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Concerted action of two dlx paralogs in sensory placode formation

Department of Biology, Emory University, Atlanta, Georgia 30322, USA

View larger version (98K): [in a new window]   Fig. 1. dlx3 and dlx7 are co-expressed by the same otic and olfactory cells. (A,B) Whole-mount RNA in situ hybridization with probes for (A) dlx3 and (B) dlx7 at 18 somites. Lateral views, anterior towards the left. (C,D) Flat-mount double in situ hybridization showing (C) olfactory and (D) otic expression of dlx3 and dlx7 at 18 somites. dlx3 expression is shown in red in both panels with dlx7 expression shown in purple. Anterior is towards the top. ot, otic vesicle; olf, olfactory placode; va, visceral arches. Scale bars: in A, 100 µm for A,B; in C, 25 µm for C,D.

View larger version (46K): [in a new window]   Fig. 2. Loss of dlx3 and dlx7 function leads to a reduction in otic vesicle size. Lateral views, anterior towards the left, of otic vesicles in (A-E) 24 hour and (F-J) 27 hour embryos. (A,F) Wild-type, (B) dlx7 MO, (C,G) dlx3 MO, (D,I) dlx3+7 MO, (E) b380 mutant, (H) dlx3 MO + dlx3 RNA and (J) dlx3+7 MO + dlx7 RNA embryos. otl, otolith. Scale bars: in A, 25 µm for A-G; in F, 75 µm for F-J.

View larger version (21K): [in a new window]   Fig. 3. Morphological data for morpholino injection and RNA rescue. Embryos were injected with morpholinos alone or with RNA, as indicated, and otic vesicles were examined in individual embryos. Severity of otic defect was scored by counting the number of otoliths visible in each otic vesicle: 0:0, no otoliths detectable in either otic vesicle of embryo; 0:1, one otolith detectable in one otic vesicle, etc.

View larger version (54K): [in a new window]   Fig. 4. Expression of otic and olfactory differentiation markers is reduced by loss of dlx3 and dlx7 function. (A,G,M) Wild-type, (B,H,N) control MO, (C,I,O) dlx7 MO, (D,J,P) dlx3MO, (E,K,Q) dlx3+7 MO and (F,L,R) b380 mutant embryos. (A-L) Expression of eya1 at 18 somites in (A-F) whole-mount and (G-L) flat-mount embryos. (M-R) Flat-mount in situ hybridization with anxV at 24 hours. (A-F) Lateral views, anterior towards the left. (G-R) Dorsal view, anterior towards the top of the panels. ot, otic vesicle; olf, olfactory placode; ap, anterior pituitary. Scale bars: in A, 100 µm in A-F; in G, 25 µm in G-L; in M, 25 µm in M-R.

View larger version (43K): [in a new window]   Fig. 5. dlx3 and dlx7 affect otic placode formation before the onset of pax2.1 otic expression but after that of pax8. Whole-mount RNA in situ hybridization at 3 somites with (A,F) wild-type, (B,G) dlx7 MO, (C,H) dlx3MO, (D,I) dlx3+7 MO and (E,J) b380 mutant embryos. (A-E) pax8 is expressed in the presumptive otic primordia (ot) and the pronephros (pn) at this stage. Expression of this gene is unaffected by a loss of dlx3 or dlx7 function. (F-J) pax2.1 is expressed in the presumptive otic primordia (ot), pronephros (pn), midbrain-hindbrain boundary (mhb) and optic stalk (not shown) at this stage. Expression is reduced specifically in the otic primordia of (H) dlx3 MO-injected embryos, and is undetectable in (I) embryos injected with dlx3+7 MO and in (J) b380 mutants. All panels show dorsal views with anterior towards the left. Scale bar: 100 µm.

View larger version (91K): [in a new window]   Fig. 6. Cross- and autoregulatory mechanisms monitor expression levels of dlx3 and dlx7. Whole-mount in situ hybridization with (A,E,I,M) wild-type, (B,F,J,N) dlx7 MO, (C,G,K,O) dlx3 MO and (D,H,L,P) dlx3+7 MO embryos. (A-D) dlx3 and (E-F) dlx7 expression at 6 somites, dorsal views, anterior towards the left. (I-P) dlx3 expression at 21 somites. (I-L) Lateral views with anterior towards the left, (M-P) dorsoanterior views with anterior towards the top. ot, otic region; olf, olfactory region; va, visceral arches. Scale bar: 100 µm.