Author's Accepted Manuscript Canalization of The Urethral Plate Precedes Fusion of The Urethral Folds During Male Penile Urethral Development: The Double Zipper Hypothesis Yi Li, Adriane Sinclair, Mei Cao, Joel Shen, Shweta Choudhry, Gerald Cunha, Laurence Baskin
PII: DOI: Reference:
S0022-5347(14)04589-3 10.1016/j.juro.2014.09.108 JURO 11854
To appear in: The Journal of Urology Accepted Date: 19 September 2014 Please cite this article as: Li Y, Sinclair A, Cao M, Shen J, Choudhry S, Cunha G, Baskin L, Canalization of The Urethral Plate Precedes Fusion of The Urethral Folds During Male Penile Urethral Development: The Double Zipper Hypothesis, The Journal of Urology® (2014), doi: 10.1016/j.juro.2014.09.108. DISCLAIMER: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our subscribers we are providing this early version of the article. The paper will be copy edited and typeset, and proof will be reviewed before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to The Journal pertain.
Embargo Policy All article content is under embargo until uncorrected proof of the article becomes available online. We will provide journalists and editors with full-text copies of the articles in question prior to the embargo date so that stories can be adequately researched and written. The standard embargo time is 12:01 AM ET on that date. Questions regarding embargo should be directed to [email protected].
ACCEPTED MANUSCRIPT
Revised 9182014
Canalization of The Urethral Plate Precedes Fusion of The Urethral
Hypothesis
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Folds During Male Penile Urethral Development: The Double Zipper
Yi Li, Adriane Sinclair, Mei Cao, Joel Shen, Shweta Choudhry, Gerald Cunha, and
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Laurence Baskin*
Division of Pediatric Urology Correspondence:
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Laurence S. Baskin, MD, Frank Hinman, Jr., MD, Distinguished Professorship in Pediatric Urology Chief Pediatric Urology Professor of Urology and Pediatrics 400 Parnassus Ave. San Francisco, CA 94143 415-476-1611 Fax 415-476-8849 [email protected]
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UCSF Benioff Children’s Hospital
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Abbreviations:
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Supported by NIH grant DK058105
OPT = Optical Projection Tomography UP = Urethral Plate
UG = Urethral Groove
UM = Urethral Meatus EB = Erectile Body Canal = Canalization
Key Words: Urethra, Development, Fusion, Canalization, and Hypospadias
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Abstract Purpose: We describe the “double zipper”, mechanism of human male urethral formation
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where the distal zipper opens the urethral groove through canalization of the urethral plate, and a second closing zipper follows behind and closes the urethral groove to form the tubular urethra.
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Methods: Anonymous human fetal genital specimens were acquired and gender determined by PCR of the Y chromosome. Specimens were processed for Optical
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Projection Tomography (OPT), stained with E-cadherin, Ki67, Caspase 3 and imaged.
Results: Eight developing male fetal specimens from 6.5 weeks gestation to 16.5 weeks gestation were analyzed by OPT, and an additional 5 specimens by serial sections. Length of the phalluses ranged from 1.3mm to 3.7mm. The urethral plate canalized into a groove with two epithelial edges that subsequently fused. Ki67 staining was localized to
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the dorsal aspect of the urethral plate. In contrast, Caspase 3 staining was not observed. The process was completely over a 10-week period. Discussion: The human male urethra appears to form by two mechanisms: (1) An initial
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“opening zipper” that facilitates distal canalization of the solid urethral plate to form the urethral groove, which involves a high rate of epithelial proliferation (apoptosis not observed) and (2) A “closing zipper” facilitating fusion of the two epithelial surfaces of
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the urethral groove, and thus extending the penile urethra distally.
A better
understanding of the molecular mechanisms of these processes is critical to understanding the mechanism of abnormal urethral development such as hypospadias.
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Introduction
Understanding normal human urethral development is the first step is unraveling
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abnormal urethral development for which the most common abnormality is hypospadias. Hypospadias is defined by an abnormality in location of the urethral meatus, impaired
differentiation of the corpus spongiosum and failure of closure of the ventral foreskin (1).
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The urethral defects result from failure of fusion of two epithelial surfaces of the urethral
groove, which forms via canalization of the solid urethra plate (2, 3). Hypospadias
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results from events that may involve (1) formation of the urethral plate, (2) failure of canalization of the urethral plate and thus failure of formation of the urethral groove, (3) failure of medial growth of the urethral fold (the lateral edges of the urethral groove), or (4) failure of midline fusion of the urethral folds (4, 5).
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Previous studies examining urethral development have relied upon gross examination (6) serial sections (7) and 3D reconstruction of images of serial sections (5). With the advent of optical projection tomography (OPT), whole mount 3D imaging of the
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developing human urethra can now be realized (8). Herein, we describe the “double
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zipper”, hypothesis of urethral formation where the “opening zipper” facilitates formation of the urethral groove distally through canalization of the urethral plate, and a “closing zipper” follows behind and closes the urethral groove to form the tubular urethra. Midline epithelial proliferation within the urethral plate is a key feature of development of the urethral groove, while apoptosis does not appear to be involved in canalization of the urethral plate.
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Methods The study was performed after approval from the Committee on Human Research
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at UCSF. Anonymous human fetal genital specimens from elective terminations were acquired and gestational age estimated by heel-toe length (9, 10). Sex of the fetuses was
determined by two methods: (1) visual inspection of internal genitalia (gonads, Wolffian
and Mullerian ducts) using a dissecting microscope and (2) PCR for X and Y-
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chromosomal genes (11). For the later, DNA was isolated from tissue sample of the fetus
premiers of the X and Y chromosome. Optical Projection Tomography
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using QIAamp DNA Mini Kit (Qiagen). Isolated DNA was then amplified for specific
Optical Projection Tomography (OPT) is a technique for volumetric
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visualization of transparent or slightly opaque objects on the cellular level up to small organisms (8). The principle of OPT is similar to computed tomography except for the use of visible light instead of X-rays.
