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The pronephros is the first kidney to develop and is the functional embryonic kidney in lower vertebrates. It has previously been shown that pronephric tubules can be induced to form ex vivo in ectodermal tissue by treatment with activin A and retinoic acid. In this study, we investigated the role of Ca(2+) signaling in the formation of the pronephric tubules both in intact Xenopus embryos and ex vivo. In the ex vivo system, retinoic acid but not activin A stimulated the generation of Ca(2+) transients during tubule formation. Furthermore, tubule differentiation could be induced by agents that increase the concentration of intracellular Ca(2+) in activin A-treated ectoderm. In addition, tubule formation was inhibited by loading the ectodermal tissue with the Ca(2+) chelator, BAPTA-AM prior to activin A/retinoic acid treatment. In intact embryos, Ca(2+) transients were also recorded during tubule formation, and photo-activation of the caged Ca(2+) chelator, diazo-2, localized to the pronephric domain, produced embryos with a shortened and widened tubule phenotype. In addition, the location of the Ca(2+) transients observed, correlated with the expression pattern of the specific pronephric tubule gene, XSMP-30. These data indicate that Ca(2+) might be a necessary signal in the process of tubulogenesis both ex vivo and in intact embryos.
Fig. 2. Effect of blocking the Ca2+ increase on tubule differentiation. (A) The animal caps removed at stages 8–9 were incubated in the presence (right panels) or absence (left panels) of 20 µM BAPTA-AM for 30 min prior to incubation with activin A (10 ng/mL) and retinoic acid (10− 4 M) for 3 h. Animal caps were then cultured until stage 39, fixed and immunolabeled with the tubule-specific monoclonal antibody, 3G8 (upper panels) or with the duct-specific monoclonal antibody, 4A6 (lower panels). Scale bar is 100 µm. (B) A representative example of a PMT trace obtained from an aequorin-loaded animal cap pre-incubated in 50 µM La3+ prior to the addition of activin A and retinoic acid. Light emission, expressed in photons per second and reflecting changes in [Ca2+]i, is inhibited in the presence of La3+. Inset, increase in light emission upon addition of activin A and retinoic acid on a sibling control animal cap. (C) The expression of the early pronephros marker XPax8, and of the pronephric tubule marker XSMP-30 in 10 animal caps measured by RT-PCR. Animal caps were pre-incubated (+) or not (−) for 30 min in 20 µM BAPTA-AM or 50 µM La3+ prior to incubation with activin A and retinoic acid. (D) Expression of XMyoD, a marker for somitic mesoderm, in animal caps measured by RT-PCR. RNA from one sibling embryo at stage 35 served as positive control and PCR on RNA without reverse transcription was done to check the absence of genomic DNA (−RT). H4 was used as a loading control.
Fig. 3. Ca2+ is sufficient to trigger tubulogenesis following activin A treatment in animal caps. (A) Animal caps, excised at stages 8–9, were incubated for 3 h with activin A (10 ng/mL) and then for an additional 3 h with either retinoic acid (10− 4 M; upper left panel), caffeine (10 mM; upper right panel), ionomycin (1 µM; lower left panel), or NH4Cl (10 mM; lower right panel). The animal caps were then cultured until stage 41, fixed and immunolabeled with the tubule-specific monoclonal antibody, 3G8. Scale bar is 100 µm. (B) RT-PCR analysis of the expression of the early pronephros marker XPax8, and of the pronephric tubule marker XSMP-30. Animal caps were untreated (−) or treated for 3 h (+) with activin A (10 ng/mL) and then for an additional 3 h with either retinoic acid (10− 4 M) or caffeine (10 mM). RNA from one sibling embryo at stage 35 served as positive control and PCR on RNA without reverse transcription was done to check the absence of genomic DNA (−RT). H4 was used as a loading control.
Fig. 4. Ca2+ is necessary for tubulogenesis in intact embryos. (A) f-aequorin was co-injected with EGFP mRNA (as lineage tracer), into the lateral marginal zone of one ventral and one dorsal blastomere of stage 3 embryos (upper left panel). Embryos were screened at late gastrula/early neurula stage and those exhibiting expression of EGFP in the presumptive pronephric territory were selected for subsequent examination via the PMT. These embryos were screened again at the end of the recording period to confirm EGFP expression in the pronephric tubules (upper right panel, white arrowheads). Anterior is to the left. Scale bar is 1 mm. A representative PMT trace indicating an increase in light emission, expressed in photons per second (integration time 30 s) and reflecting an increase in [Ca2+]i, was recorded during the development of an embryo from late gastrula (stage 13) up to stages 37/38 when the tubules are fully differentiated (n = 7). The main developmental stages, indicated on the red bar below the trace, are as follows: stage 13, late gastrula; stage 18, neural groove stage; stage 21, neural tube completely closed and first indication of pronephric anlagen; stage 23, 24-h old larva; stages 37/38, entire pronephros functional, 32-h old larva. (B) The inactive caged Ca2+ chelator diazo-2 was co-injected with EGFP mRNA as in (A) and photo-activation of diazo-2 was performed at stages 16–17 (18 hpf). Dorsal view, anterior is on top. Scale bar is 0.5 mm. (C) The effect of diazo-2 uncaging on pronephric tubule formation was recorded when the larvae reached stages 37/38 (middle panel). Larvae were fixed and whole-mount immunolabeled with the 3G8 monoclonal antibody to label the pronephric tubules. Scale bar is 0.5 mm. In this example, the left side of the larva displays normal differentiated tubules (left panel) while the tubules on the diazo-2-injected/photoactivated side are shorter and wider (right panel). Scale bar is 100 µm for left and right panels. (D) Example of an uncaging experiment (n = 6) performed at gastrulation stage (stage 12.5). The uninjected (control) side of the larva (left pair of panels) displays normal development, as shown by the correct morphology of the head and eye (bright-field image) and a normal pronephric tubule (3G8 antibody fluorescent image). However, the diazo-2-injected/photoactivated side of the same larva (right pair of panels) shows that while the overall morphology of the larva is normal; see the morphology of the head and eyes (bright-field image), the morphology of the tubule is significantly altered (3G8 antibody fluorescent image). Scale bar is 0.5 mm.
Fig. 5. The expression of XSMP-30 correlates with domains of Ca2+ transients. (A) The pronephric territory was isolated from late gastrula aequorin-injected embryos and cultured in Steinberg's solution until sibling control embryos reached stage 29. A representative example of a PMT trace (n = 7) obtained from an aequorin-loaded explant (integration time 30 s). Light emission, expressed in photons per second and reflecting changes in [Ca2+]i, starts just prior to stage 14. Differentiated pronephric tubules can be visualized with the tubule-specific monoclonal antibody, 3G8 (see inset panel; scale bar is 100 µm). (B) Representative example (n = 3) of the spatial distribution of a sequence of Ca2+ transients that occur over a period of  4.5 h after stage 14. Each aequorin-generated image represents 120 s of accumulated light. These have been superimposed on to the corresponding bright-field images of the explant. Color scale indicates luminescent flux in photons/pixel. Scale bar is 100 µm. (C) Accumulated increase in [Ca2+]i during 6 h of data acquisition, which was superimposed on the bright-field imaged grabbed at the end of acquisition (left panel). In the same explant, the expression pattern of XSMP-30 was determined at stage 34 by in situ hybridization (right panel). The domain of gene expression is located within the domain of high Ca2+ signals. Scale bar 100 µm.
