Initiation Of Cell Differentiation Research Articles

Signal transduction of phorbol 12-myristate 13-acetate (PMA)-induced growth inhibition of human monocytic leukemia THP-1 cells is reactive oxygen dependent

Human monocytic THP-1 cells can be induced to differentiate to macrophages when treated with phorbol 12-myristate 13-acetate (PMA). It is understood that before initiating cell differentiation, PMA treatment must first induce an inhibition of cell growth. Since the initial biochemical and molecular events that are associated with this growth inhibition have not been characterized, the present study was carried out to elucidate the molecular mechanisms associated with the PMA-induced growth arrest of THP-1 cells. Our results indicate that PMA inhibits THP-1 cells at G 1-phase of the cell cycle, via a complex mechanism associated with the modulation of the expression of several cell cycle regulators, initiated by the cellular generation of reactive oxygen species (ROS). Both p21 WAF1/CIP1 mRNA and protein were upregulated 24 h post PMA treatment as demonstrated by ribonuclease protection assay and Western blotting, respectively. Because these cells lack functional p53, this effect was independent of p53 activity. Electrophoretic mobility shift assay showed that the PMA-induced activation of the p21 WAF1/CIP1 promoter was driven by the specific protein 1 (Sp1) transcription factor through Sp1-binding sites. Additionally, our study demonstrates that PMA-induces the upregulation of p21 through a protein kinase C (PKC)-mediated ROS-dependent signaling mechanism involving MAP kinase activation.

Characterization of a mutation in the lens-specific CP49 in the 129 strain of mouse.

The 129 strain of mouse carries a mutation in the gene for CP49 (phakinin), an intermediate filament protein thus far demonstrated only in the lens fiber cell. As such, these mice represent naturally occurring mutants of interest in the study of the lens cytoskeleton. However, this strain of mouse is also widely used as a source of embryonic stem cells in gene-targeting studies. The presence of a mutation in a lens-specific gene can confound interpretation of studies in which lens genes have been knocked out. In the present study, both the genotype and phenotype of these mice were characterized, to permit an evaluation of the biological impact of this mutation and to facilitate the discrimination between wild-type and mutant animals that have been derived from this strain in gene-targeting studies. The CP49 cDNA and, when relevant, the genomic DNA sequences were determined for the 129/SvJ and C57BL/6J mice and from a commercially available 129/OLa P1 genomic clone. PCR primers were screened for their capacity to discriminate between the mutant and wild-type CP49 genes. Northern blot analysis was used to assess mRNA levels for CP49, filensin, and gammaS-crystallin (control). Western blot analysis was used to identify changes in protein size and abundance. The impact of the mutation on lens architecture was evaluated at the light-microscope level. Lens fiber cell ghosts from mutant and wild-type mice were examined in the electron microscope for the presence of beaded filaments. Lens clarity was assessed by slit lamp. The 129 strain of mice exhibited a 6303-bp deletion from the end of intron B, and the beginning of exon 2. This deletion results in the loss of the exon 2 splice acceptor site, absence of exon 2 from the CP49 mRNA, and dramatically reduced levels of CP49 mRNA. The CP49 protein was undetectable by Western blot analysis. Messenger RNA levels for filensin, CP49's assembly partner, were normal, but protein levels were sharply reduced. Light microscopy established that the initial differentiation and elongation of the fiber cells proceeded normally. Electron microscopy showed the absence of beaded filaments, whereas slit lamp microscopy showed a slowly emerging and progressive loss of optical clarity. The 129/SvJ and 129/OLa strains of mice harbor a mutation that sharply reduces CP49 mRNA levels and essentially eliminates both CP49 and the beaded filament. These lenses exhibited a slow but progressive loss of optical clarity with age. Thus, the 129 strain of mouse behaves as a functional CP49 knockout. The loss of clarity in the lenses of these animals and the absence of beaded filaments (and any attendant interactions that may exist between beaded filaments and other lens proteins/structures) suggest that gene-targeting studies of lens proteins in which the 129 strain was used as a source of embryonic stem cells may need reevaluation.

