Proliferation and regeneration of retrogradely labeled adult rat corticospinal neurons in culture.

These are the first studies to demonstrate that adult rat corticospinal tract (CST) neurons, which were identified by retrograde neuronal labeling, retain regenerative and proliferative potential. To determine if adult CST neurons undergo cell division, we tested if these retrogradely labeled cells synthesize DNA by adding BrdU to the cultures 24 h prior to fixation of the cells. The result shows that adult corticospinal tract neurons are capable of DNA synthesis, and our total cell counts with labeled cells counts further suggest that these cells undergo cell division.

Overexpression of E2F1 in astrocytes leads to neoplastic transformation and changes in expression of retinoblastoma family members.

Overexpression of mouse E2F1 full-length but not truncated forms results in neoplastic transformation of astrocytes in vitro. This neoplastic transformation is accompanied with changes in cell morphology and expression of cell cycle regulators. Transformed astrocytes have higher expression of cdk2, pRb, and p107 than control astrocytes. However, expression of glial fibrillary acidic protein (GFAP) and p130 is reduced in transformed astrocytes.

Cell type specific regulation of COUP-TF II promoter activity.

COUP-TF family orphan receptors regulate activity of ligand-activated nuclear hormone receptors or function independently in the regulation of gene expression. COUP-TF II has a complex expression pattern suggesting that different mechanisms are involved in the regulation of its expression. We isolated the 5' regulatory region of the mouse COUP-TF II gene and demonstrated that the basal promoter is localized in a -200 bp region 5' from the transcription start site. All-trans retinoic acid and dibutyryl cyclic AMP have cell type specific effects on COUP-TF II promoter activity. The effect of cyclic AMP is mediated by the cyclic AMP response element that is localized 74 nucleotides upstream from the major transcriptional start. In vitro promoter analyses also demonstrated that the effect of all-trans RA is not directly mediated by the binding of RARs or RXRs to the promoter sequence.

All-trans retinoic acid affects the expression of orphan receptors COUP-TF I and COUP-TF II in the developing neural tube.

The effect of all-trans retinoic acid (RA) on chicken ovalbumin upstream promoter-transcription factor (COUP-TF) I and COUP-TF II expression in the developing cervical spinal cord and telencephalon was examined using embryonic day 11 and 13 mice. All-trans RA treatment results in changes in expression of COUP-TF I and COUP-TF II genes in the spinal cord and telencephalon. COUP-TF I mRNA levels were reduced in the spinal cord ventricular zone (VZ) after treatment, whereas in the telencephalon, mRNA levels were unaffected in the VZ and increased in the intermediate zone (IZ). COUP-TF II mRNA levels were increased in the spinal cord VZ after treatment, while in the telencephalon, IZ mRNA levels were decreased in the E11 embryos and increased in E13 embryos.

Helix-loop-helix transcription factors regulate Id2 gene promoter activity.

Id-like helix-loop-helix (HLH) transcription factors are involved in the regulation of proliferation and differentiation of several cell types. We isolated 5' regulatory region of mouse Id2 gene and demonstrated that it contains several E-box clusters. These E-boxes mediate stimulatory effects of basic-HLH (bHLH) transcription factors ME1, ME2, and NSCL1 on Id2 promoter activity. Co-expression of Id2 blocks the stimulatory effect of bHLH transcription factors which suggests the presence of feedback loops in Id2 transcriptional regulation. Overexpression of NSCL1 in F9 cells blocks the downregulation of Id2 gene expression during retinoic acid induced differentiation. Our data demonstrate that bHLH transcription factors regulate Id2 gene expression.

Orphan receptor COUP-TF I antagonizes retinoic acid-induced neuronal differentiation.

Chicken ovalbumin upstream promoter-transcription factors (COUP-TF) are expressed in the developing nervous system and interact with nuclear hormone receptors to regulate expression of different genes. The role of COUP-TF orphan receptors in neurogenesis is virtually unknown. To study the possible function of COUP-TF I during neuronal differentiation, we generated COUP-TF I overexpressing teratocarcinoma PCC7 cell lines and analyzed retinoic acid (RA)-induced neuronal differentiation of these cells. COUP-TF I overexpression results in the blockade of morphological differentiation after induction to differentiate. COUP-TF I represses expression of microtubule-associated protein 2 (MAP2) gene and delays induction of growth-associated protein 43 (GAP43) gene expression. In contrast, expression of the neurofilament light subunit (NF-L) gene is not affected by COUP-TF I overexpression during neuronal differentiation. Also, cells overexpressing COUP-TF I do not stop proliferating after RA and dBcAMP treatment and possess suppressed transcriptional activation from different RA response elements. These results suggest that COUP-TF I plays an important role in regulating RA-induced neuronal differentiation.

