Pax3 and Zic1 trigger the early neural crest gene regulatory network by the direct activation of multiple key neural crest specifiers
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ABSTRACT:
ORGANISM(S): Xenopus laevis
SUBMITTER: Anne-Helene Monsoro-Burq
PROVIDER: E-GEOD-53679 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Publications
Pax3 and Zic1 trigger the early neural crest gene regulatory network by the direct activation of multiple key neural crest specifiers.
Plouhinec Jean-Louis JL Roche Daniel D DD Pegoraro Caterina C Figueiredo Ana Leonor AL Maczkowiak Frédérique F Brunet Lisa J LJ Milet Cécile C Vert Jean-Philippe JP Pollet Nicolas N Harland Richard M RM Monsoro-Burq Anne H AH
Developmental biology 20131217 2
Neural crest development is orchestrated by a complex and still poorly understood gene regulatory network. Premigratory neural crest is induced at the lateral border of the neural plate by the combined action of signaling molecules and transcription factors such as AP2, Gbx2, Pax3 and Zic1. Among them, Pax3 and Zic1 are both necessary and sufficient to trigger a complete neural crest developmental program. However, their gene targets in the neural crest regulatory network remain unknown. Here, t ...[more]
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Project description:Neural crest development is orchestrated by a complex and still poorly understood gene regulatory network. Premigratory neural crest is induced at the lateral border of the neural plate by the combined action of signaling molecules and transcription factors such as AP2, Gbx2, Pax3 and Zic1. Among them, Pax3 and Zic1 are both necessary and sufficient to trigger a complete neural crest developmental program. However, their gene targets in the neural crest regulatory network remain unknown. Here, through a transcriptome analysis of frog microdissected neural border, we identified an extended gene signature for the premigratory neural crest, and we defined novel potential members of the regulatory network. This signature includes 34 novel genes, as well as 44 known genes expressed at the neural border. Using another microarray analysis which combined Pax3 and Zic1 gain-of-function and protein translation blockade, we uncovered 25 Pax3 and Zic1 direct targets within this signature. We demonstrated that the neural border specifiers Pax3 and Zic1 are direct upstream regulators of neural crest specifiers Snail1/2, Foxd3, Twist1, and Tfap2b. In addition, they may modulate the transcriptional output of multiple signaling pathways involved in neural crest development (Wnt, Retinoic Acid) through the induction of key pathway regulators (Axin2 and Cyp26c1). We also found that Pax3 could maintain its own expression through a positive autoregulatory feedback loop. These hierarchical inductions, feedback loops, and pathway modulation provide novel tools to understand the neural crest induction network. Transcriptomes of animal caps overexpressing Pax3+/-Zic1 in the presence/absence of cycloheximide, a translation inhibitor, were compared to control animal caps to identify direct Pax3 and Zic1 targets 2-4 cells stage embryos were injected with inducible Pax3-GR+/-Zic1-GR constructs. Animal caps were cut at stage 9. Cycloheximide (Chx, 0.1mg/ml) was then applied to the healed animal caps, from stage 10 to 10.5 (i.e. for 30 min at 23*C), then dexamethasone (Dex) was added at stage 10.5 to the cycloheximide-containing medium. Explants were rinced and lysed after two additional hours at 23*C, i.e. when sibling embryos reached stage 11.5-12. Total RNA was then extracted and hybridized on Affymetrix microarrays. Transcriptomes were compared to determine Pax3 and Zic1 targets.
