Insm1 promotes neurogenic proliferation in delaminated otic progenitors

Abstract

INSM1 is a zinc-finger protein expressed throughout the developing nervous system in late neuronal progenitors and nascent neurons. In the embryonic cortex and olfactory epithelium, Insm1 may promote the transition of progenitors from apical, proliferative, and uncommitted to basal, terminally-dividing and neuron producing. In the otocyst, delaminating and delaminated progenitors express Insm1, whereas apically-dividing progenitors do not. This expression pattern is analogous to that in embryonic olfactory epithelium and cortex (basal/subventricular progenitors). Lineage analysis confirms that auditory and vestibular neurons originate from Insm1-expressing cells. In the absence of Insm1, otic ganglia are smaller, with 40% fewer neurons. Accounting for the decrease in neurons, delaminated progenitors undergo fewer mitoses, but there is no change in apoptosis. We conclude that in the embryonic inner ear, Insm1 promotes proliferation of delaminated neuronal progenitors and hence the production of neurons, a similar function to that in other embryonic neural epithelia. Unexpectedly, we also found that differentiating, but not mature, outer hair cells express Insm1, whereas inner hair cells do not. Insm1 is the earliest known gene expressed in outer versus inner hair cells, demonstrating that nascent outer hair cells initiate a unique differentiation program in the embryo, much earlier than previously believed.

Keywords: Basal progenitors; Otocyst; Outer hair cell; Spiral ganglion; Subventricular zone; Vestibular ganglion.

Fig. 1. Schematic of otocyst proliferation and differentiation

(A) Position of the otocyst in an E10.5 mouse embryo (left panel, highlighted in blue), and relationship between delaminated progenitor region (purple) to the otocyst (center panel). (B) Morphological structure through a cross section of the otocyst along the red line in (A) with its arrangement of cell nuclei shown to the right. Within the otocyst uncommitted progenitors undergo interkinetic nuclear migration and divide apically. Neuronally-committed progenitors delaminate into the mesenchyme where some or all of them will divide an undetermined number of times to produce neurons. These neurons will then coalesce to form both spiral and vestibular ganglia. Cells that remain within the otocyst will form auditory and vestibular hair cells as well as support and structural cells. The arrangement of mature cells in the organ of Corti is more complex than we have illustrated in this figure. For a more detailed and accurate representation of the arrangement, ratios and cell types present in the organ of Corti see (Jahan et al., 2015a). Left panel in (A) from emouseatalas.org. See figure for abbreviations.

Fig. 2. Insm1 lineage analysis shows that Insm1 is expressed early in otic neurons and OHCs and/or in their progenitors, but is no longer expressed by P7

Immunohistochemistry for β-gal (magenta), GFP (green), and nuclear label with DAPI (white) in inner ear sections of P7 Insm1^GFP.Cre/+; ROSA:lacZ (A,C) and Insm1^+/+; ROSA:lacZ (B) littermate controls. (A) Insm1 is not expressed in the ear at P7; however, before P7 it was expressed by SpG and VG neurons or progenitors and OHCs or their progenitors. (B) In the absence of the Insm1^GFP.Cre allele, lacZ is not expressed. The tectorial membrane appears green in both the presence and absence of Insm1^GFP.Cre so we conclude that this is nonspecific binding of the GFP antibody or autofluorescence. (C) Close up of boxed region in (A) showing β-gal in OHCs and not IHCs. (C′ and C″) High magnification of dotted box region in (C) where C“ includes bright field to show the structure of the organ of Corti with scale bar of 20 μm. Arrows indicate IHC nucleus. Brackets designate OHC nuclei. CD: cochlear duct, Sa: saccule, SpG: spiral ganglion, VG: vestibular ganglion, Ut: utricle. Scale bar in (A) and (C): 200 μm.

