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Downstream genes of Pax6 revealed by comprehensive transcriptome profiling in the developing rat hindbrain.

Numayama-Tsuruta K, Arai Y, Takahashi M, Sasaki-Hoshino M, Funatsu N, Nakamura S, Osumi N - BMC Dev. Biol. (2010)

Bottom Line: In situ staining of Fabp7, Dbx1, Unc5h1 and Cyp26b1 mRNAs showed that expression of these transcripts not only overlapped with that of Pax6 in the hindbrain of wild-type and Pax6 heterozygous mutants, but also was clearly reduced in the hindbrain of the Pax6 homozygous mutant.These results indicate that Unc5h1 and Cyp26b1 are novel candidates for target genes transactivated by Pax6.Furthermore, our results suggest the interesting possibility that Pax6 regulates anterior-posterior patterning of the hindbrain via activation of Cyp26b1, an enzyme that metabolizes retinoic acid.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Developmental Neuroscience, Center for Translational and Advanced Animal Research, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan.

ABSTRACT

Background: The transcription factor Pax6 is essential for the development of the central nervous system and it exerts its multiple functions by regulating the expression of downstream target molecules. To screen for genes downstream of Pax6, we performed comprehensive transcriptome profiling analyses in the early hindbrain of Pax6 homozygous mutant and wild-type rats using microarrays.

Results: Comparison of quadruplicate microarray experiments using two computational methods allowed us to identify differentially expressed genes that have relatively small fold changes or low expression levels. Gene ontology analyses of the differentially expressed molecules demonstrated that Pax6 is involved in various signal transduction pathways where it regulates the expression of many receptors, signaling molecules, transporters and transcription factors. The up- or down-regulation of these genes was further confirmed by quantitative RT-PCR. In situ staining of Fabp7, Dbx1, Unc5h1 and Cyp26b1 mRNAs showed that expression of these transcripts not only overlapped with that of Pax6 in the hindbrain of wild-type and Pax6 heterozygous mutants, but also was clearly reduced in the hindbrain of the Pax6 homozygous mutant. In addition, the Pax6 homozygous mutant hindbrain showed that Cyp26b1 expression was lacked in the dorsal and ventrolateral regions of rhombomeres 5 and 6, and that the size of rhombomere 5 expanded rostrocaudally.

Conclusions: These results indicate that Unc5h1 and Cyp26b1 are novel candidates for target genes transactivated by Pax6. Furthermore, our results suggest the interesting possibility that Pax6 regulates anterior-posterior patterning of the hindbrain via activation of Cyp26b1, an enzyme that metabolizes retinoic acid.

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Gene ontology (GO) analyses of the genes that were differentially expressed between rSey2/rSey2 and WT rat hindbrains at E11.5. Annotated genes were selected by t-test using the cross-gene error model (p < 0.05). The genes are categorized by the following GO terms: cellular component (144, A), molecular function (160, B) and biological process (160, C), and are displayed in doughnut charts. Internal color arcs indicate the total rates for each category: (A) membrane (green, 52.1%), cytoplasm (blue, 29.9%) and nucleus (red, 16.0%); (B) signal transducer (green, 25.6%), catalytic (blue, 25.6%), binding (red, 25.0%) and transporter (yellow, 16.9%) activities; and (C) development (red, 26.9%), signal transduction (green, 20.0%), transport (yellow, 18.1%), and metabolism (blue, 16.9%). The details of the annotated genes and GO terms are shown in Additional file 3.
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Figure 2: Gene ontology (GO) analyses of the genes that were differentially expressed between rSey2/rSey2 and WT rat hindbrains at E11.5. Annotated genes were selected by t-test using the cross-gene error model (p < 0.05). The genes are categorized by the following GO terms: cellular component (144, A), molecular function (160, B) and biological process (160, C), and are displayed in doughnut charts. Internal color arcs indicate the total rates for each category: (A) membrane (green, 52.1%), cytoplasm (blue, 29.9%) and nucleus (red, 16.0%); (B) signal transducer (green, 25.6%), catalytic (blue, 25.6%), binding (red, 25.0%) and transporter (yellow, 16.9%) activities; and (C) development (red, 26.9%), signal transduction (green, 20.0%), transport (yellow, 18.1%), and metabolism (blue, 16.9%). The details of the annotated genes and GO terms are shown in Additional file 3.

