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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
Although it is well known that patterning in the retina of vertebrates is essential for retina formation and for the retinotopic projection of axons in the embryo, knowledge of molecular and cellular mechanisms of retina patterning is limited. We have previously identified the Xenopus Tbx3 gene (XTbx3) which is expressed in the dorsal retina but not in the ventral retina 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 that the manipulation of the amount and location of its products could be informative for understanding its normal function. Here we report that ectopic expression of Tbx3 by mRNA injection suppressed formation of the ventral retina. Furthermore, Tbx3 injection led to inhibition of molecular markers for the ventral retina including Pax-2 and netrin, indicating that Tbx3 plays an important role in retina dorsal/ventral patterning in vertebrates by inhibition of gene expression for ventral retina specification. © 2002 Elsevier Science Key Words: Tbx3; Xenopus; retina; dorsal/ventral patterning.
A fundamental aspect of eye development is the establishment of the anterior–posterior (A–P) and dorsal–ventral (D–V) axes within retina. Different cell types are aligned along these axes in patterns that are essential for vision. However, our knowledge of the molecular and cellular mechanism is very limited. Recent studies have begun to reveal this complicated process. Molecules expressed in dorsal or ventral retina have been shown to play important roles in vertebrate retina dorsal-ventral patterning. Vax2, a homeobox gene expressed in the ventral retina region, ventralizes the retina and perturbs the retinotectal mapping when it is misexpressed (3–7). Loss-of-func1
To whom correspondence and reprint requests should be addressed. Fax: (752) 2817-1006. E-mail: [email protected].
tion mutants have revealed crucial roles for Pax2 (a ventral marker) in the generation of the optic stalk and for Pax6 (an entire eye marker) in the development of the optic cup. Ectopic expression of Pax6 in the optic stalk under the control of Pax2 promoter elements resulted in a shift of the optic cup/optic stalk boundary through 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, are strictly expressed in the dorsal retina (9 –12). Misexpression of Tbx5 leads to dorsalize the retina and also perturb retinotectum projection correlated with the loss of the ventral marker Pax2 and Vax (10). However, the roles of other T box genes, such as Tbx3, in eye formation have not been reported. Tbx3 is expressed in the dorsal retina in mouse and chicken (11, 12). In frog, a pseudovariant of TBX3 (Xltbx3) is also expressed in dorsal retina. Compared with Tbx2, Tbx4, and Tbx5, Xltbx3 is the one of earliest expressed genes in Xenopus embryo (9). The presence of frog, mouse and chick Tbx3 in the dorsal retina suggests that its function in retina formation may be conserved in vertebrates. We have previously identified a new Xenopus T box gene ET, which is first expressed in retina primordia as in a single band across the midline in the anterior neural plate. It is expressed in the dorsal retina but not in the ventral retina at later stages in Xenopus embryo (1). Further studies have revealed that ET is Xenopus Tbx3 (XTbx3) (2) and its expression is much earlier than that of TBX5 (1, 9, 13). Opposite to TBX5, its gene product TBX3 is a transcription repressor rather than activator (2, 13, 14). Mutations in TBX3 and TBX5 cause dosage-sensitive phenotypes (15–20), suggesting that manipulating the amount and location of Tbx3 products will provide useful information in elucidating its normal functions. Here we report that ectopic expression of Tbx3 by mRNA injection in Xenopus embryos suppressed the formation of morphological visible ventral retina. In addition, Tbx3 injection resulted in inhibition of molecular markers expressed in the ventral retina.
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FIG. 1. The expression pattern of XTbx3 is colocalized with that of Xrx1 in the Xenopus embryos by in situ hybridization. XTbx3 transcripts are stained in purple and Xrx1 transcripts are stained in red. (A) At st. 13, XTbx3 is colocalized with Xrx1 in anterior neural plate region. (B) At st. 17, XTbx3 transcripts are located in the distinct eye promodia. (C) At st. 20, the expression pattern of Xrx is a band across the anterior neural plate, XTbx3 is expressed in the dorsal lateral region of Xrx1 field. (D) At st. 25, XTbx3 is strong expressed in the dorsal lateral region of xrx1 field. (E) At st. 28, XTbx3 expression is restricted only at dorsal retinal region, while Xrx1 is expressed in the whole eye.
METHODS AND MATERIALS Cloning and constructions. Tbx3 and Tbx3-GR were cloned into p64T vector. Nuclear -gal (-gal fused with nuclear localization signal) in pGEMblue was kindly provided by Ali HemmatiBtivanlou. Capped mRNA was transcribed by in vitro transcription using appropriate RNA polymerases. XNet-1 was cloned by PCR using forward and reverse primers: 5⬘-GCGCAACCGGATCCCT GC-3⬘ and 5⬘-CGCCTTCTTGCATTTGCC-3⬘, respectively. The resulting 1.2-kb fragment was cloned in pBluescipt KS. XPax-2 was cloned from stage 17 Xenopus cDNA library by degenerate PCR under conditions containing 2% formamide at 60°C annealing temperature. Forward primer: 5⬘-GGIGTIAAYCAR YTIGGIGGIGT-3⬘. Reverse primer: 5⬘-RAAIACRTCIGGRTAN SWIGG-3⬘. The 700-bp PCR product was used to screen a stage 17 Xenopus cDNA library. A clone containing a 1.2-kb fragment encoding for Xpax2 was obtained and used as template for generating probe used in situ hybridization. Microinjection of capped mRNA. Xenopus embryos were obtained by standard in vitro fertilization method and staged according to Nieuwkoop and Faber (21). Capped mRNA were synthesized by in vitro transcription. Four hundred picograms to 1 ng was injected into the animal pole of one blastomere in two-cell stage embryos. Embryos injected with Tbx3-GR were devitellinized at appropriate stages and raised in 1⫻ MMR containing 10 M dexamethasone (Sigma). Eye phenotype was scored at stage 37/38. In situ hybridization (22). Embryos were raised in 1⫻ MMR at room temperature or at 4°C. At desired stages, embryos were devitellinized, fixed in MEMFA at 4°C overnight, and stored in 100% methanol at ⫺20°C. In situ hybridization was performed essentially as described in Harland (22). Digotinxin-labeled mRNAs were generated by in vitro transcription in the presence of either digoxygeninUTP or fluorescein-UTP. For double in situ hybridization, embryos were hybridized with both probes simultaneously, washed, and incubated in blocking buffer (5% sheep serum, 2 mg/ml BSA, 1% DMSO) containing 1:2000 diluted anti-fluorescein antibody conjugated to alkaline phosphatase Fab fragments (BM). After developing
in fast-red substrate (BM), embryos were rinsed three times in PBTw (0.1% Tween 20 in 1⫻ PBS) and fixed in 4% paraformaldehyde for 30 min. Embryos were then washed in PBTw and incubated for 10 min in antibody buffer (0.1 M glycine–HCl, pH 2.2, 0.1% Tween 20). After washing in PBTw, samples were blocked in blocking buffer and incubated for 2 h in 1:2000 diluted anti-digoxygenin-alkaline phosphatase Fab fragments (BM). X-phosphate and NBT (BM) were used as substrates for the second color reaction. Sections and photography. After in situ hybridization, embryos were embedded in 4% low-melting point agarose and sectioned with vibratome at 30-m increments. Other embryos were processed and embedded in paraffin using standard protocol and sections of 10-m thickness were cut using a microtome (Olympus). Images were captured using Olympus microscope with a CCD camera (CCD-IRIS, Sony).
