Neuroectodermal Genes Research Articles

Identification of an embryonic differentiation stage marked by Sox1 and FoxA2 co-expression using combined cell tracking and high dimensional protein imaging

Pluripotent mouse embryonic stem cells (ESCs) can differentiate to all germ layers and serve as an in vitro model of embryonic development. To better understand the differentiation paths traversed by ESCs committing to different lineages, we track individual differentiating ESCs by timelapse imaging followed by multiplexed high-dimensional Imaging Mass Cytometry (IMC) protein quantification. This links continuous live single-cell molecular NANOG and cellular dynamics quantification over 5-6 generations to protein expression of 37 different molecular regulators in the same single cells at the observation endpoints. Using this unique data set including kinship history and live lineage marker detection, we show that NANOG downregulation occurs generations prior to, but is not sufficient for neuroectoderm marker Sox1 upregulation. We identify a developmental cell type co-expressing both the canonical Sox1 neuroectoderm and FoxA2 endoderm markers in vitro and confirm the presence of such a population in the post-implantation embryo. RNASeq reveals cells co-expressing SOX1 and FOXA2 to have a unique cell state characterized by expression of both endoderm as well as neuroectoderm genes suggesting lineage potential towards both germ layers.

CTCF deletion alters the pluripotency and DNA methylation profile of human iPSCs

Pluripotent stem cells are characterized by their differentiation potential toward endoderm, mesoderm, and ectoderm. However, it is still largely unclear how these cell-fate decisions are mediated by epigenetic mechanisms. In this study, we explored the relevance of CCCTC-binding factor (CTCF), a zinc finger-containing DNA-binding protein, which mediates long-range chromatin organization, for directed cell-fate determination. We generated human induced pluripotent stem cell (iPSC) lines with deletions in the protein-coding region in exon 3 of CTCF, resulting in shorter transcripts and overall reduced protein expression. Chromatin immunoprecipitation showed a considerable loss of CTCF binding to target sites. The CTCF deletions resulted in slower growth and modest global changes in gene expression, with downregulation of a subset of pluripotency-associated genes and neuroectodermal genes. CTCF deletion also evoked DNA methylation changes, which were moderately associated with differential gene expression. Notably, CTCF-deletions lead to upregulation of endo-mesodermal associated marker genes and epigenetic signatures, whereas ectodermal differentiation was defective. These results indicate that CTCF plays an important role in the maintenance of pluripotency and differentiation, especially towards ectodermal lineages.

Genes Involved in DNA Repair and Mitophagy Protect Embryoid Bodies from the Toxic Effect of Methylmercury Chloride under Physioxia Conditions.

The formation of embryoid bodies (EBs) from human pluripotent stem cells resembles the early stages of human embryo development, mimicking the organization of three germ layers. In our study, EBs were tested for their vulnerability to chronic exposure to low doses of MeHgCl (1 nM) under atmospheric (21%O2) and physioxia (5%O2) conditions. Significant differences were observed in the relative expression of genes associated with DNA repair and mitophagy between the tested oxygen conditions in nontreated EBs. When compared to physioxia conditions, the significant differences recorded in EBs cultured at 21% O2 included: (1) lower expression of genes associated with DNA repair (ATM, OGG1, PARP1, POLG1) and mitophagy (PARK2); (2) higher level of mtDNA copy number; and (3) higher expression of the neuroectodermal gene (NES). Chronic exposure to a low dose of MeHgCl (1 nM) disrupted the development of EBs under both oxygen conditions. However, only EBs exposed to MeHgCl at 21% O2 revealed downregulation of mtDNA copy number, increased oxidative DNA damage and DNA fragmentation, as well as disturbances in SOX17 (endoderm) and TBXT (mesoderm) genes expression. Our data revealed that physioxia conditions protected EBs genome integrity and their further differentiation.

Patched-Related Is Required for Proper Development of Embryonic Drosophila Nervous System.