Fig. 3. Insm1 is expressed in delaminating and delaminated neuronal progenitors that will produce the SVG

Representative horizontal sections of (A–D) wild type and (E–L) Insm1^GFP.Cre/+ E10.5 embryos demonstrating the Insm1 expression pattern in the otocyst during delamination. (A) In situ hybridization with an Insm1 antisense probe on a section through the anterior portion of the otocyst. Insm1 mRNA is expressed in DPs within the anteroventral aspect of the otic epithelium and in the mesenchyme. (B) Nuclear pattern of panel (A). (C) Close up of boxed region in (A). Arrowhead indicates an Insm1 positive cell located more apically in the epithelium, presumably prior to delamination. (D) Control in situ hybridization with an Insm1 sense probe shows no signal. (E) Immunohistochemistry for GFP (green) representing Insm1^GFP.Cre expression in epithelial and delaminated DPs of the anteroventral otocyst. (F) Nuclear pattern corresponding to panel (E). (G) Immunohistochemistry for DP marker Isl1 (magenta) in the same section as (E,F). (H) Close up of merge of boxed region in panels (E) and (G) shows that the GFP and Isl1 patterns overlap (white), except for a few GFP positive cells not positive for Isl1 (arrows) and even fewer Isl1 positive cells not expressing GFP (open arrow). (I,J) Immunohistochemistry co-staining for Insm1^GFP.Cre expression (I, green) and PH3 (J, magenta) shows that Insm1 is not expressed in apically-dividing cells within the epithelium (examples indicated by open arrows), but is expressed by delaminated cells in mitosis (arrows). (K,L) Immunohistochemistry co-staining for Insm1^GFP.Cre expression (K, green) and Ki67 (L, magenta) shows that while some Insm1 expressing cells are proliferating (Ki67+; examples indicated by arrows), many are not (examples indicated by open arrows). Dotted lines in all panels delineate the basal lamina of the otocyst. Solid lines delineate the apical edge of the otic epithelium. Boxes indicate regions magnified in panels (C) and (H). (A,D,E) Scale bar: 200 μm. (C,H,I,K) Scale bar: 50 μm. Ot: otocyst.

Fig. 4. Insm1 expression is maintained in nascent SpG and VG neurons

Representative coronal sections demonstrating Insm1 expression at E14.5 in the cochlea (A–D) and the vestibule (E–H) of (A,B,E,F) wild type and (C,D,G,H) Insm1^GFP.Cre/+; ROSA:lacZ embryos. (A) In situ hybridization with an Insm1 antisense probe. Section is through the two turns of the cochlea and shows Insm1 mRNA expression in the SpG and not within the epithelium. (B) Nuclear pattern corresponding to (A). (C) Immunohistochemistry for GFP (green) representing Insm1^GFP.Cre expression in Insm1^GFP.Cre/+; ROSA:lacZ embryos. Section is through one turn of the cochlea and shows Insm1^GFP.Cre expression in spiral ganglion neurons, but not within the epithelium. (D) Nuclear pattern corresponding to (C) with immunohistochemistry for lineage marker β-gal (magenta) showing no descendant cells of Insm1 progenitors within the epithelium. (E) In situ hybridization with an Insm1 antisense probe. Section is through basal cochlea, utricle and saccule and shows Insm1 mRNA expression in SpG, iVG, and faintly in sVG. (F) Nuclear pattern corresponding to panel (E). (G) Immunohistochemistry for GFP (green) representing Insm1^GFP.Cre expression in Insm1^GFP.Cre/+; ROSA:lacZ embryos. Section through utricle and saccule shows Insm1^GFP.Cre is expressed in the iVG. (H) Nuclear pattern corresponding to panel (G) with immunohistochemistry for lineage marker β-gal (magenta) showing no descendant cells of Insm1 progenitors within the epithelium. Solid lines in all panels delineate basal lamina of all epithelia. Dotted lines in all panels delineate ganglia. Scale bar: 200 μm. Co: cochlea, Sa: saccule, SpG: spiral ganglion, iVG: inferior vestibular ganglion, sVG: superior vestibular ganglion, Ut: utricle.

