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6@z

Freg Embrye,s
secupper left-hand corner of the slide,and successive tions are mounted in the following way (the numbers only;the actualnumshownarefor illustrativepurposes in bersof sections eachrow,and the number of rowsper slide,vary):

A. INTRODUCTION
Amphibian eggsare relatively large and canbe readily obtained. Many outstandingexperimentalembryRossHarrison, and ologists(including Hans Spemann, of severalstudents)took advantage thesefacts their when they beganto experimenton developingvertebrate embryosby removing parts,addingparts,and recombiningparts by microsurgery.Becauseso much of involvedexperiments embryology experimental classical that it amphibianembryos, is essential you have on early of the structureof theseembryosand an understanding how this sffuctureoriginatedin order to appreciateexThe study of early amphibiandeperimental analyses. when comparedto that of other organisms velopment, coveredin this laboratory guide and atlas'will also demonstratethe basic similaritiesin developmental amongdifferent vertebratessuch eventsand processes and betweenthese birds,and mammals, asamphibians, and invertebratessuch as seaurchins. For vertebrates hands-onstudiesusing frog embryos,see Chapter 6 (Exercises 2.1-2.4).

910

1.1. 12 13 14 15 16 17 18 19 20 21. 22 23 24 25 26 27 28 29 30 to If there aretoo many sections mount on one slide,the more posterior onesare mounted on slide #2 of the series in the following way:

31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
and so forth.

exOne other type of serialsectionwill be encountered the tensivelyin your laboratory studies: sagittalsection. cuts parallel to the long A set of serial sagittalsections axis of the embryo. A midsaginalsectionis cut exactly down the midline of the embryo,whereasa parasagittal sectionis cut either to the right or left of the midline. expass SECTIONS Thussagittalsections throughthe dorsal-ventral B. HOW TO USESERIAL tent of the embryo,either to the pightor left of the midin Laboratorywork in introductory courses develop- line or on the midline. mentalbiology and embryologyusuallyincludesstudies ers Fig. 2.1b illustrates four representativetransv e secyou anatomy.Suppose obtainedsome of developmental after they tions. Fig.2.l-cillustratesthesesamesections preservedfrog embryosat the stageillustrated by Fig. were transferredto a glassslide and mounted from left 2.La andwantedto study their anatomy.With a microto right in the order in which they werecut. Exactlyhow you could identify a few poorly defined external scope, much of the anatomyof the embryo can one expectto dorsal(cranial-caudal, featuresaswell asthe body axes that section?Suppose your seein anyonerepresentative t'entral,andright-left). To study internal featuresin deset of serial sectionscontained a total of 100 sections. tail,you could slice(section)the entire embryointo thin The sixth sectionof the set might cut through the level sectionsof a given thickness;sectionscut perpendicu- of the developingeyes; this sectionyou could deterin axisof the embryo are called larly to the cranial-caudal mine the relationshipof the eyesto other structures tansverse(cross)sections.A slide or a collectionof You might then examinea more pos(Figs. 2.1b,2.1c). sectionfrom the first slidescontainingeverytransverse throughthe heart,orstill more suchas#1-5 terior section, one (the mostanteriorone)to the lastone (the mostpos- posterior sections(#50,#80). Unfortunately,the study T}:re sections. rcrior one)is calleda setof serial transverse individual sectionsprovidesonly a two-dimensional (#1) is mounted at the of most anterior sectionof this set picture of the embryo. To understandthe anatomy of

26

Chapter 2 by providing three types of visual aids in this laboratory manual: (1) line drawings (called "Figs."); (2) section "orientators," placed on most photo legends (these orientators show the exact levels where embryos were sectioned, sliced, or cryofractured); and (3) scanning electron micrographs, which portray a more three-dimensional image than do flat, two-dimensional serial sections. Methods have been included in Chapter 6 (Advanced Hands-on Studies) to help you understand how embryos are prepared for light microscopy, scanning electron microscopy, immunocytochemistry, and in situ hybridization, as well as to help you understand why different processing procedures result in different types of images.

the embryo inthree dimensionsyou must visualizeeach section as again part of the whole embryo. For example, the notochord can be identified in sections #15,#50, and #80. By connecting the section of the notochord at the level of section #15 with the section of the notochord at the next level (section #50) and those at successive levels, you get an accurate picture of the craniocaudal extent of the notochord, as well as its relationship to other structures. In the same way, you can get an accurate picture of the craniocaudal extent of the neural tube (brain and spinal cord) and digestive tube, as well as their relationship to each other and to other structures. The most dfficult task facing the beginning student is to learn to visualize relationships of parts of an embryo to one another in three dimensions. We have attempted to help you with this difficult task

cranial

caudal

sectionnumbers

ventral

cranial

caudal

EYE

BRAIN

(NEURALTUBE)

SPINALCORD

anterior

posterior

sectionnumbers
HEART DIGESTIVETUBE

Fig. 2.1. Drawingsillustratingthe relationships betweena preserved 4-mm frog embryoviewedfrom the left side(Fig.Z.Ia) andfour representative transverse sections throughthis embryo (Figs. 2.1b,2.1c).Sectionnumbers are for example only,

FrogEmbryos 27

C. O O G EN E S I S D AN FERTILIZATION
Oryenesis (development of the ovum) begins in thd :i.1.-:;dor-aries of the mature female frog. During the rricoJins season(which begins in the spring), each ovary :r-csxts of a sac containing a cluster of spherical struc*;.s r-alled follicles (F19.2.2). Each follicle consists of r -::ge central cell containing a lot of yolk in its cyto:L:s:r. the primary oocyte, surrounded by a layer of n,;h >maller, flattened cells called foilicle cells. The :r::rn ooclte contains a large nucleus, the germinal ,nqritCe. The vitelline membrane lies between the folli: e :,eLisand the plasmalemma of the primary oocyte. r- ::;r sheath ofconnective tissue,the thecafolliculi exmcrnraforms the surface layer of the ovary. Another rir::ih. the theca folliculi interna, partially surrounds r r':i lollicle but is lacking in the region where the folllj:: J{,rntactsthe theca folliculi externa; at this region e,muftrtion (the rupture of the follicle and release of its :,-ri::.ined oocyte) occurs. :,t:: qlr-3ryalso contains cells called oogonia. These :r*. '.mdergorapid mitotic divisions, increasing in numT'i'i -{ller the breeding season is completed (that is, in .[T.: iiumn). a few thousand oogonia within each ovary ,:5- --he ability to divide mitotically. Each enlarges ,,Lir.:--r'as a primary ooclte and becomes surrounded "r:,r lmgle layer of follicle cells, forming an ovarian l. frultrhde. s'hich slowly enlarges due to the accumulation t' "'- These primary ooqtes enter the prophase stage '-r. r: :-:; ftrst meiotic division but remain there until the

followingspring. Fully grown primary oocytesundergoovulation in responseto hormonessecretedby the anterior pituitary gland (adenohypophysis). Each oocyte is slowly squeezed throughthe follicular wallat the regionwhere the thecafolliculi interna is lacking (Fig.2.2) andenters the body cavity (coelom) of the female. Many oocytes (2,000-20,000, dependingupon the species) or,ulatare ed by a singlefemaleeachbreedingseason.primary oocytescompletethe first meiotic division during ovu_ lation, with each forming a first polar body and secondary oocy'te.Both of thesestructuresare contained within the vitelline membraneformed earlier,while the primary oocytewasin the ovary. Cilia on the lining of the coelom beat toward the ostium of the oviduct and propel the secondary oocytes into this opening.The second meiotic division is initiated by each secondary oocyteat about the time that it entersthe oviduct,but it then arrests the metaphase in stage. the secondary As oocytes passthrough the oviduct,a multilayered, gelati_ nous egg capsuleis secretedoutside of the vitelline membraneby the cellslining the oviduct. 'Fertilization occursexternallyasthe secondary oocytes are spawned(shed)by the female into the water. The secondmeiotic division is completed as a sperm con_ tactsandpenetrates eachsecondary oocyte, resultingin the formation of a secondpolar hody and a mature ovum containingthe female pronucleus. The nucleus' of the penetratingsperm enlargeswithin the ovum as the male pronucleus,and the male and female pronu_ clei unite to completethe processof fertilization.

yolk-laden cytopl
germinal vesicle of

ot pnmary oocyte vitelline membrane follicle cells

prrmaryoocyte

thecafolliculi externa
\--/-----J

thecafolliculi interna

site of ovulation

Eimi

.}

-)

Schematic drawing of a section through three ovarian follicles of a mature female frog.

