Expression of ptc1 and hoxd genes in wildtype and syu pectoral fin buds. Whole-mount in situ hypbridizations. Anterior is to the the left and distal is to the top. (A-D,G,H,K,L,O,P) 32 hour pectoral fin buds. (E,F,I,J,M,N,Q,R) 38 hour pectoral fin buds. (A,C,E,G,I,K,M,O,Q) Wild-type fin buds; (B,D,F,H,J,L,N,P,R) syu fin buds. (A,B) ptc1 RNA; (C-F) hoxd-10 RNA; (G-J) hoxd-11 RNA; (K-N) hoxd- 12 RNA; (O-R) hoxd-13 RNA. At 32 hours, ptc1 and hoxd-13 are not expressed in syu buds, while hoxd-10 appears normal and hoxd-11 and hoxd-12 show a partial reduction. At 38 hours only expression of hoxd-10 remains in syu mutant buds.

Expression of hoxa genes in wild-type and syu pectoral fin buds. Wholemount in situ hybridizations. Anterior is to the left and distal is to the top. (A,B,E,F,I,J,M,N) 32 hour pectoral fin buds; (C,D,G,H,K,L,O,P) 38 hour pectoral fin buds. (A,C,E,G,I,K,M,O) Wildtype fin buds; (B,D,F,H,J,L,N,P) syu fin buds. (A-D) hoxa-9 RNA; (E-H) hoxa-10 RNA; (I-L) hoxa-11 RNA; (M-P) hoxa- 13 RNA. At 32 hours, hoxa- 13 is not expressed in syu mutant buds, while hoxa-9 and hoxa-11 are expressed normally and hoxa-10 shows a partial reduction. At 38 hours, only a strong proximal central expression of hoxa-10 and hoxa-11 and a weak expression of hoxa-9 is detectable in syu mutant buds.

Expression of msx-c and hoxc-6 in wild-type and syu pectoral fin buds. Whole-mount in situ hybridizations. Anterior is to the left and distal to the top. (A,B,E,F) 32 hour pectoral fin buds; (C,D,G,H) 38 hour pectoral fin buds. (A,C,E,G) Wild-type fin buds; (B,D,F,H) syu fin buds. (A-D) msx-c RNA; (E-H) hoxc-6 RNA. msx-c and hoxc-6, which are normally restricted to the anterior mesenchyme, are expressed throughout the mesenchyme of syu mutant fin buds.

Effect of cyclopamine on midline ptc1 expression and on fin development. Whole-mount in situ hybridizations. Anterior to the left. (A-C) Dorsal to the top, 25 hour embryos; (D-L) distal to the top: 32 hour pectoral fin buds (D-J); 48 hour pectoral fin buds (K,L). (A,D-F,L) Wild type; (B) syu; (C,G-K) wild type treated with cyclopamine. (A-C,H) ptc1 RNA; (D,G,J-L) shh RNA; (E) ehh RNA; (F) twhh RNA; (I) hoxd-11 RNA. Note the partial reduction of midline ptc1 expression in syu mutants and the complete absence in cyclopaminetreated embryos (A-C). ehh and twhh are not expressed in 32 hour pectoral fin buds (E,F). Cyclopamine-treated fin buds fail to express ptc1 (H), but activate shh and hoxd-11 (G,I). shh expression subsequently becomes weaker (J) and is lost (K). cyc, cyclopamine.

Effect of retinoic acid treatment on wild-type and syu pectoral fin buds. (A-F) retinoic acid-treated embryos. (A) Embryo at 52 hours; (B-F) whole-mount in situs of embryos at 32 hours of development. (A,B,C,E) Wild-type embryos treated with retinoic acid; (D,F) syu embryos treated with retinoic acid. (B) shh RNA; (C,D) hoxd-10 RNA; (E,F) hoxd-11 RNA. The pectoral fin buds are elongated and located next to the heavily pigmented anterior end of both wild-type and syu embryos treated with retinoic acid (arrows in A to E). Note also that the posterior markers shh and hoxd-11 are expressed throughout the fin bud mesenchyme in wild-type embryos (arrows in B and E), but not in syu embryos treated with retinoic acid, which instead show a patch of hoxd-11 expression at the posterior margin of the fin bud (arrow in F).

Expression of apical epidermal fold markers and en-1 in wildtype and syu mutant pectoral fin buds. Wholemount in situ hybridizations. Anterior is to the left and distal to the top, except in (Q-T), where dorsal is to the left and distal the top. (A,B,K,L,Q,R) 32 hour pectoral fin buds; (C,D,G,H,M,N) 38 hour pectoral fin buds; (E,F,I,J,O,P,S,T) 48 hour pectoral fin buds. (A,C,E,G,I,K,M,O,Q,S) Wild-type fin buds; (B,D,F,H,J,L,N,P,R,T) syu fin buds. (A-F) dlx-2 RNA; (G-J) fgf-8 RNA; (K-P) bmp-2 RNA; (Q-T) en-1 RNA. dlx-2 and bmp- 2 expression appears normal in the apical epidermis of syu buds at 32 hours, but is reduced at 38 hours and 48 hours. Note that dlx-2 and bmp-2 expression is stronger in the posterior apical epidermis of wild-type, but not syu buds at 38 hours. bmp-2 is expressed in posterior mesenchyme at 32 hours in syu buds, but this expression is lost by 38 hours. fgf-8 expression is not detectable in syu fin buds. en-1 is expressed normally in syu fin buds at 32 and 48 hours. Note the apical thickening in the wild-type bud (arrowhead in S), which is not visible in the syu bud (arrowhead in T).

Morphology of wild-type and syu pectoral fin buds. Cross sections through fin buds performed perpendicular to the P/D axis of the fin bud. (A,B) Pectoral fin buds at 32 hours; (C,D) pectoral fin buds at 38 hours; (E,F) pectoral fin buds at 72 hours. (A,C,E) Wildtype fin buds; (B,D,F) syu fin buds. An apical fold (af) is not present in syu buds at 38 hours (D), but is present in some syu buds at 72 hours (F). Note the reduced size of syu buds compared to wild-type buds. The syu apical fold does not develop into a fin fold, which is well developed in wild-type fins at 72 hours (E,F). Note the absence of differentiated tissues in syu buds at 72 hours. ae, apical epicermis; af, apical fold; dm, dorsal musculature; en, endoskeletal disc; ff, fin fold; mbv, marginal blood vessel; mes, mesenchyme; vm, ventral musculature.

Reduced proliferation in syu pectoral fin buds. Dorsal view of embryos at the level of the pectoral fin buds. Anterior is to the top. (A,C) Wild type; (B,D) syu. (A,B) Injected with BrdU at 28 hours and stained at 30 hours; (C,D) injected with BrdU at 39 hours and stained at 41 hours. Note the reduced BrdU incorporation in syu fin buds.

Larval pectoral fin endoskeleton formed in wild-type and syu mutants. Alcian blue stains of the pectoral girdle and fin of larvae on day 6. (A) Wild-type pectoral fin endoskeleton. (B) syu hypomorphic allele tq252. (C) syu null allele t4. syu null mutants do not form any elements of the fin endoskeleton and show a reduced cleithrum (cl). syu^tq252 hypomorhic mutants variably show reductions of the fin endoskeleton and are able to form all elements including the scapulocoracoid (sco), endoskeletal disc (ed) and the actinotrichs (ac), but show a reduction in cell number, which is most apparent in the endoskeletal disc. ac, actinotrichs; cl, cleithrum; ed, endoskeletal disc; sco, scapulocoracoid.