A Neanderthal/Denisovan GLI3 variant contributes to anatomical variations in mice.

Changes in genomic structures underlie phenotypic diversification in organisms. Amino acid-changing mutations affect pleiotropic functions of proteins, although little is known about how mutated proteins are adapted in existing developmental programs. Here we investigate the biological effects of a variant of the GLI3 transcription factor (GLI3R1537C) carried in Neanderthals and Denisovans, which are extinct hominins close to modern humans. R1537C does not compromise protein stability or GLI3 activator-dependent transcriptional activities. In contrast, R1537C affects the regulation of downstream target genes associated with developmental processes. Furthermore, genome-edited mice carrying the Neanderthal/Denisovan GLI3 mutation exhibited various alterations in skeletal morphology. Our data suggest that an extinct hominin-type GLI3 contributes to species-specific anatomical variations, which were tolerated by relaxed constraint in developmental programs during human evolution.

Disruption of the anterior commissure in Olig2 deficient mice.

In the present study, we examined neural circuit formation in the forebrain of the Olig2 knockout (Olig2-KO) mouse model and found disruption of the anterior commissure at the late foetal stage. Axon bundles of the anterior commissure encountered the wall of the third ventricle and ceased axonal extension. L1-CAM immunohistochemistry showed that Olig2-KO mice lose decussation formation in the basal forebrain. DiI tracing revealed that the thin bundles of the anterior commissure axons crossed the midline but ceased further extension into the deep part of the contralateral side. Furthermore, some fractions of DiI-labelled axons were oriented dorsolaterally, which was not observed in the control mouse forebrain. The rostral part of the third ventricle was much wider in the Olig2-KO mice than in wild-type mice, which likely resulted in the delay of midline fusion and subsequent delay and malformation of the anterior commissure. We analysed gene expression alterations in the Olig2-KO mice using a public database and found multiple genes, which are related to axon guidance and epithelial-mesenchymal transition, showing subtle expression changes. These results suggest that Olig2 is essential for anterior commissure formation, likely by regulating multiple biological processes.

Histochemical Characterization of the Dorsal Raphe-Periaqueductal Grey Dopamine Transporter Neurons Projecting to the Extended Amygdala.

The dorsal raphe (DR) nucleus contains many tyrosine hydroxylase (TH)-positive neurons which are regarded as dopaminergic (DA) neurons. These DA neurons in the DR and periaqueductal gray (PAG) region (DADR-PAG neurons) are a subgroup of the A10 cluster, which is known to be heterogeneous. This DA population projects to the central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BNST) and has been reported to modulate various affective behaviors. To characterize, the histochemical features of DADR-PAG neurons projecting to the CeA and BNST in mice, the current study combined retrograde labeling with Fluoro-Gold (FG) and histological techniques, focusing on TH, dopamine transporter (DAT), vasoactive intestinal peptide (VIP), and vesicular glutamate transporter 2 (VGlut2). To identify putative DA neurons, DAT-Cre::Ai14 mice were used. It was observed that DATDR-PAG neurons consisted of the following two subpopulations: TH+/VIP- and TH-/VIP+ neurons. The DAT+/TH-/VIP+ subpopulation would be non-DA noncanonical DAT neurons. Anterograde labeling of DATDR-PAG neurons with AAV in DAT-Cre mice revealed that the fibers exclusively innervated the lateral part of the CeA and the oval nucleus of the BNST. Retrograde labeling with FG injections into the CeA or BNST revealed that the two subpopulations similarly innervated these regions. Furthermore, using VGlut2-Cre::Ai14 mice, it was turned out that the TH-/VIP+ subpopulations innervating both CeA and BNST were VGlut2-positive neurons. These two subpopulations of DATDR-PAG neurons, TH+/VIP- and TH-/VIP+, might differentially interfere with the extended amygdala, thereby modulating affective behaviors.

Thermal Proteome Profiling Reveals Glutathione Peroxidase 4 as the Target of the Autophagy Inducer Conophylline.

