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First published online 22 October 2003
doi: 10.1242/dev.00833

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Bowl is required downstream of Notch for elaboration of distal limb patterning

Department of Anatomy, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK

View larger version (96K): [in a new window]   Fig. 1. The bowl and odd genes give similar phenotypes to Notch when mutated but are expressed at only a subset of Notch-dependent boundaries. (A,B) Organization of segments in wild-type prothoracic leg. The femur, tibia, tarsomeres (t1-t5), pretarsus (pt) and sex comb (sc) are indicated. (C) Notch mutations result in fusion and truncation of tarsomeres (Nts/Y prothoracic leg, t1-t5 segments are fused). (D-H) Legs containing clones of cells mutant for bowl (bowl2; D-F) or odd (odd5; G-H) result in similar defects to Notch. (D,G) Melanotic tissue at femur/tibia joint (arrow) and (D,D') fused tibia/t1 (arrowhead; inset D' shows higher magnification), reduced t1 and aberrant sex-combs. (E) Severe fusion and truncation of tarsus when bowl clones occupy most of the distal leg. (F,H) Single bowl (F) or odd (H) clone affecting t1-t4 in prothoracic leg (marked with forked, similar results were obtained with yellow), causing partial fusions in t2-t5 (arrowheads) and associated truncation. (I-I'') Bowl (magenta) is expressed with E(spl)mß-lacZ (green) at the t5/pretarsus boundary (arrowhead) but not at other sites within the tarsus. (J-K'') Bowl protein (magenta) and odd-lacZ expression (green) in everting pupal (J-J'') and third instar (K-K'') leg discs. Expression occurs at intersegment/joint boundaries (tibia/t1, arrows; t5/pretarsus, arrowheads), where the two genes are coexpressed (J',K'; regions of overlap appear white). Expression of Bowl appears slightly broader than odd-lacZ.

View larger version (31K): [in a new window]   Fig. 2. Phenotypes are only detected in bowl clones spanning several tarsomeres. Analysis of 62 legs with bowl2 clones marked by absence of y. Diagrams depict the segments affected in each clone (tibia and tarsomeres t1-t5). 34% of legs analysed had aberrant tarsomeres (green shading indicates extent of clone; specific tarsomeres showing defects are listed, with boxed text indicating segment fusions). 66% of legs containing clones appeared normal (yellow shading indicates extent of clone). Only clones that include t1 (or t5, data not shown) give rise to tarsomere fusions; phenotypes are not detected in clones that only span tarsomere boundaries.

View larger version (81K): [in a new window]   Fig. 3. Expression of Bowl is regulated by Notch activity. (A-C) Bowl expression (magenta, anti-Bowl) correlates with expression of Delta (green, anti-Delta). (A,A') 80-84 hour third-instar; Delta expression (green) is found adjacent to Bowl-expressing cells (magenta; presumptive femur fe). (B,B') 90 hours, a further domain of Delta (green) is intercalated within the presumptive tibia (ti) and strong expression is seen adjacent to the t5/pretarsal ring of Bowl (pretarsus, pt). (C,C') 120 hours, further rings of Delta expression are present within the tarsal region (e.g. arrows C,C'). Bowl is detected at tibia/t1 and t5/pretarsus boundaries, not at intervening sites (arrows, C). (D,D') 78-80 hours odd-lacZ expression (green, anti-ß-galactosidase) occurs adjacent to sites of Serrate (magenta, anti-Serrate). Expression of Serrate within presumptive femur is indicated (fe). (E,E') Bowl expression (magenta) at t5/pretarsus boundary is absent in N81K mutant clones (arrow, marked by absence of GFP). (F,F') Ectopic Notch activity (green; act>Gal4/UAS-Nicd) induces ectopic Bowl expression (magenta). Arrows indicate sites with ectopic Bowl (overlap between Nicd and Bowl appears white in F).

View larger version (70K): [in a new window]   Fig. 4. bowl is required for normal patterning of tarsal segments. Expression of Bab2 (anti-Bab2, blue) and E(spl)mß-CD2 (anti-CD2, red) in tarsal region of a wild-type pupal leg disc (A) and in a leg disc containing bowl2 mutant clone (B; mutant cells are marked by absence of GFP, green). Bab expression extends proximally in the mutant cells (arrowhead, B,B') and E(spl)mß-CD2 is disrupted (arrow, B,B''). Inset in B'' (boxed region in B) shows relationship between clone boundaries (green) and E(spl)mß-CD2 expression (red); mutant cells at the edge of the clone express E(spl)mß-CD2 at wild-type levels. (C,D) Expression of Bab2 (anti-Bab2, cyan) and Dac (red) in wild-type pupal leg disc (C) and in a bowl2 clone crossing t1 (absence of GFP, arrows, D-D''). The bowl2 clone results in ectopic Bab2 (blue, D,D') and decreased Dac (red, D,D''); levels appear higher near the distal edge of the clone because of the fold in the epithelium. (E,E') bowl1 clones in proximal tarsus/tibia, Bab2 (blue) is elevated in t1 (arrowhead) and Bowl staining (red) is absent from mutant clones (*). (F-F'') bowl2 clones (absence of GFP, arrows) in distal tarsus of late third-instar leg disc result in elevated Bab2 (blue) and reduced BarH1 (red). Elevated Bab2 in more proximal clone is also seen (arrowhead).

