Mahendra S. Rao

Enrichment of neurons and neural precursors from human embryonic stem cells.

Human embryonic stem (hES) cells proliferate and maintain their pluripotency for over a year in vitro (M. Amit, M. K. Carpenter, M. S. Inokuma, C. P. Chiu, C. P., Harris, M. A. Waknitz, J. Itskovitz-Eldor, and J. A. Thomson. 2000. Dev. Biol. 227: 271-278) and may therefore provide a cell source for cell therapies. hES cells were maintained for over 6 months in vitro (over 100 population doublings) before their ability to differentiate into the neural lineage was evaluated. Differentiation was induced by the formation of embryoid bodies that were subsequently plated onto appropriate substrates in defined medium containing mitogens. These populations contained cells that showed positive immunoreactivity to nestin, polysialylated neural cell adhesion molecule (PS-NCAM) and A2B5. After further maturation, these cells expressed additional neuron-specific antigens (such as MAP-2, synaptophysin, and various neurotransmitters). Calcium imaging demonstrated that these cells responded to neurotransmitter application. Electrophysiological analyses showed that cell membranes contained voltage-dependent channels and that action potentials were triggered by current injection. PS-NCAM and A2B5 immunoselection or culture conditions could be used to produce enriched populations (60-90%) which could be further differentiated into mature neurons. The properties of the hES-derived progenitors and neurons were found to be similar to those of cells derived from primary tissue. These data indicate that hES cells could provide a cell source for the neural progenitor cells and mature neurons for therapeutic and toxicological uses.

Isolation of Lineage-Restricted Neuronal Precursors from Multipotent Neuroepithelial Stem Cells

We have identified a neuronal-restricted precursor (NRP) cell that expresses E-NCAM (high polysialic-acid NCAM) and is morphologically distinct from multipotent neuroepithelial (NEP) cells (Kalyani et al., 1997) and spinal glial progenitors (Rao and Mayer-Proschel, 1997). NRP cells self renew over multiple passages in the presence of fibroblast growth factor (FGF) and neurotrophin-3 (NT-3) and differentiate in the presence of retinoic acid and the absence of FGF into postmitotic neurons. NRP cells can also be generated from multipotent E10.5 NEP cells. Clonal analysis shows that NRP cells arise from a NEP progenitor that generates other restricted CNS precursors. The NEP-derived NRPs undergo self renewal and can differentiate into multiple neuronal phenotypes. Thus, a direct lineal relationship exists between multipotential NEP cells and more restricted neuronal precursor cells present in vivo at E13.5 in the spinal cord.

Multipotent and restricted precursors in the central nervous system

Acquisition of cell type‐specific properties in the nervous system is likely a process of sequential restriction in developmental potential. At least two classes of pluripotent stem cells, neuroepithelial (NEP) stem cells and EGF‐dependent neurosphere stem cells, have been identified in distinct spatial and temporal domains. Pluripotent stem cells likely generate central nervous system (CNS) and peripheral nervous system (PNS) derivatives via the generation of intermediate lineage‐restricted precursors that differ from each other and from multipotent stem cells. Neuronal precursors termed neuronal‐restricted precursors (NRPs), multiple classes of glial precursors termed glial‐restricted precursors (GRPs), oligodendrocyte‐type 2 astrocytes (O2As), astrocyte precursor cells (APCs), and PNS precursors termed neural crest stem cells (NCSCs) have been identified. Multipotent stem cells and restricted precursor cells can be isolated from embryonic stem (ES) cell cultures providing a non‐fetal source of such cells. Analysis in multiple species illustrates similarities between rat, mouse, and human cell differentiation raising the possibility that similar factors and markers may be used to isolate precursor cells from human tissue or ES cells. Anat Rec (New Anat): 257:137–143, 1999.

Common neural progenitor for the CNS and PNS

Cultured spinal cord neuroepithelial (NEP) cells can differentiate into neurons, oligodendrocytes and astrocytes and are morphologically and antigenically distinct from neural crest stem cells (NCSCs) that generate the PNS. NEP cells, however, can generate p75/nestin-immunoreactive cells that are morphologically and antigenically similar to previously characterized NCSCs. NEP-derived p75-immunoreactive cells differentiate into peripheral neurons, smooth muscle, and Schwann cells in mass and clonal culture. Clonal analysis of NEP cells demonstrates that a common NEP progenitor cell generated both CNS and PNS phenotypes. Differentiation into NCSCs was promoted by BMP-2/4 and differentiation did not require cells to divide, indicating that BMP played an instructive role in the differentiation process. Thus, individual NEP cells are multipotent and can differentiate into most major types of cell in the CNS and PNS and that PNS differentiation involves a transition from a NEP stem to another more limited, p75-immunoreactive, neural crest stem cell.