,n:!!'-'
28 Chapter 2
hemisphere. Following contact by and entrance of the sperm, the pigmented cortex sffis relative to the lesspigmented deep_ er portion of the egg,toward and past the site of sperm entrance and away from the side of the egg opposite the sperm entrance point (Fig.2.3b). This reduces the pig_ mentation of a crescent-shaped area opposite the point of sperm entrance. This crescent-shaped area between the heavily pigmented cortex above ind the essentially nonpigmented cortex below constitutes the gray cres, cent (Figs. 2.3b,2.3c). A plane passing rhrough the animal and vegetal poles and through the center of the gray crescent corresponds to the midsagittal plane of the future embryo (Fig. 2.3c).

D. FORMATION THEGRAY OF CRES.CENT


The outer portion of the egg (the cortex) contains a dis_ tinct pattern of pigmentation at the time of ovulation (Fig.2.3a). About two-thirds of the cortex is heavily pig_ mented;the remainder contains almost no pigment. The uppermost part of the pigmented portion is the animal pole. This pole corresponds to the cranial end of the fu_ ture embryo. The half of the egg that contains the an_ imal pole is the animal hemisphere. The vegetal pole lies directly opposite the animal pole. This pole corre_ sponds tothe caudalend of the future embryo. The half of the egg that contains the vegetal pole is the vegetal

Ap

poi nt of sperm entrance direction of shift of pigmented corte

4p

midsagit t alplane of

future embryo

b gray crescent unfertilized egg (secondary oocytestage) VP fertilized egg (right side view)
gray crescent

VP fertilized egg (dorsalview)

4 micromeres

gray crescent VP 2-cellstage (dorsal-rightside view) VP 4-cellstage (dorsal-right sideview)

gray crescent

4 macromeres

VP 8-cellstage (dorsal-right sideview)

dorsal
blastoporal
ltp I

yolk plug VP blastulastage (dorsal-right sideview) VP blast dorsallip gastrula stage (dorsalview) yolk piug gastrulastage (caudalview)

Fig. 2.3. Drawingsof early developmental stages the frog. Ap, animalpole;D, aorrd sa"JJ"t of right side;V,ventral side;Vp,vegetalpole.

s0"3,

Frog Embryos 29

'il ti

E- CTEAVACE AND MLASTUTATION


til$unr,rte consists of a series of rapid mitotic divisions ltlriti: :-suh in blastulation (that is, the formation of a IttlMr- rvhich consists of a group of cells called blassurrounding a cavity, the blastocoel). Cleavage llimryrs pass through the entire egg, so cleavage is clai_ n,nil*: .s total (holoblastic). lloweve4 furrows pass tmr'trr; rhe vegetal hemisphere much more slowly than rt[Tn:r,:.h the animal hemisphere, presumably because rITi.:t:t:rer contains far more yolk than the latter. A 1lr ln(r :fotetable of the first three cleavagesfor the frog, fl*mnu ::rrens, is as follows (Hgs.2.3d-2.3f):(1) first cleav_ .'$t :eridional (that is, passesthrough both the ani_ urtu. 'l.i r-egetal poles), usually bisecting the gray ;n*s::nr (rhat is, passes through the midsagittal plane nr .:; :urure embryo), 2.5 hours postfertilization; (2) rrcr;-Edcleavage:meridional, at right angles to the first, I 5 -':ur: postfertilization; and (3) third cleavage:hor_ nr;ni;* displaced toward the animal pole due to the yolk r:mr:ir of the vegetal hemisphere,producing four small_ rr r-rrnai hemisphere cells, the micromeres, and four . lii{'r.: \ egetal hemisphere cells,the macromeres,4 hours prur:r-nilization. The egg reachesthe blastula stage near llrs ::d of cleavage (approximately 16 hours postfer_ urLi,ru.:itrn I (Fig. 2.3g). Numerous small blastomeres oc_ ;ur", rhe animal hemisphere, whereas the vegetal n*rr:sahere consists of a lesser number of larger blas_ ilme:c! The blastula is only slightly larger at the end rr :i.arage than is the newly fertilized egg. ;:r;:rne models, or preferably living eggs (see Chapter n. =u;rcise 2.1), showing early cleavagestages.Note the tlilir$irtr-rn early cleavage furrows (that is, whether of mrn:lt-rnal or horizontal), the difference in size of the Tm{":r tnneresand macromeres, and the difference in pig_ meL:rrlion of animal and vegetal hemisphere cells. Tiy :iLr i[ntifi' the gray creseent;the region of the gray cres_ i$rf,: :hat is broadest will become the dorsal surface of :lLre ;unbn'o. and the opposite side of the egg will be_ r:m; rhe ventral surface. :Tal-ne a section of the frog blastula that closely reln*r:ies Photo 2.1. Identify the main cavity, the blasmcffil- \ore that it is displaced toward one side of the rl;-N$mLla. animal pole; thus, it is contained within the nrrimel hemisphere. The blastocoel is filled with a 1lLT.c 1[n.ur,: may have coagulated during preparation of ;frs1 :,';: :nid. Note that the wall of the blastula is com_ " tr:u.;i of distinct cells, the blastomeres. Identily their mdei Spacesbetween blastomeresin the vegetal hemi@re are shrinkage spacesproduced during prepara_ tr:,n ,r; r'our slides. :a,i-ine the animal hemisphere. It has the following ;miir'neristics: (1) there is a heavily pigmented cortex, M.rr:trsrnentation being most intense at the animal pole lmc ;:ading off progressively toward the vegetal pole;

(2) it is four or five cells thick, with smaller cells and fewer cell layers at the animal pole and with a pro_ gressive increasein cell sizeand number of cell layers toward the vegetalpole;and (3) the blastomeres con_ tain very little yolk. Examine the vegetalhemisphere.Its characteristics are exactlythe opposite:(1) a pigmentedcortex,if present at all, is much lessevident than in the animal hemi_ sphere;(Z) the blastomeresare very large and few in number,indicative lessfrequentcleavage;and the of (3) blastomeres packedwith yolk. are Try to identify the gray crescent.In your sectionsit usually lies either to the left or right side of the blastocoel and alsoslightly ventral to it. It hasthe following char_ acteristics (comparesidesindicatedby lettersD andV in Photo 2.1): (1) the pigmentedcortex is thinner in the gray crescent than on the oppositeside;and(2) the blastocoellies nearer the surfaceon the grav crescent side than on the oppositeside (thar is, th"ewall of the blastulais thinner on the gray crescentside than on the opposite side). The blastulaconsists a mosaicof cellular areas, of each of which will normally producea certainstructuredur_ ing subsequent development.In other words,eacharea of cellshas a certain prospectivefate that will be real_ ized during normal development.In blastulaeof some chordates(Urochordata or tunicates) the outlines of thesecellular areascanbe determineddirectly because the cytoplasm cellswithin certainareas coloreddif_ of is ferently. But in most cases is necessary determine it to the prospectivefate of eachcellular area indirectlv bv marking experiments.Vital dyes0have been usedmost frequently for this purposein amphibians.Severalareas of the blastulaare stainedwith a vital dye,andthe struc_ ture or structuresthat are formed from each stained area are observed (see Chapter 6, Exercise 2.2\. Another techniquehasbeenusedmore recently.A cell marker (for example,the enzymehorseradishperoxi_ dase, which canbe demonstrated histochemicallv in_ bv cubating tissue containing the enzyme wittr -ttre appropriate substrate;or fluorescein- or rhodaminelabeleddextran,which can be demonstrated with a flu_ orescence microscopeafter illumination with the proper wavelengthof light) is injected into a singlecelf or groups of cells at the blastula stage. As injected cells cleave, marker is passed their descendants. the to A prospective fate map is constructed with the aid of the information gainedby thesetechniques.The amphib_ ian fate map shouldbe carefully comparedwith the ones for the chick (Fig.3.7),mouse(Fig.4.2),andseaurchin (Fig.1.1).A prospective fate map indicates location the of specificgroupsof cells prior to the onset of gastrulation. Thesegroups of cells are shifted in an olderly way into appropriate positions during gastrulation, which will enablethem to cooperateand interactin for_ mation of tissues and organs.The blastocoel appears to

