Sebox regulates mesoderm formation in early amphibian embryos

Background: Mix/Bix genes are important regulators of mesendoderm formation during vertebrate embryogenesis. Sebox, an additional member of this gene family, has been implicated in endoderm formation during early embryogenesis in zebrafish. However, it remains unclear whether Sebox plays a unique role in early Xenopus embryos. Results: In this study, we provide evidence that Sebox is uniquely required for the formation of mesoderm during early Xenopus embryogenesis. Sebox is dynamically expressed in the involuted mesoderm during gastrulation. It is activated by Nodal/Activin signaling and modulated by zygotic Wnt/β‐catenin signaling. Overexpression of Sebox perturbs movements during convergent extension and inhibits the expression of mesodermal, but not endodermal, genes induced by Nodal/Activin signaling. Depletion of Sebox using a specific morpholino increases the expression of noncanonical wnt5a, wnt5b, and wnt11b. Depletion of Sebox also up‐regulates the expression of pcdh8.2, a paraxial mesoderm‐specific protocadherin, in a Wnt11B‐dependent manner. Sebox morphants display reduced development of the head and notochord. Conclusions: Our findings illustrate that Sebox, a unique member of the Mix/Bix gene family, functions downstream of Nodal/Activin signaling and is required for the proper expression of noncanonical Wnt ligands and the normal development of mesoderm in Xenopus. Developmental Dynamics 244:1415–1426, 2015. © 2015 Wiley Periodicals, Inc.     

Genetic ablation of FLRT3 reveals a novel morphogenetic function for the anterior visceral endoderm in suppressing mesoderm differentiation

During early mouse development, the anterior visceral endoderm (AVE) secretes inhibitor and activator signals that are essential for establishing the anterior–posterior (AP) axis of the embryo and for restricting mesoderm formation to the posterior epiblast in the primitive streak (PS) region. Here we show that AVE cells have an additional morphogenetic function. These cells express the transmembrane protein FLRT3. Genetic ablation of FLRT3 did not affect the signaling functions of the AVE according to the normal expression pattern of Nodal and Wnt and the establishment of a proper AP patterning in the epiblast. However, FLRT3^−/− embryos showed a highly disorganized basement membrane (BM) in the AVE region. Subsequently, adjacent anterior epiblast cells displayed an epithelial-to-mesenchymal transition (EMT)-like process characterized by the loss of cell polarity, cell ingression, and the up-regulation of the EMT and the mesodermal marker genes Eomes, Brachyury/T, and FGF8. These results suggest that the AVE acts as a morphogenetic boundary to prevent EMT and mesoderm induction in the anterior epiblast by maintaining the integrity of the BM. We propose that this novel function cooperates with the signaling activities of the AVE to restrict EMT and mesoderm induction to the posterior epiblast.     

Induction of superficial cortical layer neurons from mouse embryonic stem cells by valproic acid

Within the developing mammalian cortex, neural progenitors first generate deep-layer neurons and subsequently more superficial-layer neurons, in an inside-out manner. It has been reported recently that mouse embryonic stem cells (mESCs) can, to some extent, recapitulate cortical development in vitro, with the sequential appearance of neurogenesis markers resembling that in the developing cortex. However, mESCs can only recapitulate early corticogenesis; superficial-layer neurons, which are normally produced in later developmental periods in vivo, are under-represented. This failure of mESCs to reproduce later corticogenesis in vitro implies the existence of crucial factor(s) that are absent or uninduced in existing culture systems. Here we show that mESCs can give rise to superficial-layer neurons efficiently when treated with valproic acid (VPA), a histone deacetylase inhibitor. VPA treatment increased the production of Cux1-positive superficial-layer neurons, and decreased that of Ctip2-positive deep-layer neurons. These results shed new light on the mechanisms of later corticogenesis.     

Differentiation of embryonic stem cells conditionally expressing neurogenin 3

Expression of the proendocrine gene neurogenin 3 (Ngn3) is required for the development of pancreatic islets. To better characterize the molecular events regulated by Ngn3 during development, we have determined the expression profiles of murine embryonic stem cells (mESCs) uniformly induced to overexpress Ngn3. An mESC line was created in order to induce Ngn3 by adding doxycycline to the culture medium. Genome-wide microarray analysis was performed to identify genes regulated by Ngn3 in a variety of contexts, including undifferentiated ESCs and differentiating embryoid bodies (EBs). Genes regulated by Ngn3 in a context-independent manner were identified and analyzed using systematic gene ontology tools. This analysis revealed Notch signaling as the most significantly regulated signaling pathway (p = .009). This result is consistent with the hypothesis that Ngn3 expression makes the cell competent for Notch signaling to be activated and, conversely, more sensitive to Notch signaling inhibition. Indeed, EBs induced to express Ngn3 were significantly more sensitive to gamma-secretase inhibitor-mediated Notch signaling inhibition (p < .0001) when compared with uninduced EBs. Moreover, we find that Ngn3 induction in differentiating ESCs results in significant increases in insulin, glucagon, and somatostatin expression.     

