Xenopus staging landmarks
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Developmental ProcessNF stage number NF stage nameLANDMARKS
n/amature egg1 cell; ovulated unfertilized egg; animal hemisphere dark, vegetal hemisphere light; animal-vegetal axis in random orientation; soft texturelarge nucleus, 'germinal vesicle' in animal hemispherenanos1: mitochondrial cloud; vegt: vegetal cortex
11-cell, fertilized egg1 cell; vitelline membrane swells; eggs rotate with dark animal hemisphere up, light vegetal hemisphere down; firm texture; sperm entry point indictated by pigment condensation; germinal vesicle breakdown leaving pale spot in animal hemispherecleavage has not begun; germinal vesicle breaks down and pronuclei fuse; visible cortical layer thickest on animal and dorsal sides.
2- first cleavage begins as a top to down furrow in animal hemispherecleavage furrow has not yet reached the vegetal pole
22-cell2 cells; 1st cleavage plane is meridional along the dorso-ventral plane dividing right and left halfs sox3, atp4a: animal hemisphere; vegt: vegetal hemisphere
34-cell4 cells; 2nd cleavage is meridional, perpendicular to the 1st cleavage; in many embryos, the 2 dorsal blastomeres (left and right) are smaller and lighter (on animal surface) than 2 larger darker ventral blastomeres (also left and right)[1]cleavage cavity present
48-cell8 cells; 3rd cleavage plane is equatorial giving 4 smaller animal blastomeres (micromeres) and 4 larger vegetal blastomeres (macromeres); animal dorsal cells are lighter and ventral cells are darker in some embryosnanos1: germ plasm; hwa: dorsal Wnt signalling
516-cell16 cells; 4th cleavage is again meridional; animal blastomeres smaller than vegetal blastomeres; dorsal blastomeres lighter than ventral.
632-cell32 cells; 5th cleavage equatorial, giving 4 rows each with 8 blastomeres; animal pole with smallest rosette of micromeres, 2 middle rows of irregular shaped cels, larger vegetal rosette of macromeres
6.5morula64 cells; 6th cleavage; cleavages becomming asynchronous; animal/dorsal blastomeres divide before vegetal blastomeresgdf1: vegetal hemisphere; shroom1, tfap2a: animal hemisphere
7early blastula128-512 cells; 7th-9th cleavages, no longer possible to count cells reliably; size of animal cells used to distinguish embryo stageclear delineation of the 3 primary germ layer tissues; notochord begins to form from mediolateral convergence of dorsal presumptive axial mesoderm under the neural ectodermnodal5 (nr5): earliest zygotic transcription
8middle blastula~1000-4000 cells; 10-12+ cleavages; cell number no longer a stage guide; animal hemisphere with small dark pigmented cells refered to as 'animal cap'; animal surface looks 'pebbly'at NF stage 8.5, after 12th cell divisions the mid-blastula transition (MBT) occurs; asynchronous internal cell division; zygotic genome activation gs17, nr1: major initiation of zygotic transcription
9late blastula animal hemisphere still darkly pigmented; animal surface more like 'grains of sand' than 'pebbles' of NF stage 8blastocoel now maximum size; 3 germ layers becoming distinct; ectoderm in the animal 1/3 of the embryo, a ring of mesoderm in the middle and yolky endoderm on the vegetal 1/3 sox17a: endoderm; tbxt: mesoderm
10initial gastruladarker pigment from bottle cells on the dorsal vegetal surface indicate dorsal blastopore lip formation, between '11 & 1 o'clock'; blastopore groove where cell ingress, is not yet visibleinitial formation of Spemann Organizer at the dorsal marginal zone/upper blaspore lipgsc: upper blastopore lip; tbxt: marginal zone
10.25early gastrulablastopore lip extends laterally ~1/4 around the circumference, from ~ '10 to 2 o'clock' ; indentation of blastopore visible as a groovemarginal zone involutes on dorsal side; internally, dorsal endomesoderm begins to migrate anteriorly along bastocoel surface; vegetal rotation of endoderm mass
10.5crescent-shaped blastoporearch of pigmented blastpore lip extends laterally from doral to lateral sides ~1/4 to 1/2 around the circumference; yolk plug almost roundmedial-lateral intercalation of dorsal mesoderm; 'Brachet's cleft' forms between internal leading edge endomesoderm and the dorsal mesoderm; neural induction begins, dorsal-ventral patterning chrd.1, lhx1: involuted dorsal mesoderm; ventx2.1, wnt8a: ventro-lateral marginal zone; hhex: anterior endomesoderm
