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Genetic and physical interaction of Meis2, Pax3 and Pax7 during dorsal midbrain development.

Agoston Z, Li N, Haslinger A, Wizenmann A, Schulte D - BMC Dev. Biol. (2012)

Bottom Line: Much less, however, is known about the regulatory relationship and functional interaction of molecules that are expressed in the tectal anlage after tectal fate specification has taken place and tectal development has commenced.Here, we provide experimental evidence for reciprocal regulation and subsequent cooperation of the paired-type transcription factors Pax3, Pax7 and the TALE-homeodomain protein Meis2 in the tectal anlage.Physical interaction with Meis2 may then confer tectal specificity to a wide range of otherwise broadly expressed transcriptional regulators, including Otx2, Pax3 and Pax7.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Neurology (Edinger Institute), J, W, Goethe University Medical School, Heinrich Hoffmannstr, 7, 50628 Frankfurt, Germany.

ABSTRACT

Background: During early stages of brain development, secreted molecules, components of intracellular signaling pathways and transcriptional regulators act in positive and negative feed-back or feed-forward loops at the mid-hindbrain boundary. These genetic interactions are of central importance for the specification and subsequent development of the adjacent mid- and hindbrain. Much less, however, is known about the regulatory relationship and functional interaction of molecules that are expressed in the tectal anlage after tectal fate specification has taken place and tectal development has commenced.

Results: Here, we provide experimental evidence for reciprocal regulation and subsequent cooperation of the paired-type transcription factors Pax3, Pax7 and the TALE-homeodomain protein Meis2 in the tectal anlage. Using in ovo electroporation of the mesencephalic vesicle of chick embryos we show that (i) Pax3 and Pax7 mutually regulate each other's expression in the mesencephalic vesicle, (ii) Meis2 acts downstream of Pax3/7 and requires balanced expression levels of both proteins, and (iii) Meis2 physically interacts with Pax3 and Pax7. These results extend our previous observation that Meis2 cooperates with Otx2 in tectal development to include Pax3 and Pax7 as Meis2 interacting proteins in the tectal anlage.

Conclusion: The results described here suggest a model in which interdependent regulatory loops involving Pax3 and Pax7 in the dorsal mesencephalic vesicle modulate Meis2 expression. Physical interaction with Meis2 may then confer tectal specificity to a wide range of otherwise broadly expressed transcriptional regulators, including Otx2, Pax3 and Pax7.

