Figure 1.
Expression of Krz mRNA and protein.
(A–A′) In situ hybridization with antisense (A) and sense (A′) krz probes in late third instar wing (w) and leg (L) discs. (B–C) Expression of Krz (red), FasIII (green) and Topro (Blue) in the wing pouch of a late third instar disc. (B) Apical focal plane of the wing pouch (B′ is the red channel showing the expression of Krz). (C) Longitudinal sections of the wing pouch at the place indicated in B by a white line. (C′) Red channel of C. (D–E) Transversal sections taken at the apical (D–D′) and medio-lateral (E–E′) level of the wing pouch. D′ and E′ show the expression of Krz (red). (F–H′) In situ hybridization with an antisense krz probe in the blastoderm (F), in stage 13 embryos (G) and in stage 17 embryos oriented laterally (H) or ventrally (H′), showing the prominent expression of krz in the CNS. (I–K′) Expression of Krz protein in the blastoderm (I), in stage 16 embryos (J) and in stage 17 embryos oriented ventrally (K) or dorsally (K′). (L) Representation of the krz gene indicating the intron-exon structure, the position of the ATG and Stop codons, the insertion sites of the e03507, e00739 and krz1 transposons and the extent of the krz deficiency (Df(3R)krz). The scale in Kb is indicated above. (M–M′) Loss of Krz expression (red) in ap-Gal4 UAS-GFP/UAS-ikrz wing discs. The expression of GFP is shown in green. (N–N′) Elimination of Krz expression (red) in clones of cells homozygous for the Df(3R)krz. The clone (arrow) is labelled by the absence of green. (O–O′) Higher magnification of the clone shown in N. (P–S) Expression of Krz (in red), FasIII (in green) and Topro (in blue) in wing imaginal discs expressing the following Krz-FLAG forms in the salEPv-Gal4 domain: UAS-krzWT (P–P′), UAS-krzV94D (Q–Q′), UAS-krzS427D (R–R′), UAS-krzLeu (S–S′). P′–S′ correspond to the red channel of P–S showing the expression of Krz. The planes of each transversal sections (shown below each picture) are indicated by white lines in P–S′.
Figure 2.
Loss-of-function phenotype of krz in the wing.
(A) Wild type control wing. (B) 638-Gal4/+; UAS-FLP/+; FRT82 Df(3R)krz/FRT82 M(3)w. In this genotype all wing cells are homozygous for the Df(3R)krz, and the wings show reduced size and incorrect folding. (C) UAS-dicer/+; nub-Gal4/+; UAS-ikrz/+. In this genotype krz iRNA is expressed in the entire wing pouch, and the wing is very similar to that shown in B. (D) wor-Gal4/UAS-krz; krz1/Df(3R)krz. The expression of krz in the CNS driven by wor-Gal4 rescues the lethality of the krz1/Df(3R)krz combination, and the wings develop without krz expression. (E) Third instar wing disc of krz1/Df(3R)krz genotype, showing a reduced size and the expression of activated-Cas3 (green) throughout the disc. (F) Third instar wing disc of wor-Gal4/UAS-krz; krz1/Df(3R)krz genotype, showing a normal expression of Smo (red) and the rescue of wing disc size. (G–H) Genetic interaction between Notch and Krz. The reduction in Notch expression (638-Gal4/+; UAS-iNotch/+; G) causes the thickening of the wing veins and the elimination of the wing margin (G). This phenotype is augmented when the expression of krz is also reduced (638-Gal4/+; UAS-iNotch/UAS-ikrz; H). (I–J) Genetic interaction between EGFR and Krz. The reduction in EGFR activity (638-Gal4/+; UAS-EGFRDN/+; I) causes the loss of veins and a reduction in wing size (I). This phenotype is not modified when the expression of krz is also reduced (638-Gal4/+; UAS-EGFRDN/UAS-ikrz; H). (K–L) Genetic interaction between Hh and Krz. The reduction in Hh expression (638-Gal4/+; UAS-ihh/+; I) causes a reduction in wing size and in the distance between the veins L3 and L4 (K). This phenotype is not modified when the expression of krz is also reduced (638-Gal4/+; UAS-ihh/UAS-ikrz; L).
Figure 3.
Expression of Notch, EGFR, and Smo in krz loss-of-function conditions.
