Retina Dorsal/Ventral Patterning by Xenopus TBX3

Biochemical and Biophysical Research Communications 290, 737–742 (2002)

doi:10.1006/bbrc.2001.6266, available online at http://www.idealibrary.com on

Retina Dorsal/Ventral Patterning by Xenopus TBX3

Kit Wong,* Ying Peng,† Hsiang-fu Kung,† and Ming-Liang He†,1

*Department of Anatomy and Neurobiology, Washington University School of Medicine, Box 8108,660 South Euclid Avenue, St. Louis, Missouri 63110; and †Institute of Molecular Biology,University of Hong Kong, Pokfulam Road, Hong Kong, People’s Republic of China

Received November 20, 2001

tion mutants have revealed crucial roles for Pax2 (a
Although it is well known that patterning in the
retina of vertebrates is essential for retina formationand for the retinotopic projection of axons in the em-bryo, knowledge of molecular and cellular mecha-nisms of retina patterning is limited. We have previ-ously identified the Xenopus Tbx3 gene (XTbx3) whichis expressed in the dorsal retina but not in the ventralretina in Xenopus embryos [H. Li, C. Tierney, L. Wen,J. Y. Wu, and Y. Rao (1997) Development 124, 603–615;M.-L. He, L. Wen, C. E. Campbell, J. Y. Wu, and Y. Rao(1999) Proc. Natl. Acad. Sci. USA 96, 10212–10217].Dosage-sensitive phenotypes in humans suggest thatthe manipulation of the amount and location of itsproducts could be informative for understanding itsnormal function. Here we report that ectopic expres-sion of Tbx3 by mRNA injection suppressed formationof the ventral retina. Furthermore, Tbx3 injection ledto inhibition of molecular markers for the ventral ret-ina including Pax-2 and netrin, indicating that Tbx3plays an important role in retina dorsal/ventral pat-terning in vertebrates by inhibition of gene expressionfor ventral retina specification. © 2002 Elsevier Science

Key Words: Tbx3; Xenopus; retina; dorsal/ventralpatterning.

A fundamental aspect of eye development is the es-tablishment of the anterior–posterior (A–P) anddorsal–ventral (D–V) axes within retina. Different celltypes are aligned along these axes in patterns that areessential for vision. However, our knowledge of themolecular and cellular mechanism is very limited.

Recent studies have begun to reveal this complicatedprocess. Molecules expressed in dorsal or ventral ret-ina have been shown to play important roles in verte-brate retina dorsal-ventral patterning. Vax2, a ho-meobox gene expressed in the ventral retina region,ventralizes the retina and perturbs the retinotectalmapping when it is misexpressed (3–7). Loss-of-func-

1 To whom correspondence and reprint requests should be ad-dressed. Fax: (752) 2817-1006. E-mail: [email protected].

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ventral marker) in the generation of the optic stalk andfor Pax6 (an entire eye marker) in the development ofthe optic cup. Ectopic expression of Pax6 in the opticstalk under the control of Pax2 promoter elementsresulted in a shift of the optic cup/optic stalk boundarythrough a reciprocal inhibition of Pax2 promoter/enhancer activity by Pax6 protein and vice versa (8).Tbx2/3/4/5, members of the T box gene family, arestrictly expressed in the dorsal retina (9–12). Misex-pression of Tbx5 leads to dorsalize the retina and alsoperturb retinotectum projection correlated with theloss of the ventral marker Pax2 and Vax (10). However,the roles of other T box genes, such as Tbx3, in eyeformation have not been reported.

Tbx3 is expressed in the dorsal retina in mouse andchicken (11, 12). In frog, a pseudovariant of TBX3(Xltbx3) is also expressed in dorsal retina. Comparedwith Tbx2, Tbx4, and Tbx5, Xltbx3 is the one of earliestexpressed genes in Xenopus embryo (9). The presenceof frog, mouse and chick Tbx3 in the dorsal retinasuggests that its function in retina formation may beconserved in vertebrates.

