Proper functioning of the adult nervous system is critically dependent on neurons adopting the correct neurotransmitter phenotype during early development. Whereas the importance of cell-cell communication in fate determination is well documented for a number of neurotransmitter phenotypes, the contributions made by early lineage to this process remain less clear. This is particularly true for gamma-aminobutyric acid (GABA)ergic and glutamatergic neurons, which are present as the most abundant inhibitory and excitatory neurons, respectively, in the central nervous system of all vertebrates. In the present study, we have investigated the role of early lineage in the determination of these two neurotransmitter phenotypes by constructing a fate map of GABAergic and glutamatergic neurons for the 32-cell stage Xenopus embryo with the goal of determining whether early lineage influences the acquisition of these two neurotransmitter phenotypes. To examine these phenotypes, we have cloned xGAT-1, a molecular marker for the GABAergic phenotype in Xenopus, and described its expression pattern over the course of development. Although we have identified isolated examples of a blastomere imparting a statistically significant bias, when taken together, our results suggest that blastomere lineage does not impart a widespread bias for subsequent GABAergic or glutamatergic fate determination. In addition, the fate map presented here suggests a general dorsal-anterior to ventral-posterior patterning progression of the nervous system for the 32-cell stage Xenopus embryo. J. Comp. Neurol. 495:645-657, 2006.(c) 2006 Wiley-Liss, Inc.

The telomeric protein TRF2 is required to prevent mammalian telomeres from activating DNA damage checkpoints. Here we show that overexpression of TRF2 affects the response of the ATM kinase to DNA damage. Overexpression of TRF2 abrogated the cell cycle arrest after ionizing radiation and diminished several other readouts of the DNA damage response, including phosphorylation of Nbs1, induction of p53, and upregulation of p53 targets. TRF2 inhibited autophosphorylation of ATM on S1981, an early step in the activation of this kinase. A region of ATM containing S1981 was found to directly interact with TRF2 in vitro, and ATM immunoprecipitates contained TRF2. We propose that TRF2 has the ability to inhibit ATM activation at telomeres. Because TRF2 is abundant at chromosome ends but not elsewhere in the nucleus, this mechanism of checkpoint control could specifically block a DNA damage response at telomeres without affecting the surveillance of chromosome internal damage.

The human telomeric protein POT1 is known to bind single-stranded telomeric DNA in vitro and to participate in the regulation of telomere maintenance by telomerase in vivo. We examined the in vitro DNA binding features of POT1. We report that deleting the oligosaccharide/oligonucleotide-binding fold of POT1 abrogates its DNA binding activity. The minimal binding site (MBS) for POT1 was found to be the telomeric nonamer 5'-TAGGGTTAG-3', and the optimal substrate is [TTAGGG](n (n >/= 2)). POT1 displays exceptional sequence specificity when binding to MBS, tolerating changes only at position 7 (T7A). Whereas POT1 binding to MBS or [TTAGGG](2) was enhanced by the proximity of a 3' end, POT1 was able to bind to a [TTAGGG](5) array when positioned internally. These data indicate that POT1 has a strong sequence preference for the human telomeric repeat tract and predict that POT1 can bind both the 3' telomeric overhang and the displaced TTAGGG repeats at the base of the t-loop.

The telomeric protein TRF2 is required to prevent mammalian telomeres from activating DNA damage checkpoints. Here we show that overexpression of TRF2 affects the response of the ATM kinase to DNA damage. Overexpression of TRF2 abrogated the cell cycle arrest after ionizing radiation and diminished several other readouts of the DNA damage response, including phosphorylation of Nbs1, induction of p53, and upregulation of p53 targets. TRF2 inhibited autophosphorylation of ATM on S1981, an early step in the activation of this kinase. A region of ATM containing S1981 was found to directly interact with TRF2 in vitro, and ATM immunoprecipitates contained TRF2. We propose that TRF2 has the ability to inhibit ATM activation at telomeres. Because TRF2 is abundant at chromosome ends but not elsewhere in the nucleus, this mechanism of checkpoint control could specifically block a DNA damage response at telomeres without affecting the surveillance of chromosome internal damage.

Proper functioning of the adult nervous system is critically dependent on neurons adopting the correct neurotransmitter phenotype during early development. Whereas the importance of cell-cell communication in fate determination is well documented for a number of neurotransmitter phenotypes, the contributions made by early lineage to this process remain less clear. This is particularly true for gamma-aminobutyric acid (GABA)ergic and glutamatergic neurons, which are present as the most abundant inhibitory and excitatory neurons, respectively, in the central nervous system of all vertebrates. In the present study, we have investigated the role of early lineage in the determination of these two neurotransmitter phenotypes by constructing a fate map of GABAergic and glutamatergic neurons for the 32-cell stage Xenopus embryo with the goal of determining whether early lineage influences the acquisition of these two neurotransmitter phenotypes. To examine these phenotypes, we have cloned xGAT-1, a molecular marker for the GABAergic phenotype in Xenopus, and described its expression pattern over the course of development. Although we have identified isolated examples of a blastomere imparting a statistically significant bias, when taken together, our results suggest that blastomere lineage does not impart a widespread bias for subsequent GABAergic or glutamatergic fate determination. In addition, the fate map presented here suggests a general dorsal-anterior to ventral-posterior patterning progression of the nervous system for the 32-cell stage Xenopus embryo. J. Comp. Neurol. 495:645-657, 2006.(c) 2006 Wiley-Liss, Inc.