Janine Zieg

Regulation of Motor Neuron Specification by Phosphorylation of Neurogenin 2

The mechanisms by which proneural basic helix-loop-helix (bHLH) factors control neurogenesis have been characterized, but it is not known how they specify neuronal cell-type identity. Here, we provide evidence that two conserved serine residues on the bHLH factor neurogenin 2 (Ngn2), S231 and S234, are phosphorylated during motor neuron differentiation. In knockin mice in which S231 and S234 of Ngn2 were mutated to alanines, neurogenesis occurs normally, but motor neuron specification is impaired. The phosphorylation of Ngn2 at S231 and S234 facilitates the interaction of Ngn2 with LIM homeodomain transcription factors to specify motor neuron identity. The phosphorylation-dependent cooperativity between Ngn2 and homeodomain transcription factors may be a general mechanism by which the activities of bHLH and homeodomain proteins are temporally and spatially integrated to generate the wide diversity of cell types that are a hallmark of the nervous system.

Regulation of gene expression by site-specific inversion

A site-specific inversion event is responsible for phase transition in Salmonella, as indicated by heteroduplex analysis of recombinant molecules carrying the gene coding for H2 flagellin in Salmonella. The inversion region corresponds to approximately 800 base pairs in length, and the inversion process does not appear to be dependent upon the E. coli RecA recombination pathway. Specific deletion derivatives of the cloned fragments no longer produce H2-specific flagella, effectively mapping the H2 gene within about 300 bp of the inversion region. Recombinant products of the hybrid molecules arose spontaneously, and they were used in the mapping of restriction sites within the inversion region. The restriction maps further demonstrate the extent and nature of the inversion.

Properties of single-step mutants of Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate

Eleven independent lines of Syrian hamster cells were selected by using very low levels of N-(phosphonacetyl)-L-aspartate (PALA), an inhibitor of aspartate transcarbamylase. The protocol employed insured that each resistant cell arose during one of the last divisions before selection was applied. Cells of each mutant line contained an amplification of the structural gene for CAD, a trifunctional protein which includes aspartate transcarbamylase and two other enzymes of UMP biosynthesis. Strikingly, despite the minimal selection employed, the degree of amplification of the CAD gene was 6 to 10 times the normal diploid number in all 11 cases. In situ hybridization indicated that the amplified CAD genes were almost always present at a single chromosomal site in each line. Therefore, one of the two alleles was amplified 11- to 19-fold. The rates at which cells became resistant to PALA, determined by fluctuation analysis, were 100 times less dependent on drug concentration than were the frequencies of resistant cells in steady-state populations. The relatively shallow dependence of this rate upon PALA concentration is consistent with our independent observation that most events gave rise to a similar degree of amplification. In six of six cell lines examined, the levels of CAD mRNA and aspartate transcarbamylase activity were elevated two- to fourfold. These lines were resistant to PALA concentrations 20- to 80-fold higher than the ones used for selection. The organization of amplified DNA was examined by hybridizing Southern blots with cloned DNA fragments containing amplified sequences, previously isolated from two cell lines resistant to high levels of PALA. A contiguous region of DNA approximately 44 kilobases long which included the CAD gene was amplified in five of five single-step mutants examined. Outside this region, these mutants shared amplified sequences with only one of the two highly resistant lines.

A chemical genetic approach reveals distinct EphB signaling mechanisms during brain development.

EphB receptor tyrosine kinases control multiple steps in nervous system development. However, it remains unclear whether EphBs regulate these different developmental processes directly or indirectly. In addition, given that EphBs signal through multiple mechanisms, it has been challenging to define which signaling functions of EphBs regulate particular developmental events. To address these issues, we engineered triple knock-in mice in which the kinase activity of three neuronally expressed EphBs can be rapidly, reversibly and specifically blocked. We found that the tyrosine kinase activity of EphBs was required for axon guidance in vivo. In contrast, EphB-mediated synaptogenesis occurred normally when the kinase activity of EphBs was inhibited, suggesting that EphBs mediate synapse development by an EphB tyrosine kinase–independent mechanism. Taken together, our data indicate that EphBs control axon guidance and synaptogenesis by distinct mechanisms and provide a new mouse model for dissecting EphB function in development and disease.