Human homolog of disc-large is required for adherens junction assembly and differentiation of human intestinal epithelial cells.

We and others have shown that phosphatidylinositol 3-kinase (PI3K) is recruited to and activated by E-cadherin engagement. This PI3K activation is essential for adherens junction integrity and intestinal epithelial cell differentiation. Here we provide evidence that hDlg, the homolog of disc-large tumor suppressor, is another key regulator of adherens junction integrity and differentiation in mammalian epithelial cells. We report the following. 1) hDlg co-localizes with E-cadherin, but not with ZO-1, at the sites of cell-cell contact in intestinal epithelial cells. 2) Reduction of hDlg expression levels by RNA(i) in intestinal cells not only severely alters adherens junction integrity but also prevents the recruitment of p85/PI3K to E-cadherin-mediated cell-cell contact and inhibits sucrase-isomaltase gene expression. 3) PI3K and hDlg are associated with E-cadherin in a common macromolecular complex in living differentiating intestinal cells. 4) This interaction requires the association of hDlg with E-cadherin and with Src homology domain 2 domains of the p85/PI3K subunit. 5) Phosphorylation of hDlg on serine and threonine residues prevents its interaction with the p85 Src homology domain 2 in subconfluent cells, whereas phosphorylation of hDlg on tyrosine residues is essential. We conclude that hDlg may be a determinant in E-cadherin-mediated adhesion and signaling in mammalian epithelial cells.

Cyclooxygenase expression is elevated in retinoic acid-differentiated U937 cells

Cyclooxygenase (COX) is the rate-limiting enzyme for the biosynthesis of prostaglandins in monocytes/macrophages. The COX-1 is constitutively expressed in most tissues and may be involved in cellular homeostasis, whereas the COX-2 is an inducible enzyme that may play an important role in inflammation and mitogenesis. When U937 monocytic cells were incubated with retinoic acid (RA) for 48 h, cell differentiation took place with concomitant increases in prostaglandin E 2 (PGE 2) production and COX activity. In this study, the mechanism of RA (all- trans- or 9- cis-RA)-induced enhancement of PGE 2 biosynthesis in U937 cells was examined. Treatment of cells with all- trans- or 9- cis-RA up to 48 h caused an increase in PGE 2 production in a time- and dose-dependent manner. Both RA isomers caused the enhancement of PGE 2 production and the up-regulation of COX-1 expression at the protein and mRNA levels. The increase in COX-1 mRNA was found to precede the increase in COX-1 protein expression. Interestingly, the COX-2 protein and COX-2 mRNA were not detected in U937 cells, and their levels remained undetectable during the entire course of RA treatment. We conclude that treatment of U937 cells by RA for 48 h caused the initiation of cell differentiation, which was found to be concomitant with a significant increase in PGE 2 production mediated via the up-regulation of COX-1 mRNA and protein expression.

Hair Cell Development in Higher Vertebrates

Mammalian inner ear morphogenesis and production of hair cells are influenced by local cellular interactions and regulated by specific genes. In particular, Notch–Notch ligand interaction–mediated lateral inhibition plays an important role in the determination of hair cell fate. Inactivation or disruption of Notch signaling can lead to the production of supernumerary hair cells. Specific basic helix-loop-helix (bHLH) transcript factors have been shown to act downstream of Notch signaling and are essential for initial hair cell differentiation. Whereas Math1, a mouse homologue of the Drosophila gene atonal, is a positive regulator of hair cell differentiation, Hes1 and Hes5, mammalian hairy and enhancer of split homologues, act as negative regulators. These bHLH transcription factors are expressed in the inner ear epithelial region at the time when hair cell differentiation occurs. Whereas targeted deletion of Math1 gene leads to failure of hair cell differentiation, misexpression of Math1 induces production of extra hair cells, and cotransfection of Hes1 and Math1 in postnatal rat cochlear explant cultures results in an inhibition on hair cell differentiation induced by Math1. In addition, specific growth factors and cell cycle regulators that influence proliferation of sensory epithelial progenitors may also affect hair cell differentiation. Furthermore, other transcription factors, including Brn3c, Barhl1, and Gfi1, are required for further maturation and maintenance of hair cells. Understanding of hair cell differentiation mechanisms may provide useful hints for stimulating hair cell regeneration in mature inner ears, which could eventually be helpful for both recovery of hearing and recovery from balance impairment induced by hair cell loss.

