= Chick Embryo: Early Development By Antone G. Jacobson. ‘This account is ftom part of a chapter by Jay E. Mittenthal and Antone G. Jacobson presented at a NATO Advanced Study Institute on Biomechanics of Active Movement and Deformation of Cells held in Istanbul, Turkey. September 3-13, 1989, ‘The proceedings were published in a book, edited by ‘Nati Akkas, titled: Biomechanics of Active Movement and Deformation of Cells Springer-Verlag Heidelberg, 1990. In cooperation with NATO Scientific Affairs Division. CLEAVAGE, BLASTODERM FORMATION, AXIS FORMATION, AND HYPOBLAST FORMATION IN THE CHICK EMBRYO Development ofthe bir egg begins with ferlizaton ofthe ovum in the mou ‘ofthe oviduct. Eaveloping ayers form around the zygote as it passes down the ‘oviduct and enters be wterus. Inthe wiru, the zygote, which is a disc of cytoplasm atop a large yolk mass, cleaves inna thick disc of cells te blastoderm, with a cavity beneath Fig, 9A,B). The future anterior poster axis detemined hile the blastoderm sheds many ells ito the cavity beneath, and while oer cele intercalate from the lower layers into the uppermost layer, forming & single layered, more translacent region, in the central region ofthe blastoderm, ‘caled the are pellucida, from which the embryo will later arise (Figs. 9C; 10) “The egg it laid inthis condition and fuer development requires incubation. In = A Bitorlesmle Cleavage Furrow Fig. 9. Sections through the foture midline of chick embryos during cleavage, sis formation, and hypoblastfonmation. Postzrir is pte let and anterior to the right, using Von Baers rule A. Eaily cleavage. Eyal Giadi and Kochay stage. B. Cleavage has produced a blastoderm five to sx cll thick. Stage VI C. Blastoden ces of the lover layers have shed into he subgermial cavity, sd others have intrcalted nto the upper layer which snow but one ell hick, Stage X, asthe ogg is nid. D. After the egg is id and incubated, the bypobast tnd blastocoel have formed. ‘Stage XII All figures are drawn tothe scale ed, (Drawn from photos in Kechay and Eyal, 1980.) 4theft few hours of incubation, some cells ingress beneath the area pellucida and form a lower ier called the hypoblast (Figs. 9D, 10), We now describe these events in more deal, “The ovum ofthe chick, surounded by its vitelline meibran, i shed into the infundibulum ofthe oviduct where i sfrilized. During the approximately five and a half hours tha it akes the aygoe to travers he oviduct, layers of albumen and two shell membrane ae enwrapped about ie egg, The chick zygote consists of an island dise of eytoplasm atop the massive sphere of yolk. Cleavage begins after the zygote enters de uterus, The embryo takes about 20 hours wo pass the length of the uterus and be laid. While inthe uterus, the egg is rotated around its long axis by uterine musles a 10 to 15 ounds per hour, and this rotation has ‘ole inaxis formation, The calcareous shell is secreted by the wens “To study development af the embryo in the uterus, the en is surfed and he enclosed eggs examined as dane by Paterson (1910) and others, or the exes may be aborted manually for study (Kochav and Eyal-Gilai, 1971). The considerable evelopment ofthe egg that takes pce during the 20 hours spent inthe uterus and the fist few hours of inebation after the egg i laid has been desribed and staged (oman numerals by Eyal Gadi and Kochav (1976) and Kochay, eal. (1980). ‘The description that follows of this period of development i derived from thse papers, and from te reviews by Eya-Giad (1984) and Vakaet (19848). CCeavage is superficial and is inially incomplete. Cells are open to the yolk ‘undemeath and peripherally (Fig, 9A). The earliest cleavage furrows are all ‘vertical, bat eventually furrows spread beneath some ofthe central eels form closed cells, The item of cleavage is variable, Stages Ito VI are cleavage ‘stages and occur during te the ist cleven hours thatthe egg is inte wer. The ise f cytoplasm of the zygote i about 2milimeter in diameter. During cleavage te diameter of he disc deeeates somewhat while the thickness increases. By he end ofthe cleavage period (stage VD, te entre disc of cytoplasm ha ben cleaved into complet cells (Fig. 9B). The central areas five to sx cells thick. Beneath the dea eubgerminal Mui filed cavity as formed that separates the Blastoise {rom the yolk. The disc can now be considered a blastodesm. From this fat ‘blastoderm, ll ofthe parts ofthe embryo and the extrasmbryonie regions will ‘stage XI Fig. 10, Views of the ventral surfaces of chick embryos after hypoblast fomnation (Stage XIN, and later, afer accumulation ofa beige of eels hat wll fniite the primitive steak (Stage XIV). Koller’ sickle has been the souree of the posterior hypo els. Ameri (A) is atthe top, and posterior (Ps atthe ‘bottom. (Drawn from photos in Eyal Gilad and Kochav, 1975) ‘ooome segregated. During cleavage stages, there is no basement membrane (no detectable ‘Sbronectn of laminin, Mirani, 1982). During early cleavage, the nuclei lace ‘meleoi, but nucleoli form and begin to function by the end of cleavage stages yal Gila, 1984), Following cleavage stages, and during the rest ofthe period inthe utes (tages VII to X, 12 to 20 hours in the uterus), the area pellacida forms. yal-Gilad and Kochav (1976), Kochav er al. (1980), and Fabian and Eyal-Giladt (1961) deseribe the shedding of cells fom the deeper layers ofthe blastoderm into ‘he subgerminal cavity during formation ofthe area pellucida. ‘The shed cells ‘degenerate in the subgerminal cavity. Up to stage VI the cells ofthe lower layer are interconnected by filopodia, but at stage VI some of these cells withdraw their filopodia and begin to roundup (Fabian and Eyal-Gilad, 1981). The cells Begin1 sed at stage VIL. Shedding begins in the future posterior end ofthe embryo and proceeds anteriorly. By the time the egg is id at stage X, the formation ofthe area pellucida is complete and the border with the thicker area opaca (ibe outer annulus of