A transgenic wnt8a:PAC reporter reveals biphasic regulation of vertebrate mesoderm development

Vertebrate wnt8a links anteroposterior and dorsoventral axis patterning, but the regulation of wnt8a expression and its relationship to mesoderm induction and maintenance pathways is unclear. To address this, we have generated zebrafish transgenic for a modified genomic PAC clone that expresses EGFP from the wnt8a locus. The EGFP reporter transgene is expressed in a pattern nearly identical to wnt8a, including maternal deposition, expression in the ventrolateral mesoderm and in the yolk syncytial layer. Loss of function studies show that wnt8a expression is under biphasic control by Nodal and No Tail/Brachyury, whereby early phase expression is Nodal‐dependent but late phase expression is Ntl/Bra dependent. EGFP fluorescence persists in cells that transcribe the reporter, thus comprising a tracer for ventrolaterally derived mesodermal lineages. We use this property to show that wnt8a expression marks Nodal‐independent tail mesoderm formation and that Ntl/Bra predominantly regulates wnt8a in paraxial mesoderm progenitors. Developmental Dynamics 240:898–907, 2011. © 2011 Wiley‐Liss, Inc.     

Whole-genome microRNA screening identifies let-7 and mir-18 as regulators of germ layer formation during early embryogenesis

Tight control over the segregation of endoderm, mesoderm, and ectoderm is essential for normal embryonic development of all species, yet how neighboring embryonic blastomeres can contribute to different germ layers has never been fully explained. We postulated that microRNAs, which fine-tune many biological processes, might modulate the response of embryonic blastomeres to growth factors and other signals that govern germ layer fate. A systematic screen of a whole-genome microRNA library revealed that the let-7 and miR-18 families increase mesoderm at the expense of endoderm in mouse embryonic stem cells. Both families are expressed in ectoderm and mesoderm, but not endoderm, as these tissues become distinct during mouse and frog embryogenesis. Blocking let-7 function in vivo dramatically affected cell fate, diverting presumptive mesoderm and ectoderm into endoderm. siRNA knockdown of computationally predicted targets followed by mutational analyses revealed that let-7 and miR-18 down-regulate Acvr1b and Smad2, respectively, to attenuate Nodal responsiveness and bias blastomeres to ectoderm and mesoderm fates. These findings suggest a crucial role for the let-7 and miR-18 families in germ layer specification and reveal a remarkable conservation of function from amphibians to mammals.     

The involvement of TGFβ1 in early avian development: gastrulation and chondrogenesis

We examined the effects of transforming growth factor-β1 (TGFβ1) and a neutralizing monoclonal antibody on two phases of early chick embryo development: gastrulation and chondrogenesis. We carried out experiments in vivo and in vitro on mesoderm cells from the gastrulating embryo at day 1, and on sclerotome cells from day 3 embryos, having previously shown that this factor is present among these cells at these stages of development. Addition of the antibody to cultures of these cells produced a dose-dependent decrease in cell outgrowth and spreading and concomitantly reduced fibronectin deposition. In vivo studies of the effects of TGFβ1 on mesoderm during gastrulation were carried out by grafting beads carrying this agent into gastrulating embryos. We used beads of ion-exchange resin as well as hydrolysed polyacrylamide, and found that the grafts produced an accumulation of mesoderm cells around the implant and, at later stages, the formation of enlarged somites. There was no effect on embryonic axis formation. Studies of bromodeoxyuridine (BrdU) incorporation indicated that the mesoderm accumulation was due, at least in part, to an increase in cell proliferation. However, examination of the effect of TGFβ1 on BrdU incorporation by mesoderm during gastrulation and sclerotome cells in vitro indicated in inhibition of cell proliferation, an inconsistency explained in terms of the variation between the in vivo and in vitro conditions. We conclude that TGFβ1 is both appropriately located, and is able, to influence cell proliferation among the mesodermal cell populations during early development, and that this effect contributes to the overall control of mesodermal morphogenesis. Chondrogenesis was studied in vitro using micromass cocultures of sclerotome cells with notochordon a permeable substratum. Under these conditions, the addition of TGFβ1 caused an increase in the deposition of Alcian blue-stainable material, indicating a stimulation of chondrogenesis. We suggest that this result, coupled with the previous demonstration that TGFβ1 is present among the sclerotome cells in the embryo at this time, supports the contention that this factor exerts a regulatory effect on sclerotome cell differentiation.     

