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The Baskin Laboratory is investigating the molecular underpinnings of bladder development. Following is a description of our bladder development research.

Hypothesis
Between gestational days 13 and 18 the mouse bladder undergoes smooth muscle differentiation from undifferentiated mesenchyme. The baskin laboratory has shown that induction of bladder smooth muscle occurs through an epithelial signal from the urothelium to the undifferentiated bladder mesenchyme. The signaling appears to be mediated by paracrine signaling molecules emanating from the epithelium to the outer mesenchymal layer. Our key question is: Which gene products from the urothelium control bladder smooth muscle induction? We believe it is relevant that bladder smooth muscle cells first develop in the periphery of the bladder away from the inducing urothelium; the undifferentiated mesenchyme adjacent to the urothelium does not differentiate into smooth muscle.

We hypothesize that select genes differentially expressed before smooth muscle differentiation, rather than those expressed after smooth muscle differentiation, are part of the mechanism that induces bladder smooth muscle formation. Our primary goals is to identify and characterize these inductive genes and their products. As part of accomplishing this goal we have mastered the techniques of isolating the bladder as early as the E12 stage of development in the mouse and of separating bladder epithelium and mesenchyme.


  signaling appears to be mediated by paracrine signaling molecules emanating from the epithelium to the outer mesenchymal layer
  series of bladders isolated at E12, E13, E14 and E15 stages of development in fetal mice with the separated mesenchyme and epithelium for E14 and E15

Genomics
We have completed in-house genomics analyses of whole bladders and of separated epithelium and mesenchyme. These analyses are part of our research to determine which genes are involved in inducing bladder smooth muscle development from the outer mesenchyme. The figures below show an example of our microarray results and a comparison of the relative expression of select genes between the E13 and E14 stages of development. These images demonstrate that expression for the serum response factor pathway is upregulated at the time that bladder smooth muscle differentiation occurs.


 
  complete microarray results comparison of the relative expression of some genes between the E13 and E14 stages of development

Specific gene characterization
The results from our microarray analyses drew our attention to several genes that were upregulated at significant time periods in bladder development. These genes were further studied by determining MRNA expression and protein localization in the developing bladder. The figures below illustrate our results with real time PCR for two markers of smooth muscle development, smooth muscle alpha actin and caldesmon. Also shown is the developmental expression of serum response factor and its co-factor genes shown by immunofluorescent studies.


 
  results with RT-PCR analysis results from fluorescent immunohistochemistry

Smooth muscle characterization and timing
We used genes specific to smooth muscle to identify when smooth muscle proteins first appear in the bladder. The images below illustrate those studies, showing that smooth muscle differentiation first occurs in the bladder at embryonic day 13.5. This is demonstrated by the expression of smooth muscle alpha actin in the periphery of the bladder. At embryonic day 14 there is an impressive increase in smooth muscle alpha actin in the peripheral aspect of the bladder illustrated in the image below. Another marker defining smooth muscle, smooth muscle gamma actin, also localizes to peripheral aspect of the bladder at embryonic day 14.

 

immunohistochemical results for smooth muscle alpha actin binding in the embryonic mouse bladder using whole body sections

(top) positive signal in the vessels flanking the bladder,
(bottom) close-up view of the positive signal just beginning to appear at the bladder periphery

 

E13 and E14 bladders reconstructed from results with immunohistochemical staining of histological sections for smooth muscle alpha actin

(left) an E13 bladder with no smooth muscle present
(right) by E14 smooth muscle protein is present

Our results suggest that bladder smooth muscle differentiation may share a similar gene expression program as that which occurs during vascular smooth muscle differentiation. The unique structure of the urinary bladder makes it an ideal model for studies of smooth muscle differentiation and epithelial-mesenchymal signaling.

 

Our results support that we can control bladder smooth muscle induction by inhibiting smooth muscle formation by placing urothelium in an ectopic location.

Our bladder research is relevant to patients with bladder augmentations where abnormal epithelial mesenchymal signaling interactions occur between intestinal and bladder cells.   Experimentally we have been able to change the urothelial phenotype  to a glandular epithelium (intestinal).   Cancer although rare in augmentations occurs  typically at the anastomotic site and may arise from these abnormal cell cell interactions.

   




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