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.

Paternally inherited gsα mutation impairs adipogenesis and potentiates a lean phenotype in vivo.

Paternally inherited inactivating mutations of the GNAS gene have been associated with a rare and disabling genetic disorder, progressive osseous heteroplasia, in which heterotopic ossification occurs within extraskeletal soft tissues, such as skin, subcutaneous fat, and skeletal muscle. This ectopic bone formation is hypothesized to be caused by dysregulated mesenchymal progenitor cell differentiation that affects a bipotential osteogenic-adipogenic lineage cell fate switch. Interestingly, patients with paternally inherited inactivating mutations of GNAS are uniformly lean. Using a mouse model of Gsα-specific exon 1 disruption, we examined whether heterozygous inactivation of Gnas affects adipogenic differentiation of mesenchymal precursor cells from subcutaneous adipose tissues (fat pad). We found that paternally inherited Gsα inactivation (Gsα(+/p-) ) impairs adipogenic differentiation of adipose-derived stromal cells (ASCs). The Gsα(+/p-) mutation in ASCs also decreased expression of the adipogenic factors CCAAT-enhancer-binding protein (C/EBP)ß, C/EBPα, peroxisome proliferator-activated receptor gamma, and adipocyte protein 2. Impaired adipocyte differentiation was rescued by an adenylyl cyclase activator, forskolin, and provided evidence that Gsα-cAMP signals are necessary in early stages of this process. Supporting a role for Gnas in adipogenesis in vivo, fat tissue weight and expression of adipogenic genes from multiple types of adipose tissues from Gsα(+/p-) mice were significantly decreased. Interestingly, the inhibition of adipogenesis by paternally inherited Gsα mutation also enhances expression of the osteogenic factors, msh homeobox 2, runt-related transcription factor 2, and osteocalcin. These data support the hypothesis that Gsα plays a critical role in regulating the balance between fat and bone determination in soft tissues, a finding that has important implications for a wide variety of disorders of osteogenesis and adipogenesis.

Shp2 acts downstream of SDF-1alpha/CXCR4 in guiding granule cell migration during cerebellar development.

Shp2 is a non-receptor protein tyrosine phosphatase containing two Src homology 2 (SH2) domains that is implicated in intracellular signaling events controlling cell proliferation, differentiation and migration. To examine the role of Shp2 in brain development, we created mice with Shp2 selectively deleted in neural stem/progenitor cells. Homozygous mutant mice exhibited early postnatal lethality with defects in neural stem cell self-renewal and neuronal/glial cell fate specification. Here we report a critical role of Shp2 in guiding neuronal cell migration in the cerebellum. In homozygous mutants, we observed reduced and less foliated cerebellum, ectopic presence of external granule cells and mispositioned Purkinje cells, a phenotype very similar to that of mutant mice lacking either SDF-1alpha or CXCR4. Consistently, Shp2-deficient granule cells failed to migrate toward SDF-1alpha in an in vitro cell migration assay, and SDF-1alpha treatment triggered a robust induction of tyrosyl phosphorylation on Shp2. Together, these results suggest that although Shp2 is involved in multiple signaling events during brain development, a prominent role of the phosphatase is to mediate SDF-1alpha/CXCR4 signal in guiding cerebellar granule cell migration.

Dual roles of Cripto as a ligand and coreceptor in the nodal signaling pathway.

The EGF-CFC gene Cripto encodes an extracellular protein that has been implicated in the signaling pathway for the transforming growth factor beta (TGF beta) ligand Nodal. Although recent findings in frog and fish embryos have suggested that EGF-CFC proteins function as coreceptors for Nodal, studies in cell culture have implicated Cripto as a growth factor-like signaling molecule. Here we reconcile these apparently disparate models of Cripto function by using a mammalian cell culture assay to investigate the signaling activities of Nodal and EGF-CFC proteins. Using a luciferase reporter assay, we found that Cripto has activities consistent with its being a coreceptor for Nodal. However, Cripto can also function as a secreted signaling factor in cell coculture assays, suggesting that it may also act as a coligand for Nodal. Furthermore, we found that the ability of Cripto to bind to Nodal and mediate Nodal signaling requires the addition of an O-linked fucose monosaccharide to a conserved site within EGF-CFC proteins. We propose a model in which Cripto has dual roles as a coreceptor as well as a coligand for Nodal and that this signaling interaction with Nodal is regulated by an unusual form of glycosylation. Our findings highlight the significance of extracellular modulation of ligand activity as an important means of regulating TGF beta signaling pathways during vertebrate development.

Stem cell therapy for hearing loss: Math1 overexpression in VOT-E36 cells.