2013-12-28 | E-GEOD-53677 | biostudies-arrayexpress
Project description:Neural crest development is orchestrated by a complex and still poorly understood gene regulatory network. Premigratory neural crest is induced at the lateral border of the neural plate by the combined action of signaling molecules and transcription factors such as AP2, Gbx2, Pax3 and Zic1. Among them, Pax3 and Zic1 are both necessary and sufficient to trigger a complete neural crest developmental program. However, their gene targets in the neural crest regulatory network remain unknown. Here, through a transcriptome analysis of frog microdissected neural border, we identified an extended gene signature for the premigratory neural crest, and we defined novel potential members of the regulatory network. This signature includes 34 novel genes, as well as 44 known genes expressed at the neural border. Using another microarray analysis which combined Pax3 and Zic1 gain-of-function and protein translation blockade, we uncovered 25 Pax3 and Zic1 direct targets within this signature. We demonstrated that the neural border specifiers Pax3 and Zic1 are direct upstream regulators of neural crest specifiers Snail1/2, Foxd3, Twist1, and Tfap2b. In addition, they may modulate the transcriptional output of multiple signaling pathways involved in neural crest development (Wnt, Retinoic Acid) through the induction of key pathway regulators (Axin2 and Cyp26c1). We also found that Pax3 could maintain its own expression through a positive autoregulatory feedback loop. These hierarchical inductions, feedback loops, and pathway modulation provide novel tools to understand the neural crest induction network. The transcriptomes of neural border samples (stage 14 and 18) were compared to the transcriptome of anterior neural fold (stage 18), early neural plate (stage 12), and animal cap explants (stage14) to identify genes expressed specifically in neural border samples. Tissue samples from Xenopus laevis embryos were dissected, then total RNA was extracted and hybridized on Affymetrix microarrays. Selected tissue samples encompass the neural crest at different stages of its induction (early neural plate at stage 12, neural border at stage 14, neural border at stage 18), as well as reference tissues (anterior neural fold at stage 18, a tissue that belongs to the neural border but does not produce neural crest, and animal cap grown until stage 14 that differentiates into epidermis).
2013-12-28 | E-GEOD-53678 | biostudies-arrayexpress
Project description:Transcriptional targets of neurogenin (Ngnr1) were identified by over-expression of an inducible form of neurogenin in Xenopus ectodermal explants. The effects of co-expressing the nucleoprotein geminin on Ngnr1-dependent target gene transactivation were defined. Regulating the transition from lineage-restricted progenitors to terminally differentiated cells is a central aspect of nervous system development. Here, we investigated the role of the nucleoprotein geminin in regulating neurogenesis at a mechanistic level during both Xenopus primary neurogenesis and mammalian neuronal differentiation in vitro. The latter work utilized both neural cells derived from embryonic stem and embryonal carcinoma cells in vitro and neural stem cells from mouse forebrain. In all of these contexts, geminin antagonized the ability of neural bHLH transcription factors to activate transcriptional programs promoting neurogenesis. Furthermore, geminin promoted a bivalent chromatin state, characterized by the presence of both activating and repressive histone modifications, at genes encoding transcription factors that promote neurogenesis. This epigenetic state restrains the expression of genes that regulate commitment of undifferentiated stem and neuronal precursor cells to neuronal lineages. Geminin is highly expressed in undifferentiated neuronal precursor cells but is downregulated prior to differentiation. Therefore, these data support a model whereby geminin promotes the neuronal precursor cell state by modulating both the epigenetic status and expression of genes encoding neurogenesis-promoting factors. Additional developmental signals acting in these cells can then control their transition toward terminal neuronal or glial differentiation during mammalian neurogenesis. A dexamethasone-inducible (GR ligand binding domain fused) form of Xenopus neurogenin-related 1, NgnrGR, was over-expressed in Xenopus embryonic ectodermal explants, in the presence or absence of over-expressed geminin. Induction of Ngnr1 activity was used to define direct targets as previously described (EMBO J. 26(24): 5093-5108). The ability of geminin to suppress Ngnr1-dependent transactivation of its target gene programs was determined.
2012-12-06 | E-GEOD-39658 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2012-12-06 | E-GEOD-39673 | biostudies-arrayexpress
Project description:The transcription factors Pax3 and Zic1 are among the earliest genes activated at the neural plate border. Pax3 and Zic1 in combination promote neural crest fate, while Zic1 alone regulate cranial placode progenitor formation. We used microarrays to identify the global repertoire of genes activated by these facors individually or in combination to gain insights into the molecular mechanisms underlying cell fate decision at the neural plate border. Xenopus laevis embryos were injected at the 2-cell stage with mRNA encoding Pax3GR and Zic1GR , the hormone-inducible version of Pax3 and Zic1, alone or in combination. At the blastula stage (stage 9), animal cap explants were dissected and cultured for 8 hours in the presence of dexamethasone. A glucocorticoid receptor (GR) mRNA was also injected as negative control.