Fig. 5. Insm1 expression subsides in SpG neurons as they differentiate and is undetectable at birth

Representative sections demonstrating Insm1 expression at E16.5-P1 in the cochlea. (A) In situ hybridization with an Insm1 antisense probe on a coronal section of E16.5 head, which results in an axial (perpendicular to the modiolus) section of the cochlea. Insm1 signal appears reduced in the SpG when compared with E14.5 (Fig. 4A). Insm1 mRNA is also expressed in the sensory epithelium. (B) Nuclear pattern corresponding to (A). (C) Immunohistochemistry for GFP (green) representing Insm1^GFP.Cre expression in the E16.5 cochlea. Insm1^GFP.Cre signal is decreased in SpG neurons when compared to Fig. 4C, and is also detected in sensory epithelium. (D) Nuclear pattern corresponding to (C). (E) Immunohistochemistry for GFP (green) representing Insm1^GFP.Cre expression in modiolar section of E18.5 cochlea. Insm1^GFP.Cre signal is further decreased in SpG neurons when compared to previous stages and epithelial pattern appears to correspond to OHCs. (F) Nuclear pattern corresponding to panel (E). (G) In situ hybridization with an Insm1 antisense probe on a modiolar section of P1 cochlea (mid-basal turn) showing no Insm1 mRNA expression. (H) Nuclear pattern corresponding to panel (G). Dotted lines in all panels delineate SpG. Brackets designate sensory epithelium. Scale bar: 200 μm. SpG: spiral ganglion, SE: sensory epithelium.

Fig. 6. Insm1 is transiently expressed by nascent OHCs, but not IHCs

(A,C,E,G,I,K,S,T) Representative sections demonstrating Insm1 expression by immunohistochemistry for GFP (green) in cochlear sections of Insm1^GFP.Cre mice at E14.5–E18.5. In (A), immunohistochemistry for lnsm1 lineage marker β-gal is represented in magenta. (A,B) At E14.5 Insm1 expressing cells or their descendants are not in the sensory epithelium (A), which is identified in an adjacent section by immunohistochemistry for P27^kip1 (B, magenta). (D,F,H,J,L) Immunohistochemistry for HC marker MyoVIIa (red) in sections adjacent to C,E,G,O,Q, respectively, with nuclear pattern (white). (C,D) At E15.5 in the most basal aspect of the organ of Corti (section provides a glancing view and hence multiple hair cells per row), Insm1^GFP.Cre and MyoVIIa are expressed in all rows of OHCs. (E,F) At E15.5 in the mid-basal turn of the organ of Corti, Insm1^GFP.Cre is expressed in 2–3 of OHCs and MyoVIIa is only expressed in the first OHC. (G,H) At E15.5 in the mid-apical turn of the organ of Corti, Insm1^GFP.Cre and MyoVIIa are not detectable. (M,N,O) Immunohistochemistry for MyoVIIa (M, white), Insm1^GFP.Cre (N, white), and the merged image (O, MyoVIIa in red and Insm1^GFP.Cre in green) in a whole mount show that, at E16.5 in the basal organ of Corti, Insm1 is expressed in all OHCs. (P, Q, R) Immunohistochemistry for MyoVIIa (P, white), Insm1^GFP.Cre (Q, white), and the merged image (R, MyoVIIa in red and Insm1^GFP.Cre in green) in a whole mount show that, at E16.5 in the apical organ of Corti, Insm1 is typically expressed in OHCs that are expressing MyoVIIa, with some exceptions where OHCs express MyoVIIa without Insm1 (filled arrow) or vise versa (open arrow). (I,J) At E18.5 throughout the cochlea, Insm1^GFP.Cre and MyoVIIa are expressed in all three rows of OHCs, and Insm1 is clearly not expressed in IHCs. (K,L) At E18.5 in the utricle and saccule, Insm1^GFP.Cre is not detectable in MyoVIIa expressing HCs. (S–T) Insm1^GFP.Cre (green) in sections through two turns of the cochlea at P1 (S) and P2 (T) demonstrating that Insm1 expression persists in apical OHCs through P1 and has mostly subsided throughout the cochlea by P2. (S′ and T′) represent boxed regions in (S and T), respectively. Brackets designate rows of OHCs. Arrow heads indicate the single row of IHCs. Scale bar: (A,Q) 200 μm, (C, I) 50 μm. SE: sensory epithelium, Sa: saccule, Ut: utricle.