Conophylline (CNP) is a vinca alkaloid extracted from the Tabernaemontana divaricata plant. It has been reported that CNP induces autophagy in a mammalian target of rapamycin-independent manner, and thereby inhibits protein aggregation. However, the mode of action of CNP in inducing autophagy remains unknown. In this study, we identified glutathione peroxidase 4 (GPX4) as a CNP-binding protein by using thermal proteome profiling. The technique exploits changes in the thermal stability of proteins resulting from ligand interaction, which is capable of identifying compound-binding proteins without chemical modification. GPX4, an antioxidant protein that uses reduced glutathione as a cofactor, directly catalyzes the reduction of hydrogen peroxide, organic hydroperoxides, and lipid peroxides. GPX4 suppresses lipid peroxide accumulation, and thus plays a key role in protecting cells from oxidative damage. We found that treatment with CNP caused accumulation of lipid reactive oxygen species (ROS) in cultured cells. Furthermore, similarly with CNP treatment, GPX4 deficiency caused accumulation of lipid ROS and induced autophagy. These findings indicate that GPX4 is a direct target of CNP involved in autophagy induction. SIGNIFICANCE STATEMENT: The present study identified glutathione peroxidase 4 (GPX4) as a binding protein of conophylline (CNP) by using thermal proteome profiling (TPP). This study showed that CNP treatment, similarly with the inhibition of GPX4, induced lipid reactive oxygen species accumulation and autophagy. The present findings suggest that GPX4 is the CNP target protein involved in autophagy induction. Furthermore, these results indicate that TPP is a useful technique for determining the mechanism of natural compounds.

Role of Decorin in Posterior Capsule Opacification and Eye Lens Development.

Decorin (DCN) is involved in a variety of physiological and pathological processes. Epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) has been proposed as a major cause for the development of posterior capsule opacification (PCO) after cataract surgery. We investigated the plausible target gene(s) that suppress PCO. The expression of Dcn was significantly upregulated in rat PCO tissues compared to that observed in the control using a microarray-based approach. LECs treated with fibroblast growth factor (FGF) 2 displayed an enhanced level of DCN expression, while LECs treated with transforming growth factor (TGF)ß-2 showed a decrease in DCN expression. The expression of tropomyosin 1 (Tpm1), a marker of lens EMT increased after the addition of TGFß-2 in human LEC; however, upregulation of Tpm1 mRNA or protein expression was reduced in human LECs overexpressing human DCN (hDCN). No phenotypic changes were observed in the lenses of 8- and 48-week-old transgenic mice for lens-specific hDCN (hDCN-Tg). Injury-induced EMT of the mouse lens, and the expression patterns of α smooth muscle actin, were attenuated in hDCN-Tg mice lenses. Overexpression of DCN inhibited the TGFß-2-induced upregulation of Tpm1 and EMT observed during wound healing of the lens, but it did not affect mouse lens morphology until 48 weeks of age. Our findings demonstrate that DCN plays a significant role in regulating EMT formation of LECs and PCO, and suggest that for therapeutic intervention, maintenance of physiological expression of DCN is essential to attenuate EMT progression and PCO formation.

Induced 2C Expression and Implantation-Competent Blastocyst-like Cysts from Primed Pluripotent Stem Cells.

Soon after fertilization, the few totipotent cells of mammalian embryos diverge to form a structure called the blastocyst (BC). Although numerous cell types, including germ cells and extended-pluripotency stem cells, have been developed from pluripotent stem cells (PSCs) in vitro, generating functional BCs only from PSCs remains elusive. Here, we describe induced self-organizing 3D BC-like cysts (iBLCs) generated from mouse PSC culture. Resembling natural BCs, iBLCs have a blastocoel-like cavity and were formed with outer cells expressing trophectoderm lineage markers and with inner cells expressing pluripotency markers. iBLCs transplanted to pseudopregnant mice uteruses implanted, induced decidualization, and exhibited growth and development before resorption, demonstrating that iBLCs are implantation competent. iBLC precursor intermediates required the transcription factor Prdm14 and concomitantly activated the totipotency-related cleavage-stage MERVL reporter and 2C genes. Thus, our system may contribute to the understanding of molecular mechanisms underpinning totipotency, embryogenesis, and implantation.

Overlapping functions of Krüppel-like factor family members: targeting multiple transcription factors to maintain the naïve pluripotency of mouse embryonic stem cells.

Krüppel-like factors (Klfs) have a pivotal role in maintaining self-renewal of mouse embryonic stem cells (mESCs). The functions of three Klf family members (Klf2, Klf4 and Klf5) have been identified, and are suggested to largely overlap. For further dissection of their functions, we applied an inducible knockout system for these Klf family members and assessed the effects of combinatorial loss of function. As a result, we confirmed that any one of Klf2, Klf4 and Klf5 was sufficient to support self-renewal, whereas the removal of all three compromised it. The activity of any single transcription factor, except for a Klf family member, was not sufficient to restore self-renewal of triple-knockout mESCs. However, some particular combinations of transcription factors were capable of the restoration. The triple-knockout mESCs were successfully captured at primed state. These data indicate that the pivotal function of a Klf family member is transduced into the activation of multiple transcription factors in a naïve-state-specific manner.