11horse-shoe shaped blastoporepigmented blastopore lip ~ half the circumference, pigmented bottle cells extend to the ventral side; yolk plug ~1/2 diameter of embryo, is slightly elongated in the dorsal-ventral directionBrachet's cleft begins to open forming the archenteron; ectoderm, mesoderm and endoderm germ layers specified; blastocoel becoming smaller, mesodermal mantle undergoes convergent extension and endoderm is internalizedsox17a: endoderm; tbxt: mesoderm; sox2: neurectoderm; krt12.4: non-neural ectoderm
11.5large yolk plugblastopore lip extends all the way around yolk plug; lower/ventral blastopore lip more darkly pigmented; yolk plug not quite round, ~1/3 of embryo diameter elongated in the dorsal-ventral directionblastocoel displaced to the ventral side; involuting 'endomesoderm' mantle continues to extent anteriorly
12medium yolk plugareas of light and dark pigment radiating from yolk plug (flower petal like); yolk plug small and circular, a little less than 1/4 of egg diameter, diameter decreasing; neurectoderm transforming into the discernible neural plateblastocoel begining to close as the archenteron expandssox2, sox3: neural plate
12.5small yolk plugdarker pigment lines on dorsal surface indicate future neural groove and neural plate; blastopore slit is slightly open, diameter decreasing and oval shapedclear delineation of the 3 primary germ layer tissues; notochord forms from mediolateral convergence of dorsal axial mesoderm under the neural ectodermchrd.1, nog, shh: axial mesoderm, notochord
13slit blastoporeblastopore completely closed to a 'slit'; neural plate on dorsal side clearly outlinedneural crest form at the neural plate border, i.e., the boundary between the neural and non-neural ectodermsox2, sox3: neural plate; ednra, msx1: neural plate border; snail2; neural crest; tbx2: cement gland primordium; myf5: paraxial medoderm; rax: optic field
13.5initial neural platesharp demarcation of anterior neural plate; anterior aspect of neural plate bent down; yolk plug internalizedtubb2b: differentating neural progenitors; msx1: anterior neural plate border
14neural plateneural plate obvious; dorsal midline thin with neural folds thickening anteriorly and laterally; neural folds begin to elevate; convergence extension begins to narrow neural plate posteriorlyblastocoel continues to close in the ventral foregut region as archenteron expands on the internal dorsal sidepax6: optic field, neural plate, lens placode; pax2: between anterior and posterior neural plate
15early neural foldneural folds distinct; anterior neural fold round; demarcation of neural plate clear caudally, narrowing in middle and caudal regions; neural groove deepens; pigmented cement gland primordium faintly visible at the ventral-anterior border of neural plate, cells stand out from epithelial layer.physical segregation of cranial neural crest from anterior (pre-chordal) neural plate; blastocoel closes in the ventral foregutpax3: neural fold/neural crest; pax8: intermediate mesoderm; rax, otx2, six3: optic field; hhex: foregut endoderm
16mid-neural foldeye primordia [2] become discernable as indentations near the lateral edges of the anterior neural plate; anterior neural plate 'rectangular' in shape; neural plate sharply constricted in the middle.right and left cardiac mesoderm migrates to anterior-ventral midline; foregut diverticulum formspax6: optic field, anterior neural plate, and lens placode; nkx2-5: cardiac progenitors; krt12.4: ectoderm, non-neural ectoderm, epidermis
17late neural foldanterior neural plate oblong, triangular, angles formed by eye primordia [2]; clear neurenteric canal along midline, posterior end of which continues over closed blastopore slit; neural folds closing in trunk regiondelineation of cranial neural crest lateral to the anterior neural plate; 1st indication of somite segregation from presomitic mesodermsox2, sox3: neural plate/neurenteric canal; otx2, rax, pax6, six1, sox2: optic field; lhx1: intermediate mesoderm
18neural grooveanterior part of neural plate narrow, club shaped; parallel neural folds very close but not touchingneural crest segregation begins; 3-4 anterior somites segregate from paraxial mesodermmyod1: early somites and presomitic mesoderm; casz: early somites; snai1, egr2, twist1: neural crest; pax8: otic placode.
