Work supported by NIH grant HD-32116, the Sandler Family Supporting Foundation, The John Bowes Stem Cell Fund, MEXT, MHLW, and HFSP. Z.M. supported by the Carlos Baldoceda Foundation and UCSF Krevans Fellowship. A.A.-B. holds the Heather and Melanie Muss Endowed Chair.

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Most studies of neurogenesis in ventricular and subventricular zones have relied on classical sectioning techniques to examine the microanatomy and cellular relationships in these regions. Here we describe an alternative technique, first used to analyze the network of migratory chains of neuroblasts generated in the SVZ 1, then used to study regeneration of the SVZ progenitor population following anti-mitotic treatment 6, and most recently used to study the precise apical and basal cell-cell interactions of adult SVZ neural stem cells 2,3,4. Interestingly, this technique has revealed that the neural stem cells, or type B1 cells, of the adult SVZ are part of a mixed neuroepithelium with differentiated non-dividing ependymal cells. En-face imaging using wholemounts has shown that this mixed neuroepithelium has pinwheel architecture consisting of the apical endings of type B1 cells surrounded by large apical surfaces of ependymal cells 4. This en-face analysis has clarified our understanding of the lineage of neural stem cells in embryonic and adult brains as consisting of cells with apical endings at the ventricle surface and basal processes contacting a vascular niche. These findings would have been nearly impossible using classical sectioning techniques. Wholemounts also facilitate the identification of neural stem cells via their ventricle-contacting apical process. As more specific markers for these stem cells are found, wholemounts will be an integral part of identifying and analyzing neural stem cell behavior.
Wholemounts of the lateral ventricle walls also provide the ideal perspective for studying the planar polarity of ependymal cells. Ependymal cells are multiciliated cells lining the ventricles that function to propel CSF in a coordinated manner. With the wholemount technique, the entire ependymal epithelium is exposed en-face and can be stained and studied comprehensively from its anterior to posterior and dorsal to ventral boundaries. Furthermore, ependymal flow assays performed on acutely dissected, live wholemounts robustly demonstrate the planar polarized flow generated by ependymal cilia. Recent work using wholemount approaches has uncovered cellular determinants of this ependymal planar polarity 5. Interestingly, wholemount studies have also suggested that ependymal-generated CSF flow establishes gradients of chemorepellents that guide the migration of young neurons in the SVZ 7. Wholemount approaches that initially identified the network of migratory neuronal chains are therefore continuing to provide insights into mechanisms regulating chain migration.
Analysis of the VZ and SVZ by wholemount imaging adds a new approach for both future studies and a way to clarify our understanding of existing studies. For example, a recent study suggested that neural stem cells in the adult SVZ were CD133+/CD24- cells in contact with the ventricle 8. Based on their immunostaining in sections, these authors claimed that these cells were a subpopulation of multiciliated ependymal cells. However, in our study using the wholemount approach, which gives a more comprehensive view of the entire ependymal epithelium, we found that all ependymal cells express CD24 and the only ventricle-contacting cells that were CD133+/CD24- were a subset of the type B1 cells 4. Furthermore, the wholemount technique promises to be useful in future studies examining the recently described mosaic organization of neural stem cells in the adult brain 9. Several studies have shown that neural stem cells in the adult brain are not a homogeneous population, but are regionally specified and normally produce only specific subtypes of olfactory bulb interneurons. These studies have proposed that different subpopulations of neural stem cells may be distinguished either by the expression of specific transcription factors 10,11,12,13,14 and/or by their regional localization along the dorsal-ventral and anterior-posterior extents of the lateral wall 9,15,16. As more molecular markers of the regionally specified subpopulations of adult neural stem cells are identified, wholemount imaging should provide a comprehensive view of the parcelation of these different progenitor domains along the ventricular wall.
The wholemount dissection and imaging techniques presented here may also be used to analyze the ventricular walls in the embryo. The dissection of the embryonic lateral wall is performed, step-by-step, in the same manner. There are only slight differences in the level of difficulty; the embryonic ventricles are relatively larger making the dissection easier, but the tissue is softer making manipulation more difficult. In particular, a similar exposure of the lateral ventricle can be used in embryos to dissect the cortical wall of the ventricle to study cortical neurogenesis. Recent evidence suggests that asymmetric centrosome inheritance maintains radial glia at the ventricular surface during cortical neurogenesis 17. En-face imaging of radial glial apical surfaces may provide insights into how centrosomes within these dividing cells are asymmetrically inherited.
As with most techniques, especially those involving precise dexterity, mastery requires practice. There are, however, a few elements in the dissection that are key to better results: 1) lighting adjusting the illumination of the sample to create shadows provides invaluable contrast during the dissection of tissue that is otherwise relatively homogeneous, 2) using the forceps like two insect pins the forceps in this technique are never used to pinch together or pick up tissue, but are used as maneuverable pins that can be continually readjusted to stabilize the tissue while cutting, 3) a balance of gentle retraction and cutting the knife should not only be used to cut but also to provide gentle retraction to separate the medial and lateral walls, remembering that the majority of this dissection is actually performed through gentle retraction with only intermittent cutting.

the subventricular zone en-face: wholemount staining and ependymal flow

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an extensively studied model system for understanding the behavior of neural stem cells and the regulation of adult neurogenesis. Traditionally, these studies have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region. Compared to sections, wholemounts preserve the complete cytoarchitecture and cellular relationships within the SVZ. This approach has recently revealed that the adult neural stem cells, or type B1 cells, are part of a mixed neuroepithelium with differentiated ependymal cells lining the lateral ventricles. In addition, this approach has been used to study the planar polarization of ependymal cells and the cerebrospinal fluid flow they generate in the ventricle. With recent evidence that adult neural stem cells are a heterogeneous population that is regionally specified, the wholemount approach will likely be an essential tool for understanding the organization and parcellation of this stem cell niche.

Watch this Scientific Journal Video about The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow at JoVE.com

The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow

The lateral ventricle walls contain the largest germinal region in the adult mammalian brain. Traditionally, studies on neurogenesis in this region have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region.

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an ...

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41.5K Views.  University of California, San Francisco - UCSF. The lateral ventricle walls contain the largest germinal region in the adult mammalian brain. Traditionally, studies on neurogenesis in this region have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region.