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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

Expression of the EYFP reporter in respiratory associated neurons of Phox2b-EYFP/CreERT2 Tg neonatal rats.A-H, Coronal sections in retrotrapezoid nucleus (RTN)/parafacial respiratory group (pFRG) of the medulla. A-D, The rostral part of RTN/pFRG which resides in the ventral surface of the facial nucleus (nVII) are fluorescent-positive (A-D). Most neurons of the facial nucleus are also fluorescent-positive but their signals vary depending on the cell groups. E-H, The caudal part of RTN/pFRG resides in the ventral and medial sides of the nucleus ambiguus (nA), and is also fluorescent-positive. All EYFP-positive cells are immunoreactive for anti-PHOX2B (C, D, G, and H). I-K, The carotid body (CB) of the Tg rat is EYFP-positive. I, Three-dimensional reconstitution using complete serial sections of surrounding CB tissues. ECC, external carotid artery; ICC, internal carotid artery; IX, glossopharyngeal nerve; occ. A., occipital artery; ant. thy. A., anterior thymic artery. J, Coronal section of the rostral part of CB stained with anti-VEGFR2 (capillary endothelial cell marker) and DAPI. K, Coronal section of the rostral part of CB is immunopositive for tyrosine hyroxylase (TH).
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pone.0132475.g005: Expression of the EYFP reporter in respiratory associated neurons of Phox2b-EYFP/CreERT2 Tg neonatal rats.A-H, Coronal sections in retrotrapezoid nucleus (RTN)/parafacial respiratory group (pFRG) of the medulla. A-D, The rostral part of RTN/pFRG which resides in the ventral surface of the facial nucleus (nVII) are fluorescent-positive (A-D). Most neurons of the facial nucleus are also fluorescent-positive but their signals vary depending on the cell groups. E-H, The caudal part of RTN/pFRG resides in the ventral and medial sides of the nucleus ambiguus (nA), and is also fluorescent-positive. All EYFP-positive cells are immunoreactive for anti-PHOX2B (C, D, G, and H). I-K, The carotid body (CB) of the Tg rat is EYFP-positive. I, Three-dimensional reconstitution using complete serial sections of surrounding CB tissues. ECC, external carotid artery; ICC, internal carotid artery; IX, glossopharyngeal nerve; occ. A., occipital artery; ant. thy. A., anterior thymic artery. J, Coronal section of the rostral part of CB stained with anti-VEGFR2 (capillary endothelial cell marker) and DAPI. K, Coronal section of the rostral part of CB is immunopositive for tyrosine hyroxylase (TH).

Mentions: Using embryos at the same stage of E12.5, we performed whole-mount in situ hybridization using Phox2b riboprobe and confirmed the fluorescent signals traced Phox2b endogenous expression followed by the flat-mount preparation (Fig 3J and 3K) (n = 3). In general, the fluorescent signals were highly similar to those of endogenous mRNA expression in E12.5 embryos, although the signals in the dorsal stripe were more apparent in endogenous expression than in fluorescence (red arrows in Fig 3J and 3K). The lateral stripe (yellow arrows in Fig 3C and 3K) was also similar. The dorsal and lateral stripes contained cells seen in the mantle layers of mice at comparable stage (Figs 2 and 5 in [29]).


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)

Expression of the EYFP reporter in respiratory associated neurons of Phox2b-EYFP/CreERT2 Tg neonatal rats.A-H, Coronal sections in retrotrapezoid nucleus (RTN)/parafacial respiratory group (pFRG) of the medulla. A-D, The rostral part of RTN/pFRG which resides in the ventral surface of the facial nucleus (nVII) are fluorescent-positive (A-D). Most neurons of the facial nucleus are also fluorescent-positive but their signals vary depending on the cell groups. E-H, The caudal part of RTN/pFRG resides in the ventral and medial sides of the nucleus ambiguus (nA), and is also fluorescent-positive. All EYFP-positive cells are immunoreactive for anti-PHOX2B (C, D, G, and H). I-K, The carotid body (CB) of the Tg rat is EYFP-positive. I, Three-dimensional reconstitution using complete serial sections of surrounding CB tissues. ECC, external carotid artery; ICC, internal carotid artery; IX, glossopharyngeal nerve; occ. A., occipital artery; ant. thy. A., anterior thymic artery. J, Coronal section of the rostral part of CB stained with anti-VEGFR2 (capillary endothelial cell marker) and DAPI. K, Coronal section of the rostral part of CB is immunopositive for tyrosine hyroxylase (TH).
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pone.0132475.g005: Expression of the EYFP reporter in respiratory associated neurons of Phox2b-EYFP/CreERT2 Tg neonatal rats.A-H, Coronal sections in retrotrapezoid nucleus (RTN)/parafacial respiratory group (pFRG) of the medulla. A-D, The rostral part of RTN/pFRG which resides in the ventral surface of the facial nucleus (nVII) are fluorescent-positive (A-D). Most neurons of the facial nucleus are also fluorescent-positive but their signals vary depending on the cell groups. E-H, The caudal part of RTN/pFRG resides in the ventral and medial sides of the nucleus ambiguus (nA), and is also fluorescent-positive. All EYFP-positive cells are immunoreactive for anti-PHOX2B (C, D, G, and H). I-K, The carotid body (CB) of the Tg rat is EYFP-positive. I, Three-dimensional reconstitution using complete serial sections of surrounding CB tissues. ECC, external carotid artery; ICC, internal carotid artery; IX, glossopharyngeal nerve; occ. A., occipital artery; ant. thy. A., anterior thymic artery. J, Coronal section of the rostral part of CB stained with anti-VEGFR2 (capillary endothelial cell marker) and DAPI. K, Coronal section of the rostral part of CB is immunopositive for tyrosine hyroxylase (TH).
Mentions: Using embryos at the same stage of E12.5, we performed whole-mount in situ hybridization using Phox2b riboprobe and confirmed the fluorescent signals traced Phox2b endogenous expression followed by the flat-mount preparation (Fig 3J and 3K) (n = 3). In general, the fluorescent signals were highly similar to those of endogenous mRNA expression in E12.5 embryos, although the signals in the dorsal stripe were more apparent in endogenous expression than in fluorescence (red arrows in Fig 3J and 3K). The lateral stripe (yellow arrows in Fig 3C and 3K) was also similar. The dorsal and lateral stripes contained cells seen in the mantle layers of mice at comparable stage (Figs 2 and 5 in [29]).

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