The evolutionally-conserved function of group B1 Sox family members confers the unique role of Sox2 in mouse ES cells.

BACKGROUND: In mouse ES cells, the function of Sox2 is essential for the maintenance of pluripotency. Since the Sox-family of transcription factors are well conserved in the animal kingdom, addressing the evolutionary origin of Sox2 function in pluripotent stem cells is intriguing from the perspective of understanding the origin of pluripotency. RESULTS: Here we approach this question using a functional complementation assay in inducible Sox2-null ES cells. Assaying mouse Sox proteins from different Groups, we found that only Group B1 and Group G proteins were able to support pluripotency. Interestingly, invertebrate homologs of mammalian Group B1 Sox proteins were able to replace the pluripotency-associated function of mouse Sox2. Moreover, the mouse ES cells rescued by the Drosophila SoxNeuro protein are able to contribute to chimeric embryos. CONCLUSIONS: These data indicate that the function of mouse Sox2 supporting pluripotency is based on an evolutionally conserved activity of the Group B1 Sox family. Since pluripotent stem cell population in developmental process could be regarded as the evolutional novelty in vertebrates, it could be regarded as a co-optional use of their evolutionally conserved function.

Zscan10 is dispensable for maintenance of pluripotency in mouse embryonic stem cells.

Zinc finger and SCAN domain-containing 10 (Zscan10, also known as Zfp206) encodes a transcription factor that has been reported to be involved in the maintenance of pluripotency in mouse embryonic stem (ES) cells. Here we generated inducible knockout ES cells for Zscan10 using the Cre-loxP system and analyzed its function. We succeeded in establishing Zscan10-null ES cells and confirmed their pluripotency by the generation of chimeric embryos. Our results clearly indicate that Zscan10 is dispensable for the ability of self-renewal and differentiation in ES cells.

Nr0b1 is a negative regulator of Zscan4c in mouse embryonic stem cells.

Nuclear receptor subfamily 0, group B, member 1 (Nr0b1, also known as Dax1) is regarded as an important component of the transcription factor network that governs pluripotency in mouse embryonic stem (ES) cells. Here we generated inducible knockout ES cells for Nr0b1 using the Cre-loxP system and analyzed its precise function. We succeeded in establishing the Nr0b1-null ES cells and confirmed their pluripotency by showing their contribution to chimeric embryos. However, they proliferated slowly with over-expression of 2-cell stage specific transcripts including Zscan4c, which is known to be involved in telomere elongation in ES cells. We revealed that over-expression of Zscan4c prevents normal self-renewal by inducing arrest at G2 phase followed by cell death and that Nr0b1 directly represses the Zscan4c promoter. These data indicated that Nr0b1 is not essential to maintain pluripotency but is involved in the proper activation of 2-cell specific transcripts for self-renewal.

The differential activation of intracellular signaling pathways confers the permissiveness of embryonic stem cell derivation from different mouse strains.

The requirement of leukemia inhibitory factor (LIF) for the establishment and maintenance of mouse embryonic stem cells (ESCs) depends on the genetic background of the ESC origin. To reveal the molecular basis of the strain-dependent function of LIF, we compared the activation of the intracellular signaling pathways downstream of LIF in ESCs with different genetic backgrounds. We found that the JAK-Stat3 pathway was dominantly activated in ESCs derived from 'permissive' mouse strains (129Sv and C57BL6), whereas the MAP kinase pathway was hyperactivated in ESCs from 'non-permissive' strains (NOD, CBA and FVB). Artificial activation of Stat3 supported stable self-renewal of ESCs from non-permissive strains. These data suggest that the difference in the balance between the two intracellular signaling pathways underlies the differential response to LIF.

LIF signal in mouse embryonic stem cells.