We have previously identified a new Xenopus T boxgene ET, which is first expressed in retina primordia asin a single band across the midline in the anteriorneural plate. It is expressed in the dorsal retina but notin the ventral retina at later stages in Xenopus embryo(1). Further studies have revealed that ET is XenopusTbx3 (XTbx3) (2) and its expression is much earlierthan that of TBX5 (1, 9, 13). Opposite to TBX5, its geneproduct TBX3 is a transcription repressor rather thanactivator (2, 13, 14). Mutations in TBX3 and TBX5cause dosage-sensitive phenotypes (15–20), suggestingthat manipulating the amount and location of Tbx3products will provide useful information in elucidatingits normal functions. Here we report that ectopic ex-pression of Tbx3 by mRNA injection in Xenopus em-bryos suppressed the formation of morphological visi-ble ventral retina. In addition, Tbx3 injection resultedin inhibition of molecular markers expressed in theventral retina.

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METHODS AND MATERIALS

Cloning and constructions. Tbx3 and Tbx3-GR were cloned intop64T vector. Nuclear b-gal (b-gal fused with nuclear localizationsignal) in pGEMblue was kindly provided by Ali Hemmati-Btivanlou. Capped mRNA was transcribed by in vitro transcriptionusing appropriate RNA polymerases. XNet-1 was cloned by PCRusing forward and reverse primers: 59-GCGCAACCGGATCCCTGC-39 and 59-CGCCTTCTTGCATTTGCC-39, respectively. The re-sulting 1.2-kb fragment was cloned in pBluescipt KS. XPax-2 wascloned from stage 17 Xenopus cDNA library by degenerate PCRunder conditions containing 2% formamide at 60°C annealing tem-perature. Forward primer: 59-GGIGTIAAYCAR YTIGGIGGIGT-39.Reverse primer: 59-RAAIACRTCIGGRTAN SWIGG-39. The 700-bpPCR product was used to screen a stage 17 Xenopus cDNA library. Aclone containing a 1.2-kb fragment encoding for Xpax2 was obtainedand used as template for generating probe used in situ hybridization.

Microinjection of capped mRNA. Xenopus embryos were obtainedby standard in vitro fertilization method and staged according toNieuwkoop and Faber (21). Capped mRNA were synthesized by invitro transcription. Four hundred picograms to 1 ng was injected intothe animal pole of one blastomere in two-cell stage embryos. Em-bryos injected with Tbx3-GR were devitellinized at appropriatestages and raised in 13 MMR containing 10 mM dexamethasone(Sigma). Eye phenotype was scored at stage 37/38.

In situ hybridization (22). Embryos were raised in 13 MMR atroom temperature or at 4°C. At desired stages, embryos were de-vitellinized, fixed in MEMFA at 4°C overnight, and stored in 100%methanol at 220°C. In situ hybridization was performed essentiallyas described in Harland (22). Digotinxin-labeled mRNAs were gen-erated by in vitro transcription in the presence of either digoxygenin-UTP or fluorescein-UTP. For double in situ hybridization, embryoswere hybridized with both probes simultaneously, washed, and in-cubated in blocking buffer (5% sheep serum, 2 mg/ml BSA, 1%DMSO) containing 1:2000 diluted anti-fluorescein antibody conju-gated to alkaline phosphatase Fab fragments (BM). After developing

FIG. 1. The expression pattern of XTbx3 is colocalized with thtranscripts are stained in purple and Xrx1 transcripts are stained in rregion. (B) At st. 17, XTbx3 transcripts are located in the distinct eythe anterior neural plate, XTbx3 is expressed in the dorsal lateral relateral region of xrx1 field. (E) At st. 28, XTbx3 expression is reswhole eye.

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in fast-red substrate (BM), embryos were rinsed three times in PBTw(0.1% Tween 20 in 13 PBS) and fixed in 4% paraformaldehyde for 30min. Embryos were then washed in PBTw and incubated for 10 minin antibody buffer (0.1 M glycine–HCl, pH 2.2, 0.1% Tween 20). Afterwashing in PBTw, samples were blocked in blocking buffer andincubated for 2 h in 1:2000 diluted anti-digoxygenin-alkaline phos-phatase Fab fragments (BM). X-phosphate and NBT (BM) were usedas substrates for the second color reaction.