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Dissected human fetal genital were fixed in formalin overnight, stored in 100% methanol and prepared for OPT (8). Samples were immunostained with anti-Ecadherin
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(diluted 1:100, BD Transduction Laboratories) using the secondary antibody, (goat antimouse diluted 1:100, Alexa Fluor 488nm). Images of the specimens were captured using Bioptonics OPT Scanner 3001M
(Bioptonics) as described previously (8). The raw data (400 projected images) from each of the two channels were reconstructed into a pair of 3D voxel data sets, using in-house
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reconstruction software. 3D OPT reconstructions were then loaded into Perkin Elmer Velocity software for visualization and analysis.
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OPT samples were isolated from the agar blocks using the standard Bioptonics OPT protocol (Bioptonics). Once isolated, the specimens were formalin fixed, paraffin embedded and serially sectioned at 7 microns. Immunohistochemistry was carried out as
previously described (5) utilizing the antibodies to Ki67 (1:100, Leica) to detect cellular
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proliferation and Caspase 3 (1:200, Cell Signaling Technology) to detect apoptosis.
Results
Optical Projection Tomography -- Gross Visualization and Morphometrics Thirteen human male fetal external genitalia specimens were evaluated following
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confirmation of male sex by PCR. Under dissecting microscopy the entire fetal specimen appeared normal. Eight developing male fetal specimens from 6.5 weeks gestation to 16.5 weeks gestation were processed for OPT and the remaining 5 male specimens
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exclusively analyzed by histology. In the OPT specimens, E-cadherin localized to the
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entire epidermis and urethral epithelial cells including the areas of canalization and fusion. Length of phalluses ranged from 1.3mm to 3.7mm, respectively (Figure 1). The urethral meatus (Figure 1, green arrows) progressed from proximal to distal as gestational age increased. Note the progression of the urethral meatus (green arrows) (defined as the most proximal location where the urethra opened into the amniotic cavity) from the scrotal folds at 6.5 weeks to a terminal position on the glans at 16.5 weeks. The open urethral groove (defined by an open groove with two epithelial edges greater than 200
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microns apart) was best seen from 9.5 to 13 weeks (Figure 1, red arrows) with clear progression of a proximal to distal fusion of the edges of urethral groove to form the tubular penile urethra (Figure 1, yellow arrows). At 13 weeks the urethra groove was
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located within the glans penis with the tubular urethra completely formed within the shaft of the penis. The epithelial tag (Figure 1, turquoise arrows) appeared at ~ 9 weeks and
disappeared by 16.5 weeks. The entire process of urethral formation was completed over
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a ten-week period.
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Indifferent Stage of Penile Development (6.5-7.5 week specimens)
In the youngest specimen estimated at 6.5 weeks (Figure 2) the urethral plate was not evident at the distal tip of the genital tubercle (1A), but was for the most part solid (2B) with evidence of canalization in the penile shaft (2C and D). The urethral meatus
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was located at the scrotal folds (2E) with the tubular urethra just proximal (F). Staining for the proliferation marker Ki67 was localized to the nuclei predominantly within the dorsal aspect of the urethral plate and in the mesenchymal condensation representing the
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forming erectile body (2G-J). Staining for apoptosis using Caspase 3 was negative (2K). A similar result was found in the 7.5 week specimen (Figure 3) with absence of the
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urethral plate in the distal most aspect of the genital tubercle (3A) and proximal canalization of the solid urethral plate (3B, C, D & E, black arrow). Cellular proliferation based on Ki67 staining was concentrated on the dorsal aspect of the urethral plate and in the mesenchymal condensation representing the forming erectile body (3G-H). Caspase 3 was not detected in any tissues (not illustrated). Early Stage of Penile Development (9.5 and 10.5 week specimens)
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The 9.5-week penis exhibited increased growth compared to the two specimens in the indifferent stage (Figure 4). The urethral groove was now well developed extending from the scrotal folds to the proximal aspect of the glans (coronal sulcus) comprising the
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entire length of the penile shaft. The width of the urethral groove was also quite large
compared to the solid urethra plate. The solid urethral plate now located exclusively within the glans exhibited ventral canalization, but remained solid dorsally (4B, F and G).
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Cellular proliferation (Ki67) was localized to the dorsal aspect of the urethral plate (4F,
G). Residual urethral plate, present in the midline of the urethral groove, also remained
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highly proliferative (4H-I).
The notable feature of the 10.5-week penis (Figure 5) is the “distal movement” of the wide urethral groove, whose proximal boundary was located at midshaft of the penis and whose distal boundary had extended into the glans. Between these two points a
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broadly opened urethral groove was evident. Proximal closure of the urethral groove created a tubular penile urethra in the penile shaft (Fig. 5E-F). Proliferation (Ki67) was localized to the dorsal aspect of the solid urethral plate within the glans (5H, I, L, M), in
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the residual urethral plate in the midline of the urethral groove (5J & N) and in the
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residual urethral plate in the dorsal midline of tubular urethra (5K & O). Within the open urethral groove there was a consistent fall off in proliferative activity from medial to lateral (the edges of the urethral folds) (5J & N). Caspase -3 activity was not observed in either specimen (data not shown). Later Stages of Penile Development (12, 13, 15 and 16.5 week specimens) At 12 weeks the proximal aspect of the urethral groove had fused and advanced further distally along the penile shaft, while the distal aspect of the wide urethral groove
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has advanced into the mid glans (Figure 6). The tubular urethra (6E) has advanced to mid shaft of the penis. The solid urethral plate has now evident in the distal aspect of the glans (6A) with associated canalization of the urethral plate at mid glans (6B). Proliferation
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(Ki67) within the urethral plate was again localized dorsally (6 G-L). In the region of the open urethral groove proliferation was highest medially and decreased laterally to
essentially zero in the edges of the urethral folds (6H, I, K, L). At 13 weeks the urethral
was still visible (7A) on the glans.
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groove was shorter in length and entirely within the glans (Figure 7). The epithelial tag The tubular urethra was completely formed
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throughout the entire penile shaft. Within the glans cellular proliferation (Ki67) remained active and localized to the dorsal aspect of the urethral plate (7G & J). More proximally within the urethral groove (7H & K) and urethral meatus (7L) the dorsal aspect of the residual urethral plate remained active for Ki67 staining but qualitatively less than in the
(Figure 8).
