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First published online May 1, 2006
doi: 10.1242/10.1242/dev.02370

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Roadkill attenuates Hedgehog responses through degradation of Cubitus interruptus

Department of Cell and Developmental Biology, University of Colorado School of Medicine, PO Box 6511, Aurora, CO 80045, USA.

View larger version (23K): [in a new window]   Fig. 1. rdx gene structure. (A) The rdx genomic locus spans 64 kb in cytological band 88A4. Exons are represented by blue boxes and numbered underneath; lines indicate splices; light blue indicates start codon; red indicates stop codon; arrows indicate direction of transcription (towards the centromere). Four alternative transcriptional start sites generate five transcripts (forms A-E) that share exons 7-13. Exons 1 and 2 are unique to the B form. Exons 3 and 4 are unique to the C/D forms. C differs from D by lacking seven nucleotides in the 5'UTR at the end of exon 3. Exon 5 is unique to the E form (represented by our cDNA 5-2; predicted to be the nested gene CG12537). Exon 6 is unique to the A form. (B) Mutants are mapped onto the gene structure. Exons are indicated by boxes, where grey indicates UTRs, red indicates coding sequence unique to each form, green indicates coding sequence for the conserved MATH domain and magenta indicates coding sequence for the conserved BTB domain. The asterisk identifies alternative termination for 3' UTRs. rdx1 is a LacW insertion 33 nt upstream of the start of exon 5. rdx4 is a complex rearrangement of the region upstream of exon 5, generated by imprecise excision of rdx1. Black arrowheads indicate the missense mutations in rdx5 (G to T in exon 5 encoding Q193H and A to T in exon 10 encoding D653V), and the splice junction mutation in rdx6 (an A to G change in the 23rd nt of the intron between exons 12 and 13). (C) Block diagrams indicating the four proteins encoded by the rdx locus. The numbers above each indicates the amino acid coordinates. Unique sequences are in red; the MATH domain is in green; the BTB domain is in magenta. The arrow indicates the convergence of amino acid sequences.

View larger version (71K): [in a new window]   Fig. 2. rdx expression in embryos. rdx mRNA was detected with probe to exons shared by all transcripts (A-I). Probes specific to rdxA or rdxE detected similar expression patterns (not shown). Lateral views show: (A) stage 1-2; (B) stage 4 with pole cells (pc); (C) stage 5; (D) stage 6; (E) stage 8 with anterior midgut (am) and posterior midgut (pm); (F) stage 10; (G-I) stage 11 with stomodeum (sto), ninth abdominal segment (A9), CNS (n) and mesoderm (ms). A ventral view of stage 10 is focused under the ectoderm to show neuroblasts in F. (H) Parasagittal optical section of an embryo homozygous for ciCe shows loss of rdx expression in the ectoderm and mesoderm, but retention in the head, in A9 ectoderm, and in the CNS. A similar view of a ptc9 homozygous embryo (I) shows extensive ectopic expression of rdx in the ectoderm and mesoderm. The gaps in rdx expression in the ectoderm correspond to posterior compartment cells expressing En (not shown). (J,K) At stage 10/11, En expression (blue) is nestled between stripes of rdx expression (brown), as detected by ß-Gal in rdx1 embryos. (L) Stage 12 with salivary gland primordium (sg). Dorsal is upwards (except in F) and anterior is towards the left in all panels.

View larger version (77K): [in a new window]   Fig. 3. rdx is necessary for the meiotic to mitotic transition. Cuticle from a wild type Canton-S embryo (A) shows evenly spaced denticle bands on the ventral surface, telson structures at the posterior end (t) and internal mouthparts at the anterior end (m). (B) Hoechst staining of DNA visualizes mitoses in a wild type Canton-S blastoderm stage embryo. The cuticle of a rdx6 GLC embryo (C) shows intact telson (t), a fusion of A2-A8, intact T3 and A1 denticle bands, and a large anterior hole extruding malformed mouthparts (m). In wild type (B), nuclei are uniformly spaced, synchronized divisions occur, and chromatin is distributed evenly between daughter nuclei. In a rdx6 GLC embryo (D), mitoses are asynchronous, nuclear spacing is uneven, mitotic figures are highly abnormal (inset) and much of the DNA has sunk into the interior of the embryo (internal staining is out of focus).