Cyclin E is expressed in neurons and forms complexes with cdk5.

Expression of cyclin dependent kinase cdk5 and its regulator p35 has been shown in the cytoplasm of adult neurons. Here we demonstrate that another potential regulator of cdk5, cyclin E, is expressed in the nervous system and forms complexes with cdk5. Western blot analyses identifies expression of two forms of cyclin E in the mouse nervous system with the 56 kDa form mainly expressed in neurons and 51 kDa form expressed in astrocytes and oligodendrocytes.

Expression of Rb, E2F1, cdc2, and D, and B cyclins in developing spinal cord.

The spatial and temporal distribution of transcripts for the tumor suppressor gene Rb, transcription factor E2F1, cdc2 kinase, cyclins D1, D2, B1 and B2 during neurogenesis of the spinal cord was determined by in situ hybridization. The Rb and E2F1 transcripts were detectable in proliferating and differentiating cells. By contrast, cdc2, cyclins D1, B1 and B2 are expressed in the ventricular zone where proliferating cells are localized. Cyclin D2 mRNA was detectable only in the marginal zone of the developing neural tube. Electrophoretic mobility shift analyses demonstrated a changing pattern of DNA/protein complexes that bind to E2F binding site. These observations suggest that Rb and E2F1 may be involved in the early stages of neuronal differentiation in addition to the cell cycle regulation.

Isolation of genes which block neuronal differentiation of teratocarcinoma PCC7 cells.

Since several viral oncoproteins block differentiation and induce proliferation of differentiated cells, we developed an expression screening method to isolate cDNAs which block neuronal differentiation and induce proliferation of teratocarcinoma cells. Mouse E2F1, RNP-1, and RNP-2 (Regulator of Neuronal Proliferation) were isolated using the developed screening method. Overexpression of E2F1, RNP-1, and RNP-2 cDNAs in neuronally differentiated teratocarcinoma PCC7 cells results in blocking differentiation and initiation of proliferation. Also, expression of RNP-1 and RNP-2 blocks the expression of neurofilament-L and GAP-43 genes in PCC7 cells.

DNA synthesis is induced in adult neurons after expression of E2F1 and E1A.

Differentiated neurons in several brain regions express the retinoblastoma (Rb) tumor suppressor gene. Changing the tumor suppressor function of Rb by expressing transcription factor E2F1 and viral oncoprotein E1A in cerebellar granular neurons in vitro and in cerebral cortical neurons in vivo results in the induction of DNA synthesis in these neurons. Immunoliposome-mediated transfection of E1A and E2F1 cDNAs into the adult cortical neurons of rats in vivo results in initiation of DNA synthesis in 5-15% of the transfected neurons. These results indicate that expression of Rb may be necessary to prevent induction of differentiated neurons to proliferate since many mitogenic growth factors are expressed in the brain.

Class A basic helix-loop-helix transcription factors in early stages of chick neural tube development: evidence for functional redundancy.

Basic Helix-Loop-Helix (bHLH) transcription factors play essential role in differentiation of several cell types including neurons. We isolated chick bHLH transcription factor E12 cDNA and demonstrated its expression in developing neural tube interneurons. To examine the function of class A bHLH transcription factors we blocked their expression using antisense oligonucleotides. Simultaneous blocking of class A bHLH transcription factors in cultured neural tube cells results in the reduction of differentiating neurons. Blocking the expression of individual class A bHLH transcription factors has no detectable effect. These results demonstrate that class A bHLH transcription factors have a functional redundancy during neuronal development.

Effects of cultured adrenal chromaffin cell implants on hindlimb reflexes of the 6-OHDA lesioned rat.

The effects of implantation of cultured adrenal medullary cells on the recovery of neurotransmitter specific reflex activity were studied in the rat spinal cord using electrophysiological testing methods. Cell suspensions of cultured neonatal adrenal medullary chromaffin (AM) cells (which produce catecholamines), or Schwann (Sc) cells (controls) were implanted into the lumbar region of the spinal cord 2 weeks after catecholamine (CA) denervation by intracisternal injection of 6-hydroxydopamine (6-OHDA). All cells were taken from 7 day neonates and cultured for 10 days in the presence of nerve growth factor (NGF). Three months after implantation, the extent of implant-associated recovery of reflex activity was determined by measuring electromyogram (EMG) activity and force associated with the long latency component of the hindlimb withdrawal reflex (which is CA modulated). After the electrophysiological testing, rats were anesthetized, and the spinal cords were rapidly removed and frozen. Spinal cords were sectioned longitudinally, and implanted cells were visualized using glyoxylic acid techniques. Labelled sections were examined to determine cell survival. Results indicate that 1) chromaffin cells survive for 3 months in the segments of the cord into which they have been implanted and 2) rats implanted with AM cells have significantly more forceful withdrawal reflexes than those that received Sc cells or received no implant after lesioning.