RESULTS Tbx3—One of the Earliest Genes Expressed in the Dorsal Retina As reported previously, Tbx3 expression is first detected by in situ hybridization at stage 12.5 embryo (1). Its transcripts are detected as a band with stronger and wider field at the lateral regions in the anterior neural plate. At stage 16, Tbx3 expression in the medial region of the retina field becomes weaker and the expression in this region totally disappears at stage 18 (1). Double in situ hybridization of Tbx3 (in purple) and Xrx1 (in red, an eye primordial marker) indicates that the expression pattern of Tbx3 is colocalized with expression field at stage 13 embryos (Fig. 1A). At stage
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becomes more apparent as the optic cup develops, Tbx3 is exclusively detected in the dorsal retina as shown in Fig. 1E.
FIG. 2. Induction of abnormal eye phenotype by over-expression of XTBX3. mRNA encoding XTBX3 was injected into one blastomere and RNA encoding for -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 with mRNA encoding for -gal). (C) Lateral view of (same embryo shown in B) abnormal eye injected with mRNA encoding XTBX3. (D) Embryos at st. 35. mRNA encoding XTBX3-GR was injected into two blastomeres of two-cell stage embryos and the function of XTBX3 was induced by dexamethasone at st. 25. (Up) Embryo injected with mRNA encoding -gal. (Down) Embryo injected with mRNA encoding TBX3-GR. (E) Section of a st. 35 embryo with injection of mRNA encoding for -gal in one blastomere and mRNA encoding XTBX3 in the other blastomere at two-cell stage. Comparing with the wild type eye (left), the PRE is lost in the ventral retinal region after injection of Xtbx3 mRNA. (F) -gal staining showed that -gal is localized only one side of embryo after injection of mRNA in one blastomere at two cell stage.
17, The expression pattern of Tbx3 is most strongly expressed in the lateral region of Xrx1 field. Tbx3 becomes apparent only as two distinct eye fields while the expression pattern of Xrx1 remains to appear as a band across the anterior neural plate (Fig. 1B). This region has been shown to form the dorsal retina (23). At stages 20 and 25, the expression pattern of Xrx1 resolves into two distinct retina regions. Double in situ of Xrx1 and Tbx3 reveals that Tbx3 specifically occupies the most lateral regions within Xrx1 expressing region, which develops the future dorsal retina (Figs. 1C and 1D). At stage 28, when dorsal/ventral retina
FIG. 3. Overexpression of XTBX3 inhibits the ventral marker expressions. (A) Whole-mount in situ hybridization. The uninjected sides of the Xenopus embryos are showed in the tight column. (B) Section after in situ hybridization with XPax2 probe. (Left) Uninjected Xenopus embryo at st. 25; (right) st. 16 embryo with injection of XTbx3 mRNA in one blastomere at the two-cell stage.
