During the embryonic period of CNS development neural stem cells (NSCs) are considered to be the proliferating cellular compartment in the neuroepithelium that both sustains and expands itself through self-renewal via symmetrical divisions and generates lineage-restricted-progenitors via asymmetrical divisions. Progenitor progeny also divide both symmetrically and asymmetrically and ultimately differentiate into the neural phenotypes composing both early and later stages of CNS development. Although the seminal biology of NSCs has come under intensive investigation, there is still no consensus regarding exactly which cells are actually NSCs, since specific markers do not exist. So, neuroepithelial cells are widely used as the primary source of NSCs, and in most studies, as if they all were NSCs. Thus, there is a general consensus that further elucidation of NSC biology and NSC differentiation along different neural lineages first requires their identification since this could provide direct experimental access to them for prospective rather than retrospective investigation. In FY2006, we expanded a surface-labeling strategy to target eight surface epitopes in order to identify all neural and non-neural cells in the embryonic cortex. Immunostaining revealed unmistakable anatomical gradients in cellular differentiation. Neuronal progenitors identified by characteristic intra- and extra-cellular markers were the first cells to differentiate in the neuroepithelium of the least developed dorsal region. Many of these expressed specific calcium-binding proteins characteristic of aptly named pioneer neurons and some expressed a glycoprotein known to be secreted by Cajal-Retzius cells. In the most developed ventral region, neuronal and marker-identified neuroglial progenitors predominated, while angiogenic endothelial cells in the extra-neural plexus invaded the cortex and formed primitive vessels. Thus, pioneer neurons and Cajal-Retzius cells precede neuroglial progenitors and both appear before angiogenesis. Furthermore, NSC proliferation and differentiation initially occurs in an avascular tissue.? ? In order to quantify the dynamically changing abundance of NSCs, which cannot be identified in sections, and neural and non-neural progenitors, we dissociated cells from dorsal and ventral regions and then used a nine-epitope surface labeling protocol together with flow cytometry to resolve multi-lineage-negative NSCs and lineage-restricted neural and non-neural progenitors. Flow cytometric analyses revealed that over 90% of cells dissociated from the dorsal telencephalon were actively proliferating NSCs in the process of expanding their compartment. Less than 1% of these cells were apoptotic, thus supporting continuing expansion. The remaining fractions (?i10%) were primarily neuronal progenitors and secondarily neuroglial progenitors. In contrast, cells derived from the ventral region were mainly neuronal and neuroglial progenitors with only ?i20% NSCs, many more of which were apoptotic than those from the dorsal region, thus curtailing continuing expansion. Endothelial cells, which included those forming the expanded extra-neural plexus as well as those undergoing angiogenesis, were ?i10 fold more abundant than in the dorsal telencephalon. These results demonstrate the existence of dorso-ventral gradients in the sequential and overlapping emergence of neurogenesis, neurogliogenesis, apoptosis and angiogenesis. The coincident and complex changes in the sequential transformation of self-renewing NSCs into neuronal and neuroglial progenitors undoubtedly involve intercellular signaling among and between both the proliferating NSCs and the emergent progenitors, which remain to be elucidated. ? ? We have focused first on the roles of basic fibroblast growth factor (bFGF) and FGF receptors (FGFRs) in the seminal biology of NSCs since all are present in the cortical neuroepithelium and deletion of either bFGF or FGFR1 dramatically compromises cortical development. Multi-epitope labeling of dorsal NSCs demonstrated that the majority co-expressed bFGF and FGFRs 1 and 3. A minority also expressed the glutamate-aspartate transporter protein GLAST. In contrast, the majority of ventral NSCs co-expressed bFGF, FGFRs 1-3 and GLAST. So, as NSCs shift from self-renewal and expansion to differentiating or dying, they up-regulate FGFR 2 and GLAST. In vitro, individual NSCs cultured at clonal density exhibited four stereotypical expansion states: 1) efficient self-renewal of GLAST-negative NSCs, thus expanding the compartment via symmetrical divisions, 2) inefficient self-renewal of GLAST-negative NSCs limited by frequent apoptoses of one daughter, thereby limiting expansion while preserving maintenance, 3) neurogenic GLAST-negative NSCs dividing asymmetrically to generate only putative pioneer neurons and 4) multi-potential GLAST-positive NSCs dividing asymmetrically to produce undifferentiated progeny and five neural progenitors in the same sequence as they appear in vivo, including more pioneer neurons followed by neuroglial progenitors, which themselves divide to generate putative Cajal-Retzius cells, then radial glial cells and finally astrocytes. The relative expression of the four states varied with bFGF concentration and the developmental stage of the NSCs in vivo. Low bFGF concentrations promoted neurogenic clones in all NSCs, while high bFGF concentrations stimulated efficient self-renewal of dorsal NSCs and both multi-potential and inefficient self-renewal of ventral NSCs. The critical role of bFGF-triggered signaling via FGFRs led us to investigate their roles in the seminal biology of NSCs. We cultured NSCs in the presence of missense constructs, which served as a control, and antisense constructs targeting specific FGFRs. Efficient self-renewal of dorsal NSCs required co-activation of FGFR 1 and 3. Inefficient self-renewal of ventral NSCs required activation of FGFR2. Neurogenic-only clones derived from dorsal and ventral NSCs were promoted by knocking down either FGFR 1 and/or 3. Multi-potential ventral NSCs required activation of either FGFR 2 or 3. These results demonstrate different roles for specific FGFRs in promoting one or another clonal expansion state.? ? We have begun to relate these results from clonal studies of isolated cells to physiological events occurring in vivo by explanting the dorsal telencephalon for incubation in vitro. Knocking down either FGFR 1 and/or 3 curtailed NSC expansion and triggered neurogenesis. These results with intact explants complement those carried out with isolated cells and further support the conclusion that specific FGFRs play critical roles in the seminal biology of NSCs. We have collaborated with Lynn Hudson!|s laboratory to profile the genes expressed by NSCs and their progenitor progeny throughout neurogenesis. We developed a multi-step isolation protocol consisting of magnetic bead and percoll gradient separation steps followed by flow cytometry. This new protocol has significantly improved NSC purity as evident by the dramatic reduction in erythroid cell-related genes. The different subpopulations sorted at different stages of development will now be microarrayed to reveal dynamical changes in specific gene expressions. This will then lead to studies on the roles of specific genes and gene products in the seminal biology of NSCs and the progressions of their progeny along different lineages.
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