PATTERNS & PHENOTYPES

Developmental Analysis of Activin-Like KinaseReceptor-4 (ALK4) Expression in Xenopus laevisYumei Chen,1 Lisha L. Whitaker,2 and Ann F. Ramsdell1–3*

The type I transforming growth factor-beta (TGF�) receptor, activin-like kinase-4 (ALK4), is an importantregulator of vertebrate development, with roles in mesoderm induction, primitive streak formation,gastrulation, dorsoanterior patterning, and left–right axis determination. To complement previous ALK4functional studies, we have analyzed ALK4 expression in embryos of the frog, Xenopus laevis. Resultsobtained with reverse transcriptase-polymerase chain reaction indicate that ALK4 is present in both theanimal and vegetal poles of blastula stage embryos and that expression levels are relatively constantamongst embryos examined at blastula, gastrula, neurula, and early tail bud stages. However, the tissuedistribution of ALK4 mRNA, as assessed by whole-mount in situ hybridization, was found to change overthis range of developmental stages. In the blastula stage embryo, ALK4 is detected in cells of the animal poleand the marginal zone. During gastrulation, ALK4 is detected in the outer ectoderm, involuting mesoderm,blastocoele roof, dorsal lip, and to a lesser extent, in the endoderm. At the onset of neurulation, ALK4expression is prominent in the dorsoanterior region of the developing head, the paraxial mesoderm, andmidline structures, including the prechordal plate and neural folds. Expression in older neurula stageembryos resolves to the developing brain, somites, notochord, and neural crest; thereafter, additional sitesof ALK4 expression in tail bud stage embryos include the spinal cord, otic placode, developing eye, lateralplate mesoderm, branchial arches, and the bilateral heart fields. Together, these results not only reflect themultiple developmental roles that have been proposed for this TGF� receptor but also definespatiotemporal windows in which ALK4 may function to modulate fundamental embryological events.Developmental Dynamics 232:393–398, 2005. © 2004 Wiley-Liss, Inc.

Key words: ActRIB; ALK4; TGF�

Received 28 May 2004; Revised 20 August 2004; Accepted 26 August 2004

INTRODUCTION

Activin-like kinase-4 (ALK4), alsotermed activin receptor IB (ActRIB),is a type I transforming growth factor-beta (TGF�) receptor that was ini-tially identified and characterized asan activin receptor (ten Dijke et al.,1993; Willis et al., 1996). In additionto activin, other TGF�s are capable ofactivating the ALK4 pathway, includ-

ing nodal/Xnr-1, Vg1, and derriere(Reissmann et al., 2001; Yeo andWhitman, 2001; Cheng et al., 2003;Chen et al., 2004). Based largely onstudies in mouse and Xenopus laevis,two animal models in which embry-onic gain-of-function and loss-of-func-tion experiments are routinely per-formed, diverse activities have beenproposed for ALK4 and its ligands

during vertebrate development, in-cluding regulation of mesoderm induc-tion, primitive streak formation, gas-trulation, primary axis formation, andleft–right axis determination (Armesand Smith, 1997; Chang et al., 1997;Gu et al., 1998; Reissmann et al.,2001; Chen et al., 2004).

Despite the accumulation of evi-dence indicating that ALK4 is a key

1Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston, South Carolina2Department of Cell and Developmental Biology and Anatomy, University of South Carolina School of Medicine, Columbia, South Carolina3Program in Women’s Studies, College of Liberal Arts, University of South Carolina, Columbia, South CarolinaGrant sponsor: National Institutes of Health; Grant number: HL73270.*Correspondence to: Ann Ramsdell, Department of Cell Biology and Anatomy, Medical University of South Carolina, Charles-ton, SC 29425. E-mail: ramsdell@musc.edu

DOI 10.1002/dvdy.20232Published online 20 December 2004 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 232:393–398, 2005

© 2004 Wiley-Liss, Inc.

