BIOLOGY OF REPRODUCTION 68, 423438 (2003) Published online before print 17 October 2002. DOI 10.1095/biolreprod.102.

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Sonic hedgehog Cascade Is Required for Penile Postnatal Morphogenesis, Differentiation, and Adult Homeostasis1
Carol A. Podlasek,2 David J. Zelner, Hong Bin Jiang, Yi Tang, John Houston, Kevin E. McKenna, and Kevin T. McVary
Department of Urology and Physiology, Northwestern University Medical School, Chicago, Illinois 60611
ABSTRACT
The penis is unique in that it undergoes morphogenesis and differentiation primarily in the postnatal period. For complex structures such as the penis to be made from undifferentiated precursor cells, proliferation, differentiation, and patterning are required. This process involves coordinated activity of multiple signals. Sonic hedgehog (Shh) forms part of a regulatory cascade that is essential for growth and morphogenesis of many tissues. It is hypothesized that the penis utilizes regulatory mechanisms similar to those of the limb and accessory sex organs to pattern penile postnatal morphogenesis and differentiation and that the Shh cascade is critical to this process. To test this hypothesis, Shh, BMP-4, Ptc, and Hoxa-10 localization and function were examined in Sprague-Dawley rat penes by means of quantitative reverse transcription polymerase chain reaction, in situ hybridization, immunohistochemistry, and Western blotting. These genes were expressed in the penis during postnatal morphogenesis in a spatially and temporally restricted manner in adjacent layers of the corpora cavernosal sinusoids. The function of Shh and BMP-4 is to establish and maintain corpora cavernosal sinusoids. The data suggest that Ptc and Hoxa-10 are also important in penile morphogenesis. The continuing function of Shh and targets of its signaling in maintaining penile homeostasis in the adult is signicant because disruption of Shh signaling affects erectile function. This is the rst report that demonstrates the signicant role that Shh plays in establishing and maintaining penile homeostasis and how this relates to erectile function. These studies provide valuable insight that may be applied to improve treatment options for erectile dysfunction.

developmental biology, male reproductive tract, male sexual function, penis

INTRODUCTION

Preliminary evidence suggests that sonic hedgehog (Shh) cascade members are active in early embryonic patterning of the penis. Posterior Hox genes such as Hoxa-13 and Hoxd-13 have been identied in the penis, and targeted loss of their function resulted in the complete absence of external genitalia [1, 2] in the developing embryo. Other members of the Shh cascade, including Shh, bone morphogenetic protein-4 (BMP-4), and patched (Ptc) are expressed in the
This work was sponsored by the National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases under grants DK54478, DK55046, and DK59071. 2 Correspondence: Carol Podlasek, Department of Urology, Northwestern University, Tarry Building 11-715, 303 E. Chicago Ave., Chicago, IL 60611. FAX: 312 908 7275; e-mail: cap325@northwestern.edu
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genital tubercle [3], the embryonic precursor of both the penis and accessory sex organs. These ndings suggest Shh cascade involvement in embryonic patterning of the penis. However, penile morphogenesis and differentiation take place primarily in the postnatal period [4]. A potential role of the Shh cascade in specication and differentiation of the penis in the postnatal period has not previously been explored. We hypothesize that the Shh cascade plays a crucial role in penile differentiation and growth and that the penis utilizes signaling mechanisms similar to those of the limb and accessory sex organs for postnatal morphogenesis. A cascade of signaling molecules that orchestrates interaction between tissue layers has been partially established in the limb, lung, gut, and accessory sex organs. This cascade consists of but is not restricted to members of diverse gene families, such as Shh, BMPs, Hox, Wnt, and the broblast growth factors (FGFs). Shh is a principal component of this conserved signaling pathway. It functions by regulating cellular proliferation and differentiation [5] either directly or through induction of secondary signaling molecules [6, 7]. The Hox genes are targets of Shh signaling that can mediate early patterning instructions. These genes dene positional identity along the anterior/posterior axis [812]. Other Shh targets are BMP-4 and Ptc. BMP-4 plays a role in interdigital and interductal space formation [13, 14] and has recently been implicated as a regulator of Shh expression [15, 16]. Ptc is the transmembrane receptor for Shh [17]. It is involved in transducing the hedgehog signal and is also a transcriptional target of Shh [18]. Shh, Hoxa-10, BMP-4, and Ptc form part of a cascade of genes that regulate mesenchymal/epithelial interactions during embryogenesis. We hypothesize that the penis utilizes similar signaling mechanisms to regulate postnatal morphogenesis and differentiation. A better understanding of signaling mechanisms that function to establish normal penile morphology may offer valuable insight into altered morphology associated with erectile dysfunction (ED). Smooth muscle and endothelial changes accompany ED resulting from diabetes mellitus [19, 20] and nerve injury after surgical intervention for prostate cancer [21]. ED is a common and devastating pathologic condition that affects 1030 million American men (1985 gures) [22]. Treatment options for individuals with ED are only partially effective [23]. A better understanding of how penile morphology is established and maintained would signicantly enhance the potential for improved treatment. With this in mind, we will examine the mechanisms that regulate penile postnatal morphogenesis and differentiation.
MATERIALS AND METHODS Animal Model
Sprague-Dawley rats from Postnatal Day 4 (P4) to P120 were obtained from Charles River (Wilmington, MA). Rats were killed, and penes were

Received: 2 May 2002. First decision: 27 May 2002. Accepted: 22 August 2002. 2003 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org

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FIG. 1.

