Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Aug 5:10:186.
doi: 10.3389/fncel.2016.00186. eCollection 2016.

LGR4 and LGR5 Regulate Hair Cell Differentiation in the Sensory Epithelium of the Developing Mouse Cochlea

Magdalena Żak  1 , Thijs van Oort  1 , Ferry G Hendriksen  1 , Marie-Isabelle Garcia  2 , Gilbert Vassart  2 , Wilko Grolman  1
Affiliations

LGR4 and LGR5 Regulate Hair Cell Differentiation in the Sensory Epithelium of the Developing Mouse Cochlea

Magdalena Żak et al. Front Cell Neurosci. .

Abstract

In the developing cochlea, Wnt/β-catenin signaling positively regulates the proliferation of precursors and promotes the formation of hair cells by up-regulating Atoh1 expression. Not much, however, is known about the regulation of Wnt/β-catenin activity in the cochlea. In multiple tissues, the activity of Wnt/β-catenin signaling is modulated by an interaction between LGR receptors and their ligands from the R-spondin family. The deficiency in Lgr4 and Lgr5 genes leads to developmental malformations and lethality. Using the Lgr5 knock-in mouse line we show that loss of LGR5 function increases Wnt/β-catenin activity in the embryonic cochlea, resulting in a mild overproduction of inner and outer hair cells (OHC). Supernumerary hair cells are likely formed due to an up-regulation of the "pro-hair cell" transcription factors Atoh1, Nhlh1, and Pou4f3. Using a hypomorphic Lgr4 mouse model we showed a mild overproduction of OHCs in the heterozygous and homozygous Lgr4 mice. The loss of LGR4 function prolonged the proliferation in the mid-basal turn of E13 cochleae, causing an increase in the number of SOX2-positive precursor cells within the pro-sensory domain. The premature differentiation of hair cells progressed in a medial to lateral gradient in Lgr4 deficient embryos. No significant up-regulation of Atoh1 was observed following Lgr4 deletion. Altogether, our findings suggest that LGR4 and LGR5 play an important role in the regulation of hair cell differentiation in the embryonic cochlea.

Keywords: LGR4; LGR5; Wnt signaling; cochlea; development; hair cells.

