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Embryonic frog epidermis: a model for the study of cell-cell interactions in the development of mucociliary disease.

Dubaissi E, Papalopulu N - Dis Model Mech (2010)

Bottom Line: These cells express high levels of ion channels and transporters; therefore, we suggest that they are analogous to ionocytes found in transporting epithelia such as the mammalian kidney.Depletion of ionocytes by foxi1e knockdown has detrimental effects on the development of multiciliated cells, which show fewer and aberrantly beating cilia.These results reveal a newly identified role for ionocytes and suggest that the frog embryonic skin is a model system that is particularly suited to studying the interactions of different cell types in mucociliary, as well as in secretory and transporting, epithelia.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life Sciences, University of Manchester, Manchester, UK.

ABSTRACT
Specialised epithelia such as mucociliary, secretory and transporting epithelia line all major organs, including the lung, gut and kidney. Malfunction of these epithelia is associated with many human diseases. The frog embryonic epidermis possesses mucus-secreting and multiciliated cells, and has served as an excellent model system for the biogenesis of cilia. However, ionic regulation is important for the function of all specialised epithelia and it is not clear how this is achieved in the embryonic frog epidermis. Here, we show that a third cell type develops alongside ciliated and mucus-secreting cells in the tadpole skin. These cells express high levels of ion channels and transporters; therefore, we suggest that they are analogous to ionocytes found in transporting epithelia such as the mammalian kidney. We show that frog ionocytes express the transcription factor foxi1e, which is required for the development of these cells. Depletion of ionocytes by foxi1e knockdown has detrimental effects on the development of multiciliated cells, which show fewer and aberrantly beating cilia. These results reveal a newly identified role for ionocytes and suggest that the frog embryonic skin is a model system that is particularly suited to studying the interactions of different cell types in mucociliary, as well as in secretory and transporting, epithelia.

