Limits...
A Phox2b BAC Transgenic Rat Line Useful for Understanding Respiratory Rhythm Generator Neural Circuitry.

Ikeda K, Takahashi M, Sato S, Igarashi H, Ishizuka T, Yawo H, Arata S, Southard-Smith EM, Kawakami K, Onimaru H - PLoS ONE (2015)

Bottom Line: Here we describe the generation of a novel transgenic (Tg) rat harboring fluorescently labeled Pre-I neurons in the RTN/pFRG.In addition, the Tg rat showed fluorescent signals in autonomic enteric neurons and carotid bodies.Because the Tg rat expresses inducible Cre recombinase in PHOX2B-positive cells during development, it is a potentially powerful tool for dissecting the entire picture of the respiratory neural network during development and for identifying the CO2/O2 sensor molecules in the adult central and peripheral nervous systems.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan; Division of Biology, Center for Molecular Medicine, Jichi Medical University, Shimotsuke, Tochigi, Japan.

ABSTRACT
The key role of the respiratory neural center is respiratory rhythm generation to maintain homeostasis through the control of arterial blood pCO2/pH and pO2 levels. The neuronal network responsible for respiratory rhythm generation in neonatal rat resides in the ventral side of the medulla and is composed of two groups; the parafacial respiratory group (pFRG) and the pre-Bötzinger complex group (preBötC). The pFRG partially overlaps in the retrotrapezoid nucleus (RTN), which was originally identified in adult cats and rats. Part of the pre-inspiratory (Pre-I) neurons in the RTN/pFRG serves as central chemoreceptor neurons and the CO2 sensitive Pre-I neurons express homeobox gene Phox2b. Phox2b encodes a transcription factor and is essential for the development of the sensory-motor visceral circuits. Mutations in human PHOX2B cause congenital hypoventilation syndrome, which is characterized by blunted ventilatory response to hypercapnia. Here we describe the generation of a novel transgenic (Tg) rat harboring fluorescently labeled Pre-I neurons in the RTN/pFRG. In addition, the Tg rat showed fluorescent signals in autonomic enteric neurons and carotid bodies. Because the Tg rat expresses inducible Cre recombinase in PHOX2B-positive cells during development, it is a potentially powerful tool for dissecting the entire picture of the respiratory neural network during development and for identifying the CO2/O2 sensor molecules in the adult central and peripheral nervous systems.

No MeSH data available.


Related in: MedlinePlus

Analysis of CreERT2 recombinase activity.A-D, Cre recombinase activity induced by estrogen analog tamoxifen (CreERT2, estrogen receptor T2, [38]) was examined by fluorescence in fetus harboring both the Phox2b-EYFP-2A-CreERT2 transgene and the ROSA26-tdTomato transgene born from pregnant rat treated with tamoxifen. The tdTomato red signals are detected in the facial nucleus (nVII). E-H, No red fluorescence is present in fetus harboring both the Phox2b-EYFP-2A-CreERT2 transgene and the ROSA26-tdTomato transgene born from pregnant rat not treated with tamoxifen. I-L, The absence of red fluorescence in the fetus harboring only the ROSA26-tdTomato transgene born from pregnant rat treated with tamoxifen. C, G, and K, Immunofluorescence using anti-PHOX2B antibody. P0-P2 fetuses are used. For EYFP excitation (panels A, E, and I), a multi-argon laser is applied and emission bandwith is 500–530 nm. For tdTomato (panels B, F, and J) and AlexaFluor633 (panels C, G, and K) excitation, a helium-neon laser is used and emission bandwith is 560–620 nm and 650- nm, respectively. All images in the panels (A-L) were taken under the same condition on the same day.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4492506&req=5

