Frog Embryos

6@zFreg Embrye,s

A. INTRODUCTION

Amphibian eggs are relatively large and can be read-ily obtained. Many outstanding experimental embry-ologists (including Hans Spemann, Ross Harrison, andtheir several students) took advantage of these factswhen they began to experiment on developing verte-brate embryos by removing parts, adding parts, and re-combining parts by microsurgery. Because so much ofclassical experimental embryology involved experimentson early amphibian embryos, it is essential that you havean understanding of the structure of these embryos andhow this sffucture originated in order to appreciate ex-perimental analyses. The study of early amphibian de-velopment, when compared to that of other organismscovered in this laboratory guide and atlas' will alsodemonstrate the basic similarities in developmentalevents and processes among different vertebrates suchas amphibians, birds, and mammals, and between thesevertebrates and invertebrates such as sea urchins. Forhands-on studies using frog embryos, see Chapter 6(Exercises 2.1-2.4).

B. HOW TO USE SERIAL SECTIONS

Laboratory work in introductory courses in develop-mental biology and embryology usually includes studiesof developmental anatomy. Suppose you obtained somepreserved frog embryos at the stage illustrated by Fig.2.La andwanted to study their anatomy. With a micro-scope, you could identify a few poorly defined externalfeatures as well as the body axes (cranial-caudal, dorsal-t'entral, andright-left). To study internal features in de-tail,you could slice (section) the entire embryo into thinsections of a given thickness; sections cut perpendicu-larly to the cranial-caudal axis of the embryo are calledtansverse (cross) sections. A slide or a collection ofslides containing every transverse section from the firstone (the most anterior one) to the last one (the mostpos-rcrior one) is called a set of serial transverse sections. T}:remost anterior section of this set (#1) is mounted at the

upper left-hand corner of the slide, and successive sec-tions are mounted in the following way (the numbersshown are for illustrative purposes only;the actual num-bers of sections in each row, and the number of rows perslide, vary):

r 2 3 4 5 6 7 8 9101.1. 12 13 14 15 16 17 18 19 20

21. 22 23 24 25 26 27 28 29 30

If there are too many sections to mount on one slide, themore posterior ones are mounted on slide #2 of the se-ries in the following way:

31 32 33 34 35 36 37 38 39 4041 42 43 44 45 46 47 48 49 50

and so forth.

One other type of serial section will be encountered ex-tensively in your laboratory studies: the sagittal section.A set of serial sagittal sections cuts parallel to the longaxis of the embryo. A midsaginal section is cut exactlydown the midline of the embryo, whereas a parasagittalsection is cut either to the right or left of the midline.Thus sagittal sections pass through the dorsal-ventral ex-tent of the embryo, either to the pight or left of the mid-line or on the midline.

Fig. 2.1b illustrates four representative transv ers e sec-tions. Fig.2.l-c illustrates these same sections after theywere transferred to a glass slide and mounted from leftto right in the order in which they were cut. Exactly howmuch of the anatomy of the embryo can one expect tosee in any one representative section? Suppose that yourset of serial sections contained a total of 100 sections.The sixth section of the set might cut through the levelof the developing eyes; in this section you could deter-mine the relationship of the eyes to other structures(Figs. 2.1b,2.1c). You might then examine a more pos-terior section, such as #1-5 through the heart,or still moreposterior sections (#50, #80). Unfortunately, the studyof individual sections provides only a two-dimensionalpicture of the embryo. To understand the anatomy of

26 Chapter 2

the embryo inthree dimensions you must visualize eachsection as again part of the whole embryo. For exam-ple, the notochord can be identified in sections #15, #50,and #80. By connecting the section of the notochordat the level of section #15 with the section of the noto-chord at the next level (section #50) and those at suc-cessive levels, you get an accurate picture of thecraniocaudal extent of the notochord, as well as its re-lationship to other structures. In the same way, youcan get an accurate picture of the craniocaudal extentof the neural tube (brain and spinal cord) and digestivetube, as well as their relationship to each other and toother structures. The most dfficult task facing the be-ginning student is to learn to visualize relationships ofparts 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 laborato-ry manual: (1) line drawings (called "Figs."); (2) sec-tion "orientators," placed on most photo legends (theseorientators show the exact levels where embryos weresectioned, sliced, or cryofractured); and (3) scanningelectron micrographs, which portray a more three-di-mensional image than do flat, two-dimensional serialsections.

Methods have been included in Chapter 6 (AdvancedHands-on Studies) to help you understand how embryosare prepared for light microscopy, scanning electron mi-croscopy, immunocytochemistry, and in situ hybridiza-tion, as well as to help you understand why differentprocessing procedures result in different types of im-ages.

cranial

section numbers

cranial

caudal

caudal

anterior

section numbers

posterior

HEART DIGESTIVETUBE

ventral

EYE BRAIN (NEURALTUBE) SPINALCORD

Fig. 2.1. Drawings illustrating the relationships between a preserved 4-mm frog embryo viewed from the leftside (Fig. Z.Ia) and four representative transverse sections through this embryo (Figs. 2.1b,2.1c). Section num-bers are for example only,

Frog Embryos 27

C. OOGENESIS ANDFERTILIZATION

Oryenesis (development of the ovum) begins in thd:i.1.-:;d or-aries of the mature female frog. During therricoJins 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 ofr -::ge central cell containing a lot of yolk in its cyto-:L:s:r. the primary oocyte, surrounded by a layer ofn,;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 :,eLis and the plasmalemma of the primary oocyte.r- ::;r sheath ofconnective tissue,the thecafolliculi ex-mcrnra- forms the surface layer of the ovary. Anotherrir::ih. the theca folliculi interna, partially surroundsr r':i lollicle but is lacking in the region where the fol-llj:: J{,rntacts the theca folliculi externa; at this regione,muftrtion (the rupture of the follicle and release of its:, - ri::.ined oocyte) occurs.:,t:: qlr-3ry also contains cells called oogonia. These:r*. '.mdergo rapid mitotic divisions, increasing in num-T'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 l. lmgle layer of follicle cells, forming an ovarianfrultrhde. s'hich slowly enlarges due to the accumulationt' "'- '-r. These primary ooqtes enter the prophase stager: :-:; ftrst meiotic division but remain there until the

yolk-laden cytopl ot pnmary oocyte

germinal vesicle offollicle cellsprrmary oocyte vitelline

membrane

theca folliculi externa\--/-----J

site of ovulation

theca folliculi interna

following spring.