(A–D) Expression of Notch (in red) in ap-Gal4 UAS-GFP/+; UAS-ikrz/+. A and B show two different focal planes in the ventral (A) and dorsal (B) wing disc surfaces. Below each panel are transversal sections in the ventral (“V” in A′) and dorsal (“D” in B′) disc surfaces. The bottom panels are the corresponding red channels (Notch protein expression) in these transversal sections. The white lines indicate the position of the different sections. (C) Longitudinal section of the wing blade showing the dorsal (green) and ventral (lack of GFP) surfaces showing the expression of Notch (red) Topro (blue) and GFP (green). The individual red channel is shown below. (D) Projection of 10 optical sections (indicated by a white vertical line in B′ and white horizontal line in C) spanning the apical side of an ap-Gal4 UAS-GFP/+; UAS-ikrz/+ wing disc showing Notch (red) and GFP (green) expression. Note in all cases the modest difference between wild type cells (ventral) and ikrz-expressing cells (dorsal). (E) Expression of Notch in third instar wing discs bearing clones of cells homozygous for the krz deficiency. Below each focal plane are the transversal sections showing the expression of Notch (red) and GFP (green). (E′) corresponding red channel of E. Clones were induced in hsFLP1.22/+; FRT82 Df(3R)krz/FRT82 Ubi-GFP M(3)w larvae 48–72 h. AEL (in panels E, H and J). (F) Expression of EGFR (in red), GFP (in green) and Topro (in blue) in ap-Gal4 UAS-GFP/+; UAS-ikrz/+ third instar discs. Below are transversal sections in the ventral (V, middle panel) and dorsal (D, bottom panel) disc surfaces. (F′) Red channel of F showing only EGFR expression. (F″) Longitudinal section of the wing blade showing the expression of EGFR (red) Topro (blue) and GFP (green). The individual red channel is shown below. (G) Projection of 10 optical sections spanning the apical side of a ap-Gal4 UAS-GFP/+; UAS-ikrz/+ disc showing EGFR (red) and GFP (green) expression. (H) Expression of EGFR in third instar wing discs bearing clones of cells homozygous for the krz deficiency. Below and to the right are the corresponding transversal and longitudinal sections showing the expression of EGFR (red) and GFP (green). (H′) Corresponding red channel of I. (I–I′) Expression of Smo (red) in ap-Gal4 UAS-GFP/+; UAS-ikrz/+. Below are transversal sections in the ventral (V, middle panel) and dorsal (D, bottom panel) disc surfaces. To the right are longitudinal sections in the posterior (p) and anterior (a) compartments. The individual red channels are shown in I′. (J) Expression of Smo in third instar wing discs bearing clones of cells homozygous the krz deficiency. Below each focal plane are the transversal sections showing the expression of Smo (red) and GFP (green). (J′) corresponding red channel of J. Note that in all genotypes tested the reduction or loss of krz does not modify EGFR (G–I) or Smo (J–L) expression.
Figure 4.
Analysis of the krz gain-of-expression phenotype and its relationship with Smo signalling.
(A) Wild type control wing grown at 29°C showing the positions of the longitudinal veins L2 to L5. (B–D) Weak (B), moderate (C) and strong (D) phenotypes resulting from krz over-expression in the wing blade (638-Gal4), using three different UAS-krz lines (2a2 in B, 2b2 in C and 2a1 in D). All flies were grown at 29°C. (E) Phenotype of krz over-expression in the domain of dpp expression (dpp-Gal4/UAS-krz2a1). (F–G) Strong Hh loss-of-function phenotypes obtained in smo2 homozygous wings (F; 638-Gal4/+; FRT42 smo2/FRF42 M(2)l2; UAS-FLP/+) and in Cos2 over-expressing wings (638-Gal4/+; UAS-Cos2/+; G). (H–I) Genetic interaction between hh loss of expression and krz gain of expression. The hh loss-of-function phenotype (638-Gal4/+; UAS-ihh/+; H) is strongly increased by the over-expression of Krz (638-Gal4/+; UAS-ihh/UAS-krz; I). (J–M) Expression of the Hh-target gene ptc in different Krz over-expression conditions. (J) Control third instar wing disc showing the normal expression of Ptc (red). (K) Krz over-expression in the wing blade (638-Gal4/+; UAS-krz2b2/+) reduces Ptc expression. (L) Krz over-expression in the dorsal compartment (ap-Gal4 UAS-GFP/+; UAS-krz2a3/+) reduces Ptc expression in dorsal cells (labelled in green). L′ is the red channel of L. (M) Clones of cells over-expressing Krz (labelled in green) show a cell-autonomous reduction in Ptc expression. M′ is the red channel of M. (O–Q) Expression of the Hh-target gene en in different Krz over-expression conditions. (N) Control third instar wing disc showing the normal expression of En (red). (O) Krz over-expression in the wing blade (638-Gal4/+; UAS-krz2b2/+) eliminates En expression in anterior cells (labelled by a white line in N). (P) Krz over-expression in the dorsal compartment (ap-Gal4 UAS-GFP/+; UAS-krz2a3/+) eliminates En expression in dorsal cells (labelled in green). P′ is the red channel of L. (Q) Clones of cells over-expressing Krz (labelled in green) show a cell-autonomous elimination of En expression. M′ is the red channel of M. As expected, the expression of Ptc and En is detected in wild type cells anterior to the clones of Krz over-expressing cells (M′ and Q′).
