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Precise control of neuronal migration is essential for the development of the neocortex. However, the molecular mechanisms underlying neuronal migration remain largely unknown. Here we identified helix-loop-helix transcription factor Ebf3 as a Prdm8 target gene, and found that Ebf3 is a key regulator of neuronal migration via multipolar-to-bipolar transition. Ebf3 knockdown cells exhibited severe defects in the formation of leading processes and an inhibited shift to the locomotion mode. Moreover, we found that Ebf3 knockdown represses NeuroD1 transcription, and NeuroD1 overexpression partially rescued migration defects in Ebf3 knockdown cells. Our findings highlight the critical role of Ebf3 in multipolar-to-bipolar transition via positive feedback regulation of NeuroD1 in the developing neocortex.

To elucidate roles of any particular gene or genetic element in higher-order biological processes, a general method(s) for genome editing and creation of such genome-modified species is essential. Therefore, the approach of genome editing in mouse embryonic stem cells (ESCs) and ESC-based genome-modified animal production is commonly used. For gene targeting via homologous recombination (HR), a targeting vector possessing a drug-resistant gene plus 5’ and 3’ homology sequence arms is introduced into the cells. Although there is a well-established general protocol and many genes are already targeted by this method in ESCs, the targeting efficiency varies at different target loci and sometimes is too low. Recently, other genome editing technologies such as zinc finger nuclease (ZFN) [1, 2], TAL effector nuclease (TALEN) [3, 4] and CRISPR/Cas9 [5, 6] systems have been developed. If specific and highly competent nuclease can be designed, all these systems would work well for gene targeting in mammals [7, 8]. The CRISPR/Cas9 system is the newest of the systems, but it is extremely useful. Unlike the ZFN and TALEN systems, the CRISPR/Cas9 system uses RNA as a guide molecule and a 20-nt RNA sequence specifies the target DNA site. Therefore, preparation of specific targeting materials is much easier and simpler. Furthermore, if Cas9 mRNA is delivered along with guide RNA into mouse zygotes, genome-edited mice can be easily obtained. However, even for the CRISPR/Cas9 system, some technical challenges still exist. An obvious one is the off-target mutagenesis risk due to the 20-nt sequence restriction of the target specificity. Furthermore, because homology-directed repair (HDR) is less efficient in mammals, targeted gene replacement or insertion mediated by HDR is inefficient and the zygote injection method with Cas9 RNA, guide RNA and a targeting template construct is generally not practical for creating such genome-edited mice. For genome editing using a standard targeting vector, various trials have been applied to improve gene targeting frequencies by HR [9]. Among them, knockdown of the Bloom syndrome gene, BLM, has been shown to enhance gene targeting efficiencies in various human cell lines [10]. BLM encodes RecQ type DNA helicase [11] and plays a role in the suppression of HR [12]. However, it has not yet been investigated whether this approach in ESCs is applicable for knockout mouse production. Therefore, in this study, we targeted multiple different gene loci with or without Blm knockdown in ESCs and used the targeted ESC clones obtained with Blm knockdown for chimeric mouse production and germline transmission. For Blm knockdown in ESCs, we designed three different Blm siRNAs (siBlm1-3). At 48 h post transfection, the amount of Blm mRNA was significantly decreased in ESCs transfected with all independent Blm siRNAs (Fig. 1a). Western blot analysis also showed significant reduction of the Blm protein amount specifically by Blm siRNA treatment (Fig. 1b). Among them, siBlm-2 and siBlm-3 induced higher knockdown efficiency than