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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

Position of conserved non-coding sequences around Phox2b exons.A, B, The VISTA plot of approximately 300 kb (A) span around the mouse Phox2b gene and the 20 kb (B) region closest to the gene are shown. The plot shows conserved sequences between mouse and rat, human, opossum, chicken, painted turtle, Xenopus, coelacanth, spotted gar, and zebrafish. Abscissa: mouse sequence, ordinate: percent identity in a 50-bp window. The conserved regions above the level of 60%/50 bp are highlighted under the curve, with pink indicating conserved non-coding sequence (CNS), blue indicating conserved exon, and cyan indicating untranslated region. The approximate position of the mouse BAC clone RP24-95M11 is shown in grey at the top of panel A. In A and B, numbers at the bottom of the plot indicate positions relative to the analyzed 540-kb mouse genome fragment (chromosome:GRCm38:5:66826857 (set as position 1)-67366858).
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pone.0132475.g001: Position of conserved non-coding sequences around Phox2b exons.A, B, The VISTA plot of approximately 300 kb (A) span around the mouse Phox2b gene and the 20 kb (B) region closest to the gene are shown. The plot shows conserved sequences between mouse and rat, human, opossum, chicken, painted turtle, Xenopus, coelacanth, spotted gar, and zebrafish. Abscissa: mouse sequence, ordinate: percent identity in a 50-bp window. The conserved regions above the level of 60%/50 bp are highlighted under the curve, with pink indicating conserved non-coding sequence (CNS), blue indicating conserved exon, and cyan indicating untranslated region. The approximate position of the mouse BAC clone RP24-95M11 is shown in grey at the top of panel A. In A and B, numbers at the bottom of the plot indicate positions relative to the analyzed 540-kb mouse genome fragment (chromosome:GRCm38:5:66826857 (set as position 1)-67366858).

Mentions: The unique expression pattern of Phox2b has been reported in mice, zebrafish, and lamprey during embryonic development [29, 30, 48–51], and in adult rat brain [13, 17]. Furthermore, the clinical symptoms of disease caused by mutations in the coding sequence of human PHOX2B are consistent with the expression patterns observed in rodents [31]. Previous studies reported that enhancers critical for temporal and spatial expressions of genes during development are frequently conserved among species [52]. In this regard, the transgenic mouse line that exploits a Phox2b-containing BAC (RP24-95M11) containing the Phox2b coding region together with upstream and downstream sequences in mice was confirmed to mirror the endogenous Phox2b expression pattern [53–55]. Since evaluation of the RP24-95M11 BAC clone could be useful for driving expression in transgenic rats, we compared the evolutionarily conserved non-coding sequences (CNSs) surrounding the Phox2b exons in mammals, together with related species, such as painted turtle, chicken, Xenopus, coelacanth, spotted gar, and zebrafish using mVISTA (Fig 1). The results showed that some CNSs were conserved only in placental mammals (rat, mouse, and human; Fig 1A). The flanking sequence of the mouse Phox2b gene showed remarkable resemblance to that of the rat Phox2b gene. The fact that RP24-95M11 BAC clone contained all the highly conserved regions between mouse and rat encouraged us to use the RP24-95M11 BAC clone in a transgenic rat model.


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)

Position of conserved non-coding sequences around Phox2b exons.A, B, The VISTA plot of approximately 300 kb (A) span around the mouse Phox2b gene and the 20 kb (B) region closest to the gene are shown. The plot shows conserved sequences between mouse and rat, human, opossum, chicken, painted turtle, Xenopus, coelacanth, spotted gar, and zebrafish. Abscissa: mouse sequence, ordinate: percent identity in a 50-bp window. The conserved regions above the level of 60%/50 bp are highlighted under the curve, with pink indicating conserved non-coding sequence (CNS), blue indicating conserved exon, and cyan indicating untranslated region. The approximate position of the mouse BAC clone RP24-95M11 is shown in grey at the top of panel A. In A and B, numbers at the bottom of the plot indicate positions relative to the analyzed 540-kb mouse genome fragment (chromosome:GRCm38:5:66826857 (set as position 1)-67366858).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0132475.g001: Position of conserved non-coding sequences around Phox2b exons.A, B, The VISTA plot of approximately 300 kb (A) span around the mouse Phox2b gene and the 20 kb (B) region closest to the gene are shown. The plot shows conserved sequences between mouse and rat, human, opossum, chicken, painted turtle, Xenopus, coelacanth, spotted gar, and zebrafish. Abscissa: mouse sequence, ordinate: percent identity in a 50-bp window. The conserved regions above the level of 60%/50 bp are highlighted under the curve, with pink indicating conserved non-coding sequence (CNS), blue indicating conserved exon, and cyan indicating untranslated region. The approximate position of the mouse BAC clone RP24-95M11 is shown in grey at the top of panel A. In A and B, numbers at the bottom of the plot indicate positions relative to the analyzed 540-kb mouse genome fragment (chromosome:GRCm38:5:66826857 (set as position 1)-67366858).
Mentions: The unique expression pattern of Phox2b has been reported in mice, zebrafish, and lamprey during embryonic development [29, 30, 48–51], and in adult rat brain [13, 17]. Furthermore, the clinical symptoms of disease caused by mutations in the coding sequence of human PHOX2B are consistent with the expression patterns observed in rodents [31]. Previous studies reported that enhancers critical for temporal and spatial expressions of genes during development are frequently conserved among species [52]. In this regard, the transgenic mouse line that exploits a Phox2b-containing BAC (RP24-95M11) containing the Phox2b coding region together with upstream and downstream sequences in mice was confirmed to mirror the endogenous Phox2b expression pattern [53–55]. Since evaluation of the RP24-95M11 BAC clone could be useful for driving expression in transgenic rats, we compared the evolutionarily conserved non-coding sequences (CNSs) surrounding the Phox2b exons in mammals, together with related species, such as painted turtle, chicken, Xenopus, coelacanth, spotted gar, and zebrafish using mVISTA (Fig 1). The results showed that some CNSs were conserved only in placental mammals (rat, mouse, and human; Fig 1A). The flanking sequence of the mouse Phox2b gene showed remarkable resemblance to that of the rat Phox2b gene. The fact that RP24-95M11 BAC clone contained all the highly conserved regions between mouse and rat encouraged us to use the RP24-95M11 BAC clone in a transgenic rat model.

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