Sections and photography. After in situ hybridization, embryoswere embedded in 4% low-melting point agarose and sectioned withvibratome at 30-mm increments. Other embryos were processed andembedded in paraffin using standard protocol and sections of 10-mmthickness were cut using a microtome (Olympus). Images were cap-tured using Olympus microscope with a CCD camera (CCD-IRIS,Sony).

RESULTS

Tbx3—One of the Earliest Genes Expressedin the Dorsal Retina

As reported previously, Tbx3 expression is first de-tected by in situ hybridization at stage 12.5 embryo (1).Its transcripts are detected as a band with strongerand wider field at the lateral regions in the anteriorneural plate. At stage 16, Tbx3 expression in the me-dial region of the retina field becomes weaker and theexpression in this region totally disappears at stage 18(1). Double in situ hybridization of Tbx3 (in purple) andXrx1 (in red, an eye primordial marker) indicates thatthe expression pattern of Tbx3 is colocalized with ex-pression field at stage 13 embryos (Fig. 1A). At stage

of Xrx1 in the Xenopus embryos by in situ hybridization. XTbx3. (A) At st. 13, XTbx3 is colocalized with Xrx1 in anterior neural plateomodia. (C) At st. 20, the expression pattern of Xrx is a band across

n of Xrx1 field. (D) At st. 25, XTbx3 is strong expressed in the dorsalted only at dorsal retinal region, while Xrx1 is expressed in the

ated

e prgiotric

becomes more apparent as the optic cup develops, Tbx3


17, The expression pattern of Tbx3 is most stronglyexpressed in the lateral region of Xrx1 field. Tbx3 be-comes apparent only as two distinct eye fields while theexpression pattern of Xrx1 remains to appear as a bandacross the anterior neural plate (Fig. 1B). This regionhas been shown to form the dorsal retina (23).

At stages 20 and 25, the expression pattern of Xrx1resolves into two distinct retina regions. Double in situof Xrx1 and Tbx3 reveals that Tbx3 specifically occu-pies the most lateral regions within Xrx1 expressingregion, which develops the future dorsal retina (Figs.1C and 1D). At stage 28, when dorsal/ventral retina

FIG. 2. Induction of abnormal eye phenotype by over-expressionof XTBX3. mRNA encoding XTBX3 was injected into one blastomereand RNA encoding for b-gal was injected in the other blastomere. (A)Dorsal view of st. 34 embryo. Notice the size of wild type eye (left)and a reduced eye (right). (B) Lateral view of WT eye (injected withmRNA encoding for b-gal). (C) Lateral view of (same embryo shownin B) abnormal eye injected with mRNA encoding XTBX3. (D) Em-bryos at st. 35. mRNA encoding XTBX3-GR was injected into twoblastomeres of two-cell stage embryos and the function of XTBX3was induced by dexamethasone at st. 25. (Up) Embryo injected withmRNA encoding b-gal. (Down) Embryo injected with mRNA encod-ing TBX3-GR. (E) Section of a st. 35 embryo with injection of mRNAencoding for b-gal in one blastomere and mRNA encoding XTBX3 inthe other blastomere at two-cell stage. Comparing with the wild typeeye (left), the PRE is lost in the ventral retinal region after injectionof Xtbx3 mRNA. (F) b-gal staining showed that b-gal is localized onlyone side of embryo after injection of mRNA in one blastomere at twocell stage.

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is exclusively detected in the dorsal retina as shown inFig. 1E.

FIG. 3. Overexpression of XTBX3 inhibits the ventral markerexpressions. (A) Whole-mount in situ hybridization. The uninjectedsides of the Xenopus embryos are showed in the tight column. (B)Section after in situ hybridization with XPax2 probe. (Left) Unin-jected Xenopus embryo at st. 25; (right) st. 16 embryo with injectionof XTbx3 mRNA in one blastomere at the two-cell stage.

