Imaging the Developing Human External and Internal Urogenital Organs with Light Sheet Fluorescence Microscopy – Beyond the Abstract

Despite advances in our understanding of urologic pathology, much of what we know about human urogenital development in the embryonic and early fetal periods remains based on morphologic studies performed in the late 19th and early 20th centuries. In this era, embryos were incidental findings after hysterectomy and few restrictions existed on their use in research.1 While contemporary methods have evolved past staining histologic sections with alum carmine or hematoxylin and eosin as was done in the 1900s, refinements in obstetric and gynecological techniques and restrictions on the use of fetal tissue in research preclude similar collection today. As a result, normal urogenital development in utero and the pathogenesis of congenital urologic abnormalities remain poorly characterized at the molecular level.

In recent years, our laboratory has been uniquely equipped to study developing human fetal urogenital organs. The application of three-dimensional imaging methods has led to novel findings that previously eluded us through decades of study of histologic sections. We have characterized molecular mechanisms of development of the penile urethra2 and homologous structures in the clitoris.3 More recently, we described a second distinct method of urethral formation in the glans penis featuring direct canalization through the glanular urethral plate.4 These findings are steps closer to understanding normal urethral development and the molecular pathogenesis of hypospadias.

Light sheet fluorescence microscopy is a recently refined technique that uses thin sheets of planar light illumination to excite biomolecules within optically cleared, transparent specimens. Fluorescence is either the “autofluorescence” of naturally occurring NADH, FAD or, more commonly, from fluorescently tagged proteins and nucleic acids. Light emissions are recorded within the microscope and digitally reconstructed into models demonstrating in vivo gene expression in three dimensions. Light sheet microscopes have been commercially available since 2012 and have since been widely applied in biologic research.

This work presents our method for clearing, immunostaining and light sheet microscopy of human fetal urogenital organs. We include at least one figure highlighting gene expression within every organ of the urinary and genital systems. At the time of our publication, these represented, to the extent of our knowledge, the first examples of light sheet images of the developing human fetal kidney, bladder, prostate, testis, penis and clitoris.

This is just a start – in our experience, the main advantages of the light sheet as compared to older 3D imaging techniques are rapid speed of data acquisition allowing for imaging of entire whole-mount specimens in minutes, and the ability to clear specimens of immunolabels and re-stain them, enabling conservation of valuable fetal organs and the comparison of many more antigens within a single specimen. The collection of full ontogenies of developing organs can and should lead to data collection and discovery at a scale and level of detail not previously seen in the study of human urogenital development.
Written by: Dylan Isaacson, MD, MPH, Laurence Baskin MD, Department of Urology, Northwestern Feinberg School of Medicine, Department of Urology, University of California, San Francisco, San Francisco, California, USA.


  1. Morgan, Lynn M. "A social biography of Carnegie embryo no. 836." The Anatomical Record Part B: The New Anatomist: An Official Publication of the American Association of Anatomists 276, no. 1 (2004): 3-7.
  2. Li, Yi, Adriane Sinclair, Mei Cao, Joel Shen, Shweta Choudhry, Sisir Botta, Gerald Cunha, and Laurence Baskin. "Canalization of the urethral plate precedes fusion of the urethral folds during male penile urethral development: the double zipper hypothesis." The Journal of urology 193, no. 4 (2015): 1353-1360.
  3. Overland, Maya, Yi Li, Mei Cao, Joel Shen, Xuan Yue, Sisir Botta, Adriane Sinclair, Gerald Cunha, and Laurence Baskin. "Canalization of the vestibular plate in the absence of urethral fusion characterizes development of the human clitoris: the single zipper hypothesis." The Journal of urology 195, no. 4 (2016): 1275-1283.
  4. Liu, Xin, Ge Liu, Joel Shen, Aaron Yue, Dylan Isaacson, Adriane Sinclair, Mei Cao, Aron Liaw, Gerald R. Cunha, and Laurence Baskin. "Human glans and preputial development." Differentiation 103 (2018): 86-99.
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