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S1.3 Phox2b and the autonomic control of breathing
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Abstracts / Autonomic Neuroscience: Basic and Clinical 149 (2009) 1–126
biology, Department of Neurochemistry, MPI for Brain Research, Frankfurt/M, Germany), Kazuto Kobayashi (Department of Molecular Genetics, Fukushima University School of Medicine, Fukushima, Japan), Marthe J. Howard (Department of Neurosciences, University of Toledo Health Sciences Center, Toledo, OH 43614, USA), Hermann Rohrer (RG Developmental Neurobiology, Department of Neurochemistry, MPI for Brain Research, Frankfurt/M, Germany) The specification and initial noradrenergic differentiation of sympathetic neurons is induced by bone morphogenetic proteins (BMPs). The effect of BMPs is mediated by a group of transcription factors that includes Ascl1, Phox2a/b, Hand2 and Gata3. These factors induce, directly or indirectly, the expression of characteristic noradrenergic marker genes as well as generic neuronal genes. Parasympathetic neuron development is also initiated by BMPs, but Hand2 and Gata3 are not present and noradrenergic properties are expressed only transiently in this lineage. This raised the issue whether Hand2 and Gata3 that are expressed in sympathetic neurons up to the adult stage, are required for the maintenance of noradrenergic characteristics in autonomic neurons. To address this question, the function of Hand2 and Gata3 was analyzed in differentiated sympathetic neurons by several loss-of-function approaches. The in vitro knockdown of Hand2 in postmitotic differentiated neurons from E12 chick embryos by siRNA resulted in a strong decrease in the expression of the noradrenergic marker genes dopamine-β-hydroxylase (DBH) and tyrosine hydroxylase (TH), whereas the expression of generic neuronal genes TuJ1, HuC and SCG10 were not affected. The in vivo role of Hand2 in differentiated sympathetic neurons was analyzed in mice harboring a conditional Hand2-null allele, excising the gene by the expression of Cre recombinase under the control of the DBH promotor. Mouse embryos homozygous for Hand2 deletion showed decreased sympathetic neuron numbers due to effects on neuron proliferation and TH expression was strongly reduced in the residual neuronal population. In contrast, DBHCre-mediated elimination of Gata3 resulted in a massively reduced neuron number due to increased apoptosis, but did not affect TH or DBH in the remaining neuronal population. Taken together, these findings demonstrate that Gata3 displays different functions in developing and mature sympathetic neurons, controlling TH-expression and neuron survival, respectively, whereas the Hand2 transcription factor plays a key role both in the induction and maintenance of noradrenergic properties in sympathetic neurons. doi:10.1016/j.autneu.2009.05.007
S1.3 Phox2b and the autonomic control of breathing C. Goridis, V. Dubreuil, J.-F. Brunet (Département de Biologie, École normale supérieure and CNRS UMR8542, 75005 Paris, France) In all vertebrates, the Phox2b transcription factor is specifically expressed and required in a limited set of neuronal types, which match for the most part the neurons of the visceral reflex circuits that regulate cardio-vascular, respiratory and digestive functions. Until recently, the role of Phox2b in the circuits that control breathing in mammals seemed patchier than in those that control blood pressure and digestion. This changed with the discovery that dominant mutations in Phox2b cause congenital central hypoventilation syndrome (CCHS). CCHS is a rare, but severe congenital disease characterized by central hypoventilation in sleep and an absent response to hypercapnia, which have been attributed to a defect in the autonomic regulation of breathing. We have modeled the most frequent human mutation in the mouse. The heterozygous Phox2b27Ala/+ mice breathe irregularly, do not respond to hypercapnia and die in the first hours after birth from
central apnoea. The sole anatomical defect we found was the lack of CO2-sensitive, Phox2b-expressing glutamatergic cells in the retrotrapezoid nucleus (RTN). The location and molecular phenotype of RTN neurons characterized by their chemosensitivity matches that of the parafacial respiratory group (pFRG) identified as intrinsically rhythmic pre-inspiratory neurons. We found that the respiratory-like spontaneous rhythmic activity of the pFRG was lost in Phox2b27Ala/+ embryos, while the pre-Bötzinger complex, the best studied respiratory rhythm generator, functioned normally when tested in isolation. We thus propose that, in addition to the unresponsiveness to CO2, the lack of the pFRG oscillator contributes to the severe phenotype of the Phox2b27Ala/+ pups.pFRG/RTN neurons not only succumb to the dominant Phox2b27Ala mutation, but are also depleted in the absence of Phox2b. Using genetic tracing to study the ontogeny of pFRG/RTN neurons, we found that they originate from a population of Lbx1+ dorsal interneurons in rhombomeres 3 or 5. This gave us the opportunity to generate conditional Phox2b mutants