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Pax7 induces Pax3 expression in the mesencephalic vesicle. (A) Representative example of pMES-Pax7 electroporation into the mesencephalic vesicle. GFP expression, indicative of transgene expression, is restricted to the right half of the neural tube 4-6 hours later. (B-B") Flat mount preparation of a HH14 chick neural tube, 16 hours after electroporation of 2 μg/μl pMES-Pax7 into the mesencephalic vesicle; Pax3 expression is in dark blue, Nkx6.1 expression in pink, GFP in green. The dotted lines mark the dorso-ventral boundary, the arrow heads indicate representative examples of ectopic Pax7 positive cells co-expressing Pax3. Ectopic patches of Pax3 expression are restricted to the right, electroporated half of the mesencephalic vesicle. (B') is a higher magnification of (B) with the GFP fluorescent image superimposed onto the preparation. (B") shows the distribution of Pax7-GFP expressing cells in the specimen shown in (B'). (C) Quantification of the results: percent specimens with induced (green bars) and unaltered (blue bars) Pax3 expression following targeted electroporation of pMES-Pax7. (D) Flat mount preparation of a HH18 chick neural tube, 24 hours after electroporation of 2 μg/μl pMES-Pax7. (E) Targeted misexpression of pMES-Pax7 into the ventral mesencephalic vesicle showing cells ectopically expressing Pax3 within the Nkx6.1 domain. (E') is a higher magnification of (E). fb: forebrain; fp: floor plate; mes: mesencephalic vesicle; met: metencephalic vesicle; ov: optic vesicle; otv: otic vesicle. Scale bar (B, B'): 100 μm.
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Figure 2: Pax7 induces Pax3 expression in the mesencephalic vesicle. (A) Representative example of pMES-Pax7 electroporation into the mesencephalic vesicle. GFP expression, indicative of transgene expression, is restricted to the right half of the neural tube 4-6 hours later. (B-B") Flat mount preparation of a HH14 chick neural tube, 16 hours after electroporation of 2 μg/μl pMES-Pax7 into the mesencephalic vesicle; Pax3 expression is in dark blue, Nkx6.1 expression in pink, GFP in green. The dotted lines mark the dorso-ventral boundary, the arrow heads indicate representative examples of ectopic Pax7 positive cells co-expressing Pax3. Ectopic patches of Pax3 expression are restricted to the right, electroporated half of the mesencephalic vesicle. (B') is a higher magnification of (B) with the GFP fluorescent image superimposed onto the preparation. (B") shows the distribution of Pax7-GFP expressing cells in the specimen shown in (B'). (C) Quantification of the results: percent specimens with induced (green bars) and unaltered (blue bars) Pax3 expression following targeted electroporation of pMES-Pax7. (D) Flat mount preparation of a HH18 chick neural tube, 24 hours after electroporation of 2 μg/μl pMES-Pax7. (E) Targeted misexpression of pMES-Pax7 into the ventral mesencephalic vesicle showing cells ectopically expressing Pax3 within the Nkx6.1 domain. (E') is a higher magnification of (E). fb: forebrain; fp: floor plate; mes: mesencephalic vesicle; met: metencephalic vesicle; ov: optic vesicle; otv: otic vesicle. Scale bar (B, B'): 100 μm.

Mentions: To investigate a possible relationship between Pax3 and Pax7 in the midbrain, we ectopically delivered an expression plasmid carrying Pax7 together with GFP (pMES-Pax7) by in ovo electroporation into the right lateral wall of the mesencephalic vesicle at HH9-HH11 (7-13 somite stage, Figure 2A). Due to the shot-gun nature of this gene delivery method, random patches of GFP expression, indicative of groups of cells forced to express the GFP and Pax7 transgenes, were seen across the right mesencephalic wall (Figure 2A, B"). Consistent with a previous report, we found that in these patches Pax3 expression was strongly induced as early as 16 hours following Pax7 misexpression (HH14, n = 15/19 GFP expressing specimens exhibited strong ectopic Pax3 expression following electroporation of 2 μg/μl pMES-Pax7 (79%); Figure 2B, B', C) [11]. Robust upregulation of Pax3 expression was still visible 24 hours and 36 hours after pMES-Pax7 transfection (HH18 and HH21 respectively, Figure 2D and data not shown). Ectopic Pax3 expression was also observed when Pax7 misexpression was specifically targeted to the ventral midbrain (n = 8/14 GFP positive specimens exhibited ectopic Pax3 expression after targeted Pax7 expression into the ventral mesencephalic vesicle (57%); Figure 2E). Scattered groups of cells expressing elevated levels of Pax3 transcripts were not only located within the territory of the endogenous Pax3 domains, but also reached ventrally towards the Nkx6.1 expression domain (Figure 2B', E'). A comparison of Figure 2B' and Figure 2B" revealed that apparently not all Pax7-GFP positive cells also express Pax3 mRNA, especially not in the ventral midbrain. This may be due to the short incubation time of the embryos after electroporation (HH9-11 to HH14) and may thus reflect incomplete upregulation of Pax3 by Pax7. In addition, gene delivery by in ovo electroporation causes targeted cells to take up varying amounts of DNA. Considering that ectopic induction of Pax3 may need a certain threshold of Pax7 protein (especially in the ventral neural tube, where Sonic hedgehog signaling promotes the induction of ventral cell fates [25,26]), it is possible that the ectopic Pax7 levels may not have reached the threshold necessary for Pax3 induction in all GFP-positive cells. In any case however, ectopic Pax3 expressing cells in the ventral mesencephalon were consistently positive for Pax7.