Mentions: To categorize the differentially expressed genes, we performed gene ontology (GO) analyses. GO annotations were collected for the 1,421 selected genes, and 144, 160 and 160 genes were assigned one or more GO terms: cellular component (CC), molecular function (MF), and biological process (BP), respectively (see Additional file 3). Downstream molecules corresponding to most of the EST probe sets were neither annotated nor included in the analyses. Because the GO terms provided by the GO consortium are based on a hierarchical clustering, we chose the most suitable GO term for each gene in order to simply sort them into the GO categories shown in Figure 2. Categorization by the GO term CC revealed that 75/144 (52%) genes encoded membrane components (green in Figure 2A). Regarding the MF term, 41/160 (26%) and 12/160 (7.5%) genes coded for molecules related to signal transducer (green in Figure 2B) and channel (orange in Figure 2B) activities, respectively. Genes (26/32, 81%) sorted into the "signal transduction" category of the BP term (green in Figure 2C) overlapped with the genes categorized as signal transducer and channel activities of the MF term. These results suggest that many downstream molecules of Pax6 that are located in or near the membrane function as signal transducers (e.g., Htr4, Gabrr2, Gna15 and Fgf9) that regulate various signaling cascades. Similarly, 23/144 (16%) of the gene products were found to localize in the nucleus (red in the CC, Figure 2A), and 21/160 (13%) genes encoded transcription factors or DNA binding molecules (purple in the MF, Figure 2B). As expected, 15/21 (71%) of these transcription factors or DNA binding molecules were classified in the "development" category of the BP term (e.g., Nkx6-1, Ascl1, Neurog1 and Klf5, red in Figure 2C). Thus, the GO analyses demonstrated that Pax6 is involved in various signaling pathways where it regulates the expression of many receptors, receptor binding molecules, transporters and transcription factors.


Downstream genes of Pax6 revealed by comprehensive transcriptome profiling in the developing rat hindbrain.

Numayama-Tsuruta K, Arai Y, Takahashi M, Sasaki-Hoshino M, Funatsu N, Nakamura S, Osumi N - BMC Dev. Biol. (2010)

Gene ontology (GO) analyses of the genes that were differentially expressed between rSey2/rSey2 and WT rat hindbrains at E11.5. Annotated genes were selected by t-test using the cross-gene error model (p < 0.05). The genes are categorized by the following GO terms: cellular component (144, A), molecular function (160, B) and biological process (160, C), and are displayed in doughnut charts. Internal color arcs indicate the total rates for each category: (A) membrane (green, 52.1%), cytoplasm (blue, 29.9%) and nucleus (red, 16.0%); (B) signal transducer (green, 25.6%), catalytic (blue, 25.6%), binding (red, 25.0%) and transporter (yellow, 16.9%) activities; and (C) development (red, 26.9%), signal transduction (green, 20.0%), transport (yellow, 18.1%), and metabolism (blue, 16.9%). The details of the annotated genes and GO terms are shown in Additional file 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Gene ontology (GO) analyses of the genes that were differentially expressed between rSey2/rSey2 and WT rat hindbrains at E11.5. Annotated genes were selected by t-test using the cross-gene error model (p < 0.05). The genes are categorized by the following GO terms: cellular component (144, A), molecular function (160, B) and biological process (160, C), and are displayed in doughnut charts. Internal color arcs indicate the total rates for each category: (A) membrane (green, 52.1%), cytoplasm (blue, 29.9%) and nucleus (red, 16.0%); (B) signal transducer (green, 25.6%), catalytic (blue, 25.6%), binding (red, 25.0%) and transporter (yellow, 16.9%) activities; and (C) development (red, 26.9%), signal transduction (green, 20.0%), transport (yellow, 18.1%), and metabolism (blue, 16.9%). The details of the annotated genes and GO terms are shown in Additional file 3.
Mentions: To categorize the differentially expressed genes, we performed gene ontology (GO) analyses. GO annotations were collected for the 1,421 selected genes, and 144, 160 and 160 genes were assigned one or more GO terms: cellular component (CC), molecular function (MF), and biological process (BP), respectively (see Additional file 3). Downstream molecules corresponding to most of the EST probe sets were neither annotated nor included in the analyses. Because the GO terms provided by the GO consortium are based on a hierarchical clustering, we chose the most suitable GO term for each gene in order to simply sort them into the GO categories shown in Figure 2. Categorization by the GO term CC revealed that 75/144 (52%) genes encoded membrane components (green in Figure 2A). Regarding the MF term, 41/160 (26%) and 12/160 (7.5%) genes coded for molecules related to signal transducer (green in Figure 2B) and channel (orange in Figure 2B) activities, respectively. Genes (26/32, 81%) sorted into the "signal transduction" category of the BP term (green in Figure 2C) overlapped with the genes categorized as signal transducer and channel activities of the MF term. These results suggest that many downstream molecules of Pax6 that are located in or near the membrane function as signal transducers (e.g., Htr4, Gabrr2, Gna15 and Fgf9) that regulate various signaling cascades. Similarly, 23/144 (16%) of the gene products were found to localize in the nucleus (red in the CC, Figure 2A), and 21/160 (13%) genes encoded transcription factors or DNA binding molecules (purple in the MF, Figure 2B). As expected, 15/21 (71%) of these transcription factors or DNA binding molecules were classified in the "development" category of the BP term (e.g., Nkx6-1, Ascl1, Neurog1 and Klf5, red in Figure 2C). Thus, the GO analyses demonstrated that Pax6 is involved in various signaling pathways where it regulates the expression of many receptors, receptor binding molecules, transporters and transcription factors.