2015-05-06 | E-GEOD-68546 | biostudies-arrayexpress
Project description:The Xenopus POU class V transcription factor XOct-25 has been shown to inhibit BMP-dependent epidermal differentiation and promote neural induction in the ectoderm during early embryogenesis. In order to identify genes that act downstream of XOct-25, transcriptional profile of Xenopus ectodermal cells overexpressing XOct-25 was compared with control cells by using a DNA microarray method. Two independent experiments. Each experiment contains ectodermal cells overexpressing XOct-25 and corresponding control cells. Xenopus embryos were injected at the 8-cell stage with mRNA encoding GR-XOct-25, a hormone-inducible version of XOct-25. Explants from stage 9 embryos were treated with or without dexamethazone (DEX) until the sibling embryos reached stage 10.5, when they were used for RNA extraction. The explants cultured without DEX was used as a control sample. biological replicates: Sample name XOct-exp 1, Sample name XOct-exp 2
2012-10-05 | E-GEOD-24562 | biostudies-arrayexpress
Project description:Analysis of epithelial explants injected with the intracellular domain of Notch (ICD) to block the formation of multi-ciliate and proton secreting cells or with dominant negative human Mastermind (HMM) to induce the formation of ectopic multi-ciliate and proton secreting cells. Results show which genes are up or down-regulated when HMM is compared to ICD. Specialized epithelial cells in the amphibian skin play important roles in ion transport, but how they arise developmentally is largely unknown. Here we show that proton-secreting cells (PSCs) differentiate in the X. laevis larval skin soon after gastrulation, based on the expression of “kidney” specific form of the H+v-ATPase that localizes to the plasma membrane, orthologs of the Cl-/HCO3- antiporters ae1 and pendrin, and two isoforms of carbonic anhydrase. Like PSCs in other species, we show that the expression of these genes is likely to be driven by an ortholog of foxi1, which is also sufficient to promote the formation of PSC precursors. Strikingly, the PSCs form in the skin as two distinct subtypes that resemble the alpha- and beta-intercalated cells of the kidney. The alpha-subtype expresses ae1 and localizes H+v-ATPases to the apical plasma membrane, whereas the beta-subtype expresses pendrin and localizes the H+v-ATPase cytosolically or basolaterally. These two subtypes are specified during early differentiation, and can be distinguished based on the expression of ubp1, a transcription factor in the grainyhead family. Finally, we show that ectopic expression ubp1 is sufficient to promote the differentiation of beta-subtypes while repressing alpha-subtypes. These results have implications for how PSCs are specified in vertebrates and become functionally heterogeneous. 2-cell stage Xenopus embryos were injected with synthetic mRNA encoding ICD or HMM. At stage 9/10 the presumptive ectoderm was cut off the embryo and cultured on a fibronectin coated coverslip. At stage 22 RNA was harvested from explants and used as the starting material for arrays.
2010-08-28 | E-GEOD-23844 | biostudies-arrayexpress
Project description:Analysis of epithelial explants injected with the intracellular domain of Notch (ICD) to block the formation of multi-ciliate and proton secreting cells or with dominant negative human Mastermind (HMM) or a DNA binding mutant of Mastermind (DBM) to induce the formation of ectopic multi-ciliate and proton secreting cells. Results show which genes are up or down-regulated when DBM/HMM are compared to ICD. Epithelial cells projecting hundreds of motile cilia are prominent in the respiratory system, brain ependyma and female reproductive tract where they generate a vigorous fluid flow along luminal surfaces. Despite their importance for organ function, how multiciliate cells arise developmentally in diverse epithelia is poorly understood. Notch signaling has been shown in a variety of tissues, including the Xenopus epithelium, to regulate the formation of multiciliate cell precursors. In order to identify early downstream targets of Notch signaling which contribute to multiciliate cell fate we blocked or activated Notch signaling in explants of Xenopus epithelium and looked at changes in gene expression to identify candidates that regulate multiciliate cell fate. Here we identify a novel coiled-coil protein, called multicilin (MCI), whose expression is Notch-regulated and restricted to epithelia where multiciliate cells forms in Xenopus embryos. Inhibiting MCI activity blocks the formation of multiciliate cell precursors while ectopic MCI activity is sufficient to induce multiciliate cell differentiation within epithelial progenitors. 2-cell stage Xenopus embryos were injected with synthetic mRNA encoding ICD, DBM or HMM. At stage 9/10 the presumptive ectoderm was cut off the embryo and cultured on a fibronectin coated coverslip. At stage 12 RNA was harvested from explants and used as the starting material for arrays.