19initial neural tubeneural folds moslty closed forming a neural tube, except for an open slit in the anterior neural plate; neurenteric canal deepening; dark pigmented cement gland primordium immediately ventral to anterior neural plate; laterla view of embryo convex oval, not elongatedmigration of 4 neural crest streams begins; 4-6 anterior somites segregated ag1, agr2: cement gland primordium
20fused neural tubeneural tube fused anteriorly; no pharyngeal bulge visible; dark oval cement gland primordium below border of anterior neural plate; embryo starts to elongate; optic cup (eye primordium) forms; oral evagination (mouth) visibleneural crest extends to front to eye; paired glomus primordia present [2] ; anterior 6-7 somites; thickening of cardiac mesodermrax, pax6, otx2, six3: optic vesicle; wt1, rgn: glomus; nkx2-5: cardiac progenitor cells; egr2: rhombomeres R3, R5, and neural crest; en2: midbrain-hindbrain boundary; snai2: neural crest; chrd.1, nog, ssh: notochord
21neural tubeembryo has a dorsal curvature, with flat ventral surface; one pharyngeal arch bulge; neural tube completely closed; optic vesicle ('eyes') begin protuding, forming 2 oblique 'oval spots'; multiciliated cells form on the surface of the epidermis8-9 somites; first indication of pronephric mesenchyme; otic placodes form posterior to optic vesicle pax8, lim1: pronephric mesenchyme; nrp1: neural tube; pax3: hatching gland; tuba4b, cfap206, foxj1: multiciliated epidermal cell
22early tailbudembryo begins to elongate (convergent extension); ventral surface slightly concaved; two pharyngeal arch bulges; distinct eye protrusion; anal opening displaced to ventral side 9-10 somites; ventral blood island forms; segreation of forebrain, midbrain and hindbraingsx1, sox3: brain segments; gata1, hba3, tal1: ventral blood island; nodal1: lateral plate mesoderm on the left side only; tbx6, foxd4l1.1: early tail bud
23early tailbudventral surface concave giving embryo a 'coffee bean' look; two pharyngeal arch bulges; olfactory placodes thicken between eyes; slight depression of otic placode; jaw and gills separated by groove; "inverted Y shaped" hatching gland between eyes to cement gland12 somites; forebrain regions telencephalon and diencephalon distinguishable cxcl14, astl3a.1, pax3: hatching gland; myod1: somites and presomitic mesoderm; six1: olfactory placode; pax2, pax6, vax2: diencephalon; foxg1: telencephalon
24early tailbudnoticable elongation of the embryo and tail bud outgrowth; in dorsal view, eyes protruding out laterally less than gills; gill primordium area smooth (ungrooved) 15 somites; primary germ cells detecable in cell trunk endoderm; primary and secondary heart fields indictated in heart primordiuminitial motor reactions to external stimulitbxt, lmo2, aplnr: tail bud; tnni3, nkx2-5: primary heart field; bmp4: secondary heart field; grip2, pgat: primordial germ cells; pcdh8.2: otic vesicle and tail bud; eya2: otic vesicle
25early tailbudembryo still convex dorsally and concave ventrally; eyes protruding out laterally equal to or more than gills, gills now grooved; otic vesicle pigmented16 somites; head somite 1 diminished; brain flexure ~90omulti-cilliated cells in the epidermis become active ag1: cement gland; tubb2b: brain and spinal cord
26tailbud If liberated from the vitelline membrane the embryo is straight, not convex dorsally; if the embryo remains in the vitelline membrane it is curved laterally; tail bud obvious; otic (ear) vesicle protruding 17 somites; head somite I disintegrated; pronephros distinct; myotomes distinctspontaneous movements beginpax8, hnf1b, irx3: pronephric mesenchyme
27tailbud tail bud defined in lateral view; fin translucent; lens begins to form, eyes flatten laterally; otic vesicle closesheart fields merge forming a triangular-shape at ventral midline, behind the cement gland primordium and anterior to liver diverticulum; 19 somitespax6, sox3, prox1, foxe3, nrl: lens; neurod1, sox3: epibranchial placodes
