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Isotocin controls ion regulation through regulating ionocyte progenitor differentiation and proliferation.

Chou MY, Hung JC, Wu LC, Hwang SP, Hwang PP - Cell. Mol. Life Sci. (2010)

Bottom Line: Double-deionized water treatment for 24 h significantly stimulated isotocin mRNA expression in zebrafish embryos.Whole-body Cl(-), Ca(2+), and Na(+) contents, mRNA expressions of ion transporters and ionocyte-differentiation related transcription factors, and the number of skin ionocytes decreased in isotocin morphants.Taken together, isotocin stimulates the proliferation of epidermal stem cells and differentiation of ionocyte progenitors by regulating the P63 and Foxi3a transcription factors, consequently enhancing the functional activities of ionocytes.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cellular and Organismic Biology, Academia Sinica, Nankang, Taipei, Taiwan.

ABSTRACT
The present study using zebrafish as a model explores the role of isotocin, a homolog of oxytocin, in controlling ion regulatory mechanisms. Double-deionized water treatment for 24 h significantly stimulated isotocin mRNA expression in zebrafish embryos. Whole-body Cl(-), Ca(2+), and Na(+) contents, mRNA expressions of ion transporters and ionocyte-differentiation related transcription factors, and the number of skin ionocytes decreased in isotocin morphants. In contrast, overexpression of isotocin caused an increase in ionocyte numbers. Isotocin morpholino caused significant suppression of foxi3a mRNA expression, while isotocin cRNA stimulated foxi3a mRNA expressions at the tail-bud stage of zebrafish embryos. The density of P63 (an epidermal stem cell marker)-positive cells was downregulated by isotocin morpholinos and was upregulated by isotocin cRNA. Taken together, isotocin stimulates the proliferation of epidermal stem cells and differentiation of ionocyte progenitors by regulating the P63 and Foxi3a transcription factors, consequently enhancing the functional activities of ionocytes.

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Effects of itnp MO on ionocyte-related gene expressions in zebrafish embryos. One to two-cell stage embryos were injected with itnp MO (1 ng/embryo) and a mismatched-MO (Mis MO), respectively, and the mRNA expressions at 72 hpf were analyzed by qRT-PCR. a The mRNA expressions of ionocyte-related genes were significantly downregulated by an itnp MO injection. b In situ hybridization analysis indicated that the mRNA signals of V-ATPase subunit A (atp6v1a), epithelial Ca2+ channel (trpv6), and Na+-Cl− cotransporter (slc12a10.2) were lower in itnp morphants. c Cell densities of atp6v1a-, trpv6-, and slc12a10.2-expressing cells in itnp morphants were significantly lower than those in mismatched-MO-injected embryos. qRT-PCR values were normalized to β-actin. Mean ± SD (n = 6). Asterisks indicates a significant difference from the Mis MO group (Student’s t test, p < 0.05). Scale bar 200 μm
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Fig3: Effects of itnp MO on ionocyte-related gene expressions in zebrafish embryos. One to two-cell stage embryos were injected with itnp MO (1 ng/embryo) and a mismatched-MO (Mis MO), respectively, and the mRNA expressions at 72 hpf were analyzed by qRT-PCR. a The mRNA expressions of ionocyte-related genes were significantly downregulated by an itnp MO injection. b In situ hybridization analysis indicated that the mRNA signals of V-ATPase subunit A (atp6v1a), epithelial Ca2+ channel (trpv6), and Na+-Cl− cotransporter (slc12a10.2) were lower in itnp morphants. c Cell densities of atp6v1a-, trpv6-, and slc12a10.2-expressing cells in itnp morphants were significantly lower than those in mismatched-MO-injected embryos. qRT-PCR values were normalized to β-actin. Mean ± SD (n = 6). Asterisks indicates a significant difference from the Mis MO group (Student’s t test, p < 0.05). Scale bar 200 μm

Mentions: As described above, isotocin appears to be involved in zebrafish ion regulation mechanism. Subsequent loss-of-function experiments were designed to test a hypothesis if isotocin is involved in the development of zebrafish skin/gill ionocytes, which are major cells responsible for ion regulation mechanisms. The mRNA expressions of ionocyte-related genes were downregulated by the itnp MO. As shown in Fig. 3a, mRNA expressions of atp6v1a, atp1b1b, trpv6, slc12a10.2, and nhe3b were reduced in itnp morphants. In whole-mount in situ hybridization experiments, RNA signals of atp6v1a, trpv6, and slc12a10.2 were also decreased by itnp MO knockdown consistent with the qRT-PCR data (Fig. 3b, c). In the whole-mount immunocytochemistry experiments, NaRCs and HRCs were recognized. The cell densities of NaRC and HRC were reduced in itnp morphants compared with those in Mis MO-injected embryos (Fig. 4a–c). In contrast, increased cell densities of NaRC and HRC were detected in itnp-overexpressed embryos compared with those in the control injected with 1× Danieau solution (Fig. 4d–f).Fig. 3


Isotocin controls ion regulation through regulating ionocyte progenitor differentiation and proliferation.

