LncRNA-Smad7 mediates cross-talk between Nodal/TGF-β and BMP signaling to regulate cell fate determination of pluripotent and multipotent cells

Transforming growth factor β (TGF-β) superfamily proteins are potent regulators of cellular development and differentiation. Nodal/Activin/TGF-β and BMP ligands are both present in the intra- and extracellular milieu during early development, and cross-talk between these two branches of developmental signaling is currently the subject of intense research focus. Here, we show that the Nodal induced lncRNA-Smad7 regulates cell fate determination via repression of BMP signaling in mouse embryonic stem cells (mESCs). Depletion of lncRNA-Smad7 dramatically impairs cardiomyocyte differentiation in mESCs. Moreover, lncRNA-Smad7 represses Bmp2 expression through binding with the Bmp2 promoter region via (CA)₁₂-repeats that forms an R-loop. Importantly, Bmp2 knockdown rescues defects in cardiomyocyte differentiation induced by lncRNA-Smad7 knockdown. Hence, lncRNA-Smad7 antagonizes BMP signaling in mESCs, and similarly regulates cell fate determination between osteocyte and myocyte formation in C2C12 mouse myoblasts. Moreover, lncRNA-Smad7 associates with hnRNPK in mESCs and hnRNPK binds at the Bmp2 promoter, potentially contributing to Bmp2 expression repression. The antagonistic effects between Nodal/TGF-β and BMP signaling via lncRNA-Smad7 described in this work provides a framework for understanding cell fate determination in early development.     

A Combination of Culture Conditions and Gene Expression Analysis Can Be Used to Investigate and Predict hES Cell Differentiation Potential towards Male Gonadal Cells

Human embryonic stem cell differentiation towards various cell types belonging to ecto-, endo- and mesodermal cell lineages has been demonstrated, with high efficiency rates using standardized differentiation protocols. However, germ cell differentiation from human embryonic stem cells has been very inefficient so far. Even though the influence of various growth factors has been evaluated, the gene expression of different cell lines in relation to their differentiation potential has not yet been extensively examined. In this study, the potential of three male human embryonic stem cell lines to differentiate towards male gonadal cells was explored by analysing their gene expression profiles. The human embryonic stem cell lines were cultured for 14 days as monolayers on supporting human foreskin fibroblasts or as spheres in suspension, and were differentiated using BMP7, or spontaneous differentiation by omitting exogenous FGF2. TLDA analysis revealed that in the undifferentiated state, these cell lines have diverse mRNA profiles and exhibit significantly different potentials for differentiation towards the cell types present in the male gonads. This potential was associated with important factors directing the fate of the male primordial germ cells in vivo to form gonocytes, such as SOX17 or genes involved in the NODAL/ACTIVIN pathway, for example. Stimulation with BMP7 in suspension culture resulted in up-regulation of cytoplasmic SOX9 protein expression in all three lines. The observation that human embryonic stem cells differentiate towards germ and somatic cells after spontaneous and BMP7-induced stimulation in suspension emphasizes the important role of somatic cells in germ cell differentiation in vitro.     

Specific induction of cranial placode cells from Xenopus ectoderm by modulating the levels of BMP,Wnt, and FGF signaling

The neural–epidermal boundary tissues include the neural crest and preplacodal ectoderm (PPE) as primordial constituents. The PPE region is essential for the development of various sensory and endocrine organs, such as the anterior lobe of the pituitary, olfactory epithelium, lens, trigeminal ganglion, and otic vesicles. During gastrulation, a neural region is induced in ectodermal cells that interacts with mesendodermal tissue and responds to several secreted factors. Among them, inhibition of bone morphogenetic protein (BMP) in the presumptive neuroectoderm is essential for the induction of neural regions, and formation of a Wnt and fibroblast growth factor (FGF) signaling gradient along the midline determines anterior–posterior patterning. In this study, we attempted to specifically induce PPE cells from undifferentiated Xenopus cells by regulating BMP, Wnt, and FGF signaling. We showed that the proper level of BMP inhibition with an injection of truncated BMP receptor or treatment with a chemical antagonist triggered the expression of PPE genes. In addition, by varying the amount of injected chordin, we optimized specific expression of the PPE genes. PPE gene expression is increased by adding an appropriate dose of an FGF receptor antagonist. Furthermore, co‐injection with either wnt8 or the Wnt inhibitor dkk‐1 altered the expression levels of several region‐specific genes according to the injected dose. We specifically induced PPE cell differentiation in animal cap cells from early‐stage Xenopus embryos by modulating BMP, Wnt, and FGF signaling. This is not the first research on placode induction, but our simple method could potentially be applied to mammalian stem cell systems. genesis 53:652–659, 2015. © 2015 Wiley Periodicals, Inc.     