Since the establishment of mouse embryonic stem cells (mESCs) in the 1980s, a number of important notions on the self-renewal of pluripotent stem cells in vitro have been found. In serum containing conventional culture, an exogenous cytokine, leukemia inhibitory factor (LIF), is absolutely essential for the maintenance of pluripotency. In contrast, in serum-free culture with simultaneous inhibition of Map-kinase and Gsk3 (so called 2i-culture), LIF is no longer required. However, recent findings also suggest that LIF may have a role not covered by the 2i for the maintenance of naïve pluripotency. These suggest that LIF functions for the maintenance of naïve pluripotency in a context dependent manner. We summarize how LIF-signal pathway is converged to maintain the naïve state of pluripotency.

The C-terminal region of Xpc is dispensable for the transcriptional activity of Oct3/4 in mouse embryonic stem cells.

The transcription factor Oct3/4 is essential to maintain pluripotency in mouse embryonic stem (ES) cells. It was reported that the Xpc DNA repair complex is involved in this process. Here we examined the role of Xpc on the transcriptional activation of the target genes by Oct3/4 using the inducible knockout strategy. We found that the removal of the C-terminal region of Xpc, including the interaction sites with Rad23 and Cetn2, showed faint impact on the gene expression profile of ES cells and the functional Xpc-ΔC ES cell lines retained proper gene expression profile as well as pluripotency to contribute chimeric embryos. These data indicated that the C-terminal region of Xpc is dispensable for the transcriptional activity of Oct3/4 in mouse ES cells.

Context-dependent wiring of Sox2 regulatory networks for self-renewal of embryonic and trophoblast stem cells.

Sox2 is a transcription factor required for the maintenance of pluripotency. It also plays an essential role in different types of multipotent stem cells, raising the possibility that Sox2 governs the common stemness phenotype. Here we show that Sox2 is a critical downstream target of fibroblast growth factor (FGF) signaling, which mediates self-renewal of trophoblast stem cells (TSCs). Sustained expression of Sox2 together with Esrrb or Tfap2c can replace FGF dependency. By comparing genome-wide binding sites of Sox2 in embryonic stem cells (ESCs) and TSCs combined with inducible knockout systems, we found that, despite the common role in safeguarding the stem cell state, Sox2 regulates distinct sets of genes with unique functions in these two different yet developmentally related types of stem cells. Our findings provide insights into the functional versatility of transcription factors during embryogenesis, during which they can be recursively utilized in a variable manner within discrete network structures.

Maintenance of pluripotency in mouse ES cells without Trp53.

Tumor suppressor Trp53 works as a guardian of the genome in somatic cells. In mouse embryonic stem (ES) cells, it was reported that Trp53 represses pluripotency-associated transcription factor Nanog to induce differentiation. However, since Trp53-null mice develop to term, Trp53 is dispensable for both the maintenance and differentiation of the pluripotent stem cell population in vivo, suggesting the differential functions of Trp53 in ES cells and embryos. To reveal the basis of this discrepancy, here we established a new line of Trp53-null ES cells by sequential gene targeting and evaluated their ability to differentiate in vitro and in vivo. We found that Trp53-null ES cells had defects in differentiation in vitro as reported previously, whereas they were able to contribute to normal development in chimeric embryos. These data indicated that the requirement of Trp53 for maintaining and executing the ES pluripotency is not absolute.

E-cadherin promotes incorporation of mouse epiblast stem cells into normal development.

Mouse epiblast stem cells (mEpiSCs) are pluripotent stem cells derived from epiblasts of postimplantation mouse embryos. Their pluripotency is distinct from that of mouse embryonic stem cells (mESCs) in several cell biological criteria. One of the distinctions is that mEpiSCs contribute either not at all or at much lower efficiency to chimeric embryos after blastocyst injection compared to mESCs. However, here we showed that mEpiSCs can be incorporated into normal development after blastocyst injection by forced expression of the E-cadherin transgene for 2 days in culture. Using this strategy, mEpiSCs gave rise to live-born chimeras from 5% of the manipulated blastocysts. There were no obvious signs of reprogramming of mEpiSCs toward the mESC-like state during the 2 days after induction of the E-cadherin transgene, suggesting that mEpiSCs possess latent ability to integrate into the normal developmental process as its origin, epiblasts.

Esrrb is a pivotal target of the Gsk3/Tcf3 axis regulating embryonic stem cell self-renewal.