28tailbud tail bud elongates distally and extends downward to cloaca; fin divided into outer transparent (outer fin) and inner translucent bands (inner fin); black cement gland fully formed; otic vesicle separates from epidermis heart primordium and pericardial cavity discernible; pronephric nephrostomes form; 20-22 somites; epibranchial placodes first segregateembryos liberated from the vitelline membrane glide around due to multi-cilliated cell fluid flowhand2, actc1: endocardial tube; dlx2, sox9, sox10: cranial neural crest; pax2, lhx1: nephrostomes; neurog2, eya1: epibranchial placodes; fgf8, sox9: otic vesicle
29 & 30late tailbudtail bud distinct; outer fin edge transparent over entire length; gray disc of the eye cup now visible 23-25 somites segregated to end of tail; lumen in pronephric kidney collecting duct; appearance of glomus and thyroid; neural tube closure has formed the spinal cordrunx1: olfactory placode; lhx9: brain segments; foxa2, tubb2b: spinal cord; nphs1, wt1: glomus; nkx2-1: thyroid primordium; not: tail tip
31late tailbudtail bud equal in length and height; nasal/olfactory pits first indicatedheart primordium extends ventrally and bends slightly to right; 22-23 post-otic somites; midbrain-hindbrain boundary distincthey1, myod1, actc1: somites; en2, pax2, fgf8: midbrain-hindbrain boundary
32late tailbudtail bud ~1.5x longer than height; eye cup distinct, U-shaped (open); mouth primordium not visibleheart a linear tube with anterior outflow tract, left ventricle, atrioventricular canal and atrium; 26 post-otic somites; pronephric nephrostomes form; lung buds visiblepax2, vax1: optic stalk; cfap161: nephrostomes and multiciliated epidermal cells; nkx2-1: lung and thyroid progenitors; aldh1a3, agr2: otic vesicle
33 & 34late tailbudtail bud ~2x longer than height; gut ~3 x longer than tail; eye cup open C-shape with darker pigmentation dorsally; mouth primordium a shallow vertical groove; 32 post-otic somites; pigmented cells (melanophores) first appear on head (near hindbrain) and anterior trunk (near pronephric kidney)heart looping begins; heart beat clearly observable; foregut begin to constrict at trachea-eophagus boundary; cranial nerves distinct; thyroid primordium discernible [2]; pronephric kidney and duct formed with surrounding vasculature; thyroid primordium detectabledab2: pronephric sinus, posterior cardinal vein; sox2: dorsal foregut; tubb2b: cranial nerves; myl2, bves: heart; actc1, mybpc3: heart and somites; pax2, lhx1: pronephric kidney and pronephric nephrostomes [2]
35 & 36free swimming tadpole [3]tail bud ~3x longer than height; gut ~2 x longer than tail; outline of the proctodeum still curved; optic vesicle/retina completely black, choroidal fissure open; cardiac mesoderm starts to spontaneously contract; mouth invagination not quite round; 2 gill lobes; 36 post-otic somites; melanophores extend over top of head and along dorsal trunk heart S-shaped, with distinct atrium lying dorsal to ventricle; vasculature to head and tail developing; liver bud visible posterior to heart; pronephric duct fused with rectal diverticulum, pronephric nephrostomes obviousembryos naturally hatch from vitelline membrane to become free swimmingaplnr, hbz: blood vessels and heart; onecut, nr1h5, hhex: liver; aldh1a1: pronephric kidney/duct, choroidal fissure and olfactory bulb; tal1, hba3: ventral blood island; nkx3.2: mouth primordium
37 & 38free swimming tadpolegut almost same length as tail; eye's choroid fissure closing ventrally but remain open; mouth invagination deep, round-shaped; heart contractions obvious, blood flow visible; proctodeum at obtuse angle (~140 degrees) to tail somites; 40 post-otic somites; melanophores extend over tailpaired lymph hearts; entire pronephric kidney functioning; ventral bud of pancreas formed; tnni3: heart; sftpc: lung buds; myh6: lymph heart; atp1a1: pronephric kidney and pronephric duct; nkx2-1: thyroid and lungs; foxe3: thyroid and lens; ptf1a, pdia2: pancreatic buds
39free swimming tadpolegut equal in length to tail somites; melanophores around nasal pits & along ventral edge of tail somites; ventral choroid fissure nearly closed; proctodeum at ~125 degree angle to tail somites; 43 post-otic somitesretinal ganglion cell axons reach optic tectum; mesonephric kidney begins to formbmp4, hoxa13: proctodeum; map2, pou4f1; retinal ganglion cell layer; insm1, nos1: optic tectum
