Gary Ciment

The Metalloproteinase Stromelysin-1 (Transin) Mediates PC12 Cell Growth Cone Invasiveness through Basal Laminae

Matrix metalloproteinases have been implicated in various extracellular matrix remodeling events that occur during normal development and in a number of pathologies. In previous work with PC12 rat pheochromocytoma cells, we found that the matrix metalloproteinase stromelysin-1 (ST1) was highly induced by nerve growth factor (NGF), but not by epidermal growth factor (EGF). Here, we show that ST1 immunoreactivity is present in growth cones of NGF-treated PC12 cells, but not EGF-treated or untreated cells. To determine whether ST1 expression confers neurite invasiveness, three lines of PC12 cells were produced that constitutively express ST1 antisense mRNA. These lines expressed and secreted significantly reduced levels of ST1 protein, as determined by immunoblot and immunocytochemical methods, but otherwise responded normally to NGF-treatment by elaborating neurites. We found, however, that the neurites of these ST1 antisense cells showed a significantly reduced ability to penetrate a Matrigel reconstituted basal lamina, as compared to the parental cells, suggesting that ST1 confers neurite invasiveness. Finally, we show that ST1 is also expressed in vivo in sections through Embryonic Day 15 rat embryos, including neurons of both the peripheral and central nervous systems. These data indicate that ST1 may play a role in axonal growth in vivo, including a role in growth cone invasiveness.

Cellular distribution, subcellular localization and possible functions of basic and acidic fibroblast growth factors

The distribution in the rat nervous system of acidic and basic fibroblast growth factors (FGFs) was analysed by a combination of biochemical and anatomical methods. Acidic FGF (aFGF) was found to be present exclusively in specific neuronal populations, such as motor neurons and basal forebrain cholinergic neurons. Basic FGF (bFGF) was found in astrocytes and in neurons in hippocampal area CA2. Within labelled astrocytes and CA2-neurons, bFGF was detected in both the cytoplasm and the nucleus. The levels of intracellular bFGF were manipulated by antisense oligonucleotide treatment of cultures of developing neural crest cells. Results indicated that the amount of melanogenesis in the cultures is likely to be regulated by intracellular, possibly nuclear bFGF.

Chicken growth-associated protein (GAP)-43: primary structure and regulated expression of mRNA during embryogenesis

Growth-associated protein (GAP)-43 is a neuron-specific phosphoprotein whose expression is associated with axonal outgrowth during neuronal development and regeneration. In order to investigate the expression of this gene product in the early developing nervous system we have isolated and sequenced a cDNA for chicken GAP-43. The predicted amino acid sequence for chicken GAP-43 displays extensive similarity to that of the mammalian protein, particularly in the amino-terminal region, to which functional domains of the protein have been assigned. The cDNA hybridizes with two RNAs of differing molecular weights on Northern blots; both appear to be regulated similarly. These RNAs first appear in the brain on embryonic day 3 (E3), suggesting that GAP-43 begins to be expressed when neuroblasts become post-mitotic. In situ hybridization analysis reveals that GAP-43 RNA is expressed by several neural structures in the chick embryo, including derivatives of the neural tube, neural crest, and neuroectodermal placodes.

Transient expression of GAP-43 in nonneuronal cells of the embryonic chicken limb

Growth associated protein (GAP)-43 is a membrane-bound phosphoprotein expressed in neurons and is particularly abundant during periods of axonal outgrowth in development and regeneration of the nervous system. In previous work, we cloned a full-length chicken GAP-43 cDNA and described the expression of its corresponding mRNA during early development of the chicken nervous system. We report here that the GAP-43 mRNA is also expressed transiently in developing limbs of chicken embryos, which contain axons of spinal cord and dorsal root ganglion neurons, but do not contain neuronal cell bodies. GAP-43 mRNA was first detectable by RNA blot analysis in limbs from Embryonic Day 5 (E5) embryos, reached maximal levels between E6 and E8, and diminished by E10. In situ hybridization analysis showed that the GAP-43 mRNA was localized in distal regions of developing limbs and was particularly abundant in the mesenchyme surrounding the digital cartilage. In some regions of the limb, GAP-43 immunoreactivity colocalized in cells that were also immunoreactive for meromyosin, a muscle-specific marker. These data suggest that both GAP-43 mRNA and the protein are expressed in nonneuronal cells of the developing limb, some of which may be part of the muscle cell lineage.

