Frog Embryos

6@z Freg Embrye,s

A. INTRODUCTION Amphibian eggsare relatively large and canbe readily obtained. Many outstandingexperimentalembryologists(including Hans Spemann,RossHarrison, and their severalstudents)took advantageof thesefacts when they beganto experimenton developingvertebrate embryosby removing parts,addingparts,and recombiningparts by microsurgery.Becauseso much of classicalexperimentalembryologyinvolvedexperiments on early amphibianembryos,it is essentialthat you have an understandingof the structureof theseembryosand how this sffuctureoriginatedin order to appreciateexperimental analyses.The study of early amphibiandevelopment,when comparedto that of other organisms coveredin this laboratory guide and atlas'will also demonstratethe basic similaritiesin developmental eventsand processesamongdifferent vertebratessuch asamphibians,birds,and mammals,and betweenthese vertebratesand invertebratessuch as seaurchins. For hands-onstudiesusing frog embryos,see Chapter 6 (Exercises2.1-2.4).

secupper left-hand corner of the slide,and successive tions are mounted in the following way (the numbers shownarefor illustrativepurposesonly;the actualnumbersof sectionsin eachrow,and the number of rowsper slide,vary):

r

2

3

4

5

6

7

8

910

1.1. 12 13 14 15 16 17 18 19 20 21. 22 23 24 25 26 27 28 29 30 If there aretoo many sectionsto 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.

One other type of serialsectionwill be encounteredextensivelyin your laboratory studies:the sagittalsection. A set of serial sagittalsectionscuts parallel to the long 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. SECTIONS Thussagittalsectionspassthroughthe dorsal-ventralexB. HOW TO USESERIAL tent of the embryo,either to the pightor left of the midLaboratorywork in introductory coursesin develop- line or on the midline. mentalbiology and embryologyusuallyincludesstudies Fig. 2.1b illustrates four representativetransverse secanatomy.Supposeyou obtainedsome of developmental tions. Fig.2.l-cillustratesthesesamesectionsafter they 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 scope,you could identify a few poorly defined external much of the anatomyof the embryo can one expectto featuresaswell asthe body axes(cranial-caudal,dorsalsection?Supposethat 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;in this sectionyou could deterlarly to the cranial-caudalaxisof the embryo are called mine the relationshipof the eyesto other structures tansverse(cross)sections.A slide or a collectionof (Figs.2.1b,2.1c).You might then examinea more posslidescontainingeverytransversesectionfrom the first terior section,suchas#1-5throughthe heart,orstill more one (the mostanteriorone)to the lastone (the mostpos- posterior sections(#50,#80). Unfortunately,the study sections.T}:re rcrior one)is calleda setof serial transverse of individual sectionsprovidesonly a two-dimensional (#1) is mounted at the most anterior sectionof this set picture of the embryo. To understandthe anatomy of

26

Chapter 2

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

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.

cranial

caudal

ventral

sectionnumbers

cranial

caudal

EYE

BRAIN

(NEURALTUBE)

SPINALCORD

anterior

posterior

sectionnumbers HEART

DIGESTIVETUBE

Fig. 2.1. Drawingsillustratingthe relationshipsbetweena preserved4-mm frog embryoviewedfrom the left side(Fig.Z.Ia) andfour representative transversesectionsthroughthis embryo (Figs.2.1b,2.1c).Sectionnumbers are for example only,

FrogEmbryos 27 followingspring.

C. O O G EN E S IASN D 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:,rl. lmgle layer of follicle cells, forming an ovarian 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

yolk-laden cytopl

Fully grown primary oocytesundergoovulation in responseto hormonessecretedby the anterior pituitary gland (adenohypophysis). Each oocyte is slowly squeezedthroughthe 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)are or,ulated 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 secondaryoocytesinto this opening.The second meiotic division is initiated by each secondary oocyteat about the time that it entersthe oviduct,but it then arrestsin the metaphasestage.As the secondary oocytespassthrough the oviduct,a multilayered,gelati_ nous egg capsuleis secretedoutside of the vitelline membraneby the cellslining the oviduct. 'Fertilization occursexternallyasthe secondaryoocytes are spawned(shed)by the female into the water. The secondmeiotic division is completed as a sperm con_ tactsandpenetrateseachsecondaryoocyte,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.

ot pnmary oocyte

germinal vesicle of

prrmaryoocyte

vitelline membrane

follicle cells

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

Chapter2

D. FORMATIONOF THEGRAY CRES.CENT

hemisphere.