HYPOTHESIS: VOT-E36 cells acquire mechanosensitivity after mammalian atonal homolog 1 (Math1) overexpression. BACKGROUND: VOT-E36 cells are derived from a population of epithelial cells in the ventral region of the otocyst at embryonic Day 10.5, before hair cell differentiation. These cells express a number of specific molecular markers for hair cells under both proliferation and differentiation states. Overexpression of Math1 can convert nonsensory epithelial cells into hair cells in the cochlea. Based on this information, we tested whether VOT-E36 cells can be converted into hair cells by Math1 overexpression. METHODS: Using reverse transcriptase-polymerase chain reaction-based analysis, we first compared the expression patterns of various molecular markers for hair cell development in VOT-E36 cells between proliferation and differentiation states, and also before and after overexpression of Math1. Subsequently, with a standard calcium imaging method, we examined whether VOT-E36 cells overexpressing Math1 could detect mechanical vibrations and activate spiral ganglion neurons in a coculture model. In addition, using confocal and scanning electron microscopes, we examined morphologic changes of VOT-E36 cells after Math1 overexpression. RESULTS: Consistent with previous reports, this study has shown that VOT-E36 cells express a number of specific molecular markers for hair cells in both proliferation and differentiation states. Under appropriate culture conditions, Math1 is transiently expressed in this cell line during conditional differentiation. In VOT-E36 cells overexpressing Math1, the normal expression pattern of certain molecular markers for mature hair cells is partially restored. Interestingly, after coculture with spiral ganglion neurons, VOT-E36 cells overexpressing Math1 are able to respond to mechanical vibrations and activate spiral ganglion neurons. Possible molecular mechanisms underlying this novel finding have been explored. CONCLUSION: Math1 overexpression can partially restore presumably downstream signaling cascades for normal hair cell differentiation in VOT-E36 cells, which are able to detect mechanical vibrations after being cocultured with spiral ganglion neurons.

Reprogramming of single-cell-derived mesenchymal stem cells into hair cell-like cells.

HYPOTHESIS: Adult mesenchymal stem cells (MSCs) can be converted into hair cell-like cells by transdetermination. BACKGROUND: Given the fundamental role sensory hair cells play in sound detection and the irreversibility of their loss in mammals, much research has focused on developing methods to generate new hair cells as a means of treating permanent hearing loss. Although MSCs can differentiate into multiple cell lineages, no efficient means of reprogramming them into sensory hair cells exists. Earlier work has shown that the transcription factor Atoh1 is necessary for early development of hair cells, but it is not clear whether Atoh1 can be used to convert MSCs into hair cells. METHODS: Clonal MSC cell lines were established and reprogrammed into hair cell-like cells by a combination of protein transfer, adenoviral based gene transfer, and co-culture with neurons. During transdetermination, inner ear molecular markers were analyzed using reverse transcriptase-polymerase chain reaction, and cell structures were examined using immunocytochemistry. RESULTS: Atoh1 overexpression in MSCs failed to convert MSCs into hair cell-like cells, suggesting that the ability of Atoh1 to induce hair cell differentiation is context dependent. Because Atoh1 overexpression successfully transforms VOT-E36 cells into hair cell-like cells, we modified the cell context of MSCs by performing a total protein transfer from VOT-E36 cells before overexpressing Atoh1. The modified MSCs were transformed into hair cell-like cells and attracted contacts from spiral ganglion neurons in a co-culture model. CONCLUSION: We established a new procedure, consisting of VOT-E36 protein transfer, Atoh1 overexpression, and co-culture with spiral ganglion neurons, which can transform MSCs into hair cell-like cells.

Erythroid gene suppression by NF-kappa B.

NF-kappa B/Rel transcription factors play essential roles to mediate the immune response and apoptosis, and they have also been implicated in cellular differentiation such as erythropoiesis. To elucidate the possible role(s) of NF-kappa B in erythroid gene regulation and erythropoiesis, we have carried out transient transfection studies of the human embryonic/fetal erythroid cell line K562 and mouse adult erythroid MEL cells. It is shown that tumor necrosis factor-alpha represses the transcription activity directed by either alpha or zeta globin promoter in a dose-dependent manner. Furthermore, different NF-kappa B family members could effectively repress the transfected alpha-like globin promoters in K562 as well as in MEL cells. The involvement of NF-kappa B pathway is supported by the ability of a NF-kappa B-specific, dominant negative mutant to block the tumor necrosis factor-alpha or p65-mediated suppression of the alpha-like globin promoter activities. The suppression appears to be mediated through cis-linked HS-40 enhancer. Finally, stably transfected K562 cells overexpressing p65 contain reduced amounts of the p45/NF-E2 RNA and functional NF-E2 proteins. Our studies have identified a new set of targets of NF-kappa B. We suggest that the relatively high activity of the NF-kappa B pathway in early erythroid progenitors is involved in the suppression of erythroid-specific genes. Later in differentiation, together with other changes, the decline of the amounts of the NF-kappa B family of factors leads to derepression and consequent increase of NF-E2, which in turn would activate a subset of erythroid-specific genes.

Coupling gene chip analyses and rat genetic variances in identifying potential target genes that may contribute to neuropathic allodynia development.

Genetic factors and nerve injury-induced changes of gene expression in sensory neurons are potential contributors to tactile allodynia, a neuropathic pain state manifested as hypersensitivity to innocuous mechanical stimulation. To uncover genes relevant to neuropathic allodynia, we analyzed gene expression profiles in dorsal root ganglia (DRG) of spinal nerve-ligated Harlan and Holtzman Sprague Dawley rats, strains with different susceptibilities to neuropathic allodynia. Using Affymetrix gene chips, we identified genes showing differential basal-level expression in these strains without injury-induced regulation. Of more than 8000 genes analyzed, less than 180 genes in each strain were regulated after injury, and 19-22% of that was regulated in a strain-specific manner. Importantly, we identified functionally related genes that were co-regulated post injury in one or both strains. In situ hybridization and real-time PCR analyses of a subset of identified genes confirmed the patterns of the microarray data, and the former also demonstrated that injury-induced changes occurred, not only in neurons, but also in non-neuronal cells. Together, our studies provide a global view of injury plasticity in DRG of these rat stains and support a plasticity-based mechanism mediating variations in allodynia susceptibility, thus providing a source for further characterization of neuropathic pain-relevant genes and potential pathways.