RESEARCH ARTICLE

Chordin Affects Pronephros Development inXenopus Embryos by Anteriorizing PresomiticMesodermTracy Mitchell,1† Elizabeth A. Jones,2 Daniel L. Weeks,3 and Michael D. Sheets1*

Spemann’s organizer emits signals that pattern the mesodermal germ layer during Xenopus embryogenesis.In a previous study, we demonstrated that FGFR1 activity within the organizer is required for theproduction of both the somitic muscle- and pronephros-patterning signals by the organizer and theexpression of chordin, an organizer-specific secreted protein (Mitchell and Sheets [2001] Dev. Biol.237:295–305). Studies from others in both chicken and Xenopus embryos provide compelling evidence thatpronephros forms by means of secondary induction signals emitted from anterior somites (Seufert et al.[1999] Dev. Biol. 215:233–242; Mauch et al. [2000] Dev. Biol. 220:62–75). Here we provide several lines ofevidence in support of the hypothesis that chordin influences pronephros development by directing theformation of anterior somites. Chordin mRNA was absent in ultraviolet (UV) -irradiated embryos lackingpronepheros (average DAI<2) but was always found in UV-irradiated embryos that retain pronepheros(average DAI>2). Furthermore, ectopic expression of chordin in embryos and in tissue explants leads to theformation of anterior somites and pronephros. In these experiments, pronephros was only observed inassociation with muscle. Chordin diverted somatic muscle cells to more anterior positions within the somitefile in chordin-induced secondary trunks and induced the expression of the anterior myogenic gene myf5.Finally, depletion of chordin mRNA with DEED antisense oligonucleotides substantially reduced somiticmuscle and pronephric tubule and duct formation in whole embryos. These data and previous studies onectoderm and endoderm (Sasai et al. [1995] Nature 377:757) support the idea that chordin functions as ananteriorizing signal in patterning the germ layers during vertebrate embryogenesis. Our data support thehypothesis that chordin directs the formation of anterior somites that in turn are necessary for pronephrosdevelopment. Developmental Dynamics 236:251–261, 2007. © 2006 Wiley-Liss, Inc.

Key words: Xenopus; pronephros; organizer; chordin; BMP signaling; anterior somites; mesodermal patterning

Accepted 11 October 2006

INTRODUCTIONCells of Spemann’s organizer secretesignaling proteins, such as chordinand noggin, that function nonautono-mously to influence the fate of neigh-boring cells (De Robertis and Kuroda,

2004). The formation of specific tis-sues such as the pronephros andsomitic muscle from the mesodermgerm layer depends upon these orga-nizer signals. The existence of thesesignals and their importance for in-

ducing specific tissues have been de-duced from a variety of embryologicaland molecular studies. However, inmany cases the specific tissues in-duced by the individual organizer sig-naling proteins and whether this in-

1University of Wisconsin Medical School, Department of Biomolecular Chemistry, Madison Wisconsin2Department of Biological Sciences, Warwick University, Coventry United Kingdom3Department of Biochemistry, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IowaGrant sponsor: NIH; Grant numbers: HD43996; HL062178; Grant sponsor: Beckman Foundation; Grant sponsor: Pew Scholars Program;Grant sponsor: James D. Shaw and Dorothy Shaw Fund of the Greater Milwaukee Foundation; Grant sponsor: American Heart Association.†Dr. Mitchell’s present address is OSI Eyetech, 35 Hartwell Avenue, Lexington, MA 02421.*Correspondence to: Michael Sheets, University of Wisconsin Medical School, Department of Biomolecular Chemistry,Madison, WI 53705. E-mail: mdsheets@facstaff.wisc.edu

DOI 10.1002/dvdy.21014Published online 14 November 2006 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 236:251–261, 2007

© 2006 Wiley-Liss, Inc.

duction is direct or indirect remain tobe elucidated.

Several lines of evidence indicatethat pronephros development dependsupon signals from the anteriorsomites. The pronephros consists ofthree major structures, the pronephrictubules, duct, and glomus. Thesestructures are derived from the inter-mediate mesoderm and arise in theembryo at a position ventral tosomites 3 to 5—the anterior somites.The formation of the pronephros inclose proximity to the anterior somitesis functionally significant—the ante-rior somites produce pronephros-in-ducing signals. The existence of thesesignals was demonstrated using tis-sue manipulations in frog and chickembryos. Separation of pronephrosprogenitor cells from the nearbysomitic tissue in chick embryos in-hibited pronephros differentiation(Mauch et al., 2000). Culture of chickpronephros precursor cells with quailsomites induced pronephros in thechick mesoderm. Similarly, culture ofisolated, presumptive pronephric tis-sue with anterior somites from frogembryos induced pronephric tubuleformation, whereas incubation of thistissue with notochord or neural tissuedid not (Seufert et al., 1999). Theseresults establish the anterior somitesas an important source of pronephrosinducing signals. However, very littlein known about the signals and mech-anisms that direct somitic muscle pre-cursors to form anterior somites.