Gliogenesis in the central nervous system

Multipotential neuroepithelial stem cells are thought to give rise to all the differentiated cells of the central nervous system (CNS). The developmental potential of these multipotent stem cells becomes more restricted as they differentiate into progressively more committed cells and ultimately into mature neurons and glia. In studying gliogenesis, the optic nerve and spinal cord have become invaluable models and the progressive stages of differentiation are being clarified. Multiple classes of glial precursors termed glial restricted precursors (GRP), oligospheres, oligodendrocyte‐type2 astrocyte (O‐2A) and astrocyte precursor cells (APC) have been identified. Similar classes of precursor cells can be isolated from human neural stem cell cultures and from embryonic stem (ES) cell cultures providing a non‐fetal source of such cells. In this review, we discuss gliogenesis, glial stem cells, putative relationships of these cells to each other, factors implicated in gliogenesis, and therapeutic applications of glial precursors. GLIA 30:105–121, 2000.

Regulation of eye development by frizzled signaling in Xenopus

Eye development in both invertebrates and vertebrates is regulated by a network of highly conserved transcription factors. However, it is not known what controls the expression of these factors to regulate early eye formation and whether transmembrane signaling events are involved. Here we establish a role for signaling via a member of the frizzled family of receptors in regulating early eye development. We show that overexpression of Xenopus frizzled 3 (Xfz3), a receptor expressed during normal eye development, functions cell autonomously to promote ectopic eye formation and can perturb endogenous eye development. Ectopic eyes obtained with Xfz3 overexpression have a laminar organization similar to that of endogenous eyes and contain differentiated retinal cell types. Ectopic eye formation is preceded by ectopic expression of transcription factors involved in early eye development, including Pax6, Rx, and Otx2. Conversely, targeted overexpression of a dominant-negative form of Xfz3 (Nxfz3), consisting of the soluble extracellular domain of the receptor, results in suppression of endogenous Pax6, Rx, and Otx2 expression and suppression of endogenous eye development. This effect can be rescued by coexpression of Xfz3. Finally, overexpression of Kermit, a protein that interacts with the C-terminal intracellular domain of Xfz3, also blocks endogenous eye development, suggesting that signaling through Xfz3 or a related receptor is required for normal eye development. In summary, we show that frizzled signaling is both necessary and sufficient to regulate eye development in Xenopus.

Expression of EGF receptor and FGF receptor isoforms during neuroepithelial stem cell differentiation.

To characterize the role of epidermal growth factor (EGF) and fibroblast growth factor (FGF) in regulating neuroepithelial stem cells differentiation, we have examined the expression of FGF, EGF, and their receptors by neuroepithelial (NEP) cells and their derivatives. Our results indicate that undifferentiated NEP cells express a subset of FGF receptor (FGFR) isoforms, but do not express platelet-derived growth factor receptors (PDGFRs) or epidermal growth factor receptor (EGFR). The FGFR pattern of expression by differentiated neuron and glial cells differs from that found on NEP stem cells. FGFR-4 is uniquely expressed on NEP cells, while FGFR-1 is expressed by both NEP cells and neurons, and FGFR-2 is down-regulated during neuronal differentiation. FGFRs present on astrocytes and oligodendrocytes also represent a subset of those present on NEP cells. Expression of FGF and EGF by NEP cells and their progeny was also examined. NEP cells synthesize detectable levels of both FGF-1 and FGF-2, and EGF. FGF-1 and FGF-2 synthesis is likely to be biologically relevant, as cells grown at high density do not require exogenous FGF for their survival and cells grown in the presence of neutralizing antibodies to FGF show a reduction in cell survival and division. Thus, neuroepithelial cells synthesize and respond to FGF, but not to EGF, and are therefore distinct from other neural stem cells (neurospheres). The unique pattern of expression of FGF isoforms may serve to distinguish NEP cells from their more differentiated progeny.