blastoderm sill atached to the yolk) is sharply defined (Fig. 9C). During area pellucida formation the thickness of the blastoderm is reduced from five tosx els toa layer just one cel thick. Begining at stage VIL after the cleavage sages, the dameter ofthe Blasodi begins to increase. Cell division, ofcourse, continues during this period. ‘The literature isnot clear as to when cll division leads to growth, but Downie (1976) has observed tht cell volumes in tbe extraembryonic eps are halved with ect division during te first day of incubation after the egg is Ini, but thereafter cel divisions fllowed by a growth period that restores the size ofthe daughter cell to that ofthe mother eel, Iewould sem key, therefore, tha cell proliferation does rot prodace growth during uterine stages. This leaves open the question of how the blastodenm expands during these stages. One possibility is that expansion results when cells ofthe deeper layers nterealate wit cells ofthe surface layer ‘Vakact (1984) favors the view that deeper cells intercalate with ells ofthe surface layer during area pellcia formation, leading tothe expansion ofthe surface layer. ‘Both Vaket and Eyal-Gilad are likely corec, tha is, bot interaltion and cell ‘shedding may be oeurrng during area pellucida formation, ‘The newly aid hens ‘egg has a blastoderm tat is Am in diameter, compared to about 2mm in diameter in the zygote, Thus about a doubling of diameter ofthe blastoderm and 2 (quadrupling ofits surface area occurs uring later development inthe wens ‘The citi period for axis formation is from the end of sage VI to the middle of stage VII (14 to 16 hour inthe uterus) (Vintemberger and Clavet, 1960). “This perio coincides with area pellucida formation and cell shedding from the lower surface in the posterior regions ofthe blastoderm, The enteriorpostrior sof the embryo can be predicied withthe well-known nue of Von Baer (1828). ong the epg withthe harp end of the shel o your right, the anterior-posterior axis wll be at right angles tothe log axis of the shell, and withthe posterior end toward you, Nonmally the eg is nid pointed en fist. ‘Vintemberger and Clave: correlated the dreton of rotation ofthe eg in the eras withthe polarity ofthe axis. The future anterior end form in he direction of rotation, In an egg at t,he ctcularlasoderm sts atop the attached massive yolk, When the eg is rotated, the heavy yolk remains nearly stationary and the ‘Albumin, shell membranes and shell rotate about it. Kochav and Eyal-Giladi (Q971) have demonstrated tat gravity imposes the diection of the axis. They removed werine eggs and rotated them, observing the postin ofthe blasodem. ‘The blastoderm wasted somewhat inthe direction of rotation, and the posterior end always formed on the uphill side. They also showed the axis could be imposed by gravity without rotation by hanging th egg in a beaker of normal saline by one chalaa (the spiral of thik albumen at each end ofthe yoIK), or by {incubating whole egg in vertical positions, edber blunt end up or sharp end up. Ta theae cases, the yolk i unable orotate and the blastoderm ison one side rather ‘han on op and the uppermost side ofthe blastoderm always formed the posterior ‘end ofthe embryo, Ifthe blastoderm is kept ina horizontal postion with elation to gravity, then no ais is formed (Olszansta eal 1984). “When the egg is aid at stage X, after about 20 hours of development inthe eras the area pellucida has formed and the underside of the blastoderm in the area pellucida has begun te next phise of development, the fommaton of the hypoblast, Polyinvaginaion Gngression) of small cells from the upper epielist layer results in patches of epithelium onthe lower surface. The basal suface of ‘hie new epithelium is upward toward the basal surface of the epiblast. The ‘numerous investigators that have contibuted to this understanding are cited by Eyal-Giladi (1984, p. 249) A second source of hypoblast eels, the prospective posterior portion of the hypoblast, is frst seen asa concentration of cells in a crescent form (Koller sickle, Fig 10) near the posterior end ofthe are pellucida. [A shelf of hypoblas epithelium emerges from this sickle of cells, which have emerged from what willbe the posterior marginal zone (Spratt and Haas, 1965; ‘Azar and Eyal-Gilai, 1979), and extends anteriorly, becoming confluent with the patches of ngresed cells encountered along the way until the hypobast becomes compete, The overlying upet layer of basoderm then can be vermed the epiblast “The hypoblast doesnot extend peripherally to underlie the entire area pellucida “The border of the area pellucida that snot underlain by hypoblas is termed thenargnal zone, ‘The Important conclusion is that some hypoblast ces segregate hemselves from the epiblast by ingression at multiple sites, and a shelf of| fluent hypoblast cells emerging fom the future marginal zone athe posterior order extends anteriorly, joining with the patches of ingressed hypoblast countered along the way. By stage XI the hypoblat is compete (Figs. 9D, 0). “The hypoblast becomes separated from the epiblast by a closed cavity, the astocee, Probably this cavity begins to form as soon as any portion ofthe sypablast becomes complet, so it should be expected to appear first inthe osterior end where the hypoblast i frst a complete layer. The literature is not ear on this pont. Sodium pumping might be involved in blastocoe! formation, as vell asin creating the ion currets that flow out af the primitive steak to be cussed below. Hamburger and Hamilton (1951) have staged chick embryos commencing with peubston after the egg is Ind. Reference to their sages is made in roman pamerals, Stages X to XIV of Eyal-Gilad and Kochav (1976) fll within stage 1 of Hamburger and Hamiton, EPIBOLY piboly inthe chick embryo isa monumental ask and isnot completed until fer four day of incubstion. Eventually the blastoderm spreads to encompass the aire massive sphereof yolk. Epibaly