View larger version (97K): [in a new window]   Fig. 4. rdx acts in a feedback loop to reduce levels of Ci. Wing imaginal discs are oriented with anterior towards the left and genotypes in the upper right-hand corner. (A-D) rdx expression is detected by ß-gal (green) in the enhancer trap line rdx2/+ (rdxZ). In wild type (A,B), rdx is in a strip of approximately five cells along the compartment border. En expression (red in A) marks the posterior compartment, with additional expression extending approximately three cells into the anterior compartment. rdx expression overlaps the anterior edge of the En expression (inset in A). The anterior edge of Ci155 expression (red in B,E,F,L) marks the compartment border. The anterior edge of rdx coincides with that of Ci155 (inset in B), so rdx expression abuts the compartment border. The Hh-dependence of rdx expression (green) is demonstrated by its expansion when Hh is overexpressed (C); or by its loss when Ptc overexpression suppresses Hh responses (D). rdx6 (E) and rdx5 (F) loss-of-function clones are marked by loss of GFP (green). Clones within six to ten cells of the compartment border (arrowheads near white line) accumulated Ci155 (red), while clones farther from the border (arrow) did not. Filling wings with unmarked rdx5 clones caused a slight overgrowth of the anterior compartment (G) compared with wild type (H); the arrowhead indicates the compartment border (just anterior to vein 4) while the outline of a wild type wing is superimposed for comparison. RdxA (I,K-M) or MycRdxA (J) was mis/overexpressed using the Gal4 line MS1096. The resulting expression of Rdx is demonstrated by Myc (red in J): highest in the dorsal wing pouch (large bracket), low in part of the notum (small bracket) and off in the remainder of the disc. This reduced levels of Ci155 (red in L) and prevented Hh-dependent En induction (blue in L) in the anterior compartment (marked by the edge of Ci155 expression). It reduced levels of Ptc (green in J) near the compartment border, but broadened expression of dpplacZ (K). The resulting wings (I) had small anterior compartments and ectopic veins anterior to vein 3. Western blots (10 wing discs/lane) demonstrate a modest reduction (approximately halved) in both Ci155 and Ci75 when Rdx expression is driven by MS1096 (M). (N-P) Levels of Hh, Ptc, rdx, Ci155, CiR across the anteroposterior axis (x-axis) of wing imaginal discs, with levels indicated on the y-axis. The vertical broken lines indicate the border between the anterior (Ant) and posterior (Post) compartments. In wild type (N), rdx is expressed adjacent to the compartment border, where Hh activates Ci; there it reduces Ci155. With rdx loss (O), Ci155 is elevated adjacent to the compartment border, though this has little effect on Hh target gene expression. Ectopic Rdx (P) reduces both Ci155 and CiR, resulting in dysregulation of Hh target gene expression.