Neuronal expression of regulatory helix-loop-helix factor Id2 gene in mouse.

Id-like helix-loop-helix (HLH) proteins, which lack a basic DNA binding domain, have been suggested to serve as general inhibitors of differentiation. We present data that Id2 is expressed in specific cell types during neurogenesis and in the adult. At early stages of neurogenesis, Id2 is expressed in the ventricular zone of neuroepithelium. After the first neuronal populations are born, the expression of Id2 is down regulated in neuroepithelial cells and continues to be high in Purkinje cells of the cerebellum, in mitral cells of the olfactory bulb, and in layers 2, 3, and 5 of the cerebral cortex. In neuronally differentiating cell lines, the Id2 expression is up regulated (PCC7), down regulated (NG108), or unchanged (N18) during differentiation. Analyses of promoter sequences of the Id2 gene identify the region which is responsible for the down regulation of transcription during neuronal differentiation. Our data indicate that Id2 has different functions in different cell types during neurogenesis.

ME1 and GE1: basic helix-loop-helix transcription factors expressed at high levels in the developing nervous system and in morphogenetically active regions.

Several class A basic helix-loop-helix (bHLH) transcription factors have been cloned from the developing mouse and chick nervous system. The cloned cDNAs (ME1, ME2, ME3, ME4, in the mouse and GE1, GE2 in the chick) have HLH coding regions highly homologous to other known class A bHLH genes. The genes corresponding to ME1 and GE1 are abundantly expressed during development of the central nervous system. ME1 and GE1 are expressed in proliferating neuroblasts and in cells at the initial stages of differentiation (for example in the external granule cell layer of the cerebellum and in the lateral region of the ventricular zone in the developing neural tube and cortex). They are also expressed at high levels in morphogenetically active regions such as limb buds, somites and mesonephric tubules. The expression of ME1 and GE1 decreases once cellular differentiation is over. Based on the expression of ME1 and GE1 in regions of active cellular proliferation and differentiation and on the known role of other bHLH factors in development, we suggest that ME1 and GE1 play important roles during development of the nervous system as well as in other organ systems.

Pax2, a new murine paired-box-containing gene and its expression in the developing excretory system.

The murine genome contains multiple genes with protein domains homologous to the Drosophila paired box, present in certain segmentation genes. At least one of these murine paired box (Pax) genes is associated with a developmental mutation. This report, in conjunction with the accompanying paper, describes a second member of this gene family, Pax2, that is also expressed during embryogenesis. Two overlapping cDNA clones were isolated and sequenced. At least two forms of the Pax2 protein can be deduced from the cDNA sequence. In addition to the highly conserved paired domain, an octapeptide sequence is located downstream. Expression of Pax2 is primarily restricted to the developing embryo in the excretory and central nervous systems. The transient nature of Pax2 expression during kidney organogenesis correlates with polarization and induction of epithelial structures and may indicate an important morphogenetic role for this gene.

Spatially and temporally restricted expression of Pax2 during murine neurogenesis.

The expression of the murine paired-box-containing gene, Pax2, is examined in the developing central nervous system by in situ hybridization. Pax2 expression is detected along the boundaries of primary divisions of the neural tube. Initially, Pax2 is expressed in the ventricular zone in two compartments of cells on either side of the sulcus limitans and along the entire rhombencephalon and spinal cord. At later times, Pax2 is restricted to progeny cells that have migrated to specific regions of the intermediate zone. In the eye, Pax2 expression is restricted to the ventral half of the optic cup and stalk and later to the optic disc and nerve. In the ear, expression is restricted to regions of the otic vesicle that form neuronal components. The transient and restricted nature of Pax2 expression suggests that this murine segmentation gene homologue may also establish compartmental boundaries and contribute to the specification of neuronal identity, as do certain Drosophila segmentation genes.

A neurotransmitter specific functional recovery mediated by fetal implants in the lesioned spinal cord of the rat.

In the electrophysiologic test of the hindlimb withdrawal reflex where the long latency component is found to be dependent on noradrenaline (NA), implants of fetal NA tissue in lesioned animals result in a recovery of NA dependent function proportional to the number of catecholamine fluorescent cells in the implants.

Avoidance training using an incremental vertical jump response in rats.

A procedure is described for conditioning a vertical jump avoidance response in rats. Employing an auditory signal and gradually increasing the height of the response platform, rats showed rapid acquisition of a jump response to avoid shock. While the average maximum height of the jump response increased between original training and retraining 7 days later, retraining 40 days later resulted in stable responding that was consistent with the first retraining session.