS POSTERIOR)

EXFERDCEIfT 56

the head behmd the e>-« and »pht the posterior two-thirds of sninuU Two tifls will form if the parts are kept separate b> contm “of reopening of the sht.

169


6. EXPERIMEXTAL PRODUCTION OF BIPOLAR FORilS (POLAR HETEROaiORPHOSES)

(After F S Miller)

The prcvioua expcnments have given evidence that the polanty is ufu ally maintained in rcEcneratmg plananans Cases in which the polant^ IS changed are therefore of speaal interest- hlorgan (1898) was the first to describe the regeneration of two heads from the anterior and the posterior cut surfaces of a short trans\'er3c piece and caDed this phenomenon "heteromorphosis Later on the term was used to designate all kinds of atypical regenerations for instance the regeneration of antennae m the place of amputated eye stalks in arthropods To avoid confusion Inw sions of polanty are called *^poIar heteromorphoses- They occur rather frequently in very short transvcTBC pieces of the post pharyngeal Ie^’el8. Rustla (1925) has shown that the rate of their occurrence can be con troDedandincreaiedbyvarK)U5agcnts(etber chlorelone etc,) F S JIB ler (1937) succeeded In raising the madence of bipolar heads %'er> substantially by treatment with strychnine The following expenment is based on her data An interpretation of bipolar forms of Plaitana on the basis oT the gradient theory was given by Child (1915 1941, and others) and by Rustia (1925)


ExpERnaafT 57

MaUnal

Duiena doroloccphUa 10-15 specimens per student other material os on page 166

prepare a fresh solution of ftrychnme sulphate M/ioo/xio for each ex pemnent

Procedure

Make all sections in water, not in the 8tr>clmlne solution For the fol lowing experiments only post phaiyT’geal one-eighth pieces will be used £ pieces (Fig 38 e) give the highest percentage of bipolar heads F pieces may also be used Cut in the following order a d,/e(Fig 38, r) Dacard all but the £ and F pieces Transfer these fragments immediately to the strychnine solution cover the dishes and keep them in a dark, cool place After 12-16 hours transfer all pieces to spnng or well water A longer exposure Is lethal Check the cultures frequently and rcmo^'c dead fragments Observe the de\‘clopnicnl of bipolar forms Atypical heads (sec p 188 Fig 42) will be found occasionally Note the direction of the beat of cilia (cf RusUa, 1925 and MiUcr 1937) The percentage of bl


170


polar beada can be increased further by delaying the postenor cut for 17-24 hours. Run parallel cultures of E and F pieces in well or spring water as controls

7 PRODUCTTQN OF DUPLICTTAS CRUOATA (After Silber and Hamburger)

EllCrEKIMEMT $8

Dupliatas cruoata is one of the strangest duplications occurring in animals It is a complete duplication in which two heads and two taHs are present. The two heads face m opposite directions and ha\*e a com mon median plane likewise the two tails pomt m opposite directions However the median plane of the heads and that of the tails are per pendicular to each other (Fig 38, g) Duplintas cruoata in amphibians 13 discussed on page 75 (see also Fig 20)

llaltnal

Buiwa ti^nna or D dorotocephcla lo specunens per student other material as on p 166

Procedure

^lake a longitudinal cut in the median plane through bod> and taxi to t pomt behind the aundes ip~p m Fig 38 f) After 34 hours male a transverse cut (r) at a short distance in front of the crotch, so that the tmx hah tails are held together bj a narrow connection about one-third tbeir width. If necessary reopen the longitudinal cut Make 6-10 opera Check the operated AnTmAlt on the following days Reopen the tticdian slit if necessarj

In a high percentage of cases the anterior transv’erse surface will re S^^iCTate a nor mal head and another normal head will dei'clop m the Qutch (Fig 38 g) The crotch heads are occasionally duplicated or abThe anterior heads are cither normal or in a few esses absent Ofaserv'c the movements of these monsters after completion of the regen ^Uon. Either head may ta k** the lead with the other head and the tails ^^*ihiig behind, and they may alternate m leading

BIBUOGRArm (1-7)

^***^*^ 1891 yenuebe tur fonktiooeOen Aupauung Arch f nub Aoat.

C.M 1915 Indhiduslitvinorgiiutnu Clmago bnA’emt^ of Qucajo Press i!m Pittenis tod problems of devdopment Chjago Lolversitj of Cbi

“10 Pros.


Hyicah L. iQji Studies oo tbe moipbo)o£} tsxooocny at>d dlstifbutkiQ of hortb Amcncao tridad TttrhtUan^ IV Recent European reviiioQS of the tiidadi and thm appCcaboc to the American forms «ith a kc> to the latter and itev notes on distifbatloa. Trans. Amer Uiff Soc, 50 316

1937 rianarians. InGAUsoi?, P S (ed.) Culture methods for Invertebrate

animaJi. Ithaca, N Y CocnitocL

1939 North American triclad rurWZflfio IX. Trans. Amer Ufa Soc. 58:

164.