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TBX3 in Eye Development To access the possible functions of TBX3, we have utilized a gain-of-function assay by microinjection of TBX3 mRNA into single blastomere in two-cell stage Xenopus embryos. Nuclear -gal mRNA and TBX3 mRNA are injected together in some cases to help differentiate the injected side from the un-injected side. A reduced-eye phenotype (dorsal view, Fig. 2A) is observed in association with TBX3 injection at high frequency (253 of 281, 90%). The phenotype is characterized by change in retina morphology, including aberrant ventral retinal structures such as loss of ventral retina pigment epithelium (RPE) and lamination, disorder of dorsal RPE as shown in Figs. 2C and 2E. The distance between neural tube and retina is often reduced in the injected site of embryos (Fig. 2E). Molecular Characterization of TBX3-Injected Embryos To determine whether the abnormal eye phenotype in TBX3 injected embryos is due to change of dorsal/ ventral retinal cell fate, whole-mount in situ hybridization on TBX3 injected embryos using molecular markers that specifically express in dorsal (BMP4, Xvent2, and XTbx5) (25–27) or ventral (XNet1 and XPax2) (8, 28, 29) retina was performed. Expressions of dorsal markers BMP-4, Tbx5 and Xvent-1 in stage 26 to 28 embryos are still present in the dorsal retina on the TBX3 injected side with morphological change in shape from crescent to straight band (Fig. 3A). When ventral markers Xnet1 and Xpax2 are used on stage 28 and 35 embryos, no staining (Xnet-1) or reduced staining (Xpax-2) is detected in the ventral retina while optic stalk staining remains apparent (Fig. 3A). Further section assay showed that Xpax-2 was strongly expressed in the very ventral side of retina in uninjected embryo (Fig. 3B, left panel) or uninjected 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 phenotype observed in the TBX3 injected embryos may be due to TBX3 misexpression. The loss of ventral marker staining indicates a loss of ventral retina in the TBX3 injected 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 results in abnormal eye phenotype characterized with loss of ventral retina specification. The next question we wanted to address is when TBX3 acts during the course of eye development. To approach this problem, we made use of the hormone inducible fusion protein system. mRNA encoding full-length TBX3 fused with the
glucocorticoid receptor (GR) was microinjected into one blastomere of two-cell-stage embryos, the activation of TBX3 was induced at different developmental stages by the addition of ligand dexamethasone. The results indicated that TBX3 injection is able to give rise to the abnormal eye phenotype with retina defect in the ventral side when the protein is activated as late as stage 25, after the start of evagination of the optic vesicle (Fig. 2D). DISCUSSION Retina development is a major event during the formation of the vertebrate eye. The timing of dorsal/ ventral patterning in the retina has also been defined in Xenopus by isolating and rotating the eye field at different stages and following the resulting retinotectal projection (30, 31). Although Tbx5 and Vax2 were shown to specify positional identity along the D-V axis of the retina and influence retinotectal projection, the understanding of the molecular mechanisms is very poor. More molecules involved in this complicated process should be identified. In this paper, we showed that Tbx3 is an early gene responsible for retina dorsal/ ventral patterning. TBX3 in Dorsal/Ventral Retina Specification The identification of genes that are dorsal- or ventral-retina specific, such as BMP4 and Tbx5, and netrin and XPax2, respectively, provides useful resources as molecular markers for the assessment of dorsal/ventral identity of the retina (3, 8, 26, 27). Early expression pattern of TBX3 suggests that TBX3 plays an important role in early dorsal retina specification. It is one of the earliest gene known to be expressed in the lateral anterior neural ridge, which has been fatemapped to become the future dorsal retina (1, 12). TBX3 is first detected in the retina primordia as early as stage 12.5, whereas the expression of Tbx5 does not become detectable by in situ hybridization until retina area has been specified at stage 20 (13). Tbx3 is expressed in all the layers of retina (1), while Tbx5 is strictly expressed in the retina epithelium layer (32), suggesting that the dorsal specification of retina by TBX3 is the first event. It is possible that a dorsal area is first specified by TBX3 in the retina, then other molecules, such as TBX5, specify more specific structures. Mutations in human homologue of TBX3 have recently been linked to the ulnar-mammary syndrome (15, 16). Defects in this autosomal dominant disorder include malformation of upper limb structures, possibly due to alterations of proximal/distal, anterior/ posterior and dorsal/ventral axes (16). The dosagedependent characteristic of this gene, as revealed by the haploinsufficiency linkage with the ulnar-mammary syndrome and by gain-of-function study in this
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report, suggests the function of this T-box gene in axes patterning during development. TBX3 in Dorsal Retina Development To access the possible function of TBX3 in eye development, we have employed over/misexpression of the gene by mRNA microinjection. The resulting eye defect led us to believe that the abnormal eye phenotype is mainly due to the misexpression of TBX3 in the region where it normally would become the ventral retina. The aberrant ventral retinal structures in the TBX3 injected embryos are likely to have resulted from a lack of ventral retina specification, as suggested by the loss or reduction of ventral retina markers XNet-1 and XPax-2 expression. The resulting morphological change may then lead to the observed malformed ventral retina with defects in ventral RPE and ventral retinal lamination which are developed by interdependent events. TBX3 mRNA microinjection may cause the observed eye phenotype by promoting dorsal retina cell fate or by suppressing ventral retina cell fate. Results from in situ hybridization indicate that the latter is more likely to be the case because only the dorsal region of the retina, but not the whole retina, stained positive for dorsal retina markers (BMP4 and Tbx5). This in turn suggests that the lack of ventral retina structures in the TBX3 injected embryos was a result of TBX3 misexpression. Results from the TBX3-GR experiment revealed that TBX3 misexpression as late as stage 25 can affect normal eye development. This information allowed us to further identify possible events during retina development that may be affected by increased TBX3 activity. According to previous reports on normal Xenopus eye development, optic cup formation takes place at this stage, the key process involved is invagination, and forces pushing at the ventral retina are also reported to be important for the morphogenesis of the optic cup (33–36). The morphological changes of the retina including malformed optic cup in TBX3 injected embryo suggest the likelihood that TBX3 misexpression affects retina development by affecting this process. Interestingly, TBX3 misexpression did not seem to affect optic stalk development, as indicated by the in situ hybridization with ventral markers, which also stained the optic stalk. This suggests that the region that will develop into optic stalk is specified by other sets of genes different from that of the ventral retina. In fact, TBX5 has been revealed to play an important role in specifying optic stalk (10). Unlike the conversion between ventral retina and optic stalk tissues in the case of Pax-2 overexpression, the domain that was to become the ventral retina seemed to have remained unaltered in TBX3 misexpressing embryo but failed to undergo proper differentiation and morphogenesis.