t'

i.
i'

{. 't: t

lt

30

2 Chaprer

to in be essential many species provide a spaceinto groupsof cellscan move eithet en masse which certain or individual$ during gastrulation'

the blastoporallips (seeChapter 6, Exercise2'3)' arbitrarily as The locationsof severalareas(designated areasl--10 and25-27)before and during their involuare tion over the dorsal and ventral blastoporal lips on the shownin Fig. 2.5. Allof theseareasare located surfaceat the blastulastage.Areas 1-5haveundergone lip involution over the dorsqlblastoporal by the dorsal gasffulastage. Similarly,areas1-8 have.undergone lip yolk inuitrrriott ou"i th" dorsal blastoporallip by the and areas2l and26haveundergone plug gastrulastage, over the ventral blastoporal lip' The reitru6tlntiott maining numbered areaswill undergo involution durdevelopment' O1!:t cellular areas ing subsequent in ,rrid"rgo involution over the lateralblastoporallips a similar manner. areas' the Fig.2.4shows locationsof severalprospective soire of which undergoinvolution during gastrulation' Someof the cells of the prospectiveendodermand all the cells of the prospective head mesenchlme and notochord involute over the dorsalblastoprospective porui tip. Similarly,some of the cells of the prospeciive endoderm, some of the cells of the prospective Fig. 2.a, Prospectivefate map of the frog blastula' and all the cellsof the prospec' lateral plate mesoderm, site of blastoporeformation is inTh"eapproximate tive sefrnental plate mesoderminvolute over the laterdashedline' the dicatedby a circular a/ lips"of the blastopore. The remaining cells of aswell as someof ectoderm(epidermis) l. Prospective fro.p""ti"" lateral plate mesoderm' neural Plate of the prospectiveendoderm,involute over the ifr" 2. ProsPective ""Ut Only a relatively small number notochord ventralblastop-oral1ip. 3. ProsPective 'prospective endodermal cells undergo involution segmentalplate mesoderm of 4. Prospective endodermal lips. Most prospective over ttreUlastoporal 5. Prospectivelateral plate mesoderm relativeiy stationary during gastrulation head mesenchYme cells remain 6. ProsPective endoderm forming the yolk plug and floor of the archen' stages 7. ProsPective neur' involute,the prospective (Fig.2.Sj.As areas ter6n ectoderm(epidermis)undergo at plate aiO prospective F. GASTRULATION toward the blastoporeto replace oi spieading, "piboly, aieasthat havemoved into the interior of the gastrula' During gastrulationsomeof the cells originally locatof eA on itt-esurfaceof the blastulaturn inward or underObtain a slide containingsagittalsections the dorsal go involution to move into the interior' Thesecellswill and selecta sectioncloselyresemblingPhoto lip gastrula or innermost live rise to two germ layers:the endoderm, Z'21 ldentrfy the blastopore' dorsal blastoporal lip' and or the mesoderm, middle layer;cellsthat reiaver.and blastocoel. The blastoporerepresentsthe future causurfaceform the outermost germ layer' ott the dal end. The future cranial end lies directly opposite' the -uirrectoderm. A depression, blastopore,beginsto the The blastoporeopensinto a narrow cavity,the primias just below the gray crescent cellsinitiate invoform tive gut, oiarchenteron. The floor of the archenteron the a Z.3h).Simultaneously,liplike structure, tution @'ig. is foimed by yolk-filled endodermalcells' The cranial dorsalLhstoporal lip, forms just abovethe blastopore' end of the archenteronroof is formed from endoderm With formati,onof the blastoporeand dorsal blastopothe because first cells to involute over the dorsal and blastulais transformedinto a gastmla' Cells ral lip, the blastoporallips,and thus to contribute to the wall lateral contiiue to involute over the dorsalblastoporallip with are of the archenteron, the cells of the prospectiveen' ocand involution progressively further development, (Fig.Z.q- The remainder of the archenteron doderm curs laterally and ultimately ventrally as well' This reis stage formed by mesoderm'which roof at thi gastrula sultsin formation of a circular blastoporecontaininga the dorsal and lateral blastoporallips folinvolutes over cellscalledthe yolk plug massof yolk-filled endodermal endoderm'The mesolowing involution of prospective by is surrounded con@ig.2.3i).The circularblastopore archenteronroof vill' later be coveredby derm"of the iioiorrt dorsal,tateral,and ventralblastoporallips' The endoderm,which migratesupward over the inner surcan movements be alteredexdirectionsof gastrulation face of the mesoderm(seebelow)' Thus the archenperimentally,iesulting in exogastrulation,a processdurteron is ultimately lined entirely by endoderm' Note ine which surfacecells move but fail to involute over of that the ectodermin the frog consists two layers:an

Frog Embryos 31

blastocoel
,:-t--E
r"-f "

archenteron dorsal
blastoporal

tr lip

dorsal blastoporal

rif,

blastopore
blastocoel

blastopore yolk plug

aichenteron

blastopore

\?
:i:siu1a Stage a
*il t''

-siteof dorsal blastoporal lip Iormatron

blastocoel dorsal lip gastrulastage b

ventral
blastoporal
llp

yolk plug gastrulastage


v

l-5'

l ltrnal side; V ventral side; VP, vegetal pole; numbers 1-10 indicate specific areas of the blastula that un;11i1gr'p ' -'- 'rfution over the dorsal blastoporal lip; numbers 25-27 indicate specific areas that undergo involuti
,Il llu

Drawingsof the cut surfaces right halvesof blastula and gastrulastagesof tn" of

rrog[

*i.rr.ot

ffi ;fi;;:",:":il#;:llil:"1l]o'Iu'[lon

nmm ryerficial) ectodermal layer and an inner (deep) uumdennal laver. " lirli :T i slde containing sagittal sections of a yolk plug iltlllttfir-*: and select a section closely resembling photo : : ...r=niri_tr yolk plug, a protruding mass of yolk_ the :L^rur .:-..jodermal cells located between the very promi_ urlnrurr ilursal blastoporal lip above and the less prominent qruilmdblastoporal lip below. Also identify the archenrfunlra- -ra-p1gd above the mass of poorly defined endo_ ruLi*tr,& ;ells forming its floor. The yolk plug fills the *rmnrr:;,-to the archenteron, the blastopore. Beneath Hiu r--;:Lenteronand separated from the latter by en_ jrhrurir::,al cells is the irregularly shaped blastocoel. This ilir,iilirirrilater squeezed out of existence. Note that the -i n'ululr::ed cortex now covers all cells except those of lie ' .-ft plug. The more rapidly dividing cells of the nmry*ttire ectoderm (epidermis) and prospective neuru dllire undergo epiboly between the blastula stage and ItrrL,s -ijranced gastrula stage, growing down over the dk-:l-ied cells of the vegetal hemisphere. Meanwhilq ',r 'iliru ,r-Lrri endodermal cells of the vegetal hemisphere 'r',: iJnken inward to some extent and become re_ lur:lrr-l;d to form the floor and ventrolateral walls oi trrrrr .L:;henteron. Most of the archenteron roof still con_ mn; : i mesoderm at the yolk plug gastrula stage. ilirr-i. :,.'rmparisonof gastrulation in vertebrates and ineirL.r.atel see Chapter 1, Section H.

