E2F2 converts reversibly differentiated PC12 cells to an irreversible, neurotrophin-dependent state.

E2Fs play a central role in cell proliferation and growth arrest through their ability to regulate genes involved in cell cycle progression, arrest and apoptosis. Recent studies further indicate that this family of transcriptional regulators participate in cell fate/differentiation events. They are thus likely to have a prominent role in controlling the terminal differentiation process and its irreversibility. Here we have specifically examined the role of E2F2 in neuronal differentiation using a gain-of-function approach. Endogenous E2F2 increased in PC12 cells in response to nerve growth factor (NGF) and was also expressed in cerebellar granule neurons undergoing terminal differentiation. While PC12 cells normally undergo reversible dedifferentiation and cell cycle re-entry upon NGF removal, forced expression of E2F2 inhibited these events and induced apoptosis. Thus, E2F2 converted PC12-derived neurons from a reversible to a 'terminally' differentiated, NGF-dependent state, analogous to postmitotic sympathetic neurons. This contrasts with the effects of E2F4, which enhances the differentiation state of PC12 cells without affecting cell cycle parameters or survival. These results indicate that E2F2 may have a unique role in maintaining the postmitotic state of terminally differentiated neurons, and may participate in apoptosis in neurons attempting to re-enter the cell cycle. It may also be potentially useful in promoting the terminally arrested/differentiated state of tumor cells.

Cloning and interspecies comparisons of three newt (Notophthalmus viridescens) fibroblast growth factor receptor sequences.

We report the nucleotide sequences of two fibroblast growth factor receptor (FGFR) cDNAs, FGFR1 and FGFR3, from the newt species Notophthalmus viridescens. These two cDNA sequences and a previously published newt FGFR cDNA, FGFR2, were used to derive the amino acid sequences which were then compared with their homologues from other species. This comparison shows that the intracellular tyrosine kinase domain is highly conserved across the species examined with the second half of the domain slightly more conserved than the first half. The 3' portion of the carboxyl terminal tail is not very highly conserved. The comparison of the extracellular portion of FGFR2 shows a high degree of conservation among the Ig-like domains and a low degree of conservation in the region that links the third Ig-like domain with the transmembrane domain.

Re-programming of expression of the KGFR and bek variants of fibroblast growth factor receptor 2 during limb regeneration in newts (Notophthalmus viridescens).

We have previously shown, by in situ hybridization, that fibroblast growth factor receptor 2 (FGFR2) is present in the basal layer of wound epithelium during limb regeneration in newts (Notophthalmus viridescens). In contrast, FGFR1 expression is observed throughout the blastema mesenchyme but is distinctly absent from the wound epithelium (Poulin et al. [1993] Development 119:353-361). Sequence analysis revealed that we have isolated both the KGFR and bek variants of FGFR2. These two variants differ only in the second half of the last of their three (or two) Ig-like domains. In this report, we show the expression patterns of FGFR2 variants during limb regeneration by in situ hybridization. During the pre-blastema stages of regeneration, FGFR2 expression was observed in the basal layer of the wound epithelium and in the cells of the periosteum. The wound epithelial hybridization was observed when the KGFR-specific probe was used while the bek-specific probe hybridized to mRNA in the cells of the periosteum. As regeneration progresses to the blastema stages, KGFR expression continued to be observed in the basal layer of the wound epithelium with additional hybridization seen in the blastema mesenchyme closely associated with the bisected bones. The bek-specific hybridization pattern observed at this stage corresponds specifically to the mesenchymal hybridization. In the differentiation stages of regeneration, the mesenchymal expression of FGFR2 becomes restricted to the cells of the condensing cartilage and later to the perichondrium. Interestingly, there appears to be a dorsoventral gradient of the expression of both KGFR and bek variants of FGFR2, which are opposite each other at the later stages of regeneration. Thus, re-programming of expression of the two FGFR2 variants is required during the initial wound closure of limb regeneration. Remarkably, the expression patterns of KGFR and bek mimic those observed in the mouse limb bud during early embryonic development (Orr-Urtreger et al. [1993] Dev. Biol. 18:475-486). Moreover, our results suggest that the two FGFR2 variants have distinct roles in limb regeneration. Further investigation regarding the potential sources of the FGF ligands will help establish the roles that FGFs and FGFRs play in limb regeneration.

