A genome-wide screen to identify transcription factors expressed in pelvic Ganglia of the lower urinary tract

Abstract

Relative positions of neurons within mature murine pelvic ganglia based on expression of neurotransmitters have been described. However the spatial organization of developing innervation in the murine urogenital tract (UGT) and the gene networks that regulate specification and maturation of neurons within the pelvic ganglia of the lower urinary tract (LUT) are unknown. We used whole-mount immunohistochemistry and histochemical stains to localize neural elements in 15.5 days post coitus (dpc) fetal mice. To identify potential regulatory factors expressed in pelvic ganglia, we surveyed expression patterns for known or probable transcription factors (TF) annotated in the mouse genome by screening a whole-mount in situ hybridization library of fetal UGTs. Of the 155 genes detected in pelvic ganglia, 88 encode TFs based on the presence of predicted DNA-binding domains. Neural crest (NC)-derived progenitors within the LUT were labeled by Sox10, a well-known regulator of NC development. Genes identified were categorized based on patterns of restricted expression in pelvic ganglia, pelvic ganglia and urethral epithelium, or pelvic ganglia and urethral mesenchyme. Gene expression patterns and the distribution of Sox10+, Phox2b+, Hu+, and PGP9.5+ cells within developing ganglia suggest previously unrecognized regional segregation of Sox10+ progenitors and differentiating neurons in early development of pelvic ganglia. Reverse transcription-PCR of pelvic ganglia RNA from fetal and post-natal stages demonstrated that multiple TFs maintain post-natal expression, although Pax3 is extinguished before weaning. Our analysis identifies multiple potential regulatory genes including TFs that may participate in segregation of discrete lineages within pelvic ganglia. The genes identified here are attractive candidate disease genes that may now be further investigated for their roles in malformation syndromes or in LUT dysfunction.

Keywords: Genitourinary Development Molecular Anatomy Project; autonomic nervous system; genitourinary; in situ hybridization; lower urinary tract; mouse; pelvic ganglia; transcription factor.

Figure 1

Distribution of neural elements in fetal mouse LUT. Dorsal, lateral, and anterior views of whole-mount urogenital tract (UGT) processed to detect neuronal antigens are shown. (A) Positions of peripheral ganglia and nerve fibers are labeled by whole-mount IHC in female 15.5dpc fetal LUT stained for neurofilament (NF). (B) Whole-mount IHC for PGP9.5 similarly stains both peripheral ganglia and nerve processes at this stage in female LUT. Dorsal views of the intact UGT are shown for each antigen. Lateral views of dissected bladder with attached urethra are shown (A′,B′). Dorsal views of the urethra (A″,B″) and anterior views of the bladder (A‴,B‴) show relative positions of the pelvic ganglia to these structures. (C) Schematic diagrams of fetal mouse UGT at 15.5dpc summarize elements of autonomic innervation detected by IHC in dark blue. Axonal processes of efferent innervation that are accompanied by NC-derived peripheral glia are shown in green. In dorsal views (left), the adrenal plexus extends processes into the NC-derived medulla (dark gray) of the adrenal. Sympathetic chain ganglia at this stage reside as tight clusters medially between the kidneys. Neurites from bilateral pelvic ganglia flanking the urethra extend processes toward the center of the dorsal urethra. When viewed laterally (right) neurites also extend from pelvic ganglia and out toward the bladder dome along blood vessels that flank the bladder. (a, adrenal; ap, adrenal plexus; b, bladder; bv, blood vessel; g, gonad; gn, gonadal nerve; hg, hypogastric nerve; k, kidney; pg, pelvic ganglia; sc, sympathetic chain; u, urethra; ur, ureter; scale bar = 300 μm).