Koxscbxlt £. 1937 RegeneratlaQ and Tiansplantatlooy ^ oL i Berlin Bonitiic gcr

MiLLEt F S 1937 SomeedectsofatrychnlneonreccmstltntioainEH^Mna^Mtea* Ctpkaia PhydoL Z06L 10 *76

Mcxoan T H 1898. Experimental atodlcs of the rt^eoeration of Planaria maetdata. Arch f Entw’mech. 7:364.

â–  190a Regeneration In ptanarians. Ihd le 58

X901 Regeneration. New York MaemPbn.

Rvunr 0 1936 Experimental asymmetries of the bead of Euptonuria itnioctphala Ph>'sioL ZoflL, 9 *78

RranA,C,P 19 J5 The control o/bfailaldevtlopmait in the reconstltutioo of pieces of plana na. Jour Ezper ZoOL, 4a tit

Stun, R. H. and HAiotnorR, V 1939. The production of dnpUatas cruciata and multiple beads by regenermtion hi J^^lamarU iltrOu Ph) sid Z06L, :a 385.


172


C REGENERATION IN AMPHISIAN LARVAE 1 TAIL REGENERATIOV

Tails of urodelc and aniiran larvae and of adult urodeles regenerate when amputated It is advisable to use larvae because the regeneration in adults takes several months The experiment illustrates the differentia tbnof blastema mto complex structures such as vertebral column spinal cord, musculature bloodvessels etc Ifobbque cuts are made theansof the regenerated tail will be at first perpendicular to the cut surface (Bar forth 8 rule [Barfurth 1891] see p 169) Later on the regenerate will straighten out

The ejqicnment affords no opportumly to stud> the origin of the blastema cells This question Is not yet definitely answered because it is diffi colt to trace cells trt tw The old mcw that each tissue which is exposed tt the cut surface regenerates its own kind holds true m onjv a few m stances IVa'viUe (igsi 1924) in a masterly 5tud> has traced the muscles of the rege n erated tail to mjured muscles at the cut surface which dedif fereatiate to a certain degree and then redifferentiate into muscles Other kistological and experimental studies give ^x>d e%ndeDce that most of the Wutema celli originate in a different waj partlj from mdiffcrent mesen chjine cells of the tail stump and partlj from old tissue which dedifferen hates completely then forms mdifferent cells and c\Tntuanj redifferen possiblj along new Imes This latter process is called metaplasj (*«Schott6 1939)

ExrERiUEjrr 59

Afctfrjfl/

• 4 w 4 yj/owa or Tnturiis or Rana an> spccira (stages from swimming •tages on)

1 pair of fine forceps Petn dishes

Pq^ttes chlorctone i 3 000 or MS 222

bowis or Lflj dishes i 3 000 for narcosis

^ '^arcotiae 10-20 specimens of equal sue (take measureroenls) Oper

»te In narcoUc m Petn dishes

  • V ith a pair of fine scissors amputate the tails In 5 10 specimens

the cuts tran5\-crscl) In 5-10 specimens make them obliquelj


173


Record the details of the operation Mate ahetches of rcprcscntati\-e cases

3 Feed the animals daily (pp ai ay)

4 Observe the wound healing and the daily progress of regeneration the appearance of the blastema lU outgrowth, the differentiation of the fins, pigraenUtion etc Observe in particular the direction of outgrowth In the obliquely cut animals The angle between the mam axu of the ani mal and the axis of the regenerating tail can be observed best on the first daj^ after the appearance of the blastema. Later on the regenerating Up straightens out


2. LUIB REGENERATION

Amputation of parts of forelimb or hind limbs of amphibian larvae at any level results In a regeneration of the lost ports tJrodclcj retain their regenerative power throughout life but anuran legs will not regenerate after metamorphosis Complete regeneration was observed in urodeles even after removal of the girdles Usually the amputated parts are restored completely, but occasionally the regenerate is atypical For in stance it may develop fewer digits or toes than normal (hypodoct>ly) Of special interest are duplications for example the formation of two feetorofsupeniumerarydlgits(hypcrdactyly) (Weiss, 19*5) TTiesecascs give evidence that the early regeneration blastema cannot be a rigid mosaic but that it has regulative properties The origin of the blastema cells 18 not yet definitely established The careful histological studies of Butler (1933) and Thornton (1938) give strong arguments for the view that akdetal and muscle cells at the amputation stump dedifferentiate and then redlffcrenUate possibly along entirely new lines

prelhiinaky exerosi:

In order to find out if the development of the external form of regener Qtes proceeds along the same hnes as docs normal limb development stud) the development of normal forclintbs in Antbysloma (an> speacs) Start out with 3-5 larvae of stage H40 and make a complete senes of shclehes up to stages in which all digits arc formed Narcotlic the animals during obscrv'atwns Compare with the stage series for Tntnrus b> S Glueck sohn (1931) Note in particular the appearance of the elbow the sequence of the appearance of the digits and thcir rclatixT: proportions. Take notes of the rate of differentiation under the conditions of your eipenmcnt The animals should be Lept In a constant temperature U possible


  • 74


ExpekmektCo


Mcimal

AtnhysUma larvae with 4 digits, 4-6 specimens for each student Select specimens whose limbs and toes are completely mtacL When several qieamens are kept in the same dish it happens frequentlj that the} snap and injure one another a limbs particularl> if they arc not fed adequately It is therefore advisable to rear the specimens to be used m this experiment singly in Lfly cups I pair of fine scissors pipettes

finger bowls or Lily cups chloretone (i 3000) or MS 223

a dah with Pennoplast bottom (1 3 000) for narcosis Procedure

I Narcotiac 4-5 speomenfl

f Make a sketch of the extended right arm of a rcpresentativT spea men Use the camera luada If available In order to mount the arm bon lontally use a duh with Pennoplast bottom Make a deep groove m which the body of the ammal fits and spread the arm over the edge of the groove 3 In 2-3 specimens make a trans^■er»e cut through the right humerus 39 <»)j in 2-3 specimens amputate the right hand through the wnst (ft)

Uie a pair of fine scissors Indicate the le\tl of cutting on the sketch L Gn-e each specimen a serial number and place each m a separate disL Rear m a constant temperature if possible 5 Follow the regeneration for several weeks Make a complete senes of sketches Compare the regeneration with normal de%xlopment Note that in the specimens amputated at the humerus the digits will appear first and the parts between the digits and the cut le\Tl will be restored ^ter If time per m its both the amputated ends and the full} regcncnited ihubs should be fixed and stained with meth\lene blue (after Lund\TLll P 134) in order to find out if the cartilaginous skeleton has been restored ooropletely

3 THE ^\OLFFIA^ LENS REGENERATION IN ERODELA (After H Wachs 1914)

The lens regeneration Is unique among the regeneration phenomena "olff (1S95 1901) di5co\tiTd that if the fens is carefullj extirpated


Fio 39 — Forcbmb i(i;euu&Uoo In

■alamaful fT IaTVW d ft — ol KC

tlODina (kc t£Zt)


75


In a urodde, a perfectly normal new lens will be formed Strangely enough the origin of the regenerated lens is different from that of the erabiyonic lens m that the upper margin of the iris, and not the epidermis forms the regenerate (Fig 40, c~f) This case illustrates an important pnnaplc that the same end result can be achle\'ed by two entirdy different modes of development The lens regeneration is unusual in other respects It is the dearest case of “metaplasy* hnown so far (see p 173) the differen tiated and pigmented cells of the ins undergo a dedifferentiation and a subsequent rcdifferentiation into highly speaahxed lens cells (li^achs, 1914, Sato, 1930 and others)

If the operation is done carefully the upper iris does not suffer onj in jury This observation raises an important issue. In most instances of re generation the creation of a wound surface is considered essential for the initiation of the regeneration process In the present instance the necessary stimulation for the onset of regeneration must be provided by other factors Speraonn (1905) set forth the hypothesis that the retina exerts an inductive effect on the upper iris, probably by releasuig an fnducti\*e substance mto the posterior chamber In the norma) imdisturbcd eye a chemical substance emanating from the normal lens inactivates or in hibita this retina factor If the lens is removed the balance between the “retina factor and the Tensfactori is upset and the retina factor exerts its mfluence on the ms A number of ingenious expemnents by Wachs Sato and others have confirmed this hypothesis (reviews in Wachs 1919, jMangold 1931 Speraonn 1938)

Lens r^eneration in Anura has been reported m only a few instances it IS dcfinitdj Umited to larval stages Anurans should not be used for this experiment. Of the urodeles all European Tnfunts spedes and the Japanese Tr are capable of Wolffian regeneration throughout

life Data on Ambyslcmui are scarce. Ballard (1936) in a short note re ported positive results only for young larvae of A marulaiMm and A tigrtnum up to stage H43 m later larval stages these fonns as well as A mtcr&slomum A je^ersontanum, and A opccum showed no rcgcncra tk)n Stone and Dmnean (1940) found no Wolffian regeneration In A macHletHm in stage H46 and earlier