Genetic and physical interaction of Meis2, Pax3 and Pax7 during dorsal midbrain development.

Agoston Z, Li N, Haslinger A, Wizenmann A, Schulte D - BMC Dev. Biol. (2012)

Pax7 induces Pax3 expression in the mesencephalic vesicle. (A) Representative example of pMES-Pax7 electroporation into the mesencephalic vesicle. GFP expression, indicative of transgene expression, is restricted to the right half of the neural tube 4-6 hours later. (B-B") Flat mount preparation of a HH14 chick neural tube, 16 hours after electroporation of 2 μg/μl pMES-Pax7 into the mesencephalic vesicle; Pax3 expression is in dark blue, Nkx6.1 expression in pink, GFP in green. The dotted lines mark the dorso-ventral boundary, the arrow heads indicate representative examples of ectopic Pax7 positive cells co-expressing Pax3. Ectopic patches of Pax3 expression are restricted to the right, electroporated half of the mesencephalic vesicle. (B') is a higher magnification of (B) with the GFP fluorescent image superimposed onto the preparation. (B") shows the distribution of Pax7-GFP expressing cells in the specimen shown in (B'). (C) Quantification of the results: percent specimens with induced (green bars) and unaltered (blue bars) Pax3 expression following targeted electroporation of pMES-Pax7. (D) Flat mount preparation of a HH18 chick neural tube, 24 hours after electroporation of 2 μg/μl pMES-Pax7. (E) Targeted misexpression of pMES-Pax7 into the ventral mesencephalic vesicle showing cells ectopically expressing Pax3 within the Nkx6.1 domain. (E') is a higher magnification of (E). fb: forebrain; fp: floor plate; mes: mesencephalic vesicle; met: metencephalic vesicle; ov: optic vesicle; otv: otic vesicle. Scale bar (B, B'): 100 μm.
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Figure 2: Pax7 induces Pax3 expression in the mesencephalic vesicle. (A) Representative example of pMES-Pax7 electroporation into the mesencephalic vesicle. GFP expression, indicative of transgene expression, is restricted to the right half of the neural tube 4-6 hours later. (B-B") Flat mount preparation of a HH14 chick neural tube, 16 hours after electroporation of 2 μg/μl pMES-Pax7 into the mesencephalic vesicle; Pax3 expression is in dark blue, Nkx6.1 expression in pink, GFP in green. The dotted lines mark the dorso-ventral boundary, the arrow heads indicate representative examples of ectopic Pax7 positive cells co-expressing Pax3. Ectopic patches of Pax3 expression are restricted to the right, electroporated half of the mesencephalic vesicle. (B') is a higher magnification of (B) with the GFP fluorescent image superimposed onto the preparation. (B") shows the distribution of Pax7-GFP expressing cells in the specimen shown in (B'). (C) Quantification of the results: percent specimens with induced (green bars) and unaltered (blue bars) Pax3 expression following targeted electroporation of pMES-Pax7. (D) Flat mount preparation of a HH18 chick neural tube, 24 hours after electroporation of 2 μg/μl pMES-Pax7. (E) Targeted misexpression of pMES-Pax7 into the ventral mesencephalic vesicle showing cells ectopically expressing Pax3 within the Nkx6.1 domain. (E') is a higher magnification of (E). fb: forebrain; fp: floor plate; mes: mesencephalic vesicle; met: metencephalic vesicle; ov: optic vesicle; otv: otic vesicle. Scale bar (B, B'): 100 μm.
Mentions: To investigate a possible relationship between Pax3 and Pax7 in the midbrain, we ectopically delivered an expression plasmid carrying Pax7 together with GFP (pMES-Pax7) by in ovo electroporation into the right lateral wall of the mesencephalic vesicle at HH9-HH11 (7-13 somite stage, Figure 2A). Due to the shot-gun nature of this gene delivery method, random patches of GFP expression, indicative of groups of cells forced to express the GFP and Pax7 transgenes, were seen across the right mesencephalic wall (Figure 2A, B"). Consistent with a previous report, we found that in these patches Pax3 expression was strongly induced as early as 16 hours following Pax7 misexpression (HH14, n = 15/19 GFP expressing specimens exhibited strong ectopic Pax3 expression following electroporation of 2 μg/μl pMES-Pax7 (79%); Figure 2B, B', C) [11]. Robust upregulation of Pax3 expression was still visible 24 hours and 36 hours after pMES-Pax7 transfection (HH18 and HH21 respectively, Figure 2D and data not shown). Ectopic Pax3 expression was also observed when Pax7 misexpression was specifically targeted to the ventral midbrain (n = 8/14 GFP positive specimens exhibited ectopic Pax3 expression after targeted Pax7 expression into the ventral mesencephalic vesicle (57%); Figure 2E). Scattered groups of cells expressing elevated levels of Pax3 transcripts were not only located within the territory of the endogenous Pax3 domains, but also reached ventrally towards the Nkx6.1 expression domain (Figure 2B', E'). A comparison of Figure 2B' and Figure 2B" revealed that apparently not all Pax7-GFP positive cells also express Pax3 mRNA, especially not in the ventral midbrain. This may be due to the short incubation time of the embryos after electroporation (HH9-11 to HH14) and may thus reflect incomplete upregulation of Pax3 by Pax7. In addition, gene delivery by in ovo electroporation causes targeted cells to take up varying amounts of DNA. Considering that ectopic induction of Pax3 may need a certain threshold of Pax7 protein (especially in the ventral neural tube, where Sonic hedgehog signaling promotes the induction of ventral cell fates [25,26]), it is possible that the ectopic Pax7 levels may not have reached the threshold necessary for Pax3 induction in all GFP-positive cells. In any case however, ectopic Pax3 expressing cells in the ventral mesencephalon were consistently positive for Pax7.

