Neurogenin (Ngn) 1 and Ngn2 encode basic-helix-loop-helix transcription factors expressed in the developing neocortex. Like other proneural genes, Ngns participate in the specification of neural fates and neuronal identities, but downstream effectors remain poorly defined. We set out to identify Ngn2 effectors in the cortex using a subtractive hybridization screen and identified several regionally expressed genes that were misregulated in Ngn2 and Ngn1;Ngn2 mutants. Included were genes down-regulated in germinal zone progenitors (e.g., Nlgn1, Unc5H4, and Dcc) and in postmitotic neurons in the cortical plate (e.g., Bhlhb5 and NFIB) and subplate (e.g., Mef2c, srGAP3, and protocadherin 9). Further analysis revealed that Ngn2 mutant subplate neurons were misspecified and that thalamocortical afferents (TCAs) that normally target this layer instead inappropriately projected towards the germinal zone. Strikingly, EphA5 and Sema3c, which encode repulsive guidance cues, were down-regulated in the Ngn2 and Ngn1;Ngn2 mutant germinal zones, providing a possible molecular basis for axonal targeting defects. Thus, we identified several new components of the differentiation cascade(s) activated downstream of Ngn1 and Ngn2 and provided novel insights into a new developmental process controlled by these proneural genes. Further analysis of the genes isolated in our screen should provide a fertile basis for understanding the molecular mechanisms underlying corticogenesis.

Our knowledge of the serine/threonine protein phosphatases of the mammalian nucleus is limited compared with their cytosolic counterparts. Microcystin-Sepharose chromatography and mass spectrometry were utilized to affinity purify and identify protein phosphatase-associated proteins from isolated rat liver nuclei. Far Western analysis with labeled protein phosphatase 1 (PP1) showed that many more PP1 binding proteins exist in the nucleus than were previously demonstrated. Mass spectrometry confirmed the presence in the nucleus of the mammalian PP1 isoforms ␣1, ␣2, ␤, and ␥1, plus the A␣ and several of the B and B subunits that are complexed to PP2A. Other proteins enriched on the microcystin matrix include the spliceosomal proteins known as the U2 snRNPs SAP145 and SAP155 and the U5 snRNPs p116 and p200, myosin heavy chain, and a nuclear PP1 myosin-targeting subunit related to M 110 . The putative RNA binding protein ZAP was also established as a nuclear PP1 binding protein using the criteria of co-purification with PP1 on microcystin-Sepharose, co-immunoprecipation, binding PP1 in an overlay assay, and presence of a putative PP1 binding site (KKRVRWAD). These results further support a key role for protein phosphatases in several nuclear functions, including the regulation of pre-mRNA splicing. Molecular & Cellular Proteomics 3:257-265, 2004. Protein phosphatase 1 (PP1)1 and 2A (PP2A) are highly conserved serine/threonine-specific protein phosphatases that have been identified in all eukaryotic species examined (1, 2). Dephosphorylation by PP1 is controlled by targeting or regulatory subunits that take PP1 to specific locations in the cell, potentially alter its phosphatase activity, and allow regulation by intra-or extracellular-derived signals (3-6). Biochemistry has shown that PP1 activity is highly enriched in the nucleus, and recent fluorescence microscopy studies with tagged versions of PP1 have dramatically illustrated this (7). PP1␥1 resides primarily in the nucleolar compartment, PP1␣ in the nucleoplasmic fraction, and PP1␤ in both nuclear compartments (7). Several nuclear PP1-targeting subunits have now been identified (6 -9). The two most-abundant nuclear PP1 binding subunits, p99 or PNUTS and nuclear inhibitor PP1 (NIPP-1), are RNA-binding proteins that likely play a role in pre-mRNA splicing (9 -11). Both proteins contain the PP1 binding motif R/K-V/I-X-F/W that was originally identified from studies on the glycogen and myosin PP1-targeting subunits (12-16). This motif has been shown to be present in nearly all PP1-associating proteins. Due to the number of nuclear events controlled by phosphorylation/dephosphorylation, there undoubtedly exist many more, as yet unidentified proteins that target or localize nuclear protein phosphatases. Here, we have done an extensive examination of the mammalian nucleus for protein phosphatase-associated proteins by utilizing the protein phosphatase affinity matrix microcystin-Sepharose. This matrix has been used previously to successfully purify other protein phosp...

SUMMARY A continuous schedule of reinforcement (CR) in an operant conditioning procedure results in the acquisition of associative learning and the formation of long-term memory. A 50 % partial reinforcement (PR) schedule does not result in learning. The sequence of PR—CR training has different and significant effects on memory retention and resistance to extinction. A CR/PR schedule results in a longer-lasting memory than a PR/CR schedule. Moreover,the memory produced by the CR/PR schedule is resistant to extinction training. In contrast, extinction occurs following the PR/CR schedule.