View larger version (80K): [in a new window]   Fig. 6. Ectopic bowl expression causes expansion of proximal and distal tarsal fates. (A-D) Expression of bowl within distal leg leads to fusion of segments and transformation to more proximal t1 fates as indicated by ectopic sex combs (e.g. arrowheads). (A) Wild-type prothoracic leg; arrow marks the sex comb in t1. (B-D) Prothoracic legs from lines with different levels of bowl expression: (B) weak, klumpfuss-Gal4G410/UAS-bowl[6.1]; (C) intermediate, Dll-Gal4em212/UAS-bowl[9.1]; (D) strong, Dll-Gal4em212/UAS-bowl[1.1]. Higher levels of Bowl lead to more severe phenotypes: in (D), the tarsus is completely fused with multiple ectopic sex combs. Domain of Dll expression is seen in Fig. 4A-C; klumpfuss-Gal4G410 is in patches and rings within the tarsal region (Klein and Campos-Ortega, 1997). (E,F) Expression of Dac (red), Bab2 (blue) and BarH1 (green) in pupal legs from wild-type (E) and Dll-Gal4em212/UAS-bowl[9.1] (F). Ectopic Bowl results in decreased Bab2 and expansion of Dac (red arrowhead) and BarH1 (green arrowhead). (G-G'') Levels of ectopic Bowl protein (green) are more variable than those of ß-galactosidase (red), even though both are driven by ptc-Gal4. (120 hour leg discs from Ptc-Gal4559.1/UAS-bowl[1.1], UAS-lacZ). Only low levels of Bowl are detected within the tarsus or pretarsus (arrowheads). Expression of bowl mRNA is more uniform and similar to lacZ (data not shown). (H,H') Prothoracic leg from ssa/ss114 has ectopic sex comb on t2 (H, arrowhead), ectopic joint in t1 (H', arrow) and fusion of t2-t3 (H', arrowhead). (I,I') In ssa/ss114 pupal leg discs, ectopic patches of Bowl (green) are seen in t1 and t2 (arrowheads). Dac (red) is unaffected but Bab (blue) is decreased in places (*).

View larger version (100K): [in a new window]   Fig. 5. Expression of Bowl relative to gene products involved in distal leg patterning. (A-C) Expression of Bowl (green, anti-Bowl), Bab2 (blue, anti-Bab2) and Dll (red, Dll-lacZ01092) in wild-type leg discs. (A-A'') At 80 hours, Bowl- and Bab2-expressing cells abut at the edge of the Dll domain. Most Bowl-expressing cells are adjacent to the domain of Dll-lacZ (green arrow, A') (B,B') At 86 hours, Bowl-expressing cells are within the Dll-lacZ domain (yellow arrow, B') and a gap (arrow) is appearing between Bab2 and Bowl expressing cells. (C,C') At 96 hours, Dll-lacZ-expressing cells extend more proximally than the Bowl tibia/tarsus ring (red arrow, C'). Bowl-expressing cells no longer abut Bab2 expression domain (C''; arrows, t1/t2; arrowhead, t5). (D-F) Expression of BarH1(red, anti-BarH1) Bab2 (blue) and odd-lacZ (green). (D) At 76-80 hours BarH1 expression overlaps most of the Bab2 domain. (E) By 90 hours, Bab2 expression extends more proximally and has disappeared from the central/distal region. (F) At 120 hours, odd-lacZ expression abuts BarH1. (G) At 96 hours, odd-lacZ expression persists within the presumptive tarsus (e.g. *).

View larger version (17K): [in a new window]   Fig. 7. Model of the relationship between Bowl and Bab2 expression domains and limb patterning. (1) Early stage of tarsal development: a leg imaginal disc with tarsal domains of Bab2 (blue) and Bowl (green). Dotted line outlines region shown below and in subsequent stages. Within this region (rectangle), Bab2 expression (blue) is uniform and flanked by Bowl (green). Bowl inhibits Bab2. Bab2 (or another target of Bowl) prevents upregulation of Dac and BarH1 (dashed grey lines). Below is shown the hypothetical distal leg structures correlating with this stage of expression (Ti, tibia; ta, tarsus; pt, pretarsus), the tarsus consists of a single segment. Arthropods with an unsegmented tarsus are predicted to arrest distal limb patterning at this stage. (2) Middle stage of tarsal development. The length of the tarsal territory (rectangle) has increased. Bab2 expression (blue) is now induced in a larger domain as Bowl (green) decays from the central region (top). Hypothetical distal leg structures correlating with this stage of expression, the tarsus consists of three segments. Arthropods with intermediate numbers of tarsal segments are predicted to arrest distal limb patterning at this stage (bottom). (3) Late stage of tarsal patterning. The length of the tarsal territory (rectangle) has further increased. Bab2 expression (blue) has reached its full extent; as Bowl (green) decays further, Bab2 can no longer be induced and Dac and BarH1 are upregulated in t1 and t5, respectively (top). Distal leg structures correlating with this stage of expression; the tarsus consists of five segments (bottom).