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Hindbrain interneurons and axon guidance signaling critical for breathing

Abstract

Breathing is a bilaterally synchronous behavior that relies on a respiratory rhythm generator located in the brainstem. An essential component of this generator is the preBötzinger complex (preBötC), which paces inspirations. Little is known about the developmental origin of the interneuronal populations forming the preBötC oscillator network. We found that the homeobox gene Dbx1 controls the fate of glutamatergic interneurons required for preBötC rhythm generation in the mouse embryo. We also found that a conditional inactivation in Dbx1-derived cells of the roundabout homolog 3 (Robo3) gene, which is necessary for axonal midline crossing, resulted in left-right de-synchronization of the preBötC oscillator. Together, these findings identify Dbx1-derived interneurons as the core rhythmogenic elements of the preBötC oscillator and indicate that Robo3-dependent guidance signaling in these cells is required for bilaterally synchronous activity.

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Figure 1: Disrupted breathing and rhythm generation in the preBötC of Dbx1 null mice.
Figure 2: Dbx1-derived cells in the preBötC are rhythmically active.
Figure 3: Phenotypic profiles of Dbx1-derived cells of the preBötC.
Figure 4: The preBötC derives from ventral Dbx1-positive progenitors.
Figure 5: preBötC commissural connectivity is disrupted in Dbx1 null mutants.
Figure 6: Left-right de-synchronization of the preBötC and of motor neuronal outputs in Robo3GFP/GFP embryos.
Figure 7: Left/right de-synchronization of the preBötC in the Dbx1::cre; Robo3loxP/loxP conditional mutant.

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References

  1. Feldman, J.L. & Del Negro, C.A. Looking for inspiration: new perspectives on respiratory rhythm. Nat. Rev. Neurosci. 7, 232–242 (2006).

    Article  CAS  Google Scholar 

  2. Thoby-Brisson, M. et al. Genetic identification of an embryonic parafacial oscillator coupling to the preBötzinger complex. Nat. Neurosci. 12, 1028–1035 (2009).

    Article  CAS  Google Scholar 

  3. Dubreuil, V. et al. Defective respiratory rhythmogenesis and loss of central chemosensitivity in Phox2b mutants targeting retrotrapezoid nucleus neurons. J. Neurosci. 29, 14836–14846 (2009).

    Article  CAS  Google Scholar 

  4. Rose, M.F., Ahmad, K.A., Thaller, C. & Zoghbi, H.Y. Excitatory neurons of the proprioceptive, interoceptive and arousal hindbrain networks share a developmental requirement for Math1. Proc. Natl. Acad. Sci. USA 106, 22462–22467 (2009).

    Article  CAS  Google Scholar 

  5. Smith, J.C., Ellenberger, H.H., Ballanyi, K., Richter, D.W. & Feldman, J.L. Pre-Bötzinger complex: a brainstem region that may generate respiratory rhythm in mammals. Science 254, 726–729 (1991).

    Article  CAS  Google Scholar 

  6. Gray, P.A., Rekling, J.C., Bocchiaro, C.M. & Feldman, J.L. Modulation of respiratory frequency by peptidergic input to rhythmogenic neurons in the preBötzinger complex. Science 286, 1566–1568 (1999).

    Article  CAS  Google Scholar 

  7. Stornetta, R.L. et al. A group of glutamatergic interneurons expressing high levels of both neurokinin-1 receptors and somatostatin identifies the region of the pre-Bötzinger complex. J. Comp. Neurol. 455, 499–512 (2003).

    Article  CAS  Google Scholar 

  8. Tan, W., Pagliardini, S., Yang, P., Janczewski, W.A. & Feldman, J.L. Projections of preBötzinger Complex neurons in adult rats. J. Comp. Neurol. 518, 1862–1878 (2010).

    Article  Google Scholar 

  9. Gray, P.A., Janczewski, W.A., Mellen, N., McCrimmon, D.R. & Feldman, J.L. Normal breathing requires preBötzinger complex neurokinin-1 receptor–expressing neurons. Nat. Neurosci. 4, 927–930 (2001).

