Characterizing PCDH19 in human induced pluripotent stem cells (iPSCs) and iPSC-derived developing neurons: emerging role of a protein involved in controlling polarity during neurogenesis

Abstract

PCDH19 (Protocadherin 19), a member of the cadherin superfamily, is involved in the pathogenic mechanism of an X-linked model of neurological disease. The biological function of PCHD19 in human neurons and during neurogenesis is currently unknown. Therefore, we decided to use the model of the induced pluripotent stem cells (iPSCs) to characterize the location and timing of expression of PCDH19 during cortical neuronal differentiation. Our data show that PCDH19 is expressed in pluripotent cells before differentiation in a homogeneous pattern, despite its localization is often limited to one pole of the cell. During neuronal differentiation, positional information on the progenitor cells assumes an important role in acquiring polarization. The proper control of the cell orientation ensures a fine balancing between symmetric (giving rise to two progenitor sister cells) versus asymmetric (giving rise to one progenitor cell and one newborn neuron) division. This process results in the polar organization of the neural tube with a lumen indicating the basal part of the polarized neuronal progenitor cell; in the iPSC model the cells are organized in the 'neural rosette' and interestingly, PCDH19 is located at the center of the rosette, with other well-known markers of the lumen (N-cadherin and ZO-1). These data suggest that PCDH19 has a role in instructing the apico-basal polarity of the progenitor cells, thus regulating the development of a properly organized human brain.

Keywords: Neuroscience Section; PCDH19; human iPSCs; iPSC-derived neurons; neural rosettes; neuronal polarity.

Figure 1. PCDH19 localization in iPSC colony

The confocal images in A and B represent proliferative iPSCs with the red signal indicating PCDH19 antibody staining. The arrows in A point to the focal and polarized PCDH19 signal. The arrows in B point to the specific localization of PCDH19 at the poles of mitotic cells.

Figure 2. In vitro cortical neurogenesis

Bright field images taken at different days during the differentiation of iPSCs into cortical neurons show the reorganization of the cells and their changes in cell morphology. The immunofluorecence in the bottom right corner indicate that the cells have successfully differentiated in neurons, positive to the β-III TUBULIN antibody.

Figure 3. PCDH19 localization in the lumen of the neural rosette

Confocal images of immunofluorescence for PCDH19 and ZO-1. The immunofluorescence for PCDH19 is localized at the lumen of the neural rosette, which is indicated by the localization of the known marker of this highly polarized structure ZO-1 A., B., C. The white box in C indicates the region magnified in D., E., F. The inset in E represents the merge image of D and E.

Figure 4. PCDH19 localization in the lumen of the neural rosettes

Confocal image of the neural rosette lumen A. with arrows pointing to the foci of PCDH19 localization in the mitotic cells a and a’. Confocal central plane of a and a’ cells is magnified below and the yz axes are reported on the right side. B. 3D surface rendering of the region of interest. On the right (b and b’) 3D reconstructions of mitotic cells are reported. Bars: 10 μm.

Figure 5. PCDH19 localization in mature neurons

Confocal photographs of immunofuorescence for PCDH19 (red) and SMI32 (green) on iPSC-derived cortical neurons after 30 days of differentiation. The images in A. and B. clearly show that PCDH19 protein is present at the site of cell-cell contacts in neuronal cultures (positive to the neuronal marker SMI32) as indicated by the arrows. In particular, in A the cell-cell contact is mainly at the level of cell soma, while in B the top neuron presents the PCHD19 signal between the neurite and the cell soma of the neighboring cell.

Figure 6. PCDH19 in proliferating and differentiating iPSCs

Drawing of a dividing iPSC with PCDH19 positive foci indicated in red A. Schemata of a neural rosette (on the left) and of a neural tube (on the right), showing the correspondence between their lumen (positive to ZO-1) and indicating in red mitotic cells, in green post-mitotic neurons which are migrating away from the lumen and the radial glia (or progenitor cells) B. In C. a model of the rosette photographed in Fig 3 and 4 is reported to focus the attention on the cell “a” and “b”, which present different spindle pole orientations indicated by the position of the PCDH19 positive foci. The drawing in D. represents the interkinetic nuclear migration observed during the development of the nervous system.