Embryonic retinal gene expression in sonic-you mutant zebrafish.

Hedgehog (Hh) signaling is required for proper eye development in vertebrates; known roles for Hh in the zebrafish include regulation of eye morphogenesis, ganglion cell neurogenesis, and photoreceptor differentiation. To gain insight into the mechanisms by which Hh signaling influences these developmental events, we have examined proliferation, cell death, and expression patterns of several retinal genes in the eyes of embryonic zebrafish lacking the sonic hedgehog gene. We find that features of the eye phenotype of the sonic-you (syu) mutant are consistent with multiple roles for the Hh signal during retinal development. Most interestingly, half of the mutant retinas failed to initiate cell differentiation and, instead, retained a neuroepithelial appearance. In the other half of the mutants, retinal cell differentiation was initiated, but not fully propagated. We also find that Hh signaling is important for retinal cell proliferation and retinal cell survival; together, these functions provide an explanation for progressive microphthalmia in the syu-/- mutant.

Differential activation of the IGF binding protein-3 promoter by butyrate in prostate cancer cells.

Sodium butyrate (NaB), a dietary micronutrient, is a potent growth inhibitor that initiates cell differentiation in many cell types, including prostate cancer cells. The molecular mechanisms by which these effects occur remain largely unknown. In this study, we investigated the effects of NaB on the expression of IGF binding protein (IGFBP)-3, a known growth regulator, in two human prostate cancer cell lines (PC-3 and LNCaP). Treatment with NaB (0-10 mM) caused a dose-dependent stimulation of IGFBP-3 mRNA expression and parallel increases in protein levels. A specific histone deacetylase inhibitor, trichostatin A (TSA) similarly induced IGFBP-3 expression, indicating that histone hyperacetylation may be critical in the regulation of IGFBP-3 expression. To investigate the molecular mechanism of NaB-regulated IGFBP-3 expression, 1.87 kb of the human IGFBP-3 gene promoter was cloned into the pGL2-basic luciferase reporter vector. In both PC-3 and LNCaP cells, NaB (10 mM) significantly increased luciferase activity 20- to 30-fold, compared with the untreated control. However, using 5' sequential deletion constructs of the IGFBP-3 promoter, the NaB response sequences in the IGFBP-3 promoter were different in PC-3 and LNCaP cells. Our studies identified a region, -75 to +69 from the start of transcription (+1), that is fully inducible by NaB treatment in LNCaP cells, but not in PC-3 cells. Unlike other well characterized NaB-regulated genes, Sp1 DNA sequences are not involved in NaB up-regulation of IGFBP-3 gene in LNCaP cells. Further deletion studies identified two independent regions critical for NaB-induced transactivation in LNCaP cells. These regions contain consensus binding sites for p53 and GATA, respectively, but mutational analyses and gel shift assays suggested that, while the p53 response element is required for NaB responsiveness, neither p53 nor GATA are involved. In summary, we have demonstrated that 1) NaB significantly up-regulates IGFBP-3 mRNA and protein levels in PC-3 and LNCaP prostate cancer cells; and 2) novel butyrate- responsive elements lacking consensus Sp1 sites are used in LNCaP cells.

The use of fibrin beads for tissue engineering and subsequential transplantation.