The detailed molecular mechanism for TBX3 to contribute to the development of the Xenopus retina remains unknown. As TBX3 is a transcriptional repressor (2), one possibility is that TBX3 may repress the expression of genes that are important in ventral retina development. Hence, misexpressing TBX3 in ventral domain resulted in the repression of ventral retina development. Downstream targets of TBX3 and its other interacting proteins remain to be elucidated. REFERENCES 1. Li, H., Tierney, C., Wen, L., Wu, J. Y., and Rao, Y. (1997) A single morphogenetic field gives rise to two retina primordia under the influence of the prechordal plate. Development 124, 603– 615. 2. He, M.-L., Wen, L., Campbell, C. E., Wu, J. Y., and Rao, Y. (1999) Transcription repression by Xenopus ET and its human ortholog TBX3, a gene involved in ulnar-mammary syndrome. Proc. Natl Acad. Sci. USA 96, 10212–10217. 3. Schulte, D., Furukawa, T., Peters, M. A., Kozak, C. A., and Cepko, C. L. (1999). Misexpression of the Emx-related homeobox genes cVax and mVax2 ventralizes the retina and perturbs the retinotectal map. Neuron 24, 541–553. 4. Ohsaki, K., Morimitsu, T., Ishida, Y., Kominami, and Takahashi, N. (1999) Expression of the Vax family homeobox genes suggests multiple roles in eye development. Genes Cells 4, 267–276. 5. Pannese, M., Lupo, G., Kablar, B., Boncinelli, E., Barsacchi, G., and Vignali, R. (1998) The Xenopus Emx genes identify presumptive dorsal telencephalon and are induced by head organizer signals. Mech. Dev. 73, 73– 83. 6. Barbieri, A. M., Lupo, G., Bulfone, A., Andreazzoli, M., Mariani, M., Fougerousse, F., Consalez, G. C., Borsani, G., Beckmann, J. S., Barsacchi, G., Ballabio, A., and Banfi, S. (1999) A homeobox gene, vax2, controls the patterning of the eye dorsoventral axis. Proc. Natl. Acad. Sci. USA 96, 10729 –10734. 7. Liu, Y., Lupo, G., Marchitiello, A., Gestri, G., He, R. Q., Banfi, S., and Barsacchi, G. (2001) Expression of the Xvax2 gene demarcates presumptive ventral telencephalon and specific visual structures in Xenopus laevis. Mech. Dev. 100, 115–118. 8. Schwarz, M., Cecconi, F., Bernier, G., Andrejewski, N., Kammandel, B., Wagner, M., and Gruss, P. (2000) Spatial specification of mammalian eye territories by reciprocal transcriptional repression of Pax2 and Pax6. Development 127, 4325– 4334. 9. Takabatake, Y., Takabatake, T., and Takeshima, K. (2000) Conserved and divergent expression of T-box genes Tbx2-Tbx5 in Xenopus. Mech. Dev. 91, 433– 437. 10. Koshiba-Takeuchi, K., Takeuchi, J. K., Matsumoto, K., Momose, T., Uno, K., Hoepker, V., Ogura, K., Takahashi, N., Nakamura, H., Yasuda, K., and Ogura, T. (2000) Tbx5 and the retinotectum projection. Science 287, 134 –137. 11. Chapman, D. L., Garvey, N., Hancock, S., Alexiou, M., Agulnik, S. I., Gibson-Brown, J. J., Cebra-Thomas, J., Bollag, R. J., Silver, L. M., and Papaioannou, V. E. (1996) Expression of the T-box family genes. Tbx1–Tbx5, during early mouse development. Dev. Dyn. 206, 379 –390. 12. Gibson-Brown, J. J., Agulnik, S. I., Silver, L. M., and Papaioannou, V. E. (1998) Expression of T-box genes Tbx2–Tbx5 during chick organogenesis. Mech. Dev. 74, 165–169. 13. Horb, M. E., and Thomsen, G. H. (1999) Tbx5 is essential for heart development. Development 126, 1739 –1751. 14. Hiroi, Y., Kudoh, S., Monzen, K., Ikeda, Y., Yazaki, Y., Nagai, R., and Komuro, I. (2001) Tbx5 associates with Nkx2–5 and synergistically promotes cardiomyocyte differentiation. Nat. Genet. 28, 276 –280.
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15. Bamshad, M., Lin, R. C., Law, D. J., Watkins, W. C., Krakowiak, P. A., Moore, M. E., Franceschini, P., Lala, R., Holmes, L. B., Gebuhr, T. C., et al. (1997) Mutations in human TBX3 alter limb, apocrine and genital development in ulnar-mammary syndrome. Nat. Genet. 16, 311–315. 16. Bamshad, M., Le, T., Watkins, W. S., Dixon, M. E., Kramer, B. E., Roeder, A. D., Carey, J. C., Root, S., Schinzel, A., Van Maldergem, L., et al. (1999) The spectrum of mutations in TBX3: Genotype/phenotype relationship in ulnar-mammary syndrome. Am. J. Hum. Genet. 64, 1550 –1562. 17. Basson, C. T., Bachinsky, D. R., Lin, R. C., Levi, T., Elkins, J. A., Soults, J., Grayzel, D., Kroumpouzou, E., Traill, T. A., LeblancStraceski, J., et al. (1997) Mutations in human TBX5 [corrected] cause limb and cardiac malformation in Holt–Oram syndrome. Nat. Genet. 15, 30 –35. 18. Bruneau, B. G., Logan, M., Davis, N., Levi, T., Tabin, C. J., Seidman, J. G., and Seidman, C. E. (1999) Chamber-specific cardiac expression of Tbx5 and heart defects in Holt–Oram syndrome. Dev. Biol. 211, 100 –108. 19. Bruneau, B. G., Nemer, G., Schmitt, J. P., Charron, F., Robitaille, L., Caron, S., Conner, D. A., Gessler, M., Nemer, M., Seidman, C. E., and Seidman, J. G. (2001) A murine model of Holt–Oram syndrome defines roles of the T-box transcription factor Tbx5 in cardiogenesis and disease. Cell 106, 709 –721. 20. Li, Q. Y., Newbury-Ecob, R. A., Terrett, J. A., Wilson, D. I., Curtis, A. R., Yi, C. H., Gebuhr T., Bullen, P. J., Robson, S. C., Strachan, T., et al. (1997) Holt–Oram syndrome is caused by mutations in TBX5, a member of the Brachyury (T) gene family. Nat. Genet. 15, 1–9. 21. Nieukoop, P. D., and Faber, J. (1967) Normal Table of Xenopus laevis (Daudin), North Holland, Amsterdam. 22. Harland, R. M. (1991) In situ hybridization: An improved wholemount method for Xenopus embryos. Methods Cell Biol. 36, 675– 685. 23. Casarosa, S., Andreazzoli, M., Simeone, A., and Barsacchi, G. (1997) Xrx1, a novel Xenopus homeobox gene expressed during eye and pineal gland development. Mech. Dev. 61, 187–198. 24. Andreazzoli, M., Gestri, G., Angeloni, D., Menna, E., and Barsacchi, G. (1999) Role of Xrx1 in Xenopus eye and anterior brain development. Development 126, 2451–2460.