Fully grown primary oocytes undergo ovulation in re-sponse to hormones secreted by the anterior pituitarygland (adenohypophysis). Each oocyte is slowlysqueezed through the follicular wallat the region wherethe theca folliculi interna is lacking (Fig.2.2) andentersthe body cavity (coelom) of the female. Many oocytes(2,000-20,000, depending upon the species) are or,ulat-ed by a single female each breeding season. primaryoocytes complete the first meiotic division during ovu_lation, with each forming a first polar body and sec-ondary oocy'te. Both of these structures are containedwithin the vitelline membrane formed earlier, while theprimary oocyte was in the ovary. Cilia on the lining ofthe coelom beat toward the ostium of the oviduct andpropel the secondary oocytes into this opening. The sec-ond meiotic division is initiated by each secondaryoocyte at about the time that it enters the oviduct, butit then arrests in the metaphase stage. As the secondaryoocytes pass through the oviduct, a multilayered, gelati_nous egg capsule is secreted outside of the vitellinemembrane by the cells lining the oviduct.

'Fertilization occurs externally as the secondary oocytes

are spawned (shed) by the female into the water. Thesecond meiotic division is completed as a sperm con_tacts and penetrates each secondary oocyte, resulting inthe formation of a second polar hody and a matureovum containing the female pronucleus. The nucleus'of the penetrating sperm enlarges within the ovum asthe male pronucleus, and the male and female pronu_clei unite to complete the process of fertilization.

Eimi .} -) Schematic drawing of a section through three ovarian follicles of a mature female frog.

,n:!! '- '28 Chapter 2

D. FORMATION OF THE GRAYCRES.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. Theuppermost part of the pigmented portion is the animalpole. 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 polelies directly opposite the animal pole. This pole corre_sponds tothe caudalend of the future embryo. The halfof the egg that contains the vegetal pole is the vegetal

b

gray crescent

hemisphere.

Following contact by and entrance of the sperm, the pig-mented cortex sffis relative to the less pigmented deep_er portion of the egg, toward and past the site of spermentrance and away from the side of the egg opposite thesperm entrance point (Fig.2.3b). This reduces the pig_mentation of a crescent-shaped area opposite the pointof sperm entrance. This crescent-shaped area betweenthe heavily pigmented cortex above ind the essential-ly nonpigmented cortex below constitutes the gray cres,cent (Figs. 2.3b,2.3c). A plane passing rhrough theanimal and vegetal poles and through the center of thegray crescent corresponds to the midsagittal plane of thefuture embryo (Fig. 2.3c).

Ap point of sperm entrance 4p midsagi t ta l p lane of

direction of shift offuture embryo

pigmented corte

gray crescent

gray crescent

unfertilized egg(secondary oocyte stage)

VPfertilized egg

(right side view)

VP4-cell stage

(dorsal-right side view)

VPfertilized egg(dorsal view)

gray crescent

dorsalblastoporal

ltp I

4 micromeres

4 macromeres

yolk plug

VP2-cell stage


VPblastula stage


VP blastdorsal lip gastrula stage

(dorsal view)

VP8-cell stage


yolk piug gastrula stage(caudal view)

Fig. 2.3. Drawings of early developmental stages of the frog. Ap, animal pole;D, aorrd sa"JJ"t s0"3,right side;V, ventral side;Vp, vegetal pole.

E- CTEAVACE ANDMLASTUTATION

Frog Embryos 29

(2) it is four or five cells thick, with smaller cells andfewer cell layers at the animal pole and with a pro_gressive increase in cell size and number of cell layerstoward the vegetal pole;and (3) the blastomeres con_tain very little yolk.

Examine the vegetal hemisphere. Its characteristics areexactly the opposite: (1) a pigmented cortex, if presentat all, is much less evident than in the animal hemi_sphere; (Z) the blastomeres are very large and few innumber,indicative of less frequent cleavage;and (3) theblastomeres are packed with yolk.Try to identify the gray crescent. In your sections it usu-ally lies either to the left or right side of the blastocoeland also slightly ventral to it. It has the following char_acteristics (compare sides indicated by letters D andVin Photo 2.1): (1) the pigmented cortex is thinner inthe gray crescent than on the opposite side;and (2) theblastocoel lies nearer the surface on the grav crescentside than on the opposite side (thar is, th"e wall of theblastula is thinner on the gray crescent side than onthe opposite side).

The blastula consists of a mosaic of cellular areas, eachof which will normally produce a certain structure dur_ing subsequent development. In other words, each areaof cells has a certain prospective fate that will be real_ized during normal development. In blastulae of somechordates (Urochordata or tunicates) the outlines ofthese cellular areas can be determined directly becausethe cytoplasm of cells within certain areas is colored dif_ferently. But in most cases it is necessary to determinethe prospective fate of each cellular area indirectlv bvmarking experiments. Vital dyes0have been used mostfrequently for this purpose in amphibians. Several areasof the blastula are stained with a vital dye, and the struc_ture or structures that are formed from each stainedarea are observed (see Chapter 6, Exercise 2.2\.Another technique has been used more recently. A cellmarker (for example, the enzyme horseradish peroxi_dase, which can be demonstrated histochemicallv bv in_cubating tissue containing the enzyme wittr

-ttre

appropriate substrate; or fluorescein- or rhodamine-labeled dextran, which can be demonstrated with a flu_orescence microscope after illumination with the properwavelength of light) is injected into a single celf orgroups of cells at the blastula stage. As injected cellscleave, the marker is passed to their descendants. Aprospective fate map is constructed with the aid of theinformation gained by these techniques. The amphib_ian fate map should be carefully compared with the onesfor the chick (Fig. 3.7), mouse (Fig.4.2),and sea urchin(Fig. 1.1). A prospective fate map indicates the locationof specific groups of cells prior to the onset of gastru-lation. These groups of cells are shifted in an olderlyway into appropriate positions during gastrulation,which will enable them to cooperate and interact in for_mation of tissues and organs. The blastocoel appears to

'iltii.t 'i '

lt{.'t:

ttil$unr,r€te consists of a series of rapid mitotic divisionsltlriti: :-suh in blastulation (that is, the formation of a