dorsallip of the blastopore, which sendtheir induction signal through the horizontal plane of the ectoderm. The initial signalis later reinforcedby mesodermal cells within the roof of the archenteron, which sendtheir in_ duction signalverticallyto the overlyingectoderm.The gastrulais transformedinto the neurulawith formation of the neural plate. Obtain a slide containingtransverse sectionsof a neu_ rula at the neural plate stage. Selecta sectionclosely resembling Photos2.4,2.5 identi{y the neuralptate. and The roof of the archenteron at the neurula stage is formed by endoderm. Between the gastrulaand neu_ rula stages, endodermmigratedupward over the inner surfaceof the mesodermthat previouslyformed the archenteron roof. Note that the dorsalmesodermis now organizedinto a midline rod of cells,the notochord, flankedby two bandsof cellqthe segmental platemesoderm. Thislatter mesoderm the sourceof the somites. is The segmentalplate mesodermgradually mergeslat_ erally with the lateral plate mesoderm. The coelom forms within the lateral plate mesoderm. Obtain a slide containingtransverse sectionsof a neurula at the neural fold stageand selecta sectionclose_ ly resembling Photos 2.6,2.7.Identify pairedneural the folds, which have formed at the lateral marsins of the neural plate,and the neural groove.The notochord,segmental plate mesoderm,and lateral plate mesodermare more clearly defined at this stage,and the endoderm of the roof of the archenteronexistsas a more distinct layer. During subsequent development neuralfolds the will fusein the dorsalmidline to closethe neuralsroove and thus establishthe neural tube. Neurai creJt cells will later formfrom the roof of the neuraltube and give rise to a multitude of structures, includingpigmentcells (seeChapter 6, Exercise2.4).

6- NEURUTATION
-hrr :lodermal cellsdirectly overlyirigthe archenteron "ir'1rr- rhe yolk plug gastrulastageconstitutethe neur19 n unmoderm. neural ectodermis inducedto thickThe :n ,u :he neural plate. Induction of the neural plate *= r,,o'f s'ith prospective mesodermal cellswithin the

32

Chapter2

H.
1. lntroduction at this th.e Fig.Z.rashowsthe general shape of _b-ody in a mid,t and Fig.Z.O o*, ttt" ,t*itor", visible stage, yourself withthe spatial re sagittal section. Familiarize 'uctures Detoreti^drruu - ;"'iut lationshipsof thesestructuiesbefore "*umioi"g transversesections. 2. Serial transverse sections tiew"A tlrougtr the microscope, " n:F' u" rn PhotJ 2.8. Do not placemicroscope 'l'4t, "J to on slides hold them in plac" ^ '!:::::': fi ir"i*a 'iir*rnt sein slides anteroposterror r"rtions. E'xamine otherwise (see Chapter z' unless directed A;;;;. dection B).

ep' corneal the d.ucedby lensto form the transparent cut optic cups'sections ffi;t""i. At the level of the with (veniral$) continuous ;;;;;;" ;tosencephalon

(dorsatly)' the rnesencephalon parts assections The brain constrictsinto two separate 2J0)' The ventral part is are tracJ po1t"t-t1V Gtrofl posterior pituitary the infundi:butum,the source of the The dorsal part is the gr""a t"""t"nvpophysis)' and mye' ir'o*n"*"prtoron ittt" tut*" *"tencephalon a solid ectodermal turr""pt'uloi oittre'Urain;' Identify rodaiaboutthislevel'justventraltotheinfundibulum' .t the anterior pituitary t"Oi..* from the invagination, stomodeum' ilffi;dermal (SeePhotos2'L0' originatedas an outgrowh' ;ffi; rudiment i .i-i"r ,rt" iocation of the stomodeum' The and the stomodeumare pituitary gland ;irh" ;;fu uia'section 6vel betweenthoseillustrated "orrtirrroo. Gi"ni i1U, the adiesive of the stomodeum and extending through ;i;;;rtd" many sections.

Positionyoursrideonthemicroscopestageso'l'llTl *Ll;yi';*ffiiliJnT:T:xt#;'l'rlTf;continuous eachsection ottll gland is

;i::,X,,*?a?f)#ff-T3l,"",ffi:#iitrffffi: derivatives a.Ecrodermar the tip of the head' glands(ventralsuckers)'to


The first few sectionscut through (ry,.T"i"t""t"4*l: rdentify the rurg" pror"lnlph;d of the brain), which is-enclosedby and ailnceptralon

'*,':H1"3iffi::ft$i:::ff)'ff'ilffi[i:ffi ]""i:'hi:13;111:'il1iT":t':*Hi :" 3;:;1*H[::ff*"iil1"1"'ffi'li,T:['Jii?.i:i*f#L,T;1*=*:*';:


tfri"t"t"J ment. Tiy to identify a region-ot 'ftit side ventral to ttr" proseric*til ectodermon each just u""tt,,'"tuii*of ectodermalcells(neuralcrestcells) of ganglia eachof them' Theseare the semilunar above.

the (orract'"ffi:ffii::li are nasar thickenings form the tt"iltt::]"f rhese i*,*i:ru;t,t*Tffi|:'ffi' S^y#",::":);"1!{{{ will eventually The nasulplacodes on each side,an epibranchial placode' continuetotracesec -"itlu*, fi::Filetothesegangria) the paired auditory vesi:ilL:ffi:ii'"ilX'i?J#:l5if":#,ffy"'fi:ilffi1 ;;;;;;;t""0lolntitv - ti.on: of theseplacod; tJ""#;granulesat the periph".ry iliffi; *:'$;u""ttorut"*fto.therhombencephalon(Photoof the thickenings teristic of invaginating ectodermal z'ni'-rte"evesicles originatedfrom presumably,the peripheial ends ot inuugiiliffi:,Jil subsequent$invaginatlayer'-which ,nJ piE-"", into the '1i';""l"O"tt"f become narrowed, thus concentrating rtr" u"oitory vlsicles later differentiate "0. ears' the inner ends of the cens "riturg". content, whereas inner placodesand Young newo n nt", oiinate from these producetheaxonsoitfieoractory(I)cranialnerves'Continuetotlacesectionsposterior$andnotethatthe the level of ,r"rrrui,,rb" gradual$ 1T1:*., indicating as orfactory reas well as the dendrites that function Examine the spinalcord tft" .pi""i"oiJ Gno.o 2'L4)' of smell). Nasaiplaceptors (receptors;;; d;;;e *rtnrrig5magniiication. Noteitscharacteristicthinroof some embryos. codesmay not have yet developed.in its thick lateral walls' At about this fl;;;i;and which originated Identify the pineargland (epiphysis), "rd pigmented or nonpigmentedcells lying l"""il;;;ttfy from the p'o*"t'""phu1o"' as a dorsal and beneaththe skin ectoderm; "uugi'i^tio" ao"uito trt" tpi"i and identify the "ota cells (Photo 2'14)' As you apcontinue to trace sectionsposteriorly these are neural crest is derivedfrom optic cups(photo iil."r,^[n "ptic cup the dorsal in which p.ou"t it rirralsections your set'identify ^fi" a laterar evaginationfrom the prosencephalon, " rpi""r cord (Photos z'r5-2'18\' The loose #;;" forming a dousecondarilyiruug-ui"Jut it, ufirra end ble-layeredopticcup.Thethickenedlayeroftf,eopticcellsformingitscoreareneuralcrestcells,whichinduce cupisthesensory'"ti"";thethinlayeristhepigment'formationofthisstructure'Notethatthebodynarrows identify the ventral fin J prog;lu"iv to the prosen"u"oal,levels; ed retina. The optic cupsare connected cephalonbytheopticstalks..Notethattheinneruttr'""l.,aaiendofthebody(Photo2.1.8) .Identifythe skin ectoderm pro"roa""-, a ventral invagination of retina has ectodermal layer adjacent to the sensory the ventral fin (Photo anteriir to sections 2'10)' The it'"t'it thickened to form the lens placode@hoto "1"* "oui", in2'I7)' pr,g^""ted layer of the ectodermwill laterbe