New biological technologies such as tissue engineering procedures require the transplantation of functionally active cells within supportive carrier matrices. This paper describes a sequential culture procedure for different types of cells. The technique includes the initial preparation of a mixed alginate-fibrin vehicle that guaranteed an initial cell proliferation and differentiation to establish a stable matrix structure, and the subsequent removal of the alginate component prior to transplantation to circumvent the problem of missing bioresorbability. The resulting biodegradable carrier is mechanically stable and promotes further tissue maturation. Chondrocytes, periosteal-derived cells, as well as nucleus pulposus cells were entrapped in fibrin-alginate beads and in fibrin beads. The results indicate a promising technical approach to create stable transplants for reconstructive surgery of cartilage and bone.

Cell cycle phase, cellular Ca2+ and development in Dictyostelium discoideum.

In Dictyostelium discoideum, the initial differentiation of cells is regulated by the phase of the cell cycle at starvation. Cells in S and early G2 (or with a low DNA content) have relatively high levels of cellular Ca2+ and display a prestalk tendency after starvation, whereas cells in mid to late G2 (or with a high DNA content) have relatively low levels of Ca2+ and display a prespore tendency. We found that there is a correlation between cytosolic Ca2+ and cell cycle phase, with high Ca2+ levels being restricted to cells in the S and early G2 phases. As expected on the basis of this correlation, cell cycle inhibitors influence the proportions of amoebae containing high or low Ca2+. However, it has been reported that in the rtoA mutant, which upon differentiation gives rise to many more stalk cells than spores (compared to the wild type), initial cell-type choice is independent of cell cycle phase at starvation. In contrast to the wild type, a disproportionately large fraction of rtoA amoebae fall into the high Ca2+ class, possibly due to an altered ability of this mutant to transport Ca2+.

Effects of sodium butyrate on expression of members of the IGF-binding protein superfamily in human mammary epithelial cells.

Dietary factors play an important role in both the development and prevention of human cancers, including breast carcinoma. One dietary micronutrient, sodium butyrate (NaB), is a major end product of dietary starch and fiber, produced naturally during digestion by anaerobic bacteria in the cecum and colon. NaB is a potent growth inhibitor and initiates cell differentiation for many cell types in vitro. In this study, we investigated the effects of NaB on three human mammary epithelial cells and regulation of the IGF axis, specifically, IGF-binding protein-3 (IGFBP-3), a known growth regulator in human mammary cells, and IGFBP-related protein 2 (IGFBP-rP2)/connective tissue growth factor. NaB inhibited DNA synthesis, as measured by [3H]thymidine incorporation, in estrogen-responsive (MCF-7) and estrogen-non-responsive (Hs578T) breast cancer cells, and normal human mammary epithelial cells (HMEC) to a similar degree (up to 90% inhibition at 1-10 mM concentrations). Treatment of cells with NaB induced histone hyperacetylation, suggesting that NaB exerts its biological effects, at least in part, as a histone deacetylase inhibitor in mammary epithelial cells. Treatment of Hs578T cells with NaB caused an induction of apoptotic cell death. NaB treatment resulted in increased levels of p21(Waf1/Cip1) mRNA and protein in Hs578T cells and distinct upregulation of p27(Kip1) in HMEC, suggesting that NaB activates different genes involved in cell cycle arrest, depending upon the cell type. In the same context, among the IGFBP superfamily members tested, NaB specifically upregulated the expression of IGFBP-3 and IGFBP-rP2. These two proteins are known to be involved in inhibition of mammary epithelial cell replication. Northern blot analysis showed that NaB treatment at 1-10 mM concentrations caused a dose-dependent stimulation of IGFBP-3 mRNA expression in cancerous cells and IGFBP-rP2 mRNA expression in both cancerous and non-cancerous cells. Protein data from Western ligand blot and immunoblot analyses demonstrated parallel results. In summary, we have demonstrated that NaB (i) uniformly suppresses DNA synthesis in both cancerous and non-cancerous mammary cells, and (ii) upregulates IGFBP-3 and IGFBP-rP2 mRNA and protein levels in cancerous and non-cancerous mammary cells. These results provide the first demonstration that butyrate regulates the IGFBP system in the human mammary system.