Changes in the migratory properties of neural crest and early crest-derived cells in vivo following treatment with a phorbol ester drug☆

In previous work, we found that the phorbol ester drug 12-O-tetradecanoyl phorbol acetate (TPA) reversed the developmental restriction of melanogenesis that normally occurs in neural crest-derived Schwann cell precursors around embryonic Day 5 of quail development. That is, TPA treatment of dorsal root ganglia (DRG) from 7-day quail embryos caused Schwann cell precursors to regain the ability to give rise to melanocytes. In this paper, we examine other long-term effects of TPA on the differentiative and migratory properties of neural crest and crest-derived DRG cells, using heterospecific grafting methods. We report that TPA treatment in culture increased the extent of cell migration following grafting into host embryos, including some ectopic migration into the central nervous system and other locations. TPA did not, however, seem to change the fate of these crest-derived cells, except that some DRG cells underwent pigmentation, as had been observed previously. Interestingly, graft cells associated with peripheral nerves were found to be exclusively unpigmented, whereas graft cells found in all other locations, including the central nervous system, were both pigmented and unpigmented. This suggests that peripheral nerves may act in a fashion antagonistic to the effects of TPA. These findings are consistent with the notion that TPA treatment causes early crest-derived cells to regain developmental properties lost with developmental age.

Regulated expression of the neurofibromin type I transcript in the developing chicken brain

Neurofibromatosis type 1 (NF‐1) is among the most common inherited diseases affecting cells of the central and peripheral nervous systems. A region of the NF‐1 gene is similar in sequence to the ras‐GTPase activator protein (ras‐GAP), and investigations have confirmed that the NF‐1 gene product (now known as neurofibromin) stimulates ras‐GTPase activity in vitro and in vivo. Neurofibromin modulates the ability of ras proteins to regulate cellular proliferation and/or differentiation, suggesting a possible role in normal development. An alternative form of the neurofibromin transcript with an additional 63‐bp exon inserted in the GAP‐related domain (GRD) has been described recently. To determine whether differential expression of the two forms of neurofibromin GRD mRNA plays a role in embryonic development, we have isolated and characterized the corresponding chicken cDNA. The predicted amino acid sequence for the inserted exon is identical between chick and human, as are the exon‐intron boundaries. RNase protection and RNA‐polymerase chain reaction analyses demonstrate that most tissues express predominantly type II mRNA (which contains the insert) throughout embryonic development. In contrast, whereas type II is the major form in the brain early in development, expression of the type I transcript (without the insert) in this tissue increases dramatically at later times. Analysis of primary cultures derived from chick embryo brain indicates that the type I mRNA is enriched in neurons.

Analysis of the sequence and embryonic expression of chicken neurofibromin mRNA

Neurofibromatosis type 1 (NF1) is a common inherited disorder that primarily affects tissues derived from the neural crest. Recent identification and characterization of the human NF1 gene has revealed that it encodes a protein (now called neurofibromin) that is similar in sequence to the ras-GTPase activator protein (or ras-GAP), suggesting that neurofibromin may be a component of cellular signal transduction pathways regulating cellular proliferation and/or differentiation. To initiate investigations on the role of the NF1 gene product in embryonic development, we have isolated a partial cDNA for chicken neurofibromin. Sequence analysis reveals that the predicted amino acid sequence is highly conserved between chick and human. The chicken cDNA hybridizes to a 12.5-kb transcript on RNA blots, a mol wt similar to that reported for the human and murine mRNAs. Ribonuclease protection assays indicate that NF1 mRNA is expressed in a variety of tissues in the chick embryo; this is confirmed by in situ hybridization analysis. NF1 mRNA expression is detectable as early as embryonic stage 18 in the neural plate. This pattern of expression may suggest a role for neurofibromin during normal development, including that of the nervous system.

Role of the bZIP transcription factor IREBF1 in the NGF induction of stromelysin-1 (transin) gene expression in PC12 cells.

Stromelysin-1 (ST-1) is one of the most nerve growth factor- (NGF) responsive gene products expressed in PC12 cells. In previous work, we identified a novel NGF-responsive element in the proximal promoter region of the ST-1 gene that participates in this induction, and showed that it bound a protein present in the nuclei of PC12 cells. Here, we identify a transcription factor that specifically recognizes this regulatory element—the interferon-response element binding factor-1 (IREBF1), a member of the basic leucine zipper gene family. We show that IREBF1 is constitutively expressed in PC12 cells and that overexpression of IREBF1 augments NGF-responsive ST-1 gene regulation, but does not affect basal levels of expression. On the other hand, expression of a mutated form of this transcription factor lacking the DNA binding domain attenuated NGF responsiveness, without affecting basal levels of expression. These data suggest that IREBF1 is part of the NGF-responsive transcriptional machinery necessary for the expression of ST-1 in PC12 cells.

Precocious Expression of NAPA‐73, an Intermediate Filament‐Associated Protein, during Nervous System and Heart Development in the Chicken Embryo

A monoclonal antibody was generated, against early neural crest-derived cells, which recognizes an epitope present on a novel intermediate filament-associated protein. This protein has been named NAPA-73 and is expressed by progenitor cells of the nervous system and heart. Biochemical and ultrastructural studies indicate that this protein associates with bundles of intermediate filaments and therefore may play a role in the determination of cell shape.