IttlMr- rvhich consists of a group of cells called blas-surrounding a cavity, the blastocoel). Cleavage

llimryrs pass through the entire egg, so cleavage is clai_n,nil*: .s total (holoblastic). lloweve4 furrows passtmr'trr; rhe vegetal hemisphere much more slowly thanrt[Tn:r,:.h the animal hemisphere, presumably becauserITi. :t:t:rer contains far more yolk than the latter. A1lr ln(r :fotetable of the first three cleavages for the frog,fl*mnu ::rrens, is as follows (Hgs.2.3d-2.3f): (1) first cleav_.'$t :eridional (that is, passes through both the ani_urtu. 'l.i r-egetal poles), usually bisecting the gray;n*s::nr (rhat is, passes through the midsagittal planenr .:; :urure embryo), 2.5 hours postfertilization; (2)rrcr;-Ed cleavage: meridional, at right angles to the first,I 5 -':ur: postfertilization; and (3) third cleavage:hor_nr;ni;* displaced toward the animal pole due to the yolkr:mr:ir of the vegetal hemisphere, producing four small_rr r-rrnai hemisphere cells, the micromeres, and four .lii{'r.: \ egetal hemisphere cells, the macromeres,4 hoursprur:r-nilization. The egg reaches the blastula stage nearllrs ::d of cleavage (approximately 16 hours postfer_urLi,ru.:itrn I (Fig. 2.3 g). Numerous small blastomeres oc_;ur", rhe animal hemisphere, whereas the vegetaln*rr:sahere consists of a lesser number of larger blas_ilme:c! The blastula is only slightly larger at the endrr :i€.arage than is the newly fertilized egg.;:r;:rne models, or preferably living eggs (see Chaptern. =u;rcise 2.1), showing early cleavage stages. Note thetlilir$irtr-rn of early cleavage furrows (that is, whethermrn:lt-rnal or horizontal), the difference in size of theTm{":r tnneres and 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$rf,: :hat is broadest will become the dorsal surface of:lLre ;unbn'o. and the opposite side of the egg will be_r:m; rhe ventral surface.:Tal-ne a section of the frog blastula that closely re-ln*r:ies Photo 2.1. Identify the main cavity, the blas-mcffil- \ore that it is displaced toward one side of therl;-N$mLla. the animal pole; thus, it is contained within1lLT.c nrrimel hemisphere. The blastocoel is filled with a1[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 theirmdei Spaces between blastomeres in the vegetal hemi-@re are shrinkage spaces produced 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 polelmc ;:ading off progressively toward the vegetal pole;

Fig. 2.a, Prospective fate map of the frog blastula'

Th"e approximate site of blastopore formation is in-

30 Chaprer 2

be essential in many species to provide a space into

which certain groups of cells can move eithet en masse

or individual$ during gastrulation'

the blastoporal lips (see Chapter 6, Exercise 2'3)'

The locations of several areas (designated arbitrarily as

areas l--10 and25-27) before and during their involu-

tion over the dorsal and ventral blastoporal lips are

shown in Fig. 2.5. Allof these areas are located on the

surface at the blastula stage. Areas 1-5 have undergone

involution over the dorsqlblastoporal lip by the dorsal

lip gasffula stage. Similarly, areas 1-8 have.undergone

inuitrrriott ou"i th" dorsal blastoporal lip by the yolk

plug gastrula stage, and areas2l and26 have undergone

itru6tlntiott over the ventral blastoporal lip' The re-

maining numbered areas will undergo involution dur-

ing subsequent development' O1!:t cellular areas

,rrid"rgo involution over the lateral blastoporal lips in

a similar manner.

Fig.2.4shows the locations of several prospective areas'

soire of which undergo involution during gastrulation'

Some of the cells of the prospective endoderm and all

the cells of the prospective head mesenchlme and

prospective notochord involute over the dorsal blasto-

porui tip. Similarly, some of the cells of the prospec-

iive endoderm, some of the cells of the prospective

lateral plate mesoderm, and all the cells of the prospec'

tive sefrnental plate mesoderm involute over the later-

a/ lips"of the blastopore. The remaining cells of the

fro.p""ti"" lateral plate mesoderm' as well as some of

ifr" ""Ut

of the prospective endoderm, involute over the

ventralblastop-oral1ip. Only a relatively small number

of 'prospective endodermal cells undergo involution

over ttre Ulastoporal lips. Most prospective endodermal

cells remain relativeiy stationary during gastrulation

stages forming the yolk plug and floor of the archen'

ter6n (Fig.2.Sj. As areas involute, the prospective neur'

at plate aiO prospective ectoderm (epidermis) undergo

spieading, oi "piboly,

toward the blastopore to replace

aieas that have moved into the interior of the gastrula'

Obtain a slide containing sagittal sections of the dorsal

lip gastrula and select a section closely resembling Photo

Z'21 ldentrfy the blastopore' dorsal blastoporal lip' and

blastocoel. The blastopore represents the future cau-

dal end. The future cranial end lies directly opposite'

The blastopore opens into a narrow cavity, the primi-

tive gut, oiarchenteron. The floor of the archenteron

is foimed by yolk-filled endodermal cells' The cranial

end of the archenteron roof is formed from endoderm

because the first cells to involute over the dorsal and

lateral blastoporal lips, and thus to contribute to the wall

of the archenteron, are the cells of the prospective en'

doderm (Fig.Z.q- The remainder of the archenteron

roof at thi gastrula stage is formed by mesoderm'which

involutes over the dorsal and lateral blastoporal lips fol-

lowing involution of prospective endoderm' The meso-

derm"of the archenteron roof vill' later be covered by

endoderm, which migrates upward over the inner sur-

face of the mesoderm (see below)' Thus the archen-

teron is ultimately lined entirely by endoderm' Note

that the ectoderm in the frog consists of two layers: an

dicated by a circular dashed line'

l. Prospective ectoderm (epidermis)2. ProsPective neural Plate3. ProsPective notochord4. Prospective segmental plate mesoderm

5. Prospective lateral plate mesoderm

6. ProsPective head mesenchYme7. ProsPective endoderm

F. GASTRULATION

During gastrulation some of the cells originally locat-

eA on itt-e surface of the blastula turn inward or under-

go involution to move into the interior' These cells will

live rise to two germ layers: the endoderm, or innermost

iaver. and the mesoderm, or middle layer; cells that re-

-uirr ott the surface form the outermost germ layer'

the ectoderm. A depression, the blastopore, begins to

form just below the gray crescent as cells initiate invo-

tution @'ig. Z.3h). Simultaneously, a liplike structure, the

dorsal Lhstoporal lip, forms just above the blastopore'

With formati,on of the blastopore and dorsal blastopo-

ral lip, the blastula is transformed into a gastmla' Cells

contiiue to involute over the dorsal blastoporal lip with

further development, and involution progressively oc-

curs laterally and ultimately ventrally as well' This re-

sults in formation of a circular blastopore containing a

mass of yolk-filled endodermal cells called the yolk plug

@ig.2.3i). The circular blastopore is surrounded by con-

iioiorrt dorsal, tateral, and ventral blastoporal lips' The

directions of gastrulation movements can be altered ex-

perimentally,iesulting in exogastrulation, a process dur-

ine which surface cells move but fail to involute over

,:-t--Er"-f "

blastocoel

blastocoel

-site of dorsal

Frog Embryos 31

archenteron

dorsalblastoporaltr lip

dorsalblastoporal* rif,

blasto-pore

aichenteron

blastocoel

blastopore

yolk plug

blastopore

ventral\?