Patched-related (Ptr), classified primarily as a neuroectodermal gene, encodes a protein with predicted topology and domain organization closely related to those of Patched (Ptc), the canonical receptor of the Hedgehog (Hh) pathway. To investigate the physiological function of Ptr in the developing nervous system, Ptr null mutant embryos were immunolabeled and imaged under confocal microscopy. These embryos displayed severe alterations in the morphology of the primary axonal tracts, reduced number, and altered distribution of the Repo-positive glia as well as peripheral nervous system defects. Most of these alterations were recapitulated by downregulating Ptr expression, specifically in embryonic nerve cells. Because similar nervous system phenotypes have been observed in hh and ptc mutant embryos, we evaluated the Ptr participation in the Hh pathway by performing cell-based reporter assays. Clone-8 cells were transfected with Ptr-specific dsRNA or a Ptr DNA construct and assayed for changes in Hh-mediated induction of a luciferase reporter. The results obtained suggest that Ptr could act as a negative regulator of Hh signaling. Furthermore, co-immunoprecipitation assays from cell culture extracts premixed with a conditioned medium revealed a direct interaction between Ptr and Hh. Moreover, in vivo Ptr overexpression in the domain of the imaginal wing disc where Engrailed and Ptc coexist produced wing phenotypes at the A/P border. Thus, these results strongly suggest that Ptr plays a crucial role in nervous system development and appears to be a negative regulator of the Hh pathway.

Coup-TF: A maternal factor essential for differentiation along the embryonic axes in the sea urchin Paracentrotus lividus

Coup-TF, a member of the nuclear receptor super-family, is present in the pool of maternal mRNAs and proteins in the sea urchin egg. The presence of this protein seems to be essential for the execution of the early developmental program, leading to all three embryonic layers. Our results demonstrate that Pl-Coup-TF morphants, i.e. Pl-Coup-TF morpholino knockdown embryos, resemble blastulae that lack archenteron at 24 hpf (hours post fertilization), a stage at which normal embryos reach the end of gastrulation in Paracentrotus lividus. At 48 hpf, when normal embryos reach the pluteus larva stage, the morphants are seemingly underdeveloped and lack the characteristic skeletal rods. Nevertheless, the morphant embryos express vegetal endomesodermal marker genes, such as Pl-Blimp1, Pl-Endo16, Pl-Alx1 and Pl-Tbr as judged by in situ hybridization experiments. The anterior neuroectoderm genes, Pl-FoxQ2, Pl-Six3 and Pl-Pax6, are also expressed in the morphant embryos, but Pl-Hbn and Pl-Fez mRNAs, which encode proteins significant for the differentiation of serotonergic neurons, are not detected. Consequently, Pl-Coup-TF morphants at 48 hpf lack serotonergic neurons, whereas normal 48 hpf plutei exhibit the formation of two bilateral pairs of such neurons in the apical organ. Furthermore, genes indicative of the ciliary band formation, Pl-Hnf6, Pl-Dri, Pl-FoxG and Pl-Otx, are not expressed in Pl-Coup-TF morphants, suggesting the disruption of this neurogenic territory as well. In addition, the Pl-SynB gene, a marker of differentiated neurons, is silent leading to the hypothesis that Pl-Coup-TF morphants might lack all types of neurons. On the contrary, the genes expressing signaling molecules, which establish the ventral/dorsal axis, Pl-Nodal and Pl-Lefty show the characteristic ventral lateral expression pattern, Pl-Bmp2/4, which activates the dorsal ectoderm GRN is down-regulated and Pl-Chordin is aberrantly over-expressed in the entire ectoderm. The identity of ectodermal cells in Pl-Coup-TF morphant embryos, was probed for expression of the ventral marker Pl-Gsc which was over-expressed and dorsal markers, Pl-IrxA and Pl-Hox7, which were silent. Therefore, we propose that maternal Pl-Coup-TF is essential for correct dissemination of the early embryonic signaling along both animal/vegetal and ventral/dorsal axes. Limiting Pl-Coup-TF's quantity, results in an embryo without digestive and nervous systems, skeleton and ciliary band that cannot survive past the initial 48 h of development.

