SCA of a very-low-conductance channel.

<p>(<b>A</b>) Current recording of a single very-low-conductance channel. The bath solution (panels <b>A</b>, <b>B</b>, and <b>C</b>) contained 3 M KCl. See legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g004" target="_blank">Figure 4<b>A</b></a> for other details. (<b>B</b>) Current trace of the channel in response to the shown voltage-ramp protocol. Dotted line indicates the current level at zero holding potential. Note the near linear dependence of the current on the applied voltage. (<b>C</b>) Current traces of a single channel in response to the indicated voltage-step protocol. (<b>D</b>) Ion-selectivity of the channel. See legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g004" target="_blank">Figure 4<b>D</b></a> for details.</p

SCA of a high-conductance channel.

<p>(<b>A</b>) Current trace of a single high-conductance channel. The insertion event (marked by an asterisk) was registered at +10 mV and the applied voltage was then switched to −10 mV. The dashed line indicates the current level (zero) before insertion of the channel. The data in panels <b>A</b>, <b>B</b>, and <b>C</b> were collected using 3 M KCl as the electrolyte. (<b>B</b>) Current trace of the channel in response to the indicated voltage-ramp protocol. Note the near linear dependence of the current on the applied voltage. (<b>C</b>) Single channel currents in response to the indicated voltage-step protocol. (<b>D</b>) Dependence of the single channel conductance on the KCl concentration. After detection of a single channel insertion using 3 M KCl as bath solution (holding potential +10 mV), the electrolyte was diluted and registration of the current amplitudes of the same channel was conducted at 2.0 M and 1.0 M KCl, respectively. Data points are mean±SD for at least 4 independent measurements. (<b>E</b>) Current traces of a single channel in response to a low-speed linear increase (upper trace) or decrease (lower trace) of the holding potential. The bath solution contained 1.0 M NH₄Cl, 20 mM Tris-Cl, pH 7.8, and 2 mM DTT at both sides of the membrane. Note that the channel was still open even at hyperpolarizing holding potentials of ±150 mV. (<b>F</b>) Current-voltage relationship of the high-conductance channel under asymmetric salt conditions: 3.0 M KCl <i>trans</i>/1.5 M KCl <i>cis</i> compartment. The insertion of a single channel was detected at 3 M KCl at both sides of the membrane and at a voltage of +10 mV, then the electrolyte concentration in the <i>cis</i> compartment was decreased by dilution and an initial current recording was conducted at zero potential followed by stepwise (±10 mV) change of the applied voltage. Data points are mean±SD, n = 4–5. Bars in some cases are smaller than symbols.</p

Relationship of intracellular MAPK pathway to RET receptor tyrosine kinase signaling.

<p><i>In situ</i> hybridization of the RET signaling target <i>Cxcr4</i> in E13.5 (A) control kidney shows expression in UB tips (arrow) as well as in kidney mesenchyme, while expression is lost in (B) UB tip (arrow) of dko kidney. (C–D) Another RET signaling target <i>Dusp6</i>, which is a negative regulator of MAPK activity, shows no changes (arrows) in the absence of MAPK pathway activity at E13.5. (E) Table summarizing the expression results of RET signaling targets in UB of dko kidneys. Scale bar: A–D 500 µm.</p

Localization of MAPK pathway activity in developing kidney.

<p>(A–A′) Representative cross sections of wild type E11.5 kidneys stained with anti-phospho-ERK1/2 (red) demonstrate MAPK pathway activity both in the ureteric (UB) bud, visualized by calbindin staining (green), and metanephric mesenchyme (arrow). (B–B′) In E13.5 wild type kidneys, pERK1/2 localization in the ureteric bud epithelium is concentrated to UB tips where the staining is unevenly distributed among the epithelial cells (arrowheads), which all express UB marker calbindin (green). Additional pERK1/2 staining is detected in nephron primordia (arrows). (C) Chromogenic pERK1/2 staining on E14.5 wild type kidneys shows strong but heterogeneous MAPK activity in UB tips with lack of activity in sporadic cells (red arrowheads). (D) <i>Ret</i> expression in the ureteric bud tips of E13.5 wild type kidney as detected by <i>in situ</i> hybridization of mRNA on vibratome sections. In A and B, nuclei are labeled with Hoechst. Scale bars: A–B 50 µm, C–D 500 µm.</p

Relationship of intracellular MAPK pathway to RET receptor tyrosine kinase signaling.

<p><i>In situ</i> hybridization of the RET signaling target <i>Cxcr4</i> in E13.5 (A) control kidney shows expression in UB tips (arrow) as well as in kidney mesenchyme, while expression is lost in (B) UB tip (arrow) of dko kidney. (C–D) Another RET signaling target <i>Dusp6</i>, which is a negative regulator of MAPK activity, shows no changes (arrows) in the absence of MAPK pathway activity at E13.5. (E) Table summarizing the expression results of RET signaling targets in UB of dko kidneys. Scale bar: A–D 500 µm.</p

Normal ureteric bud outgrowth in dko kidneys is followed by severely compromised branching morphogenesis.

<p>(A–F) Time lapse imaging of kidneys cultured for 48 h, and ureteric buds visualized by the green fluorescent protein tag encoded by the Hoxb7CreGFP construct. (A) In control kidney, ureteric bud is formed and swollen at E11.5, followed by branching (arrow) (B) 24 h later. (C) At 48 h, several generations of branches (average UB tip number: 10.5, n = 6) have been generated in the control kidney. (D) The UB formation in dko kidney at E11.5 is indistinguishable from the control kidney. (E) At 24 h, branching morphogenesis is progressing very slowly in the dko kidney as indicated by deepening of the cleft (arrowhead) on top of the developing T-bud, which however fails to complete and further generate secondary branches (arrows, average tip number: 3.8, n = 5) even at 48 h (F). (G–H) E13.5 ureteric epithelium visualized by calbindin (green) staining, followed by 3-dimensional reconstruction from confocal optical sections, shows the typical shape and distribution of UB tips at the surface of control kidney (G). Note that the control UBs are distributed over the entire surface area of the kidney, while in (H) dko kidney UB tips are very infrequent due to defective branch formation, leaving large areas of the kidney surface devoid of UB epithelium. (I–J) Calbindin staining on E13.5 kidneys shows several UB tips and branches (brown) in (I) control but only few in (J) dko. Scale bar: Scale bars: A–F 500 µm, G–H 50 µm, I–J 250 µm.</p