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

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).

poi nt of sperm entrance

4p

midsagit t alplane of

direction of shift of pigmented corte

future embryo

b gray crescent unfertilized egg (secondaryoocytestage)

gray crescent

VP fertilized egg (right side view)

VP fertilized egg (dorsalview)

4 micromeres

gray crescent VP 2-cellstage (dorsal-rightside view)

gray crescent VP 4-cellstage (dorsal-rightsideview)

4 macromeres

VP 8-cellstage (dorsal-rightsideview)

dorsal blastoporal ltp

I

yolk plug VP blastulastage (dorsal-rightsideview)

VP blast dorsallip gastrulastage (dorsalview)

yolk piug gastrulastage (caudalview)

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

s0"3,

Frog Embryos 29

'il ti

E- CTEAVACE AND MLASTUTATION til$unr,r€teconsists of a series of rapid mitotic divisions ltlriti::-suh in blastulation (that is, the formation of a IttlMr- rvhich consists of a group of cells called blassurrounding a cavity, the blastocoel). Cleavage llimryrs pass through the entire egg, so cleavage is clai_ n,nil*: .s total (holoblastic). lloweve4 furrows pass tmr'trr; rhe vegetal hemisphere much more slowly than rt[Tn:r,:.hthe animal hemisphere, presumably because rITi.:t:t:rer contains far more yolk than the latter. A 1lrln(r :fotetable of the first three cleavagesfor the frog, fl*mnu ::rrens, is as follows (Hgs.2.3d-2.3f):(1) first cleav_ .'$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$irtr-rn of early cleavage furrows (that is, whether mrn:lt-rnal or horizontal), the difference in size of the Tm{":r tnneresand macromeres, and the difference in pig_ meL:rrlion of animal and vegetal hemisphere cells. Tiy :iLri[€ntifi' the gray creseent;the region of the gray cres_ i$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. the animal pole; thus, it is contained within nrrimel hemisphere. The blastocoel is filled with a 1lLT.c 1[n.ur,: ;frs1 may have coagulated during preparation of :,';: :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_ gressiveincreasein cell sizeand number of cell layers toward the vegetalpole;and (3) the blastomerescon_ tain very little yolk. Examine the vegetalhemisphere.Its characteristicsare 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,indicativeof lessfrequentcleavage;and(3) the blastomeresare packedwith yolk. 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 crescentthan 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 oppositeside). The blastulaconsistsof a mosaicof cellular areas,each of which will normally producea certainstructuredur_ ing subsequentdevelopment.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 cytoplasmof cellswithin certainareasis coloreddif_ ferently. But in most casesit is necessaryto determine 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 demonstratedhistochemicallvbv in_ cubating tissue containing the enzyme wittr -ttre appropriate substrate;or fluorescein- or rhodaminelabeleddextran,which can be demonstratedwith a flu_ orescencemicroscopeafter illumination with the proper wavelengthof light) is injected into a singlecelf or groups of cells at the blastula stage. As injected cells cleave,the marker is passedto their descendants.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 prospectivefate map indicatesthe location 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 tissuesand organs.The blastocoelappearsto

i.

t'

i'