Apoptosis is involved in maintaining the character of the midbrain and the diencephalon roof plate after neural tube closure

Apoptosis, a major form of programmed cell death, is massively observed in neural plate border and subsequently in the roof plate (RP). While deficiency of apoptosis often results in brain malformations including exencephaly and hydrocephalus, the impact of apoptosis on RP formation and maintenance remains unclear. Here we described that mouse embryos deficient in Apaf1, a gene crucial for the intrinsic apoptotic pathway, in C57BL/6 genetic background exhibited narrow and discontinuous expression of RP marker genes in the midline of the midbrain and the diencephalon. Instead, cells positive for the neuroectodermal gene SOX1 ectopically accumulated in the midline. A lineage-tracing experiment suggests that these ectopic SOX1-positive cells began to accumulate in the midline of apoptosis-deficient embryos after E9.5. These embryos further displayed malformation of the subcommissural organ, which has been discussed in the etiology of hydrocephalus. Thus, the apoptosis machinery prevents ectopic emergence of SOX1-positive cells in the midbrain and the diencephalon RP, and helps in maintaining the character of the RP in the diencephalon and midbrain, thereby ensuring proper brain development.

TATA box-binding protein-related factor 3 drives the mesendoderm specification of human embryonic stem cells by globally interacting with the TATA box of key mesendodermal genes

BackgroundMesendodermal formation during early gastrulation requires the expression of lineage-specific genes, while the regulatory mechanisms during this process have not yet been fully illustrated. TATA box-binding protein (TBP) and TBP-like factors are general transcription factors responsible for the transcription initiation by recruiting the preinitiation complex to promoter regions. However, the role of TBP family members in the regulation of mesendodermal specification remains largely unknown.MethodsWe used an in vitro mesendodermal differentiation system of human embryonic stem cells (hESCs), combining with the microarray and quantitative polymerase chain reaction (qRT-PCR) analysis, loss of function and gain of function to determine the function of the TBP family member TBP-related factor 3 (TRF3) during mesendodermal differentiation of hESCs. The chromatin immunoprecipitation (ChIP) and biochemistry analysis were used to determine the binding of TRF3 to the promoter region of key mesendodermal genes.ResultsThe mesendodermal differentiation of hESCs was confirmed by the microarray gene expression profile, qRT-PCR, and immunocytochemical staining. The expression of TRF3 mRNA was enhanced during mesendodermal differentiation of hESCs. The TRF3 deficiency did not affect the pluripotent marker expression, alkaline phosphatase activity, and cell cycle distribution of undifferentiated hESCs or the expression of early neuroectodermal genes during neuroectodermal differentiation. During the mesendodermal differentiation, the expression of pluripotency markers decreased in both wild-type and TRF3 knockout (TRF3−/−) cells, while the TRF3 deficiency crippled the expression of the mesendodermal markers. The reintroduction of TRF3 into the TRF3−/− hESCs rescued inhibited mesendodermal differentiation. Mechanistically, the TRF3 binding profile was significantly shifted to the mesendodermal specification during mesendodermal differentiation of hESCs based on the ChIP-seq data. Moreover, ChIP and ChIP-qPCR analysis showed that TRF3 was enriched at core promoter regions of mesendodermal developmental genes, EOMESODERMIN, BRACHYURY, mix paired-like homeobox, and GOOSECOID homeobox, during mesendodermal differentiation of hESCs.ConclusionsThese results reveal that the TBP family member TRF3 is dispensable in the undifferentiated hESCs and the early neuroectodermal differentiation. However, it directs mesendodermal lineage commitment of hESCs via specifically promoting the transcription of key mesendodermal transcription factors. These findings provide new insights into the function and mechanisms of the TBP family member in hESC early lineage specification.

Coordination of germ layer lineage choice by TET1 during primed pluripotency.