In our doss experiments r^cncralion was found to occur in A 0pccuw% nnd A maevAx/wm larvae of stage H46 and older But the number 0/ sue cessful cases was variable and nciTr exceeded 50 per cent. Likewise the rate of regeneration ^'a^icd great)} from 10 days to o\Tr 3 weeks

\\ c recommend using only j'oung larvae and making a considerable number of experiments It wdll be found that after some practice a large number of operations can be done in a short penod

Lm nrewnUwi occon reiulaily lo Tr (pefXHul cwmntfliiatioti et Wr D

Bodfmtoa)

176


ExypUMEyr 6i


MaJenol

fwimming lamie of Tr pyrrhogasltr or Tr torosm or A opccum or inaculaium stage H43 and older 6-10 speamens for each student t straight glass needle (Hg 40, a), which should be shorter and much stronger than a needle used for carl) embryos- It should not be dasbc and should taper mto a fine pomt m order to pierce the tough cornea.


Fit. 40 — xiu ItM reftoertlKio tf»opCTaUon {•« lert) i — nomal eje »itb

lou f-fUteprocc u of irgcTKnUoo from the upper n»- dotted hae* in b (iw lot) P E ^ F^tnent epiiirftun


Ins knife

duhes with Pennoplast bottom hflger bowls or Lily cups filter paper


insect pins

chloretone (i 3 000) or MS 222 (i 3 000) for narcosis a camera luada is desirable


Pnetdure

  • Prepare a groo^ In the Pennoplast dish in vt hich the larvTJ tits w ben

bTng on its left side

2 ^^sreotize 6-10 larv'ae

3 Futen a brva nght side up in the Pennoplast groo\-c using w^de

  • tnps of filter paper and Insect pins

4 Make a slit in the cornea abo\'e the lens slightlj longer than the Qiinictcrof the lens. Proceed as follows (Fig 40 a) Careful^ pierce the ^tnea With the needle and holding it honaintall} push it along the inner


surface of the akm and out again Carefufly avoid any injury to the cyx or lens Scrape gently with the ins kmfe against the stin over the needle until the skin is cut

5 Lift the lens out intact Work the tip of the needle carefuDj between the ms and the lens and use the needle as a lever Do not pierce the lens In a successful operation the entire spherical lens slicks to the needle and can be seen easily, although it Is glass-dear Avoid hemor rhages Discard specimens in which the retina Vim been injured and opera Uons m which you have not seen the intact lens after extirpation

6 Take a protocol

Repeat the operatwn on at least 6 anunals

7 During the following weeks feed the annuals frequentl> Make observations about twice a week. Narcotize the arumal each time. Observe the collapse of the pupd after the operation and its gradual reopening The details of the regenemtjon cannot be seen without sectfoning, but the width of the pupil gives an indication of the sisc of the regenerating lens. Make sketches. Observe the gradual clearing up of the cornea, which be came opaque after wound healing, and the gradual widening of the pupn

The structural changes which occur, meanwhile, in the upper his are fllustrated dlagrammatically in Figure 40, c-f

8 After3-4 weeks narcoUze an the animals and fix them in 10 per cent formaldehyde After a few minutes the lenses will become opaque and thus very disUnct Wash the anunals m water Mount them in the Per raoplastgroow and dissect and peel off the akin from over both eyres The left eye serves as a control Make camera ludda draidiiga of both eyes and lenses. For this purpose place the animal m the Pennoplast groove so that first the right lens and then the left lens is In exactly the same plane Calculate the ratio lens diameter cyr duuneter for both eyTS and obtain thus a quantitativT estimate of the degree of regeneration

If time permits and facilities are available, a number of eipenroental anfmals should be sacrificed at 5-^5 day-s after operation and sectioned to obtain early stages of regeneration However the results arc unpredict able (at least in Ambystema) on account of the high individuel \'anxition in the speed of regeneration

BIBLIOCRArm (1-3)

Bauaj® ^ 1936. ObKrvstkms on kni rt^eoeritioo in Amhlytioma BW. BaD

7J 38S (â– bjtrtct) -V

BAa/mtTTi D 1891 \ erwebe nir funktioodkn Anpossung AtcIl 1 mill amu

E, G I9J3 The cffecU of x-ndution 00 tie txpoertiion oi lie fortimb

ot AmUjrsiffma lime Joor Eiper Zo8l C5 271 178


GuHCEStHN S 1931 A unrr e EDtiri fJclnng dfr F-TtirmltatiTi nnH ^ rtritcn^fitrnrrng dcT Ltrvtnpcilodc von Trilen taemaius Lc>d. und Triton cristaius Lanr ArcL t Entw’medi-, las 343

iIi*'COiD 0 1931 Dt 3 DttomlnmtiorBproblent, HL Du UTrbelticaa^ m der Entwlcklan* and Regeaeration. Ergelm. d- BIoL, 7 193

bivnix, A. 1932- Hl3togeni*e ct r6gfa£r«tHU) du musde cfae* Ics tnourta. Ani. d. bioL,3a 37

1914. Rccbcrcho mr ITmtogenise el U r^fafrmticm chti ks bitixaens

iiwcres (cDfde domic et teguments) /hd 34 335

Saio,T 1930 Bdtrfge mr AiuUj’k der UoUTcchen LaHenregcnetalioa. L Ardu f Entw^mcdi. laa 451

SCHOriX, 0 E. 1939 Tlie ongm end moiphoccoetic potesaes 0/ regenentes.

