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The pronephros is a transient embryonic kidney that is essential for the survival of aquatic larvae. It is also absolutely critical for adult kidney development, as the pronephric derivative the wolffian duct forms the ductal system of the adult kidney and also triggers the condensation of metanephric mesenchyme into the adult nephrons. While exploring Xenopus pronephric patterning, we observed that epidermally delivered hedgehog completely suppresses pronephric kidney tubule development but does not effect development of the pronephric glomus, the equivalent of the mammalian glomerulus or corpuscle. This effect is not mediated by apoptosis. Microarray analysis of microdissected primordia identified FGF8 as one of the potential mediators of hedgehog action. Further investigation demonstrated that SU5402-sensitive FGF signaling plays a critical role in the very earliest stages of pronephric tubule development. Modulation of FGF8 activity using a morpholino has a later effect that blocks condensation of pronephric mesenchyme into the pronephric tubule. Together, these data show that FGF signaling plays a critical role at two stages of embryonic kidney development, one in the condensation of the pronephric primordium from the intermediate mesoderm and a second in the later epithelialization of this mesenchyme into the pronephric nephron. The data also show that in Xenopus, development of the glomus/glomerulus can be uncoupled from nephron formation via ectopic hedgehog expression and provides an experimental avenue for investigating glomerulogenesis in the complete absence of tubules.
Fig. 2. Hedgehog target genes. Embryonic stage is indicated in the top right of each panel. Unilaterally injected embryos (the injected half of the embryo, identified via fluorescent lineage tracer, is oriented towards the top in all dorsal views) were fixed and processed at stages 17/18 (left), stage 21/22 (center), or stage 29/30 (right). Ectopic ectodermal hh has only a modest effect on the pronephros at stage 17/18, but by stage 21/22 expression of all markers of the pronephric tubule primordium are extinguished. Marker gene expression in other tissues is not affected, despite equivalent exposure to the hh (blue arrows). Black arrows indicate normal pronephric staining, whereas white arrows indicate abnormal and red arrows absent pronephric staining. The white arrow in the glomus at stage 21/22 indicates a more diffuse but still visible crescent staining pattern. wnt4 is only transcribed at extremely low levels in the pronephros at stage 16/17 and so is not shown. All views of stage 17/18 and 21/22 embryos are dorsal with anterior to the left. All stage 29/30 views are lateral, with anterior to the left and dorsal up. The glomus was detected using crescent at stage 17/18 and 21/22, and nephrin at stage 29/30.
Fig. 6. SU5402 blocks pronephric development. (A) Stage 36 control embryo triple labeled for somites with 12/101 (B), the pronephric distal segment with NKCC2 (C) and pronephric tubules and duct with Na+K+ ATPase (D). (E–H) Embryo treated with 50 μM SU5402 from stage 12.5 to stage 36. (I–L) Embryos treated with SU5402 from stage 15 to 36 and processes as described above. No pronephric tubules are present in treated embryos.
Fig. 1. Ectopic hh suppresses pronephric tubule and duct development but not glomeral development in Xenopus embryos. (A) Ectopic hh mRNA was delivered to the animal pole of either one (unilateral) or two (bilateral) cells at the two-cell stage. This results in the mRNA being present in the epidermis overlying the pronephric primordium, not the primordium itself. (B) Embryos injected unilaterally appear superficially normal at stage 42/43. Tadpoles that develop from bilaterally injected embryos lack all pronephric tubules and die from edema. (C, H, and E) Counterstained transverse section through 3G8 stained stage 38/39 embryo. 3G8 stains the apical surface of proximal pronephric tubules on the left. No tubules are present on the hh-injected side (right). A normal glomus is present on both sides of the injected embryo. (D) Transverse section through unilaterally injected embryo. As in C, the uninjected side has both tubules and glomus, but the injected side only has glomeral tissue. In place of tubules, a flat layer of mesoderm is observed (*). (E) Left side (uninjected) of hh unilaterally injected embryo probed with Na+K+ ATPase. Proximal tubules (white arrow), distal tubule, and duct (green arrow) are present. (F) As for panel E, except the probe is lim-1. (G) As for panel E, except staining is immunofluorescence with 3G8. (H) The opposite (right) side of the same embryo as shown in panel E. Note the complete absence of all pronephric tubules. (I) The opposite (right) side of the same embryo as shown in panel F. Note the complete absence of all lim-1-positive tissues. (J) The opposite (right) side of the same embryo as shown in panel G. No 3G8-positive tubules are present.
Fig. 3. The hh block to tubule development is not mediated by apoptosis. Embryos unilaterally injected with 100 pg of hh mRNA at the two-cell stage were grown to stage 21/22 or 28–30, fixed and processed for TUNEL staining in whole mount. Random apoptotic nuclei are detected, as expected (for examples, see white arrows). The pronephric region does not show any TUNEL-positive cells (red boxes) at any stage examined. All embryos are oriented with anterior to the left, and dorsal up.
