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Custom Antibody Highlights: Pou3f4-expressing non-neuronal cells regulate auditory sensory neuron development: Effects of non-neuronal cells on axonal bundling and trophic support

by Gary Ciment, Ph.D., Scientific Director, Aves Labs, Inc.

Auditory sensory neurons of the inner ear innervate hair cells within the cochlea, and transmit this information to auditory nuclei within the brain stem. The peripheral ganglion they form -- the cochlear ganglion -- appears next to the otic vesicle around embryonic day 10.5 in the mouse (Ruben, 1967). As this ganglion condenses, nascent sensory neurons differentiate into bipolar neurons with one process (the central axon) growing into the brain stem, and another process (the peripheral axon) growing towards nascent hair cells within the developing cochlea -- axonal guidance feats that navigate very complex anatomies both centrally and peripherally.

Non-neuronal cellular components of the auditory apparatus are critically important in the morphogenesis of the cochlea. These cells are derived from head neural crest cells, and are known to express the transcription factor Pou3f4 (named for its DNA sequence homology to the Pit, Oct and Unc transcription factors; GenBank accession number EDL14021.1) (Ryan and Rosenfeld, 1997; Coate et al., 2012). It is known, moreover, that mutations of the Pou3f4 gene in humans and other mammals are responsible for the most common form of congenital X-linked deafness -- the DFNX2 mutation (Kok et al., 1995) -- suggesting that these cells must play important morphogenic roles in cochlear ganglion development.

To understand the roles Pou3f4-positive non-neuronal cells might play, Dr. Thomas Coate (Department of Biology, Georgetown University in Washington, D.C.) asked Aves Labs for help in designing a chicken anti-mouse Pou3f4 anti-peptide antibody that would be useful in his group's immunohistochemistry (IHC) studies. By using this alternative  host for these antibodies, his thinking was that these chicken IgY antibodies would be useful alongside the many marker antibodies made in rabbits and mice.

Choosing peptide sequences within any DNA-binding nuclear protein, including transcription factors, is a little trickier than your garden-variety cytoplasmic protein due to issues of steric hindrance by the DNA and the tight quarters of the nucleus.  Epitopes involved in DNA binding, or even those far away from DNA-binding regions but masked by this huge polymer, would need to be avoided. Using our proprietary Immunogenicity Algorithm®, we identified one particularly promising peptide sequence within the mouse Pou3f4 sequence that fit our criteria for immunogencity (see Figure 1, below). This consisted of a 22-mer near the middle of this protein (residues #158-179 of the 341 residue protein). After consultation with Dr. Coate, we added a cysteine and a synthetic spacer amino acid to the N-terminus of this sequence, and synthesized the now 24-mer peptide (CZ STQ SLH PVL REP RDH GEL GSH H, where "Z" corresponds to aminocaproic acid -- a 6-carbon spacer epsilon amino acid).


Figure 1. Location of the peptide sequence (yellow box, residues #158-179) within Pou3f4 transcription factor identified by the Immunogenicity Algorithm®. The pink box represents the Pou3f4 DNA-binding region of the protein (residues #278-337).  The red double helix at the bottom of the figure represents the DNA, including the Pou3f4 transcription factor-binding domain on the gene.

A KLH-conjugate of this peptide was injected over a 7-week period into the breast muscles of two laying hens, and then eggs were collected over a 3-week period after the 4th injection. From 12 of these eggs, we purified 2178 mg of IgY using our proprietary methods, and from this IgY preparation, we further purified 29 mg of affinity purified antibody. Since the hens were unharmed during the egg collection phase, we also performed additional injections and collected additional eggs.

Using this antibody along with others, Dr. Coate's group and collaborators found a number of effects of Pou3f4 non-neuronal cells on cochlear ganglion development. To determine whether these non-neuronal cells had a trophic effect on auditory sensory neurons in the cochlear ganglion, they looked at auditory sensory neuronal numbers. Figure 2 shows the anatomical relationship between the sensory neurons of the auditory (spiral) ganglion at various post-natal ages. Panel b is a P0 mouse (i.e., neonatal); panel c is a P8 mouse (i.e., post-natal day 8); panel d is a P29 mouse.  Neurons, as identified by HuD staining, are seen in green, whereas non-neuronal mesenchymal cells expressing the Pou3/4 gene product are seen in magenta.  Panel h quantifies the data from wild-type and Pou3/4-knockout mice, showing statistically significant differences in neuronal cell numbers.


Figure 2. Immunohistochemical-staining studies of sections through the cochlea at various post-natal stages in the mouse (panel b. P0; panel c. P8; panel d, P29).  In these photomicrographs, the cochlear (spiral) ganglion with its sensory neurons expressing the HuD gene product is seen in green, and the non-neuronal head mesenchymal cells making up most of the other cell types within the cochlea are seen in magenta.  Panel e is a low-power and high-power diagram of the location of these cells, and shows the anatomical relation-ship with the hair cells. Type I sensory neurons SGN innervate inner hair cells; type II sensory neurons innervate outer hair cells;  Panel h shows neuronal cell numbers in the cochlear (spiral) ganglion of P29 and P45 mice of wild-type (black dots in bars 1 and 3) and Pou3f4-knockout mice (blue dots in bars 2 and 4). Panel h shows that Pou3/4f-knock out mice contain significantly fewer sensory neurons in the auditory ganglion.  (panels taken from Figure 1 of Brooks et al., 2020)


These studies show clear trophic interactions between the Pou3/4f-expressing non-neuronal cells of the embryonic and early post-natal cochlea and the sensory neurons that innervate the hair cells of the organ of Corti.

References

  • Brooks, P.M., Rose, K.P., Macrae, M.L., Rangoussis, K.M., Gurjar, M., Hertzano, R. and Coate, T.M.  (2020).  Pou3f4-Expressing Otic Mesenchyme Cells Promote Spiral Ganglion Neuron Survival in the Postnatal Mouse Cochlea.  Research in Systems Neuroscience DOI: 10.1002/cne.24867.
  • Coate, T.M., Raft, S., Zhao, X., Ryan, A.K., Crenshaw III, E.B. and Kelley, M.W. (2012).  Otic Mesenchyme Cells Regulate Spiral Ganglion Axon Fasciculation through a Pou3f4/EphA4 Signaling Pathway.  Neuron 73(1): 49-63.
  • Coate, T.M., Spita, N.A., Zhang, K.D., Isgrig, K.T. and Kelley, M.W.  (2015). Neuropilin-2/Semaphorin-3F-Mediated Repulsion Promotes Inner Hair Cell Innervation by Spiral Ganglion Neurons.  eLife DOI: 10.7554/eLife.07830.
  • Kok, Y. J. M. De, Maarel, S. M. Van Der, Bitner-glindzicz, M., Malcolm, S., Pembrey, M. E., Ropers, H., and Cremers, M. (1995). Association Between X-Linked Mixed Deafness and Mutations in the POU Domain Gene POU3F4.  Science 267 (5198): 685–688.
  • Ruben RJ.  (1967).  Development of the inner ear of the mouse: a radioautographic study of terminal mitoses.  Acta Otolaryngol. (Suppl 220):221–244.
  • Ryan AK, Rosenfeld MG.  (1997).  POU domain family values: flexibility, partnerships, and developmental codes. Genes Dev. 11: 1207–1225.