Early in development, neurons only express NR1/NR2B-containing N-methyl-d-aspartate (NMDA) receptors. Later, NR2A subunits are upregulated during a period of rapid synapse formation. This pattern is often interpreted to indicate that NR2A-containing receptors are synaptic and that NR2B-containing receptors are extrasynaptic. We re-examined this issue using whole cell recordings in cultured hippocampal neurons. As expected, the inhibition of whole cell currents by the NR2B-specific antagonist, ifenprodil, progressively decreased from 69.5 +/- 2.4% [6 days in vitro (DIV)] to 54.9 +/- 2.6% (8 DIV), before reaching a plateau in the second week (42.5 +/- 2%, 12-19 DIV). In NR2A-/- neurons, which express only NR1/NR2B-containing NMDA receptors, autaptic excitatory postsynaptic currents (EPSCs; > or =12 DIV) were more sensitive to ifenprodil and decayed more slowly than EPSCs in wild-type neurons. Thus NR2B-containing receptors were not excluded from synapses. We blocked synaptic NMDA receptors with MK-801 during evoked transmitter release, thus allowing us to isolate extrasynaptic receptors. Ifenprodil inhibition of this extrasynaptic population was highly variable in different neurons. Furthermore, extrasynaptic receptors in autaptic cultures were only partially blocked by ifenprodil, indicating that NR2A-containing receptors are not exclusively confined to the synapse. Extrasynaptic NR2A-containing receptors were also detected in NR2A(-/-) neurons transfected with full-length NR2A. Truncation of the NR2A C terminus did not eliminate synaptic expression of NR2A-containing receptors. Our results indicate that NR2A- and NR2B-containing receptors can be located in either synaptic or extrasynaptic compartments.

We developed a cell division-activated Cre-lox system for stochastic recombination of loxP-flanked loci in mice. Cre activation by frameshift reversion is modulated by DNA mismatch-repair status and occurs in individual cells surrounded by normal tissue, mimicking spontaneous cancer-causing mutations. This system should be particularly useful for delineating pathways of neoplasia, and determining the developmental and aging consequences of specific gene alterations.Valuable mouse cancer models exist that combine conditional expression of the Cre recombinase with various loxP-flanked tumor suppressor or oncogene alleles 1 . In these model systems, 'cancer' is induced by gene alteration ubiquitously throughout the target tissue or in a selected cell type. But this does not accurately mimic the natural process of sporadic cancer initiation and progression. Two Cre-lox models that facilitate stochastic cancer gene alterations in isolated cells rely on homologous recombination to induce activation of an oncogene 2 , or to induce inactivation of one or more tumor suppressor genes on the same chromosome 3 . The former system is restricted in that it is only applicable to activation of an oncogenic K-ras allele 2 . The latter more flexible system uses engineered loxP-FRT sites to induce mitotic recombination of individual chromosomes containing modified genes 3 . Here we report a highly versatile system that features Cre-mediated stochastic genetic changes in single cells or cell lineages in normal tissue. The system can be applied to any loxP-flanked allele, is dependent on cell division and can be modulated by DNA mismatch-repair status.To construct an inactive but revertible Cre allele, we first engineered an 11-bp A·T run in a modified version of Cre 4 without altering the nuclear localization signal (Supplementary Methods online). We added an extra A·T base pair, creating a +1 bp out-of-frame Cre allele, which we termed 12A-Cre. To ensure efficient expression at the intended target locus, we added a splice acceptor and internal ribosome entry site to 12A-Cre. We cloned the modified 12A-Cre plus a neo module between homology arms of the DNA mismatch repair gene Pms2 (ref. 5) to generate a Pms2-Cre targeting vector. Targeting resulted in an out-of-frame Cre gene under the control of the Pms2 promoter, which is expressed in several cell types, including stem cells of the mouse intestine 6 . Targeting to Pms2 removed exon 2, creating a null allele, which we refer to as Pms2 cre (Fig. 1a). Because of mismatch-repair deficiency, Pms2 cre/cre mice should have increased frequency of −1 bp frameshifts 7 and hence increased Cre reversion relative to Pms2 cre/+ mice. Therefore, Cre activation frequency can be modulated appropriately for a particular study by breeding Pms2 cre/+ or Pms2 cre/cre mice. Notably, this system should