lt

{. 't: t

30

Chaprer2

the blastoporallips (seeChapter 6, Exercise2'3)' as The locationsof severalareas(designatedarbitrarily involuareasl--10 and25-27)before and during their are tion over the dorsal and ventral blastoporal lips the on shownin Fig. 2.5. Allof theseareasare located surfaceat the blastulastage.Areas 1-5haveundergone involution over the dorsqlblastoporallip by the dorsal lip gasffulastage. Similarly,areas1-8 have.undergone yolk inuitrrriott ou"i th" dorsal blastoporallip by the plug gastrulastage,and areas2l and26haveundergone itru6tlntiott over the ventral blastoporal lip' The remaining numbered areaswill undergo involution during subsequentdevelopment' O1!:t cellular areas in ,rrid"rgo involution over the lateralblastoporallips a similar manner. Fig.2.4showsthe locationsof severalprospectiveareas' soire of which undergoinvolution during gastrulation' Someof the cells of the prospectiveendodermand all the cells of the prospective head mesenchlme and prospectivenotochord involute over the dorsalblastoporui 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' lateral plate mesoderm,and all the cellsof the prospec' is information blastopore of site Th"eapproximate tive sefrnental plate mesoderminvolute over the laterline' dashed the dicatedby a circular a/ lips"of the blastopore. The remaining cells of aswell as someof l. Prospectiveectoderm(epidermis) fro.p""ti"" lateral plate mesoderm' of the prospectiveendoderm,involute over the ifr" 2. ProsPectiveneural Plate ""Ut Only a relatively small number ventralblastop-oral1ip. 3. ProsPectivenotochord 'prospective cells undergo involution mesoderm plate endodermal segmental of 4. Prospective prospectiveendodermal Most mesoderm lips. plate Ulastoporal lateral over ttre 5. Prospective during gastrulation stationary mesenchYme relativeiy head cells remain 6. ProsPective of the archen' floor and plug yolk the endoderm forming stages 7. ProsPective neur' prospective the involute, areas As (Fig.2.Sj. ter6n at plate aiO prospectiveectoderm(epidermis)undergo F. GASTRULATION toward the blastoporeto replace spieading,oi "piboly, locatoriginally cells the into the interior of the gastrula' of some moved have gastrulation aieasthat During eA on itt-esurfaceof the blastulaturn inward or underObtain a slide containingsagittalsectionsof the dorsal go involution to move into the interior' Thesecellswill lip gastrulaand selecta sectioncloselyresemblingPhoto or innermost live rise to two germ layers:the endoderm, Z'21 ldentrfy the blastopore' dorsal blastoporal lip' and rethat cells layer; middle or mesoderm, the iaver.and blastocoel. The blastoporerepresentsthe future caulayer' germ outermost the form surface ott the dal end. The future cranial end lies directly opposite' -uirr the ectoderm. A depression,the blastopore,beginsto The blastoporeopensinto a narrow cavity,the primiinvoinitiate cells as gray crescent just the below form tive gut, oiarchenteron. The floor of the archenteron a liplike structure,the tution @'ig.Z.3h).Simultaneously, 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 blastopobecausethe first cells to involute over the dorsal and Cells gastmla' a into is transformed blastula ral lip, the lateral blastoporallips,and thus to contribute to the wall with lip blastoporal dorsal the over involute contiiue to of the archenteron,are the cells of the prospectiveen' ocprogressively involution and further development, doderm (Fig.Z.q- The remainder of the archenteron curs laterally and ultimately ventrally as well' This reroof at thi gastrulastageis formed by mesoderm'which sultsin formation of a circular blastoporecontaininga involutes over the dorsal and lateral blastoporallips folplug yolk the called cells endodermal yolk-filled massof lowing involution of prospectiveendoderm'The mesois surroundedby conby @ig.2.3i).The circularblastopore derm"of the archenteronroof vill' later be covered The lips' blastoporal ventral and tateral, iioiorrt dorsal, endoderm,which migratesupward over the inner surdirectionsof gastrulationmovementscanbe alteredexface 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 that the ectodermin the frog consistsof two layers:an

be essentialin many speciesto provide a spaceinto which certain groupsof cellscan move eithet en masse or individual$ during gastrulation'

Frog Embryos 31

blastocoel

archenteron

,:-t--E

dorsal

r"-f "

dorsal blastoporal

blastoporal

tr lip

rif,

*

blastopore

blastopore yolk plug

blastocoel

\? :i:siu1a Stage a *il

t''

l-5'

-siteof dorsal blastoporallip Iormatron

blastopore

aichenteron

blastocoel

ventral blastoporal

dorsal lip gastrulastage b

llp

yolk plug gastrulastage v

Drawingsof the cut surfacesof right halvesof blastula and gastrulastagesof tn"

rrog[

*i.rr.ot

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

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 the yolk plug, a protruding mass of yolk_ :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::,alcells is the irregularly shaped blastocoel. This ilir,iilirirri -i later squeezed out of existence. Note that the 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.

6- NEURUTATION -hrr :lodermal cellsdirectly overlyirigthe archenteron "ir'1r 19r- rhe yolk plug gastrulastageconstitutethe neurn unmoderm. The neural ectodermis inducedto thick:n ,u :he neural plate. Induction of the neural plate *= s'ith prospective r,,o'f mesodermalcellswithin the

dorsallip of the blastopore,which sendtheir induction signal through the horizontal plane of the ectoderm. The initial signalis later reinforcedby mesodermalcells 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 containingtransversesectionsof a neu_ rula at the neural plate stage. Selecta sectionclosely resemblingPhotos2.4,2.5and identi{y the neuralptate. 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 archenteronroof. Note that the dorsalmesodermis now organizedinto a midline rod of cells,the notochord, flankedby two bandsof cellqthe segmentalplatemesoderm. Thislatter mesodermis the sourceof the somites. The segmentalplate mesodermgradually mergeslat_ erally with the lateral plate mesoderm. The coelom forms within the lateral plate mesoderm. Obtain a slide containingtransversesectionsof a neurula at the neural fold stageand selecta sectionclose_ ly resemblingPhotos2.6,2.7.Identifythe pairedneural 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 subsequentdevelopmentthe neuralfolds 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).