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Impact of foxi1e knockdown on the mucociliary epidermis. (A) Diagrammatic representation of foxi1e, showing the position of the ATG MO (red) and sp MO (blue). E1, exon 1; E2, exon 2; I, intron. (B) Reverse transcriptase (RT)-PCR with ornithine decarboxylase (odc) control and foxi1e primers, showing that abnormal splicing of foxi1e is observed when a foxi1e sp MO is injected. Asterisk marks reduction in foxi1e splice product in foxi1e splice morphants compared with control. (C) Injection of foxi1e sp MO reduces (11/11) and injection of foxi1e ATG MO abolishes (11/11) the expression of v1a, whereas MOC has no effect (0/9) at early tadpole stage as shown by whole-mount in situ hybridisation. Neither foxi1e sp MO (0/13), foxi1e ATG MO (0/11) or MOC (0/10) had an effect on expression of the ciliated cell marker α-1-tubulin. Scale bars: 500 μm. (D) Embryos were injected with MOC or foxi1e ATG MO (20 ng each) and analysed as indicated. In situ hybridisation with foxi1e probe shows that the spotted expression of foxi1e is reduced (9/9) in foxi1e morphants, whereas MOC has no effect (0/11). Scale bars: 500 μm. Immunostaining of tadpole epidermis with anti-foxi1e antibody shows that the protein expression is reduced in foxi1e morphants (11/12) but not in the MOC controls (0/10). In situ hybridisation with α-1-tubulin probe (black), combined with anti-xeel antibody staining (red), as indicated, reveals that, in the foxi1e morphants, the number of cells that are not positive for either α-1-tubulin or xeel is greatly reduced compared with controls. This suggests that the missing cells are ionocytes. Actin staining (phalloidin–Alexa-Fluor-488; green) combined with anti-acetylated α-tubulin antibody (red) reveals that epidermal cells are arranged in a rosette pattern around each ciliated cell in the foxi1e-ATG-MO-injected epidermis, but in the MOC control (stained with anti-ZO-1 and acetylated α-tubulin), this rosette formation is broken by the insertion of ionocytes (inset shows higher-magnification view). See text for details. Scale bars: 50 μm. (E) Immunostaining for v1a (green), ca12 (green) and acetylated α-tubulin (red) in the combinations indicated confirms that ionocyte markers are missing in foxi1e-ATG-MO-injected embryos (34/35; n=5 experiments) but not in MOC controls (0/35; n=5 experiments). Scale bars: 50 μm. High-magnification images (zoom; scale bar: 20 μm) reveal that ciliated cells are abnormal in the foxi1e morphants. Bar graph shows quantification of the experiments looking at defective ciliated cells (n=4 experiments). Defective ciliated cells were evident in the majority of foxi1e ATG morphant embryos (42/47), but not in MOC-treated embryos (2/32). **P=0.0018.
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f6-0040179: Impact of foxi1e knockdown on the mucociliary epidermis. (A) Diagrammatic representation of foxi1e, showing the position of the ATG MO (red) and sp MO (blue). E1, exon 1; E2, exon 2; I, intron. (B) Reverse transcriptase (RT)-PCR with ornithine decarboxylase (odc) control and foxi1e primers, showing that abnormal splicing of foxi1e is observed when a foxi1e sp MO is injected. Asterisk marks reduction in foxi1e splice product in foxi1e splice morphants compared with control. (C) Injection of foxi1e sp MO reduces (11/11) and injection of foxi1e ATG MO abolishes (11/11) the expression of v1a, whereas MOC has no effect (0/9) at early tadpole stage as shown by whole-mount in situ hybridisation. Neither foxi1e sp MO (0/13), foxi1e ATG MO (0/11) or MOC (0/10) had an effect on expression of the ciliated cell marker α-1-tubulin. Scale bars: 500 μm. (D) Embryos were injected with MOC or foxi1e ATG MO (20 ng each) and analysed as indicated. In situ hybridisation with foxi1e probe shows that the spotted expression of foxi1e is reduced (9/9) in foxi1e morphants, whereas MOC has no effect (0/11). Scale bars: 500 μm. Immunostaining of tadpole epidermis with anti-foxi1e antibody shows that the protein expression is reduced in foxi1e morphants (11/12) but not in the MOC controls (0/10). In situ hybridisation with α-1-tubulin probe (black), combined with anti-xeel antibody staining (red), as indicated, reveals that, in the foxi1e morphants, the number of cells that are not positive for either α-1-tubulin or xeel is greatly reduced compared with controls. This suggests that the missing cells are ionocytes. Actin staining (phalloidin–Alexa-Fluor-488; green) combined with anti-acetylated α-tubulin antibody (red) reveals that epidermal cells are arranged in a rosette pattern around each ciliated cell in the foxi1e-ATG-MO-injected epidermis, but in the MOC control (stained with anti-ZO-1 and acetylated α-tubulin), this rosette formation is broken by the insertion of ionocytes (inset shows higher-magnification view). See text for details. Scale bars: 50 μm. (E) Immunostaining for v1a (green), ca12 (green) and acetylated α-tubulin (red) in the combinations indicated confirms that ionocyte markers are missing in foxi1e-ATG-MO-injected embryos (34/35; n=5 experiments) but not in MOC controls (0/35; n=5 experiments). Scale bars: 50 μm. High-magnification images (zoom; scale bar: 20 μm) reveal that ciliated cells are abnormal in the foxi1e morphants. Bar graph shows quantification of the experiments looking at defective ciliated cells (n=4 experiments). Defective ciliated cells were evident in the majority of foxi1e ATG morphant embryos (42/47), but not in MOC-treated embryos (2/32). **P=0.0018.

Mentions: foxi1e was knocked down using either a translation-blocking morpholino (ATG MO) that covered the ATG start site or a splice MO (sp MO) that covered the splice acceptor site (Fig. 6A). The efficiency of the splice MO was confirmed by the aberrant pattern of splicing (Fig. 6B). Ornithine decarbocylase (odc) is a ubiquitously expressed gene that was used as a loading control to show that loss of foxi1e was due to the presence of the splice morpholino rather than a difference in the amount of starting material. The efficiency of the ATG MO was shown by both the reduction of foxi1e-haemagglutinin (HA) protein level in the embryo (supplementary material Fig. S2) and reduced immunostaining for foxi1e (Fig. 6D). Both MOs gave the same phenotypic results, confirming the specificity of the phenotype.