Bottom Line: Much less, however, is known about the regulatory relationship and functional interaction of molecules that are expressed in the tectal anlage after tectal fate specification has taken place and tectal development has commenced.Here, we provide experimental evidence for reciprocal regulation and subsequent cooperation of the paired-type transcription factors Pax3, Pax7 and the TALE-homeodomain protein Meis2 in the tectal anlage.Physical interaction with Meis2 may then confer tectal specificity to a wide range of otherwise broadly expressed transcriptional regulators, including Otx2, Pax3 and Pax7.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Neurology (Edinger Institute), J, W, Goethe University Medical School, Heinrich Hoffmannstr, 7, 50628 Frankfurt, Germany.

ABSTRACT

Background: During early stages of brain development, secreted molecules, components of intracellular signaling pathways and transcriptional regulators act in positive and negative feed-back or feed-forward loops at the mid-hindbrain boundary. These genetic interactions are of central importance for the specification and subsequent development of the adjacent mid- and hindbrain. Much less, however, is known about the regulatory relationship and functional interaction of molecules that are expressed in the tectal anlage after tectal fate specification has taken place and tectal development has commenced.

Results: Here, we provide experimental evidence for reciprocal regulation and subsequent cooperation of the paired-type transcription factors Pax3, Pax7 and the TALE-homeodomain protein Meis2 in the tectal anlage. Using in ovo electroporation of the mesencephalic vesicle of chick embryos we show that (i) Pax3 and Pax7 mutually regulate each other's expression in the mesencephalic vesicle, (ii) Meis2 acts downstream of Pax3/7 and requires balanced expression levels of both proteins, and (iii) Meis2 physically interacts with Pax3 and Pax7. These results extend our previous observation that Meis2 cooperates with Otx2 in tectal development to include Pax3 and Pax7 as Meis2 interacting proteins in the tectal anlage.

Conclusion: The results described here suggest a model in which interdependent regulatory loops involving Pax3 and Pax7 in the dorsal mesencephalic vesicle modulate Meis2 expression. Physical interaction with Meis2 may then confer tectal specificity to a wide range of otherwise broadly expressed transcriptional regulators, including Otx2, Pax3 and Pax7.

Show MeSH
Related in: MedlinePlus