Futel M et al. (2015), TRPP2-dependent Ca2+ signaling in dorso-lateral...

XB-ART-50093

J Cell Sci 2015 Mar 01;1285:888-99. doi: 10.1242/jcs.155499.

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TRPP2-dependent Ca2+ signaling in dorso-lateral mesoderm is required for kidney field establishment in Xenopus.

Futel M , Leclerc C , Le Bouffant R , Buisson I , Néant I , Umbhauer M , Moreau M , Riou JF .

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In Xenopus laevis embryos, kidney field specification is dependent on retinoic acid (RA) and coincides with a dramatic increase of Ca(2+) transients, but the role of Ca(2+) signaling in the kidney field is unknown. Here, we identify TRPP2, a member of the transient receptor potential (TRP) superfamily of channel proteins encoded by the pkd2 gene, as a central component of Ca(2+) signaling in the kidney field. TRPP2 is strongly expressed at the plasma membrane where it might regulate extracellular Ca(2+) entry. Knockdown of pkd2 in the kidney field results in the downregulation of pax8, but not of other kidney field genes (lhx1, osr1 and osr2). We further show that inhibition of Ca(2+) signaling with an inducible Ca(2+) chelator also causes downregulation of pax8, and that pkd2 knockdown results in a severe inhibition of Ca(2+) transients in kidney field explants. Finally, we show that disruption of RA results both in an inhibition of intracellular Ca(2+) signaling and of TRPP2 incorporation into the plasma membrane of kidney field cells. We propose that TRPP2-dependent Ca(2+) signaling is a key component of pax8 regulation in the kidney field downstream of RA-mediated non-transcriptional control of TRPP2.