    Article  CAS  Google Scholar 

  10. Tan, W. et al. Silencing preBötzinger complex somatostatin-expressing neurons induces persistent apnea in awake rat. Nat. Neurosci. 11, 538–540 (2008).

    Article  CAS  Google Scholar 

  11. Blanchi, B. et al. MafB deficiency causes defective respiratory rhythmogenesis and fatal central apnea at birth. Nat. Neurosci. 6, 1091–1100 (2003).

    Article  CAS  Google Scholar 

  12. Wallén-Mackenzie, A. et al. Vesicular glutamate transporter 2 is required for central respiratory rhythm generation but not for locomotor central pattern generation. J. Neurosci. 26, 12294–12307 (2006).

    Article  Google Scholar 

  13. Koizumi, H. et al. Functional imaging, spatial reconstruction, and biophysical analysis of a respiratory motor circuit isolated in vitro. J. Neurosci. 28, 2353–2365 (2008).

    Article  CAS  Google Scholar 

  14. Koshiya, N. & Smith, J.C. Neuronal pacemaker for breathing visualized in vitro. Nature 400, 360–363 (1999).

    Article  CAS  Google Scholar 

  15. Jen, J.C. et al. Mutations in a human ROBO gene disrupt hindbrain axon pathway crossing and morphogenesis. Science 304, 1509–1513 (2004).

    Article  CAS  Google Scholar 

  16. Marillat, V. et al. The slit receptor Rig-1/Robo3 controls midline crossing by hindbrain precerebellar neurons and axons. Neuron 43, 69–79 (2004).

    Article  CAS  Google Scholar 

  17. Renier, N. et al. Genetic dissection of the function of hindbrain axonal commissures. PLoS Biol. 8, e1000325 (2010).

    Article  Google Scholar 

  18. Sabatier, C. et al. The divergent Robo family protein rig-1/Robo3 is a negative regulator of slit responsiveness required for midline crossing by commissural axons. Cell 117, 157–169 (2004).

    Article  CAS  Google Scholar 

  19. Zhang, Y. et al. V3 spinal neurons establish a robust and balanced locomotor rhythm during walking. Neuron 60, 84–96 (2008).

    Article  CAS  Google Scholar 

  20. Jessell, T.M. Neuronal specification in the spinal cord: inductive signals and transcriptional codes. Nat. Rev. Genet. 1, 20–29 (2000).

    Article  CAS  Google Scholar 

  21. Lumsden, A. & Krumlauf, R. Patterning the vertebrate neuraxis. Science 274, 1109–1115 (1996).

    Article  CAS  Google Scholar 

  22. Briscoe, J. & Ericson, J. Specification of neuronal fates in the ventral neural tube. Curr. Opin. Neurobiol. 11, 43–49 (2001).

    Article  CAS  Google Scholar 

  23. Garcia-Campmany, L., Stam, F.J. & Goulding, M. From circuits to behavior: motor networks in vertebrates. Curr. Opin. Neurobiol. 20, 116–125 (2010).

    Article  CAS  Google Scholar 

  24. Lanuza, G.M., Gosgnach, S., Pierani, A., Jessell, T.M. & Goulding, M. Genetic identification of spinal interneurons that coordinate left-right locomotor activity necessary for walking movements. Neuron 42, 375–386 (2004).

    Article  CAS  Google Scholar 

  25. Briscoe, J. et al. Homeobox gene Nkx2.2 and specification of neuronal identity by graded Sonic hedgehog signaling. Nature 398, 622–627 (1999).

    Article  CAS  Google Scholar 

  26. Pattyn, A., Hirsch, M.-R., Goridis, C. & Brunet, J.-F. Control of hindbrain motor neuron differentiation by the homeobox gene Phox2b. Development 127, 1349–1358 (2000).

