Npr2 null mutants show initial overshooting followed by reduction of spiral ganglion axon projections combined with near-normal cochleotopic projection

  • Hannes Schmidt
  • Bernd FritzschEmail author
Regular Article


Npr2 (natriuretic peptide receptor 2) affects bifurcation of neural crest or placode-derived afferents upon entering the brain stem/spinal cord, leading to a lack of either rostral or caudal branches. Previous work has shown that early embryonic growth of cochlear and vestibular afferents is equally affected in this mutant but later work on postnatal Npr2 point mutations suggested some additional effects on the topology of afferent projections and mild functional defects. Using multicolor lipophilic dye tracing, we show that absence of Npr2 has little to no effect on the initial patterning of inner ear afferents with respect to their dorsoventral cochleotopic-specific projections. However, in contrast to control animals, we found a variable degree of embryonic extension of auditory afferents beyond the boundaries of the anterior cochlear nucleus into the cerebellum that emanates only from apical spiral ganglion neurons. Such expansion has previously only been reported for Hox gene mutants and implies an unclear interaction of Hox codes with Npr2-mediated afferent projection patterning to define boundaries. Some vestibular ganglion neurons expand their projections to reach the cochlear apex and the cochlear nuclei, comparable to previous findings in Neurod1 mutant mice. Before birth, such expansions are reduced or lost leading to truncated projections to the anteroventral cochlear nucleus and expansion of low-frequency fibers of the apex to the posteroventral cochlear nucleus.


Natriuretic peptide receptor 2 Npr2 null mutants Cochleotopic projection Axonal branching C-type natriuretic peptide 



anterior canal crista


anteroventral cochlear nucleus




cochlear nucleus




cochlear nerve


choroid plexus


cochleo-vestibular anastomosis


dorsal cochlear nucleus


endolymphatic duct


facial nerve


floor plate


horizontal canal crista


mesencephalic trigeminal projection


nodose ganglion


NeuroVue Jade


NeuroVue Maroon


NeuroVue Red


posterior canal crista


petrosal ganglion


posteroventral cochlear nucleus




restiform body




spiral ganglion




ventral acoustic stria

(a, p) VG

(anterior, posterior) vestibular ganglion


vestibular nerve


trigeminal ganglion


trigeminal motor neurons


octaval ganglion


facial (geniculate) ganglion


proximal glossopharyngeal ganglion


proximal vagal ganglion


transient accessory ganglia


Funding information

This study was financially supported by the NIH (R01 AG060504 to BF) and by the Deutsche Forschungsgemeinschaft (Grant FOR 2060 project SCHM 2371/1 to HS).

Compliance with ethical statements

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All applicable international, national and/or institutional guidelines for the care and use of animals were followed.