Rat penis in cross section. Arrows indicate signicant morphologic features.

harvested by sharp dissection (scalpel and scissors) and either frozen in liquid nitrogen or xed in 4% paraformaldehyde. Animals were cared for in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals.

Cavernous Nerve Injury

P120 Sprague-Dawley rats were randomized into two groups: bilateral cavernous nerve (CN) resection (n 14) and sham abdominal exploration

(control, n 14). Sections (5 mm) of the cavernous nerve were removed bilaterally using a KAPS industrial microscope under direct vision through a midline abdominal incision. The prostatic capsule was manipulated in control animals without resecting the CN. Stress-related uctuations of serum testosterone were minimized at the time of abdominal exploration through bilateral epididymo-orchiectomy and s.c. placement of a 2-cm piece of medical-grade silastic tubing (Dow Corning, Midland, MI) lled with crystalline testosterone [24, 25]. This method ensures reliable, uniform serum testosterone levels for both the control and intervention groups

FIG. 2. Graph of the desnity of RT-PCR products versus age in the Sprague-Dawley rat. All penes assayed were between P4 and P120. Experiments were performed in triplicate on three sets of pooled tissue specimens (n 510), and the data are reported as the mean SEM. Where error bars are absent, the error is too minimal to be distinguishable from the data point. A ttest was used to determine signicant changes in expression. A) Ratio of RT-PCR products of Shh/GAPDH versus age. Shh expression in the penis is low during the rst month after birth, increases during adolescence (P40P60), peaks at P90, and remains abundant in the adult. B) Ratio of RT-PCR products of BMP-4/RPL-32 versus age. BMP-4 expression in the penis is abundant throughout the entire period of postnatal penile development (P4P120). C) Ratio of RT-PCR products of Ptc/ GAPDH versus age. Ptc expression in the penis is most abundant in the rst few weeks after birth but gradually decreases with age to reach a minimum at P90. A second peak in expression occurs at P100. Expression diminishes in the adult (P120). D) Ratio of RT-PCR products of Hoxa-10/ RPL-19 versus age. Hoxa-10 is most abundant in the penis immediately after birth and decreases rapidly. A second peak in Hoxa-10 expression is apparent from P40 to P60. Expression is minimal in the adult (P120).

Shh CASCADE IS REQUIRED FOR PENILE DEVELOPMENT

TABLE 1. Primers used for RT-PCR of rat genes. Gene Primer type Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense 5 5 5 5 5 5 5 5 5 5 5 5 5 5 -CAT -TTC -GTC -ACA -AGT -GTG -CAG -ACA -GGC -AGA -GTG -GAA -CTG -TGT CCC CCA GGT AAC TCC AGT CTA TGT TAC GTC AAG CAC GCC AAT Sequence AGA CCG GTC ATG AAA TCT ATC AGA AGA TTG CCC AAA AGA TGG GAA TGT AAC GGG GGG GGG TCG CGT AGA ATG AAG ACA TGT GGG TGA CAC GGA GCA GAA GCA AGA GCA GGC ATC ATC GGC TTT TGA GGT GA-3 ATT GT-3 TTT G-3 TCA G-3 AAT GC-3 GAG GC-3 CCA AC-3 CAC GA-3 TT-3 TC-3 GTC-3 ACA C-3 CTG GT-3 GTT CC-3 Product size (bp) 202 397 353 225 560 412 232

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BMP-4 GAPDH Hoxa-10

Ptc RPL-19 RPL-32 Shh

up to 28 days after placement. Penes were harvested 7 and 21 days after CN resection and were either frozen in liquid nitrogen or xed in 4% paraformaldehyde.

dehydrated with ethanol and xylene and mounted using Krystalon (Diagnostic Systems, Gibbstown, NJ).

