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doi: 10.1242/10.1242/dev.00555

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Fgf3 and Fgf10 are required for mouse otic placode induction

Department of Human Genetics, University of Utah, Salt Lake City, UT 84112-5330, USA

View larger version (46K): [in a new window]   Fig. 5. Cell proliferation and survival are not altered significantly in Fgf3-/-;Fgf10-/- embryos. Whole mount embryos were analysed for mitotic or apoptotic cells using an antibody to phosphohistone H3 (A,C) or TUNEL (E,G), respectively and sectioned in the transverse plane. A section taken through the otic region (the plane is indicated by a line through each embryo) is shown in the panel to the right of each whole embryo. Rostral is to the left. Phosphistone H3 immunoreactivity in transverse sections from an 8 somite control (B) and double mutant embryo (D). Mitotic cells (brown) can be identified in all three tissues that are involved in otic induction; neurectoderm (ne), mesoderm (m) and surface ectoderm (se). TUNEL staining in transverse sections from 7 somite control (F) and double mutant embryos (H). Apoptotic cells (brown) can be identified in all three tissues that are involved in otic induction; neurectoderm, mesoderm and surface ectoderm.

View larger version (62K): [in a new window]   Fig. 1. Fgf3 and Fgf10 are expressed in sites relevant to early otic development. Whole mount embryos were probed with labelled cDNA for Fgf3 (A,C) and Fgf10 (E,G,I,K) and sectioned in the transverse plane. A section taken through the otic region (the plane is indicated by a line through each embryo) is shown in the panel to the right of each whole embryo. Rostral is to the left. Fgf3 is expressed in the developing neurectoderm (n) and surface ectoderm (se) from 3 (A,B) to 12 (C,D) somites. Fgf10 is expressed in the mesenchyme (m) that underlies the otic ectoderm at zero somites (E,F), 7 somites (G,H) and at E8.75 (I,J). By E9.0, Fgf10 expression is induced in the otic cup (oc) (K,L). Fgf10 transcripts can also be detected in the neurectoderm (G-J) and the pharyngeal endoderm (pe) (J,L).

View larger version (70K): [in a new window]   Fig. 2. Fgfr2b and Fgfr1 are expressed in prospective otic placode. Whole mount embryos were probed with labelled cDNA for Fgfr2IIIb (A,C,E,G) and Fgfr1 (I,K) and sectioned in the transverse plane. A section taken through the otic region (the plane is indicated by a line through each embryo) is shown in the panel to the right of each whole embryo. Rostral is to the left. Fgfr2IIIb is expressed in the developing neurectoderm (n) at 3 (A,B), 6 (C,D), 8 (E,F) and 16 (G,H) somites. The onset of Fgfr2IIIb expression in the otic placode (op) coincides with placodal thickening (E,F) and persists to the otic cup (oc) stage (G,H). Fgfr1 is expressed in the developing neurectoderm (n) from at least 4 (I,J) to 7 (K,L) somites. Fgfr1 expression in the otic placode is apparent by 7 somites (K,L).

View larger version (91K): [in a new window]   Fig. 3. Morphological and in situ hybridisation analyses of E9.5 and E10.5 Fgf3/Fgf10 double mutant embryos reveal a failure of otic vesicle formation. Whole mount embryos were stained with haemotoxylin and eosin (A,C) or were probed with labelled cDNA for Pax2 (E,G) and Dlx5 (I,K) and sectioned in the transverse plane. A section taken through the otic region (the plane is indicated by a line through each embryo) is shown in the panel to the right of each whole embryo. Rostral is at the top (A,C) or to the left (E,G,I,K). Comparison of E10.5 control (A,B) and double mutant embryos (C,D) shows the absence of otic vesicles (ov), forelimbs (fl) and hindlimbs (hl) as well as truncation of the tail (t) in double mutants (C,D). In situ hybridisation with Pax2 to E9.5 control (E) and double mutant (G) embryos detects transcripts in the eye (e), kidney (k) and isthmus (i). Pax2 transcripts can be detected in the ventromedial wall of the otic vesicle in control embryos (F) but Pax2 is absent from the comparable region of double mutant embryos (H). In situ hybridisation with Dlx5 to E9.5 control (I) and double mutant (K) embryos detects transcripts in the first and second branchial arches (ba) and forebrain (f). Dlx5 transcripts can be detected in the forelimb and the dorsolateral wall of the otic vesicle in control (I,J) but not double mutant (K,L) embryos. Arrowheads indicate microvesicles (D,H).