doesnot bepin unl about eight to twelve pours of incubation (Downie, 1974). Cells ofthe blastoderm donot attach tothe jtelline membrane until iv to six hours of incubation (Vaket, 1962). Prior to en hours of incubation, cultured epiblas cells stick o one another only briefly. pat after ten hours of incubation, these cells stick to one another permanently Downie, 1974). The active cells inthe expansion of the epiblst are those that soupy the perimeter. These cells have localized adhesive sites on their outermost upper edges that adhere tothe inner surface ofthe overlying vitelline membrane, and this adhesion appears to be necessary for the migratory behavior of this ‘marginal bond of cells tha expands the blastoderm, creating consdemble tension on the ret ofthe epithelium ofthe epiblast. New (1959) found that blastoderm only expand when their margins are spreading, and a marginal band of cells Isolated from the rest of the blastoderm will expand by itself on the vitelline membrane, New (1959) also demonstrated the localization ofthe adhesive region ofthe marginal cells by placing blastoderm upside down onthe undersurface of the viteline membrane, afer which the blastoderm edge tured under itself asthe suhesive upper ede tached to and migrated onthe membrane. Atthe stat of epboly, the epiblast cells compose alow columnar epithelium, but soon the epiblast cells become flatened (squamous) and remain that way uring the period of epiboly (Downie, 1976). This atening is likely du to the tension in the blastoderm produced by the centrifuga expansion ofthe blastoderm caused by the edge cxls migrating agaist the Viteline membrane. The amount of tension would be affected by the balance between proliferation of cells within the bastoderm and the pullin ofthe edge cells (Downie, 1976). The active edge cols are a band about thre cells wide and layered two to thee cells deep (Downie and Pegram, 1971). These cells underlap one another 50 thatthe outermost perimeter consists of just the leading lamelipodia ofa single set of eutermos cells ‘These lamellipodia pull on the underside of the vitelline membrane. ‘Tyinkaus (19846) exically reviews chick eiboly and points out the problem of whether or not the underapping cells advance to tke a rum at pulling, and whether there is cll intercalation and de-interalation among the edge cells. Since the diameter of the rng of edge ces changes dramatically during epiboly, some mechani needed fr increasing the ring size until the blastoderm reaches the equator ofthe yolk, the decreasing he ring size thereafter. Individual cell behavior will have to be examined to settle these questions. Proliferation of eels cccurs in the blastoderm daring epiboly, but the edge cells arent dividing. Since these cells do not divide, itis necessary to assume that intercalation of cells occurs asthe ring of edge cells increases diameter. ‘The lack of proliferation in the edge cells is ‘consistent with the observation tat actively migrating cells donot divide.me INDUCTION OF THE PRIMITIVE STREAK Gastruaton ofthe flat blastoderms of bird embryos i accomplished through the activities of « morphological ent called the primitive steak. In this section we will examine how the primitive streak becomes induced in the epiblst by interactions with the undeying hypoblst. Waddington (1932,1933) concluded on the bass of rotation experiments that the hypoblast induces he tse movements in the epiblas that ead to formation of the primitive steak, Spratt and Has (1960) soggesed thatthe movements within the hypoblast influenced the formation of the primitive streak, In contrast, Byal-Giladi and Wolk (1970) found primitive sreak induction occured after they had interposed a milipore filter between the hypoblast and the epiblas, and they concluded that primitive steak formation sno dependent upon movements within the hypoblast. By rotation of hypoblast in relationship tothe epiblast in precisely staged blastoderms, and by heterochronc recombinations of hypoblast and epiblat, Azar and Fyal-Giladi (1981) assessed the temporal changes inthe inductive ability of hypoblst and inthe response capability ofthe epiblas to frm a primitive steak. They concluded thatthe hypoblast reaches a peak of ability to induce primitive steak at stage XIII (within stage 1). Within the hypoblast, ability to indace primitive streaks dissibuted in a gradient fashion witha maximum atthe posterior competent to respond to primitive steak induction at stage XL, with a similar maximum at the posterior end anda gradient of declining resposse Interally and anteriorly. When these two fields (the hypoblas and te epiblst) are rotated in relationship to one another at stage XI, the inductive hypoblast dominates in fixing teste of primitive steak formation, Following stage XI, competence of the epiblast to respond declines more rapidly than does te ability of hypoblas to induce primitive streak formation. Competence to form a primitive steak is limited to the posterior half ofthe epiblast at stage 2, and is gone at stage 3. The ability ofhypobas to induce a primitive steak in younger epiblas is til present atatage 3, “The ability to induce a primitive trek is restricted to that portion of the hypoblast that emerges from the posterior marginal zone. Azar and Eyal-Giladi (1979) removed the hypoblast from stage XII embryos. New hypoblasts regenerated and primitive streaks were induced, However, if bo the hypoblast andthe marginal zone were removed then the hypoblas that regenerated ould not Induce a primitive aueak, “They coneluded tha only that ponion of the hypoblast ‘hat rss from the marznal zone can induce primitive streak. Khaner and Eyal-Giladi (1986) rotted the hypoblast and epiblast in relationship tothe marginal zone, o relocated pices of the marginal zone from posterior to ltral regions and vice versa in embryos at stages X to XIL. From these experiments, they concluded that he ability to induce primitive steak resides ‘in the marginal zone at stage X, with a maximum atthe posterior end. Atstage XI, the posterior marginal zone is contributing cells to the inductive part of the Ihypoblast and the ability to induce the primitive streak is sifting from the marginal one ito the hypoblat. AL stage XI he hypoblas is maximally able to induce andthe epibas i maximally ale wo respond. Nevertheless, removal ofthe Ihypoblas at this stage is fllowed by egeneration of an inductive hypobst ifthe posterior marginal zone i il present. SUMMARY OF PREGASTRULAR EVENTS Cleavage divides a ise of cytoplasm atop the yolk int adic of cells. The cellular ise intlly becomes several cells thick, but many cells beneath the surface layer are shed in apolar way into te subgerminal cavity (between the ella ise and the yolk) where they appear to de, Other cells ofthe lower layers apparenly Imercalte into thé surface layer asthe aes of this upper layer expands. These events coincide with axis determination and occu while the egg is being rotated ‘within the uterus, ‘The yol and attached cellular die re tied with respect to _rovty daring otaton, and the axis i Fixed so thatthe posterior end ison the highest side ofthe cellular dis. After shedding and spreading, the blastodigc is Just one cell hick in the central region area pellucida), but thicker peripherallyrere itamaches ote yolk (are opaca). “The next event isthe formation of the hypeblast, which occurs when the egg is cubated after being laid. Cell ingress from the surface layer at many sites polyinvagination’) and form patches of prospective hypoblast. Other cells ers from the posterior marginal zone ofthe blastoise to frm a shelf of cells at then extends anteriorly, becoming confluent with the patches of hypoblast, fen encountered, The end esl isa fla blastoderm composed of epiblas (upper yer) and hypoblast (lower layer) with a fluid-filled cavity between (the lastocoel). These are the conditions when gastrulation begins. The posterior ypoblst induces the primitive steak which nts gstaation. STRULATION [At the beginning of gastrulation, all three prospective gexm layers of the nbryo are located inthe epiblast. The hypoblast is nota part ofthe embryo, and mosis involved in gastrulation only passively. The main oe ofthe hypoblast, to induce the primitive steak, How a flat blastoderm gastrulates is best understood inthe chick embryo. ster than a whole see of cells involutng ito the interior through a blsstopore, s in amphibian and other embryos, chick gastrulation is a more subtle savergence of epiblast calls, and ingression of endodermal and mesodermal cells om the epblast trough a primitive steak. In this section we will describe the ranizaon of morphogenetic evens that accomplish gastrulation inthis form, “Mesoderm indution an the earliest phases of nttion ofthe primitive streak cour daring stages X to XIV of Eyal-Gilad and Kochav (1976). As noted above, ese stages are early hases of incubation following the lying of the epg, and ince with Hamburger and Hamiton (1951) sage 1. Gastrulation begins after the embryo has formed an epiblast and hypoblast, ad during most of gastrulation, epiboly exens a cetifugal tension inthe epiblat. stage XIIT thee i a marginal one fee of hypoblast between the hypoblas and eaea opaca At th end of sage I at stage XIV), bre of cells aocumuates spanning the posterior marginal zone (Fg.10). These accumulating cells are the ‘beginning ofthe primitive streak (Eyal iladi and Kochav, 1976). ‘After is appearing asa massing of cells at the posterior end ofthe epibast the primitive steak extends anteriorly to is fullest length at stage 4 (Fig. 11) Daring the extension of the primitive streak, the entire blastodenn is distorted from ‘disc int a pear shape (Fig. 11). The new forms probably involve distortions of the epiblast in response to the centrifugal force of epiboly and the polar convergence occuring in the streak, and rearrangements of the cells in respect to their neighbors since a permanent record of tensions isnot reflected in te shapes ‘ofthe ces, most of which become isodamerc aftr brie distortions. “The exact mechanisms of steak formation are not known, but the movements ofthe ces have been deduced from fate mapping, sing maskers of various sorts, and from time-lapse cinematography (see Bellairs, 1971 for references). As indicated in the drawings (Fig. 11), the sea is inte as cells converge tothe posterior midline, and as convergence continues, the streak elongates slong the anterior posterior axis. This convergence-exension is much like that seen inthe ‘amphibian embryo and may be duet similar intercalation behavior ofthe cells However, a distinct difference inthe avian embryo is the massive ingression of calls that occue through te streak region even while the streak is elongating. As cells enter the steak, they deform, bocoming botle-shaped inthe steak, and camying the deformation tothe extreme of reducing apical surface oa point, then etaching as they ingress beneath the streak. Because the cells all change to a ‘ate shape, the cll density is considerably greater inthe streak than in the rest of the pias. [AS the primitive steak is forming, individual streak cells ingress through a ‘basal lamina (Mitrani, 1982; Vakaet, 1984) Steak cells begin bebbing basally ‘as they converge toward the midline ofthe streak (Vakaet, 19848). As the streak ‘forms, the bass lamina is lost beneath i (Sanders, 1984), When streak cells ingress, they detach from the epithelial epiblast and begin to migrate as ‘meseachyme ells, Coasderable behavior differences result from te conversion fom epithe cell mesenchymal cll pila cls have an pia-basl polarity and hey aac Iaerally oe to0 (botore egg is laid) Fig. 11. Fate maps onthe dorsal surfaces of chick embryos (A -F,H) at stages When te primi steak (doted areas) forms and extends. The stage numbers ‘re those of Vakiet (1962) followed (in parentheses) by the equivalent stages of “Hamburger and Hamiton (1951). In G and I the epblas is removed on the Fighthalf to reveal the underlying mesoderm and endoderm. He ectoderm, ‘Xial mesoderm, P = paraxial mesoderm, LP = lateral plate mesoderm, {endoderm ofthe embryo, X = extraembryonic mesoderm, MZ = marginal zone ‘Arrows indicat the dectons of cell movements as observed by Vakact (1984). ‘These figures area synthesis from maps of Vakat (1988) and Rosenguist (1966). Te dition of tsues win tbe primitive sueak is ourbest guess.another by specialize junctions and by eelcell adhesion. Cytoskeletal elements, including intermediate filaments and actin laments, attach within the cells othe junetons so that a structural Integrity exists across the whole epithelium. Mesenchymal cells migrate Individually or in bunches and in general ere surrounded by considerable extracellular matrix, Mesenchymal cells have no fixed spical-basl polarity. Contacts between cells arent extensive and are made and broken repeatedly, Nevertheless, cells that ingress trough the primitive steak to emerge as mesenchymal cells tat disperse beneath the epiblast are organized Immediately ino panes, “The mechanical propentes of mesenchyme and epithelium are quite different. Cells in an epithelium hae extensive interfaces and adhesive contacts between the lateral surfaces of the cells, and are tightly bound to one another by apc junctional complexes. Epithelial cells transmit ores to one anther dre, via interclilar junctions and sustain hese fores in the continuous eitelialshet by ‘way ofthe filaments aached to the junctions within the cells, Extracellular matix associated with eitelias mostly located as basal lamina atthe basl surfaces of the cells. Mesenchymal cells are largely surounded by extracellular matrix, and the cells make relatively few contacts with oe another, and such contacts are made and broken easily, Much of the mechanics of mesenchyme depends on ‘interactions between cells and extracellular mars. Traction by mesenchymal cells ‘may distort the extracellular matrix, and reciprocally, the cells may respond to {forces exerted upon them by the extracellular matic. ‘While the steak i still forming and elongating (sages 2 to 4) cells are ingressing. A population ofthe first cells to ingress itercalate into the hypoblat nd give isto the definitive endoderm. The orginal hypoblst els are displaced peripherally durin this process (Belair, 1953; Vakaet, 1962; Modak, 1966; Rosenguist, 1971; and others). As endoderm cells from the epiblast are ‘intercalated into the hypoblast, 2 masaic ofthe two cell ypes is formed and this mosaic persists forthe period of steak formation (Aza and Byal-Gilai, 1983). ‘One consequence of ti is that inductive hypoblat cells persist beneath the steak uring its formation. Eventually, aftr stage 4, these two populations sort out 30 thatthe hypoblat cells are peripheral, Such sorting out has buen observed ia explant combinations ofthe two cell ypes (Sanders, Bellas, and Portch, 1978). ‘The diplaced hypoblast becomes the endoderm ofthe yolk sae stalk (Rosenquist, 1972). By the middle of stage 3, the prechordal plate (the most anterlor axial resoderm) has formed asa round patch of cll in the midline anterior tothe streak, and to each side of the prechordal plate, the paraxial mesoderm has organized int circular domains that are the ist pir of somitomere (Triplet and ‘Meier, 1982)(Fig. 12). The cll of those strucures have just ingressed through the steak. By stage 4, a second pur of somitomeres is organized lateral to Hensen's node, and at sage 5, asthe streak begins regression (regression is a posterior displacement and shortening ofthe steak), addtional somitomeres frm in the paraxial mesoderm just Intra to the regressing node, andthe incipient notochord a the midline is laid down in segmental patches (Triplet and Meier, 1982) (Fig. 12), How the loose and wandering mesenchyme cells that compose these pattems manage to do so ssl unknowa, but similar pate ae found in nttruating embryos of six other species of amniotes, amphibia and teleosts (acobson, 1988). “The primitive steak ie wed up at egress and lays down the axial and more Interal mesoderm, As the sreak dsappeas the node moves tothe caudal end and its remnants are eventually incorporated int the al bud (se Bellas, 1986). MODELS OF CHICK GASTRULATION Ingression ofthe hypoblat could be the result of adhesive difeences between, epiblast and hypoblast cells, bu since many hypoblast cells seemingly ingress randomly, itis difficult to suggest how such adhesive differences aris. Alternatively, adhesive diferences could emerge between epilast and hypoblast cell afer ingression ofthe hypoblast cells, and the ingession coud be the result of eher factors. (Once te hypoblast i segregated and fonmed into a complete layer, a clgsed compartment is created between epiblast and hypoblst that could facilitate theStage 4 Fig. 12. Dorsal vews of chick embryos withthe epiblast removed fo show the Cistribution of axial and paraxial mesoderm at three Hamburger and Hamilton ‘sages. Tho primitive steak is indicued by the doted areas. Stage 3+, ‘extending sueak. Stage 4, maximal exeason ofthe seak. Stage 5, afer some ‘Sreakreresion. P= prechordal plat (Ine most anterior axial mesoderm, The ‘eles indicate somitomeres, andthe numbers within the icles the onder oftheir ‘appearance. At sage 5, notochord (shaded) les between prechoral plate and Hreak. (Modified from Jacobson, 1988). ‘organization of electrical currents that arse during primitive steak formation Primitive streak formation and stabilization may rely on these fon currents, The Primitive steak isthe st of exit of strong electrical currents hat enter though the epiblast (affe and Stem, 1982). These currents were measured with a vibrating probe. Stern (1982) has found thatthe epiblast has a transepithelial ‘potential such thatthe basal surface is more postive han the apical surface. ‘Sodium ions enter the apical surface passively and are pumped out