RNA Isolation and Quantication of Gene Expression by Reverse Transcription Polymerase Chain Reaction
Total RNA was extracted from penes of Sprague-Dawley rats using the TRIzol (Life Technologies, Gaithersburg, MD) method. Samples were treated with DNase (Promega, Madison, WI) to eliminate genomic DNA contamination. Primers (Table 1) were synthesized at the Northwestern University Biotechnology Facility. Reverse transcription polymerase chain reaction (RT-PCR) was performed using the Gene Amp RNA PCR Core kit (Perkin-Elmer, Branchburg, NJ), and products were digested with restriction enzymes to conrm that bands represented the sequences of interest. Quantitative RT-PCR was performed as described previously [19, 26] using noncompetitive methodology and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L-32 (RPL-32), or RPL-19 as endogenous internal standards. All measurements were made in the linear range for Shh and GAPDH, BMP-4 and RPL-32, Hoxa-10 and RPL19, and Ptc and GAPDH. Assays were performed in triplicate on three sets of pooled tissue specimens, and the product ratios are reported as the mean SEM. The data presented for each gene were normalized to 1 so results could be presented in a comparable manner, and a t-test was used to determine signicant changes in gene expression.

Bead Experiments
Af-Gel beads (100200 mesh; Bio-Rad Laboratories, Hercules, CA) were equilibrated with 5E1 anti-Shh antibody (13 g/ml; Jessel, Hybridoma Bank, University of Iowa, Ames, IA), mouse IgG (3 g/ml), recombinant mouse Shh peptide (7.5 g/animal; R&D Systems, Minneapolis, MN) [28], recombinant human BMP-4 peptide (3 g/animal; R&D Systems) [32], or recombinant mouse Noggin/Fc peptide (15 g/animal; R&D Systems) overnight before injection into P30 (n 25) and P120 (n 25) rat penes. Fifty-six-day-old (n 25) rats were also injected, but half the concentration of reagents was used. These ages were chosen for study because they represents the developmental periods before, during, and after puberty. Approximately 2030 beads were injected into each animal. Rats were killed 7 days postinjection. Penes were harvested and xed in 4% paraformaldehyde for sectioning. Bead technology has previously been used successfully for delivery of proteins and antibodies to target tissues [33, 34].

Intercavernosal Pressure Measurements

Af-Gel beads soaked in Shh inhibitor (3 g/ml; Jessel) or mouse IgG (control with beads: 3 g/ml) overnight at 4 C were injected into the corpora cavernosa of P120 penes. Seven days after injection, the intercavernosal pressure (ICP) was measured after stimulation in control (no beads, n 3), control with beads (n 3), and inhibitor-treated (n 3) penes as previously described [35]. Nerves were stimulated (intensity of 6 volts) by placing them on bipolar platinum stimulating electrodes connected to an electrical stimulator (Grass Instruments, Quincy, MA) delivering a series of square-wave pulses (1 msec duration at 30 Hz). The CN was unilaterally stimulated at a distance of 3 and 5 mm from the major pelvic ganglion. Stimulation lasted 40 sec. A resting interval of at least 5 min separated two consecutive stimulation procedures.

In Situ Hybridization
Penes that were xed in 4% paraformaldehyde overnight were used for in situ hybridization as previously described [19, 27] utilizing a mouse Shh RNA probe [28], a mouse Ptc RNA probe [29], and a mouse Bmp-4 probe [30].

Immunohistochemical Analysis
Immunohistochemical analysis (IHC) was performed as previously outlined [19]. Sections were incubated with one of the following antibodies: goat polyclonal IgG Shh, Ptc, Bmp-4, and CD31 (Santa Cruz Biotechnology, Santa Cruz, CA; 200 g/ml), Hoxa-10 (BAbCO, Richmond, CA), or alpha smooth muscle actin (Sigma, St. Louis, MO). Sections were stained with diaminobenzidine (DAB) or DAB with nickel and mounted using crystal mount (Biomedia, Foster City, CA).

RESULTS

Anatomy of the Penis

Western Analysis
Western analysis was performed on CN-injured (n 5) and control (n 5) penes as outlined previously [19]. Membranes were incubated with a goat polyclonal Shh antibody (Santa Cruz Biotechnology; 200 g/ml) for 18 h at 4 C. Protein bands were visualized using enhanced chemiluminescence detection reagent (ECL Western Blotting Analysis System; Amersham, Piscataway, NJ) according to the manufacturers directions and then exposed to Kodak (Rohester, NY) X-AR2 lm for 15 min.