TBX3 in Eye Development glucocorticoid receptor (GR) was microinjected into one


To access the possible functions of TBX3, we haveutilized a gain-of-function assay by microinjection ofTBX3 mRNA into single blastomere in two-cell stageXenopus embryos. Nuclear b-gal mRNA and TBX3mRNA are injected together in some cases to help dif-ferentiate the injected side from the un-injected side.

A reduced-eye phenotype (dorsal view, Fig. 2A) isobserved in association with TBX3 injection at highfrequency (253 of 281, 90%). The phenotype is charac-terized by change in retina morphology, including ab-errant ventral retinal structures such as loss of ventralretina pigment epithelium (RPE) and lamination, dis-order of dorsal RPE as shown in Figs. 2C and 2E. Thedistance between neural tube and retina is often re-duced in the injected site of embryos (Fig. 2E).

Molecular Characterization of TBX3-Injected Embryos

To determine whether the abnormal eye phenotypein TBX3 injected embryos is due to change of dorsal/ventral retinal cell fate, whole-mount in situ hybrid-ization on TBX3 injected embryos using molecularmarkers that specifically express in dorsal (BMP4,Xvent2, and XTbx5) (25–27) or ventral (XNet1 andXPax2) (8, 28, 29) retina was performed.

Expressions of dorsal markers BMP-4, Tbx5 andXvent-1 in stage 26 to 28 embryos are still present inthe dorsal retina on the TBX3 injected side with mor-phological change in shape from crescent to straightband (Fig. 3A). When ventral markers Xnet1 andXpax2 are used on stage 28 and 35 embryos, no stain-ing (Xnet-1) or reduced staining (Xpax-2) is detected inthe ventral retina while optic stalk staining remainsapparent (Fig. 3A). Further section assay showed thatXpax-2 was strongly expressed in the very ventral sideof retina in uninjected embryo (Fig. 3B, left panel) oruninjected side (Fig. 3B, left eye showed in right panel)but weakly expressed in the injected side of embryos(Fig. 3B, right eye showed in the right panel).

Therefore, it appears that the abnormal eye pheno-type observed in the TBX3 injected embryos may bedue to TBX3 misexpression. The loss of ventral markerstaining indicates a loss of ventral retina in the TBX3injected embryos and suggests a role of TBX3 in dorsal/ventral patterning of the retina.

TBX3 Affects Retina Developmental Event(s)prior to Optic Cup Formation

As described above, injection of TBX3 mRNA resultsin abnormal eye phenotype characterized with loss ofventral retina specification. The next question wewanted to address is when TBX3 acts during the courseof eye development. To approach this problem, wemade use of the hormone inducible fusion protein sys-tem. mRNA encoding full-length TBX3 fused with the

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blastomere of two-cell-stage embryos, the activation ofTBX3 was induced at different developmental stagesby the addition of ligand dexamethasone.

The results indicated that TBX3 injection is able togive rise to the abnormal eye phenotype with retinadefect in the ventral side when the protein is activatedas late as stage 25, after the start of evagination of theoptic vesicle (Fig. 2D).

DISCUSSION

Retina development is a major event during the for-mation of the vertebrate eye. The timing of dorsal/ventral patterning in the retina has also been definedin Xenopus by isolating and rotating the eye field atdifferent stages and following the resulting retinotec-tal projection (30, 31). Although Tbx5 and Vax2 wereshown to specify positional identity along the D-V axisof the retina and influence retinotectal projection, theunderstanding of the molecular mechanisms is verypoor. More molecules involved in this complicated pro-cess should be identified. In this paper, we showed thatTbx3 is an early gene responsible for retina dorsal/ventral patterning.