Organizer signaling affects pro-nephros development indirectly bymeans of its affect on the formation ofthe pronephros-inducing anteriorsomites. Cortical rotation and, there-fore, organizer signaling can beblocked to different extents by expos-ing fertilized eggs to varying doses ofultraviolet (UV) light. Treatment withhigh doses of UV light abolishes corti-cal rotation, and eliminates organizersignaling and the formation of tissuesthat depend upon this signaling (Ger-hart et al., 1984, 1989; Gerhart andKeller, 1986). The resulting embryoslack most mesodermal cell types, in-cluding pronephroi, somitic meso-derm, and notochord, and lack neuraltissues. Embryos treated with inter-mediate doses of UV light developwith a subset of mesodermal tissues—pronephroi and somitic muscle are

still present in these embryos, but no-tochord and anterior neural struc-tures are absent (Seufert et al., 1999).These results demonstrate that tis-sues have different requirements fororganizer signaling. The similar sen-sitivities of the pronephros andsomitic muscle to disruptions in orga-nizer function supports the idea thatpronephroi are induced by signalsfrom the anterior somites (Seufert etal., 1999). The organizer produces sev-eral different signaling molecules, andit is not known which of these areinvolved in the formation of the ante-rior somites to influence pronephrosformation (De Robertis and Kuroda,2004).

Clues to the identity or the orga-nizer signals important for both ante-rior somite formation and pronephrosdevelopment have come from studiesthat specifically block FGFR1 functionin the organizer cells. Organizer cellsexpressing DN-FGFR1 are severelydisrupted in their ability to emit sig-nals for mesodermal patterning(Mitchell and Sheets, 2001). This dis-ruption in signaling eliminates pro-nephric tubule and duct formationand reduces somatic muscle forma-tion, indicating that an FGFR1-de-pendent signal from the organizer isrequired for pronephros formationand somitogenesis. Intriguingly, dis-rupting FGFR1 function also signifi-cantly reduces the expression of chor-din mRNA (Mitchell and Sheets,2001), but not the expression of othermRNAs such as noggin or Xlim1.These observations raise the possibil-ity that chordin is an importantFGFR1-dependent component of orga-nizer signaling that is necessary foranterior somite formation that in turnaffects pronephros development.

In this study, we investigated chor-din’s role as an organizer-specific sig-naling protein important for anteriorsomite formation and pronephros de-velopment. Our results strongly sug-gest that chordin is important for theformation of anterior somites that inturn generate secondary signals thataffect pronephros development. Theseresults also suggest that chordin’s rolein pronephros development is indirectand occurs by promoting anteriorsomite formation and the pronephros-inducing signals generated from thesecells. The data provide additional sup-

port for the hypothesis that chordinand other organizer-specific bone mor-phogenetic protein (BMP) antago-nists, such as noggin, function as an-teriorizing factors that modify andpattern the mesodermal germ layers(Lane and Sheets, 2000, 2002; Lane etal., 2004).

RESULTS

Pronephros Formation andChordin Expression AreSensitive to Similar Levelsof Reduction in OrganizerFunction

One of the key observations connect-ing the anterior somites with prone-phros formation, is the result thatboth tissues are sensitive to similarreductions in organizer function(Seufert et al., 1999). We used thesame approach to examine the rela-tionship of pronephros and chordin ex-pression. Organizer function can begradually reduced in Xenopus em-bryos by incrementally disrupting cor-tical rotation with increasing amountsof UV irradiation (Seufert et al.,1999). Fertilized eggs were UV irradi-ated for different times before corticalrotation, and the treated embryoswere analyzed for chordin expressionby in situ hybridization at the gas-trula stage (stage 10.5). Sibling em-bryos cultured to stage 35 were scoredfor defects according to the DAI series(Kao and Elinson, 1988) and also an-alyzed for expression of the proneph-ric tubule-specific mRNA XSMP-30,using in situ hybridization, and formuscle using immunocytochemistrywith the muscle-specific antibody 12/101. Chordin mRNA was highly ex-pressed in embryo batches with aver-age DAI scores of 2 and above (Fig.1A,B; Table 1), and expression wassignificantly reduced in embryos withDAI scores below 2 (Fig. 1C; Table 1).In contrast, goosecoid mRNA was ab-sent from embryos with DAI scores 3.3and below (Fig. 1D,E; Table 1), indi-cating that goosecoid expression wasmore sensitive to reductions in corti-cal rotation compared with chordin. Inaddition, the vast majority of embryoswith average DAI scores of 2 andabove developed pronephric tubulesand somitic muscle (Fig. 1F–I; Table1) as previously shown (Seufert et al.,

252 MITCHELL ET AL.

Fig. 1.

Fig. 2.

Fig. 1. Chordin expression correlated with thepresence of pronephros in ultraviolet (UV) -irra-diated embryos. A–E: Fertilized eggs were irra-diated with different amounts of UV light beforecortical rotation. At stage 10.5, 1/3 of the em-bryos at each UV dose were analyzed for chor-din or goosecoid expression by in situ hybrid-ization. Chordin expression in stage 10.5: DAI5untreated embryos (A), embryos with an aver-age DAI of 3.0 (B), embryos with an average DAIof 0.5 (C). Goosecoid expression in stage 10.5:DAI5 untreated embryos (D), embryos with anaverage DAI of 3.3 (E). F–K: Stage 35 embryosrepresentative of each DAI level (5-0). Siblingembryos from each level of UV treatment werescored at stage 35 using the DAI scale andanalyzed for pronephros and somitic muscleformation using in situ hybridization forXSMP-30 (blue, black arrowhead) and immuno-cytochemistry with the 12/101 antibody (brown,red arrowhead).