The rat penis contains three erectile cylinders: the paired larger corpora cavernosa and the smaller corpus spongiosium (Fig. 1). The corpora cavernosa are composed of a meshwork of interconnected cavernosal spaces lined by vascular endothelium and separated by trabeculae containing bundles of smooth muscle in a framework of collagen, elastin, and broblasts (Fig. 1).
Measurement of Shh Expression During Penile Development

Morphology
The morpholgy of penis tissue was examined in sections stained with hematoxylin and eosin (H&E) as outlined previously [31]. Sections were

The time course of Shh expression in the penis was measured by RT-PCR at points spanning the entire range of postnatal morphogenesis (P4P120). During the rst month after birth, Shh expression was low (Fig. 2A). Expression

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FIG. 4. Analysis of Shh protein in control and CN-injured Sprague-Dawley rat penes. A) In Western blots, Shh protein is no longer detectable in the penis 7 days after CN injury. C, Control; CN7, 7 days after CN injury. B) Twenty-one days after CN injury, Shh protein is no longer detectable by IHC in the smooth muscle of the corporal cavernosal sinusoids. Arrows indicate Shh protein or where Shh protein would be if present. Magnication 20.

FIG. 3. Analysis of juvenile (P22) and adolescent (P63) Sprague-Dawley rat penes for Shh protein and RNA. Top: IHC for Shh protein. At P22, Shh protein is visible in the smooth muscle of the corpora cavernosal sinusoids, the nerves of the dorsal nerve bundle, and the epithelium of the urethra. This localization remains unchanged in the adolescent (P63) and the adult (data not shown). DAB (with or without nickel) was used to visualize Shh protein. Arrows indicate Shh protein. N, Nerve. Magnication 40. Bottom: In situ hybridization analysis for Shh RNA in the juvenile (P22) and adolescent (P63) rat. Shh is expressed in the smooth muscle of the corpora cavernosal sinusoids and the epithelium of the urethra and faintly in the nerves. The endothelial lining of the corpora cavernosal sinusoids is apparent as a thin layer adjacent to the smooth muscle (asterisk); it did not stain for Shh RNA. Arrows indicate Shh RNA. N, Nerve. Magnication 40.

then increased signicantly after P40 (P 0.046), peaked at P90, and remained abundant in the adult (P120) and in aged rats (P200, data not shown). Thus, Shh was expressed in the penis during the entire period of postnatal morphogenesis, and expression remained abundant in the adult.
Time Course of Expression of Shh Targets

The time courses of expression of Shh targets, including BMP-4, Ptc, and Hoxa-10, in the penis during postnatal morphogenesis (P4P120) were measured by RT-PCR. BMP-4 was abundant during the entire period of postnatal morphogenesis, with decreased expression observed in the

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FIG. 5. IHC of juvenile Sprague-Dawley rat (P30) penes assayed for BMP-4, Ptc, and Hoxa-10 proteins. Top: BMP-4 protein is restricted to the endothelial lining of the corpora cavernosal sinusoids and is not visible in the nerves of the dorsal nerve bundle or the urethra. The smooth muscle of the sinusoid is visible as an unstained layer adjacent to the endothelium. Middle: Ptc protein is localized in the smooth muscle of the corpora cavernosal sinusoids and the epithelium of the urethra. It is not visible in the nerves of the dorsal nerve bundle. Bottom: Hoxa-10 protein is localized in the smooth muscle of the corpora cavernosal sinusoids and faintly in the epithelium of the urethra. It is not visible in the nerves of the dorsal nerve bundle. Arrows indicate BMP-4, Ptc, and Hoxa-10. N, Nerve. Magnication 40.

adult (P100P120; Fig. 2B). Ptc expression was abundant in the rst few weeks after birth (P4P20), decreased gradually from P40 to P90, and remained abundant in the adult (P100P120; Fig. 2C). Hoxa-10 expression was most abundant immediately after birth and decreased signicantly by P40 (P 0.05; Fig. 2D). A second peak in expression was observed between P40 and P90, and Hoxa-10 expression remained measurable in the adult (Fig. 2D).
Localization of Shh Protein During Penile Postnatal Morphogenesis IHC was performed on juvenile (P12 and P22), adolescent (P63), and adult (P120) penes to determine Shh local-

ization. Shh protein was faintly observed in the corpora cavernosal sinusoids and in the epithelium of the urethra at P12 (data not shown). At P22, P63, and P120 (data not shown for the adult), protein was observed in smooth muscle of the corpora cavernosal sinusoids, the epithelium of the urethra, and the nerves of the dorsal nerve bundle (Fig. 3). The endothelial lining of the sinusoids was identiable as a thin layer of unstained tissue adjacent to the smooth muscle. These results indicate that Shh protein is restricted to specic regions of the penis during postnatal morphogenesis and in the adult.

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FIG. 6. IHC of adolescent Sprague-Dawley rat (P63) penes assayed for BMP-4, Ptc, and Hoxa-10 proteins. Top: BMP-4 protein is restricted to the endothelial lining of the corpora cavernosal sinusoids and is not visible in the nerves of the dorsal nerve bundle or the urethra. Middle: Ptc protein is localized in the smooth muscle of the corpora cavernosal sinusoids and the epithelium of the urethra. It is not visible in the nerves of the dorsal nerve bundle. The unstained endothelial lining of the sinusoid is adjacent to the smooth muscle. Bottom: Hoxa-10 protein is localized in the smooth muscle of the corpora cavernosal sinusoids and faintly in the epithelium of the urethra. It is not detectable in the nerves of the dorsal nerve bundle. Arrows indicate BMP-4, Ptc, and Hoxa-10. N, Nerve. Magnication 40.