atthe Dasolateral surface by a sodium-potassium ATPase. Water follows the osmotic sadint that the pump generates. Sodium and water accumulate in the cavity between he epbla nd the hypoblast. “The ATPare has ben localized by auloraiogrphy of plat after exposure 0 trite ouabain (Stem and Macksnzie, 1983). In the epblast, the oubain binds to ‘he basal surface, but binds basolatrally inthe forming steak, and binds onthe upper surface inthe formed primitive steak. The polity is thus reversed within ‘he steak. Experimental reversal of polaiy of electic currents through the pias results in positioning of extracellular matrix of basal lamina onthe upper ‘ther than the lower surface ofthe epiblas (Stem, 1982), These observations ‘may account forthe ingresson of cells through he steak. ‘Stem (1984) proposes thatthe epblast transpots sodium and water into the blastocoe! between its basal surface and the hypoblast, When the basal concentration of sodium or the hydrostatic pressure exceed a threshold, the soium-potassium ATPase stops pumping. This likely stars beneath the forming streak where the hypoblst is fist continuous. AS cells accumulate sodium, their junctions with adjacent cells open and they detach from the piblas. Calcium is released from intracellular pools an activates contraction of apical microfilament ‘bundles that then deform the cells oa flask shape and aids their tachment, The calcium also activates migratory activity and te cells ngres into the cavity below the eiblast. The opening ofthe junction allows sium and wate to flow tothe upper surface, a reversal of polarity in the cells, transport of soiam up trough the primitive streak, anda circulating curet tha ables and localizes the steak. ‘Transport of sodium by te epiblast could thus lead o formation and stabilization ‘ofthe primitive streak and ingresson of cells through it. Patterns of Adhesion and Fate Maps in a Chick Embryo Daring gastrulation there is considerable distin ofthe epithelial epibast. ‘These distortions ae sen in fate maps in Fig. 11. These maps, modified from Valact (1984) and Rosenquist (1966), ae synthesis fom previous maps made by following marked cells using vital staining, carbon marks, transplantation of ‘xls treated with tiated thymidine to non-treated hosts, or transplantations of calls between chick and qual embryos. Vaksct als followed movements with time-lapse video cinematography. These changes in the fate maps during ‘ustrulaton might be partly accounted for by adhesive diferences that lad 10 Sorting ou in the plane ofthe epiblat. Sorting out of eels in a two-dimensional ‘sue has been demonstrated by Garrod and Swinberg (1973). We propose a model invoking adhesive differences among the domains of prospective tissues sen on the surface ofthe epiblas inthe fat maps, The modeley generalized and modified from the differential adhesion hypothesis developed by Seinberg (1963; 1970), Many ofthe movements seen could be explained ifthe following hypotbeses were tue: 1) Te epiblast cont of ferent domains of cells, ‘The adhesive affinity between cll varies among the domain inthe fllowing hierarchy: Marginal zone (MZ) > Eetoderm (Ec)> Apical mesoderm (A) >Paraxal mesoderm (P)> Lateral Plate mesoderm (LP) > Extraembryonic mesoderm (X) > Endoderm (E). This proposed orders similar to that which hasbeen demonstnted for affinities among frog dexp cul, that is, ectoderm > mesodesm > endoderm, as discussed above. 2) The cells at particular level have a greater affinity for cells higher in the order than among themslves, and less fr ecll ower in the omer. 13) The cells ofthe epilast migrate within the plane ofthe eptelium and change contact relations with neighboring cells in ways that will increase the sengths of adesion oftheir contacts. +4) When the axi is determined, the prospective marginal zone becomes most adhesive atts posterior edge. Given the proposed adhesion hierarchy, the preincubation pattern is unstable snd neighbor exchanges may occur that wil erde the pattem and produce greater subilty. The pater tat can emerge is constrained by the original ordering to those new sors of contacts that cells can make, since cells ean interact adhesively caly with other cells hat they contact. For example, te highly adhesive marginal zone originally surrounds all the other tissues, and there is no way thatthe expected engulfment ofthe marginal zone by any of te other tissues it encounters Extraembryonie mesoderm and ectoderm both abut the marginal zone, but coder is much higher inthe adhesive hierarchy than this mesoderm, soas cells move randomly about atthe common boundary of ectoderm, extraembryonic mesoderm and marpnal zone, ectodermal wil replace mesodermal contacts with the marginal zone and the ectoderm-margial 2one boundary will increase in length. This would produce a convergence of ectodermal cells toward the posterior midline (Fig. 110), and concomitant rearrangements of cells within he mesodermal pions as thy become driven toward the posterior midline. = ‘The cells within the semi-annulus that represents the region of prospective ‘endoderm (Fig. 11A) ar ot oftheir hierarchical adbesive onder in elation bth 0 ‘he lateral plate ells to one side and the exraembxyonic mesoderm cells the ‘other, Lateral plate and extraembryonic mesoderm are higher in the adhesive birarchy than is endoderm, so they will adhere more with themselves and with ‘ne another than either will with endoderm, Similarly, the mesoderms should adhere more strongly with ectoderm than would endoderm, As cls move about, probably randomly, mesoderm cells wil replace endoderm cells a the ectoderm boundary, then the two mesoderm wll stay butted whenever they contact athe ‘expense of contacts with endoderm, The endodermal cells re inevitably driven Ito the configuration that provides the least boundary with surrounding ‘mesoderm, that i, a circle. The begining of tis process is seen in Fig. 11B, ‘This se of cellular rearrangements