PubMed Disclaimer

Figures

FIGURE 1
FIGURE 1
Overproduction of hair cells in Lgr5-eGFP mice. (A) A reconstruction of Z-stack images of the cochlear mid-basal turns stained with phalloidin. Homozygous Lgr5-eGFP mice showed overproduction of IHCs (arrows) and OHCs (arrowheads). (B) The number of IHCs was significantly increased along the cochlear duct in the homozygous Lgr5-eGFP mice. (C) The number of OHCs was also significantly increased in the mid-basal turn of the cochleae isolated from homozygous Lgr5-eGFP mice. (D) Comparing the length of basilar membrane between the Lgr5 deficient mice and their wild type littermates.
FIGURE 2
FIGURE 2
qPCR results for the E13 cochleae of Lgr5-eGFP embryos. The expression of Lgr5 significantly decreased in the cochleae isolated from homozygous and heterozygous Lgr5-eGFP mice. The expression from Lgr5 exon1 and the expression of Lgr6, Atoh1, and Nhlh1 significantly increased in homozygous and heterozygous Lgr5-eGFP mice, while the expression of Pou4f3 increased only in the homozygous Lgr5-eGFP mice. The expression of Lgr4 was unaffected.
FIGURE 3
FIGURE 3
The expression of cell cycle regulators in the E13 cochleae of Lgr5-eGFP embryos. The expression of Ccnd1, p27Kip1, and p57Kip2 did not change in the Lgr5 deficient mice.
FIGURE 4
FIGURE 4
Proliferation in the cochleae of E14.5 Lgr5-eGFP embryos. (A–B′′) SOX2-positive pro-sensory cells (green, arrow) did not incorporate EdU (red). Cell nuclei were counterstained with DAPI (blue), scale bars indicate 25 μm.
FIGURE 5
FIGURE 5
The distribution of LGR4 in the developing mouse cochlea. (A) At E14.5, strong LGR4 staining was detected in the cochlear duct and spiral ganglion cells (white arrowhead). LGR4 was detected in the pro-sensory domain, where it overlapped with SOX2 (white arrow), and non-sensory domains lateral and medial to the pro-sensory domain. (B) At E17, LGR4 (green) was detected in IHCs and OHCs (asterisks) and their surrounding supporting cells. Hair cells were labeled with phalloidin (F-actin) (red). (C) At P1, LGR4 expression was observed in the sensory epithelium (arrowhead) and spiral ganglion cells (arrow), from which projected TUJ1-positive nerves (red). (D) At P7, LGR4 was observed in hair cells (asterisks) that co-expressed Prestin (PRES) and in the surrounding supporting cells: Deiters’ cells (arrowheads) and IPCs and OPCs. (E–G) At P21, LGR4 was down-regulated in the OHCs and pillar cells, but was still expressed in the IHCs (arrowhead), Deiters’ cells (arrows), which were also positive for acetylated tubulin (ACTBA), and phalangeal processes of Deiters’ cells (arrowheads). LGR4 signals were also observed in the spiral ganglion cells. (H) At P42, LGR4 was detected in Deiters’ cells and their phalangeal processes. Cell nuclei were counterstained with DAPI (blue), asterisks indicate hair cells, scale bars indicate 20 μm in the picture (B,D,E,G,H) and 50 μm in (A,C,F). (I,J) Cochlear cryosections from Lgr4-LacZ mice at the age of P21 were stained for β-galactosidase activity. Expression was seen in Deiters’ cells (black arrow) and their phalangeal processes (gray arrow), IHCs (black arrowhead), and spiral ganglion cells.
FIGURE 6
FIGURE 6
Overproduction of hair cells in Lgr4-LacZ mice. (A) Cochlear whole mounts captured by SEM. Both heterozygous and homozygous Lgr4-LacZ mice showed supernumerary OHCs. (B,C) Counting IHCs and OHCs along the cochlear duct showed a significant increase in the number of OHCs at 50% of cochleae obtained from heterozygous and homozygous Lgr4-LacZ mice. (D) Comparing the length of basilar membrane between the Lgr4 deficient mice and their wild type littermates.
FIGURE 7
FIGURE 7
Hair cell formation in the embryonic cochlea of Lgr4-LacZ mice. (A–D′′) The cochlear cryosections obtained from Lgr4 deficient mice and their wild type counterparts were stained for a hair cell marker (MYO7a, green) and two pro-sensory cell markers (SOX2, and JAG1, both red). Developing hair cells (asterisks) were detected only in the mid-basal turns. Cell nuclei were counterstained with DAPI (blue), scale bars indicate 20 μm.
FIGURE 8
FIGURE 8
The expression of “pro-hair cell genes” and cell cycle regulators in the E13 cochleae of Lgr4 deficient mice. The expression of Atoh1, Nhlh1, and Pou4f3 did not change, while the expression of Ccnd1 significantly increased in heterozygous and homozygous Lgr4 mice.
FIGURE 9
FIGURE 9
Proliferation in the embryonic cochleae of Lgr4 deficient mice. (A–B′′) In the cochleae from heterozygous and homozygous Lgr4-LacZ mice, CCND1 (green) expression increased in the mid-basal turn, where CCND1 merged with red signal of SOX2 (arrows) in the pro-sensory domain. (C–D′′) The proliferation marker, EdU (red), was incorporated into pro-sensory cells (arrows) positive for SOX2 (green) in the mid-basal turn of the cochlea from Lgr4 deficient mice. Cell nuclei were counterstained with DAPI (blue), scale bars indicate 20 μm.
FIGURE 10
FIGURE 10
The expression of Wnt/β-catenin target genes in the E13 cochleae of Lgr4 deficient mice. The expression of Lgr4 significantly decreased in the cochleae of Lgr4 deficient mice, while the expression of Lgr5 and the expression from Lgr5 exon1 significantly increased. The expression of Lgr6 did not change, but the expression of and Axin2 significantly decreased in heterozygous and homozygous Lgr4 mice.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Adam J., Myat A., Le Roux I., Eddison M., Henrique D., Ish-Horowicz D., et al. (1998). Cell fate choices and the expression of Notch, Delta and Serrate homologues in the chick inner ear: parallels with Drosophila sense-organ development. Development 125 4645–4654. - PubMed
    1. Atkinson P. J., Huarcaya Najarro E., Sayyid Z. N., Cheng A. G. (2015). Sensory hair cell development and regeneration: similarities and differences. Development 142 1561–1571. 10.1242/dev.114926 - DOI - PMC - PubMed
    1. Barker N., Huch M., Kujala P., van de Wetering M., Snippert H. J., van Es J. H., et al. (2010). Lgr5(+ve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro. Cell Stem Cell 6 25–36. 10.1016/j.stem.2009.11.013 - DOI - PubMed
    1. Barker N., van Es J. H., Kuipers J., Kujala P., van den Born M., Cozijnsen M., et al. (2007). Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449 1003–1007. 10.1038/nature06196 - DOI - PubMed
    1. Bermingham N. A., Hassan B. A., Price S. D., Vollrath M. A., Ben-Arie N., Eatock R. A., et al. (1999). Math1: an essential gene for the generation of inner ear hair cells. Science 284 1837–1841. 10.1126/science.284.5421.1837 - DOI - PubMed

LinkOut - more resources