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solid urethral plate distally (7J). At 15 weeks the penis is almost completely formed The urethral groove is shorter in length compared to earlier stages of
development. The location of the groove is in the distal glans close to the final terminal
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position of the normal urethral meatus. Fusion of the ventral foreskin is just about complete (8G, H, & K). Where urethral formation is still active in the distal glans,
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cellular proliferation remains active in the dorsal aspect of the urethral plate (8J). In the completely formed tubular urethra, proliferation is equally distributed around the entire urethral epithelium (8K and L). Caspase 3 was rarely detected in these specimens (data not shown). In the 16.5 week specimen (Figure 1) the urethra has reached its normal position on the glans, the foreskin has fused ventrally and the epithelial tag is no longer present.
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Discussion
previous observations (3, 6, 7).
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Our observations on the developing human fetal urethra are in agreement with OPT confirms a solid urethral plate that canalizes
forming the urethral groove that progressively advances and fuses to form the tubular
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penile urethra. The process starts proximally at the scrotal folds and progresses along the penile shaft to the glans to form the terminal urethral meatus. There is no evidence of
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ectodermal intrusion of epidermal cells (2) that meet the solid and/or canalized urethral plate or urethral groove consistent with the endodermal theory of urethral development (3) (Figures 1-8).
The mechanism of penile urethral formation appears to simulate a “double zipper”.
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The first or opening zipper involves the canalization process within the urethral plate to create the urethral groove. This process is seen in the OPT images initially starting at the scrotal fold/ penile junction and is visualized in the cross sectional OPT and histological
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sections (Figure 2E, H and K, & Figure 3C, G and H). During these early stages of development at 6.5 and 7.5 weeks gestation there is already evidence of canalization of
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the urethral plate (Figure 2C, G and I) consistent with the subsequent development of the urethral groove that develops between 7.5 weeks and 9.5 weeks gestation (Figure 4). The “opening zipper” starting at the urethral meatus opens the urethral groove through canalization of the urethral plate and lateral expansion of the urethral groove, a process that may be facilitated by exceptionally high proliferative activity in the dorsal aspect of the urethral plate and the residual urethral plate in the midline of the urethral groove. Between 9.5 weeks and 15 weeks gestation the “opening zipper” constitutes the distal
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leading edge of the urethral groove, which moves towards and then into the glans to its final terminal position. The initial zipper leaves behind a wide urethral groove with lateral epithelial edges (urethral folds) that ultimately fuse together to form the tubular urethral
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as the “closing zipper” fuses the edges of the urethral groove and distally extends the tubular urethra. Between 9.5 weeks and 10.5 weeks the ‘closing zipper” extends the
tubular urethra well into the penile shaft with the midline fusion of the epithelial edges of
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the urethral groove (the urethral folds) well visualized in the OPT images of developing human penis (Figure 5D, J, N, K and O, & Figure 6E, F, I and L). The process continues
foreskin ventrally (Figure 8 G-L).
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through the glans penis (Figure 6-8) in similar fashion with ultimate fusion of the
The mechanism of action of the two zippers remains unknown.
Our data
demonstrate that cellular proliferation based on Ki67 immunostaining is an important
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component of formation and lateral expansion of the urethral groove (see Ki67 immunostaining in Figures 2-8). Pechriggi et. al. also observed Ki67 staining in the developing human male urethra (12). Proliferation was consistently highest in the dorsal
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aspect of the urethral plate as well as in the residual urethral plate in the midline of the
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urethral groove with a substantial fall off in proliferative activity laterally within the epithelium of the urethral groove. This pattern is suggestive of a centrally placed proliferative zone generating post-mitotic epithelial cells that migrate laterally to expand the width of the urethral groove in a process somewhat akin to that occurring in intestinal crypts/villi. In the opening zipper, which extends the urethral groove distally, cellular proliferation appears in conjunction with epithelial de-adhesion events that facilitate the
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canalization of the urethral plate. The process of canalization of the urethral plate does not appear to involve apoptosis as Caspase 3 positive cells were rarely observed throughout penile development (Figure 2K). The proximal or closing zipper in turn
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requires adhesion of the epithelial surfaces of the urethral folds to form the tubular penile
urethra. Utilizing a mouse model of urethral seam fusion we have shown that the distal aspect of the mouse urethra and especially the urethral meatus forms by fusion of edges
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of the urethral folds, resulting in a midline epithelial seam (5, 13).
OPT is a new innovative technique to selective stain and visualize the developing Our analysis confirms and refines the elegant work of Altemus and
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penile urethra.
Hutchins who described the development of the penile urethral using gross examination, serial sections and artistic rendering of the human fetal specimens (6). We and others have performed molecular analysis of human urethral development showing differential
14).
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expression of cytokeratins, uroplakins, E-Cadherin and mesenchymal markers (3, 4, 12, Mammalian animal studies have also defined critical genes such as Shh, FGF 10
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and FGFr2-111b that are required for urethral development (15, 16).
Our study has a number of limitations. We have made the assumption based on
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microscopic examination of the fetal tissue that the specimens we are analyzing are normal.
We are also limited by the relatively small sample of specimens available for
examination.
In summary we have defined the ontogeny of human male urethral development by OPT. We have documented two mechanisms for further study: (1) The opening
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zipper that facilitates distal canalization of the solid urethral plate to form the urethral groove, which involves a high rate of epithelial proliferation dorsally within the urethral plate. The process of canalization of the urethral plate apparently does not involve
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apoptosis. (2) The closing zipper that facilitates fusion of the two epithelial surfaces of
the urethral groove and thus extending the penile urethra distally. A better understanding of the molecular mechanisms of these processes is critical to understanding the
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mechanism of abnormal urethral development such as hypospadias.