Fig. 2. Secondary trunks resulting from ectopicchordin expression contained pronephros. Oneposterior blastomere of four- to eight-cell stageembryos was injected with chordin mRNA. A,B:Embryos analyzed form muscle and notochordusing immunocytochemistry 12/101 and Tor70antibodies. C,D: At stage 33/34, some embryoswere analyzed for pronephros (blue, yellow ar-rowhead) using in situ hybridization to detectXSMP-30 expression and analyzed for somiticmuscle (brown, red arrowhead) formation im-munocytochemistry with the 12/101 antibody.E,F: Some embryos were analyzed at stage38–40 for pronephric duct formation using the4A6 antibodies. The black arrows indicate pro-nephric ducts in the secondary and primaryaxis. G: Injected embryos analyzed at stage 38for pronephric tubule formation using the 3G8antibodies. The black arrows indicate proneph-ric tubules in the secondary and primary axis.H: One posterior blastomere of four- to eight-cell stage embryos was injected with nogginmRNA. Embryos were analyzed at stage 38–40for pronephric duct formation using the 4A6antibodies. The black arrows indicate proneph-ric ducts in the secondary and primary axis.

1999), whereas embryos with DAIscores below 2 (Fig. 1J,K; Table 1) con-tained limited amounts of muscle andpronephric tubules (Fig. 1J,K)(Seufert et al., 1999). Therefore, chor-din expression and pronephros forma-tion were sensitive to very similar lev-els of reduction in organizer function.

Chordin-Induced SecondaryTrunks Contain Pronephros

Ectopic chordin expression in Xenopusembryos induces secondary trunkscontaining muscle and neural tissue(Sasai et al., 1994, 1995). A previousstudy noted the presence of pro-nephroi in secondary axes (Carrolland Vize, 1999). To more extensivelyexamine chordin-generated secondarytrunks for pronephros, a single poste-rior blastomere of four-cell embryoswas injected with chordin mRNA, andthe resulting secondary trunks wereanalyzed for the presence of pro-nephros using in situ hybridization todetect the pronephros-specific mRNAXsmp30 or using immunocytochemis-try to detect pronephric tubules orducts with the 3G8 or 4A6 antibodies(Fig. 2C–G; Table 2). Embryos werealso analyzed for somitic muscle(stage 32) using immunocytochemistry(12/101 antibodies). The majority of thechordin-directed secondary trunks con-tained pronephros (Xsmp30 expression;Fig. 2C; Table 2), pronephric ducts andtubules (Fig. 2E,G; Table 2). As previ-ously described, chordin-derived sec-ondary trunks contained somitic mus-cle, but not notochord (Fig. 2A,B; Table2; Sasai et al., 1994, 1995). Thus, sec-ondary trunks that result from ectopicchordin expression contained ectopicpronephros.

Secondary trunks produced as theresult of ectopic expression of otherBMP antagonists, noggin and the DN-ALK3 receptor, also contained ectopicpronephros (Fig. 2H, and data notshown). We have focused on chordinbecause our previous results indicatedthat specifically blocking FGFR1 func-tion in the organizer cells disruptedpronephros and somitic muscle forma-tion and the expression of chordinmRNA (Mitchell and Sheets, 2001),but not the expression of other mR-NAs such as noggin or Xlim1. Theseobservations raise the possibility thatchordin is an important FGFR1-de-

pendent component of organizer sig-naling that is necessary for anteriorsomite formation that, in turn, affectspronephros development.

Depletion of Chordin mRNACauses Defects inPronephros Development

The experiments described above pro-vided data consistent with a role forchordin in pronephros formation.However, to test this requirement de-finitively required perturbing chordinfunction in the embryo. Toward thisend, we analyzed embryos depleted ofchordin mRNA by means of injectionof an oligonucleotide containing inter-nucleoside phosphate linkages modi-fied with the cation N,N-diethyl-ethylenediamine (DEED)—a DEED-modified antisense oligonucleotide(Gururajan et al., 1991; Bailey et al.,1998; Bailey and Weeks, 2000; Dagleet al., 2000; Dagle and Weeks, 2000;Jallow et al., 2004). The modified in-

ternucleoside phosphate linkages inthese oligonucleotides promote stabil-ity, allowing them to persist in embry-onic cells and degrade specific zygoticmRNAs by means of the endogenousRNaseH activity (Bailey et al., 1998;Bailey and Weeks, 2000; Dagle et al.,2000; Dagle and Weeks, 2000). ADEED oligonucleotide complimentaryto the 5� end of chordin mRNA wasinjected into the two anterior cells intwo- to four-cell embryos—the cellsthat will give rise to Spemann’s orga-nizer. At the gastrula stage, RNAs iso-lated from injected embryos were ex-amined for chordin mRNA by RNAblot hybridization. Chordin mRNAwas specifically depleted in a dose-de-pendent manner from the chordinDEED antisense oligonucleotide-in-jected embryos (Fig. 3A, lanes 5–7),but not from controls (Fig. 3A, lanes1–4). Goosecoid mRNA levels wereunchanged by injection of the chordinantisense oligonucleotide, consistent

TABLE 1. Chordin Expression Correlates With the Presence ofPronephros in Ultraviolet-Irradiated Embryosa

Average DAI score% expressingchordin

% withpronephros

0.5 (n � 79) 8 91.3 (n � 96) 39 482.0 (n � 97) 90 892.5 (n � 37) 88 823.0 (n � 31) 93 863.3 (n � 38) 100 833.7 (n � 36) 86 97

DAI% expressinggoosecoid

% withpronephros

2.2 (n � 151) 0 863.3 (n � 75) 25 90

aSee Figure 1.