TBX3 in Dorsal/Ventral Retina Specification

The identification of genes that are dorsal- orventral-retina specific, such as BMP4 and Tbx5, andnetrin and XPax2, respectively, provides useful re-sources as molecular markers for the assessment ofdorsal/ventral identity of the retina (3, 8, 26, 27). Earlyexpression pattern of TBX3 suggests that TBX3 playsan important role in early dorsal retina specification. Itis one of the earliest gene known to be expressed in thelateral anterior neural ridge, which has been fate-mapped to become the future dorsal retina (1, 12).TBX3 is first detected in the retina primordia as earlyas stage 12.5, whereas the expression of Tbx5 does notbecome detectable by in situ hybridization until retinaarea has been specified at stage 20 (13). Tbx3 is ex-pressed in all the layers of retina (1), while Tbx5 isstrictly expressed in the retina epithelium layer (32),suggesting that the dorsal specification of retina byTBX3 is the first event. It is possible that a dorsal areais first specified by TBX3 in the retina, then other mole-cules, such as TBX5, specify more specific structures.

Mutations in human homologue of TBX3 have re-cently been linked to the ulnar-mammary syndrome(15, 16). Defects in this autosomal dominant disorderinclude malformation of upper limb structures, possi-bly due to alterations of proximal/distal, anterior/posterior and dorsal/ventral axes (16). The dosage-dependent characteristic of this gene, as revealed bythe haploinsufficiency linkage with the ulnar-mam-mary syndrome and by gain-of-function study in this

report, suggests the function of this T-box gene in axes The detailed molecular mechanism for TBX3 to con-


patterning during development.

TBX3 in Dorsal Retina Development

To access the possible function of TBX3 in eye devel-opment, we have employed over/misexpression of thegene by mRNA microinjection. The resulting eye defectled us to believe that the abnormal eye phenotype ismainly due to the misexpression of TBX3 in the regionwhere it normally would become the ventral retina.The aberrant ventral retinal structures in the TBX3injected embryos are likely to have resulted from a lackof ventral retina specification, as suggested by theloss or reduction of ventral retina markers XNet-1and XPax-2 expression. The resulting morphologicalchange may then lead to the observed malformed ven-tral retina with defects in ventral RPE and ventralretinal lamination which are developed by interdepen-dent events.

TBX3 mRNA microinjection may cause the observedeye phenotype by promoting dorsal retina cell fate orby suppressing ventral retina cell fate. Results from insitu hybridization indicate that the latter is more likelyto be the case because only the dorsal region of theretina, but not the whole retina, stained positive fordorsal retina markers (BMP4 and Tbx5). This in turnsuggests that the lack of ventral retina structures inthe TBX3 injected embryos was a result of TBX3misexpression.

Results from the TBX3-GR experiment revealed thatTBX3 misexpression as late as stage 25 can affectnormal eye development. This information allowed usto further identify possible events during retina devel-opment that may be affected by increased TBX3 activ-ity. According to previous reports on normal Xenopuseye development, optic cup formation takes place atthis stage, the key process involved is invagination,and forces pushing at the ventral retina are also re-ported to be important for the morphogenesis of theoptic cup (33–36). The morphological changes of theretina including malformed optic cup in TBX3 injectedembryo suggest the likelihood that TBX3 misexpressionaffects retina development by affecting this process.

Interestingly, TBX3 misexpression did not seem toaffect optic stalk development, as indicated by the insitu hybridization with ventral markers, which alsostained the optic stalk. This suggests that the regionthat will develop into optic stalk is specified by othersets of genes different from that of the ventral retina.In fact, TBX5 has been revealed to play an importantrole in specifying optic stalk (10). Unlike the conver-sion between ventral retina and optic stalk tissues inthe case of Pax-2 overexpression, the domain that wasto become the ventral retina seemed to have remainedunaltered in TBX3 misexpressing embryo but failed toundergo proper differentiation and morphogenesis.

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tribute to the development of the Xenopus retina re-mains unknown. As TBX3 is a transcriptional repres-sor (2), one possibility is that TBX3 may repress theexpression of genes that are important in ventral ret-ina development. Hence, misexpressing TBX3 in ven-tral domain resulted in the repression of ventral retinadevelopment. Downstream targets of TBX3 and itsother interacting proteins remain to be elucidated.

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Retina Dorsal/Ventral Patterning by Xenopus TBX3