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Generation of Atoh1-rtTA transgenic mice: a tool for inducible gene expression in hair cells of the inner ear.

Cox BC, Dearman JA, Brancheck J, Zindy F, Roussel MF, Zuo J - Sci Rep (2014)

Bottom Line: Presence of the transgene was confirmed by alkaline phosphatase staining and rtTA activity was measured using two tetracycline operator (TetO) reporter alleles with doxycycline administered between postnatal days 0-3.Although the tetracycline-inducible system is thought to produce transient changes in gene expression, reporter positive HCs were still observed at 6 weeks of age.To confirm that Atoh1-rtTA activity was specific to Atoh1-expressing cells, we also analyzed the cerebellum and found rtTA-driven reporter expression in cerebellar granule neuron precursor cells.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pharmacology, Southern Illinois University, School of Medicine, Springfield, IL 62702 [2] Department of Surgery, Division of Otolaryngology, Southern Illinois University, School of Medicine, Springfield, IL 62702 [3] Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN 38105.

ABSTRACT
Atoh1 is a basic helix-loop-helix transcription factor that controls differentiation of hair cells (HCs) in the inner ear and its enhancer region has been used to create several HC-specific mouse lines. We generated a transgenic tetracycline-inducible mouse line (called Atoh1-rtTA) using the Atoh1 enhancer to drive expression of the reverse tetracycline transactivator (rtTA) protein and human placental alkaline phosphatase. Presence of the transgene was confirmed by alkaline phosphatase staining and rtTA activity was measured using two tetracycline operator (TetO) reporter alleles with doxycycline administered between postnatal days 0-3. This characterization of five founder lines demonstrated that Atoh1-rtTA is expressed in the majority of cochlear and utricular HCs. Although the tetracycline-inducible system is thought to produce transient changes in gene expression, reporter positive HCs were still observed at 6 weeks of age. To confirm that Atoh1-rtTA activity was specific to Atoh1-expressing cells, we also analyzed the cerebellum and found rtTA-driven reporter expression in cerebellar granule neuron precursor cells. The Atoh1-rtTA mouse line provides a powerful tool for the field and can be used in combination with other existing Cre recombinase mouse lines to manipulate expression of multiple genes at different times in the same animal.

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Atoh1-rtTA activity in the utricle.(A–E) Representative confocal images of mCherry+ve (red) cells and Myo7a-labeled HCs (green) in the utricle for each founder line breed with the TetO-mCherry reporter at P3. (F–J) Representative confocal images of LacZ+ve (red) cells and Myo7a-labeled HCs (green) in the utricle for each founder line breed with the TetO-LacZ reporter at P3. (K) Percentage of Myo7a+ve cells that express mCherry in utricular regions of each founder line, normalized to the number of total Myo7a+ve cells in the same region. (L) Percentage of Myo7a+ve cells that express LacZ in utricular regions of each founder line, normalized to the number of total Myo7a+ve cells in the same region. Data are expressed as mean ± S.E.M for an n of 3. Statistical differences between regions were obtained using a two-way ANOVA followed by a Tukey's posthoc test. *p < 0.05, **p < 0.01, ***p < 0.001. M, medial; S, striolar; L, lateral Scale bar: 100 μm.
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f4: Atoh1-rtTA activity in the utricle.(A–E) Representative confocal images of mCherry+ve (red) cells and Myo7a-labeled HCs (green) in the utricle for each founder line breed with the TetO-mCherry reporter at P3. (F–J) Representative confocal images of LacZ+ve (red) cells and Myo7a-labeled HCs (green) in the utricle for each founder line breed with the TetO-LacZ reporter at P3. (K) Percentage of Myo7a+ve cells that express mCherry in utricular regions of each founder line, normalized to the number of total Myo7a+ve cells in the same region. (L) Percentage of Myo7a+ve cells that express LacZ in utricular regions of each founder line, normalized to the number of total Myo7a+ve cells in the same region. Data are expressed as mean ± S.E.M for an n of 3. Statistical differences between regions were obtained using a two-way ANOVA followed by a Tukey's posthoc test. *p < 0.05, **p < 0.01, ***p < 0.001. M, medial; S, striolar; L, lateral Scale bar: 100 μm.

Mentions: In Atoh1-rtTA; TetO-mCherry utricles, the percentage of total mCherry+ve HCs were: F7 = 78.2 ± 0.6%, F10 = 85.6 ± 0.5%, F12 = 73.7 ± 4.1%, F23 = 48.6 ± 6.4%, and F26 = 79.4 ± 3.6% (mCherry+ve HCs expressed as a percentage of total HCs, n = 3, Figure 4A–E, K). Several of the Atoh1-rtTA; TetO-mCherry founders showed a significant difference in the percentage of mCherry+ve HCs in different regions of the utricle (Figure 4K). This included a difference between striolar and lateral regions in F7, F10, and F12 and a difference between medial and striolar regions in F10. All founders had varying numbers of mCherry+ve SCs that were found across all regions of the utricle with F7 and F10 having the fewest and F12 having the most. Lastly, the mCherry intensity varied from cell to cell across all founders (Figure 4A–E). We also observed mCherry+ve HCs in the lateral and superior cristae.