regulator of many embryonic pro-cesses, very little is known about thespatiotemporal aspects of ALK4 sig-naling during vertebrate embryogene-sis. Mice that are ALK4 null fail toform a primitive streak and are em-bryonic lethal before gastrulation, in-dicating that ALK4 signaling is re-quired very early in development (Guet al., 1998). Functional assays in Xe-nopus that use expression of a kinase-deficient, dominant-negative ALK4receptor result in defective dorsoante-rior development, mesoderm forma-tion, and left–right pattern, demon-strating that ALK4 signaling is alsonecessary for other developmentalevents (Chang et al., 1997; Chen et al.,2004). However, as with results ob-tained in mouse, the studies in Xeno-pus uncover a requirement for ALK4signaling but do not reveal when orwhere in the embryo ALK4 functionsto modulate various developmentalprocesses. To begin to address this is-sue, we have performed a develop-mental analysis of ALK4 expression inXenopus, using embryos ranging fromearly blastula stages to early tail budstages.

RESULTS

Temporal Analysis of ALK4Expression

As a prelude to performing in situ hy-bridization studies, ALK4 mRNA ex-pression was evaluated by reversetranscriptase-polymerase chain reac-tion (RT-PCR) to establish whenALK4 transcripts are present duringXenopus development. We find thatALK4 is expressed in all stages exam-ined, ranging from early blastulastages to early tail bud stages (Fig. 1).As determined by comparison to orni-thine decarboxylase-1 (ODC), an en-zyme that is uniformly expressedthroughout Xenopus development(Cao et al., 2001), maternal (stages7–8) and zygotic (stages 9�) expres-sion of ALK4 appears relatively in-variant across these stages.

To determine whether ALK4 ex-pression is restricted along the ani-mal–vegetal axis, animal or vegetalpole tissue was harvested from em-bryos collected at stages 8–9 for RT-PCR analysis. As shown in Figure 1B,ALK4 is detected in both tissue types,

as is ODC. By comparison, a mater-nally expressed mRNA, Vg1, is de-tected only in vegetal tissue, consis-tent with previous findings (Weeksand Melton, 1987).

Localization of ALK4 mRNAin Blastula Stage Embryos

Whole-mount in situ hybridization ofstage 7 embryos reveals that ALK4 isexpressed in cells of the animal pole(Fig. 2A), with faint nuclear stainingdetected in some vegetal cells posi-tioned at or near the marginal zone(Fig. 2B). At stage 8, ALK4 expressionis maintained in the animal pole andmarginal zone (Fig. 2C,D), and bystage 9, staining shifts posteriorly asanimal cells move toward the vegetalpole in a pregastrulation processcalled epiboly (Fig. 2E). Examinationof sectioned embryos (Fig. 2F) showsthat staining is present in cells of theanimal pole and the marginal zone;however, no staining is discernible inthe vegetal region, despite the detec-tion of ALK4 transcripts in this tissueby RT-PCR. Hybridization of multiplestages of embryos with a sense-strandprobe was performed as a negativecontrol to determine the extent of non-specific background signal, which wasminimal to none (Fig. 2G,H). Thesefindings, along with results obtainedby RT-PCR, indicate that, duringearly blastula stages, maternal ALK4expression is present in cells of thethree presumptive germ layers.

Localization of ALK4 mRNAin Gastrula Stage Embryos

At stage 10, ALK4 is detected in cellsof the animal pole and the marginalzone (Fig. 3A). Although cells withinthe blastopore do not express ALK4,