Localization of Shh RNA by In Situ Hybridization

Localization of Shh in the penis was also examined by in situ hybridization (P12, P22, P63, and P120). Shh RNA was abundant in the smooth muscle of the corpora cavernosal sinusoids and in the epithelium of the urethra at all ages assayed (Fig. 3, bottom; P12 and P120 data not shown). Contrary to observations made for Shh protein, little Shh RNA was observed in the nerves of the dorsal nerve bundle. However, in the nerve cell body, the pelvic ganglia, Shh was abundant (data not shown). These results conrm IHC ndings of restricted Shh protein distribution.

Shh Protein in the Nerves and Corpora Cavernosa May Be Related

Western analysis was performed on control and CN-injured penes to examine a possible realtionship between the presence of Shh protein in the nerves of the dorsal nerve bundle and in the corpora cavernosa. Shh protein was dramatically decreased in the penis (no longer detectable) 7 days after CN injury (Fig. 4A). IHC analysis revealed the absence of Shh protein in the corpora cavernosa 21 days after CN injury (Fig. 4B). These results show that the presence of Shh protein in the nerves and corpora cavernosa of

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FIG. 7. H&E staining of control (mouse IgG), 5E1 Shh inhibitor-treated, and recombinant Shh peptide-treated corpora cavernosa of the adolescent (P63) Sprague-Dawley rat. HE staining. Gross evaluation (6 ) reveals abundant corpora cavernosal sinusoids in control penes. After inhibitor treatment, sinusoids are almost completely absent. The corporal tissue resembles that of the undifferentiated newborn penis (20 ). After exogenous Shh peptide treatment, an increase in differentiated endothelial tissue is visible surrounding the beads. Arrows indicate Af-Gel beads.

the penis are related. However, the nature of this relationship remains to be determined.
Localization of Shh Targets in the Penis During Development
FIG. 8. IHC of control (mouse IgG), Shh inhibitor-treated, and Shh peptide-treated Sprague-Dawley rat penes assayed for Shh, BMP-4, Ptc, Hoxa-10, alpha actin, and CD31 proteins. Left: All of these proteins are present in the corpora cavernosa of normal penes. Middle: After inhibitor treatment, Shh protein is absent in the treated area of the corpora, thus conrming inhibitor function. BMP-4 protein localization is dramatically increased a short distance from the bead vehicle and Ptc and Hoxa-10 are downregulated after Shh disruption. Staining for alpha actin and CD31 is completely absent in the affected region. Arrows indicate Af-Gel beads and the area treated with inhibitor. Right: After addition of exogenous Shh peptide, staining for Shh protein is abundant around the bead vehicles, thus conrming treatment with Shh peptide. In contrast to inhibitor treatment, Shh peptide treatment resulted in the absence of BMP-4 protein and an increase in localization of Ptc, Hoxa-10, and CD31. Alpha actin is faintly visible in a diffuse region surrounding the beads. Arrows indicate protein localization. , Absence of protein; , presence of protein. Magnication 6.

The localization of BMP-4, Ptc, and Hoxa-10 proteins was examined by IHC in juvenile (P12 and P30), adolescent (P63), and adult (P90) penes (n 3 for each age group). BMP-4 protein was restricted to the endothelial lining of the corpora cavernosal sinusoids, whereas Ptc and Hoxa-10 were localized in the adjacent smooth muscle of the sinusoids and in the epithelium of the urethra (Figs. 5 and 6; data not shown for P12 and P90). Shh, Ptc, BMP4, and Hoxa-10 were all localized in close proximity (in adjacent tissue layers) in the corpora cavernosa of the penis.

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Effect of Shh Inhibitor on Postnatal Penile Morphology

Shh inhibitor (5E1 anti-Shh antibody), a recombinant Shh peptide, and mouse IgG (control) were preabsorbed into Af-Gel beads and injected into the corpora cavernosa of P56 penes to examine what role Shh may play in penile postnatal morphogenesis. H&E staining revealed a meshwork of corpora cavernosal sinusoids (Fig. 7) that looked normal despite the presence of Af-Gel beads used to deliver the mouse IgG (control). Shh inhibitor treatment (1.5 g/ml) resulted in gross abnormalities, including the absence of corpora cavernosal sinusoids in the affected region (Fig. 7). The resulting corpora cavernosal tissue resembled the undifferentiated tissue of the newborn (data not shown). These experiments were repeated in P30 and P120 animals (juvenile and adult, respectively) and with double the inhibitor concentration (3 g/ml). A similar but stronger response was observed (data not shown); a larger region of the corpora was affected, and corpora cavernosal sinusoids were completely absent over a larger cross section of the penis. The presence of a dose-dependent change in morphology that was observed at several different ages indicates the importance of Shh function to penile postnatal morphogenesis and adult homeostasis.
Effect of Shh Inhibitor on Targets of Shh Signaling