Figure 5.
Effects of krz on Smo expression.
(A–D) Expression of Smo in different genetic backgrounds in which Krz is over-expressed. (A) Wild type control third instar disc showing normal Smo expression (red). (B) Elimination of Smo expression of the wing blade of 638-Gal4/+; UAS-krz2a2/+ discs. (C–C′) Expression of Smo (red) in clones of cells over-expressing Krz (labelled in green in C). C′ is the red channel of C. (D) Expression of Smo in ap-Gal4 UAS-GFP/+; UAS-krz2a3/+. Below are transversal sections in the ventral (V, above) and dorsal (D, below) compartments and to the right a longitudinal section showing Smo expression. Note in C and D the cell-autonomous elimination of Smo by excess of Krz. (E, F) Krz modulates Smo stability and degradation by the proteasome pathway. (E) Krz over-expression decreases steady-state Smo levels. Stable Myc-Smo S2 cells were transiently transfected with either empty pUAS/actinGAL4 vector or pUAS-krz/actinGAL4 vector, and incubated in presence or absence of Hh conditioned medium, as indicated in the figure. The levels of Smo were determined in the whole cellular lysates by immunoblotting. The basal amount of Smo in the mock-transfected cells was defined as 1 and all the data were normalised by Tubulin protein levels. Data are the mean±SEM of five independent experiments. A representative gel is shown. * (P<0.05); *** (P<0.001) when compared to values of mock-transfected cells. (F) Smo turnover is inhibited by the proteosome inhibitor MG132, and the stability of Myc-Smo is increased in the presence of MG132 even when Krz is over-expressed. Smo protein levels were examined by immunoblot analysis in cells incubated with control or Hh conditioned medium upon treatment with Cycloheximide (Chx) at 0 and after two hours treatment, in the absence or presence of MG132 as indicated. The amount of Smo at 0 h was defined as 1 for each case, and data normalized by Tubulin protein levels. Data are the mean±SEM of three independent experiments. A representative gel is shown. * (P<0.05); *** (P<0.001) when compared to values at 0 h.
Figure 6.
Interactions between Krz mutant forms and Smo.
(A–D) Effects of different Krz-Flag mutant forms expressed in the dorsal compartment on Smo expression. (A) Control ap-Gal4 UAS-GFP/+; UAS-krz-Flag/+ showing Flag (blue) and GFP (green), and the elimination of Smo (red) in the dorsal compartment. (B) ap-Gal4 UAS-GFP/+; UAS-krzV94D-Flag/+. The mutant protein KrzV94D is over-expressed (blue staining in B), but it does not affect Smo expression (red in B′). (C) ap-Gal4 UAS-GFP/+; UAS-krzS427D-Flag/+. The mutant protein KrzS427D is over-expressed (blue staining in C), and eliminates Smo expression from dorsal cells (red in C′). (D) ap-Gal4 UAS-GFP/+; UAS-krzLeu-Flag/+. The mutant protein KrzLeu is over-expressed (blue staining in D), but it does not affect Smo expression (red in D′). A′–D′ corresponds to the single red channels showing Smo expression. (E, F) Interactions between Krz and Smo in S2 cells. Stable Myc-Smo S2 or S2 control cells were transiently transfected with either empty pUAS/actinGAL4 vector (mock lanes), pUAS-krz/actinGAL4 (Kurtz lanes) or pAWF-krz mutant constructs (pAWF-krzwt, pAWF-krzV94D, pAWF-krzS247D or pAWF-krzLeu; F-Kurtz construct lanes) as indicated in the figure, in presence of Hh conditioned medium for 18–24 hrs. Cell extracts were immunoprecipitated with anti-Myc affinity gel. Immunoprecipitates (F) and whole-cell lysates (E) were immunoblotted with antibodies against Krz (detecting both endogenous Kurtz, lower band and the Flag-Kurtz protein, upper band), Myc (detecting Myc-Smo protein) and Tubulin. In control IPs carried out in Smo-Myc non-expressing cells transfected with pAWF-krzwt, pAWF-krzV94D, pAWF-krzS247D or pAWF-krzLeu, we did not detect any Krz or FLAG-Krz protein (data not shown). The levels of Smo were determined in the whole cellular lysates by immunoblotting. The basal amount of Smo in the Krzwt transfected cells was defined as 1 and all data were normalised by tubulin protein levels. Data are the mean ±SEM of three independent experiments. A representative gel is shown. * (P<0.05); *** (P<0.001) *, P<0.05; ***, when compared to values of Krzwt transfection.