    CAS  Google Scholar 

  27. Pattyn, A. et al. Coordinated temporal and spatial control of motor neuron and serotonergic neuron generation from a common pool of CNS progenitors. Genes Dev. 17, 729–737 (2003).

    Article  CAS  Google Scholar 

  28. Pattyn, A., Vallstedt, A., Dias, J.M., Sander, M. & Ericson, J. Complementary roles for Nkx6 and Nkx2 class proteins in the establishment of motoneuron identity in the hindbrain. Development 130, 4149–4159 (2003).

    Article  CAS  Google Scholar 

  29. Pierani, A. et al. Control of interneuron fate in the developing spinal cord by the progenitor homeodomain protein Dbx1. Neuron 29, 367–384 (2001).

    Article  CAS  Google Scholar 

  30. Pierani, A., Brenner-Morton, S., Chiang, C. & Jessell, T.M. A sonic hedgehog–independent, retinoid-activated pathway of neurogenesis in the ventral spinal cord. Cell 97, 903–915 (1999).

    Article  CAS  Google Scholar 

  31. Ericson, J. et al. Pax6 controls progenitor cell identity and neuronal fate in response to graded Shh signaling. Cell 90, 169–180 (1997).

    Article  CAS  Google Scholar 

  32. Thoby-Brisson, M., Trinh, J.B., Champagnat, J. & Fortin, G. Emergence of the pre-Bötzinger respiratory rhythm generator in the mouse embryo. J. Neurosci. 25, 4307–4318 (2005).

    Article  CAS  Google Scholar 

  33. Funk, G.D., Smith, J.C. & Feldman, J.L. Generation and transmission of respiratory oscillations in medullary slices: role of excitatory amino acids. J. Neurophysiol. 70, 1497–1515 (1993).

    Article  CAS  Google Scholar 

  34. Rekling, J.C. & Feldman, J.L. PreBötzinger complex and pacemaker neurons: hypothesized site and kernel for respiratory rhythm generation. Annu. Rev. Physiol. 60, 385–405 (1998).

    Article  CAS  Google Scholar 

  35. McKay, L.C., Janczewski, W.A. & Feldman, J.L. Sleep-disordered breathing after targeted ablation of preBötzinger complex neurons. Nat. Neurosci. 8, 1142–1144 (2005).

    Article  CAS  Google Scholar 

  36. Liu, Y.Y. et al. Relationship between two types of vesicular glutamate transporters and neurokinin-1 receptor–immunoreactive neurons in the pre-Bötzinger complex of rats: light and electron microscopic studies. Eur. J. Neurosci. 17, 41–48 (2003).

    Article  Google Scholar 

  37. Del Negro, C.A., Johnson, S.M., Butera, R.J. & Smith, J.C. Models of respiratory rhythm generation in the pre-Bötzinger complex. III. Experimental tests of model predictions. J. Neurophysiol. 86, 59–74 (2001).

    Article  CAS  Google Scholar 

  38. Johnson, S.M., Koshiya, N. & Smith, J.C. Isolation of the kernel for respiratory rhythm generation in a novel preparation: the pre-Bötzinger complex “island”. J. Neurophysiol. 85, 1772–1776 (2001).

    Article  CAS  Google Scholar 

  39. Bianchi, A.L., Denavit-Saubie, M. & Champagnat, J. Central control of breathing in mammals: neuronal circuitry, membrane properties and neurotransmitters. Physiol. Rev. 75, 1–45 (1995).

    Article  CAS  Google Scholar 

  40. Moran-Rivard, L. et al. Evx1 is a postmitotic determinant of v0 interneuron identity in the spinal cord. Neuron 29, 385–399 (2001).

    Article  CAS  Google Scholar 

  41. Geisen, M.J. et al. Hox paralog group 2 genes control the migration of mouse pontine neurons through slit-robo signaling. PLoS Biol. 6, e142 (2008).