Gastrulation in the early postimplantation stage mammalian embryo begins when epiblast cells ingress to form the primitive streak or develop as the embryonic ectoderm. The DNA dioxygenase Tet1 is highly expressed in the epiblast and yet continues to regulate lineage specification during gastrulation when its expression is diminished. Here, we show how Tet1 plays a pivotal role upstream of germ layer lineage bifurcation. During the transition from naive pluripotency to lineage priming, a global reconfiguration redistributes Tet1 from Oct4-cobound promoters to distal regulatory elements at lineage differentiation genes, which are distinct from high-affinity sites engaged by Oct4. An altered chromatin landscape in Tet1-deficient primed epiblast-like cells is associated with enhanced Oct4 expression and binding to Nodal and Wnt target genes, resulting in collaborative signals that enhance mesendodermal and inhibit neuroectodermal gene expression during lineage segregation. A permissive role for Tet1 in neural fate induction involves Zic2-dependent engagement at neural target genes at lineage priming, is dependent on the signaling environment during gastrulation, and impacts neural tube closure after gastrulation. Our findings provide mechanistic information for epigenetic integration of pluripotency and signal-induced differentiation cues.

A biphasic role of non-canonical Wnt16 signaling during early anterior-posterior patterning and morphogenesis of the sea urchin embryo.

ABSTRACTA Wnt signaling network governs early anterior-posterior (AP) specification and patterning of the deuterostome sea urchin embryo. We have previously shown that non-canonical Fzl1/2/7 signaling antagonizes the progressive posterior-to-anterior downregulation of the anterior neuroectoderm (ANE) gene regulatory network (GRN) by canonical Wnt/β-catenin and non-canonical Wnt1/Wnt8-Fzl5/8-JNK signaling. This study focuses on the non-canonical function of the Wnt16 ligand during early AP specification and patterning. Maternally supplied wnt16 is expressed ubiquitously during cleavage and zygotic wnt16 expression is concentrated in the endoderm/mesoderm beginning at mid-blastula stage. Wnt16 antagonizes the ANE restriction mechanism and this activity depends on a functional Fzl1/2/7 receptor. Our results also show that zygotic wnt16 expression depends on both Fzl5/8 and Wnt/β-catenin signaling. Furthermore, Wnt16 is necessary for the activation and/or maintenance of key regulatory endoderm/mesoderm genes and is essential for gastrulation. Together, our data show that Wnt16 has two functions during early AP specification and patterning: (1) an initial role activating the Fzl1/2/7 pathway that antagonizes the ANE restriction mechanism; and (2) a subsequent function in activating key endoderm GRN factors and the morphogenetic movements of gastrulation.

Eomes and Brachyury control pluripotency exit and germ-layer segregation by changing the chromatin state.

The first lineage specification of pluripotent mouse epiblast segregates neuroectoderm (NE) from mesoderm and definitive endoderm (ME) by mechanisms that are not well understood. Here we demonstrate that the induction of ME gene programs critically relies on the T-box transcription factors Eomesodermin (also known as Eomes) and Brachyury, which concomitantly repress pluripotency and NE gene programs. Cells deficient in these T-box transcription factors retain pluripotency and differentiate to NE lineages despite the presence of ME-inducing signals transforming growth factor β (TGF-β)/Nodal and Wnt. Pluripotency and NE gene networks are additionally repressed by ME factors downstream of T-box factor induction, demonstrating a redundancy in program regulation to safeguard mutually exclusive lineage specification. Analyses of chromatin revealed that accessibility of ME enhancers depends on T-box factor binding, whereas NE enhancers are accessible and already activation primed at pluripotency. This asymmetry of the chromatin landscape thus explains the default differentiation of pluripotent cells to NE in the absence of ME induction that depends on activating and repressive functions of Eomes and Brachyury.

Down-regulation of ATF1 leads to early neuroectoderm differentiation of human embryonic stem cells by increasing the expression level of SOX2.