using cre recombinase expression in either rhombomere 3/5 or in Lbx1+ interneurons. Like the Phox2b27Ala/+ mutation, the two conditional Phox2b mutations resulted in fatal central apnoea and abolished the activity of the embryonic pFRG. Breathing is a rhythmic behaviour generated by neuronal networks in the medulla. In newborn rodents, the pre-Bötzinger complex and the pFRG display spontaneous rhythmic activity in phase with respiratory motor outflow, but which site dominates under which condition is still a matter of debate. Changes in CO2/pH provide a major drive to respiration, but the nature and location of the primary central pH sensors are still controversial issues. Our results underscore the essential role of the pFRG/RTN in the autonomic control of breathing in the perinatal period, probably by providing both, responsiveness to CO2 and adequate rhythmicity. doi:10.1016/j.autneu.2009.05.008
S1.4 The transcription factor Hand2 is necessary for differentiation and cell type-specific gene expression of sympathetic and enteric neurons in murine embryos T.J. Hendershot, J. Liu, S. Lin, M.J. Howard (Department of Neurosciences, Medical University of Ohio, 3000 Arlington Avenue, Toledo, OH 43614, USA) The basic helix–loop–helix DNA binding protein Hand2 is expressed in neural crest-derived precursors of noradrenergic sympathetic (SNS) and enteric neurons (ENS) and has been shown to affect both neurogenesis and neurotransmitter specification. The goal of our studies is to more fully understand the molecular mechanisms of Hand2 function. Systemic knock-out of Hand2 is embryonic lethal. We circumvented this problem by generating a floxed allele of Hand2. Deletion of Hand2 specifically in neural crest-derived cells results in a significant decrease in the differentiation of both SNS and ENS neurons. Based on counts of cells expressing the proliferation marker Ki67, Hand2 regulates neurogenesis by assuring the generation of the appropriate number of neural precursor cells. Hand2 also regulates expression and function of dopamine-b-hydroxylase in the SNS and vasoactive intestinal polypeptide in the ENS thus impacting neurotransmitter expression. In the ENS, loss of Hand2 appears to prevent the transition of neural progenitor to a precursor state suggesting an additional role of Hand2 in neurogenesis. Since expression of Hand2 is maintained in the SNS throughout development but is significantly decreased in the ENS we determined the effect of loss of Hand2 following neural specification. We selectively deleted Hand2 in neural precursor cells by crossing our floxed Hand2 mice to mice in which Cre recombinase expression
Abstracts / Autonomic Neuroscience: Basic and Clinical 149 (2009) 1–126
biology, Department of Neurochemistry, MPI for Brain Research, Frankfurt/M, Germany), Kazuto Kobayashi (Department of Molecular Genetics, Fukushima University School of Medicine, Fukushima, Japan), Marthe J. Howard (Department of Neurosciences, University of Toledo Health Sciences Center, Toledo, OH 43614, USA), Hermann Rohrer (RG Developmental Neurobiology, Department of Neurochemistry, MPI for Brain Research, Frankfurt/M, Germany) The specification and initial noradrenergic differentiation of sympathetic neurons is induced by bone morphogenetic proteins (BMPs). The effect of BMPs is mediated by a group of transcription factors that includes Ascl1, Phox2a/b, Hand2 and Gata3. These factors induce, directly or indirectly, the expression of characteristic noradrenergic marker genes as well as generic neuronal genes. Parasympathetic neuron development is also initiated by BMPs, but Hand2 and Gata3 are not present and noradrenergic properties are expressed only transiently in this lineage. This raised the issue whether Hand2 and Gata3 that are expressed in sympathetic neurons up to the adult stage, are required for the maintenance of noradrenergic characteristics in autonomic neurons. To address this question, the function of Hand2 and Gata3 was analyzed in differentiated sympathetic neurons by several loss-of-function approaches. The in vitro knockdown of Hand2 in postmitotic differentiated neurons from E12 chick embryos by siRNA resulted in a strong decrease in the expression of the noradrenergic marker genes dopamine-β-hydroxylase (DBH) and tyrosine hydroxylase (TH), whereas the expression of generic neuronal genes TuJ1, HuC and SCG10 were not affected. The in vivo role of Hand2 in differentiated sympathetic neurons was analyzed in mice harboring a conditional Hand2-null allele, excising the gene by the expression of Cre recombinase under the control of the DBH promotor. Mouse embryos homozygous for Hand2 deletion showed decreased sympathetic neuron numbers due to effects on neuron proliferation and TH expression was strongly reduced in the residual neuronal population. In contrast, DBHCre-mediated elimination of Gata3 resulted in a massively reduced neuron number due to increased apoptosis, but did not affect TH or DBH in the remaining neuronal population. Taken together, these findings demonstrate that Gata3 displays different functions in developing and mature sympathetic neurons, controlling TH-expression and neuron survival, respectively, whereas the Hand2 transcription factor plays a key role both in the induction and maintenance of noradrenergic properties in sympathetic neurons. doi:10.1016/j.autneu.2009.05.007