GrOKth, SoppI p 59

Smuxx H 1905. Uber lixtsenbUdung nacb cxperimcnteller Entfemnng der pnndrcQ TJrueBhndangTOllefi. ZooL Ant., 381419.

1938. Embrjtmicdevefcpment end indaction. New Haven \ale Univtnity

Pits*.

SrcTfi, L. S end Ddotean F L. 1940 Eiperimentel ttndla on the rdaden of the optic voidc end cup to lens forroitioo In AmH}siorn 3 jutnOaint. Jem. Erper Zofl 83 95

TsiBurTua C S 1938 fhe hatogcoean of musde In the refencreting fordimb of hiTtl AmUyUoma fiMnOatuM Jour Morph., 6a 17

Wacej, H. 1914- \eae \enudie *oj Wolff’aehen linsojtgenmtion. Arch, f Eatw'oech 39 383

‘ “ 19x9. Zar Angts der UlrbdtMTe. \etnrwHS.

Tiaa. 705

Woss, P 1935 Die Mithcbe RegenerttioQ der DmddeaextremltlL Arch, f mihr Aott. CL Efltw’medi., 104 395

VToup G 1895 Entwlchlungsphyiiologijdic Stadien I Die Regeneretion der Uroddenlntje. Ardi. f Entw'mech. i 380

' 19®! EntwfcMtmpphytBdoguche Stodicn. H- Wotcre ^Gtteilixnges mr

fkl cnrri bo D der Urodelenlnue, Ibtd la 307


179



PART V


THE GRADIENT THEORY



A. GENERAL REAIARKS


The gradient theory of C M Ch 3 d is an ingenious attempt to interpret development and reconstitution under a uniform concept AU structural differentiations, m the embrjt) as well as m the regenerating adult bod> In asexual reproduction of mvertebrates os well as m the buds of plants arc considered as expressions of basic physiological actiMties which pre cede these structural differences These activities follow defimte grada lions of intensity the anterior end of an animal the animnl pole of an egg or the apical end of a growing bud usually show the highest metabolic activity and the decrement of Intensity usuall> follows the mam ( ami gradients ) Similar gradients maj bepresentmdorsoventraland m mcdiolateral directions. The existence of anal gradients has been demonstrated by Chfld and his assoontes b> se\’eml different methods Foremost among them is the method which mates use of the differential aiscqitibility’ of living tissues to tone agents If a plananan or a chick enhryo u placed in e lethal concentration of potassium cj-amde the beads disintegrate first, and the further breakdown of tissue proceeds along the anterior posterior ans (Tig 41) \ high degree of susceptibflitj u considered as an mdication of high physiological actl^^t> An> number of toxic agents can be used they ail tend to shois the same basic pattern physiological activity for a given stage Another method is that of differential reduction or ondation of vital d^'es The nature of the physrological activities of which susceptibflitj etc ^ indicators is obscure According to Child the underl>Tng chemical


may be different m different animals and plants and of a dif ferent nature even m the anterior postenor dorsoi’cntral and mediolat ^ gradient systems of the same organism Therefore Child uses the “oiiconuiuttal tenus axial gradients or activity gradients The implications of thi< theorj for the problem of detemunation arc far According to the gradient theory the fate of a gn en embryonic ^ crating group of cells is primarily detenmned by its rcIaU\ e posi m a gradient system For instance in a transvTise piece of a plananan wta of highest physiological actJ\^ty will gl^'c nse to head structures ^^the area of lowest phyTuological actmty will gne nse to tad struc the intermediate levels of activity will be correlated with trunk like pharynx (the relations of lev'cls of activity and structures course relaU\'c and not absolute I’alues) If m a ^■e^} short


  • 83


tnms\Trfle piece of a plananan, both the antenor and the posterior cut surfaces are approximately on the same metabolic then both Trill regenerate heads (p 170) The "head frequency experiment (Expt 64) has been designed to demonstrate that the relative positwn of a cut surface on the main axis plaj’s a role m the determination of the regenerat mg head High le\'els give nse to a higher percentage of normal heads than do postenor levels, and a quantitative relation can be established between the frequency of head abnormalities and the position of the regenerating surface on the antenor postenor axis