TABLE 2. Secondary Trunks Resulting From Ectopic ChordinExpression Contain Pronephrosa

mRNAinjected

% secondarytrunks

Tissues in secondary trunks

% pronephros % muscle % notochord

Chordin 43 (n � 244) 47 (n � 72) 80 (n � 35) 0 (n � 19)None 0 (n � 358) Not applicable

aSee Figure 2.

254 MITCHELL ET AL.

with the DEED oligonucleotide show-ing specificity for chordin mRNA.

Most chordin mRNA-depleted em-bryos exhibited anterior defects suchas the reduction or absence of eyes(Fig. 3B; Table 3) and developed withreduced elongation of the trunk andtail (Fig. 3B). Embryos injected withthe control oligonucleotide developedwith normal morphology, similar touninjected controls (Fig. 3C,D).

To demonstrate that the biologicaleffects were specifically due to chordinmRNA depletion, we performed a res-cue experiment. In this experiment, amodified chordin mRNA that lackedcomplimentarity to the DEED anti-sense oligonucleotide was injectedalong with the oligonucleotide intoembryos. Significantly, the vast ma-jority of these embryos exhibited nomorphological abnormalities (Fig. 3E;Table 3), indicating that the co-in-jected chordin mRNA was sufficient torescue defects due to depletion of theendogenous chordin mRNA. Thesedata provided compelling evidencethat the defects observed in DEED an-tisense-injected embryos were due todepletion of the endogenous chordinmRNA. Importantly, the phenotypewe observe with the DEED antisenseoligonucleotide was similar to thatproduced by injecting chordin morpho-linos (Oelgeschlager et al., 2003).

Having established that we could

specifically deplete chordin mRNAfrom embryos, we tested the effects ofthis depletion on pronephros forma-tion. Analysis of embryos (stage 38)injected with the chordin antisenseoligonucleotide revealed that deple-tion of chordin mRNA resulted in sig-nificant reductions in formation of thepronephric ducts (4A6 staining cells)and pronephric tubules (3G8 stainingcells; compare Fig. 3F and I with 3G,H, J and K and see Table 3). Theseobservations demonstrate that Xeno-pus embryos depleted of the endoge-nous chordin mRNA develop with de-fects in pronephros formation.

In addition, analysis of embryos in-jected with the chordin antisense oli-gonucleotide revealed that depletionof chordin mRNA resulted in signifi-cant reductions in somitic muscle(compare Fig. 3L with 3N and see Ta-ble 4). Therefore, Xenopus embryosdepleted of the endogenous chordinmRNA develop with somitic muscledefects. Muscle defects were also ob-served in embryos injected with chor-din morpholinos (Oelgeschlager et al.,2003).

Chordin Anteriorizes SomiticMesoderm

The results presented above indicatethat secondary trunks due to ectopicchordin expression contained pro-

nephros (Fig. 2; Table 2) and that de-pletion of the endogenous chordinmRNA caused significant reductionsin pronephros and muscle formation(Fig. 3; Table 3). We next wanted toexamine what step in pronephros de-velopment chordin influenced.

Pertinently, studies in frog andchick demonstrate that pronephroiforms from the intermediate meso-derm in response to inducing signalsemitted from anterior somitic muscle(Seufert et al., 1999; Mauch et al.,2000). In addition, chordin influencesembryonic tissues to form anterior de-rivatives of the ectodermal andendodermal germ layers (Sasai et al.,1995). For example, chordin inducesanimal cap cells to form anteriorneurectoderm and express the ante-rior neural mRNAs xanf1, otx2, andxif3, as well as the anterior ectoder-mal mRNA cg-13 (Sasai et al., 1995).Based on these observations, a rea-sonable postulate was that chordin’srole in pronephros formation was in-direct and due to its affect on anteriorsomite formation. To test this idea, welineage-traced chordin-injected cellsand analyzed their anterior–posteriorcontribution within the ectopic somitefile. Chordin mRNA was co-injectedwith a lineage tracer (biotin-labeledmini-ruby dextran) into one C4 blas-tomere of 32-cell embryos. The biotinon the lineage tracer was detectedwith horseradish peroxidase (HRP)-coupled streptavidin (black staining)and somitic muscle was detectedby immunocytochemistry using the12/101 antibody (brown staining). Theanterior-most contribution of the lin-eage-labeled cells was analyzed in thepresence and absence of chordinmRNA. From this analysis, we foundthat the anterior-most contribution ofchordin-injected cells corresponds tosomite 3 of the secondary axis (see red

TABLE 3. Chordin mRNA Depletion Causes Pronephros Defectsa

Sample injected % Normal% Headdefects

% Pronephrosdefects

None (n � 62) 100 0 0Control DEED oligonucleotide (n � 61) 92 0 0Chordin antisense DEED oligonucleotide (n � 136) 23 62 83Chordin antisense DEED oligonucleotide�chordin mRNA (n � 40) 75 22

aSee Figure 3.

TABLE 4. Chordin mRNA Depletion Causes Somitic Muscle Defectsa

Sample injected:% Muscledefects

None (n � 82) 0Control DEED oligonucleotide (n � 66) 0Chordin antisense DEED oligonucleotide (n � 73) 80

aSee Figure 3.

CHORDIN AND PRONEPHROS DEVELOPMENT IN XENOPUS 255

Fig. 3.