Chapter2

32

H. 1. lntroduction at this th.e Fig.Z.rashowsthe general shape of _b-ody visiblein a midstage,and Fig.Z.O,t o*, ttt" ,t*itor", spatial re withthe yourself sagittal section. Familiarize 'uctures Detoreti^drruu - ;"'iut lationshipsof thesestructuiesbefore "*umioi"g transversesections. 2. Serial transverse sections

cornealep' d.ucedbythe lensto form the transparent cut sections cups' optic ffi;t""i. At the level of the continuouswith (veniral$) ;;;;;;" ;tosencephalon

(dorsatly)' the rnesencephalon parts assections The brain constrictsinto two separate ventral part is The 2J0)' are tracJ po1t"t-t1V Gtrofl pituitary posterior the of the infundi:butum,the source part is the dorsal The gr""a t"""t"nvpophysis)' and mye' ir'o*n"*"prtoron ittt" tut*" *"tencephalon ectodermal solid a turr""pt'uloi oittre'Urain;' Identify rodaiaboutthislevel'justventraltotheinfundibulum'

Positionyoursrideonthemicroscopestageso'l'llTl *Ll;yi';*ffiiliJnT:T:xt#;'l'rlTf; eachsection ottll gland is continuous tiew"A tlrougtr the microscope, " n:F' u" rn PhotJ 2.8. Do not placemicroscope 'l'4t, "J hold them in plac" to slides on ir"i*a fi'iir*rnt ^ '!:::::': ser"rtions. E'xamineslidesin anteroposterror z' unless directed otherwise (see Chapter A;;;;. dection B).

.t the anterior pituitary t"Oi..* invagination,the stomodeum'from ilffi;dermal 2'L0' originatedas an outgrowh' (SeePhotos ;ffi; rudiment The i .i-i"r ,rt" iocation of the stomodeum' pituitary gland and the stomodeumare ;irh" ;;fu uia'section 6vel betweenthoseillustrated "orrtirrroo.

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

Gi"ni i1U, the adiesive of the stomodeum and extending through ;i;;;rtd" many sections.

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

'*,':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 proseric*til ttr" to ventral side ectodermon each

just u""tt,,'"tuii*of ectodermalcells(neuralcrestcells) of ganglia above.eachof them' Theseare the semilunar

(orract'"ffi:ffii::lii*,*i:ru;t,t*Tffi|:'ffi' S^y#",::":);"1!{{{ thenasar are thickenings rhese the tt"iltt::]"f will form 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 - ti.on: ;;;;;;;t""0lolntitv of theseplacod; tJ""#;granulesat the periph".ry iliffi; *:'$;u""ttorut"*fto.therhombencephalon(Photo thickeningsof the teristic of invaginating ectodermal z'ni'-rte"evesicles originatedfrom invaginatpresumably,the peripheial ends ot inuugiiliffi:,Jil subsequent$ layer'-which piE-"", ,nJ into the concentrating thus differentiate '1i';""l"O"tt"f become narrowed, rtr" u"oitory vlsicles later cens the of ends inner "0. the content, whereas "riturg". inner ears' and placodes 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 spinalcord the Examine 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 continue to trace sectionsposteriorly As you apthese are neural crest cells (Photo 2'14)' is derivedfrom optic cups(photo iil."r,^[n "ptic cup the dorsal identify set' your it rirralsectionsin which p.ou"t ^fi" a laterar evaginationfrom the prosencephalon, The loose " z'r5-2'18\' rpi""r cord (Photos #;;" 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 ectoderm skin pro"roa""-, a ventral invagination of retina has (Photo ectodermal layer adjacent to the sensory fin ventral the sectionsanteriir to it'"t'it lens placode@hoto 2'10)' The the form to thickened "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