In a further elaboration of the gradient theory Child has Introduced the concepts of 'physiological dommance "subordmatlon,' "physiologi cal isolation, etc. and has reinterpreted organiser and inductor activities duplications individuatioa etc., in terms of the theorj His books (tee also the complete bibliography of Child s publications m Hyman and Van Cleave, 1938) are recommended for collateral reading Critical cvalu ationa of the theory will be found m Parker (1929), Needham (1931), and Spemann (1938) One of the major criticisms is that the measured differ entials of "physiologlaU activity may be the effects rather than the cause of local structural differentiations The difficulties mvolvtd In this con tro\*crsy lie m our ignorance of the chemical or physiological procooes underlying the ' metabolic gradients Apart from these cntiasms, the anal gradients must be given due consideration as important factors in the complex of conditions which deternune symmetry relaUons and struc tural differentiations of embryonic cells


184


B EXPERIMENTS

1 THE SUSCEPTIBILm GRADIENT IN PLANARIA (KCN)

(After Child)

The term * Misccptibfllty gradient has been discussed in the preceding section Planaria ha* been used widely for a demonstration of dismtegra tion gradients. The foUorsdng experiment was dodscd by Child (1913)

Exmiwx}rT63

llaUnol

Dugesta (P/ancna) dcrolocephala, 15-30 spcamens for each student small brush for handimg planarians

M/soo potassium cyanide (130 mg in i 000 cc. of spring or tap water) M/i 000 potassium cyanide (65 mg m 1 000 cc of spring or tap water) Note —The solutions must be prepared immediately before use. KCN IS poisonous. Be xtry careful to avoid spilUng It on the table or on the microscope


X JC, »r a if

rio 41 — SUgea of dubtesimllon of Phwtrte (*« tot) (frcun Aiiaai*, 1^41) Procedure

Fill one dish completely with the stronger solution another one with the weaker solution and place 8-15 animals b each dish Cover the dishes with glass plates excluding all air bubbles Observe the animals under the low power of the binocular microscope for a period of from i to several hours. Child distinguishes 5 stages of dismtegration (Fig 41) Tabulate jw results mdicatmg the tune required for the different itages Adams 185


(194O suggests assignmg the following arbitrary values to each stage I*»4o n'-30 III-«ao,rV— 10, V«o and calculating the total value for a given time of observation In the following way Multiplj the I’alue of a stage by the number of worms at that stage add these figures up and di \’ide the total by the number of worms used Make two graphs one for disintegration m M/500 KCN the other for disintegration in M/1,000 KCN Plot the time on the absassa and the disintegration values on the ordinate Compare the two disintegration cutvts

NoU — ^The time-required for complete disintegration vanes consider ably for different lots of matcnal, for different tcmpcniturcs etc It is ad iTsablc to run a test experiment with the solution to be used In the cla«  If necessary, start the dass experiment before the laboratory period be gins

Further suggationx — Dismtqpatlon experiments of the same type haiT been made on a large number of amroals and wdth a large number of toxic agents Further expenraents may be selected from the hteniturc which is listed in Child (1941, Appen in, p 734)

2. OXEDATiaV REDUCTION GRADIENTS IN THE CHICK EJIBRYO OANUS GREEN)

(After Rulon)

Physwlogictl axial gradients can be demonstrated by the differential reduction of vital dyes by Irving tissue For Instance the blue oxJdued form of methylene blue wUl be reduad to the cobrlcas Icuco form and Janus green will change to pink under conditions of low oxygen tension The decoloration proceeds m anterior posterior direction, thus indicating an axial gradient of differential cxj-i^n need If older stages arc used different organ prlmordm wUl stand out as areas of high oxygen requirement and complex patterns of differential reduction capaaty rather than simple gradients will become apparent (for details sec Rulon, 1935)


ExpzsuircKT


Ifatenal

chick embryos an> stage between 5 and so somites 4-5 cmbo'us for each student


hair loop

I pair of fine scissors watchmaker forceps scalpel

wide mouthed pipette mediane droppers heating plate


depression slides and cow glasses

melted \'ascllnc for scaling of co\cr glasses

watch glasses with cotton nests

(p 154)

filter paper


1S6


c 9 per cent sodium chlondc (Keep at 3S®-4o® C ovxr a Bunsen burner or a healing plate )

1 /50 000 oxidized Janus green made up m o 9 per cent salt solution

(Keep at 3S“-4o“ C )

Procedure

t Place an egg on a cotton nest break open the shell on top and ex pose the cnibr>*o without injuring it

2 Cut out the blastoderm at a considerable distance from the embr>*o using a pair of fine sassors Transfer the embrjx) to the depression slide with saline solution usmg a wide mouthed pipette CarefuUj a^X)ld anj Injury to the embrj'o

