Project description:In order to isolate novel genes regulating neural induction, we utilized a DNA microarray approach. As neural induction is thought to occur via the inhibition of BMP signaling, BMP signaling was inhibited in ectodermal cells by overexpression of a dominant-negative receptor. RNAs were isolated from control animal cap explants and from dominant-negative BMP receptor expressing animal caps and subjected to a microarray experiment using newly generated high-density Xenopus DNA microarray chips. Keywords = neural induction Keywords = BMP Keywords = nervous system Keywords = Xenopus Keywords = microarray
Project description:The neural fate commitment of pluripotent stem cells requires repression of extrinsic inhibitory signals and activation of intrinsic positive transcription factors. However, it remains elusive how these two events are integrated to ensure appropriate neural conversion. Here, we show that Oct6 functions as an essential positive factor for neural differentiation of mouse embryonic stem cells (ESCs), specifically during the transition from epiblast stem cells (EpiSCs) to neural progenitor cells (NPCs). Chimera analysis showed that Oct6 knockdown leads to markedly decreased incorporation of ESC in neuroectoderm. By contrast, Oct6-overexpressing ESC derivatives preferentially contribute to neuroectoderm. Genome-wide ChIP-seq and RNA-seq analyses indicate that Oct6 is an upstream activator of neural lineage genes, and also a repressor of BMP and Wnt signalings. Our results establish Oct6 as a critical regulator that promotes neural commitment of pluripotent stem cells through a dual role: activating internal neural induction programs and antagonizing extrinsic neural inhibitory signals. RNA-seq was performed to examine Oct6 function in ESC neural differentiation at Day2, Day4 and Day6 after dox induction. On Day4 EB, ChIP-seq assay was ultilized to characterize the targets of Oct6.
Project description:In order to isolate novel genes regulating neural induction, we utilized a DNA microarray approach. As neural induction is thought to occur via the inhibition of BMP signaling, BMP signaling was inhibited in ectodermal cells by overexpression of a dominant-negative receptor. RNAs were isolated from control animal cap explants and from dominant-negative BMP receptor expressing animal caps and subjected to a microarray experiment using newly generated high-density Xenopus DNA microarray chips. Keywords = neural induction Keywords = BMP Keywords = nervous system Keywords = Xenopus Keywords = microarray Keywords: parallel sample
Project description:The BMP signaling pathway regulates multiple steps of hematopoiesis, mediated through receptor-regulated Smads, including Smad1 and Smad5. Here we use loss-of-function approaches in zebrafish to compare the roles of Smad1 and Smad5 during embryonic hematopoiesis. Microarray experiments revealed that the two proteins regulate redundantly the key initiators of the hemato-vascular program, including scl, lmo2, and gfi1. However, each also regulates a remarkably distinct genetic program, with Smad5 uniquely regulating the BMP signaling pathway itself. Our results suggest that specificity of BMP signaling output, with respect to hematopoiesis, can be explained by differential functions of Smad1 and Smad5. Keywords: Gene expression transcript profiles
Project description:The neural fate commitment of pluripotent stem cells requires repression of extrinsic inhibitory signals and activation of intrinsic positive transcription factors. However, it remains elusive how these two events are integrated to ensure appropriate neural conversion. Here, we show that Oct6 functions as an essential positive factor for neural differentiation of mouse embryonic stem cells (ESCs), specifically during the transition from epiblast stem cells (EpiSCs) to neural progenitor cells (NPCs). Chimera analysis showed that Oct6 knockdown leads to markedly decreased incorporation of ESC in neuroectoderm. By contrast, Oct6-overexpressing ESC derivatives preferentially contribute to neuroectoderm. Genome-wide ChIP-seq and RNA-seq analyses indicate that Oct6 is an upstream activator of neural lineage genes, and also a repressor of BMP and Wnt signalings. Our results establish Oct6 as a critical regulator that promotes neural commitment of pluripotent stem cells through a dual role: activating internal neural induction programs and antagonizing extrinsic neural inhibitory signals.
Project description:Complex regulatory mechanisms control continuous maintenance of myeloid progenitors and renewal of differentiated cells. Transcription factors play a important role in these processes. Here we report that the activation the calcineurin-NFAT signaling pathway inhibit the proliferation of myeloid granulocyte-monocyte progenitor (GMP). Myeloid progenitor subtypes possessed different susceptibilities to Ca2+ flux induction and consequently differential engagement of the calcineurin-NFAT pathway. This study show that inhibition of the calcineurin-NFAT pathway enhanced proliferation of GMPs both in vivo and in vitro. The calcineurin-NFAT signaling in GMPs is initiated through Flt3-L. The inhibition of the calcineurin-NFAT pathway altered the expression of the cell cycle regulation genes CDK4, CDK6, and CDKN1A, thus enabling faster cell cycle progression. The extensive use of NFAT inhibitors in the clinic should take into account that, in addition to the immunosuppression role in lymphoid cells, these NFAT inhibitors also affect the maintenance of the myeloid compartment. Microarray technology was used to understand the effects of NFAT inhibitors on C-kit enriched lineage negative cells.
Project description:BMP signalling acts as an instructive cue in various developmental processes such as tissue patterning, stem cell proliferation, and differentiation. However, it is not fully understood how this signalling pathway generates different cell-specific outputs. Here we have identified PRDM16 as a key co-factor for BMP signalling. PRDM16 contributes to a repressive role of BMP signalling on neural stem cell (NSC) proliferation. We demonstrate that PRDM16 regulates the genomic distribution of BMP pathway transcription factors, the SMAD4/pSMAD complex, preventing the activation of cell proliferation genes. When Prdm16 is lost, the SMAD complex relocates to nearby genomic regions, leading to abnormal upregulation of BMP target genes. This function of PRDM16 is also required for the specification of choroid plexus (ChP) epithelial cells. Through a single-cell resolution fluorescent in situ approach, we have observed that genes co-repressed by SMAD and PRDM16, such as Wnt7b and several cell cycle regulators, become overexpressed in Prdm16 mutant ChP. Our findings elucidate a mechanism through which SMAD4 and pSMAD1/5/8 repress gene expression. Moreover, our study suggests a regulatory circuit composed of BMP and Wnt signaling, along with PRDM16, in controlling stem cell behaviors.