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First published online 29 March 2007
doi: 10.1242/dev.000760

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Opposing gradients of Gli repressor and activators mediate Shh signaling along the dorsoventral axis of the inner ear

¹ National Institute on Deafness and other Communication Disorders, Rockville, MD 20850, USA.
² Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA, USA.
³ Institute for Animal Developmental and Molecular Biology, Heinrich-Heine University, Düsseldorf, Germany.

View larger version (125K): [in this window] [in a new window]   Fig. 1. Expression patterns of Shh, Ptc1 and the Gli family transcription factors in the developing inner ear at 11 dpc. (A,A') Gli1 and (B,B') Ptc1 show a graded expression pattern in the otocyst, strongest in the ventral-medial tip of the otocyst and weaker towards the dorsal region. Weaker hybridization signals are also found in the dorsal-lateral region of the otic epithelium as well as in the mesenchyme. (C) Gli2 mRNA is broadly expressed throughout the otic epithelium but its expression in the endolymphatic duct (ed) is barely detectable. (D) Gli3 expression is evident in the entire otocyst, with weaker expression in the dorsal-lateral side. Both Gli2 and Gli3 transcripts are also detected in the mesenchymal tissues, especially in the lateral region of the otocyst. Arrowheads (C,D) indicate the margin of the otic epithelium. (E) Shh is strongly expressed in the ventral midline structures, the floor plate (arrow) and notochord (arrowhead), but its transcripts are not detectable in the otocyst. vp, vertical pouch; co, cochlea; D, dorsal; L, lateral. Scale bars: in A, 100 µm for A-E; in A', 100 µm for A',B'.

View larger version (39K): [in this window] [in a new window]   Fig. 2. Inner ear phenotypes of Gli3-/- mutants. Paint-filled inner ears of Gli3 heterozygous (A,D) and homozygous (B,C,E,F) embryos at 15.5 dpc shown in lateral (A-C) and dorsal (D-F) views. Gli3-/- inner ears are missing the lateral canal (red asterisks), but the lateral ampulla is present (arrows in E,F). The anterior canal is usually missing or truncated (white asterisks), and the caliber of the posterior canal is smaller. The saccule and the proximal tip of the cochlear duct are misshapen, but the length of the cochlear duct is normal. Inner ear phenotypes in mutants with exencephaly are more severe (C,F). aa, anterior ampulla; asc, anterior semicircular canal; cc, common crus; cd, cochlear duct; ed/s, endolymphatic duct and sac; la, lateral ampulla; lsc, lateral semicircular canal; pa, posterior ampulla; psc, posterior semicircular canal; s, saccule; u, utricle, D, dorsal; A, anterior; M, medial. Scale bar: 100 µm.

View larger version (18K): [in this window] [in a new window]   Fig. 3. Inner ears of Shh and Gli3 compound mutants. Paint-filled inner ears of (A) Shh+/-;Gli3+/+, (B) Shh-/-;Gli3+/+, (C) Shh-/-;Gli3+/-, (D) Shh-/-;Gli3-/- and (E) Gli3699 mutants at 14.75 dpc. (B) Shh-/-;Gli3+/+ inner ears lack ventral structures, including the cochlear duct and saccule (asterisk). Dorsally, the lateral semicircular canal/ampulla and endolymphatic duct are missing. (C) The dorsal structures in Shh-/-;Gli3+/- inner ears are normal, but only a small knob-like structure is present ventrally (arrow). (D) In Shh-/-;Gli3-/- ears, the dorsal structures are malformed, and a shortened cochlear duct is present. (E) Inner ears of Gli3699 homozygotes show normal dorsal inner ear phenotypes, yet the cochlear duct is shortened. (A',D',E') Ventral views of respective cochlear ducts. For abbreviations refer to Fig. 2; p, proximal; d, distal. Scale bar: 100 µm.