: i :siu1a Stagea

blastoporal lipIormatron

blastoporall lp

dorsal lip gastrula stageb

yolk plug gastrula stagev

*ilt''

,Il llu

l-5' Drawings of the cut surfaces of right halves of blastula and gastrula stages of tn" rrog[ *i.rr.otl 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 involutiffi ;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 plugiltlllttfir-*: 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 prominentqruilmd blastoporal lip below. Also identify the archen-rfunlra - -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. BeneathHiu r--;:Lenteron and separated from the latter by en_jrhrurir::,al cells is the irregularly shaped blastocoel. Thisilir,iilirirri -i later squeezed out of existence. Note that then'ululr::ed cortex now covers all cells except those oflie ' .-ft plug. The more rapidly dividing cells of thenmry*ttire ectoderm (epidermis) and prospective neur-u dllire undergo epiboly between the blastula stage andItrrL,s -i jranced gastrula stage, growing down over the',r dk-:l-ied cells of the vegetal hemisphere. Meanwhilq'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 oitrrrrr .L:;henteron. Most of the archenteron roof still con_mn; : i mesoderm at the yolk plug gastrula stage.ilirr- i. :,.'rmparison of gastrulation in vertebrates and in-eirL.r.atel see Chapter 1, Section H.

6- NEURUTATION-hrr :lodermal cells directly overlyirig the archenteron"ir'1r 19 r- rhe yolk plug gastrula stage constitute the neur-n unmoderm. The neural ectoderm is induced to thick-:n ,u :he neural plate. Induction of the neural plater,,o'f *= s'ith prospective mesodermal cells within the

dorsal lip of the blastopore, which send their induction- signal through the horizontal plane of the ectoderm.The initial signal is later reinforced by mesodermal cellswithin the roof of the archenteron, which send their in_duction signal vertically to the overlying ectoderm. Thegastrula is transformed into the neurula with formationof the neural plate.

Obtain a slide containing transverse sections of a neu_rula at the neural plate stage. Select a section closelyresembling Photos2.4,2.5 and identi{y the neural ptate.The roof of the archenteron at the neurula stage isformed by endoderm. Between the gastrula and neu_rula stages, endoderm migrated upward over the innersurface of the mesoderm that previously formed thearchenteron roof. Note that the dorsal mesoderm isnow organized into a midline rod of cells, the notochord,flanked by two bands of cellq the segmental plate meso-derm. This latter mesoderm is the source of the somites.The segmental plate mesoderm gradually merges lat_erally with the lateral plate mesoderm. The coelomforms within the lateral plate mesoderm.Obtain a slide containing transverse sections of a neu-rula at the neural fold stage and select a section close_ly resembling Photos 2.6,2.7.Identify the paired neuralfolds, which have formed at the lateral marsins of theneural plate, and the neural groove. The notochord, seg-mental plate mesoderm, and lateral plate mesoderm aremore clearly defined at this stage, and the endodermof the roof of the archenteron exists as a more distinctlayer. During subsequent development the neural foldswill fuse in the dorsal midline to close the neural srooveand thus establish the neural tube. Neurai creJt cellswill later formfrom the roof of the neural tube and giverise to a multitude of structures, including pigment cells(see Chapter 6, Exercise 2.4).

32 Chapter 2

H.d.ucedby the lens to form the transparent corneal ep'

ffi;t""i. At the level of the optic cups' sections cut

;;;;;;" ;tosencephalon (veniral$) continuous with

1. lntroduction the rnesencephalon (dorsatly)'

Fig.Z.rashows the general shape of th.e _b-ody at this The brain constricts into two separate parts as sections

stage, and Fig. Z.O ,t o*, ttt" ,t*itor", visible in a mid- are tracJ po1t"t-t1V Gtrofl 2J0)' The ventral part is

sagittal section. Familiarize yourself withthe spatial re the infundi:butum, the source of the posterior pituitary

lationships of these structuies before "*umioi"g

;"'iut gr""a t"""t"nvpophysis)' The dorsal part is the

transverse sections.

'uctures Detore ti^drruu - ir'o*n"*"prtoron ittt" tut*" *"tencephalon and mye'

turr""pt'uloi oittre'Urain;' Identify a solid ectodermal

2. Serial transverse sections rodaiaboutthislevel'justventraltotheinfundibulum'

Positionyoursrideonthemicroscopestageso'l'llTl *Ll;yi';*ffiiliJnT:T:xt#;'l'rlTf;tiew"A tlrougtr the microscope, each section

" ottll

t"Oi..* .t the anterior pituitary gland is continuous

"J u" rn PhotJ 2.8. Do not place microscope n:F'

'l'4t, ilffi;dermal invagination, the stomodeum' from

fi ir"i*a on slides to hold them in plac" ^ '!:::::': ;ffi; originated as an outgrowh' (See Photos 2'L0'

'iir*rnt r"rtions. E'xamine slides in anteroposterror se-

i .i-i"r ,rt" iocation of the stomodeum' The rudiment

A;;;;. unless directed otherwise (see Chapter z' ;irh" ;;fu pituitary gland and the stomodeum are

dection B). "orrtirrroo.

uia'section 6vel between those illustrated

a. Ecrodermar derivatives ;i::,X,,*?a?f)#ff-T3l,"",ffi:#iitrffffi:The first few sections cut through the tip of the head'

Gi"ni i1U, the adiesive glands (ventral suckers)' to

rdentify the rurg" pror"lnlph;d (ry,.T"i"t""t"4*l: ;i;;;rtd" of the stomodeum and extending through

and ailnceptralon of the brain), which is- enclosed by many sections.

