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Proc.Natl.Acad.Sci.USAVol.94,pp.1301113016,November 1997Developmental BiologyThe role of thyroid hormone in zebrafish and axolotl developmentDONALDD.BROWNDepartment of Embryology,Carnegie Institution of Washington,115 West University Parkway,Baltimore,MD 21210Contributed by Donald D.Brown,September 25,1997ABSTRACTExogenous thyroid hormone(TH)inducespremature differentiation of the zebrafish pectoral fins,whichare analogous to the forelimbs of tetrapods.It accelerates thegrowth of the pelvic fins but not precociously.Goitrogens,which are chemical inhibitors of TH synthesis by the thyroidgland,inhibit the transition from larva to juvenile fishincluding the formation of scales,and pigment pattern;theystunt the growth of both pectoral and pelvic paired fins.Inhibition by goitrogens is rescued by the simultaneous ad-dition of thyroxine.The effect of adding TH to the rearingwater of the postembryonic Mexican axolotl was reinvesti-gated under conditions that permit continued growth anddevelopment.In addition to morphological changes that havebeen described,TH greatly stimulates axolotl limb growthcausing the resulting larva to be proportioned as an adult inabout two months.This study extends the known evolutionaryrelatedness of tetrapod limbs and fish fins to include the THstimulation of salamander limb and zebrafish fin growth,andsuggests that TH is required to complete the life cycle of atypical bony fish and a salamander at the same developmentalstage that it controls anuran and flounder metamorphosis.A remarkable feature of metamorphosis in anurans and ho-lometabolous insects is the replacement of larval by adulttissues and the large number of adult tissues and organs whosedevelopment is controlled by hormones.Even insects thatdevelop directly with no larval stage are dependent on ecdys-one to transit their life cycle(1).Adult frog organs such as thelimb,intestinal tract,kidney,and skin,resemble those of othervertebrates closely yet until recently only in anurans have theybeen shown to require thyroid hormone(TH)to completetheir differentiation(2).Recently TH-dependent metamor-phosis in vertebrates was extended to include a bony fish,theflounder,which requires TH to develop beyond larval stages(3).However,the larval to juvenile transition of most bony fishand salamanders is less dramatic than that of the flounder andthe frog,and is usually described as direct development.Thezebrafish(Daniorerio)isarapidlydevelopingyettypicalbony fish(Osteichthyes)that completes embryogenesis,hatches in about 3 days and then begins to feed as a larva.Apair of pectoral fins,which will differentiate as part of thelarval to juvenile transition,is formed by 28 hr postfertilization(4).Smooth dorsal and ventral midline epithelial folds fuse ina seamless covering over the entire tail so that a zebrafish larvamore closely resembles an amphibian tadpole than an adultfish(Fig.1).Timing of the larval to juvenile transition that isthe subject of this paper depends on the growth conditions ofthe larva.The transition begins several days after the larva caneat brine shrimp and is characteristic of bony fish(5,6).Morphological changes in the zebrafish and the Mexicanaxolotl(Ambystoma mexicanum)are closely correlated withthe size of the animal.When growth is slowed so is develop-ment.With the rearing conditions used here(see Materials andMethods),the first external signs of change to a juvenile aredetectable in a 5 mm zebrafish larva about 3 weeks postfer-tilization(Fig.1).The rounded end of the tail epitheliumflattens as the first sign of its transformation into the typical,two pronged,homocercal adult spiny tail that characterizesbony fish.The midline dorsal and ventral unpaired fins pro-trude and develop,followed by the appearance of fin rays.When the larva is about 10 mm the paired pectoral fins beginto differentiate into a pair of flat,spiny fan-like paddles thatsplay laterally like outriggers(see Fig.2).Several days later,when the larva is 13 mm,the paired pelvic fins appear oneither side of the last remaining larval epithelial fold justanterior to the anus.This ventral fold resorbs as the pelvic finsgrow,the striped pattern of adult fish appears,and scales form.Thedetailedanatomyofthepairedfinsandtheirunmistakablerelationship to tetrapod limbs has been described(7).Thehomology between limbs and paired fins has been extendedrecently by the demonstration that several genes that areinvolved in the formation of limb buds are also activelyexpressed in the paired fin buds(8,9).Salamanders become sexually mature in a larval form,aphenomenon referred to as neoteny.The term metamorpho-sis is used in salamanders to refer to changes that occurnormally or can be induced by TH in sexually mature adultsand therefore are not necessary to complete the life cycle(10).Some salamanders metamorphose spontaneously.They resorbtheir external gills and tail fins,change their head shape,andundergo thickening of the