25. Gawantka, V., Delius, H., Hirschfeld, K., Blumenstock, C., and Niehrs, C. (1995) Antagonizing the Spemann organizer: Role of the homeobox gene Xvent-1. EMBO J. 14, 6268 – 6279. 26. Trousse, F., Esteve, P., and Bovolenta, P. (2001) Bmp4 mediates apoptotic cell death in the developing chick eye. J. Neurosci. 21, 1292–1301. 27. Zhang, X.-M., and Yang, X.-J. (2001) Temporal and spatial effects of sonic hedgehog signaling in chick eye morphogenesis. Dev. Biol. 223, 271–290. 28. de la Torre, J. R., Hopker, V. H., Ming, G. L., Poo, M. M., Tessier-Lavigne, M., Hemmati-Brivanlou, A., and Holt, C. E. (1997) Turning of retinal growth cones in a netrin-1 gradient mediated by the netrin receptor DCC. Neuron 19, 1211–1224. 29. Hopker, V. H., Shewan, D., Tessier-Lavigne, M., Poo, M., and Holt, C. (1999) Growth-cone attraction to netrin-1 is converted to repulsion by laminin-1. Nature 401, 69 –73. 30. Gaze, R. M., and Straznicky, C. (1980) Stable programming for map orientation in disarranged embryonic eyes in Xenopus. J. Embryol. Exp. Morphol. 55, 143–165. 31. Ide, C. F., Fraser, S. E., and Meyer, R. L. (1983) Eye dominance columns from an isogenic double-nasal frog eye. Science 221, 293–295. 32. Hatcher, C. J, Kim, M. S, Mah, C. S, Goldstein, M. M, Wong, B., Mikawa, T., and Basson, C. T. (2001) TBX5 transcription factor regulates cell proliferation during cardiogenesis. Dev. Biol. 230, 177–188. 33. Eagleson, G., Ferreiro, B., and Harris, W. A. (1995) Fate of the anterior neural ridge and the morphogenesis of the Xenopus forebrain. J. Neurobiol. 28, 146 –158. 34. Cepko, C. L. (1999) The roles of intrinsic and extrinsic cues and bHLH genes in the determination of retinal cell fates. Curr. Opin. Neurobiol. 9, 37– 46. 35. Perron, M., and Harris, W. A. (1999) Cellular determination in amphibian retina. In Cell Lineage and Fate Determination (Moody, S. A., Ed.), pp. 353–368, Academic Press, New York. 36. Reh, T. A., and Levine, E. M. (1998) Multipotential stem cells and progenitors in the vertebrate retina. J. Neurobiol. 36, 206 – 220.
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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
Although it is well known that patterning in the retina of vertebrates is essential for retina formation and for the retinotopic projection of axons in the embryo, knowledge of molecular and cellular mechanisms of retina patterning is limited. We have previously identified the Xenopus Tbx3 gene (XTbx3) which is expressed in the dorsal retina but not in the ventral retina 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 that the manipulation of the amount and location of its products could be informative for understanding its normal function. Here we report that ectopic expression of Tbx3 by mRNA injection suppressed formation of the ventral retina. Furthermore, Tbx3 injection led to inhibition of molecular markers for the ventral retina including Pax-2 and netrin, indicating that Tbx3 plays an important role in retina dorsal/ventral patterning in vertebrates by inhibition of gene expression for ventral retina specification. © 2002 Elsevier Science Key Words: Tbx3; Xenopus; retina; dorsal/ventral patterning.
A fundamental aspect of eye development is the establishment of the anterior–posterior (A–P) and dorsal–ventral (D–V) axes within retina. Different cell types are aligned along these axes in patterns that are essential for vision. However, our knowledge of the molecular and cellular mechanism is very limited. Recent studies have begun to reveal this complicated process. Molecules expressed in dorsal or ventral retina have been shown to play important roles in vertebrate retina dorsal-ventral patterning. Vax2, a homeobox gene expressed in the ventral retina region, ventralizes the retina and perturbs the retinotectal mapping when it is misexpressed (3–7). Loss-of-func1
To whom correspondence and reprint requests should be addressed. Fax: (752) 2817-1006. E-mail: [email protected].
tion mutants have revealed crucial roles for Pax2 (a ventral marker) in the generation of the optic stalk and for Pax6 (an entire eye marker) in the development of the optic cup. Ectopic expression of Pax6 in the optic stalk under the control of Pax2 promoter elements resulted in a shift of the optic cup/optic stalk boundary through 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, are strictly expressed in the dorsal retina (9 –12). Misexpression of Tbx5 leads to dorsalize the retina and also perturb retinotectum projection correlated with the loss of the ventral marker Pax2 and Vax (10). However, the roles of other T box genes, such as Tbx3, in eye formation have not been reported. Tbx3 is expressed in the dorsal retina in mouse and chicken (11, 12). In frog, a pseudovariant of TBX3 (Xltbx3) is also expressed in dorsal retina. Compared with Tbx2, Tbx4, and Tbx5, Xltbx3 is the one of earliest expressed genes in Xenopus embryo (9). The presence of frog, mouse and chick Tbx3 in the dorsal retina suggests that its function in retina formation may be conserved in vertebrates. We have previously identified a new Xenopus T box gene ET, which is first expressed in retina primordia as in a single band across the midline in the anterior neural plate. It is expressed in the dorsal retina but not in the ventral retina at later stages in Xenopus embryo (1). Further studies have revealed that ET is Xenopus Tbx3 (XTbx3) (2) and its expression is much earlier than that of TBX5 (1, 9, 13). Opposite to TBX5, its gene product TBX3 is a transcription repressor rather than activator (2, 13, 14). Mutations in TBX3 and TBX5 cause dosage-sensitive phenotypes (15–20), suggesting that manipulating the amount and location of Tbx3 products will provide useful information in elucidating its normal functions. Here we report that ectopic expression of Tbx3 by mRNA injection in Xenopus embryos suppressed the formation of morphological visible ventral retina. In addition, Tbx3 injection resulted in inhibition of molecular markers expressed in the ventral retina.