Fig. 4.

arrow Fig. 4B, average of 19 embryos),whereas in the control embryos, C4cells injected with the lineage traceralone, the anterior-most labeledsomite is somite 9 (see red arrow Fig.4A, average of 16 embryos). In addi-tion, significantly more lineage la-beled cells occupied more anterior po-sitions when chordin mRNA was co-injected as compared with the cellsinjected with lineage label alone.Therefore, ectopic chordin expressionredirected cells from the somitic mus-cle of the primary axis into a second-ary trunk, and the chordin-injectedcells now contributed to a more ante-rior position within the somite filecompared with the cells injected withlineage label alone (Fig. 4A,B).

To further examine chordin’s abilityto direct somites to more anterior

fates, we analyzed the formation ofanterior somite progenitor cells fol-lowing chordin mRNA-injection. Al-though mRNAs exclusively expressedby the anterior somites have not beenidentified, myf5 mRNA is an earlymyogenic marker that is specificallyexpressed in the anterior somite pro-genitor cells that flank the blastoporelip (the B2 & C2 derivatives) in stage10.5 gastrulae (Dale and Slack, 1987;Moody, 1987; Bauer et al., 1994; Laneand Smith, 1999; Lane and Sheets,2000, 2002). Therefore, myf5 expres-sion at stage 10.5 is an indicator ofwhether a cell has the potential toform anterior somites.

To examine whether ectopic chordinexpression affected myf5 expression,chordin and green fluorescent protein(GFP) mRNAs were injected into one

C4 blastomere of 32-cell embryos. Atstage 10.5, injected embryos exhibit-ing GFP fluorescence opposite theblastopore lip were analyzed for myf5mRNA expression by in situ hybrid-ization. Sibling embryos were ana-lyzed at stage 33 for pronephros andsomitic muscle formation. Ectopicmyf5 expression was observed in all ofthe chordin-injected embryos (Fig. 4D;Table 5), and the vast majority of theirstage 32 siblings developed a second-ary trunk containing both pronephrosand somitic muscle (Table 5). Similarresults with myf5 expression havebeen observed with other BMP inhib-itors (Dosch et al., 1997; Wardle et al.,1999; Blitz et al., 2000). In addition,injection of chordin morpholinos dis-rupted endogenous myf5 expression(Oelgeschlager et al., 2003). Togetherthese results supported the postulatethat chordin promotes anteriorsomitic muscle formation.

Chordin DirectedPronephroi Are OnlyObserved in the Presence ofSomitic Muscle

Our hypothesis is that chordin affectspronephros formation by directing theformation of anterior somites that, inturn, produce a pronephros inducingsignal. Alternatively, other evidencesuggests that chordin directly influ-ences BMP signaling that causes themesodermal germ layer to differenti-ate as pronephros (Dosch et al., 1997).A critical distinction between thesepossibilities is the association of mus-cle and pronephros. If chordin’s affecton pronephros development occurs asa secondary consequence of its affecton somitic muscle, then pronephrosand muscle tissues should alwaysform in close association. However, ifchordin influences BMP signaling

Fig. 3. Depletion of chordin mRNA in Xenopus embryos caused defects in pronephros and muscleformation. A: Blot hybridization of RNA isolated from uninjected control embryos (lane 1), embryosinjected with increasing amounts of the control N,N-diethyl-ethylenediamine (DEED) oligonucleo-tide (lanes 2–4), and embryos injected with increasing amounts of the chordin DEED-antisenseoligonucleotide (lanes 5–7). The blot was hybridized with probes to detect chordin and goosecoidmRNA. B–E: Morphology of chordin mRNA-depleted embryos. B: Embryos injected with chordin-antisense DEED oligo. C: Embryo injected with control DEED oligonucleotide. D: Uninjected controlembryo. E: Embryo injected with chordin mRNA � chordin-antisense DEED oligo. F–H: Analysis ofpronephric duct formation in chordin-depleted embryos using immunocytochemistry with the 4A6antibody. F: Injected with chordin-antisense DEED oligo. G: Injected with control DEED oligo. H:Uninjected control. I–K: Analysis of pronephric tubule formation in chordin-depleted embryos usingimmunocytochemistry with the 3G8 antibody. I: Injected with chordin-antisense DEED oligo. J:Injected with control DEED oligo. K: Uninjected control. L–N: Analysis of somitic muscle formationin chordin-depleted embryos using immunocytochemistry with the 12-101 antibody. L: Uninjectedcontrol. M: Injected with control DEED oligo. N: Injected with chordin-antisense DEED oligo.

Fig. 4. Ectopic chordin expression promoted anterior somite formation and ectopic myf-5 expres-sion. A,B: A C4 blastomere of 32-cell stage embryos was injected with biotin-labeled mini-rubydextran � chordin mRNA, and embryos were analyzed at stage 32 to detect the injected cells(black, horseradish peroxidase/streptavidin) and somitic muscle (brown, immunocytochemistrywith the 12/101antibody). The red arrow indicates the anterior-most dextran-labeled muscle and,therefore, the anterior-most somite contribution from C4 progeny � chordin mRNA. C,D: A singleC4 blastomere of 32-cell stage embryos was injected with green fluorescent protein (GFP) mRNA �chordin mRNA. Gastrulae exhibiting GFP fluorescence opposite the blastopore lip were split intotwo groups: half was analyzed at stage 10.5 for myf5 expression by in situ hybridization; the otherhalf was analyzed at stage 33 for ectopic trunk formation and by in situ hybridization for pronephrosand somitic muscle using XSMP30 and muscle actin probes (Table 5). The red arrow indicates theblastopore lip. The blue arrowhead indicates the normal myf5 expression (C). The black arrowheadindicates the ectopic myf5 expression (D).

