Platelet-derived growth factor receptor-α cells in mouse urinary bladder: a new class of interstitial cells

Abstract

Specific classes of interstitial cells exist in visceral organs and have been implicated in several physiological functions including pacemaking and mediators in neurotransmission. In the bladder, Kit(+) interstitial cells have been reported to exist and have been suggested to be neuromodulators. More recently a second interstitial cell, which is identified using antibodies against platelet-derived growth factor receptor-α (PDGFR-α) has been described in the gastrointestinal (GI) tract and has been implicated in enteric motor neurotransmission. In this study, we examined the distribution of PDGFR-α(+) cells in the murine urinary bladder and the relation that these cells may have with nerve fibres and smooth muscle cells. Platelet-derived growth factor receptor-α(+) cells had a spindle shape or stellate morphology and often possessed multiple processes that contacted one another forming a loose network. These cells were distributed throughout the bladder wall, being present in the lamina propria as well as throughout the muscularis of the detrusor. These cells surrounded and were located between smooth muscle bundles and often came into close morphological association with intramural nerve fibres. These data describe a new class of interstitial cells that express a specific receptor within the bladder wall and provide morphological evidence for a possible neuromodulatory role in bladder function.

Fig 1

Platelet-derived growth factor receptor (PDGFR)-α cells in the bladder detrusor. (A) Cryostat section of detrusor muscle showing PDGFR-α immunoreactivity throughout the entire mucularis. (B) Whole mount of the bladder muscularis. PDGFR-α⁺ cells run parallel in the direction of the smooth muscle cells in the detrusor. PDGFR-α⁺ cells possess spindle- and stellate-shaped morphology with multiple processes that formed a discrete network. Scale bars are indicated in each panel.

Fig 2

Double labelling of platelet-derived growth factor receptor (PDGFR)-α cells and smooth muscle cells in the detrusor. (A) Cryostat sections of PDGFR-α (red) and smMHC/Cre/eGFP (green) labelling within the detrusor muscle. (B) At high power, PDGFR-α⁺ cells were observed to lie interstitially along and in between smMHC/Cre/eGFP⁺ muscle bundles. (C) PDGFR-α⁺ cells were found in the muscularis (M) and in the lamina propria (LP) but absent in the urothelium (U). A high-power image of PDGFR-α⁺ cells in the lamina propria is shown in the inset. (D) Confocal image of a single slice (0.5 μm) of a whole mount revealing the complicated arrangement of PDGFR-α⁺ cells (red) and smMHC/Cre/eGFP (green) immunohistochemistry within the detrusor muscularis. Scale bars are indicated in each panel.

Fig 3

(A) Platelet-derived growth factor receptor (PDGFR)-α and vimentin immunoreactivity in the bladder wall. Cryostat section of PDGFR-α (green) double-labelled with vimentin (red). (B) Higher magnification of a double-labelled image of a cryostat section revealing the distribution of both PDGFR-α and vimentin immunoreactivity in the bladder wall. Note only partial cellular overlap of PDGFR-α and vimentin immunoreactivity (arrows; yellow). (C, D) Confocal reconstructions of whole mounts labelled with vimentin (red) showing only partial cellular localization with PDGFR-α (green) in a discrete population of cells (yellow, *). Other cells were either PDGFR-α⁺ (arrow) or vimentin⁺ (arrowhead). Scale bars are indicated in each panel. Lamina propria (LP), lumen (L) and muscularis (M) are shown in (A).

Fig 4

Lack of Kit immunoreactivity in the bladder detrusor. (A) The absence of Kit immunoreactivity within the muscularis of the urinary bladder. Only minimal autofluorescense was observed with the muscularis. (B) As a positive control, a whole mount preparation in the mouse colon revealing Kit immunoreactivity using the same antibody as in (A). These tissues were processed at the same time in an identical manner. Images were also collected using similar laser and detector settings. Scale bars are indicated in each panel.

Fig 5

Close anatomical apposition between nerve fibres and platelet-derived growth factor receptor (PDGFR)-α⁺ cells within the bladder wall. (A–D) Confocal reconstructions of cryostat sections double labelled with a pan-neuronal marker, PGP9.5 (red) and PDGFR-α⁺ cells (green). (A, C) Low-power image through the bladder wall revealing the distribution of neural elements and PDGFR-α⁺ cells. (B) At higher power, some nerve fibres can be observed to be closely associated with PDGFR-α⁺ cells while others are not in close apposition. (D) Larger nerve trucks [denoted by * in (C)] can be seen surrounded by PDGFR-α⁺ cells. (E, F) Whole mounts double labelled with PDGFR-α⁺ (green) and PGP9.5 (red) revealing the close proximity of some nerve fibres with PDGFR-α⁺ cells. (F) Many but not all nerve fibres are seen running parallel to the long axis of PDGFR-α⁺ cells. Scale bars are indicated in each panel. Lamina propria (LP), lumen (L), muscularis (M) and serosa (S) are shown in (C).

Fig 6

Double labelling of excitatory nerve fibres and platelet-derived growth factor receptor (PDGFR)-α⁺cells in the bladder detrusor. (A, B) Whole mount labelled with vesicular acetylcholine transferase (vAChT, red) to identify cholinergic nerves and PDGFR-α⁺ cells (green). The majority of vAChT⁺ nerves were not intimately associated with PDGFR-α⁺ cells but some fibres were in a similar anatomical location (*). Scale bars are indicated in each panel.

Fig 7

Histone 2B-eGFP fusion protein expression in the nuclei of cells that express platelet-derived growth factor receptor (PDGFR)-α. (A, B) Low-power images revealing eGFP expression in all the nuclei of PDGFR-α⁺ cells. (C, D) High-power images showing the exact location of eGFP fluorescent nuclei within PDGFR-α⁺ cells. Scale bars are indicated in each panel.

Fig 8

Enzymatically dispersed cells from the bladder detrusor of mice with histone 2B-eGFP fusion protein expression in platelet-derived growth factor receptor (PDGFR)-α⁺ cells. (A–C) Fluorescent (A) and differential interference contrast (B) images revealing typical morphology of freshly dispersed GFP⁺ cells. eGFP was localized to the nuclei of small rounded cells [arrowheads (C)]. (D–F) PDGFR-α⁺ cells were found to be smaller in diameter and ovoid in shape compared with typical smooth muscle cells that were not fluorescent (arrowhead). Scale bars are indicated in each panel.