Heart development before beating

During heart development at the pregastrula stage, prospective heart cells reside in the posterior lateral region of the epiblast layer. Interaction of tissues between the posterior epiblast and hypoblast is necessary to generate the future heart mesoderm. Signaling regulating the interaction involves fibroblast growth factor (FGF)-8, Nodal, bone morphogenetic protein (BMP)-antagonist, and canonical Wnt and acts on the posterior epiblast to induce the expression of genes specific for the anterior lateral mesoderm. At the early gastrula stage, prospective heart cells accumulate at the posterior midline and migrate to the anterior region of the primitive streak. During gastrulation, future heart cells leave the primitive streak and migrate anterolaterally to form the left and right anterior lateral plate mesoderm including the precardiac mesoderm. At this stage, prospective heart cells receive endoderm-derived signals, including BMP, FGF, and Wnt-antagonist, and thereby become committed to the heart lineage. At the neurula stage, the left and right precardiac mesoderm move to the ventral midline and fuse, resulting in the formation of a single primitive heart tube. Therefore, a two-step signaling cascade, which includes tissue interaction between epiblast and hypoblast at the blastula stage and endoderm-derived signals during gastrulation, is required to generate a beating heart.

HOXB4 can enhance the differentiation of embryonic stem cells by modulating the hematopoietic niche

Hematopoietic differentiation of embryonic stem cells (ESCs) in vitro has been used as a model to study early hematopoietic development, and it is well documented that hematopoietic differentiation can be enhanced by overexpression of HOXB4. HOXB4 is expressed in hematopoietic progenitor cells (HPCs) where it promotes self-renewal, but it is also expressed in the primitive streak of the gastrulating embryo. This led us to hypothesize that HOXB4 might modulate gene expression in prehematopoietic mesoderm and that this property might contribute to its prohematopoietic effect in differentiating ESCs. To test our hypothesis, we developed a conditionally activated HOXB4 expression system using the mutant estrogen receptor (ER(T2)) and showed that a pulse of HOXB4 prior to HPC emergence in differentiating ESCs led to an increase in hematopoietic differentiation. Expression profiling revealed an increase in the expression of genes associated with paraxial mesoderm that gives rise to the hematopoietic niche. Therefore, we considered that HOXB4 might modulate the formation of the hematopoietic niche as well as the production of hematopoietic cells per se. Cell mixing experiments supported this hypothesis demonstrating that HOXB4 activation can generate a paracrine as well as a cell autonomous effect on hematopoietic differentiation. We provide evidence to demonstrate that this activity is partly mediated by the secreted protein FRZB.     

Broad mesodermal and endodermal deletion of Nodal at postgastrulation stages results solely in left/right axial defects

Nodal signaling is a critical regulator of multiple aspects of early vertebrate development including asymmetry along the left/right (LR) axis. To study Nodal function occurring specifically in the postgastrulation embryo, we have used Cre/loxP based conditional mutagenesis. A floxed allele of Nodal was generated and shown to have wild‐type function. This allele was then used in conjunction with the T‐Cre line, which expresses Cre recombinase broadly in the mesodermal and definitive endodermal lineages posterior to the cranial region. T‐Cre activity leads to complete deletion of Nodal before its normal transient expression in the early somite stage lateral plate mesoderm, thereby causing severe LR developmental defects. No other abnormalities were found, suggesting that Nodal signaling has no additional essential functions in developmental patterning within the extensive mesodermal and endodermal domains marked by T‐Cre activity. Developmental Dynamics 237:3591–3601, 2008. Published 2008 Wiley‐Liss, Inc.     