Frog Embryos

33

I4
1a IJ

Fry. 2.6. Schematicdrawing of a midsagittalsectionof a 4-mm frog embryo. \otochord Subnotochordal rod .\rtus Hindgut Yolk-filled endodermalcells Lateral plate mesoderm Liver rudiment Pericardialcavity 9. Heart (ventricle) 10. Oral membrane I l. Stomodeum 12. Rudiment of the anterior pituitary gland 13. Infundibulum 14. Prosencephalon 15. Pinealgland 16. Head mesenchyme 17. Mesencephalon 18. Rhombencephalon 19. Pharynx 20. Midgut 21. Spinalcord 22. Neural crest cells 23. Dorsal fin

h. Endodermalderivatives
R.eturn to the level of the nasal placodes (photo 2.8) nd trace sections posteriorly. Identify a small cavity --,rnsbeneath the prosencephalon (Photo 2.10). This '. the cranial end of the foregut; its walls are formed :"rm endoderm. The stomodeum has invasinated toa ard the foregut, and the stomodeal ectodeim and the :",regut endoderm are in contact as the oral membrane. llris membrane later ruptttres to form the mouth openmg. The outer part of the mouth is therefore lined by stomodeal ectoderm, and the inner part by foregut en:oderm. llhe foregut expands as the pharynx as sections are :raced posteriorly (Photo 2.12). (In a few embryos the -ateral walls of the pharynx may contact the skin ecto:erm, which invaginates slightly to meet them. Such lo;alized pharyngeal expansions are the pharyngeal pouches.) Continue tracing sections posteriorly. The ioregut narrows and then forms a prominent ventral er-agination,the liver rudiment (Photo 2.13). The level of the foregut that is continuous ventrally with the liver rudiment is the duodenum. The liver rudiment separates from the duodenum and then fades out a few sec-

tions more posteriorly. The remaining portion of the gut is the midgut(Photos 2.14,2.15).It contains small a cavity boundeddorsallyby a thin layer of endodermal cellsand ventrally by a large massof yolk-filled endodermal cells.Continueto tracesections posteriorlyfollowing the midgut. It gradually moves ventrally and enlarges somewhatas the hindgut (Photo 2.16). (In someembryosthe endodermof the hindgut fuseswith the ectoderm the proctodeumin more posteriorsecof tions to form the cloacalmembrane.The cloacalmembrane ultimately rupturesto form the anus.)

c. Mesodermalderivatives
Return to the level where the infundibulum first appears (Photo 2.11) and trace sectionsposteriorly. Identify a group of mesodermalcells,the notochord, lying beneaththe rhombencephalon (photo 2.I2). The vacuolated conditionofthe notochordis apparentlyresponsible its rigidity,allowingthis structureto serve for as a longitudinal supporting rod in young embryos. Quickly trace sectionsposteriorly,noting the changes that the notochord undergoes.It is smaller at caudal levelsthan at more cranial levels,and vacuolizationis progressively reducedtoward the caudalend (compare

34

Chapter2

ventral to cus via a pair of blood vesselsthat will lie is The Photos2.1'4-1'.18). caudalend of the notochord can latter vessels the pharynx,the ventralaortae' These dethe iessdeu"lopedai this stagebecause notochord. The dorsal end .o*Ltit*. be identified at this stage') sequence'In postenor sectlons velopsin craniocaudal first aortic arch is continuous with a blood vesoi blocks laentify the somites(Photos Z'15,2'16)'paired "u"tt dorsolateralto the pharynx' the dorsal aorta sel lying AIso of mesodermlying ventrolateral to the spinalcord' aortaeextendcaudadandfade befii6r.V.nl. The dorsal identrfy u ,rnutl Juster of mesodermalcells lying i"i ut uU""t the level of the midgut (Photo 2'14)' neath ihe notochord in caudal regions' the subnoto' Its cfrorAaf rod (hypochord) (Photos 2'I5-2'I7)' of of 3. Summary the contributions the is unknown' " developmentalsignificance in present the ;;il +rs to.structures at which the liver rudiment is conReturn to the level 4-mm trog emnrYo sectinuouswith the duodenum(Photo 2'13) and trace to the iion, port"riorly. The mesodermventrolateral Ectoderm in the form of more or lessdistinct somitis is usuaily adhesiveglands vesicles(Photo 2'14)' The round vesicleon "fitn"U farthest from the spinal cord is the pronephric auditorYvesicles eachside latter, and often continuouswith duct. Just abovethe cornealePithelium and ii, ut" ttt" pronephrictubules' Thb pronephricduct dorsalfin tubulesoi eachsideconstitutethe pronephrickidney' infundibulum which is functional in amphibianlarvae' The pronephric latthe kidney when well formed causes body to bulge lens Placodes ridge' erad asthe PronePhric mesencePhalon The lateral plate mesoderm (often difficult to identify nasalPlacodes kidas a distinciarea) lies ventral to eachpronephric ectodermand the endoderm' This neuralcrestcells n"y, U"*""" ttre itin processof splitting into an outer mesodermis in the oPticcuPs adjacentto the skin ectoiuyer of somatic oPtic stalks mesoderm'ad-"*d"ttt', derm, and aninner layer of splanchnic pineal gland jaceni to the endoderm' If somatic and splanchnic identify a space(or a seriesof mesodermhaveiormed, proctodeum betweenthem, the coelom' small spaces) prosencePhalon Return to the level at which the foregut first appears rhombencePhalon posteriorly' Identify the (Photo 2.10)and tracesections (bulbus cordis) region of the developing' rudiments of the anterior pituitary gland otrorr".tt "ot lying beneaththe pharynx (PhotoZ'lL)' The heart heart semilunarganglia -as sections , enlarges the ventricle in more posterior stomodeum of heart consists an inner @hotlo2.13). Note that the surroundedby an outer'thickcord sPinal luj"r, the endocardium, Both theselayersare derived eilayer, the myocardium' ventral fin will mesoderm mesoderm (Splanchnic ' fromsplanchnic outerIater iontrrbute to a third layer of the heart: its Mesoderm Identify a thin layer of cellsenclosing most covering.) conotruncus the heart,the parietatpericardium,deriv edftom somatic dorsal aortae mesoderm. The parietal pericardium is usually sepaspace'The ectodermby a shrinkage ratedfrom the skin dorsalmesocardium spacebetweenthe heart and the parietal pericardium first aortic arches within is the pericardiatcavity. The heart is.suspended notochord the pe'ricardialcavity by a dorsal bridge of splanchnic mesocardium' the dorsal *"*d"t-, parietal pericardium are vessels in early stagesof formaThe major blood pronephrickidneYs tion at ihi, ti*", and they are thus difficult to identify somites veswith certainty in most embryos' Two major blood rod subnotochordal be selscan sometimes identified' The first aortic arch' to the pharynx (PhotoZ'I2)' esmainly lie ventrolateral ventricle with the conotrun(They will soonestabtshconnections