Nevel Function of GTP-Binding Protein and Ribosome in Initiating Cell Differentiation in Streptomyces.

Nevel Function of GTP-Binding Protein and Ribosome in Initiating Cell Differentiation in Streptomyces.

A cyclin-dependent kinase-activating kinase regulates differentiation of root initial cells in Arabidopsis.

Cell division and differentiation continue throughout the plant life cycle without significant loss of control. However, little is known about the mechanisms that allow the continuous development of meristems. Cell division is controlled by a family of cyclin-dependent kinases (CDKs). CDK-activating kinases (CAKs) are known to phosphorylate and activate almost all CDKs and thus may have a crucial role in controlling CDK activities in each cell of the meristems. Here, we show that overexpression of sense or antisense gene for Cak1At in Arabidopsis by using the glucocorticoid-mediated transcriptional induction system resulted in a reduction of CDK activities. After 14-24 h of glucocorticoid treatment, starch granules appeared in columellar initials in the root meristem, and cortical initials were periclinally divided into cortical and endodermal cells. Accumulation of the cyclin beta-glucuronidase fusion protein ceased after 72 h of glucocorticoid treatment. Our results indicate that a change of Cak1At activity leads to differentiation of initial cells, followed by cessation of cell division. Therefore, we propose that differentiation of initial cells is controlled by Cak1At but is maintained independent of cell division.

Time course of foreign gene integration and expression in transgenic fish embryos

Using a nuclear transplantation approach, the integration and expression of the green fluorescent protein (GFP) gene in the embryogenesis of transgenic loach (Misgurnus anguillicaudatus Cantor) have been studied. TheGFP gene expression is first observed at the gastrula stage, which is consistent with the initiation of cell differentiation of fish embryos. The time course of the foreign gene expression is correlated with the regulatory sequences. The expression efficiency also depends on the gene configuration: the expression of pre-integrating circular plasmid at early embryos is higher than that of the linear plasmid. The integration of theGFP gene is first detected at the blastula stage and lasts for quite a long period. When two types of different plasmids are co-injected into fertilized eggs, the behavior of their integration and expression is not identical.

Interleukin-6 and interleukin-12 participate in induction of a type 1 protective T-cell response during vaccination with a tuberculosis subunit vaccine.

We examined the role of cytokines in the development of gamma interferon (IFN-gamma)-secreting protective T cells following immunization with a culture filtrate subunit vaccine against Mycobacterium tuberculosis containing the adjuvant dimethyldioctadecylammonium bromide (DDA). Depletion of either interleukin-6 (IL-6) or IL-12 with specific neutralizing antibodies during vaccination reduced the priming of T cells for antigen-specific proliferation and IFN-gamma secretion. Such reduction was also observed in IL-6 gene-disrupted mice as compared to wild-type animals. IL-6 was found to play a role in the initial differentiation of Th1 cells but not in their expansion. The defect found after IL-6 depletion or in IL-6-knockout mice was compensated by the inclusion of recombinant mouse IL-12 in the vaccine. The induction of protective immunity against an intravenous or an aerosol challenge with live, virulent M. tuberculosis was markedly reduced by neutralizing either IL-6 or IL-12 during immunization with the vaccine. Likewise, the effects of IL-6 neutralization were partially reversed by including IL-12 in the vaccine. Our data point to an important role of IL-6 and IL-12 in the generation of cell-mediated immunity to tuberculosis.

Mouse models of congenital cataract.