Isolation and Differentiation of Chondrocytic Cells Derived from Human Embryonic Stem Cells Using dlk1/FA1 as a Novel Surface Marker

Few surface markers are available to monitor lineage differentiation during chondrogenesis. Recently, delta-like1/fetal antigen1 (dlk1/FA1), a transmembrane protein of the Notch/Delta/Serrata family, was shown to be essential for inducing early chondrogenesis. Thus, we investigated the possible use of dlk1/FA1 as a novel surface marker for chondroprogenitor cells during hESC differentiation. We found that, Dlk1/FA1 is expressed specifically in cells undergoing transition from proliferating to prehypertrophic chondrocytes during endochondral ossification of the mouse limb. In hESC cells, dlk1/FA1 was not expressed by undifferentiated hESC, but expressed during in vitro embryoid bodies (hEBs) formation upon down-regulation of undifferentiated markers e.g. Oct 3/4. Similarly, dlk1/FA1 was expressed in chondrocytic cells during in vivo teratoma formation. Interestingly, treatment of hEBs with Activin B, a member of TGF-ß family, markedly increased Dlk1 expression in association with up-regulation of the mesoderm-specific markers (e.g. FOXF1, KDR and VE-cadherin) and SOX9. dlk1/FA1⁺ cells isolated by fluorescence activated cell sorting (FACS) were capable of differentiating into chondrocytic cells when cultured as micromass pellets in a xeno-free system containing TGFβ1. In conclusion, we identified dlk1/FA1 as a novel marker of chondroprogenitor cells that undergo embryonic lineage progression from proliferation to the prehypertrophic stage. Tracking dlk1/FA1 expression as a mesoderm/chondroprogenitor surface marker provides a novel strategy for designing clinically relevant protocols to direct the differentiation of hESC into chondrocytes.     

PAX5 promotes pre-B cell proliferation by regulating the expression of pre-B cell receptor and its downstream signaling

PAX5 is indispensable for the commitment of early lymphoid progenitors to the B cell lineage as well as for the development of B cells. Although previous studies have indicated that the Pax5-conditional-knockout mouse exhibited dedifferentiation of mature B cell and the development of aggressive lymphomas, the changes of Pax5 gene expressions in pre-B cells have not been analyzed. To understand the functional importance of Pax5 gene in the proliferation and survival of pre-B cells, we established a Pax5-knockdown model using 70Z/3 pre-B cell line. Pax5 knockdown 70Z/3 cells (70Z/3-KD cells) showed down-regulations of pre-BCR compounds such as CD19, BLNK, Id2 and λ5. The signaling via pre-BCRs was significantly diminished in the 70Z/3-KD cells, and this alteration was normalized by restored Pax5 gene expression. Loss of PAX5 reduced the growth rates in the 70Z/3-KD cells, compared to the mock cells. Meanwhile, the proliferation of pre-B cells was reduced by the knockdown of Pax5 gene. Moreover, further examinations showed that PAX5 was also activated in B cell acute lymphoblastic leukemia (B-ALL) as a cell proliferation enhancer. These findings suggested that pax5 is critically important for the proliferation and survival of pre-B cells.     

Two nodal-responsive enhancers control left-right asymmetric expression of Nodal.

Asymmetric expression of Nodal in the lateral plate mesoderm (LPM) plays the major role in left-right (L-R) patterning. A Nodal-responsive enhancer located in the intron 1 (ASE) regulates asymmetric Nodal expression, but it is unknown how Nodal expression is initiated in the left LPM. Here, we have identified a second asymmetric enhancer (left side-specific enhancer, LSE) in the upstream region of mouse Nodal gene. LSE is also located in the corresponding region of human NODAL. L-R specificity of LSE is affected by iv and inv mutations. The requirement of a conserved FoxH1-binding sequence for LSE activity and the dependence of LSE activity on Nodal coreceptor Cryptic indicate that LSE is activated by Nodal signal. However, the mutant mouse lacking LSE does not show obvious L-R patterning defects. These results suggest that Nodal expression in the left LPM is induced by a combination of two Nodal-responsive autoregulatory enhancers, ASE and LSE.     

An anteroposterior Dorsal gradient in the Drosophila embryo

Dorsoventral (DV) patterning of the Drosophila embryo is initiated by a broad Dorsal (Dl) nuclear gradient, which is regulated by a conserved signaling pathway that includes the Toll receptor and Pelle kinase. We investigate the consequences of expressing a constitutively activated form of the Toll receptor, Toll^10b, in anterior regions of the early embryo using the bicoid 3′ UTR. Localized Toll^10b products result in the formation of an ectopic, anteroposterior (AP) Dl nuclear gradient along the length of the embryo. The analysis of both authentic dorsal target genes and defined synthetic promoters suggests that the ectopic gradient is sufficient to generate the full repertory of DV patterning responses along the AP axis of the embryo. For example, mesoderm determinants are activated in the anterior third of the embryo, whereas neurogenic genes are expressed in central regions. These results raise the possibility that Toll signaling components diffuse in the plasma membrane or syncytial cytoplasm of the early embryo. This study also provides evidence that neurogenic repressors may be important for the establishment of the sharp mesoderm/neuroectoderm boundary in the early embryo.     