Frog Embryos 35

ffiderm
[ttuJr(:-;[1 'linriyg-: !]rnlt.li"{i_: i.Iiii,: :!,;lnlent

2.20and2.24). The lips of the blastoporeform during gastrulastages (Photos2.25-2.27). First the dorsaltip torms,followed by the lateral lips, and finally the veniral lip. The yolk plug continuesto occupythe blastoporethro-ughout gas_ trulation. The neuralplate forms and rolls up into the neuralfube b_etween gastrula stage and early embryo stage the (Photos2.28-2.30). Concomitantwith iormation of the neural tube,the embryo beginsto elongatecraniocau_ dally. This elongation is an obviou, f"itrrr" of devel_ opingfrog embryos(photos2.3I-2.32). The neuraltube extendsthroughoutthe length of the embryo. It bulges laterad at its cranial end to form the developingeyes in conjunctionwith the overlying skin ectoderm. Note that the skin ectoderm in the ernbryosillustrated in Photos2.31,2.32is coveredwith ciliary tufts. These structffes establishcurrentsaround the embryosasthey beat, circulating fluids. Thesestructuresals-o function in primitive locomotory movementsbefore swimming begins.In 4-mm embryosthe skin ectodermat the cra_ nial end of the embryo has invaginatedin the midline forming the stomodeum. Caudally, skin ectoderm the is attenuated,demarcatingthe dorsal and ventral fins (Photo2.32).

illtlttriiul-i:d

ffiotemm and endoderm


jt..iL:. =_'mbrane --rhrane lmtir.

ffiffimoscoPY

It sC{!{NING ELECTRON

ffinryryuu* ::;rming electron micrographs (photos 2.19_ .i,,i,,,Il T{-_:"-ou r-isualizethe shapesof developing frog :r; ,,rr|llUflllru. wf :::?n'os three-dimensionally. Note at cleav_ lrullw lfiilrrs'::iFhotos 2.19-2.24) the positions and orien_ iiilllllltullll0lllnri :d'deerage furrows separating blastomeres and ;l|ilw"illm:::-ces in sizes of the micromeres and riiisu'rrFnrslf'es. Boundaries between blastomeres become ,u*erll irunlri,iNr as cleavageadvances (compare photos -:led

I TER,MS KNOW TO
I)rilii]lilfll lrt-, :5iinff

rilllrliiin.& klorv the meaning of the following iilfii;{I|': terms, which appeared in boldfacein the preceding discussion of unur,mi, r:ipophtSiS giands u,Ltriluex;-: i{m:[n"i* ::;misphere u,lrmrrr;r nlle lrmrfiE:: pituitary gland -r
L.iu} {lllJt idflll:tli;::iron

cloacalmembrane cloacalvalves coelom conotruncus cornealepithelium cortex of egg deepectodermal layer dendrites diencephalon dorsal aorta dorsalblastoporallip dorsal fin dorsal lymph sac dorsalmesocardium duodenum

epiboly epibranchialplacode epidermis epiphysis exogastrulation eyes femalepronucleus fertilization first aortic arches first meioticdivision first polar body floor plate fluorescein-labeled dextran follicles follicle cells foregut gastrula gastrulation germ layers

riuruilr:if-,-r esicles &rlr, L>

lm.rUUSf -C,:el 'lrtiliffi : ni are s llni.rtits'. -L'tlfe lrui,u5|*. lfiiiLtr-:Iion :mil -r-;i-[s1 inil:r:r- c];
"-;i, Ell]& "* i -"i.JIL\

gOnadotropin

ectoderm egg capsule endocardium endoderm

***,0 ua- furrows

36

Chapter2 germinalvesicle gray crescent hindgut holoblasticcleavage peroxidase horseradish hypochord infundibulum inner ears inner ectodermallaYer involution lateral blastoPoralliPs lateral plate mesoderm lensplacode liver rudiment macIomeres male pronucleus mature ovum mesencePhalon mesoderm metaphase metencePhalon micromeres midgut movements morphogenetic mouth opening myelencephalon myocardium nasalcavities nasalplacodes neural crestcells neural ectoderm neural folds neural groove neural plate neural tube neurohypophysis neurula notochord nuclei nuptial pads olfactory (I) cranial nerves olfactory placodes olfactory receptors oogenesis oogonia optic cups optic stalks oral membrane ostium of oviduct outer ectodermallaYer ovaries oviduct ovulation parietal pericardium pericardialcavity pharyngealpouches pharynx pigment cells pigmentedretina pineal gland plasmalemma posterior pituitary gland primary oocyte proctodeum pronephricduct pronephrickidney pronephricridge pronephrictubules prophase prosencephalon prospectiveectoderm prospectiveendoderm prospectivefate prospectivefate maP prospectivehead mesenchYme prospectivelateral Plate mesoderm prospectiveneural Plate prospectivenotochord prospectivesegmental Plate mesoderm dextran rhodamine-labeled rhombencephalon roof plate rudiment of the anterior pituitary gland secondmeiotic division secondpolar body oocytes secondary plate mesoderm segmental semilunarganglia sensoryretina skin skin ectoderm somaticmesoderm somites sperm spinalcord mesoderm splanchnic stomodeum rod subnotochordal superficialectodermallaYer surfaceectoderm telencephalon thecafolliculi externa thecafolliculi interna total cleavage trigeminal (V) cranial nerves vegetalhemisphere pole vegetal ventral aortae ventral blastoPoralliP ventral fins ventral suckers ventricle of heart vital dyes vitelline membrane yolk yolk plug young neurons

ilr

38

Chapter2

@rrtt*

2./-2.5

Frog Embryos
felend
cortex 1. Pigmented 2. Vitelline membrane 3. Blastocoel 4. Areaofgraycrescent 5. Shrinkagespaces 6. Nuclei 7. Blastomeres 8. Outer ectodermallaYer 9. Inner ectodermallaYer 10. Archenteron roof (mesoderm, except at cranial end where roof formed from endoderm) 11. Direction of epibolY liP 12. Dorsalblastoporal 13. Blastopore 14. Archenteron 15. Yolk-filled endodermalcells 16. Yolk plug 17. Ventral blastoporalliP 18. Neuralplate 19. Skin ectoderm 20. Lateral plate mesoderm 21. Directionsof cellularmigration to form endodermalroof of archenteron plate mesoderm 22. Segmental 23. Notochord 24. Archetteron roof (endoderm)

t.

2.2
pole' Photo 2.1. Frog blastula(sagittalsection).AP,Animal pole;D, Dorsal side;VVentral side;VP;Vegetal Photo 2.2. Frogdorsallip gastrula(sagittalsection).D, Dorsal side;Y Ventral side.

Frog Embryos 39

Mtmto 2.3. Frog yolk plug gastrula(sagittalsection). D, Dorsal side;V ventral side. Flimtos2-4t 2.5. Frog neuralplate neurula(transverse section).Photo 2.5 is anenlargement the dorsal of :i-nion of Photo2.4.

It|

40

Chapter2

@/ntu*2.C2J)

Frog Embryos
1. Neural groove 2. Skin ectoderm 3. Lateral plate mesoderm 4. Archenteron 5. Yolk-filled endodermal cells 6. Neural fold 7. Outer ectodermal layer 8. Inner ectodermal layer 9. Segmental plate mesoderm 10. Notochord 11. Archenteron roof (endoderm) 12. Pineal gland 13. Prosencephalon 14. Nasal placode 15. Mesencephalon

16. Pigmentedretina of the oPtic cup retina of the oPticcuP 17. Sensory 18. Optic stalk 19. Rudiment of the anterior Pituitary gland 20. Head mesenchyme 21. Stomodeum

of section).Photo2.7is an enlargement the dorsal Photos 2.6, 2.7. Frog neuralfold neurula(transverse portion of Photo 2.6.