Mouse mutants affecting lens development are excellent models for corresponding human disorders. The mutant aphakia has been characterised by bilaterally aphakic eyes (Varnum and Stevens, J Hered 1968;59:147-50); the corresponding gene was mapped to chromosome 19 (Varnum and Stevens, Mouse News Lett 1975;53:35). Recent investigations in our laboratory refined the linkage of 0.6 cM proximal to the marker D19Mit10. Several candidate genes have been excluded (Chuk1, Fgf8, Lbp1, Npm3, Pax2, Pitx3). The Cat3 mutations are characterised by vacuolated lenses caused by alterations in the initial secondary lens fibre cell differentiation. Secondary malformations develop at the cornea and iris, but the retina remains unaffected. The mutation has been mapped to chromosome 10 close to the markers D10Mit41 and D10Mit95. Several candidate genes have been excluded (Dcn, Elk3, Ldc, Mell8, Tr2-11). The series of Cat2 mutations have been mapped close to the gamma-crystallin genes (Cryg; Löster et al., Genomics 1994;23:240-2). The Cat2nop mutation is characterised by a mutation in the third exon of Crygb leading to a truncated gamma B-crystallin and the termination of lens fibre cell differentiation. The Cat2 mutants are interesting models for human cataracts caused by mutations in the human CRYG genes at chromosome 2q32-35.

Early retinal development in the zebrafish, Danio rerio: Light and electron microscopic analyses

The morphological differentiation of the zebrafish retina was analyzed by using light (LM) and transmission electron (TEM) microscopy between the time of initial ganglion cell differentiation (≈32 hours postfertilization; hpf) and shortly after the point when the retina appears functional (≈74 hpf), i.e., when all major cell types and basic synaptic connections are in place. The results show that the inner retinal neurons, like the photoreceptor and ganglion cells, differentiate first within the ventronasal region, and differentiation subsequently spreads asymmetrically into the nasal and dorsal regions before reaching the ventrotemporal retina. In addition, we show that the attenuation of the optic stalk occurs in parallel with ganglion cell differentiation between 32 and 40 hpf. The first conventional synapses appear within the inner plexiform layer simultaneously with the first photoreceptor outer segment discs at 60 hpf; functional ribbon triads arise within photoreceptor synaptic terminals at 65 hpf; and synaptic ribbons occur within bipolar cell axon terminals at the time larvae exhibit their first visual responses (≈70 hpf). Although development is initially more advanced within the ventronasal region between 50 and 60 hpf, development across the retina rapidly equilibrates such that it is relatively comparable within all quadrants of the central retina by 70 hpf. An area within the temporal retina characterized by tightly packed and highly tiered cones emerges with subsequent development. Retinal differentiation in the zebrafish corresponds with that generally described in other vertebrates and can be correlated with the development of visual and electroretinographic responses in the animal. J. Comp. Neurol. 404:515–536, 1999. © 1999 Wiley-Liss, Inc.

Early retinal development in the zebrafish,Danio rerio: Light and electron microscopic analyses

The morphological differentiation of the zebrafish retina was analyzed by using light (LM) and transmission electron (TEM) microscopy between the time of initial ganglion cell differentiation (approximately 32 hours postfertilization; hpf) and shortly after the point when the retina appears functional (approximately 74 hpf), i.e., when all major cell types and basic synaptic connections are in place. The results show that the inner retinal neurons, like the photoreceptor and ganglion cells, differentiate first within the ventronasal region, and differentiation subsequently spreads asymmetrically into the nasal and dorsal regions before reaching the ventrotemporal retina. In addition, we show that the attenuation of the optic stalk occurs in parallel with ganglion cell differentiation between 32 and 40 hpf. The first conventional synapses appear within the inner plexiform layer simultaneously with the first photoreceptor outer segment discs at 60 hpf; functional ribbon triads arise within photoreceptor synaptic terminals at 65 hpf; and synaptic ribbons occur within bipolar cell axon terminals at the time larvae exhibit their first visual responses (approximately 70 hpf). Although development is initially more advanced within the ventronasal region between 50 and 60 hpf, development across the retina rapidly equilibrates such that it is relatively comparable within all quadrants of the central retina by 70 hpf. An area within the temporal retina characterized by tightly packed and highly tiered cones emerges with subsequent development. Retinal differentiation in the zebrafish corresponds with that generally described in other vertebrates and can be correlated with the development of visual and electroretinographic responses in the animal.

