Inner ear phenotype of Noggin knockout mice
The anatomy of Noggin mutant inner ears was visualized by injecting 0.1% alkyd white paint solution to the cavity of the inner ear (). A total of 15 homozygous mutant embryos were analyzed between 15.5 to 17.5 dpc. Homozygous inner ears in a mixed C57BL/6/FVB background (; n = 4) have a milder phenotype than those in C57BL/6 background (; n = 11). Although all inner ear components are present in most Noggin mutants, the overall patterning of the inner ear appears distorted compared to heterozygotes. For example, the shape of the endolymphatic duct is abnormal (, asterisk; ), the common crus is slightly widened and appears slanted (, double asterisks), the anterior canal is extended (, double-headed arrow), and the saccule is often malformed (, arrow; ). More importantly, the cochlear duct is invariably shortened and shows aberrant coiling. The outgrowth of a normal cochlear duct extends ventrally in an anteromedial direction, and then coils laterally. shows the cochlear duct of a Noggin mutant that displays abnormal protrusions (arrowheads) and terminates prematurely in an upward direction (arrow). In more severe cases, the cochlear duct coils medially instead of laterally (, red arrowheads). In addition, these inner ears are smaller in size, and the posterior semicircular canal is truncated or not resorbed (white arrowheads) (, arrow).
Inner ear phenotypes of Noggin -/- mutants at 16.5 dpc
Frequency of malformed inner ear structures in Noggin -/- mutants
Normal expression of Noggin in the developing inner ear
In an attempt to understand the observed phenotype in Noggin knockout mice, we investigated Noggin expression in the developing inner ear using RNA probes against Noggin in wild type embryos, or performing β-gal histochemistry or detecting lacZ mRNA using in situ hybridization in Noggin heterozygous embryos. Among the three techniques used, β-gal histochemistry appears to be the most sensitive in identifying Noggin expression domains. Noggin is transiently expressed in the dorsal periotic mesenchyme at 9.5 dpc (, arrowheads). This mesenchymal expression is not detectable at 11.5 and 12 dpc ( and data not shown). At 14 dpc, Noggin expression is expressed in the cochlear duct (, arrows) and semicircular canals of the inner ear (, arrowheads), as well as the developing otic capsule surrounding the labyrinth (). By 16.5 dpc, in addition to the expression in the cochlear duct (), semicircular canals (, arrowheads) and otic capsule (), Noggin is also present in the center region of the crista ampullaris (, insert).
Gene expression patterns in Noggin +/- and -/- inner ears
In other parts of the body, Noggin
expression domains are often found intimately associated with expression domains of Bmps
(Marcelle et al., 1997
; Tonegawa and Takahashi, 1998
). Similarly, Noggin
expression domains in the cristae () and cochlear duct () are associated with that of Bmp4
(), whereas its expression domain in the canals is associated with that of Bmp2
(, arrowheads). A faint Bmp4
hybridization signal is sometimes detectable in wild type otic capsule (, arrowheads), and this expression appears upregulated in the Noggin
mutants (, arrowheads). No upregulation of Bmps
is evident in the membranous labyrinth of the mutant inner ear (data not shown).
Early appearance of inner ear defects in Noggin mutants
Since Noggin expression is not robustly detected in the otic epithelium or periotic mesenchyme until late-gestation, we investigated the onset of the gross anatomical defects in the mutants at earlier stages. All the inner ear defects observed at 16.5 dpc are already apparent at 12 and 13.5 dpc (n = 8), including defects in the cochlear duct outgrowth (), endolymphatic duct (, asterisk), and common crus (, double asterisks). The extended anterior canal phenotype is already evident at 12 dpc when the prospective canal is a pouch (, arrowheads). Since the morphological defects in the mutants are evident earlier than any robust Noggin expression domains associated with the developing inner ear, it suggests that the inner ear defects caused by the lack of Noggin may be indirect. The shortened neural axis and the kinky notochord reported in Noggin mutants prompted us to investigate the location of the otocysts in relationship to other body parts, in particular r5 and 6, which are thought to provide important signaling molecules for inner ear patterning.