40free swimming tadpoletail now longer than the abdomin; optic choroid fissure completely closed; mouth opening 'breaks through'; stomach and pancreas visible on the left side of gut; proctodeum at 90 degree angle to tail somites (lateral view), ~45 post-otic somitesgall bladder primordium formed and sometimes visible (irridescent on ventral view); blood circulation in gills visibleembryos begin taking gulps of air from the surface cela1.2: pancreas; sfrp5, klf5: stomach; onecut, hhex, sox17a: gall bladder; hhex, nr1h5: liver
41free swimming tadpoleconical shaped proctodeum formed, at angle of ~ 60 degrees to tail somitesmyocardium thickens and develops trabeculae, atrium posterior to ventricle; torsion of gut starts; post-anal gut disappears; the pancreas, now visible in ventral view, posterior to left-sided stomachclcnkb: pronephros; ins: pancreas; myod1: tail somites; s1pr1: brain segments; nkx2-5, tpm1: myocardium; hoxa13: proctodeum
42free swimming tadpoleopercular fold first visible; head somites I and II disappearedtrachea and esophagus seperatesox2: esophagus and stomach; nkx2.1: trachea and lung buds; sftpc: lung buds
43free swimming tadpolecement gland starts to lose pigmentation; lateral line pits visiblestomach has lengthened further; pancreas shifted to right side [5]; duodenum formed by 1st gut coil constriction to anterior-right; midgut and hindgut form hairpin curve, visible on left side (will become the 'apex' of future intestinal coil)foxq1, spdef, bmpr1a, cfap161: stomach; cela1.2: pancreas; aldh1a2: duodenum;
44free swimming tadpoleheart fully formed and clearly visible; barbels/tentacles start to grow; gills/branchial basket shrinkingseptum begins to form in cardiac atrium which is slightly anterior to ventricle; midgut and hindgut lengthened more; the intestinal apex visible in ventral view as a "U" shape in the upper left quadrant of the gut cavityvisual avoidance behavior beginstnni3, frzb, sox9, nkx2-5: heart; cdx2: midgut-hindgut
45feeding tadpole [3]operculum partly covers the gills, hindlimb bud not visiblemidgut and hindgut continue to lengthen; the intestinal apex begins to rotate inward in a counterclockwise trajectory; spleen forms; mesonephric kidney tadpoles to start swim continuously and begin feeding darmin, a2m: midgut, hindgut and liver; nkx2-5: spleen primordium
46feeding tadpolecrescent-shaped hindlimb bud first appears although is difficult to see; pigment cells appear on eye and around abdomen; trunk somite 1 disappearedmidgut and hindgut lengthen further- apex continues to rotate inward, forming multiple coils of intestine; blood circulation to gills diminishingfood can be seen in intestine as now feeding
47feeding tadpoleirredescent gold-coloured abdominal wall surrounds coiled gut; blood circulation visible from heart to gills, and through paired dorsal aorta; cement gland starts to degenerate; barbels/tentacles longerretinal ganglion cells have formed complex synapses with optic tectum neurons; thyroid gland begins to function; thymus gland detectablefoxn1: thymus
48hindlimb bud now clearly visible, with nearly semi-circular shaperetinal ganglion cells-optic tectum synapses more compactfgf8, spry1, sall4: hindlimb bud
49hindlimb bud length equal to it's widththyroid follicles first appear
50hindlimb bud slightly constricted at base; tiny oval forelimb buds just visiblegonads undifferentiated hoxa13: forelimb bud; spry4: hindlimb bud
51hindlimb bud is cone-shaped; forelimb bud is oval shaped (in lateral view)resorption vacuoles in thyroid follicles first appearhoxd10, hoxa13, hoxa9: hindlimb bud
52hindlimb bud with slight 'wrist' indent; forelimb bud slightly constricted at base 5 complete coils of the intestine (internal and external coils)regeneration competent [4]sox9: hindlimb digits (cartilage elements)