pronounced staining is detected in thedorsal lip (Fig. 3A). Sections of hybrid-ized embryos show that ALK4 ispresent in involuting mesoderm andin cells of the blastocoele roof (Fig.3B). In addition, endoderm and meso-derm cells that are beginning to mi-grate over the blastocoele roof showstaining for ALK4 expression (Fig.3B,F, arrowheads). These localizationpatterns persist during stage 11 (Fig.3C), with no obvious asymmetry de-tected in the left–right or dorsoante-rior axes (Fig. 3D). At stage 12–12.5,ALK4 is detected in the entire ecto-derm of whole-mount embryos (Fig.3E). Sections of hybridized embryosshow that ALK4 is expressed in cellsof the entire blastocoele roof, the en-tire outer ectoderm, the involutingdorsal and ventral mesoderm, and to alesser extent, cells of the ventralendodermal yolk mass (Fig. 3F). Bystage 13–13.5, ALK4 signal begins toresolve to broad domains encompass-ing the dorsoanterior and midline re-gions of the embryo as viewed inwhole-mount (Fig. 3G). Transverseand sagittal sections (Fig. 3H,I) showthat ALK4 is expressed in the primi-tive notochord, paraxial mesoderm,prechordal plate, and ectoderm. Noexpression is detected in endodermcells at this stage (Fig. 3H,I), and nostaining is observed in embryos hy-bridized with sense-strand probe(Fig. 3J).

Localization of ALK4 mRNAin Neurula and Tail BudStage Embryos

At stage 15 (Fig. 4A,B), ALK4 contin-ues to be detected in the dorsoanteriorand midline (arrowheads) regions ofthe embryo where expression ispresent in the neural plate, the neural

Fig. 1. Temporal analysis of ALK4 expression. A: The presence of ALK4 mRNA (top) and ornithinedecarboxylase 1 mRNA (ODC, bottom) was examined by reverse transcriptase polymerase chainreaction (RT-PCR) in blastula (stages 7–9), gastrula (stages 10–13), neurula (stages 15–20), and tailbud (stages 23–26) stage embryos. B: The presence of ALK4, ODC, and Vg1 mRNAs was examinedby RT-PCR in animal (A) or vegetal (V) pole tissue obtained from stages 8–9 embryos. ALK4,activin-like kinase-4.

394 CHEN ET AL.

and non-neural ectoderm, and theneural folds. Other tissues in this re-gion, including the somitic mesodermand the notochord, also express ALK4.At stage 18 (Fig. 4C,D), ALK4 midlineexpression (Fig. 4C, arrowheads) is lo-calized in the neural tube, neuralfolds, and the notochord. Staining alsois detected in the somitic mesodermand the dorsoanterior region of the de-veloping head. Midline expression ismaintained in stage 20 embryos (Fig.4E, arrowheads), and stronger stain-ing is detected in the outer ectoderm(Fig. 4F). Control embryos hybridizedwith a sense-orientation probe did notshow discernible staining (Fig. 4G,H).At stage 22, dorsoanterior ALK4 ex-pression is detected in all three re-gions of the developing brain as well

as in the developing eye; at the poste-rior end of the embryo, ALK4 is de-tected in the unsegmented paraxial(somitic) mesoderm and the tail blast-ema (not shown). Transverse sectionsof hybridized embryos show that mid-

line structures that express ALK4 in-clude the spinal cord, neural crest, no-tochord, and somites (Fig. 4I). Atstage 24, the midline distribution ofALK4 transcripts is maintained, andstaining is additionally detected in the

Fig. 2. ALK4 distribution in blastula stage embryos. A–H: Embryoswere collected at stages 7 (A), 8 (C), and 9 (E,G) and whole-mounthybridized with either an ALK4 antisense probe (A,C,E) or ALK4 senseprobe (G), followed by sectioning along the animal–vegetal axis(B,D,F,H). A higher magnification view of the outlined area in B is shownin the inset. ALK4, activin-like kinase-4; An, Animal; BC, blastocoele;MZ, marginal zone; Veg, vegetal.

Fig. 3. ALK4 distribution in gastrula stage embryos. A,C,E,G,J: Em-bryos were collected at stages 10 (A), 11 (C), 12 (E), and 13 (G) andwhole-mount hybridized with either an ALK4 antisense probe (A,C,E,G)or ALK4 sense probe (J). B,D,F,H,I: Hybridized embryos were sectionedalong the animal–vegetal axis (B,F), transversely (D,H), or sagittally (I,J).ALK4, activin-like kinase-4; ARC, archenteron; BC, blastocoele; BP,blastopore; DM, dorsal mesoderm; ECT, ectoderm; ENDO, endoderm;IDM, involuting dorsal marginal zone; IVM, involuting ventral marginalzone; NTC, notochord; PM, paraxial mesoderm; PCP, prechordal plate;VM, ventral mesoderm.