2012-03-01 | E-GEOD-32331 | biostudies-arrayexpress
Project description:Analysis of epithelial explants injected with the intracellular domain of Notch (ICD) alone, to block the formation of multiciliate cells, or with Dexamethasone inducible Multicilin (MCI-HGR) to induce the formation of ectopic multiciliate cells. MCI-HGR plus ICD injected samples were compared to ICD alone to identify candidate genes whose expression changes when MCI is active. Epithelial cells projecting hundreds of motile cilia are prominent in the respiratory system, brain ependyma and female reproductive tract where they generate a vigorous fluid flow along luminal surfaces. Despite their importance for organ function, how multiciliate cells arise developmentally in diverse epithelia is poorly understood. We identified a novel coiled-coil protein, we have termed multicilin (MCI), whose expression is Notch regulated and restricted to epithelia where multiciliate cells form in Xenopus embryos. In order to identify early downstream targets of multicilin we blocked the formation of endogenous multiciliate cells by injecting ICD mRNA. In a subset of samples we then misexpressed a dexamethasone inducible form of MCI (MCI-HGR) to identify specific targets of MCI in explants of Xenopus epithelium. In explants expressing MCI-HGR genes specifically expressed in ciliated cells such as FoxJ1 and alpha-tubulin were highly upregulated along with various genes known to be required for ciliogenesis, and a large number of genes likely to be part of the ciliome based on homology of potential structural motifs. 2-cell stage Xenopus embryos were injected with synthetic mRNA encoding ICD or ICD along with MCI-HGR. At stage 9/10 the presumptive ectoderm was cut off the embryo and cultured on a fibronectin coated coverslip. At stage 11.5 dexamethasone was added to all conditions to induce MCI. Explants were harvested 5 hours after adding dexamethasone (approximately stage14) and used as the starting material for arrays.
2012-03-01 | E-GEOD-32452 | biostudies-arrayexpress
Project description:We aimed to identify genes that are inhibited by Kctd15 overexpression during neural crest (NC) induction. Injection of RNAs encoding Wnt3a and chordin into the embryo followed by animal cap explant culture leads to the induction of NC marker genes as well as additional genes expressed in the neural plate border. Our previous study indicated that co-injection of kctd15 inhibits the induction of these markers. We injected four sets of RNAs, lacZ as control (L), wnt3a+chd (WC), wnt3a+chd+kctd15 (WCK), and kctd15 (K). After evaluation of marker gene expression with RT-PCR, we prepared probes with same RNA extract for Affymetrix DNA microarray. The hybridization, washing and scanning were perfomed following the manual from Affymetrix. Partek Genomics Suite was employed for data analysis. Xenopus embryos were injected with mRNAs at two-cell stage. When developing to stage 9, the animal caps were dissected for RNA extraction and hybridization on Affymetrix microarrays. According to the mRNAs injected, the animal caps (AC) were divided into four groups, including (1) AC injected with lacZ; (2) AC injected with kctd15; (3) AC injected with chordin and wnt3a; (4) AC injected with chordin, wnt3a and kctd15. The injected animal caps were cultured till stage 15 and then collected for RT-PCR and microarray assay. Each group had three repeats.
2015-08-26 | E-GEOD-72391 | biostudies-arrayexpress