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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

EYFP expression from the Phox2b-EYFP/CreERT2 Tg is comparable to in situ hybridization patterns of endogenous Phox2b in E12.5 rat embryos.A, The transgenic (Tg) rat embryo at E12.5 (somite number 39) harboring the Phox2b-EYFP-2A-Cre/ERT2 Rec BAC transgene exhibits fluorescence in known sites of Phox2b/PHOX2B expression. The EYFP signal is detected in the oculomotor (III) and trochlear (IV) motor nuclei in the central nervous system (III and IV), and in the three epibranchial sensory/distal ganglia; geniculate VIIth, petrosal IXth, and nodose Xth ganglia (gVII, gIX, and gX). Primary sympathetic chain (arrowhead) and neurons in the ventral columns of neural tubes are also positive (arrows). B, Superposition of fluorescence from A on bright field image. The positions of coronal sections are indicated as "Section a" in E and "Section b" in F. C, Flat-mount preparation of an E12.5 fetal rat midbrain and hindbrain shows transgene expression. In the mesencephalic region, the signals are found in the oculomotor (III) and trochlear (IV) motor nuclei and in the forming locus coeruleus (LoC). In the rhombencephalon, the signals are found in the ventral stripe composed of V, VII, and ventral motor neurons (vMN) and in the lateral stripe of rhombomeres 2, 3, 4, 5, and 6 (r2, r3, r4, r5, and r6, respectively). D, Superposition of figures under white-light and fluorescence of C. E, F, Coronal sections at the level of sections taken at the Sections a and b shown in B. The sympathetic ganglionic chain (s) and enteric nervous system (en) are fluorescent-positive. G-I, Magnified figures in C. The unique projection of axons from the trochlear nucleus is indicated by the sharp arrow. J, Whole-mount in situ hybridization of rat embryo at E12.5. K, Flat-mount preparation of midbrain and hindbrain of the embryo shown in J.
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pone.0132475.g003: EYFP expression from the Phox2b-EYFP/CreERT2 Tg is comparable to in situ hybridization patterns of endogenous Phox2b in E12.5 rat embryos.A, The transgenic (Tg) rat embryo at E12.5 (somite number 39) harboring the Phox2b-EYFP-2A-Cre/ERT2 Rec BAC transgene exhibits fluorescence in known sites of Phox2b/PHOX2B expression. The EYFP signal is detected in the oculomotor (III) and trochlear (IV) motor nuclei in the central nervous system (III and IV), and in the three epibranchial sensory/distal ganglia; geniculate VIIth, petrosal IXth, and nodose Xth ganglia (gVII, gIX, and gX). Primary sympathetic chain (arrowhead) and neurons in the ventral columns of neural tubes are also positive (arrows). B, Superposition of fluorescence from A on bright field image. The positions of coronal sections are indicated as "Section a" in E and "Section b" in F. C, Flat-mount preparation of an E12.5 fetal rat midbrain and hindbrain shows transgene expression. In the mesencephalic region, the signals are found in the oculomotor (III) and trochlear (IV) motor nuclei and in the forming locus coeruleus (LoC). In the rhombencephalon, the signals are found in the ventral stripe composed of V, VII, and ventral motor neurons (vMN) and in the lateral stripe of rhombomeres 2, 3, 4, 5, and 6 (r2, r3, r4, r5, and r6, respectively). D, Superposition of figures under white-light and fluorescence of C. E, F, Coronal sections at the level of sections taken at the Sections a and b shown in B. The sympathetic ganglionic chain (s) and enteric nervous system (en) are fluorescent-positive. G-I, Magnified figures in C. The unique projection of axons from the trochlear nucleus is indicated by the sharp arrow. J, Whole-mount in situ hybridization of rat embryo at E12.5. K, Flat-mount preparation of midbrain and hindbrain of the embryo shown in J.

Mentions: To examine whether EYFP fluorescence driven by Phox2b-EYFP/CreERT2 transgene conferred expression of endogenous Phox2b in the developmental nervous system, we examined the fluorescence of the whole embryo at E12.5 (n = 6, somite number 38–40) (Fig 3A and 3B). Fluorescent signals were found in the oculomotor (III) and trochlear (IV) motor nuclei (Fig 3A, III and IV), as reported previously in the developing "mouse" mRNA/protein [29]. Signals were found in the epibranchial distal VIIth, IXth, and Xth ganglia (Fig 3A, gVII, gIX, and gX). Signals were also detected in primary sympathetic chain in the whole embryo (arrowhead in Fig 3A), as well as thick coronal sections cut at the level of Sections a and b in Fig 3E and 3F (s, sympathetic chain). Progenitors of the enteric nervous system were also fluorescent-positive (Fig 3E, en). We confirmed the region of fluorescent signals in flat-mounted hindbrains of the embryos (Fig 3C and 3D). Fluorescent signals in the hindbrain were organized in the longitudinal columns, in agreement with mouse Phox2b mRNA expression [29]. Branchial motor neurons (V and VII in Fig 3C) and visceral motor neurons (vMN in Fig 3C), which reside in the ventral columns (white arrows in Fig 3A and 3C), were clearly identified. A unique dorsal projection of trochlear (IV) axons was also identified along the midbrain-hindbrain boundary (arrow in Fig 3G). The axons of V and VII motor neurons converged towards the future exit points of their cranial nerves (Fig 3C and 3I). The locus coeruleus (LoC), the location of the major noradrenergic neuronal population in the brain, situated in the rostral hindbrain, was also fluorescent-positive at this stage (Fig 3C and 3H), consistent with the fact that PHOX2B is an upstream transcription factor for dopamine-ß-hydroxylase gene [30].