BMP Induces Cochlin Expression to Facilitate Self-renewal and Suppress Neural Differentiation of Mouse Embryonic Stem Cells

BMP4 maintains self-renewal of mouse embryonic stem cells (ESCs) in collaboration with LIF. Here, we report the identification of a novel key BMP target gene, cochlin (Coch) in mouse ESCs. Coch can be significantly up-regulated by BMP4 specifically in ESCs but not in somatic differentiated cells, and this up-regulation is dependent on the BMP signaling mediators Smad1/5 and Smad4. Overexpression of Coch can partially substitute BMP4 to promote self-renewal of mouse ESCs together with LIF, whereas knockdown of Coch impairs self-renewal marker gene expression even in the presence of both BMP4 and LIF. Further studies showed that COCH could mimic BMP4 in repressing neural differentiation of mouse ESCs upon LIF withdrawal and the inhibitory effect of BMP4 on neural differentiation is compromised by Coch knockdown. Taken together, our data suggest that COCH is a part of the downstream target network of BMP signaling and serves as another important effector to fine-tune mouse ESC fates.     

Protein kinase CK2 both promotes robust proliferation and inhibits the proliferative fate in the C. elegans germ line

Stem cells are capable of both self-renewal (proliferation) and differentiation. Determining the regulatory mechanisms controlling the balance between stem cell proliferation and differentiation is not only an important biological question, but also holds the key for using stem cells as therapeutic agents. The Caenorhabditis elegans germ line has emerged as a valuable model to study the molecular mechanisms controlling stem cell behavior. In this study, we describe a large-scale RNAi screen that identified kin-10, which encodes the β subunit of protein kinase CK2, as a novel factor regulating stem cell proliferation in the C. elegans germ line. While a loss of kin-10 in an otherwise wild-type background results in a decrease in the number of proliferative cells, loss of kin-10 in sensitized genetic backgrounds results in a germline tumor. Therefore, kin-10 is not only necessary for robust proliferation, it also inhibits the proliferative fate. We found that kin-10’s regulatory role in inhibiting the proliferative fate is carried out through the CK2 holoenzyme, rather than through a holoenzyme-independent function, and that it functions downstream of GLP-1/Notch signaling. We propose that a loss of kin-10 leads to a defect in CK2 phosphorylation of its downstream targets, resulting in abnormal activity of target protein(s) that are involved in the proliferative fate vs. differentiation decision. This eventually causes a shift towards the proliferative fate in the stem cell fate decision.     

Tsukushi is essential for proper maintenance and terminal differentiation of mouse hippocampal neural stem cells

Secreted proteoglycan molecule Tsukushi (TSK) regulates various developmental processes, such as early body patterning and neural plate formation by interacting with major signaling pathways like Wnt, BMP, Notch etc. In central nervous system, TSK inhibits Wnt signaling to control chick retinal development. It also plays important roles for axon guidance and anterior commissure formation in mouse brain. In the present study, we investigated the role of TSK for the development and proper functioning of mouse hippocampus. We found that TSK expression is prominent at hippocampal regions of early postnatal mouse until postnatal day 15 and gradually declines at later stages. Hippocampal dimensions are affected in TSK knockout mice (TSK-KO) as shown by reduced size of hippocampus and dentate gyrus (DG). Interestingly, neural stem cell (NSC) density at the neural niche of DG was higher in TSK-KO compared with wild-type. The ratio of proliferating NSCs as well as the rate of overall cell proliferation was also higher in TSK-KO hippocampus. Our in vitro study also suggests an increased number of neural stem/progenitor cells residing in TSK-KO hippocampus. Finally, we found that the terminal differentiation of NSCs in TSK-KO was disturbed as the differentiation to neuronal cell lineage was increased while the percentages of astrocytes and oligodendrocytes were decreased. Overall, our study establishes the involvement of TSK in hippocampal development, NSC maintenance and terminal differentiation at perinatal stages.     