pone.0132475.g007: Analysis of CreERT2 recombinase activity.A-D, Cre recombinase activity induced by estrogen analog tamoxifen (CreERT2, estrogen receptor T2, [38]) was examined by fluorescence in fetus harboring both the Phox2b-EYFP-2A-CreERT2 transgene and the ROSA26-tdTomato transgene born from pregnant rat treated with tamoxifen. The tdTomato red signals are detected in the facial nucleus (nVII). E-H, No red fluorescence is present in fetus harboring both the Phox2b-EYFP-2A-CreERT2 transgene and the ROSA26-tdTomato transgene born from pregnant rat not treated with tamoxifen. I-L, The absence of red fluorescence in the fetus harboring only the ROSA26-tdTomato transgene born from pregnant rat treated with tamoxifen. C, G, and K, Immunofluorescence using anti-PHOX2B antibody. P0-P2 fetuses are used. For EYFP excitation (panels A, E, and I), a multi-argon laser is applied and emission bandwith is 500–530 nm. For tdTomato (panels B, F, and J) and AlexaFluor633 (panels C, G, and K) excitation, a helium-neon laser is used and emission bandwith is 560–620 nm and 650- nm, respectively. All images in the panels (A-L) were taken under the same condition on the same day.

Mentions: Next, we investigated whether Phox2b promoter/enhancer induces efficient expression of EYFP and CreERT2. The transgenic construct (Fig 2) contained coding sequences for EYFP and CreERT2 separated by the 2A peptide, whose sequence caused ribosomal skipping during translation and subsequent cleavage of the translated polypeptide at the 2A site, resulting in the generation of distinct polypeptides that were subsequently folded independently into their native structures [66]. Theoretically, the inducible CreERT2 is active only in the presence of tamoxifen [38]. We used fetuses that harbored both the Phox2b-EYFP/CreERT2 transgene and the ROSA26-tdTomato reporter transgene obtained by crossing the two Tg rat lines as the parent. Upon administration of tamoxifen to mother Phox2b-EYFP/CreERT2 rats, tdTomato red signal was observed in the facial nucleus (nVII) of the fetus (Fig 7B) and those were EYFP-positive (n = 9 pups from 4 mothers) (Fig 7A and 7D). In the absence of tamoxifen, signals were not detected in the fetus that harbored both transgenes (n = 3 pups from 2 mothers) (Fig 7F).


A Phox2b BAC Transgenic Rat Line Useful for Understanding Respiratory Rhythm Generator Neural Circuitry.

Ikeda K, Takahashi M, Sato S, Igarashi H, Ishizuka T, Yawo H, Arata S, Southard-Smith EM, Kawakami K, Onimaru H - PLoS ONE (2015)

Analysis of CreERT2 recombinase activity.A-D, Cre recombinase activity induced by estrogen analog tamoxifen (CreERT2, estrogen receptor T2, [38]) was examined by fluorescence in fetus harboring both the Phox2b-EYFP-2A-CreERT2 transgene and the ROSA26-tdTomato transgene born from pregnant rat treated with tamoxifen. The tdTomato red signals are detected in the facial nucleus (nVII). E-H, No red fluorescence is present in fetus harboring both the Phox2b-EYFP-2A-CreERT2 transgene and the ROSA26-tdTomato transgene born from pregnant rat not treated with tamoxifen. I-L, The absence of red fluorescence in the fetus harboring only the ROSA26-tdTomato transgene born from pregnant rat treated with tamoxifen. C, G, and K, Immunofluorescence using anti-PHOX2B antibody. P0-P2 fetuses are used. For EYFP excitation (panels A, E, and I), a multi-argon laser is applied and emission bandwith is 500–530 nm. For tdTomato (panels B, F, and J) and AlexaFluor633 (panels C, G, and K) excitation, a helium-neon laser is used and emission bandwith is 560–620 nm and 650- nm, respectively. All images in the panels (A-L) were taken under the same condition on the same day.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492506&req=5

