Conservation and evolution of transcriptional mechanisms in eukaryotes.

Eukaryotic transcriptional activators play key roles in controlling cell growth and specifying embryonic development. These activators can stimulate promoters from distances up to tens of kilobases by a mechanism that is remarkably conserved in eukaryotes ranging from yeast to humans. Although the primary sequence of certain activators has also been conserved in widely divergent organisms, the regulatory roles that these factors play have been altered over evolution to fit the specific needs of the host.

Expression of neurogenic basic helix-loop-helix genes in primitive neuroectodermal tumors.

The basic helix-loop-helix (bHLH) class of transcription factors plays a pivotal role in tissue-specific determination and differentiation. Moreover, dysregulated expression or loss of function of these factors contributes to leukemogenesis and solid tumor development. Neurogenesis is regulated by genes of the NEUROD/atonal and ACHAETE SCUTE families. We analyzed expression of human NEUROD1, NEUROD2, NEUROD3, and ACHAETE SCUTE 1 (HASH1) in cerebellar and cerebral primitive neuroectodermal tumors (PNETs), gliomas, and cell lines derived from a variety of neuroectodermal tumors by Northern analysis and in situ hybridization. NEUROD1 was expressed in each of the 12 medulloblastoma specimens, whereas NEUROD2 and NEUROD3/neurogenin were expressed in partly overlapping subsets of medulloblastomas. All of the tumors that presented with distant metastases expressed NEUROD3. The only other NEUROD3-positive tumor progressed early in treatment. Human ACHAETE SCUTE homologue (HASH1) was not expressed in medulloblastomas (infratentorial PNETs) but was expressed in three of five supratentorial PNETs. Neuroectodermal tumor cell lines derived from other sites (e.g., neuroblastoma and retinoblastoma) expressed NeuroD and ACHAETE SCUTE family members. No NEUROD message was detected in glial tumors or cell lines. Neurogenic bHLH transcription factor expression patterns suggest that specific family members may contribute to or reflect biological differences that arise during malignant transformation.

Expression of multiple neuregulin transcripts in postnatal rat brains.

The distribution of neuregulin transcripts in rat brains was studied by both RNA blotting and in situ hybridization. Our data demonstrate that multiple neuregulin transcripts are expressed in neurons of the basal forebrain, the hippocampus, the diencephalon, the cerebellum, the brainstem, and the spinal cord. Developmental changes in the distribution of neuregulin transcripts were observed only in the cerebellum and the hippocampus. The intense neuregulin hybridization signals in the brainstem motor and sensory nuclei and the spinal motor neurons are suggestive of a functional involvement of neuregulins in motor and sensory systems. The expression of neuregulins in other parts of the brain also indicates that these factors are involved in a variety of central nervous system functions.

Ontogenetic expression of trk neurotrophin receptors in the chick auditory system.

Neurotrophins and their cognate receptors are critical to normal nervous system development. Trk receptors are high-affinity receptors for nerve-growth factor (trkA), brain-derived neurotrophic factor and neurotrophin-4/5 (trkB), and neurotrophin-3 (trkC). We examine the expression of these three neurotrophin tyrosine kinase receptors in the chick auditory system throughout most of development. Trks were localized in the auditory brainstem, the cochlear ganglion, and the basilar papilla of chicks from embryonic (E) day 5 to E21, by using antibodies and standard immunocytochemical methods. TrkB mRNA was localized in brainstem nuclei by in situ hybridization. TrkB and trkC are highly expressed in the embryonic auditory brainstem, and their patterns of expression are both spatially and temporally dynamic. During early brainstem development, trkB and trkC are localized in the neuronal cell bodies and in the surrounding neuropil of nucleus magnocellularis (NM) and nucleus laminaris (NL). During later development, trkC is expressed in the cell bodies of NM and NL, whereas trkB is expressed in the nerve calyces surrounding NM neurons and in the ventral, but not the dorsal, dendrites of NL. In the periphery, trkB and trkC are located in the cochlear ganglion neurons and in peripheral fibers innervating the basilar papilla and synapsing at the base of hair cells. The protracted expression of trks seen in our materials is consistent with the hypothesis that the neurotrophins/tyrosine kinase receptors play one or several roles in the development of auditory circuitry. In particular, the polarized expression of trkB in NL is coincident with refinement of NM terminal arborizations on NL.

The copper, zinc-superoxide dismutase gene of Saccharomyces cerevisiae: cloning, sequencing, and biological activity.

The gene for copper, zinc-superoxide dismutase (Cu,Zn-SOD; EC 1.15.1.1) from the yeast Saccharomyces cerevisiae has been cloned, sequenced, and shown to have physiological activity. The gene was isolated from a lambda gt11 library by using a long, unique deoxyoligonucleotide probe. The probe sequence was deduced from the known amino acid sequence by using a computer-generated yeast codon preference table. The sequence of the coding and flanking regions is reported. The cloned gene was expressed and shown to be active in vivo. A 3.2-kilobase fragment containing the coding region and 160 upstream bases, subcloned in a yeast/Escherichia coli shuttle vector, was used to transform a yeast strain lacking Cu,Zn-SOD activity. The presence of the Cu,Zn-SOD gene-containing plasmid corrected the characteristic dioxygen sensitivity of this strain. Electrophoretic transfer blots with antibody to yeast Cu,Zn-SOD showed the presence of the protein in transformants and wild-type yeast but not in the mutant. The role of Cu,Zn-SOD in defense against dioxygen toxicity is discussed in the light of these findings.

