FIGURE SUMMARY
Title

Semicircular canal morphogenesis in the zebrafish inner ear requires the function of gpr126 (lauscher), an adhesion class G protein-coupled receptor gene

Authors
Geng, F.S., Abbas, L., Baxendale, S., Holdsworth, C.J., Swanson, A.G., Slanchev, K., Hammerschmidt, M., Topczewski, J., and Whitfield, T.T.
Source
Full text @ Development

Semicircular canal morphogenesis in the zebrafish ear. (A-F) Sketches of the developing semicircular canal system in the wild-type zebrafish ear, showing projection outgrowth, fusion and pillar formation. (G-L) Expression of vcana in the wild-type ear. (A,G) At 48 hpf, both anterior (A) and posterior (P) projections have begun to grow, and to express vcana at their tips. (B,H) The lateral projection, with its A and P bulges, is present by 50 hpf. The bulges and projections express vcana strongly at this time. The apparent downward growth of the A projection in H may be an artefact of fixation. (C,I) From 57-68 hpf, the A and P projections and bulges fuse to form the A and P pillars. The lateral projection now forms a ventral (V) bulge, and a V projection develops. Expression of vcana is downregulated in the A and P pillars, but is now strongly expressed in the V bulge and projection. (D,J) As fusion is completed at <70 hpf, expression of vcana is downregulated in all pillars. Expression remains in the dorsolateral septum (DLS) at 84 hpf. (E,K) At 72 hpf, all three pillars are fused [timing of fusion was slightly later than previously reported (Waterman and Bell, 1984)]. (F,L) At 4-5 dpf, only a trace of vcana expression remains in the DLS. Grey shading indicates the canal lumens. The positions of the cristae are shown. Abbreviations: A, anterior; DLS, dorsolateral septum; P, posterior; proj., projection; ssc, semicircular canal; V, ventral. Scale bar: 50 μm.

lauscher mutant zebrafish have swollen ears at 5 dpf and defects in semicircular canal formation. (A-F) Live images of wild-type sibling (A,B) and homozygous mutant (C-F) embryos at 5 dpf. The ears (arrowheads) of the mutants are swollen at 5 dpf, but otoliths appear normal. (G-J) Live images of wild-type sibling (G,H) and tb233c mutant (I,J) ears at 5 dpf. (K) Injection of hyaluronidase into the lateral projection of the left ear of a wild-type embryo results in the collapse of the projection, failure of fusion and a swollen ear. (L) Injection of PBS into the lateral projection of the left ear has no effect on ear development. Arrowheads mark the injected ears. (M,N) Comparison of ear swelling and projection fusion in the tb233c allele (M) with an ear in which the lateral projection has been injected with hyaluronidase (N) (dorsal views). Black arrows mark unfused projections; white arrows mark fused pillars. Asterisk in J marks projections that have touched but not fused correctly. Abbreviations: a and p, lumens of anterior and posterior semicircular canals; lp, enlarged lateral projection. A,C,E,F,H,J-N are dorsal views. Scale bars: in A, 200 μm for A,C,E,F,K,L; in B, 200 μm for B,D; in G, 50 μm for G-J.

Expression of genes involved in endolymph homeostasis in the lau mutant ear. (A-D) The endolymphatic duct, marked by expression of bmp4 (arrowheads, A,B) and foxi1 (C,D), appears to develop normally in lau mutants. The three cristae also express bmp4 normally (asterisks, A,B). (E-J) Expression of ion transporter genes in the lau mutant ear. (E,F) Expression of the atp1a1a.4 subunit of Na+/K+-ATPase1 is more diffuse and lacking from the ventral pillar (vp). (G-J) There is a substantial reduction in both kcnq1 and nkcc1 expression. Abbreviation: vp, ventral pillar (E) or ventral projection (F). Alleles: B,D,H,J, tk256a; F, tb233c. Scale bars: 50 μm.