pone.0132475.g007: Analysis of CreERT2 recombinase activity.A-D, Cre recombinase activity induced by estrogen analog tamoxifen (CreERT2, estrogen receptor T2, [38]) was examined by fluorescence in fetus harboring both the Phox2b-EYFP-2A-CreERT2 transgene and the ROSA26-tdTomato transgene born from pregnant rat treated with tamoxifen. The tdTomato red signals are detected in the facial nucleus (nVII). E-H, No red fluorescence is present in fetus harboring both the Phox2b-EYFP-2A-CreERT2 transgene and the ROSA26-tdTomato transgene born from pregnant rat not treated with tamoxifen. I-L, The absence of red fluorescence in the fetus harboring only the ROSA26-tdTomato transgene born from pregnant rat treated with tamoxifen. C, G, and K, Immunofluorescence using anti-PHOX2B antibody. P0-P2 fetuses are used. For EYFP excitation (panels A, E, and I), a multi-argon laser is applied and emission bandwith is 500–530 nm. For tdTomato (panels B, F, and J) and AlexaFluor633 (panels C, G, and K) excitation, a helium-neon laser is used and emission bandwith is 560–620 nm and 650- nm, respectively. All images in the panels (A-L) were taken under the same condition on the same day.
Mentions: Next, we investigated whether Phox2b promoter/enhancer induces efficient expression of EYFP and CreERT2. The transgenic construct (Fig 2) contained coding sequences for EYFP and CreERT2 separated by the 2A peptide, whose sequence caused ribosomal skipping during translation and subsequent cleavage of the translated polypeptide at the 2A site, resulting in the generation of distinct polypeptides that were subsequently folded independently into their native structures [66]. Theoretically, the inducible CreERT2 is active only in the presence of tamoxifen [38]. We used fetuses that harbored both the Phox2b-EYFP/CreERT2 transgene and the ROSA26-tdTomato reporter transgene obtained by crossing the two Tg rat lines as the parent. Upon administration of tamoxifen to mother Phox2b-EYFP/CreERT2 rats, tdTomato red signal was observed in the facial nucleus (nVII) of the fetus (Fig 7B) and those were EYFP-positive (n = 9 pups from 4 mothers) (Fig 7A and 7D). In the absence of tamoxifen, signals were not detected in the fetus that harbored both transgenes (n = 3 pups from 2 mothers) (Fig 7F).

Bottom Line: Here we describe the generation of a novel transgenic (Tg) rat harboring fluorescently labeled Pre-I neurons in the RTN/pFRG.In addition, the Tg rat showed fluorescent signals in autonomic enteric neurons and carotid bodies.Because the Tg rat expresses inducible Cre recombinase in PHOX2B-positive cells during development, it is a potentially powerful tool for dissecting the entire picture of the respiratory neural network during development and for identifying the CO2/O2 sensor molecules in the adult central and peripheral nervous systems.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan; Division of Biology, Center for Molecular Medicine, Jichi Medical University, Shimotsuke, Tochigi, Japan.

ABSTRACT
The key role of the respiratory neural center is respiratory rhythm generation to maintain homeostasis through the control of arterial blood pCO2/pH and pO2 levels. The neuronal network responsible for respiratory rhythm generation in neonatal rat resides in the ventral side of the medulla and is composed of two groups; the parafacial respiratory group (pFRG) and the pre-Bötzinger complex group (preBötC). The pFRG partially overlaps in the retrotrapezoid nucleus (RTN), which was originally identified in adult cats and rats. Part of the pre-inspiratory (Pre-I) neurons in the RTN/pFRG serves as central chemoreceptor neurons and the CO2 sensitive Pre-I neurons express homeobox gene Phox2b. Phox2b encodes a transcription factor and is essential for the development of the sensory-motor visceral circuits. Mutations in human PHOX2B cause congenital hypoventilation syndrome, which is characterized by blunted ventilatory response to hypercapnia. Here we describe the generation of a novel transgenic (Tg) rat harboring fluorescently labeled Pre-I neurons in the RTN/pFRG. In addition, the Tg rat showed fluorescent signals in autonomic enteric neurons and carotid bodies. Because the Tg rat expresses inducible Cre recombinase in PHOX2B-positive cells during development, it is a potentially powerful tool for dissecting the entire picture of the respiratory neural network during development and for identifying the CO2/O2 sensor molecules in the adult central and peripheral nervous systems.

No MeSH data available.


Related in: MedlinePlus