Enhanced BMP signaling prevents degeneration and leads to endochondral ossification of Meckel′s cartilage in mice

Meckel′s cartilage is a transient supporting tissue of the embryonic mandible in mammals, and disappears by taking different ultimate cell fate along the distal–proximal axis, with the majority (middle portion) undergoing degeneration and chondroclastic resorption. While a number of factors have been implicated in the degeneration and resorption processes, signaling pathways that trigger this degradation are currently unknown. BMP signaling has been implicated in almost every step of chondrogenesis. In this study, we used Noggin mutant mice as a model for gain-of-BMP signaling function to investigate the function of BMP signaling in Meckel′s cartilage development, with a focus on the middle portion. We showed that Bmp2 and Bmp7 are expressed in early developing Meckels′ cartilage, but their expression disappears thereafter. In contrast, Noggin is expressed constantly in Meckel′s cartilage throughout the entire gestation period. In the absence of Noggin, Meckel′s cartilage is significantly thickened attributing to dramatically elevated cell proliferation rate associated with enhanced phosphorylated Smad1/5/8 expression. Interestingly, instead of taking a degeneration fate, the middle portion of Meckel′s cartilage in Noggin mutants undergoes chondrogenic differentiation and endochondral ossification contributing to the forming mandible. Chondrocyte-specific expression of a constitutively active form of BMPRIa but not BMPRIb leads to enlargement of Meckel′s cartilage, phenocopying the consequence of Noggin deficiency. Our results demonstrate that elevated BMP signaling prevents degeneration and leads to endochondral ossification of Meckel′s cartilage, and support the idea that withdrawal of BMP signaling is required for normal Meckel′s cartilage development and ultimate cell fate.     

Epigenetic Regulation of miR-302 by JMJD1C Inhibits Neural Differentiation of Human Embryonic Stem Cells

It has been recently reported that the regulatory circuitry formed by OCT4, miR-302, and NR2F2 controls both pluripotency and neural differentiation of human embryonic stem cells (hESCs). We show here that JMJD1C, a histone 3 lysine 9 (H3K9) demethylase expressed in hESCs, directly interacts with this circuitry. hESCs with stable knockdown of JMJD1C remain pluripotent while having reduced miR-302 expression, decreased BMP signaling, and enhanced TGFβ signaling. JMJD1C binds to the miR-302 promoter and reduces H3K9 methylation. Withdrawal of basic fibroblast growth factor (bFGF) from the culture induces neural differentiation of the knockdown, but not the control, cells within 3 days, accompanied by elevated NR2F2 expression. This can be attenuated with miR-302 mimics or an H3K9 methytransferase inhibitor. Together, our findings suggest that JMJD1C represses neural differentiation of hESCs at least partially by epigenetically sustaining miR-302 expression and that JMJD1C knockdown is sufficient to trigger neural differentiation upon withdrawal of exogenous bFGF.     

Masked mRNA is stored with aggregated nuclear speckles and its asymmetric redistribution requires a homolog of mago nashi

Background

The capacity of muscle to grow or to regenerate after damage is provided by adult stem cells, so called satellite cells, which are located under the basement lamina of each myofiber. Upon activation satellite cells enter the cell cycle, proliferate and differentiate into myoblasts, which fuse to injured myofibers or form new fibers. These processes are tightly controlled by many growth factors.

Results

Here we investigate the role of bone morphogenetic proteins (BMPs) during satellite cell differentiation. Unlike the myogenic C2C12 cell line, primary satellite cells do not differentiate into osteoblasts upon BMP signaling. Instead BMP signaling inhibits myogenic differentiation of primary satellite cells ex vivo. In contrast, inhibition of BMP signaling results in cell cycle exit, followed by enhanced myoblast differentiation and myotube formation. Using an in vivo trauma model we demonstrate that satellite cells respond to BMP signals during the regeneration process. Interestingly, we found the BMP inhibitor Chordin upregulated in primary satellite cell cultures and in regenerating muscles. In both systems Chordin expression follows that of Myogenin, a marker for cells committed to differentiation.

