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Xlim-1, a LIM class homeobox gene expressed in Xenopus laevis, is one of the earliest known marker genes of pronephros development and is expressed in pronephros rudiment. In this study, we examined the role of Xlim-1 in pronephros development. Temporal expression of Xlim-1 in explants was analyzed in a series of induction assays using RT-PCR analysis. Xlim-1 was expressed 9 to 15 h after activin/retinoic acid treatment, corresponding to pronephros differentiation in explants. We further examined the role of Xlim-1 using a series of microinjection experiments. Presumptive pronephric anlagen of embryos were injected with various Xlim-1 mutants, and effects of these Xlim-1 mutants on pronephrogenesis in embryos and in explants were analyzed by RT-PCR and immunohistochemistry. Dominant-negative Xlim-1 inhibited differentiation of pronephros in activin/retinoic acid-treated animal caps. In embryos injected with a dominant-negative form of Xlim-1, development of pronephric tubules was inhibited at the late tail-bud stage. Our results suggest that Xlim-1 may not initiate differentiation of the pronephros, but that it is necessary for growth and elongation in the development of pronephric tubules.
FIG. 5. Injection of Xlim-1-enR did not inhibit development of pronephric duct. All embryos were stained with antibodies 3G8 (dark blue) and 4A6 (red). (A) Normal pronephros of a stage 40 embryo. In the illustration below, arrows indicate the three normal dorsal branches. (B and C) Inhibited pronephros in Xlim-1-enR-injected embryos. These embryos showed a normal phenotype before staining. (B) Faint staining of the posterior dorsal branch of the pronephric tubules is observed. The length of the dorsal branches is reduced, and only one anterior dorsal branch is detected (arrows). The common tubule is also shorter than normal pronephric tubules. (C) Severe inhibition of development of an Xlim-1-enR-injected embryo. Arrow indicates the reduced common tubule with a small posterior dorsal tubule. Length of S-shaped coil in pronephric duct is also reduced, but the diameter of the duct is normal (shown in E). (D and E) Transverse sections of embryos in A and C (D, normal embryo; E, Xlim-1-enR-injected embryo). Compared to normal pronephros in D, the pronephric tubule is severely reduced in size in Xlim-1-enR-injected embryos, but the structure of the pronephric duct is normal. As the appearance of these embryos suggests, somites are normal in both normal and Xlim-1-enR-injected embryos (arrow). This suggests that Xlim-1-enR can directly inhibit pronephric tubules without affecting anteriorsomites.
FIG. 4. Effects of Xlim-1-enR on pronephros development. (A and B) Xlim-1-enR-injected embryos. Xlim-1-enR and beta-galactosidase were injected into C2 and C3 blastomeres (left side) of 32-cell stage embryos. Embryos were fixed at stage 31/32 (A) and stage 33/34 (B), and then development of pronephric tubules was analyzed by staining with 3G8. (A) Pronephric tubules of st 31/32. The embryo injected with Xlim-1-enR is on the bottom. An embryo injected with beta-galactosidase alone was used for injection control (middle). Pronephric tubules in Xlim-1-enR-injected embryo have the same appearance as the control embryo (top in A). (B) Pronephric tubules of stage 33/34 embryos. Arrangement of embryos is the same as in A. The effects of Xlim-1-enR during pronephros development are detectable at this stage. In the Xlim-1-enR-injected embryo (bottom), reduced pronephric tubule development was observed. Details are described in D. (D) Xlim-1-enR inhibited tubulogenesis. Arrows indicate reduced development of the dorsal branches of pronephric tubules. Compared to C, the most anterior branch of the connective tubule is slightly reduced. The length and diameter of another anterior dorsal branch is severely reduced (black arrow), and the posterior dorsal branch of tubules (red arrow) is almost absent. (E and F) Histological analysis of reduced pronephric tubules. Dorsal is up and lateral is right. (E) Control embryo. Transverse section through a normal pronephros, pronephric tubules are stained with 3G8 in dark blue. (F) Transverse section of Xlim-1-enR shown in D. The structure of the reduced tubules shows that the common tubule is lumenized normally. These results suggest that Xlim-1-enR suppresses the growth of tubules but not lumenization during tubulogenesis.
FIG. 3. Induction experiments in Xlim-1-mutant-injected animal caps. Two-cell stage embryos were injected with 250 pg/embryo Xlim-1 mutant (Xlim-1/3m, Xlim-1-enR). (A) Immunohistochemistry of activin/retinoic acid-treated animal caps. All animal caps were cotreated with activin and retinoic acid for 3 h. Xlim-1-enR inhibited pronephros differentiation in a dose-dependent manner. Animal caps were bleached in H2O2 for 6 h after staining. (A) Differentiation of the pronephros was detected by the pronephric tubule-specific 3G8 antibody in control activin A/retinoic acid-treated animal caps. Arrows indicate signals (dark blue) of 3G8. In animal caps injected with 50 pg of Xlim-1-enR (B) and animal caps injected with 250 pg of Xlim-1-enR (C) the frequency of pronephros differentiation (41% in this experiment) was markedly decreased compared to animal caps injected with 50 pg of Xlim-1-enR (72%). (D) Differentiation patterns in Xlim-1 construct-injected animal caps. (D) Xlim-1/3m-injected untreated animal cap. Cement gland was observed (arrow). (E) Xlim-1/3m- injected animal cap with activin treatment. Arrows indicate notochord (Nc) and neural (N) tissues in animal cap. (F) Animal cap (injected with Xlim-1-enR) treated with activin A and retinoic acid. Though muscle and mesenchyme tissues were recognized (arrows), no pronephros was detected. (G) Pronephros (arrows) was recognized in Xlim-1/3m and Xlim-1-enR co-injected animal caps treated with activin A and retinoic acid. Cg, cement gland. N, neural tissue. Nc, notochord. M, muscle. Mes, mesenchyme. Pt, pronephric tubule. (H) Overexpression of Xlim-1 induced no pronephros marker genes. Lanes 1: Xlim-1/3m-injected explants. There is no expression of pronephric marker genes detected in Xlim-1/3m-injected explants with activin A (lane 1) or retinoic acid (lane 2) or in controls (lane 3). Lane 4: activin A/retinoic acid cotreated explants. Lane 5: sample from the whole embryo.