siBlm-1. Therefore, we selected siBlm-2 and siBlm-3 and combined them for further Blm knockdown experiments (Fig. 1c). Fig. 1. Blm was knocked down by siRNAs. a) 20 nM of each Blm siRNA was transfected into KY1.1. At 48 h after transfection, the Blm mRNA level was measured by quantitative RT-PCR using two primer sets. b) The expression level of Blm protein was determined by western blot. ... To validate how Blm knockdown affects gene targeting efficiency in ESCs, we targeted three different gene loci, namely, Prdm5 on chromosome 6, Prdm8 on chromosome 5 and, Arl14ep on chromosome 2, with or without Blm siRNAs pretreatment. Prdm5 and Arl14ep are expressed but Prdm8 is silent in ESCs (not shown). We used standard gene targeting vectors for these three genes (Fig. 2a) [13]. Forty-eight hours before transfection of the targeting vectors, one part of the cells was transfected with Blm siRNAs in the condition shown in Fig. 1c. Then, the ESCs transfected with each targeting vector were selected with G418. More than 200 drug-resistant colonies per transfection were screened for proper gene targeting. As summarized in Table 1, the targeting efficiencies of Prdm5, Prdm8 and Arl14ep were 8/214 (3.7%), 8/796 (1.0%) and 14/240 (5.8%) for ESCs without Blm knockdown, respectively. For the ones with Blm knockdown, the targeting efficiency was 29/232 (12.5%) for Prdm5, 15/363 (4.1%) for Prdm8 and 32/240 (13.3%) for Arl14ep. Thus, pretreatment with the Blm siRNAs pre-treatment enhanced the targeting efficiency for all three gene loci, and the fold activation enrichment was 3.4 for Prdm5, 4.1 for Prdm8 and 2.3 for Arl14ep. In another experiment for Arl14ep gene targeting, we screened cells transfected with control siRNA (siC-L) in addition to cells treated with Blm siRNAs (Table 2). This time, the targeting efficiencies were generally low but treatment with the Blm siRNAs gave higher fold activation enrichment than that with siC-L (2.6 for Blm siRNAs and 1.4 for siC-L) suggesting that the effect of Blm siRNAs is not non-specific. Fig. 2. Schematic diagram for gene targeting. a) Prdm5 targeting: lox P (shaded triangle)-frt (open triangle)-PGK-Neo-frt and another lox P site were introduced into upstream and downstream of exon (Ex) 1, respectively. Prdim8 targeting: lox P-frt-PGK-Neo-frt ... Table 1. Gene targeting efficiency with or without Blm knockdown Table 2. Influence of siRNA on gene targeting Then, we examined whether the targeted ESC clones obtained with Blm siRNA pretreatment maintained pluripotency, especially for the germline transmission potential. Since sister chromatid exchanges (SCEs) are highly increased in Blm knockout or knockdown cells [14,15,16,17,18,19], we first checked chromosome stability. As shown in Table 3, we performed a karyotype analysis of three targeted ESC clones for Arl14ep (#12, #13 and #27) with Blm knockdown and a parental ESC, KY1.1, as a control. For all targeted clones examined, the average chromosome number per cell was not changed and remained at ~40. Table 3. Karyotype analysis of the established targeted ESC lines with Blm knockdown Finally, we created chimeric mice using the targeted ESC clones obtained with Blm knockdown for the three genes, Prdm5, Prdm8 and Arl14ep. As summarized in Table 4, 2/3 to 4/4 of them generated > 80% chimeric mice as judged by the coat color contribution. Furthermore, germline transmission of the targeted allele was confirmed for all three gene loci from those good chimeric mice (more than two lines for each gene). Therefore, we concluded that Blm siRNA pretreatment does not have clear negative effects on chromosomal stability or germline transmission potential of the obtained targeted ESC clones. Table 4. Production of chimeric mice and germline transmission by the targeted ESC lines with Blm knockdown In conclusion, Blm knockdown provides a general benefit for efficient ESC-based and HR-mediated knockout mouse production.