'*,':H1"3iffi::ft$i:::ff)'ff'ilffi[i:ffi ]""i:'hi:13;111:'il1iT":t':*HitiJfifil*"r*3;:;1*H[::ff*"iil1"1"'ffi'li,T:['Jii?.i:i*f#L,T;1*=*:*';::*(i;#JilJi:"11fl :"ment. Tiy to identify a region-ot tfri"t"t"J

'ftit u""tt,,'"tuii*of ectodermalcells(neuralcrestcells) just

ectoderm on each side ventral to ttr" proseric*til above. each of them' These are the semilunar ganglia of

rhese thickenings are the nasar (orract'"ffi:ffii::li i*,*i:ru;t,t*Tffi|:'ffi' S^y#",::":);"1!{{{The nasul placodes eventually form the tt"iltt::]"f

-"itlu*, on each side, an epibranchial placode' will

:ilL:ffi:ii'"ilX'i?J#:l5if":#,ffy"'fi:ilffi1 fi::Filetothesegangria) continuetotracesec-

granules at the periph".ry of these placod; tJ""#;- - ti.on: ;;;;;;;t""0lolntitv the paired auditory vesi-

teristic of invaginating ectodermal iliffi; *:'$;u""ttorut"*fto.therhombencephalon(Photopresumably, the peripheial ends ot inuugiiliffi:,Jil z'ni'-rte"evesicles originated from thickenings of the

become narrowed, thus concentrating ,nJ piE-"", '1i';""l"O"tt"f layer'-which subsequent$ invaginat-

content, whereas the inner ends of the cens "riturg". "0.

rtr" u"oitory vlsicles later differentiate into the

Young newo n nt", oiinate from these placodes and inner ears'

producetheaxonsoitfieoractory(I)cranialnerves'Continuetotlacesectionsposterior$andnotethattheas well as the dendrites that function as orfactory re- ,r"rrrui,,rb" gradual$ 1T1:*.,

indicating the level of

ceptors (receptors ;;; d;;;e of smell). Nasaipla- tft" .pi""i"oiJ Gno.o 2'L4)' Examine the spinal cord

codes may not have yet developed.in some embryos. *rtnrrig5magniiication. Noteitscharacteristicthinroof

Identify the pinear gland (epiphysis), which originated "rd

fl;;;i;and its thick lateral walls' At about this

as a dorsal "uugi'i^tio"

from the p'o*"t'""phu1o"' l"""il;;;ttfy pigmented or nonpigmented cells lying

continue to trace sections posteriorly and identify the ao"uito trt" tpi"i "ota

and beneath the skin ectoderm;

optic cups (photo iil."r,^[n "ptic

cup is derived from these are neural crest cells (Photo 2'14)' As you ap-

a laterar evagination from the prosencephalon, which p.ou"t it "

rirral sections in your set' identify the dorsal

secondarily iruug-ui"Jut it, ufirra end forming a dou- ^fi"

#;;" rpi""r cord (Photos z'r5-2'18\' The loose

ble-layeredopticcup.Thethickenedlayeroftf,eopticcellsformingitscoreareneuralcrestcells,whichinducecupisthesensory'"ti"";thethinlayeristhepigment'formationofthisstructure'Notethatthebodynarrowsed retina. The optic cups are connected to the prosen- prog;lu"iv J

"u"oal,levels; identify the ventral fin

cephalonbytheopt icstalks. .Notethattheinnerut t r ' " " l . ,aaiendofthebody(Photo2.1.8). Ident i fytheectodermal layer adjacent to the sensory retina has pro"roa""-, a ventral invagination of skin ectoderm

thickened to form the lens placode @hoto 2'10)' The it'"t'it "1"*

sections anteriir to the ventral fin (Photo

"oui", pr,g^""ted layer of the ectoderm will laterbe in- 2'I7)'

Frog Embryos 33

I41aIJ

Fry. 2.6. Schematic drawing of a midsagittal section of a 4-mm frog embryo.

h. Endodermal derivativesR.eturn to the level of the nasal placodes (photo 2.8)nd trace sections posteriorly. Identify a small cavity--,rns beneath the prosencephalon (Photo 2.10). This'. the cranial end of the foregut; its walls are formed:"rm endoderm. The stomodeum has invasinated to-a 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 open-mg. The outer part of the mouth is therefore lined bystomodeal 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 pharyngealpouches.) Continue tracing sections posteriorly. Theioregut narrows and then forms a prominent ventraler-agination, the liver rudiment (Photo 2.13). The levelof the foregut that is continuous ventrally with the liverrudiment is the duodenum. The liver rudiment sepa-rates from the duodenum and then fades out a few sec-

\otochordSubnotochordal rod.\rtusHindgutYolk-filled endodermal cellsLateral plate mesodermLiver rudimentPericardial cavity

9. Heart (ventricle)10. Oral membraneI l . Stomodeum12. Rudiment of the anterior

pituitary gland13. Infundibulum14. Prosencephalon15. Pineal gland

16. Head mesenchyme17. Mesencephalon18. Rhombencephalon19. Pharynx20. Midgut21. Spinal cord22. Neural crest cells23. Dorsal fin

tions more posteriorly. The remaining portion of thegut is the midgut (Photos 2.14,2.15).It contains a smallcavity bounded dorsally by a thin layer of endodermalcells and ventrally by a large mass of yolk-filled endo-dermal cells. Continue to trace sections posteriorly fol-lowing the midgut. It gradually moves ventrally andenlarges somewhat as the hindgut (Photo 2.16). (Insome embryosthe endoderm of the hindgut fuses withthe ectoderm of the proctodeum in more posterior sec-tions to form the cloacal membrane. The cloacal mem-brane ultimately ruptures to form the anus.)

c. Mesodermal derivativesReturn to the level where the infundibulum first ap-pears (Photo 2.11) and trace sections posteriorly.Identify a group of mesodermal cells, the notochord,lying beneath the rhombencephalon (photo 2.I2). Thevacuolated condition ofthe notochord is apparently re-sponsible for its rigidity, allowing this structure to serveas a longitudinal supporting rod in young embryos.Quickly trace sections posteriorly, noting the changesthat the notochord undergoes. It is smaller at caudallevels than at more cranial levels, and vacuolization isprogressively reduced toward the caudal end (compare

$

II

34 Chapter 2

Photos 2.1'4-1'.18). The caudal end of the notochord is

iess deu"loped ai this stage because the notochord. de-

velops in craniocaudal sequence' In postenor sectlons

laentify the somites (Photos Z'15,2'16)'paired blocks

of mesoderm lying ventrolateral to the spinal cord' AIso

identrfy u ,rnutl Juster of mesodermal cells lying be-

neath ihe notochord in caudal regions' the subnoto'

cfrorAaf rod (hypochord) (Photos 2'I5-2'I7)' Its

developmental significance is unknown'

Return to the level at which the liver rudiment is con-

tinuous with the duodenum (Photo 2'13) and trace sec-

iion, port"riorly. The mesoderm ventrolateral to the

somitis is usuaily in the form of more or less distinct

"fitn"U vesicles (Photo 2'14)' The round vesicle on

each side farthest from the spinal cord is the pronephric

duct. Just above the latter, and often continuous with

ii, ut" ttt" pronephric tubules' Thb pronephric duct and

tubules oi eachside constitute the pronephric kidney'

which is functional in amphibian larvae' The pronephric

kidney when well formed causes the body to bulge lat-

erad as the PronePhric ridge'