Expression of extracellular matrix (ECM) genes and other semicircular canal markers is altered in the lauscher mutant ear. (A-JJ) Expression of ECM structural and enzyme genes in lau mutant ears at 76 hpf, 4 dpf and 5 dpf. Expression of the HA binding hapln1a (A-F) and the versican genes vcana and vcanb (M-X) is highly upregulated in mutant canal tissue. Expression of hapln3 is normally upregulated in wild-type ears on canal projection fusion (G,I,K); in the mutant, fusion fails, and transcript levels remain low (H,J,L). Antibody staining for type II Collagen (Y-DD) shows precocious protein accumulation at 76 hpf in mutants (Y,Z), persisting at 4-5 dpf (AA-DD); aberrant canal tissue is visible. Genes coding for enzymes for chondroitin synthesis (chsy1, EE,FF) and HA production (has3, GG,HH; ugdh, II,JJ) are upregulated in mutants at 76 hpf. (KK-PP) The canal markers aldh1a3, bmp7b and sox9b are upregulated in the unfused projections in the lau mutant ear. wt, wild type; sib, phenotypically wild-type sibling. Mutant alleles are shown on the panels. Scale bars: in A, 50 μm (applies to columns 1 and 2); in C, 50 μm (applies to all other panels).

Positional cloning, identification and confirmation of mutations in gpr126. (A) Genetic map of the lau locus. Numbers of meiotic recombinants for the flanking SSLP markers and SNP markers in slc25a27, schnurri2 and galnt14 are shown. (B) Sequence analysis of gpr126 cDNA in wild type (upper panels) and lau mutants (lower panels), with predicted changes to the coding sequence. (C) Schematic diagram of the Gpr126 protein, with its conserved domains: CUB (Complement C1r/C1s, Uegf, BMP1), PTX (Pentraxin), HBD (hormone binding domain), GAIN (GPCR autoproteolysis inducing) domain, GPS (GPCR proteolytic site) motif and 7TM (7-transmembrane) domain (not to scale). Positions of the mutations are shown. (D) Amino acid comparison of the fourth transmembrane (TMIV) region from eight zebrafish adhesion class GPCRs, showing the conserved hydrophobic residue at position 963 (mutated in tb233c), and the highly conserved proline (P) at position 969 (mutated in tk256a) (asterisks). (E) Genotyping of lau mutant fish by restriction digest of PCR-amplified genomic DNA. In tb233c, the mutation eliminated an SfaN1 site; in tk256a, the mutation eliminated a BsmF1 site; in the fr24 allele, a Bfa1 site was gained. (F,G) gpr126 morpholino injection recapitulates the lau mutant ear phenotype. (F) Wild-type (nac) embryos (left hand panels) injected with 5 ng control morpholino exhibit normal ear morphology at 5 dpf (a,c), and low vcanb expression at 5 dpf (e). Wild-type (nac) embryos injected with 5 ng gpr126 morpholino (right hand panels) have abnormal projection outgrowth (b,d). The lateral projection (lp) is enlarged, and the posterior projection (pp) in this ear has grown past the lateral projection without fusing. Expression of vcanb is upregulated (f). (G) RT-PCR analysis of gpr126 mRNA processing of the 19th exon in gpr126 morpholino-injected, or in control (mismatched) morpholino-injected, embryos at 5 dpf. Sequencing confirmed two aberrant splice variants. Panels c,d are dorsal views, anterior towards the top. Scale bars: 200 μm in a,b; 50 μm in c,d; 50 μm in e,f.