Nucleotide sequences of two newt (Notophthalmus viridescens) fibroblast growth factor receptor-2 variants.

The FGF receptor tyrosine kinase family consists of four members. We report the sequence of two newt (Notophthalmus viridescens) FGFR2 cDNAs which were isolated from a forelimb blastema cDNA library and represent the newt cognates of two different isoforms of FGFR2, one homologous to bek the other to the KGFR.

Heterogeneity in the expression of fibroblast growth factor receptors during limb regeneration in newts (Notophthalmus viridescens).

Two closely related fibroblast growth factor receptors, FGFR1 and FGFR2, have been cloned from a newt (Notophthalmus viridescens) limb blastema cDNA library. Sequence analysis revealed that we have isolated both the bek and KGFR variants of FGFR2. These two variants differ only in the second half of the last of their three Ig-like domains. The expression patterns of FGFR1 and FGFR2 during limb regeneration have been determined by in situ hybridization. During the preblastema stages of regeneration, FGFR2 expression is observed in the basal layer of the wound epithelium and in the cells of the periosteum. As regeneration progresses to the blastema stages, FGFR2 expression continues to be observed in the basal layer of the wound epithelium with additional hybridization seen in the blastema mesenchyme closely associated with the bisected bones. From the early bud to the mid-bud blastema stage, FGFR1 expression is observed throughout the blastema mesenchyme but, unlike FGFR2, is distinctly absent from the wound epithelium. In the differentiation stages of regeneration, the mesenchymal expression of FGFR2 becomes restricted to the cells of the condensing cartilage and later to the perichondrium. During these later stages of regeneration, the wound epithelium hybridization to the FGFR2 probe is no longer observed. The expression patterns of these receptors suggest that FGFR1 and FGFR2 have distinct roles in limb regeneration, despite their sharing a number of the FGF ligands. Further investigation regarding the potential sources of the FGF ligands will help establish the role that FGFs and FGFRs play in limb regeneration.

Characterization of a newt tenascin cDNA and localization of tenascin mRNA during newt limb regeneration by in situ hybridization.

We previously showed that tenascin, a large, extracellular matrix glycoprotein, exhibits a temporally and spatially restricted distribution during urodele limb regeneration. To further investigate the role of tenascin in regeneration, we cloned a newt tenascin cDNA, NvTN.1, that has 70% homology to the chicken tenascin sequence. A deduced amino acid sequence of NvTN.1 showed a modular structure unique to tenascin characterized by epidermal growth factor-like and fibronectin type III repeats. To determine the cellular origin of tenascin protein during limb regeneration, we localized tenascin transcripts by in situ hybridization using a riboprobe synthesized from NvTN.1. Transcripts could not be detected in normal limb tissues but first became detectable in the wound epithelium at 2 days and in the distal mesoderm at 5 days after amputation. These wound epithelial cells are probably the source of tenascin protein found within and immediately underneath the wound epithelium. At preblastema stages, hybridization was seen in cells associated with most of the distal mesodermal tissues but not in dermis. At blastema stages, essentially every mesenchymal cell contained tenascin transcripts. Thus, regardless of origin, blastemal mesenchymal cells may share a common regulatory mechanism that results in tenascin gene transcription. Finally, during redifferentiation stages of regeneration, tenascin gene transcription was associated with both differentiation and growth. The results show that initiation of tenascin gene expression is an early event in regeneration and continued tenascin gene transcription is associated with some of the important processes of regeneration, namely wound epithelial-mesenchymal interactions, dedifferentiation, initiation of cell cycling, blastema outgrowth, and cellular differentiation.