We reveal by high-throughput screening that activating transcription factor 1 (ATF1) is a novel pluripotent regulator in human embryonic stem cells (hESCs). The knockdown of ATF1 expression significantly up-regulated neuroectoderm (NE) genes but not mesoderm, endoderm, and trophectoderm genes. Of note, down-regulation or knockout of ATF1 with short hairpin RNA (shRNA), small interfering RNA (siRNA), or clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) was sufficient to up-regulate sex-determining region Y-box (SOX)2 and paired box 6 (PAX6) expression under the undifferentiated or differentiated conditions, whereas overexpression of ATF1 suppressed NE differentiation. Endogenous ATF1 was spontaneously down-regulated after d 1-3 of neural induction. By double-knockdown experiments, up-regulation of SOX2 was critical for the increase of PAX6 and SOX1 expression in shRNA targeting Atf1 hESCs. Using the luciferase reporter assay, we identified ATF1 as a negative transcriptional regulator of Sox2 gene expression. A novel function of ATF1 was discovered, and these findings contribute to a broader understanding of the very first steps in regulating NE differentiation in hESCs.-Yang, S.-C., Liu, J.-J., Wang, C.-K., Lin, Y.-T., Tsai, S.-Y., Chen, W.-J., Huang, W.-K., Tu, P.-W. A., Lin, Y.-C., Chang, C.-F., Cheng, C.-L., Lin, H., Lai, C.-Y., Lin, C.-Y., Lee, Y.-H., Chiu, Y.-C., Hsu, C.-C., Hsu, S.-C., Hsiao, M., Schuyler, S. C., Lu, F. L., Lu, J. Down-regulation of ATF1 leads to early neuroectoderm differentiation of human embryonic stem cells by increasing the expression level of SOX2.

Ventx1.1 as a Direct Repressor of Early Neural Gene zic3 in Xenopus laevis.

From Xenopus embryo studies, the BMP4/Smad1-targeted gene circuit is a key signaling pathway for specifying the cell fate between the ectoderm and neuro-ectoderm as well as the ventral and dorsal mesoderm. In this context, several BMP4/Smad1 target transcriptional factors have been identified as repressors of the neuro-ectoderm. However, none of these direct target transcription factors in this pathway, including GATA1b, Msx1 and Ventx1.1 have yet been proven as direct repressors of early neuro-ectodermal gene expression. In order to demonstrate that Ventx1.1 is a direct repressor of neuro-ectoderm genes, a genome-wide Xenopus ChIP-Seq of Ventx1.1 was performed. In this study, we demonstrated that Ventx1.1 bound to the Ventx1.1 response cis-acting element 1 and 2 (VRE1 and VRE2) on the promoter for zic3, which is a key early neuro-ectoderm gene, and this Ventx1.1 binding led to repression of zic3 transcription. Site-directed mutagenesis of VRE1 and VRE2 within zic3 promoter completely abolished the repression caused by Ventx1.1. In addition, we found both the positive and negative regulation of zic3 promoter activity by FoxD5b and Xcad2, respectively, and that these occur through the VREs and via modulation of Ventx1.1 levels. Taken together, the results demonstrate that the BMP4/Smad1 target gene, Ventx1.1, is a direct repressor of neuro-ectodermal gene zic3 during early Xenopus embryogenesis.

Canonical and non-canonical Wnt signaling pathways define the expression domains of Frizzled 5/8 and Frizzled 1/2/7 along the early anterior-posterior axis in sea urchin embryos