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Species referenced: Xenopus laevis
Genes referenced: arhgef7 arl13b cyp26a1 klf6 lhx1 myod1 osr1 osr2 pax8 pkd2 pmt prlhr st14 tbx2
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	Fig. 1. TRPP2 is expressed in the kidney field. (A) Temporal analysis of TRPP2 expression during development. TRPP2 is detected by immunoblotting throughout development but its expression increases during gastrulation (NF stage 10 to stage 13) to reach a maximum at the late gastrula stage (NF stage 12.5). Expression then diminishes to increase again at late tailbud and early tadpole stages (from NF stage 25 onwards). (B) Immunolocalization of TRPP2 in kidney field cells. Expression of plasma-membrane-targeted GFP (memGFP) was carried out by mRNA microinjection at the four-cell stage. Double immunostaining for TRPP2 and GFP. Immunoreactivity for the anti-TRPP2 antibody colocalizes with GFP, indicating that TRPP2 is expressed at the plasma membrane. Scale bars: 10 mm.
	Fig. 2. Pkd2 loss of function affects pax8 expression in the kidney field. (A) In situ hybridization analysis of pax8 expression in pkd2 morphant embryos. Pkd2-MO was injected into the two left blastomeres at the four-cell stage and pax8 expression was analyzed at early neurula stage (NF stage 14–15). Comparison of the left injected side with the right control side shows a strong inhibition of pax8 expression in the kidney field (arrowheads). Extinction, or a strong decrease of pax8 expression, is observed in more than 91% of the embryos analyzed. Pax8 expression in CMO-injected embryos is unaffected. When a pkd2 mRNA lacking the Pkd2-MO target sequence is co-injected with Pkd2-MO, this proportion is reduced to 62%, showing that it results to the specific loss of function of pkd2. OV, ectodermal expression of pax8 in the presumptive otic vesicle; KF, mesodermal expression of pax8 in the kidney field. Arrows indicate orientation of the antero-posterior (AP) axis. (B) RT-QPCR analysis of kidney field gene expression in dissected explants. CMO or Pkd2-MO was injected at the two-cell stage and embryos were cultured until late gastrula stage (NF stage 13) for kidney field explant dissection. Explants were further cultured until early neurula (NF stage 14) or late neurula (NF stage 18) stages for RT-QPCR analysis of pax8, lhx1, osr1 and osr2 expression. Mean6s.e.m. results from three independent experiments are displayed. As expected from the above results performed on whole embryos, there is a dramatic downregulation of pax8 at the early neurula stage. In contrast, lhx1, osr1 and osr2 expression is not affected. At the late neurula stage, pax8 expression is severely inhibited. In contrast, lhx1, osr1 and osr2 expression is not affected, indicating that Ca2+ signaling is acting upstream of pax8, but does not act in a general mechanism of kidney field induction. P,0.01, *P,0.001 (paired Student’s t-test).
	Fig. 3. Disruption of intracellular Ca2+ signaling results in the downregulation of pax8 in the kidney field. (A) Schematic illustrating the experiment. Diazo- 2 was injected at the eight-cell stage into the left V2 blastomere. Photoactivation of diazo-2 was performed at NF stage 11, and pax8 expression was analyzed at the early neurula stage (NF stage 14). (B) Ca2+ chelation causes a downregulation of pax8 in the kidney field (arrowheads). OV, ectodermal expression of pax8 in the presumptive otic vesicle; KF, mesodermal expression of pax8 in the kidney field. Arrows indicate orientation of antero-posterior (AP) axis. (C) Pkd2-MO causes impaired tubulogenesis at later stages. Pkd2-MO was injected into the two left blastomeres at the four-cell stage and 3G8 staining of pronephric tubules was carried out at NF stage 39–40. Pkd2-MO causes a severe reduction of the 3G8-positive tubule on the injected side (arrowheads).
	Fig. 4. A. Intracellular Ca2+ signaling in the kidney field requires TRPP2. (A) Schematic illustrating the procedure for Ca2+ measurements in the kidney field. CMO or Pkd2-MO morpholinos were injected in the V2 blastomere of eight-cell stage embryos along with GFP–aequorin mRNA. Kidney field (KF) explants were dissected at the late gastrula stage (NF stage 12.5) and Ca2+ transients were measured with a PMT for 4 h, until sibling developing embryos reach NF stage 18 (late neurula). (B) PMT traces from single pronephric territories dissected from control morpholino-injected embryo (CMO) and TRPP2 morpholino-injected embryo (Pkd2-MO). Data is representative of pairs of territories measured simultaneously in four independent experiments. (C) Histogram plot displaying the mean6s.e.m. number of Ca2+ transients during 4 h. The two left bars correspond to the mean6s.e.m. number of transients calculated for pairs of CMO and Pkd2-MO explants respectively (n54 pairs). In this condition, Pkd2 knockdown significantly reduces the number of Ca2+ transients (P50.014, paired Student’s ttest). The two right bar plots correspond to pairs of Pkd2-MO and rescue (Pkd2-MO + pkd2 mRNA) explants, respectively (n53 pairs). Although the number of Ca2+ transients increases in the rescue condition, it is not significant (see results for details). (D) Rescue of Ca2+ transients by pkd2 expression in Pkd2-MO explants. Pkd2-MO and GFP–aequorin mRNA were injected alone or mixed with pkd2 mRNA. PMT traces from single pronephric territories dissected from Pkd2- MO-injected embryo (Pkd2-MO) and embryo co-injected with Pkd2-MO and a Pkd2-MO resistant form of pkd2 mRNA are shown. Data is representative of pairs of territories measured simultaneously from three independent experiments.