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References:
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1. Baskin LS, Ebbers MB. Hypospadias: anatomy, etiology, and technique. J Pediatr Surg. 2006 Mar;41(3):463-72. PubMed PMID: 16516617. 2. Glenister TW. A correlation of the normal and abnormal development of the penile urethra and of the infra-umbilical abdominal wall. British journal of urology. 1958 Jun;30(2):117-26. PubMed PMID: 13546590. 3. Kurzrock EA, Baskin LS, Cunha GR. Ontogeny of the male urethra: theory of endodermal differentiation. Differentiation; research in biological diversity. 1999 Jan;64(2):115-22. PubMed PMID: 10234808. 4. Baskin LS. Hypospadias and urethral development. The Journal of urology. 2000;163(3):951-6. PubMed PMID: 0010688029. 5. Baskin LS, Erol A, Jegatheesan P, Li Y, Liu W, Cunha GR. Urethral seam formation and hypospadias. Cell and tissue research. 2001 Sep;305(3):379-87. PubMed PMID: 11572091. 6. Altemus AR, Hutchins GM. Development of the human anterior urethra. The Journal of urology. 1991 Oct;146(4):1085-93. PubMed PMID: 1895427. 7. van der Werff JF, Nievelstein RA, Brands E, Luijsterburg AJ, Vermeij-Keers C. Normal development of the male anterior urethra. Teratology. 2000 Mar;61(3):172-83. PubMed PMID: 10661906. 8. Sharpe J, Ahlgren U, Perry P, Hill B, Ross A, Hecksher-Sorensen J, et al. Optical projection tomography as a tool for 3D microscopy and gene expression studies. Science. 2002 Apr 19;296(5567):541-5. PubMed PMID: 11964482. 9. Drey EA, Kang MS, McFarland W, Darney PD. Improving the accuracy of fetal foot length to confirm gestational duration. Obstetrics and gynecology. 2005 Apr;105(4):773-8. PubMed PMID: 15802404. 10. Mercer BM, Sklar S, Shariatmadar A, Gillieson MS, D'Alton ME. Fetal foot length as a predictor of gestational age. American journal of obstetrics and gynecology. 1987 Feb;156(2):350-5. PubMed PMID: 3548369. 11. Cui KH, Warnes GM, Jeffrey R, Matthews CD. Sex determination of preimplantation embryos by human testis-determining-gene amplification. Lancet. 1994 Jan 8;343(8889):79-82. PubMed PMID: 7903778. 12. Pechriggl EJ, Bitsche M, Blumer MJ, Fritsch H. The male urethra: spatiotemporal distribution of molecular markers during early development. Annals of anatomy = Anatomischer Anzeiger : official organ of the Anatomische Gesellschaft. 2013 May;195(3):260-71. PubMed PMID: 23433589. 13. Kim KS, Liu W, Cunha GR, Russell DW, Huang H, Shapiro E, et al. Expression of the androgen receptor and 5 alpha-reductase type 2 in the developing human fetal penis and urethra. Cell and tissue research. 2002 Feb;307(2):145-53. PubMed PMID: 11845321. 14. Baskin LS, Erol A, Li YW, Cunha GR. Anatomical studies of hypospadias. The Journal of urology. 1998;160(3 Pt 2):1108-015. PubMed PMID: 0009719287. 15. Petiot A, Perriton CL, Dickson C, Cohn MJ. Development of the mammalian urethra is controlled by Fgfr2-IIIb. Development. 2005 May;132(10):2441-50. PubMed PMID: 15843416. 16. Haraguchi R, Suzuki K, Murakami R, Sakai M, Kamikawa M, Kengaku M, et al. Molecular analysis of external genitalia formation: the role of fibroblast growth factor (Fgf) genes during genital tubercle formation. Development. 2000 Jun;127(11):2471-9. PubMed PMID: 10804187.
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Figure Legends Figure 1
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Optical Projection Tomography of male urethral development from 6.5 weeks to 16.5 weeks of gestation. Note the progression of the urethral meatus (green arrows) from the
scrotal folds at 6.5 weeks to a terminal position on the glans at 16.5 weeks. The wide
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open urethral groove (red arrows) is best seen from 9.5 to 13 weeks with clear progression of a proximal to distal fusion of the edges of urethral groove to form the
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tubular urethra (yellow arrows). At 13 weeks the urethra groove is within the glans penis with the tubular urethra completely formed within the shaft of the penis consistent with the endodermal theory of urethral development.
No evidence of ectodermal
Figure 2
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intrusion is noted in any of the specimens.
Optical Projection Tomography of a 6.5-week penis at the indifferent stage of
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development. Histologic sections A –F stained with E-Cadherin correspond to the
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horizontal lines in the gross OPT specimen. I and J are corresponding high power images of G and H showing extensive proliferation (Ki67) along the urethral plate (UP), urethral meatus (UM) and areas of urethral plate canalization (canal) Opening Zipper). Note that Caspase 3 (K) was not expressed in adjacent images to E supporting the role of cellular proliferation over apoptosis as a means of urethral plate/groove development (see text for details).
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Figure 3 Optical Projection Tomography of a 7.5-week penis at the indifferent stage of development. Note the canalization of the urethral plate and cellular proliferation (Ki67)
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localized to the dorsal urethral plate (opening zipper) (D, E, G & H) (see text for details).
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Figure 4
Optical Projection Tomography of a 9.5-week penis in early stages of development. Note
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that the urethral groove is now well developed comprising the length the penile shaft (red arrows) and the canalization of the urethral plate and cellular proliferation (Ki67)
Figure 5
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localized to the dorsal urethral plate (opening zipper) (B, F-I) (see text for details).
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Optical Projection Tomography of a 10.5-week penis in early stages of development with
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the epithelium stained for E-Cadherin. Note the continued proliferation (Ki67) in the urethral plate (opening zipper) (H-J, L-N) and fusion of the urethral groove (closing zipper) (red arrows) (K and O) (see text for details).
Figure 6
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Optical Projection Tomography of a 12-week penis in the mid stages of development. Note that the proximal urethral groove has fused (closing zipper) and advanced to the
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into the glans (red arrows) (see text for details).
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distal aspect of the shaft of penis and the distal groove (opening zipper) has advanced
Figure 7
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Optical Projection Tomography of a 13-week penis in the late stages of development. The urethral groove is contained within the proximal glans and is smaller in length than
Figure 8
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earlier specimens (red arrows) (see text for details).
Optical Projection Tomography of a 15-week penis in the late stages of development.