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-

16. Head mesenchyme 17. Mesencephalon 18. Rhombencephalon 19. Pharynx 20. Midgut 21. Spinalcord 22. Neural crest cells 23. Dorsal fin

tions more posteriorly. The remaining portion of the gut is the midgut(Photos2.14,2.15).Itcontainsa small cavity boundeddorsallyby a thin layer of endodermal cellsand ventrally by a large massof yolk-filled endodermal cells.Continueto tracesectionsposteriorlyfollowing the midgut. It gradually moves ventrally and enlargessomewhatas the hindgut (Photo 2.16). (In someembryosthe endodermof the hindgut fuseswith the ectodermof the proctodeumin more posteriorsections 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 vacuolatedconditionofthe notochordis apparentlyresponsiblefor its rigidity,allowingthis structureto serve 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 progressivelyreducedtoward the caudalend (compare

34

$

I

I

Chapter2

ventral to cus via a pair of blood vesselsthat will lie The caudalend of the notochord is Photos2.1'4-1'.18). can vessels latter the pharynx,the ventralaortae' These deiessdeu"lopedai this stagebecausethe notochord. end dorsal The .o*Ltit*. be identified at this stage') sectlons velopsin craniocaudalsequence'In postenor first aortic arch is continuous with a blood vesoi blocks laentify the somites(Photos Z'15,2'16)'paired aorta "u"tt sel lying dorsolateralto the pharynx' the dorsal AIso of mesodermlying ventrolateral to the spinalcord' andfade caudad aortaeextend befii6r.V.nl. The dorsal identrfy u ,rnutl Juster of mesodermalcells lying the midgut (Photo 2'14)' of level the uU""t ut i"i neath ihe notochord in caudal regions' the subnoto' Its cfrorAaf rod (hypochord) (Photos 2'I5-2'I7)' of the 3. is unknown' " Summaryof the contributions developmentalsignificance in the present to.structures ;;il is con+rs rudiment liver the which at Return to the level 4-mm trog emnrYo sectinuouswith the duodenum(Photo 2'13) and trace to the iion, port"riorly. The mesodermventrolateral Ectoderm distinct less or more of form the in somitis is usuaily adhesiveglands vesicles(Photo 2'14)' The round vesicleon "fitn"U auditorYvesicles eachsidefarthest from the spinal cord is the pronephric with continuous often and latter, duct. Just abovethe cornealePithelium and ii, ut" ttt" pronephrictubules' Thb pronephricduct dorsalfin tubulesoi eachsideconstitutethe pronephrickidney' infundibulum which is functional in amphibianlarvae' The pronephric latkidney when well formed causesthe body to bulge lens Placodes erad asthe PronePhricridge' mesencePhalon The lateral plate mesoderm (often difficult to identify nasalPlacodes kidas a distinciarea) lies ventral to eachpronephric This endoderm' the and ectoderm neuralcrestcells n"y, U"*""" ttre itin outer an into splitting of process mesodermis in the oPticcuPs adjacentto the skin ectoiuyer of somatic oPtic stalks -"*d"ttt', layer of splanchnicmesoderm'adderm, and aninner pineal gland jaceni to the endoderm' If somatic and splanchnic of series (or a space a identify mesodermhaveiormed, proctodeum small spaces)betweenthem, the coelom' prosencePhalon Return to the level at which the foregut first appears rhombencePhalon (Photo 2.10)and tracesectionsposteriorly' Identify the developing the of region cordis) (bulbus rudiments of the anterior pituitary gland otrorr".tt ' heart "ot The (PhotoZ'lL)' pharynx the beneath heart lying-as semilunarganglia sections , enlarges the ventricle in more posterior stomodeum heart consistsof an inner @hotlo2.13). Note that the outer'thickan by surrounded sPinalcord luj"r, the endocardium, derived are layers these Both eilayer, the myocardium' ventral fin will fromsplanchnicmesoderm' (Splanchnicmesoderm outerIater iontrrbute to a third layer of the heart: its Mesoderm enclosing of cells layer thin a Identify most covering.) conotruncus the heart,the parietatpericardium,deriv edftom somatic dorsal aortae mesoderm. The parietal pericardium is usually sepaThe space' a shrinkage by ectoderm 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 formaof stages early in are vessels The major blood pronephrickidneYs tion at ihi, ti*", and they are thus difficult to identify somites veswith certainty in most embryos' Two major blood subnotochordalrod selscan sometimesbe identified' The first aortic arch' (PhotoZ'I2)' pharynx the to esmainly lie ventrolateral ventricle (They will soonestabtshconnectionswith the conotrun-

Frog Embryos 35

ffiderm

2.20and2.24).

[ttuJr(:€-;[1

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-ughoutgas_ trulation.