Generation of Atoh1-rtTA transgenic mice: a tool for inducible gene expression in hair cells of the inner ear.

Cox BC, Dearman JA, Brancheck J, Zindy F, Roussel MF, Zuo J - Sci Rep (2014)

Atoh1-rtTA activity in the utricle.(A–E) Representative confocal images of mCherry+ve (red) cells and Myo7a-labeled HCs (green) in the utricle for each founder line breed with the TetO-mCherry reporter at P3. (F–J) Representative confocal images of LacZ+ve (red) cells and Myo7a-labeled HCs (green) in the utricle for each founder line breed with the TetO-LacZ reporter at P3. (K) Percentage of Myo7a+ve cells that express mCherry in utricular regions of each founder line, normalized to the number of total Myo7a+ve cells in the same region. (L) Percentage of Myo7a+ve cells that express LacZ in utricular regions of each founder line, normalized to the number of total Myo7a+ve cells in the same region. Data are expressed as mean ± S.E.M for an n of 3. Statistical differences between regions were obtained using a two-way ANOVA followed by a Tukey's posthoc test. *p < 0.05, **p < 0.01, ***p < 0.001. M, medial; S, striolar; L, lateral Scale bar: 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4217099&req=5

f4: Atoh1-rtTA activity in the utricle.(A–E) Representative confocal images of mCherry+ve (red) cells and Myo7a-labeled HCs (green) in the utricle for each founder line breed with the TetO-mCherry reporter at P3. (F–J) Representative confocal images of LacZ+ve (red) cells and Myo7a-labeled HCs (green) in the utricle for each founder line breed with the TetO-LacZ reporter at P3. (K) Percentage of Myo7a+ve cells that express mCherry in utricular regions of each founder line, normalized to the number of total Myo7a+ve cells in the same region. (L) Percentage of Myo7a+ve cells that express LacZ in utricular regions of each founder line, normalized to the number of total Myo7a+ve cells in the same region. Data are expressed as mean ± S.E.M for an n of 3. Statistical differences between regions were obtained using a two-way ANOVA followed by a Tukey's posthoc test. *p < 0.05, **p < 0.01, ***p < 0.001. M, medial; S, striolar; L, lateral Scale bar: 100 μm.
Mentions: In Atoh1-rtTA; TetO-mCherry utricles, the percentage of total mCherry+ve HCs were: F7 = 78.2 ± 0.6%, F10 = 85.6 ± 0.5%, F12 = 73.7 ± 4.1%, F23 = 48.6 ± 6.4%, and F26 = 79.4 ± 3.6% (mCherry+ve HCs expressed as a percentage of total HCs, n = 3, Figure 4A–E, K). Several of the Atoh1-rtTA; TetO-mCherry founders showed a significant difference in the percentage of mCherry+ve HCs in different regions of the utricle (Figure 4K). This included a difference between striolar and lateral regions in F7, F10, and F12 and a difference between medial and striolar regions in F10. All founders had varying numbers of mCherry+ve SCs that were found across all regions of the utricle with F7 and F10 having the fewest and F12 having the most. Lastly, the mCherry intensity varied from cell to cell across all founders (Figure 4A–E). We also observed mCherry+ve HCs in the lateral and superior cristae.

Bottom Line: Presence of the transgene was confirmed by alkaline phosphatase staining and rtTA activity was measured using two tetracycline operator (TetO) reporter alleles with doxycycline administered between postnatal days 0-3.Although the tetracycline-inducible system is thought to produce transient changes in gene expression, reporter positive HCs were still observed at 6 weeks of age.To confirm that Atoh1-rtTA activity was specific to Atoh1-expressing cells, we also analyzed the cerebellum and found rtTA-driven reporter expression in cerebellar granule neuron precursor cells.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pharmacology, Southern Illinois University, School of Medicine, Springfield, IL 62702 [2] Department of Surgery, Division of Otolaryngology, Southern Illinois University, School of Medicine, Springfield, IL 62702 [3] Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN 38105.

ABSTRACT
Atoh1 is a basic helix-loop-helix transcription factor that controls differentiation of hair cells (HCs) in the inner ear and its enhancer region has been used to create several HC-specific mouse lines. We generated a transgenic tetracycline-inducible mouse line (called Atoh1-rtTA) using the Atoh1 enhancer to drive expression of the reverse tetracycline transactivator (rtTA) protein and human placental alkaline phosphatase. Presence of the transgene was confirmed by alkaline phosphatase staining and rtTA activity was measured using two tetracycline operator (TetO) reporter alleles with doxycycline administered between postnatal days 0-3. This characterization of five founder lines demonstrated that Atoh1-rtTA is expressed in the majority of cochlear and utricular HCs. Although the tetracycline-inducible system is thought to produce transient changes in gene expression, reporter positive HCs were still observed at 6 weeks of age. To confirm that Atoh1-rtTA activity was specific to Atoh1-expressing cells, we also analyzed the cerebellum and found rtTA-driven reporter expression in cerebellar granule neuron precursor cells. The Atoh1-rtTA mouse line provides a powerful tool for the field and can be used in combination with other existing Cre recombinase mouse lines to manipulate expression of multiple genes at different times in the same animal.

Show MeSH
Related in: MedlinePlus