TABLE 5. myf-5 Is Ectopically Expressed in Embryos Injected With Chordin mRNAa

mRNA injected% Gastrulae expressingectopic myf5

Tissues in secondary trunks of sibling embryos

% ectopicpronephros

% ectopicmuscle

Chordin 100 (n � 43) 71 100none 0 (n � 19) 0 0

aSee Figure 4.

CHORDIN AND PRONEPHROS DEVELOPMENT IN XENOPUS 257

that induces the mesoderm to differ-entiate as pronephros then these tis-sues should form relatively indepen-dent of one another; we should seepronephroi form without muscle.

To distinguish between these possi-bilities, the presence of both pro-nephros and somitic muscle was ana-lyzed in tissue explants expressingchordin. Different amounts of chordinmRNA were injected into the two pos-terior cells of four-cell Xenopus em-bryos. Tissue pieces containing the in-jected cells were excised at thegastrula stage and cultured until sib-ling embryos reached stage 33. Tis-sues were analyzed for the presence ofpronephros using in situ hybridizationto monitor XSMP-30 expression andanalyzed for muscle using immunocy-tochemistry (the 12/101 antibody).Tissue explants receiving the lowestamounts of chordin mRNA containedmuscle, but not pronephros (Fig. 5B;Table 6). As more chordin mRNA wasinjected, tissue explants formed bothmuscle and pronephros (Fig. 5C–E;Table 6). Pronephroi were never ob-served in the absence of muscle, andthe pronephros in the explants alwaysformed in very close proximity to andat one end of the elongated muscletissue (Fig. 5C–E). Analysis of pro-nephric- and muscle-specific mRNAexpression by reverse transcriptase-polymerase chain reaction (RT-PCR)also indicated that these tissuesformed together in response to similaramounts of chordin mRNA (Fig. 5F).In addition, secondary trunks fromchordin-injected embryos also re-vealed that pronephros was only de-tected when somitic muscle waspresent (Fig. 2). Thus althoughsomitic muscle could form withoutpronephros, pronephros formationwas never observed without the closeassociation with muscle. These resultssupport the idea that chordin func-tions in pronephros development bypromoting the formation of anteriorsomitic muscle and not by directly in-ducing the mesodermal germ layer toform pronephros.

DISCUSSION

In this report, we demonstrate thatchordin is an organizer signaling pro-tein important for pronephros forma-tion in Xenopus embryos. These re-

Fig. 5. Pronephros formation occurred in association with somitic muscle in explants expressingdifferent amounts of chordin. A–E: Both posterior blastomeres of four-cell embryos were injectedwith different amounts of chordin mRNA (0–500 pg). A 90-degree wedge of posterior tissue wasremoved from injected embryos at stage 10�, cultured to stage 33/34, and analyzed for bothpronephros using in situ hybridization for XSMP-30 (blue stain, black arrowheads) and somiticmuscle (brown stain, red arrowhead) using immunocytochemistry. Some of the explants expressingchordin mRNA occasionally developed a cement gland, and these are marked with an by anasterisk (*). None of the control explants from uninjected embryos ever formed a cement gland. Thedashed box shows a magnified view of a single explant from each panel. F: Chordin-expressingexplants were analyzed for pronephros (XSMP-30) and muscle (muscle actin) specific gene ex-pression using reverse transcriptase-polymerase chain reaction (RT-PCR).

TABLE 6. Pronephros Formation Occurs in Association With SomiticMuscle in Explants Expressing Chordina

Chordin mRNA injected (pgs)

Tissues in explants

% pronephros� muscle

% muscleonly

% pronephrosonly

0 (n � 59) 0 14b 050 (n � 36) 8 47 0125 (n � 37) 35 49 0250 (n � 33) 36 55 0500 (n � 40) 52 48 0

aSee Figure 5.bMost of these explants only expressed scattered 12-101–positive cells and not blocksof muscle tissue.

258 MITCHELL ET AL.

sults provide new insights into themechanisms of organizer-directed me-sodermal patterning and pronephrosdifferentiation, and provide additionalevidence that chordin functions as ananteriorizing factor for the germ lay-ers (Sasai et al., 1995; Lane andSheets, 2000, 2002; Lane et al., 2004).

Chordin Function IsImportant for PronephrosDevelopment in FrogEmbryos

Patterning of the mesodermal germlayer into specific tissue types is con-trolled by secreted signals from theorganizer (De Robertis and Kuroda,2004). We previously demonstratedthat FGFR1 regulates the organizersignals required for the pronephrosformation. Additionally, we demon-strated that FGFR1 was required forchordin mRNA expression but not theexpression of other organizer-specificgenes (Mitchell and Sheets, 2001).Based on these observations, we hy-pothesized that chordin is an orga-nizer secreted signal important forpronephros development. The resultspresented here support this hypothe-sis. Ectopic chordin expression in wholeembryos produced secondary trunksthat contained pronephric duct and tu-bules in close association with thesomitic muscle. Significant reductionsin pronephric development were ob-served upon depletion of chordin mRNAwith antisense DEED oligonucleotides.In tissue explants intermediate to highdoses of chordin directed pronephros al-ways associated with somitic muscle,whereas low doses form predominantlymuscle with very little pronephros. Fi-nally, analysis of embryos in which or-ganizer signaling was reduced incre-mentally by exposure to different dosesof UV light revealed that chordin ex-pression and pronephros formationwere reduced by the same amounts ofUV treatment.