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Note urethral groove is contained within the distal glans and is smaller in length than earlier specimens (red arrows and A) and that the prepuce has fused ventrally (I) (see text
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for details).
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PII: DOI: Reference:
S0022-5347(14)04589-3 10.1016/j.juro.2014.09.108 JURO 11854
To appear in: The Journal of Urology Accepted Date: 19 September 2014 Please cite this article as: Li Y, Sinclair A, Cao M, Shen J, Choudhry S, Cunha G, Baskin L, Canalization of The Urethral Plate Precedes Fusion of The Urethral Folds During Male Penile Urethral Development: The Double Zipper Hypothesis, The Journal of Urology® (2014), doi: 10.1016/j.juro.2014.09.108. DISCLAIMER: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our subscribers we are providing this early version of the article. The paper will be copy edited and typeset, and proof will be reviewed before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to The Journal pertain.
Embargo Policy All article content is under embargo until uncorrected proof of the article becomes available online. We will provide journalists and editors with full-text copies of the articles in question prior to the embargo date so that stories can be adequately researched and written. The standard embargo time is 12:01 AM ET on that date. Questions regarding embargo should be directed to [email protected].
ACCEPTED MANUSCRIPT
Revised 9182014
Canalization of The Urethral Plate Precedes Fusion of The Urethral
Hypothesis
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Folds During Male Penile Urethral Development: The Double Zipper
Yi Li, Adriane Sinclair, Mei Cao, Joel Shen, Shweta Choudhry, Gerald Cunha, and
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Laurence Baskin*
Division of Pediatric Urology Correspondence:
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Laurence S. Baskin, MD, Frank Hinman, Jr., MD, Distinguished Professorship in Pediatric Urology Chief Pediatric Urology Professor of Urology and Pediatrics 400 Parnassus Ave. San Francisco, CA 94143 415-476-1611 Fax 415-476-8849 [email protected]
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UCSF Benioff Children’s Hospital
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Abbreviations:
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Supported by NIH grant DK058105
OPT = Optical Projection Tomography UP = Urethral Plate
UG = Urethral Groove
UM = Urethral Meatus EB = Erectile Body Canal = Canalization
Key Words: Urethra, Development, Fusion, Canalization, and Hypospadias
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Abstract Purpose: We describe the “double zipper”, mechanism of human male urethral formation
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where the distal zipper opens the urethral groove through canalization of the urethral plate, and a second closing zipper follows behind and closes the urethral groove to form the tubular urethra.
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Methods: Anonymous human fetal genital specimens were acquired and gender determined by PCR of the Y chromosome. Specimens were processed for Optical
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Projection Tomography (OPT), stained with E-cadherin, Ki67, Caspase 3 and imaged.
Results: Eight developing male fetal specimens from 6.5 weeks gestation to 16.5 weeks gestation were analyzed by OPT, and an additional 5 specimens by serial sections. Length of the phalluses ranged from 1.3mm to 3.7mm. The urethral plate canalized into a groove with two epithelial edges that subsequently fused. Ki67 staining was localized to
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the dorsal aspect of the urethral plate. In contrast, Caspase 3 staining was not observed. The process was completely over a 10-week period. Discussion: The human male urethra appears to form by two mechanisms: (1) An initial
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“opening zipper” that facilitates distal canalization of the solid urethral plate to form the urethral groove, which involves a high rate of epithelial proliferation (apoptosis not observed) and (2) A “closing zipper” facilitating fusion of the two epithelial surfaces of
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the urethral groove, and thus extending the penile urethra distally.
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understanding of the molecular mechanisms of these processes is critical to understanding the mechanism of abnormal urethral development such as hypospadias.
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Introduction
Understanding normal human urethral development is the first step is unraveling
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abnormal urethral development for which the most common abnormality is hypospadias. Hypospadias is defined by an abnormality in location of the urethral meatus, impaired
differentiation of the corpus spongiosum and failure of closure of the ventral foreskin (1).
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The urethral defects result from failure of fusion of two epithelial surfaces of the urethral
groove, which forms via canalization of the solid urethra plate (2, 3). Hypospadias
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results from events that may involve (1) formation of the urethral plate, (2) failure of canalization of the urethral plate and thus failure of formation of the urethral groove, (3) failure of medial growth of the urethral fold (the lateral edges of the urethral groove), or (4) failure of midline fusion of the urethral folds (4, 5).
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Previous studies examining urethral development have relied upon gross examination (6) serial sections (7) and 3D reconstruction of images of serial sections (5). With the advent of optical projection tomography (OPT), whole mount 3D imaging of the
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developing human urethra can now be realized (8). Herein, we describe the “double
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zipper”, hypothesis of urethral formation where the “opening zipper” facilitates formation of the urethral groove distally through canalization of the urethral plate, and a “closing zipper” follows behind and closes the urethral groove to form the tubular urethra. Midline epithelial proliferation within the urethral plate is a key feature of development of the urethral groove, while apoptosis does not appear to be involved in canalization of the urethral plate.
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Methods The study was performed after approval from the Committee on Human Research
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at UCSF. Anonymous human fetal genital specimens from elective terminations were acquired and gestational age estimated by heel-toe length (9, 10). Sex of the fetuses was
determined by two methods: (1) visual inspection of internal genitalia (gonads, Wolffian
and Mullerian ducts) using a dissecting microscope and (2) PCR for X and Y-
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chromosomal genes (11). For the later, DNA was isolated from tissue sample of the fetus
premiers of the X and Y chromosome. Optical Projection Tomography
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using QIAamp DNA Mini Kit (Qiagen). Isolated DNA was then amplified for specific
Optical Projection Tomography (OPT) is a technique for volumetric
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visualization of transparent or slightly opaque objects on the cellular level up to small organisms (8). The principle of OPT is similar to computed tomography except for the use of visible light instead of X-rays.