abundantly for both alpha actin and CD31 (Fig. 8). Alpha actin was observed both in the sinusoid smooth muscle and in individual cells interspersed between the trabeculae (data not shown). After Shh inhibition, the corpora cavernosa was completely devoid of both cell types in the affected region. Visual inspection of the affected tissue revealed abundant undifferentiated mesenchyme. These results show that inhibition of Shh function resulted in a dedifferentiation of the penile corpora such that smooth muscle and endothelium were absent and primitive mesenchyme was abundant.
Effect of Exogenous Shh Peptide on Penile Morphology

The effects of Shh inhibition on members of the Shh cascade, including BMP-4, Ptc, and Hoxa-10, were examined by IHC. After inhibitor treatment, Shh protein was not observed in the corpora cavernosa near the bead vehicles (Fig. 8), thus conrming that the inhibitor was effective. BMP-4 protein was dramatically increased, but Ptc and Hoxa-10 proteins were completely absent after inhibitor treatment (Fig. 8). Shh inhibition experiments were repeated in juvenile and adult animals at a higher inhibitor concentration. BMP-4 and Ptc RNA and protein were dramatically increased (Fig. 9A). The increase in Ptc RNA distribution was not observed at the lower concentration of inhibitor (Fig. 8). When inhibitor-treated beads were injected in the dorsal nerve bundle, a similar increase in BMP-4 expression was observed immediately surrounding the nerves (Fig. 9B). These results demonstrate a concentration-dependent effect of Shh inhibitor on expression of Shh targets.
Morphological Markers Assayed by IHC after Shh Inhibitor Treatment

P56 penes were treated with exogenous Shh peptide and mouse IgG (control) using Af-Gel beads to gain further insight into Shh function. The results were very different from those observed after treatment with Shh inhibitor. Cavernosal sinusoids were absent in H&E-stained sections (Fig. 7), but IHC analysis revealed that the tissue was lled in with an abundance of cells that stained positively for CD31 and only faintly for alpha actin (Fig. 8). Thus, Shh peptide and inhibitor had opposing effects, with inhibition of function resulting in dedifferentiation and addition of exogenous peptide causing increased endothelial differentiation. Targets of Shh signaling also were examined after Shh peptide treatment. BMP-4 protein was completely absent, but Ptc and Hoxa-10 proteins were upregulated in the region surrounding the beads (Fig. 8). To ensure that the method of delivery was working, we used IHC to assay Shh protein. Shh was observed in the tissue surrounding the bead vehicles (Fig. 8). These results demonstrate that Shh is a mediator of differentiation in the penis and imply a function for Shh targets in postnatal penile morphogenesis.
Effect of Exogenous BMP-4 Peptide on Penile Morphology

Corpora cavernosal morphology was evaluated after Shh inhibitor treatment by IHC analysis of smooth muscle (alpha actin) and endothelium (CD31). Gross changes in penile morphology were observed. Normal penes stained

FIG. 9. In situ hybridization (P37) and immunohistochemical analysis (P127) of BMP-4 and Ptc localization in Sprague-Dawley rat penile corpora cavernosa after Shh inhibitor treatment (3.0 g/ml). A) Top: In situ hybridization at P37 reveals abundant BMP-4 (magnication 10) and Ptc (magnication 25) RNA expression in the tissue near and directly surrounding the bead vehicle. B, Bead. Bottom: Immunohistochemical analysis after Shh inhibitor treatment in the adult (P127) shows an increase in both BMP-4 and Ptc proteins, thus conrming our observations at P37 and P63 (Fig. 8) and showing the continued requirement for Shh in the adult corpora. Arrows indicate BMP-4 and Ptc RNA (magnication 10) and protein (magnication 20) localization. B) After Shh inhibitor treatment (P37), in situ hybridization reveals abundant BMP-4 RNA expression surrounding the nerves of the dorsal nerve bundle. Arrows indicate BMP-4 RNA localization at magnication 20 and 40. N, Nerve.