'linriyg-: !]rnlt.li"{i_: i.Iiii€,: :!,;lnlent

illtlttriiul-i:d

ffiotemm and endoderm jt..iL:. =_'mbrane lmtir.--rhrane

It sC{!{NING ELECTRON

ffiffimoscoPY

ffinryryuu*::;rming electron micrographs (photos 2.19_ .i,,i,,,Il:r; T{-_:"-ou r-isualizethe shapesof developing frog ,,rr|llUflllru. wf :::?n'os three-dimensionally. Note at cleav_ lrullwlfiilrrs'::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

The neuralplate forms and rolls up into the neuralfube b_etweenthe gastrula stage and early embryo stage (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-ofunction 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,the skin ectoderm is attenuated,demarcatingthe dorsal and ventral fins (Photo2.32).

I TER,MS TO KNOW rilllrliiin.& iilfii;{I|': klorv the meaning of the following terms, which appeared in boldfacein the precedingdiscussionof

I)rilii]lilfll iinff lrt-, :5-

unur,mi, r:ipophtSiS

cloacalmembrane

epiboly

giands u,Ltriluex;-:

cloacalvalves

epibranchialplacode

i{m:[n"i*::;misphere

coelom

epidermis

u,lrmrrr;r nlle

conotruncus

epiphysis

lrmrfiE:: -r pituitary gland

cornealepithelium

exogastrulation

{lllJtL.iu}

cortex of egg

eyes

idflll:tli;::iron

deepectodermallayer dendrites

femalepronucleus fertilization

&rlr, L>

diencephalon

first aortic arches

lm.rUUSf -C,:el

dorsal aorta

'lrtiliffi : ni are s

first meioticdivision

dorsalblastoporallip

llni.rtits'. -L'tlfe

first polar body

dorsal fin

floor plate

lrui,u5|*.

dorsal lymph sac

lfiiiLtr-:Iion

dorsalmesocardium

fluorescein-labeleddextran follicles

duodenum

follicle cells

ectoderm

foregut

egg capsule

gastrula

endocardium

gastrulation

endoderm

germ layers

riuruilr:- if-,-r esicles

:mil -r-;i-[s1 inil:r:r- c];

gOnadotropin

"-;i, "* i -"iEll]& .JIL\

***,0 ua- furrows

m

36

Chapter2 germinalvesicle

olfactory (I) cranial nerves

rhodamine-labeleddextran

gray crescent

olfactory placodes

rhombencephalon

hindgut

olfactory receptors

roof plate

holoblasticcleavage horseradishperoxidase

oogenesis

rudiment of the anterior pituitary gland

hypochord

optic cups

infundibulum

optic stalks

inner ears

oral membrane

inner ectodermallaYer

ostium of oviduct

involution

outer ectodermallaYer

lateral blastoPoralliPs

ovaries

lateral plate mesoderm

oviduct

lensplacode

ovulation

liver rudiment

parietal pericardium

macIomeres

pericardialcavity

male pronucleus

pharyngealpouches

mature ovum

pharynx

mesencePhalon

pigment cells

mesoderm

pigmentedretina

metaphase

pineal gland

metencePhalon

plasmalemma

micromeres

posterior pituitary gland

midgut

primary oocyte

morphogeneticmovements

proctodeum

mouth opening

pronephricduct

myelencephalon

pronephrickidney

myocardium

pronephricridge

nasalcavities

pronephrictubules

nasalplacodes

prophase

neural crestcells

prosencephalon

neural ectoderm

prospectiveectoderm

neural folds

prospectiveendoderm

neural groove

prospectivefate

neural plate

prospectivefate maP

neural tube

prospectivehead mesenchYme

neurohypophysis

prospectivelateral Plate mesoderm

neurula notochord nuclei nuptial pads

oogonia

secondmeiotic division secondpolar body secondaryoocytes segmentalplate mesoderm semilunarganglia sensoryretina skin skin ectoderm somaticmesoderm somites sperm spinalcord splanchnicmesoderm stomodeum subnotochordalrod superficialectodermallaYer surfaceectoderm telencephalon thecafolliculi externa thecafolliculi interna total cleavage trigeminal (V) cranial nerves vegetalhemisphere vegetalpole ventral aortae ventral blastoPoralliP ventral fins ventral suckers ventricle of heart vital dyes vitelline membrane yolk

prospectiveneural Plate

yolk plug

prospectivenotochord

young neurons

prospectivesegmentalPlate mesoderm

ilr

38

Chapter2

@rrtt*

2./-2.5

Frog Embryos felend 1. Pigmentedcortex 2. Vitelline membrane 3. Blastocoel 4. Areaofgraycrescent 5. Shrinkagespaces 6. Nuclei 7. Blastomeres 8. Outer ectodermallaYer 9. Inner ectodermallaYer

t.