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Dissected human fetal genital were fixed in formalin overnight, stored in 100% methanol and prepared for OPT (8). Samples were immunostained with anti-Ecadherin
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(diluted 1:100, BD Transduction Laboratories) using the secondary antibody, (goat antimouse diluted 1:100, Alexa Fluor 488nm). Images of the specimens were captured using Bioptonics OPT Scanner 3001M
(Bioptonics) as described previously (8). The raw data (400 projected images) from each of the two channels were reconstructed into a pair of 3D voxel data sets, using in-house
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reconstruction software. 3D OPT reconstructions were then loaded into Perkin Elmer Velocity software for visualization and analysis.
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OPT samples were isolated from the agar blocks using the standard Bioptonics OPT protocol (Bioptonics). Once isolated, the specimens were formalin fixed, paraffin embedded and serially sectioned at 7 microns. Immunohistochemistry was carried out as
previously described (5) utilizing the antibodies to Ki67 (1:100, Leica) to detect cellular
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proliferation and Caspase 3 (1:200, Cell Signaling Technology) to detect apoptosis.
Results
Optical Projection Tomography -- Gross Visualization and Morphometrics Thirteen human male fetal external genitalia specimens were evaluated following
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confirmation of male sex by PCR. Under dissecting microscopy the entire fetal specimen appeared normal. Eight developing male fetal specimens from 6.5 weeks gestation to 16.5 weeks gestation were processed for OPT and the remaining 5 male specimens
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exclusively analyzed by histology. In the OPT specimens, E-cadherin localized to the
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entire epidermis and urethral epithelial cells including the areas of canalization and fusion. Length of phalluses ranged from 1.3mm to 3.7mm, respectively (Figure 1). The urethral meatus (Figure 1, green arrows) progressed from proximal to distal as gestational age increased. Note the progression of the urethral meatus (green arrows) (defined as the most proximal location where the urethra opened into the amniotic cavity) from the scrotal folds at 6.5 weeks to a terminal position on the glans at 16.5 weeks. The open urethral groove (defined by an open groove with two epithelial edges greater than 200
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microns apart) was best seen from 9.5 to 13 weeks (Figure 1, red arrows) with clear progression of a proximal to distal fusion of the edges of urethral groove to form the tubular penile urethra (Figure 1, yellow arrows). At 13 weeks the urethra groove was
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located within the glans penis with the tubular urethra completely formed within the shaft of the penis. The epithelial tag (Figure 1, turquoise arrows) appeared at ~ 9 weeks and
disappeared by 16.5 weeks. The entire process of urethral formation was completed over
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a ten-week period.
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Indifferent Stage of Penile Development (6.5-7.5 week specimens)
In the youngest specimen estimated at 6.5 weeks (Figure 2) the urethral plate was not evident at the distal tip of the genital tubercle (1A), but was for the most part solid (2B) with evidence of canalization in the penile shaft (2C and D). The urethral meatus
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was located at the scrotal folds (2E) with the tubular urethra just proximal (F). Staining for the proliferation marker Ki67 was localized to the nuclei predominantly within the dorsal aspect of the urethral plate and in the mesenchymal condensation representing the
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forming erectile body (2G-J). Staining for apoptosis using Caspase 3 was negative (2K). A similar result was found in the 7.5 week specimen (Figure 3) with absence of the
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urethral plate in the distal most aspect of the genital tubercle (3A) and proximal canalization of the solid urethral plate (3B, C, D & E, black arrow). Cellular proliferation based on Ki67 staining was concentrated on the dorsal aspect of the urethral plate and in the mesenchymal condensation representing the forming erectile body (3G-H). Caspase 3 was not detected in any tissues (not illustrated). Early Stage of Penile Development (9.5 and 10.5 week specimens)
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The 9.5-week penis exhibited increased growth compared to the two specimens in the indifferent stage (Figure 4). The urethral groove was now well developed extending from the scrotal folds to the proximal aspect of the glans (coronal sulcus) comprising the
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entire length of the penile shaft. The width of the urethral groove was also quite large
compared to the solid urethra plate. The solid urethral plate now located exclusively within the glans exhibited ventral canalization, but remained solid dorsally (4B, F and G).
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Cellular proliferation (Ki67) was localized to the dorsal aspect of the urethral plate (4F,
G). Residual urethral plate, present in the midline of the urethral groove, also remained
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highly proliferative (4H-I).
The notable feature of the 10.5-week penis (Figure 5) is the “distal movement” of the wide urethral groove, whose proximal boundary was located at midshaft of the penis and whose distal boundary had extended into the glans. Between these two points a
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broadly opened urethral groove was evident. Proximal closure of the urethral groove created a tubular penile urethra in the penile shaft (Fig. 5E-F). Proliferation (Ki67) was localized to the dorsal aspect of the solid urethral plate within the glans (5H, I, L, M), in
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the residual urethral plate in the midline of the urethral groove (5J & N) and in the
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residual urethral plate in the dorsal midline of tubular urethra (5K & O). Within the open urethral groove there was a consistent fall off in proliferative activity from medial to lateral (the edges of the urethral folds) (5J & N). Caspase -3 activity was not observed in either specimen (data not shown). Later Stages of Penile Development (12, 13, 15 and 16.5 week specimens) At 12 weeks the proximal aspect of the urethral groove had fused and advanced further distally along the penile shaft, while the distal aspect of the wide urethral groove
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has advanced into the mid glans (Figure 6). The tubular urethra (6E) has advanced to mid shaft of the penis. The solid urethral plate has now evident in the distal aspect of the glans (6A) with associated canalization of the urethral plate at mid glans (6B). Proliferation
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(Ki67) within the urethral plate was again localized dorsally (6 G-L). In the region of the open urethral groove proliferation was highest medially and decreased laterally to
essentially zero in the edges of the urethral folds (6H, I, K, L). At 13 weeks the urethral
was still visible (7A) on the glans.
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groove was shorter in length and entirely within the glans (Figure 7). The epithelial tag The tubular urethra was completely formed
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throughout the entire penile shaft. Within the glans cellular proliferation (Ki67) remained active and localized to the dorsal aspect of the urethral plate (7G & J). More proximally within the urethral groove (7H & K) and urethral meatus (7L) the dorsal aspect of the residual urethral plate remained active for Ki67 staining but qualitatively less than in the
(Figure 8).