Conclusion

Our data indicate that BMP signaling plays a critical role in balancing proliferation and differentiation of activated satellite cells and their descendants. Initially, BMP signals maintain satellite cells descendants in a proliferating state thereby expanding cell numbers. After cells are committed to differentiate they upregulate the expression of the BMP inhibitor Chordin thereby supporting terminal differentiation and myotube formation in a negative feedback mechanism.     

Endogenous Wnt/β-Catenin Signaling Is Required for Cardiac Differentiation in Human Embryonic Stem Cells

Background

Wnt/β-catenin signaling is an important regulator of differentiation and morphogenesis that can also control stem cell fates. Our group has developed an efficient protocol to generate cardiomyocytes from human embryonic stem (ES) cells via induction with activin A and BMP4.

Methodology/Principal Findings

We tested the hypothesis that Wnt/β-catenin signals control both early mesoderm induction and later cardiac differentiation in this system. Addition of exogenous Wnt3a at the time of induction enhanced cardiac differentiation, while early inhibition of endogenous Wnt/β-catenin signaling with Dkk1 inhibited cardiac differentiation, as indicated by quantitative RT-PCR analysis for β-myosin heavy chain (β-MHC), cardiac troponin T (cTnT), Nkx2.5, and flow cytometry analysis for sarcomeric myosin heavy chain (sMHC). Conversely, late antagonism of endogenously produced Wnts enhanced cardiogenesis, indicating a biphasic role for the pathway in human cardiac differentiation. Using quantitative RT-PCR, we show that canonical Wnt ligand expression is induced by activin A/BMP4 treatment, and the extent of early Wnt ligand expression can predict the subsequent efficiency of cardiogenesis. Measurement of Brachyury expression showed that addition of Wnt3a enhances mesoderm induction, whereas blockade of endogenously produced Wnts markedly inhibits mesoderm formation. Finally, we show that Wnt/β-catenin signaling is required for Smad1 activation by BMP4.

Conclusions/Significance

Our data indicate that induction of mesoderm and subsequent cardiac differentiation from human ES cells requires fine-tuned cross talk between activin A/BMP4 and Wnt/β-catenin pathways. Controlling these pathways permits efficient generation of cardiomyocytes for basic studies or cardiac repair applications.     

BMP signaling in the control of skin development and hair follicle growth

Bone morphogenetic proteins (BMPs), their antagonists, and BMP receptors are involved in controlling a large number of biological functions including cell proliferation, differentiation, cell fate decision, and apoptosis in many different types of cells and tissues during embryonic development and postnatal life. BMPs exert their biological effects via using BMP-Smad and BMP-MAPK intracellular pathways. The magnitude and specificity of BMP signaling are regulated by a large number of modulators operating on several levels (extracellular, cytoplasmic, nuclear). In developing and postnatal skin, BMPs, their receptors, and BMP antagonists show stringent spatio-temporal expressions patterns to achieve proper regulation of cell proliferation and differentiation in the epidermis and in the hair follicle. Genetic studies assert an essential role for BMP signaling in the control of cell differentiation and apoptosis in developing epidermis, as well as in the regulation of key steps of hair follicle development (initiation, cell fate decision, cell lineage differentiation). In postnatal hair follicles, BMP signaling plays an important role in controlling the initiation of the growth phase and is also involved in the regulation of apoptosis-driven hair follicle involution. However, additional efforts are required to fully understand the mechanisms and targets involved in the realization of BMP effects on distinct cell population in the skin and hair follicle. Progress in this area of research will hopefully lead to the development of new therapeutic approaches for using BMPs and BMP antagonists in the treatment of skin and hair growth disorders.     