  1. Booth KT, Azaiez H, Jahan I, Smith RJ, Fritzsch B (2018) Intracellular regulome variability along the organ of Corti: evidence, approaches, challenges and perspective. Front Genet 9:156Google Scholar
  2. Chagnaud BP, Engelmann J, Fritzsch B, Glocr JC, Straka H (2017) Sensing external and self-motion with hair cells, a comparison of the lateral line and vestibular systems from a developmental and evolutionary perspective. Brain Behav Evol 90:98–116Google Scholar
  3. Dumoulin A, Ter-Avetisyan G, Schmidt H, Rathjen FG (2018) Molecular analysis of sensory axon branching unraveled a cGMP-dependent signaling cascade. Int J Mol Sci 19:e1266Google Scholar
  4. Duncan J, Kersigo J, Gray B, Fritzsch B (2011) Combining lipophilic dye, in situ hybridization, immunohistochemistry and histology. J Vis Exp 49:2451Google Scholar
  5. Duncan JS, Elliott KL, Kersigo J, Gray B, Fritzsch B (2015) Combining whole-mount in situ hybridization with neuronal tracing and immunohistochemistry. In: Situ Hybridization Methods. Springer, pp 339–352Google Scholar
  6. Elliott KL, Kersigo J, Pan N, Jahan I, Fritzsch B (2017) Spiral ganglion neuron projection development to the hindbrain in mice lacking peripheral and/or central target differentiation. Front Neural Circuits 11:25Google Scholar
  7. Farago AF, Awatramani RB, Dymecki SM (2006) Assembly of the brainstem cochlear nuclear complex is revealed by intersectional and subtractive genetic fate maps. Neuron 50:205–218CrossRefGoogle Scholar
  8. Fritzsch B (2003) Development of inner ear afferent connections: forming primary neurons and connecting them to the developing sensory epithelia. Brain Res Bull 60:423–433CrossRefGoogle Scholar
  9. Fritzsch B, Elliott KL (2017) Gene, cell and organ multiplication drives inner ear evolution. Dev Biol 431:3–156CrossRefGoogle Scholar
  10. Fritzsch B, López-Schier H (2014) Evolution of polarized hair cells in aquatic vertebrates and their connection to directionally sensitive neurons. In: Flow H. Bleckmann et al.(eds): Flow Sensing in Air and Water. Springer, New York, pp 271–294Google Scholar
  11. Fritzsch B, Muirhead K, Feng F, Gray B, Ohlsson-Wilhelm B (2005) Diffusion and imaging properties of three new lipophilic tracers, NeuroVue™ Maroon, NeuroVue™ Red and NeuroVue™ Green and their use for double and triple labeling of neuronal profile. Brain Res Bull 66:249–258CrossRefGoogle Scholar
  12. Fritzsch B, Pauley S, Feng F, Matei V, Nichols D (2006) The molecular and developmental basis of the evolution of the vertebrate auditory system. Int J Comp Psychol 19:1–25Google Scholar
  13. Fritzsch B, Pan N, Jahan I, Duncan JS, Kopecky BJ, Elliott KL, Kersigo J, Yang T (2013) Evolution and development of the tetrapod auditory system: an organ of Corti-centric perspective. Evol Dev 15:63–79CrossRefGoogle Scholar
  14. Fritzsch B, Pan N, Jahan I, Elliott KL (2015) Inner ear development: building a spiral ganglion and an organ of Corti out of unspecified ectoderm. Cell Tissue Res 361:7–24CrossRefGoogle Scholar
  15. Fritzsch B, Duncan JS, Kersigo J, Gray B, Elliott KL (2016) Neuroanatomical tracing techniques in the ear: history, state of the art and future developments. In: Sokolowski B (ed) Auditory and Vestibular Research: Methods and Protocols, vol 1427. Springer Science+Business Media, New York, pp 243–262CrossRefGoogle Scholar
  16. Fritzsch B, Elliott KL, Pavlinkova G (2019) Primary sensory map formations reflect unique needs and molecular cues specific to each sensory system. F1000Res 8:345Google Scholar
  17. Glover JC, Elliott KL, Erives A, Chizhikov VV, Fritzsch B (2018) Wilhelm His’ lasting insights into hindbrain and cranial ganglia development and evolution. Dev Biol 444:S14–S24CrossRefGoogle Scholar
  18. Goodrich LV (2016) Early development of the spiral ganglion. In: Dabdoub A, Fritzsch B, Fay R, Popper A (eds) The primary auditory neurons of the mammalian cochlea. Springer, New York, pp 11–48Google Scholar
  19. Gu C, Rodriguez ER, Reimert DV, Shu T, Fritzsch B, Richards LJ, Kolodkin AL, Ginty DD (2003) Neuropilin-1 conveys semaphorin and VEGF signaling during neural and cardiovascular development. Dev Cell 5:45–57CrossRefGoogle Scholar
  20. Jahan I, Kersigo J, Pan N, Fritzsch B (2010a) Neurod1 regulates survival and formation of connections in mouse ear and brain. Cell Tissue Res 341:95–110CrossRefGoogle Scholar