View larger version (44K): [in this window] [in a new window]   Fig. 4. Inner ears of Gli2 and Gli3 compound mutants. (A-D) Lateral views of inner ears and (E-H) ventral views of cochlear ducts of (A,E) Gli2+/-;Gli3+/-, (B,F) Gli2-/-;Gli3+/+, (C,G) Gli2-/-;Gli3+/-, (D,H) Gli2+/-;Gli3-/- embryos at 14.75 dpc. The dorsal region of the inner ears is normal (A-C), except in Gli2+/-;Gli3-/- (D). By contrast, the length of the cochlear duct in Gli2-/-;Gli3+/- is shortened (G, arrow). (I) Bar chart showing ratios of cochlear duct lengths between compound mutants and their Gli2+/-;Gli3+/- littermate controls. Asterisk indicates an average ratio that is significantly different from others (P<0.0001; Student's unpaired t-test). Scale bar: 100 µm.

View larger version (22K): [in this window] [in a new window]   Fig. 5. Inner ears of Gli2 and Gli3 compound mutants at 13.5 dpc. Lateral views of inner ears of (A) Gli2+/-;Gli3+/-, (B) Gli2-/-;Gli3-/-, (C) Shh-/-;Gli3-/-, (D) Shh+/-;Gli3-/- mutants at 13.5 dpc. (B) Gli2-/-;Gli3-/- inner ear has a shortened cochlear duct that is comparable to that of Shh-/-;Gli3-/- mutants (C). The dorsal defects shown in Gli2-/-;Gli3-/- inner ears are similar to those in Gli3-/- single (D) and Shh-/-;Gli3-/- double mutants (C). White and red asterisks indicate the lack of anterior and lateral semicircular canals, respectively. There is a developmental delay in inner ears of Gli2-/-;Gli3-/- and Shh-/-;Gli3-/- double mutants, as indicated by the lack of resorption of the posterior semicircular canal (arrows). For abbreviations refer to Fig. 2. Scale bar: 100 µm.

View larger version (136K): [in this window] [in a new window]   Fig. 6. Expression patterns of Otx2 and Msx1 in various Shh, Gli2 and Gli3 compound mutants. (A-J) Expression patterns of Otx2 (A-E) and Msx1 (F-J) in the developing cochlear duct of Shh;Gli3 compound mutants at 12.5 dpc. (A) Otx2 is expressed in the lateral wall of the developing cochlear duct in wild type. (B) There is no expression of Otx2 in Shh-/- mutants. (C) Otx2 is expressed in the ventral-lateral wall of the Shh-/-;Gli3+/- inner ears. (D,E) Strong Otx2 expression is observed in the ventral-lateral wall of Shh-/-;Gli3-/- (D) and Gli3699 (E) embryos. (F) Msx1 is expressed at the distal tip of the cochlear duct (arrow) and the endolymphatic duct of wild-type embryos. (G,H) In Shh-/- (G) or Shh-/-;Gli3+/- (H) embryos, in which cochlear duct is not developed, Msx1 expression is not observed ventrally (asterisk). (I,J) Even though the cochlear duct is partially formed in Shh-/-;Gli3-/- (I) and Gli3699 (J) mutants, Msx1 is not observed, indicating that these cochlear ducts are missing the distal region. (K-R) Expression patterns of Otx2 (K-N) and Msx1 (O-R) in the developing cochlear duct of Gli2;Gli3 compound mutants at 12.5 dpc. Strong Otx2 expression in the cochlear duct is shown in all the Gli2;Gli3 compound mutants (K-N). Msx1 expression at the distal tip of the cochlear duct is found (arrows) in Gli2+/-;Gli3+/- (O), Gli2-/-;Gli3+/- (P) and Gli2+/-;Gli3-/- (Q) embryos, but not in Gli2-/-;Gli3-/- double mutants (asterisk, R). For abbreviations refer to Fig. 1. Scale bar: 100 µm.