Characterization of the AMPA‐Activated Receptors Present on Motoneurons

Abstract: Motoneurons have been shown to be particularly sensitive to Ca2+‐dependent glutamate excitotoxicity, mediated via AMPA receptors (AMPARs). To determine the molecular basis for this susceptibility we have used immunocytochemistry, RT‐PCR, and electrophysiology to profile AMPARs on embryonic day 14.5 rat motoneurons. Motoneurons show detectable AMPAR‐mediated calcium permeability in vitro and in vivo as determined by cobalt uptake and electrophysiology. Motoneurons express all four AMPAR subunit mRNAs, with glutamate receptor (GluR) 2 being the most abundant (63.9 ± 4.8%). GluR2 is present almost exclusively in the edited form, and electrophysiology confirms that most AMPARs present are calcium‐impermeant. However, the kainate current in motoneurons was blocked an average of 32.0% by Joro spider toxin, indicating that a subset of the AMPARs is Ca2+‐permeable. Therefore, heterogeneity of AMPARs, rather than the absence of GluR2 or the presence of unedited GluR2, explains AMPAR‐mediated Ca2+ permeability. The relative levels of flip/flop isoforms of each subunit were also examined by semiquantitative PCR. Both isoforms were present, but the relative proportion varied for each subunit, and the flip isoform predominated. Thus, our data show that despite high levels of edited GluR2 mRNA, some AMPARs are Ca2+‐permeable, and this subset of AMPARs can account for the AMPAR‐mediated Ca2+ inflow inferred from cobalt uptake and electrophysiology studies.

Contrasting molecular composition and channel properties of AMPA receptors on chick auditory and brainstem motor neurons

1 Neurons in the brainstem auditory pathway exhibit a number of specializations for transmitting signals reliably at high rates, notably synaptic AMPA receptors with very rapid kinetics. Previous work has not revealed a common structural pattern shared by the AMPA receptors of auditory neurons that could account for their distinct functional properties. 2 We have used whole‐cell patch‐clamp recordings, mRNA analysis, immunofluorescence, Western blots and agonist‐evoked cobalt uptake to compare AMPA receptors on the first‐, second‐ and third‐order neurons in the chick ascending auditory pathway with those on brainstem motor neurons of the glossopharyngeal/vagal nucleus, which have been shown to have very slow desensitization kinetics. 3 The results indicate that the AMPA receptors of the cochlear ganglion, nucleus magnocellularis and nucleus laminaris share a number of structural and functional properties that distinguish them from the AMPA receptors of brainstem motor neurons, namely a lower relative abundance of glutamate receptor (GluR)2 transcript and much lower levels of GluR2 immunoreactivity, higher relative levels of GluR3 flop and GluR4 flop, lower relative abundance of the C‐terminal splice variants GluR4c and 4d, less R/G editing of GluR2 and 3, greater permeability to calcium, predominantly inwardly rectifying I–V relationships, and greater susceptibility to block by Joro spider toxin. 4 We conclude that the AMPA receptors of auditory neurons acquire rapid kinetics from their high content of GluR3 flop and GluR4 flop subunits and their high permeability to Ca2+ from selective post‐transcriptional suppression of GluR2 expression.

Characterization of avian frizzled genes in cranial placode development

To determine the possible role of Wnt signaling in cranial placode development, we have cloned several chick frizzled genes, a family of putative Wnt receptor molecules, and analyzed their expression during chick embryogenesis. Chick frizzled-2 (cFz-2) and frizzled-7 (cFz-7) are expressed broadly in cranial ectoderm, tissue that is competent to express markers of the trigeminal placode (Stark et al., 1997. Development 124, 4287-4295; Baker et al., 1999. Development 126, 147-156). In addition, cFz-2 and cFz-7 are uniquely expressed in other cranial placodes, including the olfactory, lens, and otic placodes. Chick frizzled-1 (cFz-1) is expressed in the lens, otic placode and, along with cFz-7, in epibranchial placodes. Each frizzled gene expressed in the otic placode displays a unique domain of expression: cFz-1 transcripts are detected in the medial wall of the vesicle, cFz-2 in the rostral rim of the vesicle, and cFz-7 in the lateral half of the vesicle. Other chick frizzled family members cloned that do not show striking expression in cranial placodes include frizzled-4 (cFz-4), frizzled-8 (cFz-8), frizzled-9 (cFz-9), and frizzled-10 (cFz-10). A brief summary of their expression is given, along with a brief summary of non-placodal expression of cFz-1, cFz-2, and cFz-7. In all, frizzled genes show dynamic expression at key times during embryonic development, particularly in the cranial placodes.