  21. Jahan I, Pan N, Kersigo J, Fritzsch B (2010b) Neurod1 suppresses hair cell differentiation in ear ganglia and regulates hair cell subtype development in the cochlea. PLoS One 5:e11661CrossRefGoogle Scholar
  22. Jensen-Smith H, Gray B, Muirhead K, Ohlsson-Wilhelm B, Fritzsch B (2007) Long-distance three-color neuronal tracing in fixed tissue using NeuroVue dyes. Immunol Investig 36:763–789CrossRefGoogle Scholar
  23. Jiang T, Kindt K, Wu DK (2017) Transcription factor Emx2 controls stereociliary bundle orientation of sensory hair cells. Elife 6:e23661CrossRefGoogle Scholar
  24. Kaiser A, Manley GA (1996) Brainstem cconnections of the macula lagenae in the chicken. J Comp Neurol 374:108–117CrossRefGoogle Scholar
  25. Karis A, Pata I, van Doorninck JH, Grosveld F, de Zeeuw CI, de Caprona D, Fritzsch B (2001) Transcription factor GATA-3 alters pathway selection of olivocochlear neurons and affects morphogenesis of the ear. J Comp Neurol 429:615–630CrossRefGoogle Scholar
  26. Kersigo J, Pan N, Lederman JD, Chatterjee S, Abel T, Pavlinkova G, Silos-Santiago I, Fritzsch B (2018) A RNAscope whole mount approach that can be combined with immunofluorescence to quantify differential distribution of mRNA. Cell Tissue Res 374:251–262CrossRefGoogle Scholar
  27. Kopecky B, Santi P, Johnson S, Schmitz H, Fritzsch B (2011) Conditional deletion of N-Myc disrupts neurosensory and non-sensory development of the ear. Dev Dyn 240:1373–1390CrossRefGoogle Scholar
  28. Lu CC, Appler JM, Houseman EA, Goodrich LV (2011) Developmental profiling of spiral ganglion neurons reveals insights into auditory circuit assembly. J Neurosci 31:10903–10918CrossRefGoogle Scholar
  29. Lu CC, Cao X-J, Wright S, Ma L, Oertel D, Goodrich LV (2014) Mutation of Npr2 leads to blurred tonotopic organization of central auditory circuits in mice. PLoS Genet 10:e1004823CrossRefGoogle Scholar
  30. Macova I, Pysanenko K, Chumak T, Dvorakova M, Bohuslavova R, Syka J, Fritzsch B, Pavlinkova G (2019) Neurod1 is essential for the primary tonotopic organization and related auditory information processing in the midbrain. J Neurosci 39:984–1004CrossRefGoogle Scholar
  31. Mahmoud A, Reed C, Maklad A (2013) Central projections of lagenar primary neurons in the chick. J Comp Neurol 521:3524–3540CrossRefGoogle Scholar
  32. Maklad A, Fritzsch B (2003a) Development of vestibular afferent projections into the hindbrain and their central targets. Brain Res Bull 60:497–510CrossRefGoogle Scholar
  33. Maklad A, Fritzsch B (2003b) Partial segregation of posterior crista and saccular fibers to the nodulus and uvula of the cerebellum in mice and its development. Brain Res Dev Brain Res 140:223–236CrossRefGoogle Scholar
  34. Maklad A, Kamel S, Wong E, Fritzsch B (2010) Development and organization of polarity-specific segregation of primary vestibular afferent fibers in mice. Cell Tissue Res 340:303–321CrossRefGoogle Scholar
  35. Malmierca MS (2015) Auditory system. The rat nervous system, 4th edn. Elsevier, pp 865–946Google Scholar
  36. Mann HB, Whitney DR (1947) On a test of whether one of two random variables is stochastically larger than the other. Ann Math Stat. Whitney 18:50–60Google Scholar
  37. Mao Y, Reiprich S, Wegner M, Fritzsch B (2014) Targeted deletion of Sox10 by Wnt1-cre defects neuronal migration and projection in the mouse inner ear. PLoS One 9:e94580CrossRefGoogle Scholar
  38. Maricich SM, Xia A, Mathes EL, Wang VY, Oghalai JS, Fritzsch B, Zoghbi HY (2009) Atoh1-lineal neurons are required for hearing and for the survival of neurons in the spiral ganglion and brainstem accessory auditory nuclei. J Neurosci 29:11123–11133CrossRefGoogle Scholar
  39. Matei V, Pauley S, Kaing S, Rowitch D, Beisel K, Morris K, Feng F, Jones K, Lee J, Fritzsch B (2005) Smaller inner ear sensory epithelia in Neurog1 null mice are related to earlier hair cell cycle exit. Dev Dyn 234:633–650CrossRefGoogle Scholar
  40. Matei V, Feng F, Pauley S, Beisel K, Nichols M, Fritzsch B (2006) Near-infrared laser illumination transforms the fluorescence absorbing X-gal reaction product BCI into a transparent, yet brightly fluorescent substance. Brain Res Bull 70:33–43CrossRefGoogle Scholar
  41. Muniak MA, Connelly CJ, Suthakar K, Milinkeviciute G, Ayeni FE, Ryugo DK (2016) Central projections of spiral ganglion neurons. In: A Dabdoub, B Fritzsch, R Fay, A Popper (eds). The primary auditory neurons of the mammalian cochlea. Springer, New York, pp 157–190Google Scholar