The Integrator Complex Subunit 6 (Ints6) Confines the Dorsal Organizer in Vertebrate Embryogenesis

Dorsoventral patterning of the embryonic axis relies upon the mutual antagonism of competing signaling pathways to establish a balance between ventralizing BMP signaling and dorsal cell fate specification mediated by the organizer. In zebrafish, the initial embryo-wide domain of BMP signaling is refined into a morphogenetic gradient following activation dorsally of a maternal Wnt pathway. The accumulation of β-catenin in nuclei on the dorsal side of the embryo then leads to repression of BMP signaling dorsally and the induction of dorsal cell fates mediated by Nodal and FGF signaling. A separate Wnt pathway operates zygotically via Wnt8a to limit dorsal cell fate specification and maintain the expression of ventralizing genes in ventrolateral domains. We have isolated a recessive dorsalizing maternal-effect mutation disrupting the gene encoding Integrator Complex Subunit 6 (Ints6). Due to widespread de-repression of dorsal organizer genes, embryos from mutant mothers fail to maintain expression of BMP ligands, fail to fully express vox and ved, two mediators of Wnt8a, display delayed cell movements during gastrulation, and severe dorsalization. Consistent with radial dorsalization, affected embryos display multiple independent axial domains along with ectopic dorsal forerunner cells. Limiting Nodal signaling or restoring BMP signaling restores wild-type patterning to affected embryos. Our results are consistent with a novel role for Ints6 in restricting the vertebrate organizer to a dorsal domain in embryonic patterning.     

Tumor Necrosis Factor-Receptor–associated Factor-4 Is a Positive Regulator of Transforming Growth Factor-β Signaling That Affects Neural Crest Formation

The transforming growth factor (TGF)-β superfamily regulates cell proliferation, apoptosis, differentiation, migration, and development. Canonical TGFβ signals are transduced to the nucleus via Smads in both major signaling branches, bone morphogenetic protein (BMP) or Activin/Nodal/TGFβ. Smurf ubiquitin (Ub) ligases attenuate these pathways by targeting Smads and other signaling components for degradation by the 26S proteasome. Here, we identify tumor necrosis factor (TNF)-receptor–associated factor-4 (TRAF4) as a new target of Smurf1, which polyubiquitylates TRAF4 to trigger its proteasomal destruction. Unlike other TRAF family members, which mediate signal transduction by TNF, interleukin, or Toll-like receptors, we find that TRAF4 potentiates BMP and Nodal signaling. In the frog Xenopus laevis, TRAF4 mRNA is stored maternally in the egg animal pole, and in the embryo it is expressed in the gastrula marginal zone, neural plate, and cranial and trunk neural crest. Knockdown of embryonic TRAF4 impairs signaling, neural crest development and neural folding, whereas TRAF4 overexpression boosts signaling and expands the neural crest. In human embryonic kidney 293 cells, small interfering RNA knockdown of Smurf1 elevates TRAF4 levels, indicating endogenous regulation of TRAF4 by Smurf1. Our results uncover new functions for TRAF4 as a Smurf1-regulated mediator of BMP and Nodal signaling that are essential for neural crest development and neural plate morphogenesis.     

Regulation of the neural patterning activity of sonic hedgehog by secreted BMP inhibitors expressed by notochord and somites

The secretion of Sonic hedgehog (Shh) from the notochord and floor plate appears to generate a ventral-to-dorsal gradient of Shh activity that directs progenitor cell identity and neuronal fate in the ventral neural tube. In principle, the establishment of this Shh activity gradient could be achieved through the graded distribution of the Shh protein itself, or could depend on additional cell surface or secreted proteins that modify the response of neural cells to Shh. Cells of the neural plate differentiate from a region of the ectoderm that has recently expressed high levels of BMPs, raising the possibility that prospective ventral neural cells are exposed to residual levels of BMP activity. We have examined whether modulation of the level of BMP signaling regulates neural cell responses to Shh, and thus might contribute to the patterning of cell types in the ventral neural tube. Using an in vitro assay of neural cell differentiation we show that BMP signaling markedly alters neural cell responses to Shh signals, eliciting a ventral-to-dorsal switch in progenitor cell identity and neuronal fate. BMP signaling is regulated by secreted inhibitory factors, including noggin and follistatin, both of which are expressed in or adjacent to the neural plate. Conversely, follistatin but not noggin produces a dorsal-to-ventral switch in progenitor cell identity and neuronal fate in response to Shh both in vitro and in vivo. These results suggest that the specification of ventral neural cell types depends on the integration of Shh and BMP signaling activities. The net level of BMP signaling within neural tissue may be regulated by follistatin and perhaps other BMP inhibitors secreted by mesodermal cell types that flank the ventral neural tube.