Embryonic frog epidermis: a model for the study of cell-cell interactions in the development of mucociliary disease.

Dubaissi E, Papalopulu N - Dis Model Mech (2010)

Impact of foxi1e knockdown on the mucociliary epidermis. (A) Diagrammatic representation of foxi1e, showing the position of the ATG MO (red) and sp MO (blue). E1, exon 1; E2, exon 2; I, intron. (B) Reverse transcriptase (RT)-PCR with ornithine decarboxylase (odc) control and foxi1e primers, showing that abnormal splicing of foxi1e is observed when a foxi1e sp MO is injected. Asterisk marks reduction in foxi1e splice product in foxi1e splice morphants compared with control. (C) Injection of foxi1e sp MO reduces (11/11) and injection of foxi1e ATG MO abolishes (11/11) the expression of v1a, whereas MOC has no effect (0/9) at early tadpole stage as shown by whole-mount in situ hybridisation. Neither foxi1e sp MO (0/13), foxi1e ATG MO (0/11) or MOC (0/10) had an effect on expression of the ciliated cell marker α-1-tubulin. Scale bars: 500 μm. (D) Embryos were injected with MOC or foxi1e ATG MO (20 ng each) and analysed as indicated. In situ hybridisation with foxi1e probe shows that the spotted expression of foxi1e is reduced (9/9) in foxi1e morphants, whereas MOC has no effect (0/11). Scale bars: 500 μm. Immunostaining of tadpole epidermis with anti-foxi1e antibody shows that the protein expression is reduced in foxi1e morphants (11/12) but not in the MOC controls (0/10). In situ hybridisation with α-1-tubulin probe (black), combined with anti-xeel antibody staining (red), as indicated, reveals that, in the foxi1e morphants, the number of cells that are not positive for either α-1-tubulin or xeel is greatly reduced compared with controls. This suggests that the missing cells are ionocytes. Actin staining (phalloidin–Alexa-Fluor-488; green) combined with anti-acetylated α-tubulin antibody (red) reveals that epidermal cells are arranged in a rosette pattern around each ciliated cell in the foxi1e-ATG-MO-injected epidermis, but in the MOC control (stained with anti-ZO-1 and acetylated α-tubulin), this rosette formation is broken by the insertion of ionocytes (inset shows higher-magnification view). See text for details. Scale bars: 50 μm. (E) Immunostaining for v1a (green), ca12 (green) and acetylated α-tubulin (red) in the combinations indicated confirms that ionocyte markers are missing in foxi1e-ATG-MO-injected embryos (34/35; n=5 experiments) but not in MOC controls (0/35; n=5 experiments). Scale bars: 50 μm. High-magnification images (zoom; scale bar: 20 μm) reveal that ciliated cells are abnormal in the foxi1e morphants. Bar graph shows quantification of the experiments looking at defective ciliated cells (n=4 experiments). Defective ciliated cells were evident in the majority of foxi1e ATG morphant embryos (42/47), but not in MOC-treated embryos (2/32). **P=0.0018.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3046089&req=5