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)

EYFP expression from the Phox2b-EYFP/CreERT2 Tg is comparable to in situ hybridization patterns of endogenous Phox2b in E12.5 rat embryos.A, The transgenic (Tg) rat embryo at E12.5 (somite number 39) harboring the Phox2b-EYFP-2A-Cre/ERT2 Rec BAC transgene exhibits fluorescence in known sites of Phox2b/PHOX2B expression. The EYFP signal is detected in the oculomotor (III) and trochlear (IV) motor nuclei in the central nervous system (III and IV), and in the three epibranchial sensory/distal ganglia; geniculate VIIth, petrosal IXth, and nodose Xth ganglia (gVII, gIX, and gX). Primary sympathetic chain (arrowhead) and neurons in the ventral columns of neural tubes are also positive (arrows). B, Superposition of fluorescence from A on bright field image. The positions of coronal sections are indicated as "Section a" in E and "Section b" in F. C, Flat-mount preparation of an E12.5 fetal rat midbrain and hindbrain shows transgene expression. In the mesencephalic region, the signals are found in the oculomotor (III) and trochlear (IV) motor nuclei and in the forming locus coeruleus (LoC). In the rhombencephalon, the signals are found in the ventral stripe composed of V, VII, and ventral motor neurons (vMN) and in the lateral stripe of rhombomeres 2, 3, 4, 5, and 6 (r2, r3, r4, r5, and r6, respectively). D, Superposition of figures under white-light and fluorescence of C. E, F, Coronal sections at the level of sections taken at the Sections a and b shown in B. The sympathetic ganglionic chain (s) and enteric nervous system (en) are fluorescent-positive. G-I, Magnified figures in C. The unique projection of axons from the trochlear nucleus is indicated by the sharp arrow. J, Whole-mount in situ hybridization of rat embryo at E12.5. K, Flat-mount preparation of midbrain and hindbrain of the embryo shown in J.
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Related In: Results  -  Collection

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Show All Figures
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pone.0132475.g003: EYFP expression from the Phox2b-EYFP/CreERT2 Tg is comparable to in situ hybridization patterns of endogenous Phox2b in E12.5 rat embryos.A, The transgenic (Tg) rat embryo at E12.5 (somite number 39) harboring the Phox2b-EYFP-2A-Cre/ERT2 Rec BAC transgene exhibits fluorescence in known sites of Phox2b/PHOX2B expression. The EYFP signal is detected in the oculomotor (III) and trochlear (IV) motor nuclei in the central nervous system (III and IV), and in the three epibranchial sensory/distal ganglia; geniculate VIIth, petrosal IXth, and nodose Xth ganglia (gVII, gIX, and gX). Primary sympathetic chain (arrowhead) and neurons in the ventral columns of neural tubes are also positive (arrows). B, Superposition of fluorescence from A on bright field image. The positions of coronal sections are indicated as "Section a" in E and "Section b" in F. C, Flat-mount preparation of an E12.5 fetal rat midbrain and hindbrain shows transgene expression. In the mesencephalic region, the signals are found in the oculomotor (III) and trochlear (IV) motor nuclei and in the forming locus coeruleus (LoC). In the rhombencephalon, the signals are found in the ventral stripe composed of V, VII, and ventral motor neurons (vMN) and in the lateral stripe of rhombomeres 2, 3, 4, 5, and 6 (r2, r3, r4, r5, and r6, respectively). D, Superposition of figures under white-light and fluorescence of C. E, F, Coronal sections at the level of sections taken at the Sections a and b shown in B. The sympathetic ganglionic chain (s) and enteric nervous system (en) are fluorescent-positive. G-I, Magnified figures in C. The unique projection of axons from the trochlear nucleus is indicated by the sharp arrow. J, Whole-mount in situ hybridization of rat embryo at E12.5. K, Flat-mount preparation of midbrain and hindbrain of the embryo shown in J.
Mentions: To examine whether EYFP fluorescence driven by Phox2b-EYFP/CreERT2 transgene conferred expression of endogenous Phox2b in the developmental nervous system, we examined the fluorescence of the whole embryo at E12.5 (n = 6, somite number 38–40) (Fig 3A and 3B). Fluorescent signals were found in the oculomotor (III) and trochlear (IV) motor nuclei (Fig 3A, III and IV), as reported previously in the developing "mouse" mRNA/protein [29]. Signals were found in the epibranchial distal VIIth, IXth, and Xth ganglia (Fig 3A, gVII, gIX, and gX). Signals were also detected in primary sympathetic chain in the whole embryo (arrowhead in Fig 3A), as well as thick coronal sections cut at the level of Sections a and b in Fig 3E and 3F (s, sympathetic chain). Progenitors of the enteric nervous system were also fluorescent-positive (Fig 3E, en). We confirmed the region of fluorescent signals in flat-mounted hindbrains of the embryos (Fig 3C and 3D). Fluorescent signals in the hindbrain were organized in the longitudinal columns, in agreement with mouse Phox2b mRNA expression [29]. Branchial motor neurons (V and VII in Fig 3C) and visceral motor neurons (vMN in Fig 3C), which reside in the ventral columns (white arrows in Fig 3A and 3C), were clearly identified. A unique dorsal projection of trochlear (IV) axons was also identified along the midbrain-hindbrain boundary (arrow in Fig 3G). The axons of V and VII motor neurons converged towards the future exit points of their cranial nerves (Fig 3C and 3I). The locus coeruleus (LoC), the location of the major noradrenergic neuronal population in the brain, situated in the rostral hindbrain, was also fluorescent-positive at this stage (Fig 3C and 3H), consistent with the fact that PHOX2B is an upstream transcription factor for dopamine-ß-hydroxylase gene [30].

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