1. Volume 196, Issue 1, Pages 270-278 (July 2016)
Fetal Rat Gubernaculum Mesenchymal Cells Adopt Myogenic and Myofibroblast-Like Phenotypes 
Alan K. Robbins, Abigail B. Mateson, Ashutosh Khandha, Joan E. Pugarelli, Thomas S. Buchanan, Robert E. Akins, Julia Spencer Barthold 
The Journal of Urology 
Volume 196, Issue 1, Pages (July 2016)
DOI: /j.juro
Copyright © 2016 American Urological Association Education and Research, Inc. Terms and Conditions

2. Figure 1
Immunostaining of E17 (A and B), E19 (C) and E21 (D) rat GCC sections for desmin (green areas). Distal GCC is shown on left side and more proximal GCC is shown on right side of each frame. Differentiated muscle layers stained brightly for desmin. Single thin peripheral muscle layer was visible at E17 with opening present at GCC tip (left side) (A). Desmin expression was much lower in central mesenchymal core but enriched at tip. More proximal image of same section shows second, inner muscle layer developing in proximal half of GCC and enrichment of desmin positive mesenchymal cells between muscle layers as apparent myogenic zone (bracket) (B). Thickening of both muscle layers was visible at E19 (C). Further development by E21 obliterated space between muscle layers (top) (D). Scale bar indicates 200 (A and B) and 400 μm (C and D).
The Journal of Urology  , DOI: ( /j.juro )
Copyright © 2016 American Urological Association Education and Research, Inc. Terms and Conditions

3. Figure 2
Immunostaining of E17 (A), E19 (B) and E21 (C) GCC sections for αSMA (red areas) and myogenin (green areas). Sections are adjacent to those in figure 3. Distal GCC is on right side and more proximal GCC is on left side of each frame. Differentiated muscle layers stained brightly for αSMA. DAPI counterstaining (Merge) shows position of inner mesenchyme relative to muscle layers. At E17 cells with nuclear myogenin staining were primarily in but occasionally between differentiated muscle layers (A). At E19 additional myogenin positive cells were visible between developing muscle layers (right side) that defined myogenic zone (B). Scale bar (C) indicates 200 μm.
The Journal of Urology  , DOI: ( /j.juro )
Copyright © 2016 American Urological Association Education and Research, Inc. Terms and Conditions

4. Figure 3
Immunostaining of E17 (A), E19 (B) and E21 (C) GCC sections for desmin (green areas) and PAX7 (red areas). Sections are adjacent to those in figure 2. Distal GCC is on right side and more proximal GCC is on left side of each frame. Differentiated muscle layers stained brightly for desmin. DAPI counterstaining (Merge) shows position of inner mesenchyme relative to muscle layers. At E17 cells with nuclear PAX7 staining were primarily within but occasionally between differentiated muscle layers (A). At E19 additional PAX7 positive cells were visible between developing muscle layers (right side) that defined myogenic zone (B). Scale bar (C) indicates 200 μm.
The Journal of Urology  , DOI: ( /j.juro )
Copyright © 2016 American Urological Association Education and Research, Inc. Terms and Conditions

5. Figure 4
Immunostaining of E19 GCC sections for αSMA (red areas) (A) and PECAM/CD31 (green areas) (B) shows strong αSMA expression in differentiated muscle (left upper corner) and fainter mesenchymal expression in GCC core (right lower corner). αSMA and PECAM co-expression in blood vessels appeared yellow (Merge). Inset, higher magnification of PECAM negative region in core is enhanced to better visualize mesenchymal αSMA. Scale bar indicates 100 μm.
The Journal of Urology  , DOI: ( /j.juro )
Copyright © 2016 American Urological Association Education and Research, Inc. Terms and Conditions

6. Figure 5
CellTracker fluorescent staining (green areas) of mesothelial layer after 5-minute exposure of E17 GCC was followed by immediate fixation at 0 hours (A). Dye uptake by mesothelial cells outside peripheral muscle layer is delineated by MyHC immunostaining (red areas). Note evidence of labeled mesothelial cell (green areas and arrows) incorporation in GCC deeper muscle and mesenchymal regions after 5-minute CellTracker exposure followed by 24-hour organ culture (B). Scale bar indicates 100 μm.
The Journal of Urology  , DOI: ( /j.juro )
Copyright © 2016 American Urological Association Education and Research, Inc. Terms and Conditions

7. Figure 6
Low (A) and high (B) power phase contrast images reveal 3-day cultured intact E17 GCC grown on poly-L-lysine/laminin coated plates and immobilized in Matrigel. Progressive peripheral cell migration was observed throughout culture period from all GCC borders. Note anastomosing cellular architecture outside GCC (B). Cells migrating in Matrigel were imaged after fixation in situ followed by immunostaining for myogenic markers (C to E). Note ubiquitous αSMA expression (green areas) in migrating cells with nuclear localization of myogenin in occasional cells (red areas) (C). All migrating cells expressed desmin (green areas) (D). Subset also demonstrated nuclear localization of myogenic commitment marker PAX7 (red areas). All migrating cells expressed ITGA7 (green areas) (E). Differentiated muscle was visualized by MyHC expression (red areas), showing that some migrating cells formed striated myotubes. Scale bar indicates 200 μm (A to E).
The Journal of Urology  , DOI: ( /j.juro )
Copyright © 2016 American Urological Association Education and Research, Inc. Terms and Conditions

8. Figure 7
Phase contrast images show passage 4 or 6 GCC cells grown to confluence on collagen-I coated plates visualized after transfer to low serum (2%) medium. Note early appearance of cleared areas created by contraction of cellular monolayer beginning at day 4 in low serum medium (A). More generalized contraction of cellular monolayer created elongated 3D cellular ridges after additional 5-day observation together with medium changes (B). Reduced from ×20.
The Journal of Urology  , DOI: ( /j.juro )
Copyright © 2016 American Urological Association Education and Research, Inc. Terms and Conditions