f6-0040179: Impact of foxi1e knockdown on the mucociliary epidermis. (A) Diagrammatic representation of foxi1e, showing the position of the ATG MO (red) and sp MO (blue). E1, exon 1; E2, exon 2; I, intron. (B) Reverse transcriptase (RT)-PCR with ornithine decarboxylase (odc) control and foxi1e primers, showing that abnormal splicing of foxi1e is observed when a foxi1e sp MO is injected. Asterisk marks reduction in foxi1e splice product in foxi1e splice morphants compared with control. (C) Injection of foxi1e sp MO reduces (11/11) and injection of foxi1e ATG MO abolishes (11/11) the expression of v1a, whereas MOC has no effect (0/9) at early tadpole stage as shown by whole-mount in situ hybridisation. Neither foxi1e sp MO (0/13), foxi1e ATG MO (0/11) or MOC (0/10) had an effect on expression of the ciliated cell marker α-1-tubulin. Scale bars: 500 μm. (D) Embryos were injected with MOC or foxi1e ATG MO (20 ng each) and analysed as indicated. In situ hybridisation with foxi1e probe shows that the spotted expression of foxi1e is reduced (9/9) in foxi1e morphants, whereas MOC has no effect (0/11). Scale bars: 500 μm. Immunostaining of tadpole epidermis with anti-foxi1e antibody shows that the protein expression is reduced in foxi1e morphants (11/12) but not in the MOC controls (0/10). In situ hybridisation with α-1-tubulin probe (black), combined with anti-xeel antibody staining (red), as indicated, reveals that, in the foxi1e morphants, the number of cells that are not positive for either α-1-tubulin or xeel is greatly reduced compared with controls. This suggests that the missing cells are ionocytes. Actin staining (phalloidin–Alexa-Fluor-488; green) combined with anti-acetylated α-tubulin antibody (red) reveals that epidermal cells are arranged in a rosette pattern around each ciliated cell in the foxi1e-ATG-MO-injected epidermis, but in the MOC control (stained with anti-ZO-1 and acetylated α-tubulin), this rosette formation is broken by the insertion of ionocytes (inset shows higher-magnification view). See text for details. Scale bars: 50 μm. (E) Immunostaining for v1a (green), ca12 (green) and acetylated α-tubulin (red) in the combinations indicated confirms that ionocyte markers are missing in foxi1e-ATG-MO-injected embryos (34/35; n=5 experiments) but not in MOC controls (0/35; n=5 experiments). Scale bars: 50 μm. High-magnification images (zoom; scale bar: 20 μm) reveal that ciliated cells are abnormal in the foxi1e morphants. Bar graph shows quantification of the experiments looking at defective ciliated cells (n=4 experiments). Defective ciliated cells were evident in the majority of foxi1e ATG morphant embryos (42/47), but not in MOC-treated embryos (2/32). **P=0.0018.
Mentions: foxi1e was knocked down using either a translation-blocking morpholino (ATG MO) that covered the ATG start site or a splice MO (sp MO) that covered the splice acceptor site (Fig. 6A). The efficiency of the splice MO was confirmed by the aberrant pattern of splicing (Fig. 6B). Ornithine decarbocylase (odc) is a ubiquitously expressed gene that was used as a loading control to show that loss of foxi1e was due to the presence of the splice morpholino rather than a difference in the amount of starting material. The efficiency of the ATG MO was shown by both the reduction of foxi1e-haemagglutinin (HA) protein level in the embryo (supplementary material Fig. S2) and reduced immunostaining for foxi1e (Fig. 6D). Both MOs gave the same phenotypic results, confirming the specificity of the phenotype.

Bottom Line: These cells express high levels of ion channels and transporters; therefore, we suggest that they are analogous to ionocytes found in transporting epithelia such as the mammalian kidney.Depletion of ionocytes by foxi1e knockdown has detrimental effects on the development of multiciliated cells, which show fewer and aberrantly beating cilia.These results reveal a newly identified role for ionocytes and suggest that the frog embryonic skin is a model system that is particularly suited to studying the interactions of different cell types in mucociliary, as well as in secretory and transporting, epithelia.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life Sciences, University of Manchester, Manchester, UK.

ABSTRACT
Specialised epithelia such as mucociliary, secretory and transporting epithelia line all major organs, including the lung, gut and kidney. Malfunction of these epithelia is associated with many human diseases. The frog embryonic epidermis possesses mucus-secreting and multiciliated cells, and has served as an excellent model system for the biogenesis of cilia. However, ionic regulation is important for the function of all specialised epithelia and it is not clear how this is achieved in the embryonic frog epidermis. Here, we show that a third cell type develops alongside ciliated and mucus-secreting cells in the tadpole skin. These cells express high levels of ion channels and transporters; therefore, we suggest that they are analogous to ionocytes found in transporting epithelia such as the mammalian kidney. We show that frog ionocytes express the transcription factor foxi1e, which is required for the development of these cells. Depletion of ionocytes by foxi1e knockdown has detrimental effects on the development of multiciliated cells, which show fewer and aberrantly beating cilia. These results reveal a newly identified role for ionocytes and suggest that the frog embryonic skin is a model system that is particularly suited to studying the interactions of different cell types in mucociliary, as well as in secretory and transporting, epithelia.

Show MeSH