Expression of gpr126 in wild-type and lauscher mutant ears. (A-H) Expression of gpr126 mRNA in the ear at 24-96 hpf in wild-type (A-D) and fr24 mutant embryos (E-H). Strongest expression is in canal projections prior to fusion (48-72 hpf), with some expression remaining at 96 hpf. Inset in D: higher magnification showing expression in the anterior macula supporting cell layer. (I,M) Wild-type expression of gpr126 at 26 hpf in the anterior macula (I, dorsal view; M, transverse section). (J,N) Expression at 48 hpf shows stronger staining in the projections and sensory patches (J, dorsal view; N, transverse section). (K) Expression in sensory patches at 72 hpf is restricted to supporting cells (arrowhead) (transverse section; see also inset in D). There is strong expression in the projections. (L) Alternative focus view of D, showing residual expression in the lateral projection at 96 hpf. (O,P) Expression of gpr126 (blue) and vcanb (red) in the projections of wild-type and fr24 mutant embryos at 65 hpf. In wild-type embryos (O), there is co-expression (purple) in the recently fused ventral pillar; vcanb is downregulated in the lateral projection and in the anterior and posterior pillars (out of focus), whereas gpr126 is expressed at reduced levels. In fr24 mutants (P), vcanb and gpr126 are co-expressed in the unfused projections. Expression of vcanb persists in the dorsolateral septum, which does not express gpr126, in both wild-type and mutant embryos (O, arrow). Scale bars: in A, 50 μm for A-C,E-G,M,O,P; 50 μm in D,H,L; 25 μm in K; 100 μm in I,J,N.

Comparison of expression of gpr126 and sox10 in wild-type embryos, and expression of gpr126 in colourless (sox10-/-) mutant embryos. (A-D) Expression of gpr126 and sox10 in wild-type embryos at 24 hpf (A,B) and 48 hpf (C,D). Both genes are expressed in the otic vesicle (arrowhead), post-otic region (arrow), olfactory epithelium (nose, n) and head chondrocytes (asterisks). (E-G) Dorsal views of flat-mounted 24 hpf embryos: post-otic expression of gpr126 in the wild type (E) is lost in the cls mutant (F). Comparison with foxd3 expression identifies these cells as neural crest (G). (H,I) Dorsal view of the post-otic region showing foxd3-expressing Schwann cells extending posteriorly (H, arrow) and expression of gpr126 in the same location (I, arrow). (J,K) gpr126 expression in heart (h) and posterior mesoderm (m) persists in cls mutants, whereas neural crest expression is lost (arrows). (L,M) Expression of gpr126 is reduced in the cls mutant ear at 48 hpf, with weak expression in rudimentary projections (M). (N,O) DIC images of live ears at 96 hpf, showing a representative cls mutant ear (O). Note the small overall size and rudimentary unfused canal projections (arrowheads). Abbreviations: h, heart; m, posterior mesoderm, n, nose (olfactory epithelium); nc, neural crest; ov, otic vesicle; sib, phenotypically wild-type sibling embryo. Scale bars: in D, 100 μm for A-D; in G, 50 μm for to E-G; in I, 50 μm for H,I; in K, 100 μm for J,K; in L, 50 μm for L-O.

Treatment with cAMP agonists rescues the lauscher ear phenotype. (A-X) Live images at 5 dpf in control and drug-treated embryos. Ear swelling in tb233c mutants was variable, and categorized as ‘mild’ (E-H) or ‘severe’ (I-L) compared with wild type (A-D). Arrowhead in D indicates fused pillar in the untreated wild-type ear. Treatment between 60 and 90 hpf with cAMP agonists forskolin (25 µM; Q-T) and IBMX (100 &muM; U-X) rescues the swollen ear phenotype; DMSO has no effect (M-P). Fusion of the anterior (a), posterior (p) and ventral (v) projections to form pillars is also restored: rescued pillars are present in drug-treated embryos (R,V; arrowheads in T,X) but not in DMSO-treated samples (N,P). However, small, ectopic tissue protrusions are present on the rescued pillars (asterisks, T,X). (Y-BB) Both drugs (forskolin, 50 μM; IBMX, 100 μM) can reduce expression of vcan genes in the ear (Y-BB). (CC) Graphical representation of the ear swelling data, analysed with a 3×3 (DMSO, forskolin) or 3×4 (DMSO, IBMX) chi-square contingency table; P<0.001 (both drugs). n values are in parentheses. (C,G,K,O,S,W) Dorsal views.

