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First published online 12 November 2008
doi: 10.1242/dev.029330

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Cross-repressive interactions between Lrig3 and netrin 1 shape the architecture of the inner ear

Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA.

View larger version (19K): [in this window] [in a new window]   Fig. 1. Patterning and morphogenesis of the inner ear. Diagrams of the transformation of the otic vesicle into the mature structure of the inner ear. Early in development (left), the axes of the otic vesicle are patterned, with the presumptive vestibular system expressing Dlx5 and Hmx3 (red) and the developing cochlea expressing Otx2 (yellow). The lateral pouch is defined by expression of Otx1 (blue stipple). A few hours later, during morphogenesis, discrete regions in the dorsal and lateral pouch begin to transcribe netrin 1 (blue, middle). These regions will subsequently undergo fusion and disappear, leaving the epithelium in the perimeter to form the walls of the mature canals (right). Motion is detected by hair cells housed in swellings at the base of each canal called ampullae (*). In all of the following figures, paintfilled inner ears are shown looking down onto the lateral canal, with anterior towards the right, whereas sections through the otic vesicle are in the transverse plane (as indicated by a broken line), with dorsal upwards and lateral towards the right.

View larger version (81K): [in this window] [in a new window]   Fig. 2. Lrig3 mutant mice exhibit circling behavior owing to a truncation of the lateral semicircular canal. (A) Diagrams of two independent alleles of Lrig3 illustrating insertion of the gene trap vector in LST016 mice (left) and the introduction of LoxP sites on either side of the ATG-bearing exon in the conditional Lrig3flox allele (right). (B,C) X-gal detection of the Lrig3-β-geo fusion protein in an E10.5 Lrig3 heterozygous embryo (B) and in sections through the otic vesicle (C) in the plane indicated (broken line, B). β-Galactosidase activity is high in somitic mesoderm, the branchial arches and the limb buds. In the developing inner ear, transcription of Lrig3 is enriched in the lateral otic epithelium by E10.5 (bracket). Scale bar: 50 µm. (D) A single Lrig3 mutant mouse photographed in three points of its circling trajectory. (E,F) Lrig3 homozygotes (F) have shortened snouts compared with heterozygotes (E). (G-J) Paintfilled inner ears of E14 Lrig3+/- (G,I) and Lrig3-/- (H,J) embryos. Low magnification views of the entire inner ear (G,H) reveal a truncation of the lateral semicircular canal (arrowhead, H). Other structures appear normal in size and shape. High magnification views of the vestibular apparatus confirm truncation of the lateral canal (LC) but not the anterior (AC) or posterior (PC) canals. The lateral ampullae (asterisks) are unaffected, with no change in the number or distribution of MyosinVIIa-positive hair cells in the lateral cristae (insets). Scale bar: 50 µm. Dorsal is upwards; posterior is towards the left.

View larger version (133K): [in this window] [in a new window]   Fig. 3. Lrig3 acts in the non-fusing epithelium to prevent premature and ectopic fusion. (A-F) Dorsal views of paintfilled Lrig3+/- (A-C) and Lrig3-/- (D-F) inner ears collected at 6-hour intervals from E12 to E12.5. Anterior is towards the right. In mutants, early outgrowth is normal (D). However, fusion initiates too early (E) and occurs over a larger area (asterisk) than in control embryos (B,C), resulting in truncation by E12.5 (F). (G-I') Adjacent sections of Lrig3 heterozygotes through the lateral pouch at the level indicated (broken line, A) were processed for β-galactosidase histochemistry to reveal Lrig3-βgeo activity (G-I) or for in situ hybridization with a probe to Ntn1 (G'-I'). Lrig3 is transcribed throughout the lateral pouch prior to fusion (G). Levels become reduced (H) in the nascent fusion plate (arrowhead) just as Ntn1 expression initiates here (H'). Lrig3 expression is further diminished (I) as transcription of Ntn1 expands (I'). Scale bar: 50 µm.

View larger version (42K): [in this window] [in a new window]   Fig. 4. The basement membrane undergoes early and ectopic breakdown in the inner ear of Lrig3 mutant mice. (A,F) Transverse plastic sections through inner ears of E12 Lrig3+/- (A) and Lrig3-/- (F) littermates, with magnified views of the boxed areas (A',F'). Dorsal is upwards; lateral is towards the right. In controls, epithelial cells in the fusion plate intercalate to form a single layer of cells (A'), but in mutants, this region is expanded (brackets, F'). Scale bar: 50 µm. (B,C,G,H) Immunofluorescent detection of collagen IV (B,G) and all laminins (C,H) in E12 Lrig3+/- (B,C) and Lrig3-/- embryos (G,H) sectioned in the transverse plane. In homozygotes, the basal lamina is disturbed by breaks in the laminin network and ectopic accumulation of collagen IV (arrowheads, G,H). Scale bar: 50 µm. (D,E,I,J) Electron micrographs of the regions indicated in C and H. The basement membrane is continuous in heterozygotes (arrowheads, D,E) but is absent (asterisks, I) or severely disrupted (asterisk, J) in homozygotes. Scale bar: 500 nm.