3 Flatten the blastoderm m the following wa> Cut out a nng of filter paper with a central hole of about the diameter of the area pclluada. Flattcn the blastoderm b> manipulating it with the hair loop withdraw most of the saline solution and place the nng on the flattened blastoderm so that the erabr^‘0 proper is cipo^ In this waj the embrjt) is pre%*cnted from rolling up

4 Add the Janus green solution immediatel) Stain the whole embrjT) for 8-20 minutes (limes are different for different stages) After it is thoroughlj stained blue- gr ee n withdraw the d)^ and wash the cmbrjTS with saline

5 Add salt solution coN*er the depression sbde and seal the cow glass with \-ascline

6 Return the embr)*o to the mcubalor or prtferablj place it on a heating plate Observe the gradual change of color to brilliant red Moke a senes of sketches of the color change indicating the gradient of ox)*gcn requirements Consult Rulon (1935) for all details for instance thestrue turns which change first in different stages times required for reduction etc


3 THE INFLUENCE OF THE AXIAL GRADIENT 0\

HEAD RErENTRATION IN rL.\NARLV (After Child and atanabc 1935)

HEAD rHEQUEXCV (hir )

Child has shown that m short trans\'ersc pieces of Planarta head re generation is not alwajT complete The head regenerates g'n be arranged In a graded senes ranging from normal heads to small outgrowths These were classified b> Child m frsx arbitiarj groups (sec Fig 4-’) I NonnaL 11 Teratopht hftlmi c (abnormal cjts) The shape of the head is almost normal but the ej*es show all d eg r ees of approxnnation and fusion to the 187


cut equan> Do not U5c the pharynx aa a landmari. If the pharynx i* extruded, remove it altogether

Repeat the expenraent with 10-14 other aimnala,

6 Tranafer all antenor one SLTth pieces to the dish labeled A all median pieces to dish B, and all postenor pieces to dish C. Tate a protocol Place the dishes m a cool dark place

7 After 2 days remove the dead fragments Do not feed

8 Study the regenerates 10-14 days after operation Inspect each lot separately Make sketches of representative specimens of the 5 head types If not all ore represented In your material, exchange speomens with jiiur neighbors Determine the numbers of the different head forms In each lot and tabulate the results m the following table Add the total figures for the class and plot them as a curve


I Compare head frequency In C-pieces with that in tall pieces of Ex periment 51 (p 167) The only variable hi the two expenraents is the length of the piece What condusion can be drawn?

a Compare head frequency in pieces A, B and C of the present ex penraent. The only variable is the body level WTiat conclusion can be drawn?

A DELAYED POSTERIOR SECTIONS (After Chnd and Watanabe)

Note — This experiment should be made together with Experiment 64 This experiment gives mdircct evidence that the formation of abnormal heads m short trans\Trsc pieces Is controlled by an inhibiting agent which is released at the postenor cut surface shortly after the cut is made It Is assumed that this inhibitmg agent UuxtIs m posterwr anterior direction along the \Tntral nenT cords and exerts its influence directly on the head blastema


190




Experiment 51 (p 167) has shown that level c forms normal heads provided that no other cut is made postenor to c If such a cut is made m d (EipL 64) then abnormal heads appear the head frequency is lowered It is pos^lc to detcnmne approximately how much time is re quired for the postenor depressing* agent to reach the head blastema and for how long a period the head blastema is susceptible to this inhibit mg influence This is done by delaymg the postenor cut d for a senes of tune mtcrvals It was found that the inhibitor is effective only withm the first 8-12 hours after onset of the head regeneration in c If the postenor cut is delayed longer then the head frequency is near 100 per cent

EXFERniMT 65

Ifaicnal

Dugcsia {PI ) dontocepkda or D ttinna 15-30 specimens for each stu dent (Select mdividuals of unifonn sue ) other material as in Experiment 64 Procedure

I Cut all specimens m the c-le\el (FTg 38 0) and discard all anterior pieces

3 Make cuts m level d

a) m 5-10 specimens 3 hours after cut e

b) m 5-10 specimctis 8-14 hours after cut c

c) m 5-10 specimens 24 hours after cut c

Discard the postenor pieces Keep the 3 lots m different dishes and handle the material as in Eipcnraent 64

3 After 2 weeks calculate the head frequencj for the 3 senes as in Experiment 64 Tabulate and plot the results on graph paper Compare your curves with those of Child and W atanabc (1935) for D dorofocephala and with Watanabe (1935) for D lignma Note that these authors wurked with one-eighth and one tenth pieces. Also temperature etc ha\e an effect on the results


BIBLIOGRAPHY ( 1 - 4 )

Adami, a. E. 1941 Studies In eipcrimoital zoology Ann Arbor Mich Ed»irdi Bros.