The lateral plate mesoderm (often difficult to identify

as a distinciarea) lies ventral to each pronephric kid-

n"y, U"*""" ttre itin ectoderm and the endoderm' This

mesoderm is in the process of splitting into an outer

iuyer of somatic -"*d"ttt',

adjacent to the skin ecto-

derm, and aninner layer of splanchnic mesoderm' ad-

jaceni to the endoderm' If somatic and splanchnic

mesoderm have iormed, identify a space (or a series of

small spaces) between them, the coelom'

Return to the level at which the foregut first appears

(Photo 2.10) and trace sections posteriorly' Identify the

"ot otrorr".tt (bulbus cordis) region of the developing

heart lying beneath the pharynx (PhotoZ'lL)' The heart '

, enlarges -as

the ventricle in more posterior sections

@hotlo 2.13). Note that the heart consists of an inner

luj"r, the endocardium, surrounded by an outer'thick-

eilayer, the myocardium' Both these layers are derived

from splanchnic mesoderm ' (Splanchnic mesoderm will

Iater iontrrbute to a third layer of the heart: its outer-

most covering.) Identify a thin layer of cells enclosing

the heart, the parietat pericardium, deriv edftom somatic

mesoderm. The parietal pericardium is usually sepa-

rated from the skin ectoderm by a shrinkage space' The

space between the heart and the parietal pericardium

is the pericardiat cavity. The heart is.suspended within

the pe'ricardial cavity by a dorsal bridge of splanchnic

*"*d"t-, the dorsal mesocardium'

The major blood vessels are in early stages of forma-

tion at ihi, ti*", and they are thus difficult to identify

with certainty in most embryos' Two major blood ves-

sels can sometimes be identified' The first aortic arch'

es mainly lie ventrolateral to the pharynx (PhotoZ'I2)'

(They will soon estabtsh connections with the conotrun-

cus via a pair of blood vessels that will lie ventral to

the pharynx, the ventral aortae' These latter vessels can

.o*Ltit*. be identified at this stage') The dorsal end

oi "u"tt

first aortic arch is continuous with a blood ves-

sel lying dorsolateral to the pharynx' the dorsal aorta

fii6r.V.nl. The dorsal aortae extend caudad andfade

i"i ut uU""t the level of the midgut (Photo 2'14)'

3. Summary of the contributions of the"

;;il +rs to.structures present in the4-mm trog emnrYo

Ectodermadhesive glands

auditorY vesicles

corneal ePithelium

dorsal fin

infundibulum

lens PlacodesmesencePhalon

nasal Placodesneural crest cells

oPtic cuPs

oPtic stalks

pineal gland

proctodeum

prosencePhalon

rhombencePhalon

rudiments of the anterior pituitary gland

semilunar ganglia

stomodeum

sPinal cord

ventral fin

Mesodermconotruncus

dorsal aortae

dorsal mesocardium

first aortic arches

notochord

parietal pericardium

pronephric kidneYs

somites

subnotochordal rod

ventricle

ffiderm[ttuJr(:€-;[1

'linriyg-:

!]rnlt.li"{i_:

i.Iiii€,: :!,;lnlent

illtlttriiul-i:d

ffiotemm and endodermj t . . iL:. =_'mbrane

lmtir. --rhrane

Frog Embryos 35

2.20 and2.24).

The lips of the blastopore form during gastrula stages(Photos 2.25-2.27). First the dorsal tip torms, followedby the lateral lips, and finally the veniral lip. The yolkplug continues to occupy the blastopore thro-ughout gas_trulation.

The neural plate forms and rolls up into the neural fubeb_etween the gastrula stage and early embryo stage(Photos 2.28-2.30). Concomitant with iormation of theneural tube, the embryo begins to elongate craniocau_dally. This elongation is an obviou, f"itrrr" of devel_oping frog embryos (photos 2.3I-2.32). The neural tubeextends throughout the length of the embryo. It bulgeslaterad at its cranial end to form the developing eyesin conjunction with the overlying skin ectoderm. Notethat the skin ectoderm in the ernbryos illustrated inPhotos 2.31,2.32 is covered with ciliary tufts. Thesestructffes establish currents around the embryos as theybeat, circulating fluids. These structures als-o functionin primitive locomotory movements before swimmingbegins. In 4-mm embryos the skin ectoderm at the cra_nial end of the embryo has invaginated in the midlineforming the stomodeum. Caudally, the skin ectodermis attenuated, demarcating the dorsal and ventral fins(Photo 2.32).

It sC{!{NING ELECTRONffiffimoscoPYffinryryuu* ::;rming electron micrographs (photos 2.19_.i,,i,,,Il :r; T{-_: "- ou r-isualize the shapes of developing frog,,rr|llUflllru. wf :::?n'os three-dimensionally. Note at cleav_lrullw lfiilrrs'::i Fhotos 2.19-2.24) the positions and orien_iiilllllltullll0lllnri :d' deerage furrows separating blastomeres and;l|ilw "illm:::-ces in sizes of the micromeres andriiisu'rrFnrslf'es. Boundaries between blastomeres becomeirunlri,iNr ,u*erll

-:led as cleavage advances (compare photos

I TER,MS TO KNOWrilllrliiin.& iilfii;{I|': klorv the meaning of the following terms, which appeared inI)rilii]lilfll iinff lrt-, :5-