View larger version (127K): [in this window] [in a new window]   Fig. 5. Lrig3 and Ntn1 participate in cross-repressive interactions that define the fusing and non-fusing domains of the lateral pouch. (A,E) In situ hybridization of Ntn1 on transverse sections through E12 Lrig3+/- (A) and Lrig3-/- (E) embryos. In Lrig3 mutants, Ntn1 expression is expanded to fill the lateral pouch (outlined). (B,F) β-galactosidase histochemistry of E12 Lrig3+/- (B) and Lrig3-/- (F) littermates. As previously demonstrated (Fig. 3), Lrig3-βgeo levels are reduced in fusion plate cells (arrowhead, B) compared with the surrounding epithelium. However, in Lrig3 mutants (F) reporter activity is present at high levels throughout the pouch. (C,G) Lrig3 and Ntn1 mutant mice were generated with two different gene trap vectors, so only Lrig3LST016 mice carry a placental alkaline phosphatase (PLAP) reporter. Hence, PLAP histochemistry reveals Lrig3 transcription in Ntn1+/+;Lrig3+/- (B) and Ntn1-/-; Lrig3+/- embryos (G) at E12.5. Like β-geo, PLAP staining of Lrig3 heterozygotes (C) is absent from the fusion plate at E12.5 (arrowhead). By contrast, Lrig3 transcription is sustained in the fusion plate of age-matched Ntn1 homozygotes (G). Note that these embryos are 12 hours older than those in A,B,E,F. (D,H) β-Galactosidase histochemistry of E12 Ntn1+/- (D) and Ntn1-/- (H) littermates. Ntn1-βgeo is active in the fusion plate (arrowhead) in heterozygotes (D), consistent with in situ hybridization results (see Fig. 3). However, no activity is detected in the lateral pouch of Ntn1 homozygotes (H). Ntn1-βgeo expression is unchanged in the dorsal pouch (asterisks). Scale bar: 50 µm.

View larger version (49K): [in this window] [in a new window]   Fig. 6. The Lrig3 mutant lateral canal truncation is rescued by removal of one copy of Ntn1. (A-F) Paintfilled E14.5 inner ears from transheterozygous intercross littermates. Canals (arrowheads) are normal in wild-type (A) and transheterozygous (B) embryos, whereas the lateral canal is truncated in Lrig3 homozygotes (C). The truncation is fully rescued when one wild-type copy of Ntn1 is removed from Lrig3 homozygotes (D). Fusion does not occur in Ntn1 mutants (E) or in Ntn1;Lrig3 double mutants (F). (G) Table summarizing the proportion of ears with normal, truncated or unfused semicircular canals for each genotype in offspring from Ntn1+/-; Lrig3+/- intercrosses. The rescued population is highlighted in green. Note that the Ntn1 phenotype is not influenced by the absence of Lrig3.

View larger version (29K): [in this window] [in a new window]   Fig. 7. Proposed model for the Lrig3/Ntn1 feedback loop. (A) A diagrammatic view of the lateral pouch during canal morphogenesis. Cross-repressive interactions between Lrig3 and Ntn1 define the boundary between fusing (blue) and non-fusing (red) regions of the otic epithelium. When the regulatory loop is interrupted by loss of Lrig3, fusion is expanded, whereas in Ntn1 mutants, fusion does not occur. Interactions between Lrig3 and Ntn1 ensure that these two genes become confined to distinct domains of the lateral pouch. (B) We propose the following model for the Lrig3/Ntn1 feedback loop. Lrig3 is present throughout the canal pouch before fusion and inhibits activity of a receptor tyrosine kinase (RTK) signaling pathway (1). Subsequently, fusion is initiated by an unknown fusion plate signal, which we hypothesize acts through the RTK pathway by inhibiting Lrig3 (2), resulting in transcription of Ntn1 (3). Lrig3 initially continues to be transcribed, as expected for a feedback-induced antagonist. However, the increased levels of Ntn1 eventually inhibit Lrig3 expression, either by inhibiting Lrig3 or by potentiating activity of the fusion plate signal. For example, Ntn1 protein may augment activity of the RTK pathway by promoting basal lamina breakdown (4).

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