10. Archenteron roof (mesoderm, except at cranial end where roof formed from endoderm) 11. Direction of epibolY

18. Neuralplate 19. Skin ectoderm 20. Lateral plate mesoderm

13. Blastopore

21. Directionsof cellularmigration to form endodermalroof of archenteron

14. Archenteron

22. Segmentalplate mesoderm

15. Yolk-filled endodermalcells

23. Notochord

16. Yolk plug

24. Archetteron roof (endoderm)

12. DorsalblastoporalliP

17. Ventral blastoporalliP

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(transversesection).Photo 2.5 is anenlargementof the dorsal :i-nion of Photo2.4.

It|

40

Chapter2

@/ntu*2.C2J)

Frog Embryos 1. Neural groove

9. Segmental plate mesoderm

2. Skin ectoderm

10. Notochord

3. Lateral plate mesoderm

11. Archenteron roof (endoderm)

4. Archenteron

12. Pineal gland

5. Yolk-filled endodermal cells

13. Prosencephalon

6. Neural fold

14. Nasal placode

7. Outer ectodermal layer

15. Mesencephalon

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

8. Inner ectodermal layer

Photos 2.6, 2.7. Frog neuralfold neurula(transversesection).Photo2.7is an enlargementof the dorsal 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

9. RhombencePhalon 10. Skin ectoderm 11. Semilunarganglion 12. Infundibulum

1-8.Conotruncus 19. Duodenum 20. Liver rudiment 21. Dorsal mesocardium

5. Foregut

14. Notochord

22. Peicardial cavitY 23. Endocardium of ventricle

6. Oral membrane 7. Adhesive gland

15. Dorsal aorta

24. Myocardium of ventricle

1-6.Pharynx

8. Stomodeum

17. First aortic arch

25. P arietalPericardium 26. Shrinkagespace

13. Auditory vesicle

Photos2.1O,2.1"1.ffifrogembryoserialtransversesectionsnumberedinanteriorto posteriorsequence.

FrogEmbryos 43

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

44

Chapter2

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

Frog Embryos fgle',il 1. Roof plate

8. Pronephricridge

15. Notochord

2. Neural crestcells

9. Midgut

16. Subnotochordalrod

3. Spinalcord

10. Yolk-filled endodermalcells

17. Hindgut

4. Floor plate

11. Skin ectoderm

l-8. Proctodeum

5. Dorsal aorta

12. Liver rudiment

19. Ventral fin

6. Pronephrictubule

13. Dorsalfin

7. Pronephricduct

1-4.Somite

2.15 Photos 2.14, 2.15. Continuationof 4-mm frog embryoserialtransversesectionsnumberedin anteriorto posterior sequence.

Frog Embryos 45

Wflrllmlilrt : n m-1.18. Continuation of 4-mm frog embryo serial transverse sectionsnumbered in anterior to lll "1" l l l l l l l l :1

*

,:--;l C€.

46

Chapter2

@Dtu*2./3-2.21

Frog Embryos 1. Cleavagefurrow 2. Blastomere

3. Micromere (blastomere) 4. Macromere (blastomere)

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

FrogEmbryos

lrlllllllltttnrrlt! -ln

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

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

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

47

48

Chapter2

-&otot

2.2o-2.3O

Frog Embryos 1. Yolk plug

8. Blastopore

2. Dorsal blastoporalliP

9. Future brain level of neural plate

3. Lateral blastoporalliP 4. Archenteron 5. Ectoderm 6. Roof of archenteron 7. Blastocoel

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

i

t i

i I

l 7 I I

Photo 2.25. Scanningelectronmicrographof a frog dorsallip gastrula.D, Dorsal side.

I

t ] I

I

l

I I l"L

photo 2.26. Scanningelectronmicrographof a parasagittalslicethrougha frog yolk plug gastrula.D, Dorsal side:V.Ventralside.

50

Chapter2

@tttu*2.3/-2.32

Frog Embryos fgretu{ 1. Neural tube

3. Developing eye

5. Ventral fin

2. Ciliary tufts

4. Dorsal fin

6. Stomodeum

I

I

I I

,. i

I t

h' 6 I

Photo 2.31. Scanningelectronmicrographof a frog early embryo (dorsolateralview). R, Right side.