Figure 2

Distribution of cholinergic and noradrenergic elements in fetal mouse LUT. Whole-mount images of male 15.5dpc fetal UGT samples stained by AChE histochemistry localizes primarily cholinergic innervation and some other neuronal subtypes (A). IHC staining of female 15.5dpc fetal LUT detects specifically cholinergic VAChT+ neurons (B). IHC staining of female LUT detects sympathetic noradrenergic innervation labeled by tyrosine hydroxylase immunoreactivity (C). (A–C) Consist of whole-mount images of 15.5dpc urogenital tissues including dorsal views (A–C), lateral views of bladder (A′–C′), dorsal views of urethra (A″–C″), and anterior view of bladder (A‴–C‴). (a, adrenal; ap, adrenal plexus; b, bladder; bv, blood vessel; g, gonad; k, kidney; pg, pelvic ganglia; sc, sympathetic chain; u, urethra; scale bar = 300 μm).

Figure 3

Distribution of Sox10+ NC-derived progenitors in fetal mouse LUT. Whole-mount images of 15.5dpc UGT hybridized with anti-sense probes to detect Sox10 expression are shown. NC-derived cells are strongly labeled in dorsal view of entire UGT (A), dorsal view of urethra (B), and lateral view of bladder (C). Arrowheads highlight stained single cells visible in the dorsal view of the urethra and lateral view of the bladder. (GUDMAP Probe MTF511, MGI:3506241, scale bar = 300μm). Images of 15.5dpc UGT samples developed for a shorter period of time to discriminate regions of high versus low Sox10 expression are shown (D,E). Sox10+ progenitors are present in dorsal urethra [(D), 50×] and in pelvic ganglia viewed laterally (E). Note the high intensity of Sox10 expression within the interior region of the pelvic ganglia (arrow). GUDMAP probe MTF511, MGI:3506241. Scale bar = 300 μm.

Figure 4

Genes expressed in pelvic ganglia and urethral epithelium in the fetal mouse LUT. A schematic diagram of LUT represents dorsal views of the urethra (u), including the epithelial layer (blue), mesenchymal layer (peach), muscular layer (pink), and pelvic ganglia (pg). Distinct expression patterns in the urethral epithelium and pelvic ganglia are evident in dorsal views at 15.5dpc for a subset of WISH samples surveyed. All images are 50× magnification with a 300 μm scale bar in the first panel. ed, Ejaculatory duct (arrow); sv, seminal vescicles (arrowhead). Gene abbreviation/MGI gene ID/gender of sample: Hand1/103577/male; Satb1/105084/male; Sox2/98364/female; Gata3/95663/male; Lmo1/102812/male; Trim9/2137354/female.

Figure 5

Pelvic ganglia and urethral mesenchyme expression of genes in the fetal LUT identified by WISH. Lateral views of gene expression patterns detected for a subset of genes expressed in both pelvic ganglia and urethral mesenchyme. All images are 50× magnification with a 300 μm scale bar. Gene abbreviation/MGI gene ID/gender of sample: Epas1/109169/female; Tacc2/1928899/male; Gata2/95662/female; Sqstm1/107931/female.

Figure 6

WISH Gene expression patterns within and around fetal pelvic ganglia. Representative lateral views of individual gene expression patterns that exhibit distinct regional expression patterns within the pelvic ganglia are shown. Accompanying diagrams emphasize area of highest LUT expression detected for each gene. All images are 50× magnification with a 300 μm scale bar. Gene abbreviation/MGI gene ID/gender of sample: Rtn4/1915835/male; Gata2/95662/female; Ndrg2/1352498/female; Ndrg4/2384590/female. (b, bladder; bv, blood vessel; u, urethra; h, horizontal domain; v, vertical domain).