SnapShot: Ca2+-Dependent Transcription in Neurons

Synaptic activity stimulates the influx of calcium ions into the postsynaptic neuron and thereby sets in motion a cascade of signaling events that lead to changes in gene expression. These changes in gene expression affect many aspects of nervous system development including dendritic morphogenesis, neuronal survival, and synapse development as well as the adaptive responses that underlie learning and memory in the mature nervous system. Mutations in components of the signal-ing pathways that participate in the process of experience-dependent brain development have been found to give rise to a variety of disorders of cognitive function including autism spectrum disorders.The initial contact between the axon and the dendrite during synapse development is mediated by cell surface-associated proteins on the pre- and postsynaptic membranes. For example, the binding of presynaptic β-neurexin to its postsynaptic receptor, Neuroligin1, leads to the recruitment of PSD-95 at nascent excitatory synapses. Ephrin/Eph signaling leads to the recruitment of additional proteins and the potentiation of NMDA receptor signaling. Release of the excitatory neurotrans-mitter glutamate from the presynaptic membrane and its binding to NMDA receptors and AMPA receptors on the postsynaptic membrane lead to the opening of these glutamate-gated ion channels. This is followed by membrane depolarization, opening of the L-type voltage-gated calcium ion channel (L-VSCC), and a rapid rise in calcium ions in the postsynaptic neuron as well as other local changes including protein recruitment and activation. Calcium entry through L-VSCCs leads to the recruitment of AKAP79/150, which then recruits PKA to the channel. PKA phosphorylates the calcium channel, thereby increasing its ability to allow calcium ions to flow into the cell. Calcium ion influx through L-VSCCs is sensed by calmodulin (CaM). Activated calmodulin initiates a cascade of events including stimulation of the guanine nucleotide exchange factor RasGRF, followed by activation of the Ras-MAPK signaling cascade. Calcium-activated calmodulin also activates the CaM kinase signaling pathway. Once activated these pathways trigger the phosphorylation and activation of a wide range of transcription factors such as CREB and NeuroD2. The phosphorylation of these transcription factors occurs in the nucleus and can be triggered by a cascade of events that begins at the site of calcium entry at the mouth of the calcium channel (that is, the Ras, Raf, MEK, ERK, RSK/MSK signaling pathway). Alternatively, channel activation can trigger an elevation of calcium ions directly in the nucleus that leads to activation of nuclear CaMKII by calcium/calmodulin, which in turn phosphorylates CREB and NeuroD2. In addition, dephosphorylation-dependent signaling through calcium/calmodulin activation of calcineurin leads to the activation of the transcripton factors NFAT and MEF2.Once activated and localized to the nucleus, calcium-activated transcription factors and modulators of transcription bind to the regulatory regions of activity-regulated genes to orchestrate finely tuned levels of gene expression. The most extensively studied activity-regulated gene is

The Mechanism of Phase Variation

INTRODUCTION Studies of the properties of the lac operon have formed the basis for our understanding of a number of regulatory mechanisms. The lac operon model focuses attention on systems that involve the binding of repressor or activator proteins to sites adjacent to structural genes, leading to enhanced expression or to repression of gene activity. There are, however, many observations involving the regulation of gene activity which cannot be explained by mechanisms analogous to the lac operon. For example in yeast, the interconversion of mating types (Hicks et al. 1977) is best explained by a model involving site-specific recombination, i.e., gene expression requires the transposition of genetic material from one chromosomal locus to another. In the Zea mays genetic system, McClintock (1957) recognized and identified transposable chromosomal elements that control the functions of a variety of genes. Many other observations in genetic systems as varied as Drosophila (Green 1977), Paramecium (Sonnenborn 1977), and Escherichia coli (Starlinger and Saedler 1972) are most readily explained by invoking a specific recombination event, e.g., an inversion or transposition, or some other kind of modification that changes the state of the gene and thus affects its expression. However, very little is known about the exact molecular events that occur in any of these systems. Just as the development of genetic systems in E. coli allowed an analysis of the mechanism of regulation of the lac operon, so the introduction of gene cloning techniques allows a direct examination of DNA that might be involved in site-specific...

The right end of MudI(Ap,lac)

Stable derivatives of the bacteriophage MudI(Ap,lac) were used to generate operon fusions in S. typhimurium which exhibit a sectoring phenotype with respect to lacZ expression. The Lac- to Lac+ conversion was shown to be the result of small deletions involving the right end of the MudI element. DNA sequence analysis of several different fusions revealed that this end of MudI(Ap,lac) contains an assymetric inverted repeat of the attR site found in the wild-type Mu phage. A model is presented which explains how such a structure was formed in the construction of MudI(Ap,lac). In addition, this model explains the observed deletion formation and the Lac- to Lac+ conversion in the sectoring fusions.