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solid urethral plate distally (7J). At 15 weeks the penis is almost completely formed The urethral groove is shorter in length compared to earlier stages of
development. The location of the groove is in the distal glans close to the final terminal
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position of the normal urethral meatus. Fusion of the ventral foreskin is just about complete (8G, H, & K). Where urethral formation is still active in the distal glans,
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cellular proliferation remains active in the dorsal aspect of the urethral plate (8J). In the completely formed tubular urethra, proliferation is equally distributed around the entire urethral epithelium (8K and L). Caspase 3 was rarely detected in these specimens (data not shown). In the 16.5 week specimen (Figure 1) the urethra has reached its normal position on the glans, the foreskin has fused ventrally and the epithelial tag is no longer present.
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Discussion
previous observations (3, 6, 7).
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Our observations on the developing human fetal urethra are in agreement with OPT confirms a solid urethral plate that canalizes
forming the urethral groove that progressively advances and fuses to form the tubular
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penile urethra. The process starts proximally at the scrotal folds and progresses along the penile shaft to the glans to form the terminal urethral meatus. There is no evidence of
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ectodermal intrusion of epidermal cells (2) that meet the solid and/or canalized urethral plate or urethral groove consistent with the endodermal theory of urethral development (3) (Figures 1-8).
The mechanism of penile urethral formation appears to simulate a “double zipper”.
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The first or opening zipper involves the canalization process within the urethral plate to create the urethral groove. This process is seen in the OPT images initially starting at the scrotal fold/ penile junction and is visualized in the cross sectional OPT and histological
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sections (Figure 2E, H and K, & Figure 3C, G and H). During these early stages of development at 6.5 and 7.5 weeks gestation there is already evidence of canalization of
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the urethral plate (Figure 2C, G and I) consistent with the subsequent development of the urethral groove that develops between 7.5 weeks and 9.5 weeks gestation (Figure 4). The “opening zipper” starting at the urethral meatus opens the urethral groove through canalization of the urethral plate and lateral expansion of the urethral groove, a process that may be facilitated by exceptionally high proliferative activity in the dorsal aspect of the urethral plate and the residual urethral plate in the midline of the urethral groove. Between 9.5 weeks and 15 weeks gestation the “opening zipper” constitutes the distal
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leading edge of the urethral groove, which moves towards and then into the glans to its final terminal position. The initial zipper leaves behind a wide urethral groove with lateral epithelial edges (urethral folds) that ultimately fuse together to form the tubular urethral
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as the “closing zipper” fuses the edges of the urethral groove and distally extends the tubular urethra. Between 9.5 weeks and 10.5 weeks the ‘closing zipper” extends the
tubular urethra well into the penile shaft with the midline fusion of the epithelial edges of
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the urethral groove (the urethral folds) well visualized in the OPT images of developing human penis (Figure 5D, J, N, K and O, & Figure 6E, F, I and L). The process continues
foreskin ventrally (Figure 8 G-L).
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through the glans penis (Figure 6-8) in similar fashion with ultimate fusion of the
The mechanism of action of the two zippers remains unknown.
Our data
demonstrate that cellular proliferation based on Ki67 immunostaining is an important
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component of formation and lateral expansion of the urethral groove (see Ki67 immunostaining in Figures 2-8). Pechriggi et. al. also observed Ki67 staining in the developing human male urethra (12). Proliferation was consistently highest in the dorsal
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aspect of the urethral plate as well as in the residual urethral plate in the midline of the
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urethral groove with a substantial fall off in proliferative activity laterally within the epithelium of the urethral groove. This pattern is suggestive of a centrally placed proliferative zone generating post-mitotic epithelial cells that migrate laterally to expand the width of the urethral groove in a process somewhat akin to that occurring in intestinal crypts/villi. In the opening zipper, which extends the urethral groove distally, cellular proliferation appears in conjunction with epithelial de-adhesion events that facilitate the
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canalization of the urethral plate. The process of canalization of the urethral plate does not appear to involve apoptosis as Caspase 3 positive cells were rarely observed throughout penile development (Figure 2K). The proximal or closing zipper in turn
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requires adhesion of the epithelial surfaces of the urethral folds to form the tubular penile
urethra. Utilizing a mouse model of urethral seam fusion we have shown that the distal aspect of the mouse urethra and especially the urethral meatus forms by fusion of edges
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of the urethral folds, resulting in a midline epithelial seam (5, 13).
OPT is a new innovative technique to selective stain and visualize the developing Our analysis confirms and refines the elegant work of Altemus and
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penile urethra.
Hutchins who described the development of the penile urethral using gross examination, serial sections and artistic rendering of the human fetal specimens (6). We and others have performed molecular analysis of human urethral development showing differential
14).
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expression of cytokeratins, uroplakins, E-Cadherin and mesenchymal markers (3, 4, 12, Mammalian animal studies have also defined critical genes such as Shh, FGF 10
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and FGFr2-111b that are required for urethral development (15, 16).
Our study has a number of limitations. We have made the assumption based on
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microscopic examination of the fetal tissue that the specimens we are analyzing are normal.
We are also limited by the relatively small sample of specimens available for
examination.
In summary we have defined the ontogeny of human male urethral development by OPT. We have documented two mechanisms for further study: (1) The opening
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zipper that facilitates distal canalization of the solid urethral plate to form the urethral groove, which involves a high rate of epithelial proliferation dorsally within the urethral plate. The process of canalization of the urethral plate apparently does not involve
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apoptosis. (2) The closing zipper that facilitates fusion of the two epithelial surfaces of
the urethral groove and thus extending the penile urethra distally. A better understanding of the molecular mechanisms of these processes is critical to understanding the
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mechanism of abnormal urethral development such as hypospadias.
