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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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Double in situ staining of mRNAs for down-regulated genes and Pax6 in the E11.5 rat hindbrain. The expression sites of Dbx1, Unc5h1 and Cyp26b1 (blue-violet in A-I and red in J-L) were compared with that of Pax6 (pink in A-I and blue in J-L) in WT (A, D, G, J), rSey2/+ (B, E, H, K) and rSey2/rSey2 (C, F, I, L) hindbrains in 'open-book' preparations. (A-C) Dbx1 expression is decreased throughout the interneuron progenitor domain of the rSey2/rSey2 hindbrain with the exception of r1, where Pax6 is not expressed (asterisk in C). (D-F) Unc5h1 expression disappears in the somatic motor neuron progenitor domain and in the dorsal region of r7 in the rSey2/rSey2 hindbrain (F). (G-L) Cyp26b1 expression is specifically altered in the dorsolateral region of r5 and r6 in the rSey2/rSey2 hindbrain (arrowheads in I and L). r1 to r7 indicate the individual rhombomeres of the hindbrain. Scale bar: 500 μm.
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Figure 5: Double in situ staining of mRNAs for down-regulated genes and Pax6 in the E11.5 rat hindbrain. The expression sites of Dbx1, Unc5h1 and Cyp26b1 (blue-violet in A-I and red in J-L) were compared with that of Pax6 (pink in A-I and blue in J-L) in WT (A, D, G, J), rSey2/+ (B, E, H, K) and rSey2/rSey2 (C, F, I, L) hindbrains in 'open-book' preparations. (A-C) Dbx1 expression is decreased throughout the interneuron progenitor domain of the rSey2/rSey2 hindbrain with the exception of r1, where Pax6 is not expressed (asterisk in C). (D-F) Unc5h1 expression disappears in the somatic motor neuron progenitor domain and in the dorsal region of r7 in the rSey2/rSey2 hindbrain (F). (G-L) Cyp26b1 expression is specifically altered in the dorsolateral region of r5 and r6 in the rSey2/rSey2 hindbrain (arrowheads in I and L). r1 to r7 indicate the individual rhombomeres of the hindbrain. Scale bar: 500 μm.

Mentions: The above results indicate that not only Fabp7 and Dbx1 but also Unc5h1 and Cyp26b1 are good candidates for Pax6 downstream target molecules in the developing CNS. To ascertain the detailed expression of these downstream genes in the E11.5 hindbrain, we prepared 'open-book' samples of this tissue (Figure 5). Double staining for Dbx1 and Pax6 demonstrated that expression of Dbx1 in the interneuron progenitor domain along the anterior-posterior axis overlapped with Pax6 in both WT and rSey2/+ hindbrains (Figure 5A, B). Dbx1 expression was dramatically reduced throughout the rSey2/rSey2 hindbrain with the exception of r1 (asterisk in Figure 5C), in which Pax6 was not expressed. Unc5h1 expression disappeared in the rSey2/rSey2 hindbrain in the ventral neuron progenitor domains and in the dorsal part of r7, where Pax6 was not detected at E11.5 (Figure 5F). Cyp26b1 expression was altered specifically in the dorsolateral region of r5 and r6 in the rSey2/rSey2 hindbrain (indicated by arrowheads in Figure 5I, L). Thus, we consider these three genes to be very promising candidates for downstream targets of Pax6. We performed further detailed analyses focusing on Cyp26b1 because this gene product is involved in RA metabolism, which is an important element in the anterior-posterior patterning of the hindbrain.


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)

Double in situ staining of mRNAs for down-regulated genes and Pax6 in the E11.5 rat hindbrain. The expression sites of Dbx1, Unc5h1 and Cyp26b1 (blue-violet in A-I and red in J-L) were compared with that of Pax6 (pink in A-I and blue in J-L) in WT (A, D, G, J), rSey2/+ (B, E, H, K) and rSey2/rSey2 (C, F, I, L) hindbrains in 'open-book' preparations. (A-C) Dbx1 expression is decreased throughout the interneuron progenitor domain of the rSey2/rSey2 hindbrain with the exception of r1, where Pax6 is not expressed (asterisk in C). (D-F) Unc5h1 expression disappears in the somatic motor neuron progenitor domain and in the dorsal region of r7 in the rSey2/rSey2 hindbrain (F). (G-L) Cyp26b1 expression is specifically altered in the dorsolateral region of r5 and r6 in the rSey2/rSey2 hindbrain (arrowheads in I and L). r1 to r7 indicate the individual rhombomeres of the hindbrain. Scale bar: 500 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2818624&req=5

Figure 5: Double in situ staining of mRNAs for down-regulated genes and Pax6 in the E11.5 rat hindbrain. The expression sites of Dbx1, Unc5h1 and Cyp26b1 (blue-violet in A-I and red in J-L) were compared with that of Pax6 (pink in A-I and blue in J-L) in WT (A, D, G, J), rSey2/+ (B, E, H, K) and rSey2/rSey2 (C, F, I, L) hindbrains in 'open-book' preparations. (A-C) Dbx1 expression is decreased throughout the interneuron progenitor domain of the rSey2/rSey2 hindbrain with the exception of r1, where Pax6 is not expressed (asterisk in C). (D-F) Unc5h1 expression disappears in the somatic motor neuron progenitor domain and in the dorsal region of r7 in the rSey2/rSey2 hindbrain (F). (G-L) Cyp26b1 expression is specifically altered in the dorsolateral region of r5 and r6 in the rSey2/rSey2 hindbrain (arrowheads in I and L). r1 to r7 indicate the individual rhombomeres of the hindbrain. Scale bar: 500 μm.
Mentions: The above results indicate that not only Fabp7 and Dbx1 but also Unc5h1 and Cyp26b1 are good candidates for Pax6 downstream target molecules in the developing CNS. To ascertain the detailed expression of these downstream genes in the E11.5 hindbrain, we prepared 'open-book' samples of this tissue (Figure 5). Double staining for Dbx1 and Pax6 demonstrated that expression of Dbx1 in the interneuron progenitor domain along the anterior-posterior axis overlapped with Pax6 in both WT and rSey2/+ hindbrains (Figure 5A, B). Dbx1 expression was dramatically reduced throughout the rSey2/rSey2 hindbrain with the exception of r1 (asterisk in Figure 5C), in which Pax6 was not expressed. Unc5h1 expression disappeared in the rSey2/rSey2 hindbrain in the ventral neuron progenitor domains and in the dorsal part of r7, where Pax6 was not detected at E11.5 (Figure 5F). Cyp26b1 expression was altered specifically in the dorsolateral region of r5 and r6 in the rSey2/rSey2 hindbrain (indicated by arrowheads in Figure 5I, L). Thus, we consider these three genes to be very promising candidates for downstream targets of Pax6. We performed further detailed analyses focusing on Cyp26b1 because this gene product is involved in RA metabolism, which is an important element in the anterior-posterior patterning of the hindbrain.

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