View larger version (98K): [in this window] [in a new window]   Fig. 7. The lateral canal defects in Shh-/- and Gli3-/- mutants are caused by different mechanisms. The inner ears of Gli3+/- (A-D), Gli3-/- (E-H), and Shh-/- (I-L) at 11.75 dpc examined by the paint-fill technique (A,B,E,F,I,J) and in situ hybridization (C,D,G,H,K,L). Red lines in A,E,I represent the levels of sections shown in C,D, G,H, and K,L, respectively. (A,B) At 11.75 dpc, the inner ear shows a vertical pouch (vp; develops into the anterior and posterior semicircular canals) dorsally and a horizontal pouch (hp; develops into the lateral semicircular canal) laterally. The developing cochlear duct (cd) is also shown ventrally. (C,D) The location of the prospective lateral crista (lc) is marked by the co-expression of Bmp4 and Otx1 (arrows). Otx1 is also expressed in the prospective lateral canal region. (E,F) The vertical pouch of Gli3-/- mutants is smaller than that of heterozygotes. The horizontal pouch is absent or rudimentary. (G,H) The primordial lateral crista is present based on the overlap of expression between Bmp4 and Otx1 (arrows). Yet, the Otx1 expression domain in the canal pouch is much reduced (arrowheads). (I,J) The inner ears of Shh-/- mutants show the presence of the horizontal pouch. However, Otx1 expression, which is normally found in the horizontal pouch, has shifted to the medial side (arrowheads), such that the Otx1 expression domain no longer overlaps with that of Bmp4 (K,L). Orientations: D, dorsal; A, anterior; L, lateral. Scale bars: 100 µm.

View larger version (140K): [in this window] [in a new window]   Fig. 8. Cell proliferation and programmed cell death in the developing lateral canal region of Gli3-/- and Shh-/- mutants. Bmp4 expression (A,D,G), BrdU labeling (B,E,H) and TUNEL staining (C,F,I) in Gli3+/- (A-C), Gli3-/- (D-F), and Shh-/- (G-I) inner ears at 11.75 dpc. (A-C) Robust cell proliferation and little programmed cell death are observed in the primordial lateral canal region in Gli3+/- ears. The presumptive lateral crista is Bmp4 positive. (D-F) Markedly decreased cell proliferation and increased programmed cell death (arrowheads) are found in the lateral wall of the inner ears of Gli3-/- mutants. (G-I) In Shh-/- mutants, no changes in cell proliferation (H) and programmed cell death (I) are observed in the primordial lateral canal area. Yet, there is increased cell death in the medial and anterior wall of the inner ear (I,I', arrowheads). Orientations: A, anterior; L, lateral. Scale bar: 100 µm.

View larger version (22K): [in this window] [in a new window]   Fig. 9. A summary of how different levels of Shh mediate the formation of inner ear structures in the wild type and various mutants. A wild-type inner ear receives graded levels of Shh protein during development, highest in the ventral region and decreasing towards the dorsal region. This graded Shh signaling results in various levels of Gli activator (blue triangle) and repressor (red triangle) activities within the otocyst that are responsible for mediating the formation of different inner ear structures. The distal region of the cochlear duct (blue) requires the activator function of Gli proteins that is redundantly shared by Gli2 and Gli3, and possibly Gli1. The proximal region of the cochlear duct and the saccule (pale red and pale blue) requires relatively low levels of Shh signaling, as compared with the distal region, to remove Gli3R. The dorsal region of the inner ear (red) requires a correct dose of Gli3R balanced by the least amount of Shh signaling. In the various mutants, the missing inner ear structures are outlined in grey. The absence of GliA (blue) affected the distal region of the cochlear duct in all mutants analyzed. For most mutants, the formation of the saccule and the proximal region of the cochlear duct are affected in those situations in which Gli3R is not properly alleviated by Shh signaling (dark red rectangle, Shh-/-; pink rectangle, Shh-/-;Gli3+/-). In addition, the semicircular canal region is also partially affected in the absence of Gli3R (white, Gli3-/- and Shh-/-;Gli3-/-) or in the presence of too much Gli3R (dark red, Shh-/-). The exception is the normalcy of the vestibule and proximal cochlear duct region in the Gli3699 mutant, in which Gli3R levels cannot be alleviated. We attributed the absence of vestibular and proximal cochlear defects in this mutant to the presumably normal function of Gli1/Gli2A. There is a temporal difference in the requirement of Gli3R and Shh for lateral canal formation (asterisks).

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