FrogEmbryos 41

Fhotos2.B,2.9.4-mmfrogembryoserialtransversesections,,,,

ilr-

42

Chapter2

@hrtug2.1O-2./3

Frog Embryos
'Yge/"{
L. Mesencephalon 2. Future corneal ePithelium 3. Lens placode 4. Rudiment of the anterior Pituitary gland 5. Foregut 6. Oral membrane 7. Adhesive gland 8. Stomodeum 9. RhombencePhalon 10. Skin ectoderm 11. Semilunarganglion 12. Infundibulum 13. Auditory vesicle 14. Notochord 15. Dorsal aorta 1-6.Pharynx 17. First aortic arch 1-8.Conotruncus 19. Duodenum 20. Liver rudiment 21. Dorsal mesocardium 22. Peicardial cavitY 23. Endocardium of ventricle 24. Myocardium of ventricle 25. P arietalPericardium 26. Shrinkagespace

ffifrogembryoserialtransversesectionsnumberedinanteriorto 2.1"1. 2.1O, Photos sequence. posterior

FrogEmbryos 43

lflflhmnnri 2.13. Continuation of 4-mm frog embryo serial transverse I ll. sectionsnumberedin anterior to
)i lllll|lilufl' - r:*--OC.

44

Chapter2

@hrbs,2./1-2./B

Frog Embryos
fgle',il
1. Roof plate 2. Neural crestcells 3. Spinalcord 4. Floor plate 5. Dorsal aorta 6. Pronephrictubule 7. Pronephricduct 8. Pronephricridge 9. Midgut 10. Yolk-filled endodermalcells 11. Skin ectoderm 12. Liver rudiment 13. Dorsalfin 1-4.Somite 15. Notochord 16. Subnotochordalrod 17. Hindgut l-8. Proctodeum 19. Ventral fin

2.15 numberedin anteriorto sections Photos 2.14, 2.15. Continuationof 4-mm frog embryoserialtransverse posterior sequence.

Frog Embryos 45

Wflrllmlilrtn m-1.18. Continuation of 4-mm frog embryo serial transverse sectionsnumbered in anterior to :
lll l l l l l l l l :1 " 1 " * ,:--;l C.

46

Chapter2

@Dtu*2./3-2.21

Frog Embryos
furrow 1. Cleavage 2. Blastomere 3. Micromere (blastomere) 4. Macromere (blastomere)

ofafrogtwo-ce1lstage(viewedfromtheside).AP,Animalpo1e; VP, VegetalPole. ffigelectronmicrographofafrogfour-ce1lstage(viewedfromanimalpoleandside).

FrogEmbryos

47

lrlllllllltttnrrlt! -ln

S;anrring electron micrograph of a frog eight-cell stage (viewed from animar pole and side).

. rlllllllrnmrna. S;rnning electron micrographof a frog sixteen-cellstage(viewed from animal pole and side; " all I Il lllllllillililiiit"' : -: : i:= :tot visible).
tllllllllnnrnrum S;anning * I l electron micrograph of a frog early blastula (viewed from animal pole and side).

,UfillN*n,i* S;anningelectronmicrograph :-:.1" of a frog late blastula(viewedfrom animalpole).

48

Chapter2

-&otot

2.2o-2.3O

Frog Embryos
1. Yolk plug 2. Dorsal blastoporalliP 3. Lateral blastoporalliP 4. Archenteron 5. Ectoderm 6. Roof of archenteron 7. Blastocoel 8. Blastopore 9. Future brain level of neural plate 10. Future spinal cord level of neural plate 11. Neural fold 12. Skin ectoderm 13. Future brain level of neural groove 14. Future spinal cord level of neural groove 15. Future brain level of earlY neural tube 16. Future spinal cord level of early neural tube

t
i
I

l 7
I I
I

electronmicrographof a frog dorsallip gastrula.D, Dorsal side. Photo 2.25. Scanning photo 2.26. Scanning slicethrougha frog yolk plug gastrula.D, Dorsal electronmicrographof a parasagittal side:V.Ventralside.

t
]
I

I I l"L

50

Chapter2

@tttu*2.3/-2.32

Frog Embryos
fgretu{
1. Neural tube 2. Ciliary tufts 3. Developing eye 4. Dorsal fin 5. Ventral fin 6. Stomodeum

I I
i

,.

I
t

h'
6 I
I

Photo 2.31. Scanning electronmicrographof a frog early embryo (dorsolateral view). R, Right side. Photo 2.32. Scanning electronmicrographof a frog 4-mm embryo(lateralview). H, Head (cranial)end;! Tail (caudal) end.

r
I I

I i
olfactory pit
-:

AmphibianDevelopment:Frog4-mm Embryo(Stage1g) g.l

prosencephalon epiphysis mesencephalon


optic cup lensplacode pharynx branchial cleft2 otic vesicle branchialcleft 3 pericardialcavity rhombencephalon

stonrocleurn

'!:r-..r!

ad

gland

pronephric area somites

spinal cord

notochord

liver clivc'r'tiL'u IunT endoderm


:.

bud

Figure103
4-mm frog embryo(stage18),whole mount (60K.

(Stage18) 82 AmphibianDevelopment:Frog4-'nm Embryo

epiphysis
stomodeum adenohypoPhYsis oral evagination prosencephalon mesencephalon infundibulum rhombencePhalon gland adhesive thyroid rucliment cavitY pericardial heart notochord

pharynx

liver diverticulum spinalcord


endoderm

midgut

mesoderm vcntt'al

rod subttotochordal

t/ proctocleum

hindgut tail bud

Figure104
1

| ,l
il

'18), (60K' (stage sagittal section frogembryo +-mm

fl I

I--

A mphi bi an evel opment: D (St Frog l O-rnmTadpol e age 24) 101

lip nror-rth

exterL.tnl nares internal rrares letina

pharynx thyloid heart

le n s . otic vesicle

pronephros liver

stontacl-r intestine

colcl

cloaca

notochorcl

anus

firr ltail

Figure130
1 O- m mr og t adpo l e(s ta g e 4 ),w h o l em o u n t(5 5 X. f 2

.l 24) Tadpole(Stage Development;Frog 1O-mm 02 Amphibian

lip tooth pharynx thyroid

telencephalon diencephalon metencephalou myelencephalon

infundibulum trachea heart intestine notochord dorsalaorta myotomes esophagus

myotomes

hindgut

spinalcord

tail fin

Figure13L
'lO-mmfrog tadpole(stage24), sagittal section(35K.

104 AmphibianDevelopment:Frog .tO-mmTadpole(Stage 24)

telencephalon

olfactoly organ

tootir

Figure133
( l 0 n rm I r og t ac llt olesta g e 4 ),Ira n Sv c rss e c ti o nL h rough ol factory 2 e the organ(5OX ).

diencephalon pignrentccl layerof l'ctitra sensory layel of retina

cranialcartilage

cranialcartilatge

ly rn p hs in u s rnuscle thyroid

Figure134
10-nrmfrog tadpole(stage24), transverse sectionthroughthe eyes (SoX).