Lack of Association between ORAI1/CRACM1 Gene Polymorphisms and Kawasaki Disease in the Taiwanese Children

Objective

Kawasaki disease (KD) is characterized by systemic vasculitis of an unknown cause. A previous study has indicated that a polymorphism of the inositol 1,4,5-trisphosphate 3-kinase C (ITPKC) gene is involved in the susceptibility to KD. ORAI (also known as CRACM1) is one of the components of store-operated calcium channels involved in regulating immune and inflammatory reactions. This study was conducted to investigate if polymorphisms in ORAI1/CRACM1, a gene downstream from ITPKC, are associated with KD susceptibility and clinical outcomes.

Materials and Methods

A total of 1,056 subjects (341 KD patients and 715 controls) were investigated to identify five tagging single nucleotide polymorphisms (tSNPs) in ORAI1/CRACM1 (rs12313273, rs6486795, rs7135617, rs12320939, and rs712853) by using the TaqMan Allelic Discrimination assay.

Results

No significant associations between genotype and allele frequency of the five ORAI1/CRACM1 tSNPs were observed in the KD patients and controls. In KD patients, no significant associations between ORAI1/CRACM1 polymorphisms and coronary artery lesion (CAL) formation or intravenous immunoglobulin (IVIG) treatment response were observed. The results from haplotype analysis were insignificant.

Conclusions

This study showed for the first time that ORAI1/CRACM1 polymorphisms are not associated with KD susceptibility, CAL formation, or IVIG treatment response in the Taiwanese population.     

Association of Interleukin-10 A-592C Polymorphism in Taiwanese Children with Kawasaki Disease

Elevated serum levels of interleukin-10 (IL-10) have been reported in patients with Kawasaki disease (KD). IL-10 reduces the inflammatory actions of macrophages and T cells and it may play a significant role in the regulation of inflammatory vascular damage associated with systemic vasculitis. The aim of this study was to examine whether -592 IL-10 promoter polymorphism is a susceptibility or severity marker of KD in Chinese patients in Taiwan. The study included 105 KD patients and 100 normal controls. Genotype and allelic frequencies for the IL-10 gene polymorphism in both groups were compared. There were no significant between-group differences in the genotype distribution of IL-10 A-592C gene polymorphism (P=0.08). However, the frequency of the -592*A allele was significantly increased in the patients with KD compared with controls (71.9% vs. 61.0%, P=0.019). The odds ratio for developing KD in individuals with IL-10-592*A allele was 1.64 (95% confidence interval, 1.06-2.52) compared to individuals with the IL-10-592*C allele. No significant difference was observed in the genotype and allelic frequencies for the IL-10 A-592C polymorphism between patients with and without coronary artery lesions. The IL-10-592*A allele may be involved in the development of KD in Taiwanese children.     

Phosphatase and tensin homolog regulates the pluripotent state and lineage fate choice in human embryonic stem cells

Understanding the intrinsic and extrinsic signals that regulate the molecular basis of the pluripotent state may improve our understanding of mammalian embryogenesis, different states of pluripotency, and our ability to tailor lineage differentiation. Although the role of the PI3K/Akt pathway in the self-renewal and maintenance of mESCs is well-established, the specific contribution of the pathway or of its negative regulator, PTEN, in the maintenance of the human pluripotent state is less understood. To explore the PI3K/AKT pathway in human embryonic stem cell (hESC) pluripotency and differentiation, we generated stable PTEN knockdown (KD) hESCs using short hairpin RNA. Similar to mESCs, we found that PTEN KD hESCs have increased self-renewal, cell survival, and proliferation over multiple passages compared to control cells. However, in contrast to mESCs, in vitro, PTEN KD hESCs differentiated inefficiently in directed differentiation assays, in part due to the continued maintenance of OCT4 and NANOG expression. In teratoma assays, PTEN KD hESCs generated tissues from the three germ layers, although with a bias toward neuroectoderm differentiation. These results demonstrate that PTEN is a key regulator of hESC growth and differentiation, and manipulation of this pathway may improve our ability to regulate and understand the pluripotent state.