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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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The rhombomere 5 region is broadened in the rSey2/rSey2 hindbrain. (A-F) The expression sites of Hoxb1 and Krox20 (blue) were compared with that of Cyp26b1 (red) in WT (A, D), rSey2/+ (B, E) and rSey2/rSey2 (C, F) hindbrains. (A-C) Hoxb1 expression (as a marker of r4) is unchanged in the rSey2/rSey2 hindbrain (C). (D-F) Krox20 expression (as a marker of r5; cannot detect in r3 at E11.5) overlaps with Cyp26b1 expression in the dorsolateral domain of the WT and rSey2/+ hindbrain (D and E). The r5 region is slightly broadened in the rSey2/rSey2 hindbrain along the anterior-posterior axis (F). (G-I) EphA4 expressed in both r3 and r5 of the E11.5 hindbrain. EphA4 expression domains (blue) are broadened only within the r5 region of the rSey2/rSey2 hindbrain (I) as compared to the WT (G). (J-L) The expression site of RARβ (blue) was compared with that of Krox20 (red) in WT (J), rSey2/+ (K) and rSey2/rSey2 (L) hindbrains. Krox20 expression is posteriorly expanded in the rSey2/rSey2 hindbrain (L), although the anterior border of RARβ expression (anterior edge of r7 in L) is not altered. Scale bar: 500 μm.
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Figure 6: The rhombomere 5 region is broadened in the rSey2/rSey2 hindbrain. (A-F) The expression sites of Hoxb1 and Krox20 (blue) were compared with that of Cyp26b1 (red) in WT (A, D), rSey2/+ (B, E) and rSey2/rSey2 (C, F) hindbrains. (A-C) Hoxb1 expression (as a marker of r4) is unchanged in the rSey2/rSey2 hindbrain (C). (D-F) Krox20 expression (as a marker of r5; cannot detect in r3 at E11.5) overlaps with Cyp26b1 expression in the dorsolateral domain of the WT and rSey2/+ hindbrain (D and E). The r5 region is slightly broadened in the rSey2/rSey2 hindbrain along the anterior-posterior axis (F). (G-I) EphA4 expressed in both r3 and r5 of the E11.5 hindbrain. EphA4 expression domains (blue) are broadened only within the r5 region of the rSey2/rSey2 hindbrain (I) as compared to the WT (G). (J-L) The expression site of RARβ (blue) was compared with that of Krox20 (red) in WT (J), rSey2/+ (K) and rSey2/rSey2 (L) hindbrains. Krox20 expression is posteriorly expanded in the rSey2/rSey2 hindbrain (L), although the anterior border of RARβ expression (anterior edge of r7 in L) is not altered. Scale bar: 500 μm.

Mentions: To explore the rhombomere-specific alteration of Cyp26b1 expression, several markers were examined by double-color WISH in E11.5 WT, rSey2/+ and rSey2/rSey2 hindbrains (Figure 6). Hoxb1 expression in r4 did not show any change in the three genotypes (Figure 6A-C), suggesting that r4 was intact. Krox20 expression was observed in r5 at this stage in the rat embryo (Figure 6D-F). In the rSey2/rSey2 hindbrain, this Krox20-positive r5 region seemed to expand along the anteroposterior axis (Figure 6F; also see Figure 6J-L). A similar change in r5 morphology was also detected in the rSey2/rSey2 hindbrain using a probe for EphA4, which was expressed in r3 and r5 (Figure 6G-I). r6 was found to be intact in the rSey2/rSey2 hindbrain through the use of an EphrinB2 probe (data not shown).


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)

The rhombomere 5 region is broadened in the rSey2/rSey2 hindbrain. (A-F) The expression sites of Hoxb1 and Krox20 (blue) were compared with that of Cyp26b1 (red) in WT (A, D), rSey2/+ (B, E) and rSey2/rSey2 (C, F) hindbrains. (A-C) Hoxb1 expression (as a marker of r4) is unchanged in the rSey2/rSey2 hindbrain (C). (D-F) Krox20 expression (as a marker of r5; cannot detect in r3 at E11.5) overlaps with Cyp26b1 expression in the dorsolateral domain of the WT and rSey2/+ hindbrain (D and E). The r5 region is slightly broadened in the rSey2/rSey2 hindbrain along the anterior-posterior axis (F). (G-I) EphA4 expressed in both r3 and r5 of the E11.5 hindbrain. EphA4 expression domains (blue) are broadened only within the r5 region of the rSey2/rSey2 hindbrain (I) as compared to the WT (G). (J-L) The expression site of RARβ (blue) was compared with that of Krox20 (red) in WT (J), rSey2/+ (K) and rSey2/rSey2 (L) hindbrains. Krox20 expression is posteriorly expanded in the rSey2/rSey2 hindbrain (L), although the anterior border of RARβ expression (anterior edge of r7 in L) is not altered. Scale bar: 500 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 6: The rhombomere 5 region is broadened in the rSey2/rSey2 hindbrain. (A-F) The expression sites of Hoxb1 and Krox20 (blue) were compared with that of Cyp26b1 (red) in WT (A, D), rSey2/+ (B, E) and rSey2/rSey2 (C, F) hindbrains. (A-C) Hoxb1 expression (as a marker of r4) is unchanged in the rSey2/rSey2 hindbrain (C). (D-F) Krox20 expression (as a marker of r5; cannot detect in r3 at E11.5) overlaps with Cyp26b1 expression in the dorsolateral domain of the WT and rSey2/+ hindbrain (D and E). The r5 region is slightly broadened in the rSey2/rSey2 hindbrain along the anterior-posterior axis (F). (G-I) EphA4 expressed in both r3 and r5 of the E11.5 hindbrain. EphA4 expression domains (blue) are broadened only within the r5 region of the rSey2/rSey2 hindbrain (I) as compared to the WT (G). (J-L) The expression site of RARβ (blue) was compared with that of Krox20 (red) in WT (J), rSey2/+ (K) and rSey2/rSey2 (L) hindbrains. Krox20 expression is posteriorly expanded in the rSey2/rSey2 hindbrain (L), although the anterior border of RARβ expression (anterior edge of r7 in L) is not altered. Scale bar: 500 μm.
Mentions: To explore the rhombomere-specific alteration of Cyp26b1 expression, several markers were examined by double-color WISH in E11.5 WT, rSey2/+ and rSey2/rSey2 hindbrains (Figure 6). Hoxb1 expression in r4 did not show any change in the three genotypes (Figure 6A-C), suggesting that r4 was intact. Krox20 expression was observed in r5 at this stage in the rat embryo (Figure 6D-F). In the rSey2/rSey2 hindbrain, this Krox20-positive r5 region seemed to expand along the anteroposterior axis (Figure 6F; also see Figure 6J-L). A similar change in r5 morphology was also detected in the rSey2/rSey2 hindbrain using a probe for EphA4, which was expressed in r3 and r5 (Figure 6G-I). r6 was found to be intact in the rSey2/rSey2 hindbrain through the use of an EphrinB2 probe (data not shown).

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