Intestinal stem cells (ISCs) are maintained by a niche mechanism, in which multiple ISCs undergo differential fates where a single ISC clone ultimately occupies the niche. Importantly, mutations continually accumulate within ISCs creating a potential competitive niche environment. Here we use single cell lineage tracing following stochastic transforming growth factor β receptor 2 (TgfβR2) mutation to show cell autonomous effects of TgfβR2 loss on ISC clonal dynamics and differentiation. Specifically, TgfβR2 mutation in ISCs increased clone survival while lengthening times to monoclonality, suggesting that Tgfβ signaling controls both ISC clone extinction and expansion, independent of proliferation. In addition, TgfβR2 loss in vivo reduced crypt fission, irradiation-induced crypt regeneration, and differentiation toward Paneth cells. Finally, altered Tgfβ signaling in cultured mouse and human enteroids supports further the in vivo data and reveals a critical role for Tgfβ signaling in generating precursor secretory cells. Overall, our data reveal a key role for Tgfβ signaling in regulating ISCs clonal dynamics and differentiation, with implications for cancer, tissue regeneration, and inflammation.T he intestinal epithelium is constantly renewed by proliferating, multipotent, and self-renewing intestinal stem cells (ISCs) (1). There are two main populations of ISCs: (i) a proliferating ISC population that is important for homeostasis of the niche residing below the +4 position and expressing a set of markers [e.g., leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) and Olfm4] and (ii) a quiescent ISC population residing near the +4 position and expressing a different set of markers (e.g., Bmi1 and Hopx) (2). Proliferating ISCs are the workhorses during normal homeostasis and are maintained within the niche by a close relationship with Paneth cells (3) and the stroma (4). The proliferating ISC population can be further divided into a smaller number (4-8) of functional ISCs (5,6

APC is considered a gatekeeper for colorectal cancer (CRC). Cells with heterozygous APC mutations have altered expression profiles suggesting that the first APC hit may help set the stage for subsequent transformation. Therefore, we measured transformation efficiency following what we have designated as “simultaneous” versus “stepwise” Apc loss. We combined a conditional Apc allele (ApcCKO) with a Cre reporter gene and an out-of-frame Cre allele (Pms2cre) that stochastically becomes functional by a frameshift mutation in single cells. Loss of one Apc allele (ApcCKO/+) had little consequence, whereas simultaneous loss of both Apc alleles (ApcCKO/CKO) resulted in increased clonal expansion (crypt fission), consistent with the gatekeeper function of Apc. Interestingly, our analyses showed that most of the Apc-deficient crypts in ApcCKO/CKO mice appeared normal, with morphologic transformation, including β-catenin deregulation, occurring in only 17% of such crypts. To determine whether transformation efficiency was different following stepwise Apc loss, we combined ApcCKO with a germline mutant allele, either ApcMin or Apc1638N. Transformation efficiency following stepwise Apc loss (ApcMin/CKO or Apc1638N/CKO) was increased 5-fold and essentially all of the Apc-deficient cells were dysplastic. In summary, our data suggest that the gatekeeper function of Apc consists of two roles, clonal expansion and morphologic transformation, because simultaneous Apc loss frequently leads to occult clonal expansion without morphologic transformation, whereas stepwise Apc loss more often results in visible neoplasia. Finally, that Apc-deficient cells in certain scenarios can retain a normal phenotype is unexpected and may have clinical implications for surveillance strategies to prevent CRC.

SUMMARY1. A study has been made of the effects of the selective N-methyl-D-aspartate receptor antagonist, 2-amino-5-phosphonovalerate (APV), and the broad spectrum excitatory amino acid antagonists, y-D-glutamylglycine (y-DGG), y-D-glutamylaminomethylsulphonate (GAMS), 4(p-chlorobenzoyl)-cis-piperazine-2, 3-dicarboxylate (pCB-PzDA) and kynurenate, have been examined on excitation evoked on neurones in the magnocellular red nucleus (m.r.n.) of the anaesthetized cat by stimulation of the interpositus nucleus (i.p.n.) and sensorimotor cortex, and by ionophoresed excitant amino acid agonists.2. The profile of activity of the excitatory amino acid antagonists on m.r.n. neurones was similar to that described on neurones in other areas of the central nervous system. APV selectively depressed responses to N-methyl-D-aspartate (NMDA), whereas the broader spectrum antagonists reduced responses to kainate and quisqualate as well as to NMDA. Neuronal responses to L-glutamate and L-aspartate were depressed by all the antagonists tested.3. I.p.n.-evoked monosynaptic responses ofm.r.n. neurones were reversibly reduced by the broad spectrum antagonists, but were unaffected by APV.4. Cortically evoked mono-and polysynaptic excitatory responses were reversibly depressed by APV and the broad spectrum antagonist, pCB-PzDA. The action of APV corresponded with its ability to antagonize responses to NMDA. However, the cortically evoked responses appeared to be more sensitive to the actions ofpCB-PzDA than to those of APV, although the former is a less effective antagonist of NMDA-induced excitation compared with APV.5. APV depressed excitation induced by cortical stimuli and L-glutamate and L-aspartate. However, there was no obvious correlation between the actions of the broad spectrum amino acid antagonists on synaptically evoked responses and those induced by L-glutamate or L-aspartate on the few neurones tested.6. These results are consistent with an amino acid being the transmitter in the interposito-rubral and cortico-rubral excitatory pathways which interacts with non-NMDA and both NMDA and non-NMDA receptors respectively. However, the identity of the transmitter acting at these receptors remains to be determined.