Bottom Line: In situ staining of Fabp7, Dbx1, Unc5h1 and Cyp26b1 mRNAs showed that expression of these transcripts not only overlapped with that of Pax6 in the hindbrain of wild-type and Pax6 heterozygous mutants, but also was clearly reduced in the hindbrain of the Pax6 homozygous mutant.These results indicate that Unc5h1 and Cyp26b1 are novel candidates for target genes transactivated by Pax6.Furthermore, our results suggest the interesting possibility that Pax6 regulates anterior-posterior patterning of the hindbrain via activation of Cyp26b1, an enzyme that metabolizes retinoic acid.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Developmental Neuroscience, Center for Translational and Advanced Animal Research, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan.

ABSTRACT

Background: The transcription factor Pax6 is essential for the development of the central nervous system and it exerts its multiple functions by regulating the expression of downstream target molecules. To screen for genes downstream of Pax6, we performed comprehensive transcriptome profiling analyses in the early hindbrain of Pax6 homozygous mutant and wild-type rats using microarrays.

Results: Comparison of quadruplicate microarray experiments using two computational methods allowed us to identify differentially expressed genes that have relatively small fold changes or low expression levels. Gene ontology analyses of the differentially expressed molecules demonstrated that Pax6 is involved in various signal transduction pathways where it regulates the expression of many receptors, signaling molecules, transporters and transcription factors. The up- or down-regulation of these genes was further confirmed by quantitative RT-PCR. In situ staining of Fabp7, Dbx1, Unc5h1 and Cyp26b1 mRNAs showed that expression of these transcripts not only overlapped with that of Pax6 in the hindbrain of wild-type and Pax6 heterozygous mutants, but also was clearly reduced in the hindbrain of the Pax6 homozygous mutant. In addition, the Pax6 homozygous mutant hindbrain showed that Cyp26b1 expression was lacked in the dorsal and ventrolateral regions of rhombomeres 5 and 6, and that the size of rhombomere 5 expanded rostrocaudally.

Conclusions: These results indicate that Unc5h1 and Cyp26b1 are novel candidates for target genes transactivated by Pax6. Furthermore, our results suggest the interesting possibility that Pax6 regulates anterior-posterior patterning of the hindbrain via activation of Cyp26b1, an enzyme that metabolizes retinoic acid.

Show MeSH
Related in: MedlinePlus