boldface in the preceding discussion of

epiboly

epibranchial placode

epidermis

epiphysis

exogastrulation

eyes

female pronucleus

fertilization

first aortic arches

first meiotic division

first polar body

floor plate

fluorescein-labeled dextranfollicles

follicle cells

foregut

gastrula

gastrulation

germ layers

unur,mi, r:ipophtSiS

u,Ltriluex;-: giands

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

u,lrmrrr;r nlle

lrmrfiE:: -r pituitary gland

{lllJt L.iu}

idflll:tli;::iron

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]; gOnadotropin

"- ; i , "* -" i -El l ]& i .JIL\

***,0 ua- furrows

cloacal membrane

cloacal valves

coelom

conotruncus

corneal epithelium

cortex of egg

deep ectodermal layer

dendrites

diencephalon

dorsal aorta

dorsal blastoporal lip

dorsal fin

dorsal lymph sac

dorsal mesocardium

duodenum

ectoderm

egg capsule

endocardium

endoderm

m36 Chapter 2

germinal vesicle

gray crescent

hindgut

holoblastic cleavage

horseradish peroxidase

hypochord

infundibulum

inner ears

inner ectodermal laYer

involution

lateral blastoPoral liPs

lateral plate mesoderm

lens placode

liver rudiment

macIomeres

male pronucleus

mature ovum

mesencePhalon

mesoderm

metaphase

metencePhalon

micromeres

midgut

morphogenetic movements

mouth opening

myelencephalon

myocardium

nasal cavities

nasal placodes

neural crest cells

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 ectodermal laYer

ovaries

oviduct

ovulation

parietal pericardium

pericardial cavity

pharyngeal pouches

pharynx

pigment cells

pigmented retina

pineal gland

plasmalemma

posterior pituitary gland

primary oocyte

proctodeum

pronephric duct

pronephric kidney

pronephric ridge

pronephric tubules

prophase

prosencephalon

prospective ectoderm

prospective endoderm

prospective fate

prospective fate maP

prospective head mesenchYme

prospective lateral Platemesoderm

prospective neural Plateprospective notochord

prospective segmental Platemesoderm

rhodamine-labeled dextran

rhombencephalon

roof plate

rudiment of the anteriorpituitary gland

second meiotic division

second polar body

secondary oocytes

segmental plate mesoderm

semilunar ganglia

sensory retina

skin

skin ectoderm

somatic mesoderm

somites

sperm

spinal cord

splanchnic mesoderm

stomodeum

subnotochordal rod

superficial ectodermal laYer

surface ectoderm

telencephalon

theca folliculi externa

theca folliculi interna

total cleavage

trigeminal (V) cranial nerves

vegetal hemisphere

vegetal pole

ventral aortae

ventral blastoPoral liP

ventral fins

ventral suckers

ventricle of heart

vital dyes

vitelline membrane

yolk

yolk plug

young neurons

ilr38 Chapter 2

1. Pigmented cortex

2. Vitelline membrane

3. Blastocoel

4. Areaofgraycrescent

5. Shrinkage spaces

6. Nuclei

7. Blastomeres

8. Outer ectodermal laYer

9. Inner ectodermal laYer

@rrtt* 2./-2.5

Frog Embryos

felend10. Archenteron roof (mesoderm,

except at cranial end whereroof formed from endoderm)

11. Direction of epibolY

12. Dorsal blastoporal liP

13. Blastopore

14. Archenteron

15. Yolk-filled endodermal cells

16. Yolk plug

17. Ventral blastoporal liP

18. Neural plate

19. Skin ectoderm

20. Lateral plate mesoderm

21. Directions of cellular migrationto form endodermal roof ofarchenteron

22. Segmental plate mesoderm

23. Notochord

24. Archetteron roof (endoderm)

2.2t.

Photo 2.2. Frogdorsal lip gastrula (sagittal section). D, Dorsal side;Y Ventral side.

Photo 2.1. Frog blastula (sagittal section).AP, Animal pole;D, Dorsal side;VVentral side;VP;Vegetal pole'

Frog Embryos 39

Mtmto 2.3. Frog yolk plug gastrula (sagittal section). D, Dorsal side; V ventral side.

Flimtos 2-4t 2.5. Frog neural plate neurula (transverse section). Photo 2.5 is anenlargement of the dorsal:i-nion of Photo 2.4.

It|40 Chapter 2

1. Neural groove

2. Skin ectoderm

3. Lateral plate mesoderm

4. Archenteron


6. Neural fold

7. Outer ectodermal layer

8. Inner ectodermal layer

@/ntu*2.C2J)

Frog Embryos

9. Segmental plate mesoderm

10. Notochord

11. Archenteron roof (endoderm)

12. Pineal gland

13. Prosencephalon

14. Nasal placode

15. Mesencephalon

16. Pigmented retina of the oPticcup

17. Sensory retina of the oPtic cuP

18. Optic stalk

19. Rudiment of the anterior Pi-tuitary gland

20. Head mesenchyme

21. Stomodeum

Photos 2.6, 2.7. Frog neural fold neurula (transverse section). Photo 2.7 is an enlargement of the dorsal

portion of Photo 2.6.

Frog Embryos 41

Fhotos2.B,2.9.4-mmfrogembryoserial t ransversesect ions,, , ,

ilr-42 Chapter2

L.

2.

3.

4.

5.

6.

7.

8.

Mesencephalon

Future corneal ePithelium

Lens placode

Rudiment of the anterior Pi-tuitary gland

Foregut

Oral membrane

Adhesive gland

Stomodeum

@hrtug2.1O-2./3

Frog Embryos

'Yge/"{9. RhombencePhalon

10. Skin ectoderm

11. Semilunar ganglion

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 arietal Pericardium

26. Shrinkage space

Photos 2.1O, 2.1"1.posterior sequence.

ffifrogembryoserialtransversesectionsnumberedinanteriorto

Frog Embryos 43

lflflhmnnri I l l. 2.13.)i l l l l l | l i lufl ' - r:*--OC€.

Continuation of 4-mm frog embryo serial transverse sections numbered in anterior to

44 Chapter 2

1. Roof plate

2. Neural crest cells

3. Spinal cord

4. Floor plate

5. Dorsal aorta

6. Pronephric tubule

7. Pronephric duct

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

Frog Embryos

fgle',il

8. Pronephricridge

9. Midgut


11. Skin ectoderm

12. Liver rudiment

13. Dorsal fin

1-4. Somite

15. Notochord

16. Subnotochordalrod

17. Hindgut

l-8. Proctodeum

19. Ventral fin

2.15

Photos 2.14, 2.15. Continuation of 4-mm frog embryo serial transverse sections numbered in anterior to

posterior sequence.

Frog Embryos 45

Wflrllmlilrt : n m-1.18. Continuation of 4-mm frog embryo serial transverse sections numbered in anterior tol l l l l l l l l l l :1"1" * , : - - ; lC€.

46 Chapter2

@Dtu*2./3-2.21

Frog Embryos

1. Cleavage furrow

2. Blastomere

3. Micromere (blastomere)

4. Macromere (blastomere)

ofafrogtwo-ce1lstage(viewedfromtheside).AP,Animalpo1e;

VP, Vegetal Pole.

ffigelectronmicrographofafrogfour-ce1lstage(viewedfromanimalpoleandside).

Frog Embryos 47

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

rlllllllrnmrn . " a.

lllllllillililiiit"' I Il : -: :

S;rnning electron micrograph of a frog sixteen-cell stage (viewed from animal pole and side; alli:= :tot visible).