View larger version (90K): [in a new window]   Fig. 4. Expression of placodal and hindbrain marker genes in double mutant embryos at E8.0 reveals a disturbance of placodal patterning in the absence of hindbrain patterning defects. Whole mount embryos were probed with labelled cDNA for Pax2 (A,C), Pax8 (E,G), Dlx5 (I,K), Gbx2 (M,O), HoxB1 (Q,R), kr (S,T) and Krox20 (U,V). Embryos probed with placodal markers were sectioned in the transverse plane. A section taken through the otic region (the plane is indicated by a line through each embryo) is shown in the panel to the right of each whole embryo. Rostral is to the left. In situ hybridisation with Pax2 to 8 somite control (A) and double mutant (C) embryos detects transcripts in the eye (e), kidney (k) and isthmus (i). Pax2 transcripts can be detected in the otic placode (op) in control (B) but not double mutant (D) embryos. At 8 somites, Pax8 transcripts can be detected in control embryos in the otic placode and more ventrally in the surface ectoderm (F). In double mutant embryos, Pax8 transcripts can only be detected in the more ventrally located surface ectoderm (se, H). In situ hybridisation with Dlx5 to 10 somite control (I) and double mutant (K) embryos detects transcripts in the forebrain (fb). Dlx5 transcripts can be detected in control embryos in the otic cup (oc) (J), but in double mutant embryos the region of Dlx5 expressing thickened ectoderm is located more ventrally (L). At 6 somites, Gbx2 is expressed throughout the surface ectoderm including the surface ectoderm and in the underlying mesoderm (m) (N). In double mutant embryos, Gbx2 transcripts are excluded from the most dorsal regions of the surface ectoderm and from the underlying mesoderm of the otic region (P). At E9.0 HoxB1, Mafb/kreisler and Krox20 are expressed in rhombomeres 4 (r4; Q), rhombomeres 5/6 (r5/6; S) and rhombomeres 3 and 5 (r3, r5; U), respectively. Expression of HoxB1, Mafb/kreisler and Krox20 is unchanged in double mutant embryos (R,T,V). A bracket marks the location of the dorsal surface ectoderm of double mutant embryos, from which otic marker genes are excluded.

View larger version (115K): [in a new window]   Fig. 6. Pax2 and Dlx5 expression in Fgf3-/- embryos, Fgf10-/- embryos and embryos with three mutant Fgf alleles reveals quantitative and unequal roles for Fgf3 and Fgf10 in otic development. Whole mount embryos were probed with labelled cDNA for Pax2 (A,C,E,G) and Dlx5 (I,K,M,O) and sectioned in the transverse plane. A section taken through the otic region (the plane is indicated by a line through each embryo) is shown in the panel to the right of each whole embryo. Rostral is to the left. The control and double mutant embryos for comparison are located in Fig. 3 and are shown at the same magnification. Pax2 and Dlx5 transcripts can be detected in the ventromedial (B,D,F,H) and dorsolateral (J,L,N,P) wall of the otic vesicle (ov), respectively. The size and location of the otic vesicle as well as the pattern of Dlx5 expression are altered in Fgf3-/- mutants (D,L). In Fgf3-/-;Fgf10+/- and Fgf3+/-;Fgf10-/- embryos, the otic vesicles are reduced in size when compared to Fgf3-/- or Fgf10-/- embryos (F,H,N,P). In Fgf3-/-;Fgf10+/- embryos (H,P), the otic vesicles are also located ventrally relative to the otic vesicles in Fgf3-/- (D,L) or Fgf10-/- (B,J) embryos and Pax2 expression is expanded both dorsally and laterally (H). In embryos carrying either combination of three mutant alleles, Dlx5 expression is reduced relative to that seen in the branchial arches (N,P).

View larger version (15K): [in a new window]   Fig. 7. Analysis of the area and position of the otic vesicle in embryos with three mutant Fgf alleles reveals quantitative differences between Fgf3+/-;Fgf10-/- and Fgf3-/-;Fgf10+/- embryos. (A) Quantitative comparisons between the ratio of the area of the central otic vesicle section to the area of the central eye section in Fgf3+/-;Fgf10+/-, Fgf3+/-;Fgf10-/- and Fgf3-/-;Fgf10+/- embryos (n=6 ears and eyes per genotype). The average ear area/eye area (in %) is shown on the y-axis. The genotypes analysed are shown on the x-axis. The otic to optic area ratios of the Fgf3+/-;Fgf10-/- and Fgf3-/-;Fgf10+/- samples were significantly lower than that of the Fgf3+/-;Fgf10+/- embryos (P=0.005 and P=0.001, respectively). (B) Quantitative analysis of the dorsal-ventral position of the otic vesicle. The vertical distance from the dorsal surface of the neural tube to the top of the otic vesicle was measured and compared to the dorsoventral length of the neural tube in Fgf3+/-;Fgf10+/-, Fgf3+/-;Fgf10-/-, and Fgf3-/-;Fgf10+/- embryos (n=6 ears per genotype). The dorsoventral (DV) distance to the otic vesicle/dorsoventral length of the neural tube is shown on the y-axis. The genotypes analysed are shown on the x-axis. The position of the otic vesicle in Fgf3-/-;Fgf10+/- embryos was significantly ventralised relative to that in Fgf3+/-;Fgf10+/- embryos (P=0.001). The asterisk indicates a significant paired t test result between the genotype shown and Fgf3+/-;Fgf10+/- embryos. The bar above each data cell indicates one s.d. 3, Fgf3; 10, Fgf10.