S1.3 Phox2b and the autonomic control of breathing C. Goridis, V. Dubreuil, J.-F. Brunet (Département de Biologie, École normale supérieure and CNRS UMR8542, 75005 Paris, France) In all vertebrates, the Phox2b transcription factor is specifically expressed and required in a limited set of neuronal types, which match for the most part the neurons of the visceral reflex circuits that regulate cardio-vascular, respiratory and digestive functions. Until recently, the role of Phox2b in the circuits that control breathing in mammals seemed patchier than in those that control blood pressure and digestion. This changed with the discovery that dominant mutations in Phox2b cause congenital central hypoventilation syndrome (CCHS). CCHS is a rare, but severe congenital disease characterized by central hypoventilation in sleep and an absent response to hypercapnia, which have been attributed to a defect in the autonomic regulation of breathing. We have modeled the most frequent human mutation in the mouse. The heterozygous Phox2b27Ala/+ mice breathe irregularly, do not respond to hypercapnia and die in the first hours after birth from
central apnoea. The sole anatomical defect we found was the lack of CO2-sensitive, Phox2b-expressing glutamatergic cells in the retrotrapezoid nucleus (RTN). The location and molecular phenotype of RTN neurons characterized by their chemosensitivity matches that of the parafacial respiratory group (pFRG) identified as intrinsically rhythmic pre-inspiratory neurons. We found that the respiratory-like spontaneous rhythmic activity of the pFRG was lost in Phox2b27Ala/+ embryos, while the pre-Bötzinger complex, the best studied respiratory rhythm generator, functioned normally when tested in isolation. We thus propose that, in addition to the unresponsiveness to CO2, the lack of the pFRG oscillator contributes to the severe phenotype of the Phox2b27Ala/+ pups.pFRG/RTN neurons not only succumb to the dominant Phox2b27Ala mutation, but are also depleted in the absence of Phox2b. Using genetic tracing to study the ontogeny of pFRG/RTN neurons, we found that they originate from a population of Lbx1+ dorsal interneurons in rhombomeres 3 or 5. This gave us the opportunity to generate conditional Phox2b mutants using cre recombinase expression in either rhombomere 3/5 or in Lbx1+ interneurons. Like the Phox2b27Ala/+ mutation, the two conditional Phox2b mutations resulted in fatal central apnoea and abolished the activity of the embryonic pFRG. Breathing is a rhythmic behaviour generated by neuronal networks in the medulla. In newborn rodents, the pre-Bötzinger complex and the pFRG display spontaneous rhythmic activity in phase with respiratory motor outflow, but which site dominates under which condition is still a matter of debate. Changes in CO2/pH provide a major drive to respiration, but the nature and location of the primary central pH sensors are still controversial issues. Our results underscore the essential role of the pFRG/RTN in the autonomic control of breathing in the perinatal period, probably by providing both, responsiveness to CO2 and adequate rhythmicity. doi:10.1016/j.autneu.2009.05.008
S1.4 The transcription factor Hand2 is necessary for differentiation and cell type-specific gene expression of sympathetic and enteric neurons in murine embryos T.J. Hendershot, J. Liu, S. Lin, M.J. Howard (Department of Neurosciences, Medical University of Ohio, 3000 Arlington Avenue, Toledo, OH 43614, USA) The basic helix–loop–helix DNA binding protein Hand2 is expressed in neural crest-derived precursors of noradrenergic sympathetic (SNS) and enteric neurons (ENS) and has been shown to affect both neurogenesis and neurotransmitter specification. The goal of our studies is to more fully understand the molecular mechanisms of Hand2 function. Systemic knock-out of Hand2 is embryonic lethal. We circumvented this problem by generating a floxed allele of Hand2. Deletion of Hand2 specifically in neural crest-derived cells results in a significant decrease in the differentiation of both SNS and ENS neurons. Based on counts of cells expressing the proliferation marker Ki67, Hand2 regulates neurogenesis by assuring the generation of the appropriate number of neural precursor cells. Hand2 also regulates expression and function of dopamine-b-hydroxylase in the SNS and vasoactive intestinal polypeptide in the ENS thus impacting neurotransmitter expression. In the ENS, loss of Hand2 appears to prevent the transition of neural progenitor to a precursor state suggesting an additional role of Hand2 in neurogenesis. Since expression of Hand2 is maintained in the SNS throughout development but is significantly decreased in the ENS we determined the effect of loss of Hand2 following neural specification. We selectively deleted Hand2 in neural precursor cells by crossing our floxed Hand2 mice to mice in which Cre recombinase expression