Inner ear phenotypes of Noggin mutants at 12 and 13.5 dpc
Abnormal alignment of otic vesicles with the hindbrain in Noggin mutants
At 8.5 dpc, the otic placode region, as indicated by the expression domain of Pax8
, is broad and its anterior half corresponds to the position of Hoxb1
-positive, rhombomere 4 (). At 9.5 dpc, the location of the newly formed otocysts corresponds to the same axial positions of rhombomeres 5 and 6 in the hindbrain (Kiernan et al., 2002
). illustrate the anterior limit of normal otocysts at 9.5 dpc corresponding to the junction of r4 and 5 based on the expression domains of Hoxb1
. In Noggin
mutants, the relative relationship between the otic placode and r4 appears normal at 8.5 dpc (; n
= 3). By 9.5 dpc, the size of the homozygous otocysts is generally slightly smaller than heterozygotes (, black brackets), and the alignment of otocysts with the rhombomeres in the hindbrain is abnormal (n
= 8). All specimens show either an alignment with r4 and 5 () or with r4 alone (data not shown) instead of the normal alignment with r5 and 6. However, the relative position between the otocysts and the second branchial arch remains unchanged (data not shown). This caudal shift of the hindbrain alignment with the otocysts could affect the ability of normal signaling molecules emanating from r5 and 6 to reach the otic epithelium and may be the cause of Noggin
-/- malformed inner ears.
Alignment of inner ears with hindbrain rhombomeres in Noggin -/- mutants at 8.5 and 9.5 dpc
Caudal shift of rhombomeres in chicken embryos affects inner ear morphogenesis
To test the hypothesis that this abnormal alignment between otocysts and hindbrain could indeed result in dysmorphogenesis of the inner ear, we generated an experimental paradigm in developing chicken embryos similar to the Noggin mutants, in which the hindbrain is shifted caudally by one rhombomere in relation to the otocyst. We tried to accomplish this by duplicating some rhombomeres rostral to the inner ear position using transplantation techniques when the inner ear is a shallow cup. For reasons that are not clear, duplicating r2 or r3 by transplanting r3-6 into the space of r4-7 or transplanting r2-6 into the space of r3-7, respectively, did not result in a caudal shift of r5 and 6 in relationship with the otocysts, 24 hr after surgery. On the other hand, duplicating both r2 and r3 by replacing r4-7 with r2-6, or duplicating both r1 and r2 by replacing r3-7 with r1-6 resulted in too big a shift. The otocysts were aligned with r3 and 4, rather than r4 and 5 as we had intended (data not shown). The only permutation that resulted in a stable caudal shift of rhombomere by one position in relationship to the inner ear was replacing r5-7 with r3-6 (). illustrates an operated embryo where the otocysts are aligned with r4 and 5 instead of the normal r5 and 6, based on the expression patterns of EphA4 (n = 18) and Hoxb1 (n = 4). Ectopic otocysts formed as a result of transplantation were surgically removed 24 hrs after transplantation. Then, embryos were further incubated and harvested at E9 and processed for paint-fill analyses. Our results show that inner ears in rhombomere-shifted embryos display relatively normal dorsal vestibular structures that are not extended like those in Noggin -/- mutants (). However, both the saccule and cochlear duct are malformed in the rhombomere-shifted embryos. The saccule is usually under-developed and not clearly separated from the utricle and cochlear duct (; n = 8/8). The cochlear development is severely affected, either widened or shortened (; n = 8/8). Inner ears of control embryos in which r3-6 were replaced by r3-6 from an age-matched donor did not show any obvious morphological defects (; n = 6). These results indicate that the normal alignment between otocysts and hindbrain is important for normal morphogenesis of the chicken inner ear, especially the saccule and cochlear duct, and further suggest that these defects in Noggin mutants are likely due to the abnormal signaling from the hindbrain caused by the changes in the spatial relationship between otocysts and hindbrain.
Misalignment of inner ears with hindbrain after rhombomere transplantations in chicken embryos
Inner ear phenotypes of rhombomere-shifted chicken embryos
Increase in otic capsule size in Noggin mutants
Since Bmp4 expression appears to be upregulated in the otic mesenchyme of Noggin mutants, we examined the consequence of this effect on otic capsule formation. Our results show that the chondrogenic region in the otic capsule is expanded in Noggin mutants based on the expression patterns of lacZ and Aggrecan (). Noggin and Aggrecan are normally expressed in the chondrocytes only and their expression is not detected in the perichondrium (, arrowheads). However, in the Noggin mutants, Aggrecan expression is upregulated in the perichondrium (; arrowheads), whereas Noggin expression was limited to the chondrocytes (). In addition, Bmp4 expression in the mesenchymal cells along the membranous side of the perichondrium is quite apparent (). The increase in chondrogenesis in Noggin mutants suggests that BMP4 and Noggin normally regulate each other's activity in mediating the chondrogenic process in the otic capsule.
Upregulation of chondrogenesis in the otic capsule of Noggin mutants at 16.5 dpc