Semaphorin3a inhibits ureteric bud branching morphogenesis

Abstract

Class 3 semaphorins are guidance proteins involved in axon pathfinding, vascular patterning and lung branching morphogenesis in the developing mouse embryo. Semaphorin3a (Sema3a) is expressed in renal epithelia throughout kidney development, including podocytes and ureteric bud cells. However, the role of Sema3a in ureteric bud branching is unknown. Here we demonstrate that Sema3a plays a role in patterning the ureteric bud tree in both metanephric organ cultures and Sema3a mutant mice. In vitro ureteric bud injection with Sema3a antisense morpholino resulted in increased branching, whereas recombinant SEMA3A inhibited ureteric bud branching and decreased the number of developing glomeruli. Additional studies revealed that SEMA3A effects on ureteric bud branching involve downregulation of glial cell-line derived neurotrophic factor (GDNF) signaling, competition with vascular endothelial growth factor A (VEGF-A) and decreased activity of Akt survival pathways. Deletion of Sema3a in mice is associated with increased ureteric bud branching, confirming its inhibitory role in vivo. Collectively, these data suggest that Sema3a is an endogenous antagonist of ureteric bud branching and hence, plays a role in patterning the renal collecting system as a negative regulator.

Figure 1. Antisense Sema3a morpholinos increase ureteric bud branching

A and B) Explant showing FITC-labeled morpholino injection into the ureteric bud by phase and IF microscopy. C) Autoradiogram showing that S³⁵-labeled SEMA3A in vitro translation is blocked by Sema3a antisense morpholino (3A AS MO) whereas full-length SEMA3A is synthesized in control conditions and in the presence of Sema3a mismatched morpholino (3A MM MO). D) control E11.5 explant injected with ΔMPG peptide, shown after 24 hours culture; E and F) Sema3a mismatched morpholino (3A MM MO) injected explant at the time of morpholino injection and after 24 hours culture; G and H) Sema3a antisense morpholino (3A AS MO) injected paired explant showing significantly increased ureteric bud branching 24 hours later; I through N) FITC-DBA staining ureteric buds and Rhodamine-PNA staining glomeruli in explants exposed to MO in the media for 48 hours: I and L) control explant; J and M) 3A MM MO; K and N) 3A AS MO-treated paired explants showing increased ureteric bud branching, glomerular development and size.

Figure 2. SEMA3A inhibits ureteric bud branching in a concentration dependent manner

A) E11.5 explant at the beginning of the experiment; B) Control explant after 48 hours in culture; C) paired explant exposed to rSEMA3A [250 ng/ml]; D) explant exposed to rSEMA3A [500 ng/ml]; E) quantitation of ureteric bud branchings in control conditions and upon exposure to sema 3A after 72 h in culture. Data are expressed as mean±s.e.m., * p<0.05 sema 3A vs. control; F-K explants examined after 48 h in culture: F and I) FITC-Dolichos biflora agglutinin (DBA) staining ureteric buds; Sema 3A inhibits glomerular development: G and J) Rhodamine-peanut agglutinin (PNA) staining glomeruli; H and K) merged DBA and PNA images; F-G-H) control explants; I-J-K) paired explants exposed to sema 3A [300 ng/ml].

Figure 3. VEGF₁₆₅ partially rescues SEMA3A-induced ureteric bud defects

FITC-DBA staining ureteric buds: A and B) control and SEMA3A -treated paired explants; C and D) SEMA3A -treated and SEMA3A + VEGF₁₆₅ paired explants.

Figure 4. A-D) Ureteric buds express neuropilin-1

A and E) negative controls (no primary antibody); B and F) E15 kidney section showing immunoreactive neuropilin-1 (NP-1) in ureteric bud cells and collecting tubules, and isolated cells in developing glomeruli; C and G) immunoreactive calbindin labels ureteric buds and collecting tubules; D and H) NP-1/calbindin merge shows co-localization in ureteric buds. Scale bars = 50 μm. I) Sema 3A downregulates GDNF expression and signaling in whole explants: representative western blots showing GDNF expression and RET tyr¹⁰⁶² phosphorylation in control and sema 3A- treated explants, actin expression levels are shown as loading control. Mr is indicated on the right side of the blots (kDa); J) Sema3A downregulates Akt phosphorylation: representative western blots showing a decrease in phosphorylated pS⁴⁷²Akt upon explant exposure to sema 3A, whereas total Akt remains unchanged.

Figure 5. Sema 3A deletion results in increased ureteric bud branching

Three dimensional confocal microscopy image of the ureteric trees in H7-GFP:Sema3a mice at E12, E13, E14 and E15, showing increased UB branching in H7-GFP:Sema3a -/- kidneys (bottom panel), as compared to H7-GFP:Sema3a +/- kidneys (top panel).