amounts toa convergence toward the posterior midline, augmented by the effects ofthe ectodemn rearranging along the marginal zone boundary Fig. 11C). ‘As the lateral-posterior ectoderm converges toward the posterior midline, a tension would be exeried onthe remaining ectoderm tat would draw the ectoderm ina wheeling motion away from the snterior midline and also pall onthe midline ofthe mesoderm, seicing i toward the anterior pole (arrows, Fig. 11C, D. ‘As cells converge toward he posterior midline, some cells pile up atthe ‘midline, forming the ineipient primitive steak (Fig, 11C). The arrival of excess cells atthe midline may relax locally some ofthe constant centrifugal tension ‘produced by epiboly, allowing the posterior edge ofthe blastoderm to bulge. The stretching of the forming primitive streak inthe anterir-posteror axis may be partly due to relaxation of tome ofthe tensions of epiboly, and partly due to polling aterorly by the ectoderm inthe midline there (but this topic is discussed further below). "The process of convergence produces a rounding up ofthe mesodermal and ‘endodermal areas that would reduce the lengths of some boundaries Between domains of cells of different adhesiveness, a should be expected, The oundiag 1p is distorted from circles by stretching in the anterior-posteror axis ashe primitive steak elongates (Fig. 11D, H). ‘This stretching inthe anterior-posterior0 tretione not accounted for by the adhesion model and willbe discussed further below. ‘Some distortion from a circle of the endodermal area is posible without increasing lis boundary with surrounding tissues because ingression of endodermal cells through the streak starting at least by Vakaet stage 4 (Hamburger and Hanslton sage 3) (Fig. 11D), reduces the aea of endoderm that remains on the surface. Likewise, some reduction ofthe area composed of endoderm inthe steak occurs when these endodermal steak cells elongate and reduce their apical areas, Extrammbryonie mesoderm ingresses trough teste possibly beginning even eae than the endoderm, ‘Praxil mesoderm and axial mesoderm begin ingressing through the streak between the stages shown in Figs. IE and 11H, The prechordal pat (xa) and te frst par of somitomeres (paraxial) are established during the stage shown in Fig. LIE, anda second pair of somitomeres is established bythe stage shown in Fig 11H (ile and Meier, 1982). ‘Maximum length ofthe primitive streak is reached a the stage shown in Fig. LIFE The steak then represses posteriorly while ingresson reduces it volume and length. Notochord is laid down in segmental units in the axis by the repressing sreak (Triplet and Meier 1982), and additonal paraxial somitomeres and lateral, pte are laid down laterally During streak regression, as ingession of cell reduces its volume, the boundary of mesodemn wit the more adhesive ectoderm is reduced as shouldbe expected. Eventually the eetderm converges posterior fo the regressing streak so the whole steak i surrounded by etoderm. ‘The hypotesis atthe axis is defined by an adhesion gradient highest inthe most posterior marginal ze is one simple way of relating the ste of primitive steak formation tothe determinative events of sxs formation that occurred in the wemus, ‘The bridge of cells that gather atthe posterior marginal zone (Fig. 10) 35 the forerunners ofthe primitive streak could gather thereby following an adhesion gradient. As the ectoderm converges toward the posterior midline, is cells would be running up an adhesion gradient. Until replacement by the ectoderm, the prospective extrembryonic mesoderm should also converge along the adhesion gradicot ofthe posterior marginal zone. ‘This hypotesis is not essential othe ‘ener argument ofthis model, ut it als some greater efficiency tit. Events That May Contribute to the Elongation of Streak Primitive ‘As noted above, the adbesion model does not account for the distortion of the endodermal region into a rod extending between lateral plate and pas ‘mesoderm inthe anteior sueak (Fig. 11D). Vakuet(1984a) states that endoderm ingresss through the aneror steak beginning ahs stage 4 (Fig. 11E). He says that the anterior srek is a mixture of endoderm and mesoderm with endoderm dominating at his stage 4, and mesoderm dominating at his stage 6. We have raw the tissues within the streak at these stages (Figs. 1C-H) as we interpret ‘hey may be. Thee isn fim data on their actual distbution, Since Triplett and ‘Meier (1982) have shown that some axial and paraxial mesoderm has sleeady ingressed by Vakaet stage 5 (Fg. 11F) tere must be at eat some ofthese tissues Jn the anterior steak atthe previous stage (Vakaet stage 4). In any event, the endoderm protrudes cranally between lateral plate and aranial mesoderm at some stages, and this is contrary to expectations from the adhesion model. Other events are occurring that may contribute to this arrangement ‘As the primitive steak forms, the cells that occupy the steak become densely ‘ecked because they assume botle shapes with greatly reduced apical areas. Itis in these cells thatthe ion curent isthe reverse ofthat inthe res ofthe epiblast (Giscussed above) Tis reverse on current, besides perhaps influencing the cells to ingress, might atleast emporaily moderate the adhesion characteristics of the streak culls. The streak calls lack the basal lamina found under the rest of the pila, so they can adhere only Iaterally, while theres f the epiblast cells adhere ‘basally to ahasal lamina as wel as to neighboring cells ‘Another factor affecting epiblat cells as tbey enter the streak i a possible response to chemical signaling. A microelectrode loaded with cAMP (cyclic !Menosine monophosphate) placed onthe ental surface of an explanted chek ‘embryo causes the elongating primitive steak to diver toward the source(Roberson snd Gingle, 1977). Robertson, Grtsh and Gingle (1978) also found that cick embryo calls rele cAMP when tinued with cAMP within a limited physiological