Molecular Genetic and Immunophenotypical Analysis of Pax6 Transcription Factor and Neural Differentiation Markers in Human Fetal Neocortex and Retina In Vivo and In Vitro

Abstract

Neurotransplantation of various cells, including heterotransplantation of fetal cerebral stem/progenitor cells into the eye is used in experimental studies of central nervous tissue repair during neurodegeneration. For evaluation of this approach, human fetal (weeks 9-20) stem/progenitor cells of the neocortex and retina were studied in vivo and in vitro by quantitative PCR and immunohistochemical staining. Native tissues and cultures were characterized by expression of Pax6 transcription factor (critical for the development of the retina and neocortex) and differentiation markers (nestin, betaIII-tubulin, glial fibrillary acidic protein, recoverin, NeuN, neurofilaments, Ki-67). The expression of Pax6 gene in the retina during active neurogenesis was stable and much higher than in the neocortex. In primary cultures, the pattern of Pax6 gene expression is retained and repeats that in native tissues. Immunohistochemical analysis revealed similarity of nestin and betaIII-tubulin expression in the neocortex and retina during the early (9-10 weeks) and later (20 weeks) periods and differences in cell phenotypes and their distribution. Culture studies showed that neocortical and retinal stem/progenitor cells are determined and exhibit specific differentiation characteristic of the corresponding native tissues. It can be hypothesized that heterotransplantation of the cerebral progenitor cells into the retina of experimental animals can lead to realization of their neurotrophic effect, but not to their functional integration.

Expression patterns of Pax6 and Pax7 in the adult brain of a urodele amphibian, Pleurodeles waltl

“…Unlike in mammals (Stoykova and Gruss, 1994; Duan et al, 2012), no differences in their distribution in the two bulbs were noted. The presence of Pax6 in the olfactory bulbs is a conserved feature in vertebrates and has been reported from lampreys through mammals, including humans (Stoykova and Gruss, 1994; Hauptmann and Gerster, 2000; Puelles et al, 2000; Franco et al, 2001; Murakami et al, 2001; Derobert et al, 2002; Hauptmann, 2002; Moreno et al, 2008a; Verdiev et al, 2009; Moreno et al, 2010, 2012a; Duan et al, 2012; Ferreiro‐Galve et al, 2012). Most data obtained in mice demonstrate that Pax6 is essential for the formation of the olfactory placode, olfactory bulb, and olfactory cortex (Nomura et al, 2007).…”

NeuN As a Neuronal Nuclear Antigen and Neuron Differentiation Marker

“…Other specific proteins are present in the vast majority of neurons. One of them is the neuronal nuclear protein NeuN, which is often used as a marker of postmitotic neurons due to some of its properties (primarily nuclear localization) [ 4 - 7 ]. Monoclonal antibodies to the NeuN protein have been actively used in immunohistochemical studies of neuronal differentiation to assess the functional state of neurons in norm and pathology for more than 20 years.…”

“…Although the structure of the antigenic determinant that binds A60 antibodies and the conditions of this binding are not fully understood, antibodies to the NeuN protein are widely used in scientific research and in histopathologic diagnosis. Thus, during the last decade, the NeuN protein has been used as a universal neuron-specific marker for studying the differentiation of stem cells [ 7 , 26 - 28 ]. The presence of some specific marker proteins, whose immunocytochemical detection allows for selective identification of cells belonging to the nervous system tissues, in postmitotic cells provides evidence of neuronal differentiation.…”

Verification of the Cross Immunoreactivity of A60, a Mouse Monoclonal Antibody against Neuronal Nuclear Protein

“…Using repeated immunization with purified nuclei from neurons, a mouse monoclonal antibody was generated and named A60 (Mullen et al, 1992 ). The mouse monoclonal antibody is capable of labeling neurons out of mitotic stages in different species of vertebrates (Mullen et al, 1992 ; Rodriguez et al, 2002 ; Tonchev et al, 2003 ; Kumar and Buckmaster, 2007 ; Korzhevskii et al, 2009 ; Verdiev et al, 2009 ). However, A60 has shown a lack of labeling for cerebellar Purkinje cells, olfactory mitral cells and retinal photoreceptor cells (Mullen et al, 1992 ).…”

“…However, A60 has shown a lack of labeling for cerebellar Purkinje cells, olfactory mitral cells and retinal photoreceptor cells (Mullen et al, 1992 ). Further neuronal exceptions may include cerebellar interneurons (Weyer and Schilling, 2003 ) and a subset of substantia nigral neurons (Weyer and Schilling, 2003 ; Kumar and Buckmaster, 2007 ; Cannon and Greenamyre, 2009 ; Korzhevskii et al, 2009 ; Verdiev et al, 2009 ).Highly specific for labeling postmitotic neurons, A60 has been most widely used neuronal marker in neuroscience research and neuropathological assays (Gusel’nikova and Korzhevskiy, 2015 ). The spectrum of A60 use is very broad including labeling to monitor neuronal development (Wynder et al, 2005 ), neurogenesis (Magavi et al, 2000 ) and stem cell differentiation (Brazelton et al, 2000 ).…”