A self-avoidance mechanism in patterning of the urinary collecting duct tree

Abstract

Background: Glandular organs require the development of a correctly patterned epithelial tree. These arise by iterative branching: early branches have a stereotyped anatomy, while subsequent branching is more flexible, branches spacing out to avoid entanglement. Previous studies have suggested different genetic programs are responsible for these two classes of branches.

Results: Here, working with the urinary collecting duct tree of mouse kidneys, we show that the transition from the initial, stereotyped, wide branching to narrower later branching is independent from previous branching events but depends instead on the proximity of other branch tips. A simple computer model suggests that a repelling molecule secreted by branches can in principle generate a well-spaced tree that switches automatically from wide initial branch angles to narrower subsequent ones, and that co-cultured trees would distort their normal shapes rather than colliding. We confirm this collision-avoidance experimentally using organ cultures, and identify BMP7 as the repelling molecule.

Conclusions: We propose that self-avoidance, an intrinsically error-correcting mechanism, may be an important patterning mechanism in collecting duct branching, operating along with already-known mesenchyme-derived paracrine factors.

Figure 1

Development of the renal ureteric bud/collecting duct system. The ureteric bud begins as an unbranched epithelial tube that invades the metanephrogenic mesenchyme, and recruits cells of that mesenchyme to form a `cap¿ (this cap becomes the stem cell population that produces nephrons). The ureteric bud then bifurcates, and the cap splits along with it. As the branches of the ureteric bud grow, pieces of distal cap left behind differentiate to make first a nephrogenic condensate and then to become an epithelial early nephron. This process repeats to form a ureteric bud tree (the future collecting duct) and the nephrons that will later connect to it. A much more detailed, illustrated account of renal development can be found at www.gudmap.org.

Figure 2

Divergence angles of branching tubules are governed not by sequence but by the presence of other tips. Normal kidneys cultured intact (a) show a wide angle of first branching (`1¿), and narrower second (`2¿) and subsequent branches. Real angles from this specimen are indicated on the figure and mean values can be seen in the green bars of Figure 1d. An unbranched ureteric tip cultured with its own mesenchyme (b) shows a similar wide-then-narrower pattern. An already-branched tip (c), which would naturally go on to produce a narrow branch angle, begins by producing a wide angle characteristic of the first branch when it is cultured alone (with its own mesenchyme). In the images, the area behind the red dotted line, labelled `retro¿, is a branching system that develops from the bladder end of the cut ureteric bud, behaviour that has already been described [46]: data were not gathered from the `retro¿ branching system because its first branch occurred later then the normal ones, with very variable timing. (d) Shows branching angles quantitatively, a,b, and c referring to the culture methods shown in (a), (b) and (c) and colours in the graph matching the colour bars under each micrograph: error bars represent standard error of the mean. In all cases, second branch angles differ from first branch angles with p?<?0.05 (p values are given in the main text).

Figure 3

Patterning of tubule trees by self-avoidance, in a simple model. Beginning with an unbranched trunk (a), secreting the repulsive factor horrid, the model produces a tree (b), in which the first angle of branching is wide and subsequent angles narrower although this change is not written explicitly into the model, but emerges from self-avoidance. If two trunks are aimed at one another, either directly (c) or offset (d), they each produce a tree that is distorted but that avoids collision with the other tree.

Figure 4

Evidence for self-avoidance in the developing tubule trees of real cultured kidneys. Single ureteric buds, isolated, surrounded by mesenchyme and cultured, generate reniform trees in both the model (a) and reality (d). Pairs of ureteric buds cultured on collision courses with one another are predicted by the model to produce trees that are distorted but that avoid collision (b): this does indeed happen in reality (e). Where three ureteric buds are aimed towards one another, the model (c) and real cultures (f) generate straight `no-man¿s land¿, tip-free zones between them: these can be seen between the arrows in (f). Additional file 11: Figure S4f shows the same image false-coloured to indicate more clearly which branches belong to which tree. Ureteric buds trees are stained with anti-calbindinD28^k. Scale bars are 100 ?m.

Figure 5

Approaching branches slow and avoid contact even in intact cultured kidneys. (a) shows an example frame from Additional file 2: Movie S1, a Hoxb7-cre x ROSA-eYFP kidney developing for a total of 6 days in culture (this construct causes the ureteric bud to fluoresce). (b) shows the speed of advance of branch tips that were not approaching other branches (the majority were of this type), at different times of culture. The mean speed is nearly constant over the culture, falling by only 12%. This is important for interpretation of panel (c), which plots the closing speed of tips that are approaching one another against their separation. There is an inverse relationship between approach speed and log of distance, suggesting that the closing speed of branches decreases, even to a stop, as separation decreases (82 observations; R?=?0.67; significance?=?4.8x10^?12). The change is is much larger than the 12% reduction of scalar speed in (b), so cannot be accounted for simply by the culture ageing at the same time that tips approach other branches.

Figure 6

Involvement of the BMP signalling in self-avoidance. (a) In the presence of the inhibitor of TGF?-superfamily signalling, AlkiII, collision avoidance between co-cultured ureteric buds fails and branches make contact (arrowheads). In addition, branches become long and spindly. (b) Gremlin, a more specific inhibitor of signalling by BMPs, also causes collision avoidance between ureteric bud trees to fail, but the branch pattern is generally more normal than in AlkiII. (c) In some ureteric buds cultured in Gremlin, branches run almost parallel rather than diverging, and show inter-tree collisions. Anti-BMP7 also causes collision avoidance to fail between trees (d) and within trees (e). (f) Shows the frequency of collisions quantitatively: none of the inhibitors causes collisions in every case, but each is significantly different from the controls, in which collisions were never seen (p values are in the main text). (g) Shows the incidence of parallel branches in Gremlin-treated buds. This was the only treatment to produce this effect reliably. (h, i) show the Six2-positive caps (green) over ureteric bud tips (red) in control and AlkiII-treated kidneys respectively; (j) shows quantitatively what is apparent visually from (h, i); the drug makes no detectable difference to the thickness of this cap, so the ability of AlkiII-treated tips to collide does not result from disappearance of a Six2+ `fender¿.

Figure 7

Expression of Alk receptors and their ligands, according to the GUDMAP database.

Figure 8

Ureteric buds and their cells eschew sources of BMP7. Placing control, BSA-soaked Affigel beads near ureteric buds (a) shows no obvious repulsive effect and branches will run into beads that happen to be in their way. In contrast, branches do not make contact with BMP7-soaked Affigel beads (b), nor do they approach them very closely: these data are shown quantitatively in (c), which shows the distance between the branch and bead that are closest in each culture. (d) In a completely different assay, ureteric bud-derived cells were cultured on filters, with medium supplemented with different concentrations of BMP7 under the filter. The graph shows the frequency of cells crossing the filter: filter crossing is inhibited by BMP7.