skin.Exogenously administered THinduces these changes in facultative neotenes or paedomorphicsalamanders(11)suchastheaxolotlthatdonotmetamorphosenormally in nature(11,12).Obligatory neotenes such as themud puppy,Necturus maculosus,do not metamorphose(ac-cording to this definition)in nature and cannot be induced todo so by administering TH(10).The axolotl develops external gills late in the tailbud stage(day 5)followed two days later by forelimb buds(12).Theembryo hatches at about day 11 and then begins to feed.Atabout 4 weeks postfertilization a 25-mm axolotl has developedforelimbs,but the hindlimb buds are just detectable as bean-shaped structures on either side of the posterior intestine justanterior to the anus(see Fig.3).Over the next 3 weeks thehindlimbs elongate posteriorly,then splay laterally,developdigits,and differentiate.This report describes the effect of exogenous TH andinhibitors of the thyroid gland on the growth and developmentof the zebrafish and axolotl at a stage in their life cyclecomparable to anuran and flounder metamorphosis.Thesuggestion is that TH plays a role and may even be required forthe transit from a postembryonic larva to an adult.MATERIALS AND METHODSZebrafish(strain AB,ref.14)were raised in running dechlo-rinated tap water(system water)at 27C and fed parameciafor two weeks.Then about seven larvae were placed in 2-lThe publication costs of this article were defrayed in part by page chargepayment.This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C.1734 solely to indicate this fact.1997 by The National Academy of Sciences 0027-8424y97y9413011-6$2.00y0PNAS is available online at http:yywww.pnas.org.Abbreviations:TH,thyroid hormone;T4,thyroxine;T3,3,5,39-L-triiodothyronine;TU,thiourea.13011tanks containing thyroid gland inhibitors andyor TH.Themedium was changed 3 times a week.Zebrafish were fed brineshrimp for the remainder of the experiment.Results were thesame if inhibitors or TH were added as early as the fourth daypostfertilization.Under these conditions about five larvaewere raised in 100 ml beakers of system water containing theappropriate hormone andyor thyroid gland inhibitor with dailychanges.They were diluted to the larger volume when theywere capable of eating brine shrimp.Wild-type axolotl embryos were purchased from the axolotlfacility at the University of Indiana,and raised in system water(500 ml per animal)at room temperature.Feeding with brineshrimp began at day 14 postfertilization.When the larvae wereabout3cm(1monthold)theirdietwaschangedtoTubifexandtheir volume was increased to 1 l per animal.Final concentrations of hormones added to the system waterof either zebrafish or axolotl,unless otherwise indicated,were5 nM 3,5,39-L-triidothyronine(T3)or 30 nM thyroxine(T4).The lowest dose tried,10 nM T4,also stimulated limbyfindevelopment.However,30 nM T4was used routinely becauseit was remarkably nontoxic.Zebrafish grown in 0.5 mM orhigher methimazole died at 12 mm having formed theirunpaired fins but before differentiation of the paired pectoralfins(see Fig.5).A concentration of 0.3 mM methimazole wasthe highest nontoxic dose.Even 0.1 mM methimazole arrestedthe development of axolotl.The animals stopped feeding anddeveloped limb abnormalities.Zebrafish and axolotl wereraised in 0.025%and 0.05%KClO4,respectively.The higherdose was toxic for zebrafish.Concentrations of 0.005%and0.003%,respectively,for 6-n-propyl-2-thiouracil and thiourea(TU),werenottoxicforeitheranimal,buttheyfailedtoinhibit125I uptake(Fig.4).Inhibitors and thyroid hormone werepurchased from Sigma.Iodide uptake was carried out in system water.A stocksolution of 125I-NaI(NEZ-033A from DuPontyNEN)wasdiluted to 1 mcyml in 0.01 mM NaOH and stored at 4C.Zebrafish were incubated in 12 ucyml of radioactive NaI for26 hr at room temperature in 515 ml of system water,depending on their size.Axolotl were incubated overnight inthe same concentration(30 ml per animal).Following theirexposure to radioiodide,animals were cooled in an ice bathand fixed in 10%formaldehyde in PBS overnight.The radio-active animals were washed with many changes of PBS untilthere was no more soluble radioactivity released(at least 48 hrfor zebrafish and twice that long for axolotl).The animals wereplaced on Schleicher&Schuell medium thick gel blot paper,covered with Saran wrap,dried in a gel drier,and autoradio-graphed.Incorporation of radioactive iodine in the thyroidgland was quantified with a PhosphorImager.Larva and adults were stained with alcian blue and thenalizarin red after fixation in 10%formaldehyde in PBS for 24hr.RESULTSThe Effect of Exogenous TH on the Development of theZebrafish and Axolotl.TH added to the water of zebrafishlarvae throughout the first 2 weeks of larval development hasno visible effect on larval development until the juveniletransition begins.T4treated larvae undergo premature differ-entiation of their pectoral fins so that they develop as early asthe appearance of the unpaired fins(Fig.2).Exogenous T3causes