Chordin Affects PronephrosDevelopment by Promotingthe Formation of AnteriorSomites

Previous studies in frog and chick pro-vided evidence that anterior somiticmuscle generates inducing signals nec-essary for the formation of the prone-

phros in both frog and chick (Seufert etal., 1999; Mauch et al., 2000). Severalpieces of evidence support this idea.Pronephros development could be pre-vented by surgically eliminating ante-rior somites, by placing a physical bar-rier between the anterior somites andintermediate mesoderm, or by reducingthe organizer function with UV lighttreatments. Similarly, combining com-petent progenitor cells with anteriorsomites, but not notochord or neuraltube, leads to pronephros formation. Inaddition, these results indicate that thepronephros is the result of inductive in-teractions between organizer-inducedanterior somitic muscle and intermedi-ate mesoderm and not the direct resultof inducing signals from the organizer.However, the events responsible for an-terior somite formation and the role oforganizer signaling molecules in theformation of anterior somites were notaddressed in previous studies.

The importance of anterior somitesfor pronephros formation led us to in-vestigate whether chordin affected pro-nephros development by means of itseffect on anterior somites. Ectopic chor-din expression directed posterior cells tosignificantly more anterior positionswithin the ectopic somite file and alsoinduced ectopic myf5 expression atstage 10.5—the stage at which myf5 isspecifically expressed in the cells thatgive rise to anterior somites (Dale andSlack, 1987; Moody, 1987; Hopwood etal., 1991; Bauer et al., 1994). Also, re-ductions in chordin translation by mor-pholino injection eliminate myf5 ex-pression (Oelgeschlager et al., 2003)and cause reductions in somitic muscleformation. Collectively our data and theresults from previous studies lead us tosuggest that chordin is an organizer sig-naling protein that patterns the meso-derm into anterior somites, and this tis-sue then produces an unknowninducing signal(s) that direct(s) inter-mediate mesoderm to form pronephros(Seufert et al., 1999; Mauch et al.,2000). These results lead us to proposea model where chordin directly affectsanterior muscle development and indi-rectly affects pronephros development.

Our data are inconsistent with theidea that chordin directly affects prone-phros development (Dosch et al., 1997).In this model differentiation of noto-chord and somitic muscle tissues re-sults from low levels of BMP signaling

(high chordin expression), whereas dif-ferentiation of pronephros and blood tis-sues results from high levels of BMPsignaling (low chordin expression).However, we observed the opposite intissue explants. Specifically, prone-phros formation required higher or atleast equivalent amounts of chordincompared with the amounts requiredfor muscle differentiation. In addition,pronephros formation was always ob-served in association with muscle andnever in isolation, supporting the inter-dependence of these tissues. These re-sults argue against the idea that prone-phros formation is determined by theposition of the pronephric precursorcells within a BMP gradient.

BMP Antagonists; Regulatorsof Anterior Cell Fates

Ectopic chordin induces myf5, amarker of anterior somite progenitorsat stage 10.5, suggesting that chordinpatterns the mesoderm into anteriorsomitic fates. Similar results for myf5have been observed with noggin(Dosch et al., 1997) and dominant-negative BMPR1 (Wardle et al., 1999;Blitz et al., 2000). Additionally, reduc-tions in chordin translation by mor-pholino injection eliminate myf5 ex-pression (Oelgeschlager et al., 2003),indicating that myf5 expression de-pends on chordin. In this report, welineage traced chordin-injected cellsand observed that chordin divertscells that normally contribute to pos-terior somites to populate more ante-rior somites in the ectopic axis. Simi-lar results have been obtained withthe BMP antagonist noggin (Lane etal., 2004). Previous studies suggestedthat chordin is a positive regulator ofanterior cell fate and anterior genes.Expression of chordin in animal capcells results in the expression of theanterior neural genes xanf1, otx2, andxif3, as well as the anterior ectoder-mal gene cg-13 (Sasai et al., 1995).Analysis of the zebrafish mutantschordino and swirl reveals that ex-pression of the anterior endodermgene her1 is reduced in the absence ofchordin and expanded in the absenceof bmp2b (Tiso et al., 2002), again sug-gesting a role for chordin and BMPs inanterior–posterior patterning. Theseobservations support the revised fatemap proposed for Xenopus embryos in

CHORDIN AND PRONEPHROS DEVELOPMENT IN XENOPUS 259

which cells of the organizer contributeto anterior tissues of the embryo, andsupport the view that BMP antago-nists promote anterior cell fates in theembryo (Lane and Smith, 1999; Laneand Sheets, 2000, 2002; Lane et al.,2004).

Chordin Expression IsGoverned by Unique Modesof Regulation

Studies in Xenopus, zebrafish, and as-cidians indicate that FGFR1 controlschordin expression, but not the expres-sion of several other organizer-specificmRNAs (Mitchell and Sheets, 2001;Imai et al., 2004; Londin et al., 2005).Our current results demonstrate thatchordin expression is minimally af-fected by intermediate doses of UVlight, conditions that totally eliminategoosecoid expression. Furthermore,chordin, noggin, follistatin, and cerbe-rus all exhibit two distinct temporalphases of expression: initiation andmaintenance (Wessely et al., 2001).Therefore, it is clear that the chordingene is subject to distinct modes of reg-ulation that may be unique or sharedwith only a small subset of organizer-specific genes. This unique regulationmay be critical for precisely controllingchordin levels that affect BMP depen-dent events in the embryo.

