Unknown,Transcriptomics,Genomics,Proteomics

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Identification of Kctd15 target genes in Xenopus animal caps injected with wnt3a and chordin

ABSTRACT: 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.

ORGANISM(S): Xenopus laevis

SUBMITTER: Igor Dawid

PROVIDER: E-GEOD-72391 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

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Identification of Kctd15 target genes in Xenopus animal caps injected with wnt3a and chordin

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