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Retinoic acid (RA) signaling is important for the early steps of nephrogenic cell fate specification. Here, we report a novel target gene of RA signaling named XPteg (Xenopus proximal tubules-expressed gene) which is critical for pronephric development. XPteg starts to be expressed at the earliest stage of embryonic kidney specification and was restricted to the pronephric proximal tubules during kidney development. Anti-sense morpholino (MO)-mediated knockdown of XPteg perturbed formation of pronephros as demonstrated by reduced expression of pronephric tubule markers. Conversely, overexpression of XPteg promoted endogenous and ectopic expression of those markers and expanded pronephric tubules. Treatment of retinoic acid induced the expression of XPteg in the pronephric field without protein synthesis. Furthermore, we found that the pronephric defects caused by a dominant negative RA receptor could be rescued by coexpression of XPteg. Taken together, these results suggest that XPteg functions as a direct transcriptional target of RA signaling to regulate pronephric tubulogenesis in Xenopus early development.
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19909807
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Fig. 1. Expression patterns of XPteg (A) RT-PCR showing temporal expression pattern of XPteg. Numbers above the lanes indicate the embryonic stages. ODC serves as a loading control. –RT, control in the absence of reverse transcriptase. W.E., whole embryo. (B) Spatial expression pattern of XPteg analyzed by in situ hybridization. (a) Dorsal view of expression of XPteg in dorsolateral mesoderm (dlm) in late gastrulae. A, anterior; P, posterior. (b and c). Dorso-anterior and lateral views of XPteg expressed in the pronephric anlage (pa) at stage 14 and 16, respectively. (d) XPteg was observed in the intermediate mesoderm at stage 21 (dorsal view). (e) Transverse section of the embryo shown in panel (d). (f) Ventro-anterior view showing XPteg expressed in the eye placode (ep) and blood islands (bi) at stage 23. (g) Transverse section of the embryo in panel (f), which shows XPteg-positive blood islands. (h–j) Pronephros (pn)-specific expression of XPteg at the tadpole stages.(k–m). Enlarged views of XPteg localized to proximal tubules (pt) at stage 32, 36, and 38, respectively. (n) Transverse section of the tubules shown in panel (m). (o and p). Transverse and sagittal sections in situ hybridization of a stage 40 embryo showing XPteg-expressing proximal tubules.
Fig. 3. Effects of XPteg knockdown on pronephric mesoderm formation. Depletion of XPteg inhibits the expression of Lim1, Pax2, SMP30 and Pax8 that are related to pronephric tubules but not that of Nephrin and MyoD, a pronephric glomus and somatic mesoderm marker, respectively. These effects of MO were limited to the proximal tubules (white arrowheads) and rescued by coexpression of XPteg RNA. Eight-cell stage embryos were injected with β-galactosidase RNA as a lineage tracer along with MO (10 ng) and/or RNA into the presumptive pronephros region and later, stained with Red-Gal and subjected to in situ hybridization. One representative data are shown from three independent experiments.
Fig. 4. XPteg promotes pronephric mesoderm formation (A and B). Overexpression of XPteg stimulates the expression of Pax2, SMP30, Lim1 and Pax8 in a dose-dependent manner. Eight-cell stage embryos were injected with various doses of XPteg RNA ranging from 37.5 pg to 500 pg in the presumptive pronephric field and then in situ hybridized against these markers. Intermediate doses of XPteg RNA (125–250 pg) were optimal to activate pronephric marker expression without defects in lateral medoserm. Arrowheads point to ectopic enlarged proximal tubules. (C) XPteg has no effects on the proximal–distal patterning of pronephric tubules. Expression of Gremlin, a marker for distal and connecting tubules was not affected by overexpression or depletion of XPteg. (D) RT-PCR analysis showing the activatory effects of XPteg on pronephric marker expression. Ectodermal explants were dissected at stage 9 from XPteg RNA (1 ng)-injected embryos, incubated in media containing activin A (10 ng/ml) and all-trans retinoic acid (10−4 M) for 3 h, and then cultured for 3 days in Steinberg’s solution and analyzed by RT-PCR. (E) Quantification of the band signals shown in panel (D).
Fig. 5. PDZ binding motif of XPteg is important for pronephros formation. PDZ-binding motif (STAM)-truncated XPteg (XPtegδSTAM) inhibited the expression of SMP30 and Pax2 but not that of Nephrin and MyoD. XPtegδSTAM RNA (500 pg) was injected in the presumptive pronephric region of embryos along with or without wild-type XPteg RNA and pronephric marker expression was analyzed by in situ hybridization.
Fig. 6. XPteg acts upstream of Lim1 and Pax8 in inducing pronephric mesoderm (A). XPteg induced more ectopic expression of pronephric markers including Lim1 and Pax8 in response to activin RNA. Animal cap explants were dissected at stage 9 from embryos injected with Lim1, Pax8 or XPteg RNAs with or without activinβB RNA (5 pg), cultured for 3 days and analyzed by RT-PCR. Co AC, control animal caps. (B) Enhancement of SMP30 expression by Lim1, Pax8 and XPteg. Ectoderml explants isolated from Lim1, Pax8 or XPteg-injected embryos were treated with activin protein (10 ng/ml) for 3 h, cultured in Steinberg’s media for 3 days and processed for in situ hybridization with SMP30. (C) Coinjection of Lim1 or Pax8 could recover defective proximal expression of SMP30 and Pax2 in XPteg-depleted embryos (white arrowheads). Eight-cell stage embryos were injected with XPteg MO (10 ng) along with or without Lim1 (250 pg) or Pax8 (125 pg) RNA and later analyzed for SMP30 and Pax2 expression.
Fig. 7. XPteg is induced directly by RA signaling. XPteg and Lim1 are induced by retinoic acid even in the presence of cycloheximide (CHX), a protein synthesis inhibitor. Whole embryos, which were treated with DMSO, ethanol, retinoic acid (10 μM) and/or CHX (10 μg/ml) for 2 h from stage 12.5, were in situ hybridized against XPteg or Lim1 at the indicated stages. In the case of RA + CHX (E, E′, J, J′ and J′), embryos were treated with CHX for 30 min prior to co-treatment of RA and CHX. DMSO and EtOH serve as negative controls for RA and CHX, respectively. White arrowheads in C′, E′ and red dots in H′, J′ denote enlarged ectopic pronephric tubules.
Fig. 8. XPteg regulates pronephric mesoderm formation downstream of RAR signaling. (A) XPteg could restore disrupted expression of SMP30 and Pax2 in embryos expressing a dominant negative retinoic acid receptor α (dnRARα) (white arrowheads). (B) VP16-RARα, a constitutively active form of RARα could not rescue reduced expression of SMP30 and Pax2 caused by XPteg MO (black arrowheads). Eight-cell stage embryos were injected in the presumptive pronephric region with the combination of dnRARα (500 pg), VP16-RARα (500 pg), XPteg (250 pg), XPteg MO (10 ng) as indicated, cultured until stage 36 and subjected to in situ hybridization against SMP30 and Pax2.
Fig. 1. Pteg expression at stg 23
Xenopus laevis pdzk1ip1 /pdzk1 interacting protein 1 gene expression in stage 38 tadpole