View larger version (90K): [in a new window]   Fig. 5. rdx regulates Ci behind the MF. Third instar eye-antennal imaginal discs (B-F) are oriented with dorsal upwards, anterior towards the right, an arrowhead marking the MF and an asterisk marking the ocellar region of the head capsule. A schematic (A) shows Ci155 levels (red) across the disc, with the Hh-secreting photoreceptors posterior to the MF in grey. Anterior to the MF, in the absence of Hh, Ci155 is processed via Cul1 (blue arrow) to CiR. Posterior to the MF, Ci155 is degraded (red blobs) via Cul3 (green arrow). (B) rdx as detected by ß-gal activity (blue) in rdx2/+ discs (rdxZ), is found posterior to the MF, in the ocellar region, and at the compartment border of the antennal portion (arrow). At higher resolution (C), nuclear-localized ß-gal protein in rdxZ (green) co-localized with Elav (not shown) in all photoreceptors and cone cells, while Ci155 (red) accumulated in the intervening cells. ß-Gal protein is also found in many basal nuclei, corresponding to the position of lattice cell nuclei (not shown). (D,E) rdx clones are marked by loss of GFP (green), while Ci155 is in red. The three arrows (left to right) indicate, respectively, clones behind, within and infront of the MF. The hypomorphic allele rdx1 was used as a control to illustrate normal patterning (D). In clones of the loss-of-function allele rdx5 (E), Ci155 accumulated behind but not in front of the MF. Rdx overexpression (F, marked by GFP in green) reduced levels of Ci155 (left panel, red in right panel) within or just in front of the MF (arrow). (G) When control hsGal4 embryos (+) or hsGal4/MycRdxA embryos (^R) were immunoprecipitated for Myc (IP Myc), Ci155 but not Ci75 was detected in the immunoprecipitates. Neither Ci155 nor Ci75 were detected in control immunoprecipitates using Flag antibody (IP control). Lysates (1% and 0.2% loaded) give estimates of sensitivity. (H) The relationship of Hh, Rdx, Cul3 and Ci. Hh induces Ci155 to activate transcription of rdx (curved red arrow). Then Rdx acts with Cul3 (blue curved arrow) to degrade Ci155 (red blobs). The resulting reduction in Ci155 then attenuates Hh responses (red outlined arrow).

View larger version (85K): [in a new window]   Fig. 6. rdx regulates ommatidial packing. Eyes comprised of mostly rdx mutant cells were generated by mitotic recombination, where the wild-type daughter cells were eliminated by a cell-lethal mutation (A,B,E). SEM of hypomorphic rdx1 mutant eyes (A, left and middle panels) showed wild type patterning, including an ordered hexagonal array of ommatidia with bristles at alternate interstices; rdx5 loss-of-function eyes had disrupted ommatidial organization and loss of bristles (B, left and middle panels). Cross-sections of rdx1 mutant eyes (A, right panel) showed normal patterning; the rhabdomeres of seven photoreceptors (dark spots) per ommatidium are arranged in a distinctive trapezoidal pattern. Cross-sections of rdx5 eyes had seven rhabdomeres in most ommatidia, but their packing was looser and not so clearly trapezoidal (B, right panel). The cellular architecture of apical surface of pupal eyes 36 hours after puparium formation is visualized with Armadillo (Arm) (C,E) and schematized (D,F). Wild type (C,D) shows the regularly packed hexagonal array with the appropriate arrangement of four cone cells (c), two primary pigment cells (1°) and 12 lattice cells – six secondary pigment cells (2°) along the faces of the hexagon, with bristle (b) or tertiary pigment cells (3°) at alternating vertices. The lattice cells are shared between adjacent ommatidia. The junctions between 1° cells always align perpendicular to the anteroposterior axis and always contact 2° cells. rdx5 mutant eyes (E,F) showed misaligned 1° cells (green diamonds), 1° cells failing to wrap around cone cells (yellow stars) and displaced bristles (red triangles).

View larger version (133K): [in a new window]   Fig. 7. Hh signaling contributes to organization of the ommatidial array. (A-D) Scanning electron micrographs of the surface of adult eyes. (E-H) Arm staining used to show the cellular structure of pupal eyes, 36-42 hours (E-G) or 28 hours (H). Anomalous structures are indicated as follows: red stars for missing or duplicated cone cells; the double red star in H indicates a cone cell cluster with a round cell characteristic of apoptotic morphology; yellow stars indicate 1° cells failing to enclose cone cells; green diamonds indicate 1° cells intersecting with tertiary cells; red triangles indicate missing or misplaced bristles. (I-L) Schematics of a pupal ommatidium (see Fig. 6 legend for details). Dorsal is upwards and posterior is towards the left; genotypes are indicated under each panel.