ALK4 EXPRESSION IN XENOPUS 395

cement gland, branchial arches, eyevesicle, otic placode, lateral plate me-soderm, and in the bilateral heartfields (Fig. 4J–L).

DISCUSSION

In the early blastula stage embryo,ALK4 expression is detected in boththe animal and vegetal pole regions ofthe embryo. These results are not un-expected, as longstanding models ofgerm layer formation in Xenopus haveshown that TGF�-related signaling(most likely Xnr-1 and/or Vg1) ema-nating from vegetal cells induces ad-jacent cells at the marginal zone tobecome mesoderm (Joseph and Doug-las, 1998; Kofron et al., 1999; Osadaand Wright, 1999; Agius et al., 2000).Cells that are located at the anterior-most region of the animal pole are nor-mally fated to become ectoderm, al-though they too are competent torespond to the inductive signal ifbrought into proximity with vegetalcells. Our finding that ALK4 is ex-pressed by the presumptive ectodermand mesoderm indicates that ALK4 is

expressed at an appropriate site tofunction as a receptor for the TGF�-related signal(s) emitted from the veg-etal cells. Moreover, the ALK4 expres-sion in blastula stage embryos isconsistent with functional studies inwhich widespread expression of adominant-negative ALK4 receptor inXenopus causes axial defects resultingfrom deficient mesoderm formation(Chang et al., 1997).

In addition to mesoderm formation,endoderm formation is dependentupon TGF�-related signaling emittedby vegetal cells. A role for ALK4 inmediating the latter is supported byfunctional studies showing that ALK4can induce endodermal marker ex-pression (endodermin, GATA-4) in an-imal caps (Armes and Smith, 1997;Reissmann et al., 2001) and that ex-pression of constitutively active ALK4in zebrafish blastomeres convertsthem to an endodermal fate (Davidand Rosa, 2001). Consistent withthese studies, we find by RT-PCR thatALK4 is expressed in vegetal pole tis-sue at levels comparable to those de-

tected in animal pole tissues. How-ever, because the sensitivity of whole-mount in situ hybridization indetecting endodermally expressedtranscripts in Xenopus whole-mountsis much lower than RT-PCR (Smithand Harland, 1992; Ku and Melton,1993), we cannot report its precise lo-calization.

As gastrulation begins, ALK4 expres-sion continues to be detected in the ec-todermal and mesodermal germ layers,and a little later, in the ventralendoderm. These findings are similar toa report of ALK4 expression by all threegerm layers in the early mouse embryo(Gu et al., 1998), and it is likely thatALK4 signaling is necessary for migra-tion of cells as they involute. Mouse em-bryos that are ALK4 null fail to form aprimitive streak, and chimeric embryosshow that ALK4 null cells cannot con-tribute to the mesodermal layer duringgastrulation (Gu et al., 1998). Consis-tent with these findings, we show thatALK4 is expressed in sites correspond-ing to the involuting dorsal and ventralmesoderm as well as in nascent mesen-

Fig. 4. ALK4 distribution in neurula stage embryos. A,C,E,G,J: Embryos were collected at stages 15 (A), 18 (C), 20 (E,G), 22 (I), and 24 (J) andwhole-mount hybridized with either an ALK4 antisense probe (A,C,E,J) or ALK4 sense probe (G). B,D,F,H,I,K,L: Hybridized embryos were sectionedtransversely (B,D,F,H,I) or sagittally (K,L). BA, branchial arches; CG, cement gland; ECT, ectoderm; EV, eye vesicle; HF, heart field; LPM, lateral platemesoderm; NF, neural fold; NT, neural tube; NTC, notochord; OP, otic placode; SM, somitic mesoderm; SC, spinal cord.