EXPERIMENTALPROCEDURES

Embryos and Injections

Embryos were obtained by standardmethods and staged according to Nieu-wkoop and Faber. mRNAs encodingchordin and GFP were generated aspreviously described (Sasai et al., 1994;Zernicka-Goetz et al., 1996). Four-cellembryos received two injections in oneposterior blastomere just above and be-low where the medial cleavage furrowwas expected to form. The 32-cell em-bryos were injected into one C4 blas-tomere.

For surgical experiments, both pos-terior blastomeres of four-cell em-bryos were injected in the marginalzone with 100 pg of either GFP and 25,50, 125, 250, or 500 pg of chordinmRNA. For experiments examiningmyf5 expression, one C4 blastomere of32-cell embryos was injected with 260

pg of chordin mRNA. For chordin de-pletion experiments, 2, 4, or 6 nL(0.4–1.2 ng) of DEED oligonucleotideswere injected into the marginal zonesof two- to four-cell embryos.

UV Irradiation

Embryos were dejellied 15 min afterfertilization, irradiated with 1,000 –2,000 �J of ultraviolet light inquartz cuvettes, and left undis-turbed for 3 hr. One third of the ir-radiated embryos from each UV dosewere fixed with MEMFA at stage10.5. The remaining embryos werescored for defects at stage 33– 40 ac-cording to the DAI scale (Kao andElinson, 1988) and fixed.

Tissue Dissections

Cleavage stage embryos were injectedwith various amounts of chordin andGFP mRNAs. Only embryos exhibit-ing a GFP signal opposite the upperblastopore lip were used. Posterior tis-sue was excised from injected stage10� embryos in agarose-coated dishescontaining DFA/BSA solution (Sateret al., 1993) using a hairknife and aneyepiece protractor to remove a 90-degree wedge of tissue. The next daytissues were examined for elongationand fixed with MEMFA when unin-jected siblings reached stage 33–34.Pronephros formation was deter-mined by in situ hybridization for thetubule-specific mRNA, XSMP-30 (Satoet al., 2000); somitic muscle differen-tiation was determined by either im-munocytochemistry with the muscle-specific antibody 12/101 (Kintner andBrockes, 1984) or in situ hybridizationfor muscle actin.

Immunocytochemistry

Stage 30–33 injected embryos wereevaluated for somitic muscle using themuscle-specific antibody 12/101 (Kint-ner and Brockes, 1984) and goat anti-IgG secondary antibody coupled to ei-ther HRP or alkaline phosphatase (AP).Notochord was detected in stage 30–32embryos using the notochord-specificantibody Tor70 and an HRP-coupledanti-IgM antibody. Pronephric tubuleswere detected in stage 38–40 embryosusing the tubule-specific antibody 3G8(Vize et al., 1995), and an anti-IgG AP-

coupled secondary antibody. Pronephricducts were detected in stage 38–40 em-bryos using the duct-specific antibody4A6 (Vize et al., 1995), and an anti-IgGAP-coupled secondary antibody. HRP-coupled antibodies were detected withdiaminobenzidene substrate; 5-bromo-4-chloro-3-indolyl phosphate (BCIP) orBM purple substrates were used to de-tect the AP-coupled antibody. Prone-phros defects were assessed by visualcomparison of tubule and/or duct massto control embryos. Pronephros forma-tion was considered defective if the tu-bules and/or ducts were either entirelyabsent or greatly reduced in mass, asindicated by the absence of or strongreduction in 3G8- or 4A6-positive cells.

In Situ Hybridization

In situ hybridization was performedas described (Harland, 1991). Myf 5probe was synthesized by linearizingthe Xmyf-5-2 template (Hopwood etal., 1991) with HindIII and transcrib-ing with SP6 RNA polymerase.

RT-PCR

RNA isolation and cDNA synthesiswas performed using standard proto-cols. PCR amplification was per-formed with the following primerpairs ODC (F:GCCATTGTGAAGAC-TCTCTCCATTC, R:TTCGGGTGAT-TCCTTGCCAC [Heasman et al.,2000]), muscle actin (F:TCCCTG-TACGCTTCTGGTCGTA, R:TCTCAA-AGTCCAAAGCCACATA[Stutz andSpohr, 1986]), and XSMP-30 (F:GG-CAAAAGCTCAAATCGAAGG, R:AT-TGAGGCTGGCGTTTCTTCC).

Oligonucleotide Synthesis

Partially modified DEED oligonucleo-tides were synthesized as described(Dagle et al., 2000; Dagle and Weeks,2000). The chordin antisense se-quence used was as follows: T�T�C�T�A�TGGACCAT�G�A�G�C�C� where “�” indicates a modificationof the phosphate linkage with DEED.The control oligonucleotide sequencewas identical to the chordin sequencebut contained additional DEED modi-fications that sterically prevent recog-nition of these oligonucleotides byRNase-H.

260 MITCHELL ET AL.

ACKNOWLEDGMENTSThe authors thank Connie Lane formany helpful discussions and advice.We thank members of the Sheets andFox labs for their lively discussions. Wethank Richard Harland and FabrizioSerluca for technical advice, and theFallon lab for the use of their digitalcamera. John Gurdon, Bill Smith, Rich-ard Harland, Eddy De Robertis, andRyuicchi Nishinakamura generouslyprovided plasmids. M.S. and D.W. werefunded by grants from the NIH andT.M. was supported by a fellowshipfrom the American Heart Association.

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