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References:
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1. Baskin LS, Ebbers MB. Hypospadias: anatomy, etiology, and technique. J Pediatr Surg. 2006 Mar;41(3):463-72. PubMed PMID: 16516617. 2. Glenister TW. A correlation of the normal and abnormal development of the penile urethra and of the infra-umbilical abdominal wall. British journal of urology. 1958 Jun;30(2):117-26. PubMed PMID: 13546590. 3. Kurzrock EA, Baskin LS, Cunha GR. Ontogeny of the male urethra: theory of endodermal differentiation. Differentiation; research in biological diversity. 1999 Jan;64(2):115-22. PubMed PMID: 10234808. 4. Baskin LS. Hypospadias and urethral development. The Journal of urology. 2000;163(3):951-6. PubMed PMID: 0010688029. 5. Baskin LS, Erol A, Jegatheesan P, Li Y, Liu W, Cunha GR. Urethral seam formation and hypospadias. Cell and tissue research. 2001 Sep;305(3):379-87. PubMed PMID: 11572091. 6. Altemus AR, Hutchins GM. Development of the human anterior urethra. The Journal of urology. 1991 Oct;146(4):1085-93. PubMed PMID: 1895427. 7. van der Werff JF, Nievelstein RA, Brands E, Luijsterburg AJ, Vermeij-Keers C. Normal development of the male anterior urethra. Teratology. 2000 Mar;61(3):172-83. PubMed PMID: 10661906. 8. Sharpe J, Ahlgren U, Perry P, Hill B, Ross A, Hecksher-Sorensen J, et al. Optical projection tomography as a tool for 3D microscopy and gene expression studies. Science. 2002 Apr 19;296(5567):541-5. PubMed PMID: 11964482. 9. Drey EA, Kang MS, McFarland W, Darney PD. Improving the accuracy of fetal foot length to confirm gestational duration. Obstetrics and gynecology. 2005 Apr;105(4):773-8. PubMed PMID: 15802404. 10. Mercer BM, Sklar S, Shariatmadar A, Gillieson MS, D'Alton ME. Fetal foot length as a predictor of gestational age. American journal of obstetrics and gynecology. 1987 Feb;156(2):350-5. PubMed PMID: 3548369. 11. Cui KH, Warnes GM, Jeffrey R, Matthews CD. Sex determination of preimplantation embryos by human testis-determining-gene amplification. Lancet. 1994 Jan 8;343(8889):79-82. PubMed PMID: 7903778. 12. Pechriggl EJ, Bitsche M, Blumer MJ, Fritsch H. The male urethra: spatiotemporal distribution of molecular markers during early development. Annals of anatomy = Anatomischer Anzeiger : official organ of the Anatomische Gesellschaft. 2013 May;195(3):260-71. PubMed PMID: 23433589. 13. Kim KS, Liu W, Cunha GR, Russell DW, Huang H, Shapiro E, et al. Expression of the androgen receptor and 5 alpha-reductase type 2 in the developing human fetal penis and urethra. Cell and tissue research. 2002 Feb;307(2):145-53. PubMed PMID: 11845321. 14. Baskin LS, Erol A, Li YW, Cunha GR. Anatomical studies of hypospadias. The Journal of urology. 1998;160(3 Pt 2):1108-015. PubMed PMID: 0009719287. 15. Petiot A, Perriton CL, Dickson C, Cohn MJ. Development of the mammalian urethra is controlled by Fgfr2-IIIb. Development. 2005 May;132(10):2441-50. PubMed PMID: 15843416. 16. Haraguchi R, Suzuki K, Murakami R, Sakai M, Kamikawa M, Kengaku M, et al. Molecular analysis of external genitalia formation: the role of fibroblast growth factor (Fgf) genes during genital tubercle formation. Development. 2000 Jun;127(11):2471-9. PubMed PMID: 10804187.
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Figure Legends Figure 1
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Optical Projection Tomography of male urethral development from 6.5 weeks to 16.5 weeks of gestation. Note the progression of the urethral meatus (green arrows) from the
scrotal folds at 6.5 weeks to a terminal position on the glans at 16.5 weeks. The wide
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open urethral groove (red arrows) is best seen from 9.5 to 13 weeks with clear progression of a proximal to distal fusion of the edges of urethral groove to form the
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tubular urethra (yellow arrows). At 13 weeks the urethra groove is within the glans penis with the tubular urethra completely formed within the shaft of the penis consistent with the endodermal theory of urethral development.
No evidence of ectodermal
Figure 2
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intrusion is noted in any of the specimens.
Optical Projection Tomography of a 6.5-week penis at the indifferent stage of
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development. Histologic sections A –F stained with E-Cadherin correspond to the
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horizontal lines in the gross OPT specimen. I and J are corresponding high power images of G and H showing extensive proliferation (Ki67) along the urethral plate (UP), urethral meatus (UM) and areas of urethral plate canalization (canal) Opening Zipper). Note that Caspase 3 (K) was not expressed in adjacent images to E supporting the role of cellular proliferation over apoptosis as a means of urethral plate/groove development (see text for details).
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Figure 3 Optical Projection Tomography of a 7.5-week penis at the indifferent stage of development. Note the canalization of the urethral plate and cellular proliferation (Ki67)
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localized to the dorsal urethral plate (opening zipper) (D, E, G & H) (see text for details).
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Figure 4
Optical Projection Tomography of a 9.5-week penis in early stages of development. Note
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that the urethral groove is now well developed comprising the length the penile shaft (red arrows) and the canalization of the urethral plate and cellular proliferation (Ki67)
Figure 5
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localized to the dorsal urethral plate (opening zipper) (B, F-I) (see text for details).
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Optical Projection Tomography of a 10.5-week penis in early stages of development with
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the epithelium stained for E-Cadherin. Note the continued proliferation (Ki67) in the urethral plate (opening zipper) (H-J, L-N) and fusion of the urethral groove (closing zipper) (red arrows) (K and O) (see text for details).
Figure 6
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Optical Projection Tomography of a 12-week penis in the mid stages of development. Note that the proximal urethral groove has fused (closing zipper) and advanced to the
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into the glans (red arrows) (see text for details).
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distal aspect of the shaft of penis and the distal groove (opening zipper) has advanced
Figure 7
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Optical Projection Tomography of a 13-week penis in the late stages of development. The urethral groove is contained within the proximal glans and is smaller in length than
Figure 8
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earlier specimens (red arrows) (see text for details).
Optical Projection Tomography of a 15-week penis in the late stages of development.
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Note urethral groove is contained within the distal glans and is smaller in length than earlier specimens (red arrows and A) and that the prepuce has fused ventrally (I) (see text
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for details).
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