  42. Nichols DH, Pauley S, Jahan I, Beisel KW, Millen KJ, Fritzsch B (2008) Lmx1a is required for segregation of sensory epithelia and normal ear histogenesis and morphogenesis. Cell Tissue Res 334:339–358CrossRefGoogle Scholar
  43. Osen KK (1969) Cytoarchitecture of the cochlear nuclei in the cat. J Comp Neurol 136:453–483CrossRefGoogle Scholar
  44. Oury F, Murakami Y, Renaud J-S, Pasqualetti M, Charnay P, Ren S-Y, Rijli FM (2006) Hoxa2-and rhombomere-dependent development of the mouse facial somatosensory map. Science 313:1408–1413CrossRefGoogle Scholar
  45. Pataskar A, Jung J, Smialowski P, Noack F, Calegari F, Straub T, Tiwari VK (2016) NeuroD1 reprograms chromatin and transcription factor landscapes to induce the neuronal program. EMBO J 35:24–45CrossRefGoogle Scholar
  46. Pauley S, Lai E, Fritzsch B (2006) Foxg1 is required for morphogenesis and histogenesis of the mammalian inner ear. Dev Dyn 235:2470–2482CrossRefGoogle Scholar
  47. Ruben RJ (1966) Development of the inner ear of the mouse: a radioautographic study of terminal mitoses. Acta Otolaryngol Suppl 220:221–244Google Scholar
  48. Schmidt H, Werner M, Heppenstall PA, Henning M, Moré MI, Kühbandner S, Lewin GR, Hofmann F, Feil R, Rathjen FG (2002) cGMP-mediated signaling via cGKIα is required for the guidance and connectivity of sensory axons. J Cell Biol 159:489–498CrossRefGoogle Scholar
  49. Schmidt H, Stonkute A, Jüttner R, Schäffer S, Buttgereit J, Feil R, Hofmann F, Rathjen FG (2007) The receptor guanylyl cyclase Npr2 is essential for sensory axon bifurcation within the spinal cord. J Cell Biol 179:331–340CrossRefGoogle Scholar
  50. Schmidt H, Stonkute A, Jüttner R, Koesling D, Friebe A, Rathjen FG (2009) C-type natriuretic peptide (CNP) is a bifurcation factor for sensory neurons. Proc Natl Acad Sci 106:16847–16852CrossRefGoogle Scholar
  51. Schultz JA, Zeller U, Luo ZX (2017) Inner ear labyrinth anatomy of monotremes and implications for mammalian inner ear evolution. J Morphol 278:236–263CrossRefGoogle Scholar
  52. Stefanini M, de Martino C, Zamboni L (1967) Fixation of ejaculated spermatozoa for electron microscopy. Nature 216:173CrossRefGoogle Scholar
  53. Ter-Avetisyan G, Rathjen FG, Schmidt H (2014) Bifurcation of axons from cranial sensory neurons is disabled in the absence of Npr2-induced cGMP signaling. J Neurosci 34:737–747CrossRefGoogle Scholar
  54. Ter-Avetisyan G, Dumoulin A, Herrel A, Schmidt H, Strump J, Afzal S, Rathjen FG (2018) Loss of axon bifurcation in mesencephalic trigeminal neurons impairs the maximal biting force in Npr2-deficient mice. Front Cell Neurosci 12:153CrossRefGoogle Scholar
  55. Tonniges J, Hansen M, Duncan J, Bassett M, Fritzsch B, Gray B, Easwaran A, Nichols MG (2010) Photo-and bio-physical characterization of novel violet and near-infrared lipophilic fluorophores for neuronal tracing. J Microsc 239:117–134Google Scholar
  56. Tröster P, Haseleu J, Petersen J, Drees O, Schmidtko A, Schwaller F, Lewin GR, Ter-Avetisyan G, Winter Y, Peters S (2018) The absence of sensory axon bifurcation affects nociception and termination fields of afferents in the spinal cord. Front Mol Neurosci 11:19CrossRefGoogle Scholar
  57. Tsuji T, Kunieda T (2005) A loss-of-function mutation in natriuretic peptide receptor 2 (Npr2) gene is responsible for disproportionate dwarfism in cn/cn mouse. J Biol Chem 280:14288–14292CrossRefGoogle Scholar
  58. Wolter S, Möhrle D, Schmidt H, Pfeiffer S, Zelle D, Eckert P, Krämer M, Feil R, Pilz PK, Knipper M (2018) GC-B deficient mice with axon bifurcation loss exhibit compromised auditory processing. Front Neural Circuits 12:65CrossRefGoogle Scholar
  59. Xiang M, Maklad A, Pirvola U, Fritzsch B (2003) Brn3c null mutant mice show long-term, incomplete retention of some afferent inner ear innervation. BMC Neurosci 4:2CrossRefGoogle Scholar
  60. Yang T, Kersigo J, Jahan I, Pan N, Fritzsch B (2011) The molecular basis of making spiral ganglion neurons and connecting them to hair cells of the organ of Corti. Hear Res 278:21–33CrossRefGoogle Scholar
  61. Yang T, Kersigo J, Wu S, Fritzsch B, Bassuk AG (2017) Prickle1 regulates neurite outgrowth of apical spiral ganglion neurons but not hair cell polarity in the murine cochlea. PLoS One 12:e0183773CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Interfaculty Institute of BiochemistryUniversity of TübingenTübingenGermany
  2. 2.Department of Biology & Department of Otolaryngology, CLASUniversity of IowaIowa CityUSA

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