The potential role of BMP-4 in penile postnatal morphogenesis was examined in the penis because BMP-4 localization was profoundly altered by the presence/absence of Shh function. Af-Gel beads pretreated with recombinant BMP-4 peptide, recombinant Noggin peptide (antagonist of BMP-4), or mouse IgG (control) were injected into P56 rats. H&E staining after BMP-4 peptide treatment revealed an increase in size of the corpora cavernosal sinusoids (Fig. 10), which appeared normal in all other aspects (intact endothelial lining and blood cells within the sinusoids). IHC analysis revealed increased CD31 and Ptc proteins surrounding the beads but an absence of Shh and alpha actin proteins (Fig. 11). BMP-4 protein was identied around the beads after exogenous BMP-4 treatment, conrming peptide delivery. Thus, BMP-4 appears to downregulate Shh and upregulate Ptc. Opposing effects of BMP-4 and Noggin were observed in the corpora cavernosa. Noggin treatment resulted in a dramatic decrease in the number of sinusoids (Fig. 10) in the corpora cavernosa in a manner similar to that observed after the addition of Shh peptide. IHC analysis revealed normal CD31 staining, the absence of alpha actin and Ptc proteins, and an abundance of Shh protein near the beads (Fig. 11). IHC analysis for BMP-4 protein revealed the absence of BMP-4 after Noggin treatment, conrming antagonist delivery (Fig. 11). These results indicate that BMP-4

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FIG. 10. H&E analysis of control (mouse IgG), recombinant BMP-4 peptide-treated, and recombinant Noggin-treated corpora cavernosa from SpragueDawley rats (P63). Top: Gross evaluation (6 ) reveals abundant corpora cavernosal sinusoids in control penes. Middle: After BMP-4 treatment, sinusoidal spaces are larger upon visual observation (20 ). The increased size is not an artifact of sectioning because sinusoids have a normal appearance, with an intact endothelial lining and blood cells identiable within the sinusoids. Bottom: After Noggin treatment (BMP-4 antagonist), sinusoids are absent in the treated area and abundant endothelium is evident. Arrows indicate Af-Gel beads.

is necessary to establish normal corpora cavernosal morphology in the adolescent penis.

ICP Measurement ICP was measured in adult rats that had been either treated with Shh inhibitor, mouse IgG (bead control) or were left untreated (control). The presence of the bead vehicle did not alter the maximum pressure attained after stimulation (Fig. 12). However, the presence of Shh inhibitor dramatically decreased the ICP. These experiments demonstrate that inhibition of Shh function can alter corpora cavernosal morphology enough to result in ED.
DISCUSSION To make complex structures such as the penis from undifferentiated precursor cells, both proliferation and orga-

nization (patterning) are necessary, requiring coordinated activity of multiple signals to achieve the proper arrangement of adult tissues. Shh, Ptc, BMP-4, and Hoxa-10 form a small part of a cascade that acts coordinately to achieve postnatal penile morphogenesis and differentiation. These genes are abundant in the penis after birth, and their localization in the smooth muscle and endothelial lining of the corpora cavernosal sinusoids implies their involvement in differentiation of the sinusoidal tissue. This hypothesis was examined by inhibiting Shh function in targeted regions of the corpora cavernosa. This inhibition resulted in gross anatomical changes in corpora cavernosal morphology, including the absence of sinusoids and the presence of large amounts of undifferentiated tissue closely resembling the primitive architecture of the neonate (unpublished observations). We conrmed the absence of differentiated

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FIG. 11. IHC of control (mouse IgG), BMP-4-treated, and Noggin peptide-treated Sprague-Dawley penes assayed for Shh, BMP-4, Ptc, alpha actin, and CD31 proteins. Left: All proteins are identiable in the corpora of normal penes. Middle: After BMP-4 treatment, staining for Shh and alpha actin is absent, but Ptc and CD31 proteins are abundant in the corpora cavernosa. Right: After Noggin treatment, BMP-4, Ptc, and alpha actin proteins are absent, but Shh and CD31 are abundant. Arrows indicate protein localization. Magnication 6.

smooth muscle and endothelium by IHC (Fig. 8). The observed morphological changes are not likely to be artifacts attributable simply to the presence of the bead vehicle because the morphology of the controls was completely normal despite the presence of Af-Gel beads. Administration

of inhibitor in the juvenile, adolescent, and adult rat resulted in strikingly similar changes in morphology and thus provided evidence that Shh is not only required to establish penile morphology but continues to function in the adult to maintain corpora cavernosal homeostasis. How Shh acts to

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FIG. 12. ICP measurement in control (untreated), mouse IgG (control with beads), and Shh inhibitor-treated adult (P120) Sprague-Dawley rat penes after electrical stimulation of the CN for 40 sec. Seven days after treatment, the presence of the bead vehicle has not altered the maximum pressure attained after stimulation. However, the presence of Shh inhibitor dramatically decreased the ICP.