Distinct expression patterns of notch family receptors and ligands during development of the mammalian inner ear

The cochlea and vestibular structures of the inner ear labyrinth develop from the otic capsule via step-wise regional and cell fate specification. Each inner ear structure contains a sensory epithelium, composed of hair cells, the mechanosensory transducers, and supporting cells. We examined the spatio-temporal expression of genes in the Notch signaling pathway, Notch receptors (Notch1-4) and two ligands, Jagged1 and Delta1, in the developing mammalian inner ear. Our results show that Notch1 and Jagged1 are first expressed in the otic vesicle, likely involved in differentiation of the VIIIth nerve ganglion neurons, and subsequently within the inner ear sensory epithelia, temporally coincident with initial hair cell differentiation. Notch1 expression is specific to hair cells and Jagged1 to supporting cells. Their expression persists into adult. Notch2, Notch3, Notch4, and Delta1 are excluded from the inner ear epithelia. These data support the hypothesis that Notch signaling is involved in hair cell differentiation during inner ear morphogenesis.

Differential Expression of Protein Kinases in Cultured Primary Neurons Derived from the Cerebral Cortex, Hippocampus, and SympatheticGanglia

Protein kinases play pivotal roles in the development of the nervous system. They are involved in almost every stage of neuronal development, from initial proliferation and differentiation of progenitor cells to pathfinding of neurites and formation of synapses. Activation of protein kinases is also critical for neuronal cell survival. To gain further insights into kinases in neural development, we studied the expression patterns of protein kinases in three cultured primary neurons by degenerate primer-based reverse transcription-polymerase chain reaction (PCR) and DNA sequencing, taking advantage of all known kinases containing a conserved catalytic domain. Our data demonstrated that the expression patterns of kinases in various cultured neurons are not only different from those of non-neural tissues, but also distinct among neurons derived from discrete origins. For example, FGF receptor 1 is predominantly expressed in hippocampal neurons. As this approach may be biased during PCR and cloning steps, an RNase protection assay was employed to verify the expression levels of six kinases in cultured neurons. Results from the RNase protection assay did generally confirm those obtained by the PCR-based method. However, quantitative nature of the latter was dependent on numbers of clones analyzed, and discrepancy of expression levels of kinases detected by the two methods was sometimes observed.

Differential expression of protein kinases in cultured primary neurons derived from the cerebral cortex, hippocampus, and sympathetic ganglia.

Protein kinases play pivotal roles in the development of the nervous system. They are involved in almost every stage of neuronal development, from initial proliferation and differentiation of progenitor cells to pathfinding of neurites and formation of synapses. Activation of protein kinases is also critical for neuronal cell survival. To gain further insights into kinases in neural development, we studied the expression patterns of protein kinases in three cultured primary neurons by degenerate primer-based reverse transcription-polymerase chain reaction (PCR) and DNA sequencing, taking advantage of all known kinases containing a conserved catalytic domain. Our data demonstrated that the expression patterns of kinases in various cultured neurons are not only different from those of non-neural tissues, but also distinct among neurons derived from discrete origins. For example, FGF receptor 1 is predominantly expressed in hippocampal neurons. As this approach may be biased during PCR and cloning steps, an RNase protection assay was employed to verify the expression levels of six kinases in cultured neurons. Results from the RNase protection assay did generally confirm those obtained by the PCR-based method. However, quantitative nature of the latter was dependent on numbers of clones analyzed, and discrepancy of expression levels of kinases detected by the two methods was sometimes observed.