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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 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 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 ventralretina. Furthermore, Tbx3 injection led to inhibition of molecular markers for the ventralretina 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 ventralretina specification.
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.
FIG. 2. Induction of abnormal eye phenotype by over-expression
of XTBX3. mRNA encoding XTBX3 was injected into one blastomere
and 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 with
mRNA encoding for b-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 b-gal. (Down) Embryo injected with mRNA encoding
TBX3-GR. (E) Section of a st. 35 embryo with injection of mRNA
encoding for b-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) b-gal staining showed that b-gal is localized only
one side of embryo after injection of mRNA in one blastomere at two
cell stage.
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.