Figure 7.
Interactions between Krz and different Smo mutant forms.
(A–C) Control phenotypes caused by the over-expression of wild type Smo (638-Gal4/+; UAS-smo/+; A), and phosphorylation-defective forms in the CK1 (638-Gal4/+; UAS-smoCK1/+; B) and PKA (638-Gal4/+; UAS-smoPKA/+; C) sites. (D) Rescue of the Krz over-expression phenotype by increased levels of Smo (638-Gal4/+; UAS-smo/UAS-krz2a3). (E–F) Synergistic effects of increased Krz expression in smo mutant backgrounds (638-Gal4/+; UAS- smoCK1/UAS-krz in E and 638-Gal4/+; UAS-smoPKA/UAS-krz2a3 in F). (G–J) Expression of Engrailed in Smo-Krz combinations. (G) 638-Gal4/+; UAS-smo/+. (H) 638-Gal4/+; UAS-smo/UAS-krz2a3. (I) 638-Gal4/+; UAS-smoSD123/+. (J) 638-Gal4/+; UAS-smoSD123/UAS-krz2a3. (K–N) Expression of Ptc in Smo-Krz combinations. (K) 638-Gal4/+; UAS-smo/+. (L) 638-Gal4/+; UAS-smo/UAS-krz2a3. (M) 638-Gal4/+; UAS-smoSD123/+. (N) 638-Gal4/+; UAS-smoSD123/UAS-krz2a3. Expression of Smoothened in Smo-Krz combinations. Transversal sections along the dorso-ventral boundary of 638-Gal4/+; UAS-smo/+ (O); 638-Gal4/+; UAS-smo/UAS-krz2a3 (P); 638-Gal4/+; UAS-smoSD123/+ (Q) and 638-Gal4/+; UAS-smoSD123/UAS-krz2a3 (R) third instar discs.
Figure 8.
Interactions between Gprk2 and Krz in Smo signalling.
(A) Wild type control wing. (B) Gprk2 loss-of-function phenotype resulting from iGprk2 over-expression in the wing blade (638-Gal4/+; UAS-iGprk2/+). (C) Krz gain-of-function phenotype resulting from UAS-krz over-expression in the wing blade (638-Gal4). (D) Genetic interaction between Gprk2 loss of expression and krz gain of expression. Over-expression of Krz when Gprk2 levels are reduced cause a strong Hh los-of-function (638-Gal4/+; UAS-iGprk2/+; UAS-krz/+). (E–F) Third instar wing discs over-expressing Krz and reducing Gprk2 levels in the dorsal compartment (ap-Gal4 UAS-GFP/+; UAS-iGprk2/+; UAS-krz/+) have a complete loss of Ptc (red in E) and En (red in F) expression in anterior-dorsal cells. E′–F′ correspond to the single red channels showing Ptc (E′) and En (F′) expression. (G–G′) Expression of Smo (in red) in wing discs reducing Gprk2 expression in the dorsal compartment (ap-Gal4/UAS-GFP; UAS-iGprk2/+; GFP in green). (H–H′) Expression of Smo (in red) in wing discs over-expressing Krz and reducing Gprk2 levels in the dorsal compartment (ap-Gal4 UAS-GFP/+; UAS-iGprk2/+; UAS-krz/+). Note the difference in Smo expression between G′ and H′. (I–J and O) Expression of Smo in third instar wing discs bearing clones of cells homozygous for the Gprk2 deficiency (I–I′) and for the Gprk2 deficiency in cells that also over-express Krz (J–J′). Panels O–O′ correspond to the same genotype as J–J′ at higher magnification. Clones were induced in hsFLP1.22 actin-Gal4 UAS-GFP/+; FRT82 Df(3R)Gprk2/FRT82 tub-Gal80 (I–I′) and in hsFLP1.22 actin-Gal4 UAS-GFP/+; FRT82 Df(3R)Gprk2/FRT82 tub-Gal80; UAS-krz/+ (J–J′ and O–O′). (K–M) Expression of Ptc (K), En (L) and Smo (M) in wing imaginal disc over- expressing an active form of Smo (SmoSD123) and Krz and reducing Gprk2 expression in the wing blade (638-Gal4/SmoSD123; UAS-iGprk2/+; UAS-krz/+). (N) Expression of Smo in wing imaginal disc over-expressing an active form of Smo (SmoSD123) in the wing blade (638-Gal4). Note the reduction in Smo levels in M compared to N. Below are transversal sections and to the right longitudinal sections showing Smo expression.