Castie ^ A 1940 hlctbods for cNiJuztloo of head !>!)«* In pl»n«rl«ni Phjnfcii. Zool 13 309

Child C. M rorj Studies oa the djTuuaia of morphogenesis, and Inhentaocc m erpenmenUl reproduclloo \T The nature of the axial grtdlenti in PtanaHa and their relation to anteropostenor doeninanct, polant\ and i)ininelr\ Arch- f Enta mcdi 37 io 3

1915a. Indh-iduahtj b organisms. Chicago Lmmritj of Chicago Pros.

  • 915^ Sotictncc and rtjurenescence. Chicago Unl\ cm tv of Chicago Press


191


Hrmp CiL i9>i Tbe on|;ia and development of the Dervotaa>’stem£ram apIi}iIolocJaU viewpoint Chicago Univernty of Chicago Pres*.

1914. PhjiiologicalfoandatKJca of behavior h.ew\ork Holt

1941 Pattena and problemi of da?t!opm«it Chicago Unfverrity of Chi

cago Preaa.

Cmu C hL and \\AtAXABE, Y 1935 The head fiequeno' CraiUent in fv/fonaHtf ioroioctpkala PfayiloL ZobL, B i

IhiUM L.R and\AKCiZA\’z,C.D 1938 Annotated b{bUograpb> of the scientific pobllcatioiB of Profcasor Chariei iftnning Child, Fh)iIoL ZotJ. iz>io5

Ne£ 33HAJU,J 1931 Chemical einbr3'o2ogy VoL X Cambridge, England Cambridge Univeraity Press.

Pajuox G IL 1919. The metab^c gradient and ita apphcatjons. Brit Jour Expo* BIoL, 6 41a

Ruxon O 1935 iHfrercntlal reduction of Janos grtoi during development of the chick. Protoplaama 24:346

SratAWM H. 1938, Embryonic development and induction. New Haven Yale Unlvenlty Press.

\\ATAHAB£,\ 1935 Head frequencv ID UModa/a ifl rtlatioD to the nov ous i>’stem PbyiioL ZobL 81374.


APPENDIX PLAN FOR A ONE SEMESTER COURSE


As This stated in the Introduction the experiments arc not arranged m a definite sequence in which they should be scheduled No expenment or technique Is based on anj expenence gamed m a preceding experiment The choice and arrangement of the experiments is left to the discretion of the instructor To facflitatc the organization of a one semester course, we gi\*e the schedule which we foOowed approximate!) but with man) ranations from )*ear to ) ear Normal devtkjpmtQt of or Jtima

Dcvdopmcit of beha\ior (Ejpt. ao)

Development of embr\ oa Lo oarcems (Ezpt. 41)

Mul sUinIn( of amphibun embrios (sdcctioiu from Expo. i-S according to stages availible)

Balancer and Gmb Iramplaiititioas (Eipt 14-16)

Artificial parthenosenesis (Expt o)

QaTige under preutue (Eipt 10)

Depliabocts b> laversloa of cgp (EipL 11)

Partial Emb and ey e extupanona (sekedoo from Expta. i8-ai and jo-j i)

Production of double hearts (Eipt. sa)

Lens IndncOon (selecdan frtim Expti. 3 >-j 4 )

PirabKab (Expt 37)

Tan and limb regeneration m tmphJbUn larvae (EipU 5p-6o)

Lens r egeneration in salamander larvwe (Erpt 61)

Regeneratioti m plaoanans (selection from Eipts. 51-sS)

Axial gradients and bead regenerabon (Expt 64)

Sosceptibllit) gradient In planaxlans (Expt 61)

Mtal itafnlng of the cbIcL blastoderm (Expt 41-44}

Chorio-aBantoic grafts In the chIcL (ElxpL 43-46)

TransphmtatioQ of p rosp e eb t e meUcophores In the chick (Expt 50)

Emergenaes cannot be entirely aNxiided m a labonitorj course which uses hving embr^’ornc material ciclusivel) They can be met eas3y b) using fixed material of normal embryos and laiTp-ae for dissections with a ^as» needle Also hiing plananans can be supplied b) the large suppl) houses almost all ) ear around

In the following list the experiments arc arranged In three groups ac cording to the still required to perform them

Experiments 5-10 ix 13 i8-xi 33 a9.34»37-4r 51-60,63 64,63 iffirt difficuU biti tvUabU as chssroom txpmmKTUs Eipenmenta 1-4, 14, 15 17 33 30:33 40-46, 50, 61 63

Jy\^ctdt rttpssnnt ccnsidtrabU aftnntct Experiments ii 16 33 36 47-49


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