S;anning electron micrograph of a frog early blastula (viewed from animal pole and side).tllllllllnnrnrum * I l

,UfillN*n,i* :-:.1" S;anning electron micrograph of a frog late blastula (viewed from animal pole).

48 Chapter2

1. Yolk plug

2. Dorsal blastoporal liP

3. Lateral blastoporal liP

4. Archenteron

5. Ectoderm

6. Roof of archenteron

7. Blastocoel

-&otot 2.2o-2.3O

Frog Embryos

8. Blastopore

9. Future brain level of neuralplate

10. Future spinal cord level ofneural plate

11. Neural fold

12. Skin ectoderm

13. Future brain level of neuralgroove

14. Future spinal cord level ofneural groove

15. Future brain level of earlYneural tube

16. Future spinal cord level ofearly neural tube

Photo 2.25. Scanning electron micrograph of a frog dorsal lip gastrula. D, Dorsal side.

i

tiiI

l7III

t]

IIlIIl "L

photo 2.26. Scanning electron micrograph of a parasagittal slice through a frog yolk plug gastrula. D, Dorsal

side:V.Ventral side.

50 Chapter 2

1. Neural tube

2. Ciliary tufts

@tttu*2.3/-2.32

Frog Embryos

fgretu{

3. Developing eye

4. Dorsal fin

5. Ventral fin

6. Stomodeum

I

I

II,.i

It

h'6II

tPhoto 2.31. Scanning electron micrograph of a frog early embryo (dorsolateral view). R, Right side.

Photo 2.32. Scanning electron micrograph of a frog 4-mm embryo (lateral view). H, Head (cranial) end;!Tail (caudal) end.

rII

Ii Amphibian Development: Frog 4-mm Embryo (Stage 1g) g.l

olfactory pit prosencephalon

epiphysismesencephalon

optic cup

lens placode

pharynx

branchial cleft2

otic vesicle

branchial cleft 3

pericardial cavity

rhombencephalon

pronephric area

somites

spinal cord

notochord

endoderm

- :'!:r-.. r!stonrocleurn

adgland

l ivercl ivc'r ' t iL'u Iu nT

Figure 103

4-mm frog embryo (stage 18), whole mount (60K.

: .

bud

82 Amphibian Development: Frog 4-'nm Embryo (Stage 18)

stomodeum

adenohypoPhYsis

oral evagination

adhesive gland

thyroid rucliment

pericardial cavitY

heart

endoderm

vcntt'al mesoderm

Figure 104

epiphysis

prosencephalon

mesencephalon

infundibulum

rhombencePhalon

notochord

pharynx

liver diverticulum

spinal cord

midgut

subttotochordal rod

hindgut

tail bud

proctocleum t/

1

| +-mm frog embryo (stage '18), sagittal section (60K'

,lilflI

t

I--

Amphibian Development: Frog lO-rnm Tadpole (Stage 24) 101

l ip

nror-rth

pharynx

thyloid

heart

l iver

intestine

cloaca

anus

Figure 130

1O-mm frog tadpole (stage 24), whole mount (55X.

exterL.tnl nares

internal rrares

letina

lens .

otic vesicle

pronephros

stontacl-r

colcl

notochorcl

l tai l f i rr

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

lip

tooth

pharynxthyroid

infundibulum

heart

intestine

myotomes

telencephalon

diencephalon

metencephalou

myelencephalon

trachea

esophagus

notochord

dorsal aorta

myotomes

hindgut

spinal cord

tail fin

Figure 13L

' l O-mm frog tadpole (stage 24), sagittal section (35K.

104 Amphibian Development: Frog .t O-mm Tadpole (Stage 24)

telencephalon

olfactoly organ

tootir

Figure 133

l0 nrm Irog tacl l to le (stage 24), I ranSvcrse sect ion Lhrough the ol factory organ (5OX).

diencephalon

pignrentccl layer of l 'ct i tra

sensory layel of retina

cranial cartilage

cranial cartilatge

lyrnph sinus

Figure 134

10-nrm frog tadpole (stage 24), transverse section through the eyes (SoX).

rnuscle thyroid

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

myelencephalon

velar plate

atrium

notochorcl

opcrcLrla

Figure 135

lO-rrrnr l rog tar lpolc (sf . . tge 24), t t .anSVerSc sect ion through the heart (5OX).

ganglion, cranial nerve VII

gangl ion, cranial r rcrve V

pharynx

myelencephalon

ganglion, cranial nerve IX

pharynx

glottis

sinus venosus

opercular chamber

ot ic vesic le

notochorcl

gi l ls

intestine

Figure 136

lO-nrm frog taclpole (stage 24), transverse section througlr the glottis (sox).

106 AmPhibian DeveloPment:Frog ' l O-mm TaclPole (Stage 24)

myelen lion, cranial nerve X

pfonephfos

lung

esophagus

opercular chamber

liver

intestine

gallbladder

Fig=ure 137 .

1O-mnr frog tadpole (stage 24)' transverse section through ihe pronephros (50X'

esophagusmYeie

notochordpronePhros

Figure 138

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

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

spinalganglion

notoch

Figure 139

mesonephros

mesonephric duct

hindgut

peritoneal cavity

i O-mrn frog tadpole (stage 24), transverse section through the mesonephros (50rc.

spinal cord

spinal ganglion

notochordi

dorsal aorta

somite

hindleg bud

loaca

\ Figure 140\

' l O-mm frog tadpole (stage 24), transverse section through the cloaca (50K.

fin

t{,4Lq,

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Tooth germ

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CLEAVACE STAG"ES

lst cleavage funow

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MIDDLE GAST'RULA

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Macromliies

Micromeres

Blastobbel

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NIUITAL PLN'I 'E STACL:

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68 Arnphibian Deveiol : r r rentrFtog Cleovfrge/GnslrLrloliotl (St(r[|s$ *. I I l

outcr l i lyet .ol 'ectclc lernr

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Figure 87

Frog erlbryo, late gastr"ula (stage

lQn

1einner lnyer archerrtcron r.ocl [ 'of 'cctocle rnr (chclcla rncsodcr- lu)

rulcherr lcr 'on (glst rocoel)

b l l ts to l rorc

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veu tral of blastopor:e

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10), sagi t la l sect ior . r (100X)

2Frog EmbryosA. Introduction 25

B. How to Use Serial Sections 25

C. Oogenesis and Fert i l ization 27

D. Formation of the Cray Crescent

E. Cleavage and Blastulation 29

F. Castrulation 30

C. Neurulation 3' l

H. 4-mm Frog Embryos 32

l. Scanning Electron Microscopy 35

J. Terms to Know 35

K. Photos 2.1-2.32 37

2B

Documents

Frog Embryos