53hindlimb bud paddle-like, with wrist constriction, hindlimb digits not discernable; forelimb bud with slight wrist constrictiononset of sexual differentiation of gonadsregeneration competent; athyroid animals have arrested development
54hindlimb bud length (not including foot) 2x the width; foot paddle splayed with 5 digits and thinner inter-digital webbing; forelimb paddle with 4 digits and thinner inter-digital membranespronephric kidney begins to atrophy
thyroid hormone detectable in blood
tbx4, sall4: interdigital mesenchyme
55hindlimb length (not including foot) 3x width; forelimb hand rotates 90 degress, free parts of fingers as long as they are wideall major muscles of hindlimb developedregeneration restricted [4]
56hindlimbs visible from above as they can rotate away from body; hindlimb length = ~ 5 tail somites; larval pigmentation pattern established sexual differentiation of gonads into ovary or testis; hindlimb skeleton completey chondrifiedregeneration restricted [4]
57hindlimb length = ~ 9 tail somites; forelimb remains enclosed in operculum; lip folds form
58hindlimb length = 11-12 tail somites; claws form on toes 1-3 (mostly always still white); forelimb emerges from operculum, elbows first; tail tip begins to atrophymelanin/pigment deposited in under skin especially in tailregeneration incompetent [4]
59hindlimb muscular, claws start to harden and turn black, shortest toes first; finger tips reach base of hindlimb when forelimb is positioned along the abdomen; tentacles/barbels regressmelanin/pigment surrounds intersomitic blood vessels and between fibres of somites; forelimb muscles differentiated; pronephros no longer functionalregeneration incompetent [4]
60gill chamber opening still wide; fingertips reach beyond base of hindlimb (almost to 'knee') when forelimb/arm is positioned along side of the abdomen; forelimb held posterior to heart; tail fins greatly reducedpigmentation across body increasesregeneration incompetent [4]; animal switches from tail to leg swimming
61first sign of gill resorption, openings to gill chamber much narrower; hindlimb and forelimb fully formed; forelimb at level of posterior half of heartlateral finger-like protrusions from olfactory organcessation of feeding (due to oral and intestinal remodeling)obp: olfactory organ
62tailed froglethead slightly broader than abdomen; corner of mouth still infront of eye; forelimb reaches middle of heart; ventral tail fin gone from abdomen; adult skin on hindlimbs; only tiny nubs of barbels/tentacles remain.tiny 'stirnorgan' (light detecting cells/part of pineal gland) appears; notochord atrophies along length of tailpeak levels of thyroid hormone in plasma
63tailed froglethead narrower than abdomen; barbels/tentacles (most often) completely gone; forelimb at level of anterior half of heart; tail shortens as tail somites are rapidly resorbed, tail still slightly longer than body
64corner of mouth behind eye; tail length is about 1/3 of body length, at level of ankle when legs are in typical neutral position; body completely covered in adult skin, but 'border lines' clearly visible thymus gland ventral-lateral to otic capsule
65tail length a few millimeters, all tail somites have disappeared; body completely covered in adult skin, but 'border lines' still visible in some areasfeeding resumes
66froglettail very nearly gone, not visible from ventral view; adult skin 'border lines' have disappeared, froglet body ~ 10mm longskin remodelled with underlying dermis and secretory glandsthyroid hormone in plasma returns to prometamorphic levels
1Dorsal-ventral pigment variation only occurs in some batches of embryos. Select 2-4-cell embryos with clear pigment variation - otherwise only accurate about 70% of time.
2In the Normal Table and other texts, organ primordia are often called 'anlage'; they are visualized by as a thickening of specific cells via histology or by molecular markers. Search specific XAO terms on Xenbase for more molecular markers
3NF stages 41-66 are not referred to by specific 'stage names' by Nieuwkoop and Faber
4Regeneration classes from Aztekin et al 2021 PMID:34105722.
5Left and right sides refer to that of the tadpole/embryo, and not the viewer.