Upper-layer (UL) neocortical neurons are the most prominent distinguishing features of the mammalian neocortex compared with those of the avian dorsal cortex and are vastly expanded in primates. However, little is known about the identities of the genes that control the specification of UL neurons. Here, we found that Prdm8, a member of the PR (PRDI-BF1 and RIZ homology) domain protein family, was specifically expressed in the postnatal UL neocortex, particular those in late-born RORß-positive layer IV neurons. We generated homozygous Prdm8 knockout (Prdm8 KO) mice and found that the deletion of Prdm8 causes growth retardation and a reduced brain weight, although the brain weight-to-body weight ratio is unchanged at postnatal day 8 (P8). Immunohistochemistry showed that the relative UL thickness, but not the thickness of the deep layer (DL), was significantly reduced in Prdm8 KO mice compared with wild-type (WT) mice. In addition, we found that a number of late-born Brn2-positive UL neurons were significantly decreased in Prdm8 KO mice. To identify genes regulated by Prdm8 during neocortical development, we compared expression profiling analysis in Prdm8 KO and WT mice, and identified some candidate genes. These results suggest that the proper expression of Prdm8 is required for the normal development and construction of UL neurons in the mammalian neocortex.

Background Immunocytochemistry (ICC) is an advanced diagnostic technique used in the field of veterinary cytology. We recently developed a rapid ICC method for the detection of cytokeratin and vimentin in dogs, which helps to determine whether tumor cells are of epithelial or nonepithelial origin. However, the diagnostic value of this rapid ICC method in neoplastic diseases of dogs has not been assessed yet. Objectives The aim of the present study was to assess the diagnostic accuracy of rapid ICC compared to standard immunohistochemistry (IHC). Methods Air-dried smear samples and formalin-fixed paraffin sections were prepared from tumors excised from dogs (n = 30). Immunosignals for cytokeratin and vimentin were detected in smear samples by rapid ICC, and in paraffin sections by standard IHC. Signals in smear samples detected by rapid ICC were compared with positive staining in paraffin sections detected by standard IHC and analyzed for statistical significance (kappa statistic). Results Rapid ICC detected specific immunosignals in 25/30 cases (83.3%), and nonspecific signals were detected in 5/30 cases. Statistical analysis revealed fair agreement in epithelial tumors (n = 16) with cytokeratin (κ = 0.236) and vimentin (κ = 0.294). In nonepithelial tumors (n = 14), almost perfect agreement was demonstrated with cytokeratin (κ = 0.857) and vimentin (κ = 0.857). Conclusions The rapid ICC method can be a useful tool for the diagnostic cytology of neoplastic tissues in dogs.

The precise control of neuronal migration and morphological changes during differentiation is essential for neocortical development. We hypothesized that the transition of progenitors through progressive stages of differentiation involves dynamic changes in mitochondrial reactive oxygen species (mtROS) levels, depending on cell requirements. We found that progenitors had higher levels of mtROS, but that these levels were significantly decreased with differentiation. The Prdm16 gene was identified as a candidate modulator of mtROS using microarray analysis, and was specifically expressed by progenitors in the ventricular zone. However, Prdm16 expression declined during the transition into NeuroD1-positive multipolar cells. Subsequently, repression of Prdm16 expression by NeuroD1 on the periphery of ventricular zone was crucial for appropriate progression of the multipolar phase and was required for normal cellular development. Furthermore, time-lapse imaging experiments revealed abnormal migration and morphological changes in Prdm16-overexpressing and -knockdown cells. Reporter assays and mtROS determinations demonstrated that PGC-1α is a major downstream effector of Prdm16 and NeuroD1, and is required for regulation of the multipolar phase and characteristic modes of migration. Taken together, these data suggest that Prdm16 plays an important role in dynamic cellular redox changes in developing neocortex during neural differentiation.