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FIG. 1. The expression pattern of XTbx3 is colocalized with that of Xrx1 in the Xenopus embryos by in situ hybridization. XTbx3 transcripts are stained in purple and Xrx1 transcripts are stained in red. (A) At st. 13, XTbx3 is colocalized with Xrx1 in anterior neural plate region. (B) At st. 17, XTbx3 transcripts are located in the distinct eye promodia. (C) At st. 20, the expression pattern of Xrx is a band across the anterior neural plate, XTbx3 is expressed in the dorsal lateral region of Xrx1 field. (D) At st. 25, XTbx3 is strong expressed in the dorsal lateral region of xrx1 field. (E) At st. 28, XTbx3 expression is restricted only at dorsal retinal region, while Xrx1 is expressed in the whole eye.
METHODS AND MATERIALS Cloning and constructions. Tbx3 and Tbx3-GR were cloned into p64T vector. Nuclear -gal (-gal fused with nuclear localization signal) in pGEMblue was kindly provided by Ali HemmatiBtivanlou. Capped mRNA was transcribed by in vitro transcription using appropriate RNA polymerases. XNet-1 was cloned by PCR using forward and reverse primers: 5⬘-GCGCAACCGGATCCCT GC-3⬘ and 5⬘-CGCCTTCTTGCATTTGCC-3⬘, respectively. The resulting 1.2-kb fragment was cloned in pBluescipt KS. XPax-2 was cloned from stage 17 Xenopus cDNA library by degenerate PCR under conditions containing 2% formamide at 60°C annealing temperature. Forward primer: 5⬘-GGIGTIAAYCAR YTIGGIGGIGT-3⬘. Reverse primer: 5⬘-RAAIACRTCIGGRTAN SWIGG-3⬘. The 700-bp PCR product was used to screen a stage 17 Xenopus cDNA library. A clone containing a 1.2-kb fragment encoding for Xpax2 was obtained and used as template for generating probe used in situ hybridization. Microinjection of capped mRNA. Xenopus embryos were obtained by standard in vitro fertilization method and staged according to Nieuwkoop and Faber (21). Capped mRNA were synthesized by in vitro transcription. Four hundred picograms to 1 ng was injected into the animal pole of one blastomere in two-cell stage embryos. Embryos injected with Tbx3-GR were devitellinized at appropriate stages and raised in 1⫻ MMR containing 10 M dexamethasone (Sigma). Eye phenotype was scored at stage 37/38. In situ hybridization (22). Embryos were raised in 1⫻ MMR at room temperature or at 4°C. At desired stages, embryos were devitellinized, fixed in MEMFA at 4°C overnight, and stored in 100% methanol at ⫺20°C. In situ hybridization was performed essentially as described in Harland (22). Digotinxin-labeled mRNAs were generated by in vitro transcription in the presence of either digoxygeninUTP or fluorescein-UTP. For double in situ hybridization, embryos were hybridized with both probes simultaneously, washed, and incubated in blocking buffer (5% sheep serum, 2 mg/ml BSA, 1% DMSO) containing 1:2000 diluted anti-fluorescein antibody conjugated to alkaline phosphatase Fab fragments (BM). After developing
in fast-red substrate (BM), embryos were rinsed three times in PBTw (0.1% Tween 20 in 1⫻ PBS) and fixed in 4% paraformaldehyde for 30 min. Embryos were then washed in PBTw and incubated for 10 min in antibody buffer (0.1 M glycine–HCl, pH 2.2, 0.1% Tween 20). After washing in PBTw, samples were blocked in blocking buffer and incubated for 2 h in 1:2000 diluted anti-digoxygenin-alkaline phosphatase Fab fragments (BM). X-phosphate and NBT (BM) were used as substrates for the second color reaction. Sections and photography. After in situ hybridization, embryos were embedded in 4% low-melting point agarose and sectioned with vibratome at 30-m increments. Other embryos were processed and embedded in paraffin using standard protocol and sections of 10-m thickness were cut using a microtome (Olympus). Images were captured using Olympus microscope with a CCD camera (CCD-IRIS, Sony).
RESULTS Tbx3—One of the Earliest Genes Expressed in the Dorsal Retina As reported previously, Tbx3 expression is first detected by in situ hybridization at stage 12.5 embryo (1). Its transcripts are detected as a band with stronger and wider field at the lateral regions in the anterior neural plate. At stage 16, Tbx3 expression in the medial region of the retina field becomes weaker and the expression in this region totally disappears at stage 18 (1). Double in situ hybridization of Tbx3 (in purple) and Xrx1 (in red, an eye primordial marker) indicates that the expression pattern of Tbx3 is colocalized with expression field at stage 13 embryos (Fig. 1A). At stage
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becomes more apparent as the optic cup develops, Tbx3 is exclusively detected in the dorsal retina as shown in Fig. 1E.
FIG. 2. Induction of abnormal eye phenotype by over-expression of XTBX3. mRNA encoding XTBX3 was injected into one blastomere and RNA encoding for -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 with mRNA encoding for -gal). (C) Lateral view of (same embryo shown in B) abnormal eye injected with mRNA encoding XTBX3. (D) Embryos at st. 35. mRNA encoding XTBX3-GR was injected into two blastomeres of two-cell stage embryos and the function of XTBX3 was induced by dexamethasone at st. 25. (Up) Embryo injected with mRNA encoding -gal. (Down) Embryo injected with mRNA encoding TBX3-GR. (E) Section of a st. 35 embryo with injection of mRNA encoding for -gal in one blastomere and mRNA encoding XTBX3 in the other blastomere at two-cell stage. Comparing with the wild type eye (left), the PRE is lost in the ventral retinal region after injection of Xtbx3 mRNA. (F) -gal staining showed that -gal is localized only one side of embryo after injection of mRNA in one blastomere at two cell stage.