Fig. 7. Mice lacking Insm1 (Insm1^−/−) have fewer SpG and VG neurons

(A–F) Immunohistochemistry for nascent neuron marker β-tubulin III (labeled with the TuJ1 antibody). Representative anatomically matched sections of E14.5 SVG from Insm1^+/+ (A) and Insm1^−/− (B) littermate embryos, E18.5 SpG from Insm1^+/+ (C) and Insm1^−/− (D) littermate embryos, and E18.5 VG from Insm1^+/+ (E) and Insm1^−/− (F) littermate embryos. (A–F) Solid lines delineate boundaries of ganglia, defined by neuronal cell bodies. (A–B) Dotted lines delineate basal lamina of the otocyst based off of nuclear DAPI pattern (not shown). (G) Average cross sectional area (in multiples of 100 μm²) of Insm1^+/+ and Insm1^−/− otic ganglia from littermate pairs at E10.5, E12.5, E14.5 and E18.5. There is no significant difference in the average cross sectional area of Insm1^+/+ and Insm1^−/− SVGs at E10.5 (1.39;SEM=0.12 and 1.40;SEM=0.19, respectively. P=0.47), but there is a significant difference at E12.5 (6.16;SEM=0.26 and 5.29;SEM=0.23), and E14.5(4.53;SEM=0.18 and 3.36;SEM=0.42). At E18.5 there is a significant difference between the average cross sectional areas of Insm1^+/+ and Insm1^−/− SpGs (2.90;SEM=0.18 and 1.90;SEM=0.19) and VGs (3.10;SEM=0.51 and 1.49;SEM=0.13). (H) Average Z-length in μm of Insm1^+/+ and Insm1^−/− otic ganglia from littermate pairs at E10.5, E12.5, E14.5 and E18.5. There is no significant difference in the average Z-length of Insm1^+/+ and Insm1^−/− SVGs at E10.5 (472;SEM=21.84 and 472;SEM=21.84. P=0.5), E14.5 (405;SEM=77.02 and 357.5;SEM=123.65. P=0.3), and E18.5 SpGs (450.8;SEM=37.41 and 450;SEM=36.46. P=0.47) and VGs(333.2;SEM=78.19 and 411.3;SEM=87.83. P=0.1). The only significant difference in Z-length is at E12.5 (452;SEM=23.53 for Insm1^+/+ and 388;SEM=10.12 for Insm1^−/−), when Insm1^−/− may transiently have a shorter ganglion. (I) Average number of delaminated neurons or progenitors per cross section of Insm1^+/+ and Insm1^−/− otic ganglia at E10.5, E14.5, and E18.5. There is no significant difference in the average number of neurons in Insm1^+/+ and Insm1^−/− SVGs at E10.5 (125.97;SEM=11.24 and 119.29;SEM=18.49, respectively. P=0.38), but there is a significant difference at E14.5 (360.00;SEM=24.33 and 216.23;SEM=24.02). At E18.5 there is a significant difference between the average number of neurons in Insm1^+/+ and Insm1^−/− SpGs (193.96;SEM=38.08 and 116.79;SEM=15.13) and VGs (114.05;SEM=18.93 and 48.46;SEM=3.02). Sample size in the number of ears for each condition is indicated by the number within the column representing that group. The number of mice in each sample is written in parentheses below the number of ears. Error bars indicate SEM. *P≤0.05, **P≤0.01, ***P≤0.005, ****P≤0.0005. Scale bars: 100 μm. Ot: otocyst.

Fig. 8. A change in apoptosis does not accompany the reduction of SVG neurons in Insm1^−/−

Representative anatomically matched sections of E12.5 ears from Insm1^+/+ (A–B) and Insm1^−/− (C–D) littermate embryos. (A,C) Immunohistochemistry for DP marker Isl1 (magenta) and apoptosis marker ACC3 (green). Colabeled cells appear white. (B,D) Nuclear pattern for A and C, respectively. Dashed lines delineate the otic epithelia. Solid lines delineate SVG and do not include nearby facial ganglion which is also expresses Isl1. (E) There is no significant difference in the number of delaminated neurons undergoing apoptosis per section between Insm1^−/− and Insm1^+/+ at E12.5 (3.87;SEM=1.6 and 1.98;SEM=0.32, respectively. P=0.14) or E14.5 (3.59;SEM=0.83 and 5.37;SEM=1.06, respectively. P=0.11). Sample size in the number of ears for each condition is indicated by the number within the column representing that group. The number of mice in each sample is written in parentheses below the number of ears. Error bars indicate SEM. Scale bar: 200 μm. gg: geniculate ganglion, Ot: otocyst.

