Immunohistochemical analysis of Sonic hedgehog signalling in normal human urinary tract development

Abstract

Studies of mouse mutants have demonstrated that Sonic hedgehog (SHH) signalling has a functional role in morphogenesis and differentiation at multiple sites within the forming urinary tract, and urinary tract malformations have been reported in humans with mutations that disrupt SHH signalling. However, there is only strikingly sparse and fragmentary information about the expression of SHH and associated signalling genes in normal human urinary tract development. We used immunohistochemistry to demonstrate that SHH protein was localised in distinct urinary tract epithelia in developing normal humans, in the urothelium of the nascent bladder and in kidney medullary collecting ducts. The expression patterns of the SHH-transducing proteins Patched (PTCH) and Smoothened (SMO) were consistent with long-range paracrine signalling associated with detrusor smooth muscle differentiation in the urogenital sinus. In the developing kidney, SHH and PTCH were expressed in epithelia of the collecting system between 16-26 weeks--surprisingly, SMO was not detected. Analysis of cell proliferation and Cyclin B1 immunohistochemistry at 26 weeks, as compared with a 28 week sample in which SHH expression was down-regulated, was consistent with the idea that SHH and PTCH might influence medullary collecting duct growth by regulating the subcellular localisation of Cyclin B1 independently of SMO. Collectively, these descriptive results generate new hypotheses regarding SHH signal transduction in human urinary tract development and help to explain the varied urinary tract malformation phenotypes noted in individuals with mutations in the SHH pathway.

Fig. 1

Differentiation of the 7-week urogenital sinus. Expression of UP (brown signal) and αSMA (pink/red signal) in a series of transverse sections of the urogenital sinus (ugs) from most caudal (A) to most cranial (E) axial level, showing a graded intensity of UP and αSMA, in the urothelium and mesenchyme respectively, in the cranial-to-caudal axis. A’–E’, high-power views of the urothelium as indicated by the hashed boxes in A–E. hg, hindgut; ugs, urogenital sinus; pc, peritoneal cavity. A–E, ×12.5 magnification; A’–E’, ×100 magnification. Sections counterstained with hematoxylin.

Fig. 2

Differentiation of the 13-week bladder. Expression of UP (brown signal) and αSMA (pink/red signal) in a series of transverse sections in the caudal to cranial axis of the 13 week bladder (A–E). A′–E′, high-power views of the urothelium as indicated by the hashed boxes in A–E. Note that both differentiation markers are now expressed at all axial levels, both ventrally and dorsally, and that αSMA expression has resolved into muscle bundles. Arrowheads indicate αSMA expression in muscle bundles in the most caudal section in A, which corresponds to an axial level situated between that which is indicated in Fig. 1B and C, and in the dorsal mesenchyme in C, which corresponds to an axial level equivalent to Fig. 1D. A–E, ×12.5 magnification; A′–E′, ×100 magnification. Sections counterstained with hematoxylin.

Fig. 3

Expression of Sonic hedgehog in the 7-week urogenital sinus. Expression of SHH (brown signal) and αSMA (pink/red signal) in a series of transverse sections of the urogenital sinus (ugs) from most caudal (A) to most cranial (E) axial level. A′–E′, high-power views of the urothelium as indicated by the hashed boxes in A–E. Note the graded intensity of SHH immunostaining of urothelium in the cranial to caudal axis, and the onset of its expression correlates with the onset of αSMA expression. hg, hindgut; ugs, urogenital sinus; pc, peritoneal cavity. A–E, ×12.5 magnification; A′–E′, ×100 magnification. Sections counterstained with hematoxylin.

Fig. 4

Expression of SHH signal transduction molecules in the 7-week urogenital sinus. Immunolocalisation of PTCH (B), SMO (C) and BMP4 (D) in the peripheral mesenchyme of the 7-week urogenital sinus. (A) Negative control; antibody replaced with anti-rabbit IgG. Note the very close overlap of expression of all three proteins in adjacent sections, in condensing mesenchymal cells that express the differentiation marker αSMA (see Figs 1E and 3E). A–E, ×12.5 magnification; A′–E′, ×100 magnification. Sections counterstained with hematoxylin.

Fig. 5

Expression of SHH and PTCH in the 16-week kidney. (A) Low power view of PTCH expression in the 16-week kidney showing overall morphology. Inset dashed boxes refer to fields of view shown in B–G. B, D, F – expression of SHH; C, E, G – expression of PTCH. SHH and PTCH are expressed in the renal pelvis (B, C), and medullary collecting ducts (D, E). PTCH (G), but not SHH (F), is also expressed in collecting ducts extending out to the capsule (*) of the kidney. Neither SHH nor PTCH were detectable in the nephrogenic mesenchyme zone or in nephron precursors (F, G). Note that PTCH is localised predominantly at the apical surface of collecting ducts. H, I – fields of view equivalent to those shown in panels B, C and D, E respectively, showing no expression of SMO in the kidney; no immunoreactivity was detected for SMO in any of the kidney samples analysed between 16–28 weeks. As a positive control for SMO immunohistochemistry, SMO protein was detected in the neuroepithelium of the developing forebrain (J, high magnification of the region indicated by the hashed box is inset) and ventricular mesenchyme of the heart (not shown) in a mid-sagittal section of a 10-week gestation embryo; SMO was also detected in the developing urogenital sinus at seven weeks (Fig. 4C,C′). BMP4 was detected in glomerular parietal epithelia at 13 weeks’ gestation (K). A, ×12.5 magnification; B–J, ×50 magnification; K, ×100. Sections in J, K counterstained with hematoxylin.

Fig. 6

Expression of SHH, PTCH, Cyclin B1 and PCNA in medullary collecting ducts of the 26- and 28-week kidneys. SHH is expressed in medullary collecting ducts at 26 weeks (A), but not at 28 weeks (E), and PTCH is expressed at both ages and is localised to the apical cell surface (B, F); both were absent from intervening interstitial cells and tubules of smaller diameter (presumably mainly representing loops of Henle). Absence of SHH at 28 weeks corresponds with a redistribution of Cyclin B1, from a diffuse cytoplasmic pattern (C) to the apical cell surface (G) in a similar location to that observed for PTCH (F). There is reduced proliferation of medullary epithelial cells at 28 weeks, compared to the 26-week kidney, as evidenced by the proportion of PCNA-positive nuclei (D, H); note that this effect is limited to collecting duct cells, since no equivalent decrease in cell proliferation was observed in intervening epithelia. Arrows in C indicate occasional cell nuclei positive for Cyclin B1. Images at ×100 magnification; sections counterstained with hematoxylin.

Fig. 7

Distinct molecular and cellular responses to SHH signalling in human upper and lower urinary tract development. Different colours in A and B illustrate the different structures in which various proteins were immunolocalised in this study, according to the key. A. SHH is expressed by urogenital sinus urothelium and signals to distant mesenchymal cells which express PTCH, SMO and BMP4, and which will form smooth muscle. These results are consistent with the regulation of smooth muscle differentiation by canonical SHH signalling. B. Epithelial cells of medullary kidney collecting ducts express SHH and PTCH, but not SMO. Descriptive results in vivo suggest that SHH signalling operates independently of SMO at short range in these cells to regulate cell proliferation according to the subcellular distribution of Cyclin B1.