17, The expression pattern of Tbx3 is most strongly expressed in the lateral region of Xrx1 field. Tbx3 becomes apparent only as two distinct eye fields while the expression pattern of Xrx1 remains to appear as a band across the anterior neural plate (Fig. 1B). This region has been shown to form the dorsal retina (23). At stages 20 and 25, the expression pattern of Xrx1 resolves into two distinct retina regions. Double in situ of Xrx1 and Tbx3 reveals that Tbx3 specifically occupies the most lateral regions within Xrx1 expressing region, which develops the future dorsal retina (Figs. 1C and 1D). At stage 28, when dorsal/ventral retina
FIG. 3. Overexpression of XTBX3 inhibits the ventral marker expressions. (A) Whole-mount in situ hybridization. The uninjected sides of the Xenopus embryos are showed in the tight column. (B) Section after in situ hybridization with XPax2 probe. (Left) Uninjected Xenopus embryo at st. 25; (right) st. 16 embryo with injection of XTbx3 mRNA in one blastomere at the two-cell stage.
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TBX3 in Eye Development To access the possible functions of TBX3, we have utilized a gain-of-function assay by microinjection of TBX3 mRNA into single blastomere in two-cell stage Xenopus embryos. Nuclear -gal mRNA and TBX3 mRNA are injected together in some cases to help differentiate the injected side from the un-injected side. A reduced-eye phenotype (dorsal view, Fig. 2A) is observed in association with TBX3 injection at high frequency (253 of 281, 90%). The phenotype is characterized by change in retina morphology, including aberrant ventral retinal structures such as loss of ventral retina pigment epithelium (RPE) and lamination, disorder of dorsal RPE as shown in Figs. 2C and 2E. The distance between neural tube and retina is often reduced in the injected site of embryos (Fig. 2E). Molecular Characterization of TBX3-Injected Embryos To determine whether the abnormal eye phenotype in TBX3 injected embryos is due to change of dorsal/ ventral retinal cell fate, whole-mount in situ hybridization on TBX3 injected embryos using molecular markers that specifically express in dorsal (BMP4, Xvent2, and XTbx5) (25–27) or ventral (XNet1 and XPax2) (8, 28, 29) retina was performed. Expressions of dorsal markers BMP-4, Tbx5 and Xvent-1 in stage 26 to 28 embryos are still present in the dorsal retina on the TBX3 injected side with morphological change in shape from crescent to straight band (Fig. 3A). When ventral markers Xnet1 and Xpax2 are used on stage 28 and 35 embryos, no staining (Xnet-1) or reduced staining (Xpax-2) is detected in the ventral retina while optic stalk staining remains apparent (Fig. 3A). Further section assay showed that Xpax-2 was strongly expressed in the very ventral side of retina in uninjected embryo (Fig. 3B, left panel) or uninjected 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 phenotype observed in the TBX3 injected embryos may be due to TBX3 misexpression. The loss of ventral marker staining indicates a loss of ventral retina in the TBX3 injected 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 results in abnormal eye phenotype characterized with loss of ventral retina specification. The next question we wanted to address is when TBX3 acts during the course of eye development. To approach this problem, we made use of the hormone inducible fusion protein system. mRNA encoding full-length TBX3 fused with the
glucocorticoid receptor (GR) was microinjected into one blastomere of two-cell-stage embryos, the activation of TBX3 was induced at different developmental stages by the addition of ligand dexamethasone. The results indicated that TBX3 injection is able to give rise to the abnormal eye phenotype with retina defect in the ventral side when the protein is activated as late as stage 25, after the start of evagination of the optic vesicle (Fig. 2D). DISCUSSION Retina development is a major event during the formation of the vertebrate eye. The timing of dorsal/ ventral patterning in the retina has also been defined in Xenopus by isolating and rotating the eye field at different stages and following the resulting retinotectal projection (30, 31). Although Tbx5 and Vax2 were shown to specify positional identity along the D-V axis of the retina and influence retinotectal projection, the understanding of the molecular mechanisms is very poor. More molecules involved in this complicated process should be identified. In this paper, we showed that Tbx3 is an early gene responsible for retina dorsal/ ventral patterning. TBX3 in Dorsal/Ventral Retina Specification The identification of genes that are dorsal- or ventral-retina specific, such as BMP4 and Tbx5, and netrin and XPax2, respectively, provides useful resources as molecular markers for the assessment of dorsal/ventral identity of the retina (3, 8, 26, 27). Early expression pattern of TBX3 suggests that TBX3 plays an important role in early dorsal retina specification. It is one of the earliest gene known to be expressed in the lateral anterior neural ridge, which has been fatemapped to become the future dorsal retina (1, 12). TBX3 is first detected in the retina primordia as early as stage 12.5, whereas the expression of Tbx5 does not become detectable by in situ hybridization until retina area has been specified at stage 20 (13). Tbx3 is expressed in all the layers of retina (1), while Tbx5 is strictly expressed in the retina epithelium layer (32), suggesting that the dorsal specification of retina by TBX3 is the first event. It is possible that a dorsal area is first specified by TBX3 in the retina, then other molecules, such as TBX5, specify more specific structures. Mutations in human homologue of TBX3 have recently been linked to the ulnar-mammary syndrome (15, 16). Defects in this autosomal dominant disorder include malformation of upper limb structures, possibly due to alterations of proximal/distal, anterior/ posterior and dorsal/ventral axes (16). The dosagedependent characteristic of this gene, as revealed by the haploinsufficiency linkage with the ulnar-mammary syndrome and by gain-of-function study in this
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report, suggests the function of this T-box gene in axes patterning during development. TBX3 in Dorsal Retina Development To access the possible function of TBX3 in eye development, we have employed over/misexpression of the gene by mRNA microinjection. The resulting eye defect led us to believe that the abnormal eye phenotype is mainly due to the misexpression of TBX3 in the region where it normally would become the ventral retina. The aberrant ventral retinal structures in the TBX3 injected embryos are likely to have resulted from a lack of ventral retina specification, as suggested by the loss or reduction of ventral retina markers XNet-1 and XPax-2 expression. The resulting morphological change may then lead to the observed malformed ventral retina with defects in ventral RPE and ventral retinal lamination which are developed by interdependent events. TBX3 mRNA microinjection may cause the observed eye phenotype by promoting dorsal retina cell fate or by suppressing ventral retina cell fate. Results from in situ hybridization indicate that the latter is more likely to be the case because only the dorsal region of the retina, but not the whole retina, stained positive for dorsal retina markers (BMP4 and Tbx5). This in turn suggests that the lack of ventral retina structures in the TBX3 injected embryos was a result of TBX3 misexpression. Results from the TBX3-GR experiment revealed that TBX3 misexpression as late as stage 25 can affect normal eye development. This information allowed us to further identify possible events during retina development that may be affected by increased TBX3 activity. According to previous reports on normal Xenopus eye development, optic cup formation takes place at this stage, the key process involved is invagination, and forces pushing at the ventral retina are also reported to be important for the morphogenesis of the optic cup (33–36). The morphological changes of the retina including malformed optic cup in TBX3 injected embryo suggest the likelihood that TBX3 misexpression affects retina development by affecting this process. Interestingly, TBX3 misexpression did not seem to affect optic stalk development, as indicated by the in situ hybridization with ventral markers, which also stained the optic stalk. This suggests that the region that will develop into optic stalk is specified by other sets of genes different from that of the ventral retina. In fact, TBX5 has been revealed to play an important role in specifying optic stalk (10). Unlike the conversion between ventral retina and optic stalk tissues in the case of Pax-2 overexpression, the domain that was to become the ventral retina seemed to have remained unaltered in TBX3 misexpressing embryo but failed to undergo proper differentiation and morphogenesis.