AmphibianDevelopment:Frog 10-nrnr Tadpole(Stage 24\ 1Os

myelencephalon ganglion,cranialnerveVII g a n g lio nc ra n ia rrc rv e , l V pharynx

velarplate

atrium notochorcl opcrcLrla Figure 135


(s f l O - r r r nr og t ar lp o l c ..tg e 4 ), tt.a n SV e rScc ti o n lr 2 se through the heart(5OX ).

myelencephalon
o ti c vesicle

ganglion, cranial nerve IX


notochorcl

pharynx glottis sinus venosus

opercular chamber gills intestine

Figure136
(stage24), transverse lO-nrm frog taclpole sectionthrouglrthe glottis(sox).

(Stage 24) Frog 'lO-mmTaclPole DeveloPment: 106 AmPhibian

myelen

lion, cranialnerveX
pfonephfos

lung

esophagus opercularchamber liver intestine

gallbladder

137 Fig=ure

. (50X' sectionthroughihe pronephros (stage24)' transverse frog tadpole 1O-mnr

esophagus mYeie notochord

pronePhros

Figure138
;;;;.;;oo"'"tlttn" the through liver(sox' section 24)'transverse

(Stage 24) 107 Tadpole AmphibianDevelopment:Frog 1O-mm

spin a l

ganglion

mesonephros notoch duct mesonephric hindgut

peritoneal cavity

Figure139
(50rc. sectionthroughthe mesonephros frog tadpole(stage24), transverse i O-mrn

fin spinalcord spinalganglion notochord


i

dorsal aorta somite

hindlegbud

loaca

\ Figure 140
\ 'lO-mm tadpole (50K. (stage transverse section through cloaca the 24), frog

t{,4
-..7

Lq,
Buccole avilv
'., \.

Toothgerm

':,

.;Fig. l{,.'Cross ;,,Fig. 1{'. Crosssectionof 10,,mm.frog larva at the level of teleir"r:., icephalon:and '' ' nasal ',,. organs"

,l
I|lo|ltfto?l! Orll 9o9llb . OtEcol covllY Ogllc tac!.r
l I

,t

9uclrr Phoryru Irardthl ottloOr


Lt'|9lt ,9oc. tnfindbgftn

Hnoptda
Ogrrcdl.!

\rrrttrd oorlo '


At.h{ Frdcordd ccdtY . Slns rtroal

M.rhr
Loo

c!f!

Lhrr
lbdtl t Orodlrrill
"i' I

Ponsg

lbft-loalr hbrha
tl.Dhrlc dal

Z lfotono

ij.'l..:'.1]..I.l',li.:,.'.lil;:'l..,...,':'''.,'''

o! nn. seqdm 1-0 {g,+ry;} PE* tudinet


," .:"

. :: ffiffifiiil#;.fil3'ur"ffi1

,.:

.;.', i ;.,

. .r ,":"

- l ,,.,t.i , , , -1, :, -; I'r;.:;il_it,.r;,: : -1,', 'l : ,'.;.:,t;-1'. r'' ,, l'::,. .,ii.,,i]i

,i;tlir.;j ;;;'':.;1,

ili'iil.iii;
.,f;l$::r,1i
lr;

itj:iitii:",

If'',
tll'ventricle'
i

t. t{hiie rnotter

Trobeculor, r corliloge , Hypobrrnchiol ,t',,. , co r lilo g e.' ' ,' ; |,


Thyroidglohd,i:

.1 ..

.t,. i .:

Anlrrlor rc. contl Horl:onlol

,llrd. oorflc
grch

, Ly;rglr r rgocf

and. O
gill

rratdr

Gilb: Ogrrculul; 0ill ehombrr Prricordtot (pgilctolprrlcordtriml roc

of -- '--- lgl IJ. Cto:t section lp'rttn. frog larvartithe lovcl,of l-':: cphelon andear.s.

'Lymphheqrt

f ubules

Foslerior cordinol
,. , Y8lll +.--_ ' /

i ,--:_ ,,

t -

-. -: " r. : . '. t ' t |,..: 1, "' . t'.,. . i'l t


'.r: !. 'i,'l.t _,' ; : . ,'

Slomqch 'Goqlr.ic ' g l o nd s

t,'i. .;r,:, '] .1i, Yolk loden ' In{dsfine'

,:;li'r1.1
.1.
.- ,i :\. fr r :'

:i."

- l-r',,',.;,ii*iiiri[i'iilirffi *ffirrt.,',,,,iri''lii

.,,.,' , l ''S P C C C ; r,

l/

r iyiteLline V i te l l i n e . : nembrane
Vegetalpole

.'

STAG"ES CLEAVACE
funow lst cleavage
1115Blastomere.-$

TWO{ELL ST.ACE WHOIE

TWO{ELL STAGE SECTIONED

.t

F.OUR{ELL STAGE i.
'I

Sect ioned

( L .P.o . )

Micromeres 3rd cleavage , furrow Macromeres


.a .g:

MIDD
i
-

STAGE EIGHT.CELL
t

.\t-:roU r

'Sectione cl. lr r n' \


J

l'

Micromeres
h cleavage

furrows
Macromliies

.,. ''i t lr,clclrvlg,e , lirrrtrrv.l;

Micromeres Blastobbel

Ma

BLASTULA WHOLE
.1.

BLASTULA Section Cross (L.P,O.;

:Blastoporal groovb Yolk plug

E A R L Y GA S T R U L A .

Sectloned ( L.P.0..)

Donal lip ,
I

,Yolk plug Ventrallip i

,:,'\

MIDDLEGAST'RULA

Sectloned ( L .P.o . ) ' : '

2J

I I

I
i ii

ri
I

ii ii
i

f,

Yolk plug

t'

.' NEUITAT Sl AGES

i ..' l :

I ri
Nsural Plate
Notoclrord ,i

,rl.rlifr" -

Ncural lrt c p

le s o d e rn l
rnl Eeterd: Guf;' Encloclerrrr

N IUITAL PLN'I'E TA CL: S

{ iecU.tonad

( L .P.o . )

,::--Ncrtral

groove

lllg----Nctrrar

rorcl

lrlesoc'ernl ' l.loto clto t tl Neurulgrooye Neuralfolcl


i trt F,qtocl et D rrdoderrrt

uf

Secl.ionect
(L.It.0, ),.. .' ,.;i'i
"t'',:|.:

t Neural rrtre E llirrr0 rc Notochorcl Me s e n rc re ' Cu t


5planclililc l l l c s o ( l cr l l l

l.le ur;rltrrbe

tlesodcrtrr Sornatie Cirelorti \ Ictbclerrr r in Errd',:rler

) r)

I
\

NL UIIALT TJIJE]'A CF S

Sectioned

(L.F. o. ) ',

!.q

6 8 Arn p hibian Dev eiol :rrre nFtog Cleovfrge/GnslrLrloliotl tr (St(r[|s$ I Il *.

lQn

1e

outcr lilyet. ol'ectclclernr

in n e rln y e r
of'cctocle rnr

archerrtcron r.ocl[' (chclcla rncsodcr-lu)

rulcherr lcr'on(glst rocoel)

b lltstolrorc

t.|u
',:{ rt I

:'-'{i, '' l4l

\ .),.{ Tr ii

l'i' *:

Fii$
blas tocoel
i: i'

fi'j1 i')l

ffrit81
l r 'b " l l ,lt

#r'i

-{:

',1$$
,'i':*til

b la s t llot'c <

veutral

of blastopor:e

Figure87
Frogerlbryo, late gastr"ula (stage1 0 ),s a g i tl a l (1 s e c ti o r.r 0 0 X )

yoIr(p t,..:

2
Frog Embryos
A. Introduction 25 25 27 B. How to UseSerial Sections C. Oogenesis Fertilization and D. Formation the Cray Crescent of E. Cleavage Blastulation and F. Castrulation C. Neurulation H. 4-mm FrogEmbryos 30 3'l 32 35 29

2B

l. Scanning Electron Microscopy to J. Terms Know 35

K. Pho tos 2.1-2.32 37