Antibody-promoted dimerization bypasses the regulation of DNA binding by the heme domain of the yeast transcriptional activator HAP1.

The yeast transcriptional activator HAP1 contains a DNA-binding domain homologous to the zinc finger of GAL4 and an adjacent regulatory domain that blocks DNA binding in the absence of the inducer heme. We show that short HAP1 fragments containing the zinc finger are unable to bind to DNA but can be rescued by antibody to the HAP1 zinc finger. These fragments are missing a coiled-coil sequence similar to that within the dimerization domain of GAL4 and dimerization domains of myosin heavy chain. We surmise that the antibody promotes DNA binding by bringing together two monomers. Interestingly, the antibody will also promote DNA binding of a larger HAP1 fragment containing the DNA-binding and the heme-regulatory domains. This suggests that the regulatory domain acts by preventing dimerization of HAP1 in the absence of heme. Consistent with this view is an in vivo assay that also reveals that heme promotes HAP1 dimerization in yeast cells.

Effects of GGF/neuregulins on neuronal survival and neurite outgrowth correlate with erbB2/neu expression in developing rat retina.

We have examined the expression of neuregulin and its putative receptors, erbB2/neu, erbB3 and erbB4/tyro2 during retinal development, and tested several potential functions of this class of molecules in dissociated rat retinal cell cultures. At least one form of neuregulin is expressed in the retina, from the earliest stages of retinal development examined; in addition, all three of the known receptors are expressed by retinal neurons in a developmentally regulated manner. When added to cultures of embryonic or neonatal rat retinal cells, neuregulin (rhGGF2) promotes survival and neurite extension from retinal neurons in a dose-dependent manner. These results indicate that in addition to their well described effects on glia, the neuregulins also have direct effects on central nervous system neurons.

FGFR3 expression during development and regeneration of the chick inner ear sensory epithelia.

Several studies suggest fibroblast growth factor receptor 3 (FGFR3) plays a role in the development of the auditory epithelium in mammals. We undertook a study of FGFR3 in the developing and mature chicken inner ear and during regeneration of this epithelium to determine whether FGFR3 shows a similar pattern of expression in birds. FGFR3 mRNA is highly expressed in most support cells in the mature chick basilar papilla but not in vestibular organs of the chick. The gene is expressed early in the development of the basilar papilla. Gentamicin treatment sufficient to destroy hair cells in the basilar papilla causes a rapid, transient downregulation of FGFR3 mRNA in the region of damage. In the initial stages of hair cell regeneration, the support cells that reenter the mitotic cycle in the basilar papilla do not express detectable levels of FGFR3 mRNA. However, once the hair cells have regenerated in this region, the levels of FGFR3 mRNA and protein expression rapidly return to approximate those in the undamaged epithelium. These results indicate that FGFR3 expression changes after drug-induced hair cell damage to the basilar papilla in an opposite way to that found in the mammalian cochlea and may be involved in regulating the proliferation of support cells.

Growth factors in the treatment of degenerative retinal disorders.

There are currently a number of degenerative conditions, both inherited and acquired, that affect the retina and lead to blindness. Retinal photoreceptors degenerate from inherited conditions, such as retinitis pigmentosa or as a result of light damage or normal ageing; retinal ganglion cells degenerate from optic nerve injury or glaucoma. Current research in this field includes the use of growth factors to: (1) inhibit the degenerative processes; (2) promote regeneration of the retina from the pigmented epithelium; and (3) improve the conditions for transplantation of new cells to the retina by expanding the photoreceptor cell populations in vitro. The results to date have shown that a number of different growth factors promote survival of retinal cells in vitro and in vivo. In addition, some of the same factors can stimulate regeneration in the developing retina and act as mitogens for the retinal progenitor cells. It is likely that a combination of these approaches will ultimately be important for the treatment of the various retinal degenerations.

Neuregulin expression in PNS neurons: isoforms and regulation by target interactions.

Neuregulins have several important functions in the development of the peripheral nervous system, acting on both developing Schwann cells and muscle fibers. To determine whether these factors are also important for peripheral nerve regeneration, we have analyzed neuregulin expression in motor and sensory neurons by Northern blots and in situ hybridization. The results of this analysis show that the predominant neuregulin isoform expressed in these neurons is a novel transmembrane splice variant. After axotomy, there is a rapid decline in neuregulin expression in both motor and sensory neurons, but following reinnervation of target tissues, neuregulin expression returns to near normal levels. These results indicate that the normal expression of neuregulins in these neurons is maintained by the interactions with target tissues.

Glial growth factors are alternatively spliced erbB2 ligands expressed in the nervous system.

Glial growth factors, proteins that are mitogenic for Schwann cells, and several ligands for the p185erbB2 receptor, are products of the same gene. Alternative splicing of the messenger RNA generates an array of putative membrane-attached, intracellular and secreted signalling proteins, at least some of which are expressed in the developing spinal cord and brain. These factors are probably important in the development and regeneration of the nervous system.