The detailed molecular mechanism for TBX3 to contribute to the development of the Xenopus retina remains unknown. As TBX3 is a transcriptional repressor (2), one possibility is that TBX3 may repress the expression of genes that are important in ventral retina development. Hence, misexpressing TBX3 in ventral domain resulted in the repression of ventral retina development. Downstream targets of TBX3 and its other interacting proteins remain to be elucidated. REFERENCES 1. Li, H., Tierney, C., Wen, L., Wu, J. Y., and Rao, Y. (1997) A single morphogenetic field gives rise to two retina primordia under the influence of the prechordal plate. Development 124, 603– 615. 2. He, M.-L., Wen, L., Campbell, C. E., Wu, J. Y., and Rao, Y. (1999) Transcription repression by Xenopus ET and its human ortholog TBX3, a gene involved in ulnar-mammary syndrome. Proc. Natl Acad. Sci. USA 96, 10212–10217. 3. Schulte, D., Furukawa, T., Peters, M. A., Kozak, C. A., and Cepko, C. L. (1999). Misexpression of the Emx-related homeobox genes cVax and mVax2 ventralizes the retina and perturbs the retinotectal map. Neuron 24, 541–553. 4. Ohsaki, K., Morimitsu, T., Ishida, Y., Kominami, and Takahashi, N. (1999) Expression of the Vax family homeobox genes suggests multiple roles in eye development. Genes Cells 4, 267–276. 5. Pannese, M., Lupo, G., Kablar, B., Boncinelli, E., Barsacchi, G., and Vignali, R. (1998) The Xenopus Emx genes identify presumptive dorsal telencephalon and are induced by head organizer signals. Mech. Dev. 73, 73– 83. 6. Barbieri, A. M., Lupo, G., Bulfone, A., Andreazzoli, M., Mariani, M., Fougerousse, F., Consalez, G. C., Borsani, G., Beckmann, J. S., Barsacchi, G., Ballabio, A., and Banfi, S. (1999) A homeobox gene, vax2, controls the patterning of the eye dorsoventral axis. Proc. Natl. Acad. Sci. USA 96, 10729 –10734. 7. Liu, Y., Lupo, G., Marchitiello, A., Gestri, G., He, R. Q., Banfi, S., and Barsacchi, G. (2001) Expression of the Xvax2 gene demarcates presumptive ventral telencephalon and specific visual structures in Xenopus laevis. Mech. Dev. 100, 115–118. 8. Schwarz, M., Cecconi, F., Bernier, G., Andrejewski, N., Kammandel, B., Wagner, M., and Gruss, P. (2000) Spatial specification of mammalian eye territories by reciprocal transcriptional repression of Pax2 and Pax6. Development 127, 4325– 4334. 9. Takabatake, Y., Takabatake, T., and Takeshima, K. (2000) Conserved and divergent expression of T-box genes Tbx2-Tbx5 in Xenopus. Mech. Dev. 91, 433– 437. 10. Koshiba-Takeuchi, K., Takeuchi, J. K., Matsumoto, K., Momose, T., Uno, K., Hoepker, V., Ogura, K., Takahashi, N., Nakamura, H., Yasuda, K., and Ogura, T. (2000) Tbx5 and the retinotectum projection. Science 287, 134 –137. 11. Chapman, D. L., Garvey, N., Hancock, S., Alexiou, M., Agulnik, S. I., Gibson-Brown, J. J., Cebra-Thomas, J., Bollag, R. J., Silver, L. M., and Papaioannou, V. E. (1996) Expression of the T-box family genes. Tbx1–Tbx5, during early mouse development. Dev. Dyn. 206, 379 –390. 12. Gibson-Brown, J. J., Agulnik, S. I., Silver, L. M., and Papaioannou, V. E. (1998) Expression of T-box genes Tbx2–Tbx5 during chick organogenesis. Mech. Dev. 74, 165–169. 13. Horb, M. E., and Thomsen, G. H. (1999) Tbx5 is essential for heart development. Development 126, 1739 –1751. 14. Hiroi, Y., Kudoh, S., Monzen, K., Ikeda, Y., Yazaki, Y., Nagai, R., and Komuro, I. (2001) Tbx5 associates with Nkx2–5 and synergistically promotes cardiomyocyte differentiation. Nat. Genet. 28, 276 –280.
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