396 CHEN ET AL.

doderm cells that are migrating overthe roof of the blastocoele. It is notablethat ALK4 expression also was detectedin the dorsal lip, a structure that isfunctionally analogous to the node inavians and mammals and the shield infish. We find that the dorsal lip in Xe-nopus shows prominent ALK4 expres-sion, suggesting that ALK4 signalingcould be involved in dorsoventral pat-terning of the body plan. Because ani-mal cap assays have thus far only beenperformed by using pan-mesodermalmarkers (Armes and Smith, 1997; Re-issmann et al., 2001), it is not knownwhether this is the case; however, con-sistent with this possibility, ectopic ex-pression of dominant-negative ALK4 re-sults in embryos that exhibit highlydisorganized axial structure with re-spect to dorsoanterior development(Chang et al., 1997).

Mesodermal ALK4 expression duringneurula and tail bud stages is observedin midlines structures, including thenotochord, neural tube, paraxial(somitic) mesoderm, and the neuralcrest. Ectodermal ALK4 expression isdetected in both neural and non-neuralregions and the significance of this ex-pression is not known, as it has beensuggested that ALK4 is not involved inneural induction (Chang et al., 1997).Nevertheless, the prominent ALK4 ex-pression in the neural tube and its de-rivatives—brain and spinal cord—aswell as its conserved expression in thesetissue types in mouse (Verschueren etal., 1995) suggests that this issue is stillopen to investigation. Whether ALK4additionally plays a role in induction ofnon-neural ectodermal tissue types alsoremains to be determined. With regardto ALK4 expression by paraxial(somitic) mesoderm, and later by devel-oping somites, these results indicate apossible role for ALK4 signaling insomitogenesis. Although this role hasnot yet been directly investigated, a re-cent report indicates that activin, aknown ALK4 ligand, is capable of in-ducing ectopic somite formation in thechick embryo (Patwardhan andGhaskadbi, 2001) and analysis of ALK4expression in later stage mouse em-bryos indicates that ALK4 is expressedin tissues that are derived from somites,e.g., ribs, limb muscles, and the bodywall (Verschueren et al., 1995). More-over, studies in Xenopus indicate thatTGF-�5 is expressed in somites and

other areas that overlap with the sitesof ALK4 expression reported here (Kon-daiah et al., 2000). Additional sites ofALK4 expression in neurula stage em-bryos are the neural folds, the neuralcrest, and the branchial arches, throughwhich neural crest cells migrate. Al-though this is the first report of ALK4expression in these embryonic regions,there is abundant evidence that TGF�signaling regulates neural crest cell em-igration (Chai et al., 2003), and by anal-ogy to ALK4 expression in other migrat-ing cell types (involuting mesendo-derm), our results suggest that ALK4could play a role in this process.

Other notable sites of ALK4 expres-sion in tail bud stage embryos includethe developing eye, otic placode, and thebilateral heart fields. ALK4 expressionin the retina of the embryonic rat eyehas been reported previously and de-scribed in more detail (Yamada et al.,1999). With regard to expression in theotic placode, no requirement has beenidentified for ALK4 in ear morphogene-sis, although studies do indicate notonly that TGF�s are expressed in theear but also that TGF� signaling is nec-essary in promoting its normal develop-ment (Ito et al., 2001; Chang et al.,2002; Somi et al., 2003). The findingthat ALK4 additionally is expressed inthe bilateral heart fields is consistentwith the expression of Vg1 in the linearheart tube of the chick (Somi et al., 2003)and raises the possibility of yet anotherrole for ALK4 signaling—cardiogen-esis—in vertebrate embryogenesis.