Inhibition of glycogen synthase kinase-3 (Gsk3) supports mouse embryonic stem cells (ESCs) by modulating Tcf3, but the critical targets downstream of Tcf3 are unclear. We analyzed the intersection between genome localization and transcriptome data sets to identify genes repressed by Tcf3. Among these, manipulations of Esrrb gave distinctive phenotypes in functional assays. Knockdown and knockout eliminated response to Gsk3 inhibition, causing extinction of pluripotency markers and loss of colony forming capability. Conversely, forced expression phenocopied Gsk3 inhibition or Tcf3 deletion by suppressing differentiation and sustaining self-renewal. Thus the nuclear receptor Esrrb is necessary and sufficient to mediate self-renewal downstream of Gsk3 inhibition. Leukaemia inhibitory factor (LIF) regulates ESCs through Stat3, independently of Gsk3 inhibition. Consistent with parallel operation, ESCs in LIF accommodated Esrrb deletion and remained pluripotent. These findings highlight a key role for Esrrb in regulating the naive pluripotent state and illustrate compensation among the core pluripotency factors.

Signaling network crosstalk in human pluripotent cells: a Smad2/3-regulated switch that controls the balance between self-renewal and differentiation.

A general mechanism for how intracellular signaling pathways in human pluripotent cells are coordinated and how they maintain self-renewal remain to be elucidated. In this report, we describe a signaling mechanism where PI3K/Akt activity maintains self-renewal by restraining prodifferentiation signaling through suppression of the Raf/Mek/Erk and canonical Wnt signaling pathways. When active, PI3K/Akt establishes conditions where Activin A/Smad2,3 performs a pro-self-renewal function by activating target genes, including Nanog. When PI3K/Akt signaling is low, Wnt effectors are activated and function in conjunction with Smad2,3 to promote differentiation. The switch in Smad2,3 activity after inactivation of PI3K/Akt requires the activation of canonical Wnt signaling by Erk, which targets Gsk3ß. In sum, we define a signaling framework that converges on Smad2,3 and determines its ability to regulate the balance between alternative cell states. This signaling paradigm has far-reaching implications for cell fate decisions during early embryonic development.

Amphioxus connectin exhibits merged structure as invertebrate connectin in I-band region and vertebrate connectin in A-band region.

Connectin is an elastic protein found in vertebrate striated muscle and in some invertebrates as connectin-like proteins. In this study, we determined the structure of the amphioxus connectin gene and analyzed its sequence based on its genomic information. Amphioxus is not a vertebrate but, phylogenetically, the lowest chordate. Analysis of gene structure revealed that the amphioxus gene is approximately 430 kb in length and consists of regions with exons of repeatedly aligned immunoglobulin (Ig) domains and regions with exons of fibronectin type 3 and Ig domain repeats. With regard to this sequence, although the region corresponding to the I-band is homologous to that of invertebrate connectin-like proteins and has an Ig-PEVK region similar to that of the Neanthes sp. 4000K protein, the region corresponding to the A-band has a super-repeat structure of Ig and fibronectin type 3 domains and a kinase domain near the C-terminus, which is similar to the structure of vertebrate connectin. These findings revealed that amphioxus connectin has the domain structure of invertebrate connectin-like proteins at its N-terminus and that of vertebrate connectin at its C-terminus. Thus, amphioxus connectin has a novel structure among known connectin-like proteins. This finding suggests that the formation and maintenance of the sarcomeric structure of amphioxus striated muscle are similar to those of vertebrates; however, its elasticity is different from that of vertebrates, being more similar to that of invertebrates.

Choice of random rather than imprinted X inactivation in female embryonic stem cell-derived extra-embryonic cells.

In female mammals, one of two X chromosomes is epigenetically inactivated for gene dosage compensation, known as X inactivation (Xi). Inactivation occurs randomly in either the paternal or maternal X chromosome in all embryonic cell lineages, designated as random Xi. By contrast, in extra-embryonic cell lineages, which are segregated from somatic cell lineages in pre-implantation development, the paternal X chromosome is selectively inactivated, known as imprinted Xi. Although it is speculated that erasure of the imprinted mark on either the maternal or paternal X chromosome in somatic cell lineages might change the mode of Xi from imprinted to random, it is not known when this event is completed in development. Here, we tested the mode of Xi during the differentiation of female mouse embryonic stem (ES) cells derived from the inner cell mass (ICM) of blastocyst-stage embryos toward trophectoderm (TE) and primitive endoderm (PrE) lineages induced by artificial activation of transcription factor genes Cdx2 and Gata6, respectively. We found that random Xi occurs in both TE and PrE cells. Moreover, cloned embryos generated by the transfer of nuclei from the female ES cells showed random Xi in TE, suggesting the complete erasure of all X imprints for imprinted Xi in ICM-derived ES cells.