the same precocious stimulation of pectoral fin devel-opment as T4,but it is more toxic and slows subsequent growthand development.The pectoral fins of T4-treated larvae growlarger than control fins at the same stage.Development of theunpaired fins,resorption of the epithelial folds,and theappearance of paired pelvic fins is not accelerated by exoge-nous TH.However,the paired pelvic fins grow more rapidlyin TH-treated animals than controls.The continuous addition of T4to the water of axolotlsbeginning 14 days postfertilization induces noticeable resorp-tion of the gills by the 28th day(two weeks after the additionof T4)as described(15).The first morphological differencebetween T4-treated and control axolotl forelimbs occurs afterthe forelimb has begun digit formation(Fig.3).The hindlimbbudsappearatthesametimeinT4treatedandcontrolanimals,but when they begin to splay laterally and develop digits theT4-treated limbs grow much more rapidly.T3added to therearing water also enhanced the growth of axolotl limbs,but itseffect was not followed beyond a month because of toxicity.Histological sections(not shown)reveal that the T4-inducedlimbs have much greater muscle mass and long bone diameterthan control limbs.Initially the larval gills do shrink,but T4added to the rearing water does not induce their completeresorption(as is reported to happen when the larvae areinjected with TH,ref.15).Instead,the large flowing larval gillschange to resemble adult gills and then grow proportionatelywith the body length.Under these conditions the dorsal findoes not resorb and skin pigmentation changes prematurely inTH-treated animals to an adult pattern.Although the T4treated animals resemble miniature adults in 2 months,they donot have mature gonads.Thyroid Gland Function and Inhibition.Although therehave been measurements of whole body levels of both T3andFIG.1.(Upper)Two-week-old(4 mm)larva.(Lower)Three-week-old juvenile(6 mm)beginning unpaired fin development.Stained withalcian blue and alizarin red.Ossification has not begun.The larvalpaired pectoral fins are not visible in this side view.FIG.2.Premature development of the paired pectoral fins inducedby exogenous T4.Larvae were treated continuously with 30 nM T4beginning 11 days postfertilization.Upper,the two animals(length,6.5mm)were fixed 23 days postfertilization.Lower,the two animals(8.5mm)were fixed several days later.13012Developmental Biology:BrownProc.Natl.Acad.Sci.USA 94(1997)FIG.3.The effect of thyroxine on the early larval development of the axolotl.The same control and 30 nM T4-treated(TH)sibling animalswere photographed at the days postfertilization noted.T4was added from day 14.(Bar 5 1 cm.)Developmental Biology:BrownProc.Natl.Acad.Sci.USA 94(1997)13013T4throughout larval development and metamorphosis of theflounder(16,17),there are no such reports for the zebrafish.I chose to assess thyroid gland function by its ability toconcentrate radioiodide presumably as iodinated thyroglobu-lin.Although levels of circulating hormone,and most impor-tantly,the concentration of TH that reaches its receptors incellnucleiisregulatedbymanysteps,theuptakeofradioiodideis a sensitive monitor of the thyroid glands ability to synthesizehormone.The reduction of radioiodide uptake by inhibitors ofthyroid gland function(goitrogens)estimates the effectivenessof their inhibition.Zebrafish at various stages of developmentwere incubated with radioactive 125I-NaI,fixed and washed,the animals dried on filter paper,and then radioautographed.The single zebrafish thyroid gland fixes radioiodide for the firsttime at the third day of development after fertilization,anduptakeincreasesthroughoutthelarvalandtransitionalperiodsto the juvenile stage(Fig.4A).The pituitary has been impli-cated in the control of fish thyroid gland function(18)just asit has in frogs and other vertebrates.Exogenous TH depressesthe uptake of radioiodide by the zebrafish thyroid gland(Fig.4B)as it does in Xenopus laevis(19)presumably by shuttingdown thyrotropin synthesis by the pituitary.The other half ofthe negative feedback loop between the pituitary and thethyroid in zebrafish,reflected by goiter formation in thethyroid gland following long-term interruption of thyroidhormone synthesis,was not observed in the zebrafish or theaxolotl.However,this might require a longer exposure to aninhibitor of TH synthesis than the several weeks used in theseexperiments.Typical of amphibians,the axolotl has paired thyroid glands,but the uptake of radioiodide is much less active and variablein the time of its onset in axolotls than in zebrafish or X.laevis.Two sibling animals exactly the same size and at the samedevelopmental stage took up very different amounts of radio-iodide(data not shown).Variability of serum TH levels hasbeen reported for another salamander(20).The earliestunequivocal incorporation of radioiodide into the pair ofaxolotl thyroid gland was 3540 days postfertilization when thelarvae were 3 cm.This is about the developmental stage