Figure 7

Regional segregation of NC-derived progenitors and neurons within fetal pelvic ganglia. (A) Cryosection through the lateral sagittal plane of pelvic ganglia collected from Phox2b-H2BCFP, Sox10-H2BVenus double transgenic female fetal mouse stained for PGP9.5 and Hu shows the overall triangular shape of the pelvic ganglia at 15.5dpc. Merged confocal image at 200× magnification captures nuclear Sox10-H2BVenus (green), nuclear Phox2b-H2BCFP (blue), cytoplasmic PGP9.5 (red), and Hu (gold). Differentiating neurons in the bladder wall identified by residual nuclear H2BVenus expression are co-labeled by up-regulation of cytoplasmic PGP9.5 (arrows). (A′) Confocal image showing channels only for Venus and CFP from the cryosection presented in (A) is shown. An oval domain comprised of densely packed Sox10+ cells (green nuclei, encircled by red dotted line) is evident with Phox2b+ neurons (blue nuclei) being most numerous outside this region. (A″) A confocal image shows channels for PGP9.5 and Hu from the cryosection presented in (A). PGP9.5 (red) labels numerous cell bodies at the dorsal aspect of the pelvic ganglia, a small area of differentiating neurons nearest the anterior bladder neck, and extrinsic nerve fibers entering the ganglia. Hu (gold) labels numerous cell bodies with the greatest density being around the perimeter of the pelvic ganglia. (B) Higher magnification confocal image (400×) from the boxed region in (A) shows high density of H2BVenus+ progenitors in the core of the oval progenitor domain with Phox2b+, PGP9.5+, Hu+ neurons clustered at the dorsal aspect of the pelvic ganglia. Single confocal channels at 630× magnification from boxed area in (B) are shown for Sox10-H2BVenus [(C), green], Phox2b-H2BCFP [(C′), blue], Hu+ neuronal soma [(C″), gold], PGP9.5+ differentiating neurons [(C‴), red]. Progenitor cells labeled by H2BVenus nuclear fluorescence (arrowheads) exhibit low or no expression of Phox2b-H2BCFP and no expression of PGP9.5 and Hu. Differentiating neurons lack H2BVenus label, exhibit bright Phox2b-H2BCFP nuclear fluorescence as well as cytoplasmic Hu and PGP9.5 labeling (arrows). Dorsal (d), ventral (v), anterior (a), and posterior (p) orientations are indicated on (A′). Scale bar: 25 μm.

Figure 8

Validation of gene expression patterns in pelvic ganglia by sectional in situ hybridization (SISH). (A) Schematic diagram of 15.5dpc fetal mouse LUT viewed dorsally illustrates pelvic ganglia (dark blue clusters), neurites extending from pelvic ganglia across the dorsal urethral surface (blue lines), and axonal processes (green lines) relative to LUT structures. Red vertical lines indicate the relative planes of cryosections taken through mid-sagittal (B) and lateral sagittal regions (C) for SISH processing. Colorometric micrographs of SISH signals obtained in cryosections for individual genes (D). Images for Crip2, Ndrg4, Satb1, and Sqstm1 are from data deposited at the GUDMAP database (probe IDs: MGI:3507380, MGI:3506573, MGI:3506384, MGI:3506375, respectively). Images for Ndrg2, Gata2, Klf7, Lmo1, Rtn2, and Rtn4 derive from data posted on Eurexpress.org (probe IDs: euxassay_010581, section 14; euxassay_018037, section 14; euxassay_003485, section 16; euxassay_017874, section 07; euxassay_018264, section 14; and euxassay_004344, section 14, respectively). Dotted line outlines the pelvic ganglia border in SISH panel for Crip2 as the expression for this gene is not homogeneous within cells but was observed as faint punctate signal within some cells and not in others.

Figure 9

Identification of genes that maintain expression in pelvic ganglia across multiple developmental stages. (A) NC derivatives labeled by Sox10 expression are evident in pelvic ganglia, bladder wall, and urethra of a Sox10-H2BVenus transgenic embryo viewed laterally at 14.5dpc. (B) Schematic diagram illustrating the dissection technique to harvest pelvic ganglia based on Sox10-H2BVenus transgene fluorescence. (C) Relative size, shape, and position of pelvic ganglia compared to the overall bladder morphology are illuminated by Sox10-H2BVenus expression in lateral images of 15.5dpc, P2, P10, P21 lower urinary tracts. Scale bar = 1000 μm. (D) Gel electrophoresis images of RT-PCR products relative to a standard molecular weight marker identifies individual genes expressed in fetal development and post-natal maturation of pelvic ganglia. Sample order: no template water control, genomic DNA control, no RT control (15.5dpc RNA sample), 15.5dpc intact LUT (consisting of distal ureter, bladder, urethra, pelvic ganglia, and genital tubercle) and Sox10-H2BVenus pelvic ganglia RNA isolates harvested at 14.5dpc, 15.5dpc, P2, P10, and P21. A housekeeping control gene (Hprt) is included for comparison.