EXPRESSION / LABELING:
Gene:
Fish:
Anatomical Term:
Stage: Day 5
PHENOTYPE:
Fish:
Observed In:
Stage: Day 5

Expression of early otic patterning markers is normal in lauscher mutants. (A-J) Expression of early patterning markers in the otic vesicle is indistinguishable between wild-types and homozygous lautb233c mutants. Normal expression is seen for fgf8a (fgf8) anteriorly (A,B), tbx1 posteriorly (C,D), eya1 ventrally (E,F) and pax2a medially (G,H) at 27 hpf. Scale bars: A, 50μm (applies to B-F); G, 50μ (applies to H).

Quantitation of the lauscher swollen ear phenotype. (A,B) Sample micrographs showing dorsal views of live wildtype sibling (A) and laufr24 mutant (B) embryos at 5 dpf. Measurements were taken of ear-to-ear width (straight white line) and ear perimeter (white line outlining ear) using CELLB software (Olympus). Embryos were treated with 1-phenyl 2-thiourea (PTU) to suppress development of pigmentation. Embryos were photographed at a focal plane that highlighted the largest visible dimensions for the parameters shown, and the ear chosen for measurement in each micrograph was the one with the most clearly visible outline. Ear cross-sectional area was calculated from the perimeter drawn, using CELLB software (Olympus). (C-F) Quantitation of ear-to-ear width (C,D), ear perimeter (E) and ear area (F); data were plotted using Prism 6 (GraphPad software). Ne13 for each data set. Error bars indicate standard deviation; ****P<0.0001 (unpaired, two-tailed t-test); ns, not significant.

Expression of the myelin basic protein gene (mbpa) is lost or reduced in the posterior lateral line ganglion and nerve in lauscher mutants. (A-H) Whole mount in situ hybridisation at 5 days post fertilisation shows a reduction in mbpa (mbp) expression in Schwann cells of the posterior lateral line ganglion (asterisks) and nerve (arrowheads) in the hypomorphic tb233c homozygotes (B) and tb233c/tk256a transheterozygotes (D); expression is lost in tk256a (F) and fr24 homozygotes (H). Lateral views. Scale bar: 100μm throughout.

Expression of gpr126 mRNA at other sites during zebrafish development. (A-C) Flat mounted embryos at 5 somites (A), 14 somites (B) and 23 somites (C). Patchy expression of gpr126 is seen the anterior of the embryo and also in presumed neural crest (A). At 14 somites, weak expression is seen in the presumed developing heart fields, neural crest and intermediate mesoderm (im; precursor of the pronephric ducts) (B). At 23 somites, expression increases in the migrating heart tube (h) and neural crest (nc) (C). (D,G,J) Expression at 24 hpf in dorsal (D), lateral (G) and detailed (J) view, showing expression in the post-otic neural crest (nc), heart tube (h) and olfactory epithelium (nose, n). J shows staining of individual cells within the olfactory epithelium. (E,H,K) 48 hpf embryos in dorsal (E), lateral (H) and detailed (K) view. Expression continues in the nose (arrow, H), but is reduced in the heart and lost in the postotic ganglia. New expression is seen in the ear (ov), pectoral fin (f) and tail fin (not shown), and chondrocytes of the head (arrowheads). K shows expression of gpr126 in the chondrocytes of the pectoral fin. (F,I,L) 72 hpf embryos in dorsal (F), lateral (I) and detailed (L) view. Expression continues in the ear, nose, fins and head chondrocytes (arrowheads and line). Detailed view of gpr12 expression in posterior ceratobranchials is shown in L. By 96 hpf, expression of gpr126 remains primarily in the ear (not shown). Abbreviations: b, basihyal; cb, ceratobranchials; ep, ethmoid plate; ch, ceratohyal; f, pectoral fin; gc, gill cartilages; h, heart; im, intermediate mesoderm; m, Meckel’s cartilage, n, nose (olfactory epithelium); nc, neural crest; ov, otic vesicle. Scale bars: A-I, 100 μm; J-L, 50 μm.