Finally, with regard to formation ofthe body plan, it should be noted thatthere are several sites of ALK4 expres-sion that are interesting in the contextof left–right axis determination.These locations include involutingmesendoderm cells; midline struc-tures such as the dorsal lip, notochord,and neural tube; and the lateral platemesoderm. Because these tissues areinvolved in relaying left–right patternin the embryo and moreover, becausethey are thought to do so, at least inpart, by means of TGF�-related sig-naling (Yost, 1998; Mercola andLevin, 2001; Whitman and Mercola,2001; Hamada et al., 2002), the detec-tion of ALK4 at these sites suggeststhat this TGF� receptor could playmultiple, important roles in left–rightdevelopment in addition to its otherroles in vertebrate embryogenesis.

METHODS

Embryo Collection

Eggs were obtained from PMSG- andhCG-injected adult albino Xenopuslaevis females (fertilized with mincedtestes) and were de-jellied with 2.5%cysteine. Embryos were reared in1/3� MMR and staged according toNieuwkoop and Faber (1967).

RT-PCR

Total RNA was extracted from em-bryos of stages 7–24 as indicated inthe figure legends using TriReagent(Molecular Research Center, Inc.) ac-cording to the manufacturer’s proto-col. Two micrograms of total RNAfrom each stage was used for first-strand cDNA synthesis using Super-Script III first-strand synthesis kitfrom Invitrogen, and one tenth of thecDNA served as template for PCR.Twenty-two cycles (94°C, 30 sec; 55°C,1 min; 72°C, 30 sec) were carried outfor both ODC and Xenopus ALK4(XALK4). Primers for ODC (forward5�-CAA CGT GTG ATG GGC TGGAT-3� and reverse 5�-CAT AAT AAAGGG TTG GTC TCT GA-3�) were syn-thesized according to Altmann et al.(2002). Primers for XALK4 (forward5�-GCG GAG CTA CCG GCC TTCTTC-3� and reverse 5�-TGG GAT TGCAAT AAC AGC TAC-3�) were synthe-sized according to Chang et al. (1997).The primer sequences for Vg1 (for-ward 5�-GAT GTT GGA TGG CAAAAC TGG-3� and reverse 5�-CCA CACTCA TCT ACT GCC ATG-3�) weresynthesized according to Alarcon andElinson (2001). For comparison oftranscripts between animal and vege-tal regions, animal caps and vegetalcells were dissected out separatelyfrom 40 stage 8–9 embryos by using#5 forceps. Total RNA extraction andRT-PCR were performed as describedabove except that the PCR was run for25 cycles.

Whole-Mount In SituHybridization

A BglII insert from truncated ALK4(tALK4; Chang et al., 1997) encodingthe extracellular, transmembrane, andkinase-deficient cytoplasmic domains ofXenopus ALK4 was ligated into BamHIdigested CS2�. Digoxigenin-labeled ri-

ALK4 EXPRESSION IN XENOPUS 397

boprobes were synthesized with theMAXIscript kit from Ambion using Hin-dIII linearized CS2-tALK4 templateand T7 RNA polymerase for antisenseprobe and XbaI linearized CS2-tALK4template and SP6 RNA polymerase forsense probe. Whole-mount in situ hy-bridization was performed as describedpreviously (Harland, 1991) with the fol-lowing modifications: preabsorption ofthe anti–digoxigenin-alkaline phospha-tase antibody with matched stage em-bryos for 24 hr at 4°C and probe hybrid-ization at 68°C to reduce background.The BCIP/NBT color reaction wasstopped just before correspondingsense-hybridized embryos began tostain.

Embryo Sectioning

After whole-mount in situ hybridiza-tion, the stained embryos were photo-graphed and then sectioned as de-scribed (Germroth et al., 1995) withthe following modifications: embryoswere soaked in 15% polyacrylamidefor 30 min and oriented according tothe plane of sectioning after the addi-tion of ammonium persulfate. Sec-tions were cut with a Lancer Vi-bratome at 200 �m thickness.

ACKNOWLEDGMENTSWe thank Carlene Brandon (MUSC)for photographic assistance, CaraBaldwin from Dr. Joseph Yost’s lab(University of Utah) for assistancewith in situ hybridization, andChiffvon Stanley and Dr. RobertThompson (MUSC) for assistancewith polyacrylamide sectioning.

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