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induce and maintain sinusoid structure is not yet clear; however, clues to its function were obtained after treatment with exogenous Shh peptide, which disrupted the balance of proliferation to favor terminal differentiation into endothelial cells. Shh is normally localized in the smooth muscle of the corporal sinusoids (Fig. 3) adjacent to the endothelium. Shh may exert its inuence directly on the endothelium of the sinusoids or through induction of targets in the adjacent endothelium (BMP-4). Other members of the Shh cascade are required for establishing normal penile morphology. This requirement was rst suspected when we disrupted Shh signaling, resulting in dramatic and consistent changes in expression of Shh targets (most notably of BMP-4). The role of BMP-4 in penile differentiation was conrmed by treatment of the corpora cavernosa with recombinant BMP-4 protein or a BMP-4 antagonist (Noggin). BMP-4 protein treatment resulted in corpora cavernosal sinusoids that were large but otherwise normal (lined by endothelium and containing blood cells visible within the interior). The enlarged sinusoids were unlikely to be an artifact of the sectioning process because a normal endothelial lining was visible inside the enlarged cavernae. Likewise, morphological changes could not be attributed to the presence of the bead vehicle because control corpora cavernosa appeared normal. Treatment with Noggin resulted in the absence of sinusoids and increased endothelial tissue surrounding the beads. This result was very similar to that obtained after Shh protein administration and is consistent with elevated Shh expression surrounding the Noggin-treated beads (Fig. 11). Our experimental ndings indicate that BMP-4 plays a crucial role in establishing normal sinusoid morphology in the penile corpora cavernosa and that at least one other member of the Shh cascade is essential for postnatal morphogenesis of the penis.

Clues to how the Shh cascade regulates sinusoid differentiation were gleaned when we disrupted Shh cascade function. Disruption of either Shh or BMP-4 signaling signicantly increased expression of the other (inverse relationship). Many researchers have suggested that Shh and BMP-4 expression are related, but the exact nature of this relationship remains controversial. For example, Shh-producing cells grafted to the neural tube inhibited BMP-4 expression during neural tube development [36], and inhibition of BMP-4 expression by Shh was also observed in somite patterning [37]. In other studies, BMP-4 repressed Shh expression in dental epithelium [38] and during hair growth induction [39]. The relationship between Shh and BMP-4 clearly is complex, and both BMP-4 and Shh proteins have the ability to negatively regulate each others transcription [16]. The process of penile differentiation also is complex and most likely involves other factors that have yet to be determined. In our studies of the penis, Shh and BMP-4 were expressed in adjacent layers of the sinusoid, they negatively regulated each others expression, and they had different effects on sinusoid morphogenesis. In addition, both Shh and BMP-4 were able to induce Ptc expression and to increase endothelial differentiation. The results presented here dene a functional role for Shh cascade members in penile postnatal morphogenesis and suggest some of the regulatory mechanisms involved in this process. Shh protein localization in neural tissue was unique among the developmental genes assayed. Contrary to observations made with Shh protein, little Shh RNA was observed in the nerves of the dorsal nerve bundle; however, Shh RNA was abundant in the nerve cell body, the pelvic ganglia (data not shown). Because RNA synthesis is not possible in the nerve axon, it is likely that little Shh RNA travels down the nerve from the cell body to reside in the

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dorsal nerve bundle. Shh protein is not restricted in this manner. It is made in the pelvic ganglia but can easily travel down the axons to reside in the nerves of the dorsal nerve bundle. A potential connection between Shh signaling in the nerves and its expression in the corpora cavernosal sinusoids has been examined using rats with experimentally induced CN injury. Severing the nerves bilaterally resulted in decreased Shh protein in the smooth muscle of the corpora cavernosal sinusoids and extensive morphological changes, including altered expression of endothelial and smooth muscle markers [21], increased apoptosis [40], and ED (Fig. 12). The Shh cascade acts to establish normal penile morphology. Nerve injury disrupts the Shh cascade and corpora cavernosal homeostasis such that morphological changes in sinusoid structure ensue. ED can result when Shh signaling is abrogated. This is the rst study demonstrating the signicant role that Shh plays in establishing and maintaining penile homeostasis and how this relates to erectile function. Shh, BMP-4, Ptc, and Hoxa-10 form part of a regulatory cascade that is essential for postnatal morphogenesis and differentiation of the penis. The function of Shh and BMP4 is to establish and maintain corporal sinusoids. The data suggest that Ptc and Hoxa-10 are also important in penile morphogenesis. The continuing function of Shh and of the targets of its signaling in maintaining penile homeostasis in the adult is important because disruption of Shh signaling affects erectile function. Thus, these observations have potential application to disease states that impact erectile function.
ACKNOWLEDGMENTS
The authors thank Andrew McMahon, Matthew Scott, and Brigid Hogan for supplying Shh, Ptc, and BMP-4 constructs, respectively, to synthesize riboprobes, Alfred Rademaker for assistance with statistical analysis, Michael Pins for help in differentiating penile morphology, and Philip Hockberger for aid in manuscript preparation.

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