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| Status |
Public on Sep 04, 2012 |
| Title |
The protein kinase MLTK Is a key regulator for chondrogenesis. |
| Organism |
Xenopus laevis |
| Experiment type |
Expression profiling by array
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| Summary |
Sox9 acts together with Sox5 or Sox6 as a master regulator for chondrocyte differentiation; however, how these transcription factors functionally interact and collaborate to regulate chondrogenesis remains unclear. Here we show that the protein kinase MLTK plays an essential role in the onset of chondrogenesis through triggering the induction of Sox6 by Sox9. Knockdown of MLTK in Xenopus embryos results in drastic loss of craniofacial cartilages without defects in neural crest formation. We also find that Sox6 is specifically induced during craniofacial chondrogenesis and this induction is inhibited by MLTK knockdown. Remarkably, Sox6-knockdown embryos display essentially the same phenotype as the MLTK-knockdown embryos; the drastic loss of cartilages and the marked down-regulation of genes involved in chondrogenesis. Microarray analysis reveals a remarkable similarity between Sox6-depleted and MLTK-depleted embryos in their gene expression pattern. Moreover, we find that ectopic expression of MLTK can induce Sox6 expression in a Sox9-dependent manner. These results identify a novel, key regulator for chondrogenesis.
We used microarrays to describe the genome-wide gene expression profiles of xMLTK-depleted and xSox6-depleted embryos.
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| Overall design |
CoMO, xMLTK-MO, or xSox6-MO was injected into the animal pole of all blastomeres (10 ng/cell) at the four-cell stage. The heads of embryos were cut out and harvested at 72 hours after fertilization (corresponding to about stage 41). Total RNA was extracted using TRIsol reagent, treated with DNase (TURBO DNase, Ambion), and then purified using RNeasy Mini Kit (QIAGEN) according to the manufacturer’s instruction. The quality of total RNA was assessed using the Agilent 2100 BioAnalyzer. Reverse transcription to synthesize first-strand cDNA, second-strand cDNA synthesis, in vitro transcription to synthesize Biotin-modified aRNA, aRNA purification and fragmentation of the labeled aRNA were performed using the GeneChip 3’ IVT Express Kit and hybridization to the Affymetrix GeneChip Xenopus laevis Genome 2.0 Array was performed using the GeneChip Hybridization Wash and Stain Kit according to the manufacturer’s instruction. Hybridized arrays were scanned using an Affymetrix GeneChip Scanner. Scanned Chip images were analyzed with GeneChip operating Software v.1.4 (GCOS) and GeneSpring GX 11.0.2. (Agilent technologies).
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| Contributor(s) |
Suzuki T, Kusakabe M, Nishida E |
| Citation(s) |
22764049 |
| |
| Submission date |
Nov 17, 2011 |
| Last update date |
Oct 02, 2015 |
| Contact name |
Eisuke Nishida |
| E-mail(s) |
nishida@lif.kyoto-u.ac.jp
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| Phone |
+81-75-753-4230
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| Organization name |
Graduate School of Biostudies, Kyoto University
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| Department |
Department of Cell and Developmental Biology
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| Street address |
Kitashirakawa, Sakyo-ku
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| City |
Kyoto |
| ZIP/Postal code |
606-8502 |
| Country |
Japan |
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| Platforms (1) |
| GPL10756 |
[X_laevis_2] Affymetrix Xenopus laevis Genome 2.0 Array |
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| Samples (3) |
| GSM835213 |
Head of stage 41 embryo-CoMO injected-rep1 |
| GSM835214 |
Head of stage 41 embryo-xMLTK-MO injected-rep1 |
| GSM835215 |
Head of stage 41 embryo-xSox6-MO injected-rep1 |
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| Relations |
| BioProject |
PRJNA148377 |