Fig. 9. Reduction in proliferation of DPs precedes decrease of neurons in Insm1^−/−

(A,C) Immunohistochemistry for DP marker Isl1 (magenta) and mitosis marker PH3 (green) in representative, anatomically matched, coronal sections of E10.5 Insm1^+/+ (A) and Insm1^−/− (C) heads. Colabeled cells appear white. (B,D) Nuclear pattern for (A) and (C), respectively. Dotted lines delineate the basal lamina of the otic epithelia. Solid lines delineate SVG and do not include nearby facial ganglion which is also expresses Isl1. (E) Average number of DPs in mitosis per cross section of Insm1^+/+ and Insm1^−/− otic ganglia at E10.5 and E12.5. There are approximately 30% fewer DPs in mitosis in Insm1^−/− than in Insm1^+/+ ganglia at E10.5 (13.63;SEM=2.33 and 20.32;SEM=2.37) and E12.5 (3.87;SEM=0.46 and 5.55;SEM=0.41). (F) Average number of DPs per cross section of Insm1^+/+ and Insm1^−/− otic ganglia at E10.5. There is no significant difference in the average number of DPs in Insm1^+/+ and Insm1^−/− SVGs at E10.5 (125.97;SEM=11.24 and 119.29;SEM=18.49, respectively. P=0.38). (G) Average percent of DPs that are in mitosis per cross section of Insm1^+/+ and Insm1^−/− otic ganglia at E10.5. Of the total number of Isl1 cells, approximately 30% fewer are in mitosis in Insm1^−/− than Insm1^+/+ ganglia at E10.5 (11.29%;SEM=0.28% and 16.06%;SEM=0.92%). (H) Average number of mitotic progenitors within the epithelium per cross section of Insm1^+/+ and Insm1^−/− otic ganglia at E10.5 and E12.5. There is no significant difference in the average number of epithelial mitotic progenitors in Insm1^+/+ and Insm1^−/− SVGs at E10.5 (19.25;SEM=2.01 and 20.83;SEM=2.39. P=0.31) and at E12.5 (10.05;SEM=1.54 and 11.53;SEM=1.53. P=0.25). Sample size in the number of ears for each condition is indicated by the number within the column representing that group. The number of mice in each sample is written in parentheses below the number of ears. Error bars indicate SEM. *P≤0.05, **P≤0.01, ****P≤0.0005. Scale bars: 100 μm. gg: geniculate ganglion, Ot: otocyst.

Fig. 10. Insm1 expression in the developing inner ear, the effects of Insm1 ablation in otic neurogenesis, and a suggested common mechanism of neurogenesis among neural epithelia

(A) During the embryonic development of the ear, uncommitted, amplifying progenitors within the otocyst undergo interkinetic nuclear migration whereby mitosis occurs apically within the epithelium and synthesis basally. From E9.5–E12.5 neuronally-committed progenitors are generated by amplifying progenitors, express Insm1 (green), and delaminate from the otocyst. Once they have delaminated, these progenitors undergo an unknown number of divisions to produce SVG neurons. Nascent neurons maintain expression of Insm1. As they differentiate, Insm1 levels subside, and ultimately Insm1 is undetectable mature neurons. Following neurogenesis and the cessation of amplification in the organ of Corti, the OHCs initiate Insm1 expression in accordance with the wave of hair cell differentiation. As in neurons, OHCs decrease Insm1 expression as they mature. (B) In the absence of Insm1, the DPs undergo fewer divisions, and therefore, produce fewer neurons resulting in smaller spiral and vestibular ganglia. (C, D) The expression pattern of Insm1 in otic neurogenesis (A) is very similar to Insm1 expression in the embryonic cortex (Duggan et al., 2008)(C) and olfactory epithelium (Rosenbaum et al., 2011) (D). In each case, uncommitted progenitors undergo interkinetic nuclear migration. When a progenitor commits to neuronal cell fate it expresses Insm1, migrates basally, either staying within the epithelium or delaminating into the mesenchyme, and divides to produce neurons. This schematic also highlights differences between epithelia. The neurons migrate to different regions within or outside of the epithelium, interacting differently with the basal lamina as they do so. Also, those cells that continue to divide apically produce different types of epithelial cells specific to their organ. Circles represent cell nuclei. Abbreviations are defined in figure key.