I

t Photo 2.32. Scanningelectronmicrographof a frog 4-mm embryo(lateralview). H, Head (cranial)end;! Tail (caudal) end.

r II

I i

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

olfactory pit -: '!:r-..r!

stonrocleurn

prosencephalon epiphysis mesencephalon optic cup lensplacode pharynx

ad

branchial cleft2

gland

otic vesicle branchialcleft 3 pericardialcavity rhombencephalon

pronephricarea 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

adhesivegland

notochord

thyroid rucliment pericardialcavitY

pharynx

heart

liver diverticulum spinalcord endoderm

vcntt'almesoderm

t/ proctocleum

midgut

subttotochordalrod

hindgut tail bud

Figure104 1

| ,l il

flI

t

I--

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

A mphi bi anD evel opment:Frog l O-rnmTadpol e(St age24) 101

lip

exterL.tnl nares internalrrares

nror-rth

letina pharynx

le n s .

thyloid

otic vesicle

heart pronephros liver

stontacl-r intestine

colcl

cloaca

notochorcl

anus

firr ltail

Figure130 1 O- m mf r og t adpo l e(s ta g e2 4 ),w h o l em o u n t(5 5 X.

.l 02 AmphibianDevelopment;Frog 1O-mmTadpole(Stage24)

lip tooth pharynx thyroid

telencephalon diencephalon metencephalou myelencephalon

infundibulum trachea heart

esophagus

intestine notochord dorsalaorta myotomes

myotomes

hindgut

spinalcord

tail fin

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

104 AmphibianDevelopment:Frog .tO-mmTadpole(Stage24)

telencephalon

olfactoly organ

tootir

Figure133 ( sta g e2 4 ),Ira n Sv c rssee c ti o nL h rough l 0 n rm I r og t ac llt ole the ol factoryorgan(5OX ).

diencephalon pignrentccl layerof l'ctitra sensorylayel of retina

cranialcartilage

cranialcartilatge

ly rn p hs in u s rnuscle

thyroid

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

AmphibianDevelopment:Frog 10-nrnrTadpole(Stage24\ 1Os

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

velarplate

atrium notochorcl opcrcLrla Figure 135 f e2 4 ), tt.a n SV e rSc l O - r r r nr lr og t ar lp o l c(s ..tg s e c ti o nthroughthe heart(5OX ).

myelencephalon o ti c vesicle

ganglion, cranialnerveIX notochorcl

pharynx glottis sinusvenosus

opercularchamber gills intestine

Figure136 lO-nrmfrog taclpole(stage24), transversesectionthrouglrthe glottis(sox).

(Stage24) Frog 'lO-mmTaclPole 106 AmPhibianDeveloPment:

myelen

lion, cranialnerveX pfonephfos

lung

esophagus opercularchamber liver intestine

gallbladder

137 Fig=ure

. pronephros(50X' (stage transversesectionthroughihe 24)' 1O-mnrfrog tadpole

esophagus mYeie

pronePhros

notochord

Figure138 ;;;;.;;oo"'"tlttn"

(sox' sectionthroughthe liver 24)'transverse

AmphibianDevelopment:Frog 1O-mmTadpole(Stage24) 107

ganglion

spin a l

mesonephros notoch

duct mesonephric hindgut

peritoneal cavity

Figure139 i O-mrnfrog tadpole(stage24), transversesectionthroughthe mesonephros(50rc.

fin spinalcord spinalganglion notochord i

dorsal aorta somite

hindlegbud

loaca

\ Figure140 \

'lO-mm sectionthroughthe cloaca(50K. frogtadpole(stage24),transverse

t{,4

Lq,

-..7

Buccole avilv '., \. I

Toothgerm

':,

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

,l l I

I|lo|ltfto?l!

,t

Orll 9o9llb . OtEcol covllY Ogllc tac!.r

9uclrr Phoryru Irardthl €ottloOr -

,9oc.

Lt'|9lt

tnfindbgftn

Hnoptda Ogrrcdl.!

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of blastopor:e

l

yoIr(p t,..:

2 Frog Embryos A. Introduction

25

B. How to UseSerialSections

25

C. Oogenesis and Fertilization

27

2B

D. Formationof the Cray Crescent E. Cleavage and Blastulation F. Castrulation

30

C. Neurulation

3'l

H. 4-mm FrogEmbryos

32

l. Scanning Electron Microscopy J. Termsto Know

29

35

K. Pho tos 2.1-2.32 37

35