    Article  Google Scholar 

  42. Wilson, S.I., Shafer, B., Lee, K.J. & Dodd, J. A molecular program for contralateral trajectory: Rig-1 control by LIM homeodomain transcription factors. Neuron 59, 413–424 (2008).

    Article  CAS  Google Scholar 

  43. Srour, M. et al. Mutations in DCC cause congenital mirror movements. Science 328, 592 (2010).

    Article  CAS  Google Scholar 

  44. Kiehn, O. Locomotor circuits in the mammalian spinal cord. Annu. Rev. Neurosci. 29, 279–306 (2006).

    Article  CAS  Google Scholar 

  45. Morgado-Valle, C., Baca, S.M. & Feldman, J.L. Glycinergic pacemaker neurons in preBötzinger complex of neonatal mouse. J. Neurosci. 30, 3634–3639 (2010).

    Article  CAS  Google Scholar 

  46. Cheng, L. et al. Tlx3 and Tlx1 are post-mitotic selector genes determining glutamatergic over GABAergic cell fates. Nat. Neurosci. 7, 510–517 (2004).

    Article  CAS  Google Scholar 

  47. Bielle, F. et al. Multiple origins of Cajal-Retzius cells at the borders of the developing pallium. Nat. Neurosci. 8, 1002–1012 (2005).

    Article  CAS  Google Scholar 

  48. Keller, C., Hansen, M.S., Coffin, C.M. & Capecchi, M.R. Pax3:Fkhr interferes with embryonic Pax3 and Pax7 function: implications for alveolar rhabdomyosarcoma cell of origin. Genes Dev. 18, 2608–2613 (2004).

    Article  CAS  Google Scholar 

  49. Cohen-Tannoudji, M. et al. I-SceI-induced gene replacement at a natural locus in embryonic stem cells. Mol. Cell. Biol. 18, 1444–1448 (1998).

    Article  CAS  Google Scholar 

  50. Bouvier, J. et al. Brain-derived neurotrophic factor enhances fetal respiratory rhythm frequency in the mouse preBötzinger complex in vitro. Eur. J. Neurosci. 28, 510–520 (2008).

    Article  Google Scholar 

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Acknowledgements

We thank J.-F. Brunet, C. Goridis and A. Lumsden for comments on the manuscript; M. Tessier-Lavigne for providing the Robo3 knockout; S. Karaz for providing specimens; and S. Autran and V. Mézières for technical assistance with genotyping. J.B. is supported by Région Ile-de-France and the Fondation pour la Recherche Médicale. This work was supported by grants from Fondation pour la Recherche Médicale Equipe (A.C.), the Association Française contre les Myopathies (ASS-SUB06-00123, A.C.), the Ville de Paris (2006 ASES 102, A.P.) and the Agence Nationale de la Recherche (ANR-05-NEUR-007-01 BIS to A.P., ANR-08-MNPS-030-01 to A.C. and ANR-07-NEUR-007-01 to G.F.). This work benefited from the facilities and expertise of the Imagif Cell Biology Unit and the Anicampus mouse facility of the Gif-sur-Yvette campus. This work was supported by Centre National de la Recherche Scientifique and the Institut de la Santé et de la Recherche Médicale.

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J.E., A.P., J.C. and G.F. conceived the study. N.R. and A.C. designed the Robo3 experiments. J.B. and M.T.-B. performed the experiments. V.D. carried out Vglut2 in situ hybridization. J.B., M.T.-B. and G.F. analyzed the data. J.E., A.C. and A.P. provided reagents and mice. G.F. wrote the paper. All of the authors discussed the results and implications and commented on the manuscript at all stages.

Corresponding authors

Correspondence to Alessandra Pierani, Alain Chédotal or Gilles Fortin.

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Split left/right breathing in Robo3 mutants. (MOV 735 kb)

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Bouvier, J., Thoby-Brisson, M., Renier, N. et al. Hindbrain interneurons and axon guidance signaling critical for breathing. Nat Neurosci 13, 1066–1074 (2010). https://doi.org/10.1038/nn.2622

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