The effects of 5-hydroxytryptamine (5-HT) on the muscle tonus of the ampulla and isthmus of the oviduct isolated from nonpregnant proestrus pigs were investigated, and the 5-HT receptor subtype and mechanisms of the responses were analyzed. 5-HT (1 nM-10 microM) caused a relaxation of longitudinal and circular muscles of the isthmus in a concentration-dependent manner. Tetrodotoxin did not change the relaxation, indicating a direct action of 5-HT on smooth muscle cells. The EC(50) value in the longitudinal muscle was significantly lower than that in the circular muscle but the maximum relaxations were similar. 5-HT also caused a relaxation of both muscle layers in the ampulla but the maximum relaxation of both muscles was smaller than that of the isthmus. 5-Carboxamidotryptamine (5-CT), 5-methoxytryptamine (5-MeOT) and (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) mimicked the relaxation of the isthmic longitudinal muscle by 5-HT, and the ranking order was 5-CT5-HT5-MeOT8-OH-DPAT. On the other hand, oxymethazoline, 2-methyl-5-hydroxytryptamine (2-methyl-5-HT), alpha-methyl-5-hydroxytryptamine (alpha-methyl-5-HT), [endo-N-8-methyl-8-azabicyclo-(3,2,1) oct-3-yl]-2,3-dihydro-3-ethyl-2-oxo-1H-benzimidazol-1-carboxamide (BIMU-1), ergotamine and dihydroergotamine were less effective. The relaxation by 5-HT was not decreased by ketanserin, 2-methoxy-4-amino-5-chlorobenzoic acid 2-(diethylamino)ethyl ester (tropisetron) or [1[2-(methylsulphonyl) amino ethyl]-4-piperidinyl]methyl-1-methyl-1H-indole-3-carboxylate (GR113808) but was antagonized by the following compounds in a competitive manner (with pK(b) values in parentheses): 2a-[4-(4-phenyl-1,2,3,6-tetrahydropyridyl)butyl]-2a,3,4,5-tetrahydro-benzo[cd]indol-2(1H)-one (DR4004, 9.31), methiothepin (8.91), methysergide (7.95), metergoline (7.98), mianserin (7.69), mesulergine (8.4), spiperone (6.86) and clozapine (7.4). The correlation of these pK(b) values with pK(i) values of cloned 5-HT(7) receptor or pA(2) values of porcine uterus was high and significant. 4-(3-Butoxy-4-methoxybenzyl)-imidazolidin-2-one (Ro20-1724) significantly enhanced the relaxation by 5-HT but zaprinast, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and L-nitroarginine methylester (L-NAME) did not change the responses to 5-HT. 5-HT increased cyclic AMP in the isthmic oviduct. Ampulla and isthmus contained a single class of [3H]5-CT binding sites with a similar K(d) value (0.4 nM), but the density of the receptors in the isthmus was 2.4 times higher than that in the ampulla. A significant correlation was found between the pK(i) values in the oviduct and those of the cloned 5-HT(7) receptors. Isoprenaline, sodium nitroprusside, vasoactive intestinal peptide and pituitary adenylate cyclase activating peptide were less effective in causing the relaxation of the oviduct. In conclusion, the 5-HT receptor, functionally correlated to the 5-HT(7) type, mediates the relaxation of the porcine oviduct by 5-HT through an increase in intracellular cyclic AMP. The degrees of 5-HT-induced relaxation in the isthmus and ampulla of the oviduct were different due to the heterogeneous distribution of 5-HT(7) receptors. The strongest relaxation through 5-HT(7) receptor activation suggests that 5-HT plays an important physiological role in the regulation of porcine oviduct contractility.

Here, we found that the PR domain protein Prdm8 serves as a key regulator of the length of the multipolar phase by controlling the timing of morphological transition. We used a mouse line with expression of Prdm8-mVenus reporter and found that Prdm8 is predominantly expressed in the middle and upper intermediate zone during both the late and terminal multipolar phases. Prdm8 expression was almost coincident with Unc5D expression, a marker for the late multipolar phase, although the expression of Unc5D was found to be gradually down-regulated to the point at which mVenus expression was gradually up-regulated. This expression pattern suggests the possible involvement of Prdm8 in the control of the late and terminal multipolar phases, which controls the timing for morphological transition. To test this hypothesis, we performed gain- and loss-of-function analysis of neocortical development by using in utero electroporation. We found that the knockdown of Prdm8 results in premature change from multipolar to bipolar morphology, whereas the overexpression of Prdm8 maintained the multipolar morphology. Additionally, the postnatal analysis showed that the Prdm8 knockdown stimulated the number of early born neurons, and differentiated neurons located more deeply in the neocortex, however, majority of those cells could not acquire molecular features consistent with laminar location. Furthermore, we found the candidate genes that were predominantly utilized in both the late and terminal multipolar phases, and these candidate genes included those encoding for guidance molecules. In addition, we also found that the expression level of these guidance molecules was inhibited by the introduction of the Prdm8 expression vector. These results indicate that the Prdm8-mediated regulation of morphological changes that normally occur during the late and terminal multipolar phases plays an important role in neocortical development.

Genomic sequences encoding the 3′ exonuclease (proofreading) domains of both replicative DNA polymerases, pol delta and pol epsilon, were explored simultaneously in human colorectal carcinomas including six established cell lines. Three unequivocal sequence alterations, including one previously reported, were found, and all these were considered as dysfunctional mutations in light of the local amino-acid sequences. In particular, the F367S mutation found in the POLE gene encoding the pol epsilon catalytic subunit, which includes the proofreading domain, is the first found in human diseases. Surprisingly, the tumours carrying these proofreading domain mutations were all defective in DNA mismatch repair (MMR). In addition to the two cell lines with acknowledged MMR gene mutations, the third tumour was also demonstrated to harbour a distinct mutation in MLH1, and indeed exhibited a microsatellite-unstable phenotype. These findings suggest that, in concert with MMR deficiency, defective polymerase proofreading may also contribute to the mutator phenotype observed in human colorectal cancer. Our observations may suggest previously unrecognised complexities in the molecular abnormalities underlying the mutator phenotype in human neoplasms.