range of concentration. Chemical signalling may thus have a roe in cell behavior during primitive streak formation, and this could override dbesive characteristes, If so, one would expect thatthe normal signalling source would be Hensen's node, ‘The directions of movements that would retult from the processes proposed ‘here snd illustrate in Fig. 11 are those hat are actually seen inthe epiblast during ‘astalation (Vakae,1984a, p. 78). Cell Migration Following Ingression, and Regression of the Primitive Streak After cells ingress through the primitive sueak, they then encounter a complex environment that includes variations in extracellular matrix and in adhesive properties of matrix and neighboring cols. The means by which the mesodemm cet become properly distributed beneath the ectoderm (Fig. 11G, 1) are still ‘unresolved (ee review by Bellars, 1986), but many studies have begun o define the nature of the extracellular matrix at these stages. Certainly one or more ‘components ofthe extracellular matrix, if properly distributed, could hep guide the migrating mesodermal cells 1 thelr proper positions. The cells could, for ‘example, be fllowing an adhesion gradient. The mesodermal cells could also be sorting out amongst themselves, or they could be moving laterally due to ‘population pressure’ and their fates beset by their order of ingression andlor by Interactions with the local envionment. These possibilities are not mutually ‘exclusive, 0 any combination of them, and possibly of others as well, could be in operation, ‘The primitive steak reaches its maximum length at Vakaet stage 6, ot Hamburger and Hamilton stage 4 (Fig. 11H). This stage is followed by 2 regression ofthe primitive streak posterally and a laying down of the body ais anterior to the steak as it regresss, Regression of the primitive streak i & posterior displacement ofthe entre steak, ad a simultaneous decrease in steak 30 length and volume. Steak regression probably Begins when the amount of ingresson of cells begins w exceed the adding of new eels to the streak from convergence of lateral epihast, ‘As axial mesoderm is put into place anterior to the steak, it begins or ‘continues its typical convergent extension movements and this may help drive the streak caudad. As explained below, the notoplate inthe midline of the emerging, ‘neural plate also does converpent extension, helping to elongate the axis. ‘The steak may also be pulled exuded by the centrifugal tensions of epiboly. COMPARISON OF CLEAVAGE THROUGH GASTRULATION IN ‘THE AMPHIBIAN AND THE CHICK ‘Axis determination bepin calor in the frog than in the chick. In both, an essential radial symmetry is converted to bilateral symmetry by processes that omally involve gravity ‘The entre egg cleaves complete cleavage") in many amphibian embryos, but eavage in the larger chick eg is limited tothe patch of segregated cytoplasm on the upper surface ofthe massive yolk, and cleavage of the patch is superficial and incomplete, Inthe amphibian with complete cleavage, a spherical blasula with an ‘internal cavity, the blstocoel, results. The end result of cleavage inthe chick eg ‘sa flat disc of cellular blastoderm over a subgerminal cavity. In the chick, the ‘astooel is formed later betwen a later forming bottom layer, the hypoblst, and the overlying epiblast. ‘Sodium pumping by the prospective bastocoel roof may have a role in ‘biatocce! formation inthe frog embryo, and posibly also inthe chick embryo. ‘Once the eck forms a closed blastocoe, sodium pumping creates fon currets that ‘could help form and stabilize te primitive steak. There ae ion currents in amphibian gatula that may also affect blastopoe formation and gastrulation. [Nieuvikoop (1968) points out thatthe timely appearance ofthe blatocoe! of the amphibian embryo prevents the entre ectoderm from being induced to fogn ‘mesoderm by induction from the vegetal endoderm. Once the blastocoe i incece nly the ectoderm tha abus the vege endoderm around the equator is induced to form mesoderm, and one special region of that endodera (iewwo's organza) induces the axa wea. Inte chick embryo the mesoderm is induce by hypobls tat emerges asa sheet fom the postelor prospective marginal zoe rom the region of Koll icles Fig, 10), The leading edge of he posterior hypoblst (exirembyenic ‘doderm) i comparable inthe chick o Neuwkoop' ngnizr nthe amphibian, “This leading edge induces axial mesoderm from areas closest to he center ofthe blastoderm, and paraxial mesoderm, lateral plate, embryonic endoderm, tnd ‘extraembryonic mesoderm ae Induced from regions successively more piper ‘Spemanas organizer, located onthe dos ip of the Mastopore in th arphibiar carly gastro, i locted in Hensen's node atthe anterior end of dh primitive streak inthe chick embryo, In both cass, transplantation ofthe organizer tc others in the early embryo may induce a second axis. ‘The amphibian embryo has boule coll that nate Bastopore formation. The capable cell in the chick embryo woald be the anterior endoderm cels, These calls ingress eather than fvaginate, except they may a5 they shrink heir plea surfaces before ingressing, elp form the pit and furrow ofthe steak. Whether cells that becomes bot shaped ingress cr cause the eptbellum fo invagiat ely eto ight dtferencsin he adhesive reladonsips wi thei neighbors. Daring gastlation in both ampibin and chick, rearrangements of the ‘rorpectve ectoderm, mesoderm and endoderm may be guided by adhesive Altfereaces among the diferent domains of cells. Once the mesoderm ani endoderm are internal, by invagination and involution of cells trough th asopore in the amphibian, orby convergence and ingresson of el rough b= primive sueak inthe chick, the mesoderm migrates extensively under the ‘lastooel roof ar the epblast Migration ia etch exer it on an extracel ‘erae, he basal Tamina of the amphibian Mastoeoel roof or te basal lamina cf ‘he chick epblast. 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