The spatiotemporal expression of Frizzled receptors is critical for patterning along the early anterior-posterior axis during embryonic development in many animal species. However, the molecular mechanisms that regulate the expression of Frizzled receptors are incompletely understood in any species. In this study, I examine how the expression of two Frizzled receptors, Fzl1/2/7 and Fzl5/8, is controlled by the Wnt signaling network which directs specification and positioning of early regulatory states along the anterior-posterior (AP) axis of sea urchin embryos. I used a combination of morpholino- and dominant negative-mediated interference to knock down each Wnt signaling pathway involved in the AP Wnt signaling network. I found that the expression of zygotic fzl5/8 as well as that of the anterior neuroectoderm gene regulatory network (ANE GRN) is activated by an unknown broadly expressed regulatory state and that posterior Wnt/β-catenin signaling is necessary to down regulate fzl5/8's expression in posterior blastomeres. I show that zygotic expression of fzl1/2/7 in the equatorial ectodermal belt is dependent on an uncharacterized regulatory mechanism that works in the same cells receiving the TGF-β signals patterning this territory along the dorsal-ventral axis. In addition, my data indicate that Fzl1/2/7 signaling represses its own expression in a negative feedback mechanism. Finally, we discovered that a balance between the activities of posterior Wnt8 and anterior Dkk1 is necessary to establish the correct spatial expression of zygotic fzl12/7 expression in the equatorial ectodermal domain during blastula and gastrula stages. Together, these studies lead to a better understanding of the complex interactions among the three Wnt signaling pathway governing AP axis specification and patterning in sea urchin embryos.

Nucleosome eviction along with H3K9ac deposition enhances Sox2 binding during human neuroectodermal commitment

Neuroectoderm is an important neural precursor. However, chromatin remodeling and its epigenetic regulatory roles during the differentiation of human neuroectodermal cells (hNECs) from human embryonic stem cells (hESCs) remain largely unexplored. Here, we obtained hNECs through directed differentiation from hESCs, and determined chromatin states in the two cell types. Upon differentiation, H2A.Z-mediated nucleosome depletion leads to an open chromatin structure in promoters and upregulates expression of neuroectodermal genes. Increase in H3K9ac signals and decrease in H3K27me3 signals in promoters result in an active chromatin state and activate neuroectodermal genes. Conversely, decrease in H3K9ac signals and increase in H3K27me3 signals in promoters repress pluripotency genes. Moreover, H3K9ac signals facilitate the pluripotency factor Sox2 binding to target sites unique to hNECs. Knockdown of the acetyltransferase Kat2b erases H3K9ac signals, disrupts Sox2 binding, and fails the differentiation. Our results demonstrate a hierarchy of epigenetic regulation of gene expression during the differentiation of hNECs from hESCs through chromatin remodeling.

Abstract 2528: Mechanism and role of SOX2 repression in human germline specification

Abstract The core pluripotency regulatory master transcription factor SOX2 is repressed in human primordial germ cells (PGCs) and in the seminoma (SEM) subset of human germ cell tumors (GCTs), which arises by transformation of PGCs. Neither the mechanism of this repression not its significance to GC and GCT development have so far been clarified. Here we show that SOX2 repression in TCam-2 SEM cells is mediated by the presence of the Polycomb repressive complex (PcG) and by the H3K27me3 chromatin mark at the SOX2 transcription start site. Moreover, repression can be abrogated by recruitment of the H3K27 demethylase UTX to the SOX2 promoter through retinoid signaling. SOX2-activated TCam-2 cells express neuronal and other lineage genes, consistent with the gene's function as a neurectodermal effector. Based on the recent discovery that SOX17 initiates human PGC specification, with its downstream target PRDM1 acting to suppress mesendodermal genes, we propose that SOX2 repression is required for PGC specification to suppress neuroectodermal genes. Overt pluripotency is initiated in latently pluripotent transformed PGCs presenting as the SOX2 upregulated embryonal carcinoma (EC) subset of GCTs; by gene expression profiling analysis we characterize the functional pathways that distinguish PGC-like unipotent SEM from blastocyst-like pluripotent EC. Citation Format: Kushwaha Ritu, Nirmala Jagadish, George Bosl, Raju Chaganti. Mechanism and role of SOX2 repression in human germline specification. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2528.