Treatment with cAMP agonists can rescue fusion plate formation in lauscher mutant embryos. (A-D′) Confocal images of ears stained with FITC-phalloidin (marking F actin) to show morphology of the canal projections and pillars. Concentrations of actin mark the fusion plates (shown at higher magnification in A′-D′). (A, A′) Wild-type ear. Three fusion plates are clearly visible (asterisks). (B,B′) Untreated ear from a homozygous tb233c mutant. Canal tissue is very disorganised. Although projections are touching in this ear, analysis of the z-stack indicated that this was not a true fusion plate. (C-D′) Treatment of homozygous tb233c embryos with either 50μM forskolin or 100μM IBMX can restore pillar formation. Fusion plates appear relatively normal. (E) Quantitation of the number of fusion plates present. Data were analysed with a 433 chi-square contingency table (DMSO, forskolin) or a 432 chi-square contingency table (DMSO, IBMX); P<0.001 for both drugs. N numbers are shown in parentheses. Lateral views. Scale bars: A-D 50μm; A′-D′μ.

Incubation of lautb233c mutants at a lower temperature throughout the fusion period has a rescuing effect on otic versican expression and projection fusion. (A-I) Expression of vcanb in 4dpf wild-type (A-C), and tb233c (B-I) embryos, incubated at 28°C (A,D,G), 24°C (B,E,H) and 22°C (C,F,I). Embryos were initially grown for 24 hours at 28°C. In wild-type embryos, vcanb expression is down-regulated in the fused pillars; staining only remains in the dorsolateral septum (DLS) (A-C). Mutant embryos retain strong vcanb expression in the unfused projections (D-F). Some mutant embryos show weaker expression (G) or partial expression where there are fused and unfused pillars in the same ear (H,I). The proportion of embryos showing decreased vcanb expression and rescue of fusion increases with a decrease in temperature. Percentages for each phenotypic class are shown on the panels; N numbers (number of mutant ears analysed) are shown in the boxes at the left. (J) Graphical representation of ear swelling data for wild-type and mutant embryos grown at different temperatures. The rescue of fusion in one or more pillars results in a decrease of swelling in the ear. Embryos grown at lower temperatures have reduced swelling in the ear. N numbers (number of mutant ears analysed) are shown in parentheses.

Treatment of wild-type embryos with cAMP agonists results in semicircular canal projection defects. (A-L) Live DIC images of 5dpf wild-type embryos incubated with DMSO, 25μM forskolin or 100μM IBMX between 60 and 90hpf show a gradation in phenotype. A low dose (25μM) of forskolin has little effect on the ear (B,E,H,K). However, treatment with 100μM IBMX can give rise to a severe phenotype, with a swollen ear and unfused projections (C,F,I,L). This is similar to the ‘severe’ category for the tb233c allele, but the swelling is not as extreme as in tk256a or fr24 mutants. The dorsolateral septum (black arrowheads, J-L) and the ventral pillar (asterisks, D-F) have failed to form correctly in the presence of 100mM IBMX. (M-O) In situ hybridisation shows an upregulation of the ECM marker vcana in unfused canal projections (white arrows, O). Lateral views. Scale bars: A-C, G-I 200μm; D-F, J-L, M-O 50μm. (P) Graphical representation of the dataset. Ears swell as a graded response to both forskolin and IBMX concentration. Data were analysed with a 333 chi-square contingency table for each drug; P<0.001 for both drugs. N numbers are shown in parentheses.

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