Fig. 4. Hedgehog is not required for establishment of tubule or glomeral primordia in the zebrafish. 26 hpf wild-type (panels A and B) and smu mutant (panel C) zebrafish embryos stained with pax2.1 (brown) and wt1 (blue) probes (A and C) or pax2.1 only (panel B). Although glomera fail to migrate to the midline in smu mutants, the primordia for both glomus and tubules are clearly present. In panel A pax2.1 expression in the overlying neural tube makes the pronephric expression difficult to visualize from the dorsal view shown in panel A, but it is clearly visible, and of similar size to that in smu mutants, in control panel B.
Fig. 5. Microarray analysis of hh response in the pronephric region at stage 24. (A) Schematic of microsurgical removal of the pronephric primordium. (B) RNA prepared from pooled explants was used to probe microarrays. The response of the 75 most highly regulated genes is shown. (C) Scatter plot illustrating the distribution of significantly regulated genes according to a SAM analysis. All data have been submitted to GEO, series GSE3712, sample numbers GSM85709 through GSM85714.
Fig. 7. SU5402 treatment blocks pronephric primordium development by stage 22 but does not block glomeral development. (A, B) Control and SU5402 treated (from stage 15 to 22) embryos stained with lim1. SU5402 treatment has completely blocked lim1 expression in the pronephros. (C–F) Nephrin expression in controls and embryos treated with 50 μM SU5402 from stage 15 to 32. The glomus is abnormally shaped but is still present, as it is in embryos injected with hh. A second, previously unrecorded site of nephrin expression was observed in the branchial arches. This expression is also retained in SU5402-treated embryos but is in an abnormal position indicating an alteration in neural crest migration (red arrows). Panels E and F are enlarged views of the same embryos as shown in panels C and D.
Fig. 8. The MEK inhibitor U0126 blocks pronephric tubule development. (A, B) Untreated control embryos at stage 32/33 stained for Na+K+ ATPase. Expression of Na+K+ ATPase is much stronger in the pronephros (white arrows) than it is in the otic vesicle (red arrows). (C, D) U0126-treated (from stage 14/15 to stage 32) embryos stained for Na+K+ ATPase. Strong expression is retained in the otic vesicle, but no pronephric staining is observed. (E, F) Stage 39 control embryos stained for Na+K+ ATPase (E) and NKCC2 (F) expression. (G, H) 50 μM U0126-treated embryos (from stage 12 to stage 39) stained for Na+K+ ATPase (G) and NKCC2 (H) expression. Very faint expression of both markers is visible (white arrows). This embryo was fixed, processed, and stained under identical conditions to the embryo shown in panels E and F.
Fig. 9. FGF8-MO blocks the pronephric mesenchymal to epithelial transition. Normal epithelialization and acquisition of form in tailbud embryos visualized via Pax8 fluorescent in situ hybridization (A, B) or SEM (C) are illustrated in the top row. Stage 25 illustrates the mesenchymal stage of pronephric development, and stage 30 illustrates the early epithelialization stage. Test embryos were microinjected with FGF8-MO (14 ng) plus FLDx into blastomere V2.2 then raised to stage 30 or 33, fixed and stained for either Pax8 with a purple substrate (D–I) or 3G8 using a TRITC-coupled secondary antibody (J–L). The pronephric primordium forms (panels E and H) but fails to epithelialize. Note how the MO-injected pronephroi at stage 30 resemble the control pronephroi at the stage 25 mesenchymal period whereas the contralateral control side resembled the epithelialized controls shown in panels B and C. In both MO-injected examples, the lineage tracer (blue) is located in the pronephric mesenchyme (F, I, K, L) and is not present in the overlying somites. In panels H, I, and K, some epithelialization is occurring in the portion of the primordium that lack the tracer and the FGF8-MO. (M) Confocal optical transverse section through FGF8-MO unilateral stage 30/31 embryo stained for Pax8 via FISH. The left control side shows a Pax8-positive pronephros beginning to epithelialize (note forming lumen, white arrow), whereas the right side shows the FGF8-MO containing primordium as a Pax8-positive mesenchymal mass.
Fig. 10. Inhibition of FGF8 translation leads to retention of the pronephric mesenchymal marker Pax8 but blocks activation of the pronephric epithelial marker Na+K+ ATPase. (A–C) Pax8 expression on the contralateral control (A) and FGF8-MO-injected (B, C) sides of an embryo. The morpholino results in pronephric cells retaining Pax8 expression and appearing to be mesenchymal. (D–F) FGF8-MO blocks expression of the epithelial marker Na+K+ ATPase on the injected side of the embryo (Table 4).