Mechanism and Role of SOX2 Repression in Seminoma: Relevance to Human Germline Specification

SummaryHuman male germ cell tumors (GCTs) are derived from primordial germ cells (PGCs). The master pluripotency regulator and neuroectodermal lineage effector transcription factor SOX2 is repressed in PGCs and the seminoma (SEM) subset of GCTs. The mechanism of SOX2 repression and its significance to GC and GCT development currently are not understood. Here, we show that SOX2 repression in SEM-derived TCam-2 cells is mediated by the Polycomb repressive complex (PcG) and the repressive H3K27me3 chromatin mark that are enriched at its promoter. Furthermore, SOX2 repression in TCam-2 cells can be abrogated by recruitment of the constitutively expressed H3K27 demethylase UTX to the SOX2 promoter through retinoid signaling, leading to expression of neuronal and other lineage genes. SOX17 has been shown to initiate human PGC specification, with its target PRDM1 suppressing mesendodermal genes. Our results are consistent with a role for SOX2 repression in normal germline development by suppressing neuroectodermal genes.

Zinc finger protein 521 overexpression increased transcript levels of Fndc5 in mouse embryonic stem cells.

Zinc finger protein 521 is highly expressed in brain, neural stem cells and early progenitors of the human hematopoietic cells. Zfp521 triggers the cascade of neurogenesis in mouse embryonic stem cells through inducing expression of the early neuroectodermal genes Sox1, Sox3 and Pax6. Fndc5, a precursor of Irisin has inducing effects on the expression level of brain derived neurotrophic factor in hippocampus. Therefore, it is most likely that Fndc5 may play an important role in neural differentiation. To exhibit whether the expression of this protein is under regulation with Zfp521, we overexpressed Zfp521 in a stable transformants of mESCs expressing EGFP under control of Fndc5 promoter. Increased expression of Zfp521 enhanced transcription levels of both EGFP and endogenous Fndc5. This result was confirmed by overexpression the aforementioned vectors in HEK cells and indicated that Zfp521 functions upstream of Fndc5 expression. It is most likely that Zfp521 may act through the binding to its response element on Fndc5 core promoter. Therefore it is concluding that an enhanced expression of Fndc5 in neural progenitor cells is stimulated by Zfp521 overexpression in these cells.

Negative neuronal differentiation of human adipose-derived stem cell clones.

Adipose mesenchymal stem cells are a heterogeneous population. Therefore, the question posed in this study is whether the heterogenic differentiation potential exhibited by the different clones toward mesodermic lineages can be extended to nonmesodermic lineages, such as the neuroectoderm. Different single cell clones of human adipose mesenchymal stem cells from the same donor were isolated. Neuronal plasticity of the clones was assessed according to the pattern DNA methylation, gene expression and intracellular calcium responses. Under neurogenic culture conditions, clones presented variable expression of neuronal-specific genes, but still expressed osteogenic markers. No calcium response was exhibited in response to KCl incubation. The DNA methylation profile presented a very similar pattern in neuroectoderm gene promoters. Data indicate that there are no significant differences between the undifferentiated and supposedly neuronal-differentiated mesenchymal cells.

G1 Compartmentalization and Cell Fate Coordination

Pauklin and Vallier show that, whereas early G1 is permissive for TGF-β-dependent endoderm differentiation, cyclin D restricts the activity of Smad2/3 in late G1, resulting in a switch from endoderm to neuroectoderm potential in pluripotent stem cells. These findings provide insight into how signaling, the cell cycle, and lineage specification are coordinated.

Pax6 Is a Human Neuroectoderm Cell Fate Determinant

The transcriptional regulation of neuroectoderm (NE) specification is unknown. Here we show that Pax6 is uniformly expressed in early NE cells of human fetuses and those differentiated from human embryonic stem cells (hESCs). This is in contrast to the later expression of Pax6 in restricted mouse brain regions. Knockdown of Pax6 blocks NE specification from hESCs. Overexpression of either Pax6a or Pax6b, but not Pax6triangle upPD, triggers hESC differentiation. However, only Pax6a converts hESCs to NE. In contrast, neither loss nor gain of function of Pax6 affects mouse NE specification. Both Pax6a and Pax6b bind to pluripotent gene promoters but only Pax6a binds to NE genes during human NE specification. These findings indicate that Pax6 is a transcriptional determinant of the human NE and suggest that Pax6a and Pax6b coordinate with each other in determining the transition from pluripotency to the NE fate in human by differentially targeting pluripotent and NE genes.