Chou MY, Hung JC, Wu LC, Hwang SP, Hwang PP - Cell. Mol. Life Sci. (2010)

Effects of itnp MO on ionocyte-related gene expressions in zebrafish embryos. One to two-cell stage embryos were injected with itnp MO (1 ng/embryo) and a mismatched-MO (Mis MO), respectively, and the mRNA expressions at 72 hpf were analyzed by qRT-PCR. a The mRNA expressions of ionocyte-related genes were significantly downregulated by an itnp MO injection. b In situ hybridization analysis indicated that the mRNA signals of V-ATPase subunit A (atp6v1a), epithelial Ca2+ channel (trpv6), and Na+-Cl− cotransporter (slc12a10.2) were lower in itnp morphants. c Cell densities of atp6v1a-, trpv6-, and slc12a10.2-expressing cells in itnp morphants were significantly lower than those in mismatched-MO-injected embryos. qRT-PCR values were normalized to β-actin. Mean ± SD (n = 6). Asterisks indicates a significant difference from the Mis MO group (Student’s t test, p < 0.05). Scale bar 200 μm
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3142547&req=5

Fig3: Effects of itnp MO on ionocyte-related gene expressions in zebrafish embryos. One to two-cell stage embryos were injected with itnp MO (1 ng/embryo) and a mismatched-MO (Mis MO), respectively, and the mRNA expressions at 72 hpf were analyzed by qRT-PCR. a The mRNA expressions of ionocyte-related genes were significantly downregulated by an itnp MO injection. b In situ hybridization analysis indicated that the mRNA signals of V-ATPase subunit A (atp6v1a), epithelial Ca2+ channel (trpv6), and Na+-Cl− cotransporter (slc12a10.2) were lower in itnp morphants. c Cell densities of atp6v1a-, trpv6-, and slc12a10.2-expressing cells in itnp morphants were significantly lower than those in mismatched-MO-injected embryos. qRT-PCR values were normalized to β-actin. Mean ± SD (n = 6). Asterisks indicates a significant difference from the Mis MO group (Student’s t test, p < 0.05). Scale bar 200 μm
Mentions: As described above, isotocin appears to be involved in zebrafish ion regulation mechanism. Subsequent loss-of-function experiments were designed to test a hypothesis if isotocin is involved in the development of zebrafish skin/gill ionocytes, which are major cells responsible for ion regulation mechanisms. The mRNA expressions of ionocyte-related genes were downregulated by the itnp MO. As shown in Fig. 3a, mRNA expressions of atp6v1a, atp1b1b, trpv6, slc12a10.2, and nhe3b were reduced in itnp morphants. In whole-mount in situ hybridization experiments, RNA signals of atp6v1a, trpv6, and slc12a10.2 were also decreased by itnp MO knockdown consistent with the qRT-PCR data (Fig. 3b, c). In the whole-mount immunocytochemistry experiments, NaRCs and HRCs were recognized. The cell densities of NaRC and HRC were reduced in itnp morphants compared with those in Mis MO-injected embryos (Fig. 4a–c). In contrast, increased cell densities of NaRC and HRC were detected in itnp-overexpressed embryos compared with those in the control injected with 1× Danieau solution (Fig. 4d–f).Fig. 3

Bottom Line: Double-deionized water treatment for 24 h significantly stimulated isotocin mRNA expression in zebrafish embryos.Whole-body Cl(-), Ca(2+), and Na(+) contents, mRNA expressions of ion transporters and ionocyte-differentiation related transcription factors, and the number of skin ionocytes decreased in isotocin morphants.Taken together, isotocin stimulates the proliferation of epidermal stem cells and differentiation of ionocyte progenitors by regulating the P63 and Foxi3a transcription factors, consequently enhancing the functional activities of ionocytes.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cellular and Organismic Biology, Academia Sinica, Nankang, Taipei, Taiwan.

ABSTRACT
The present study using zebrafish as a model explores the role of isotocin, a homolog of oxytocin, in controlling ion regulatory mechanisms. Double-deionized water treatment for 24 h significantly stimulated isotocin mRNA expression in zebrafish embryos. Whole-body Cl(-), Ca(2+), and Na(+) contents, mRNA expressions of ion transporters and ionocyte-differentiation related transcription factors, and the number of skin ionocytes decreased in isotocin morphants. In contrast, overexpression of isotocin caused an increase in ionocyte numbers. Isotocin morpholino caused significant suppression of foxi3a mRNA expression, while isotocin cRNA stimulated foxi3a mRNA expressions at the tail-bud stage of zebrafish embryos. The density of P63 (an epidermal stem cell marker)-positive cells was downregulated by isotocin morpholinos and was upregulated by isotocin cRNA. Taken together, isotocin stimulates the proliferation of epidermal stem cells and differentiation of ionocyte progenitors by regulating the P63 and Foxi3a transcription factors, consequently enhancing the functional activities of ionocytes.

Show MeSH
Related in: MedlinePlus