	Fig. 5. Disruption of RA signaling inhibits intracellular Ca2+ signaling in the kidney field. (A) PMT traces from single pronephric territories dissected from control embryos injected with GFP–aequorin mRNA (Control) and embryos injected with GFP-æquorin mRNA plus Cyp26 mRNA (Cyp26). Representative data of pairs of territories measured simultaneously from three independent experiments. (B) Mean6s.e.m. number of Ca2+ transients during 4 h in control and Cyp26 conditions calculated for three independent pairs of experiments. Disruption of RA signaling significantly reduces the number of Ca2+ transients (P50.008, paired Student’s t-test).
	Fig. 6. Disruption of RA signaling relocates TRPP2 away from the plasma membrane. (A) RA disruption in lateral mesoderm does not affect pkd2 expression. Isolated LMZ explants were taken from embryos previously injected with GFP mRNA (control) or with a mix of GFP and Cyp26 mRNA, and cultured until siblings reached the early neurula stage (NF stage 14) for RT-QPCR analysis. Mean6s.e.m. results from three independent experiments are shown. Although disruption of RA signaling causes a strong inhibition of pax8 and lhx1 expression, pkd2 expression is not significantly affected. P,0.05; *P,0.01 (paired Student’s t-test) (B) Schematic representation of TIRF microscopy. TIRF microscopy exclusively images signals arising close to the cell membrane. TIRF works by directing excitation light through a glass substrate towards an aqueous specimen at an angle to obtain total internal reflection due to the refractive index decrease at the glass–water interface. In these conditions, an evanescent wave is created in the liquid with the same wavelength as the incident light. This evanescent wave decreases exponentially with distance. The wave is able to excite fluorophores only near the interface. It provides an ‘optical sectioning’ effect similar to, but even narrower, than that achieved by a confocal microscope (Axelrod, 2008). (C) Representative views of the imaging field for a hTRPP2–GFP mRNA-injected explant (left) and for an explant coinjected with hTRPP2–GFP mRNA and Cyp26 mRNA (right). (D) Histogram (mean6s.e.m.) displaying the calculated area of GFP spots per field of view (50650 mm) in control kidney fields (hTRPP2–GFP) and in kidney fields overexpressing the RA-catabolizing enzyme Cyp26. 350 and 700 pg of Cyp26 mRNA was injected. Disruption of RA signaling induces the relocation of TRPP2 proteins away from the plasma membrane. This effect is dose-dependent.
	Fig. 7. A working model illustrating the mechanism by which TRPP2- dependent Ca2+ signaling controls pax8 expression in the kidney field. Model is based on present data and previously published results (Cartry et al., 2006; Leclerc et al., 2008). The present work shows that pax8 expression requires an increase in intracellular Ca2+ concentration [Ca2+]i. This increase in [Ca2+]i might be due to the incorporation of TRPP2 channel, possibly under the control of RA signaling. We propose that RA is able to induce a [Ca2+]i in the kidney field, as it does in embryo explants (Leclerc et al., 2008), and then indirectly controls pax8 expression. The mechanism by which Ca2+ regulates pax8 transcription is still unknown.
	Fig. S1 Characterization of the anti-TRPP2 antibody. A. comparison of the Xenopus laevis and human TRPP2 sequence used to generate the anti human TRPP2 antibody. (B). The anti human TRPP2 antibody recognizes a major Mr 110kDa band and two lower mobility components in NF St.12.5 Xenopus embryo extracts. (C). Expression of exogenous Xenopus TRPP2 by injection of pkd2 mRNA results in a strong increase of the Mr 110 kDa band.
	Fig. S2 Cilia-like structures are not detected in KF cells. Schematics of kidney field (KF) and gastrocoel roof plate (GRP) explants dissections at early neurula stage are shown. Attempts to detect cilia in KF explants were performed by immunolocalization of acetylated tubulin and expression of the axoneme component arl13b fused to the mcherry. GRP explants dissected at the same stages were used as positive controls for primary cilia staining. Cilialike structures are not detected in KF explants although they are clearly visible in control GRP explants. (Scale bars, 20 μm).
	Fig. S3. Pax8 inhibition does not result from an indirect effect of pkd2 loss of function on somitic mesoderm. MO-Pkd2 was injected into the two left blastomeres at the 4-cell stage. Embryos were fixed at early neurula stage (NF St.14-15) or tailbud stage (NF St.28). Although pax8 expression is inhibited in the KF, neither neurula stage expression of myod nor mlc expression at tailbud stage are affected. Arrows indicate orientation of AP axis.
	Fig. S4 Ionomycin can partially rescue pax8 expression in Pkd2-MO morphant embryos. MO-Pkd2 was injected into the two left blastomeres at the 4-cell stage, and embryos cultured until late gastrula stage (NF st12.5) at which time ionomycin- or control DMSO-soaked beads were implanted into dorso-lateral mesoderm. Beads stayed in contact with dorso-lateral mesoderm until neurula stage (NF st14-15) after which they were generally expelled out of the embryo or fell into the archenteron. Embryos were further cultured until late gastrula stage for ISH analysis of pax8 expression. Three kind of patterns can be observed on injected sides, grading from very weak (Gr1), intermediate (Gr2) and strong (Gr3) pax8 expression. Scoring of these patterns reveals that the proportion of Gr3 patterns is significantly increased in ionomycin-bead implanted embryos (Pearson’s chi-square test of independence p<0,05). Arrows indicate orientation of AP axis.