1. Cell Biology
  2. Developmental Biology
Download icon

Drosophila hedgehog can act as a morphogen in the absence of regulated Ci processing

  1. Jamie C Little
  2. Elisa Garcia-Garcia
  3. Amanda Sul
  4. Daniel Kalderon  Is a corresponding author
  1. Department of Biological Sciences, Columbia University, United States
Research Article
Cite this article as: eLife 2020;9:e61083 doi: 10.7554/eLife.61083
8 figures, 1 table and 1 additional file

Figures

Wild-type genomic ci transgene and CRISPR ci alleles are fully functional.

(A) The ‘genomic Ci’ transgene (gCi) was derived from a 16 kb genomic region of ci cloned into an att-Pacman vector and inserted at att ZH-86FB located at 86F on the third chromosome. It includes a 6.7 kb upstream promoter region, exons1-6 (blue boxes), introns (blue line), and 0.7 kb of the 3’UTR. (A’–A”) CRISPR ci (crCi) alleles were generated in two rounds. (A’) The first round inserted a mini-white gene (pink box) into the first intron of endogenous ci using two guide RNAs (orange) in the first intron and altered PAM sites on the donor template (green star). (A”) The second round replaced intron 1 and exons 2–6 (blue lines and boxes) including the mini-white gene; the donor template had mutated PAM sites (purple stars) corresponding to the gRNA4 site, approximately 30 bp outside the mutated PAM site for gRNA1, and in the 3’UTR 5 kb away from gRNA 2, labeled gRNA 3. (B–E) Third instar wing discs showing ptc-lacZ reporter gene expression, visualized by Beta-galactosidase antibody staining (red), with the posterior edge of AP border expression marked by yellow arrowheads, and (B’–E’) full-length Ci-155, visualized by 2A1 antibody staining (gray-scale). Anterior is left and ventral is up. (B) Two copies and (D) one copy of crCi-WT, or (C) one copy of gCi-WT supported normal patterns of elevated ptc-lacZ and Ci-155 at the AP border but (D, E, I’) sporadic ectopic posterior ptc-lacZ expression (white arrows) was seen whenever a single ci94 allele was present, even (E) in discs with no synthetic ci transgene or allele (Dp[y+] has wild-type ci). (F–I) Induction of En (green) at the AP border was detected by using the posterior boundary (yellow dashed line) of ptc-lacZ (red) to distinguish anterior (left) from posterior compartment cells, which express En independent of Hh signaling. En induction was normal in the presence of (F) two copies of cr-Ci-WT, (H) one copy of crCi-WT or (I) one wild-type ci allele and (G) was slightly reduced in the presence of one copy of gCi-WT. Scale bars are (B–E) 100 μm and (F–I) 40 μm.

Figure 2 with 2 supplements
Processing-resistant Ci variants reveal the gradients of Hh-stimulated Ci-155 degradation and Hh inhibition of Ci-155 processing.

(A–J) ptc-lacZ (red) and (A’–J’) Ci-155 (gray-scale) in wing discs with one copy of the indicated crCi alleles (with ci94) at (A–F) low (20x objective) and (G–J) high (63x objective) magnification, with AP boundary (dotted yellow line at posterior ptc-lacZ boundary). Scale bars are (A–F) 100 μm and (G–J) 40 μm. (G”–J”) Intensity profiles for ptc-lacZ (red) and Ci-155 (black) from anterior (left) to posterior. Vertical lines indicate ptc-lacZ peak (green), initial rise (orange) and 50% increase to peak (brown). Profiles are from two wing discs for crCi-Δ1270–1370 and three discs for all other samples, aligned and measured as described in Materials and methods. Note that the green line corresponding to maximal ptc-lacZ effectively represents the AP compartment boundary. The profile of ptc-lacZ posterior to that location does not decline precipitously but the decline is not informative (it likely results in part because the columnar cells are not uniformly shaped, so that the measured z-sections include portions of anterior and posterior cells). Territory posterior to the ptc-lacZ peak has yellow shading in (G”–J”) and (K, L) to indicate that it does not contain useful information. The profiles of ptc-lacZ and Ci-155 that report responses to Hh are in the territory anterior to the ptc-lacZ peak. (K) Normalized Ci-155 profiles for indicated crCi alleles derived from G’-J’ but with a smoothing function that calculates average intensity for five successive locations centered on each x-axis location. Arrows indicated locations of ptc-lacZ initial rise, 50% increase and peak for crCi-WT discs. (L) ptc-lacZ (red) and Ci-155 (black) smoothened profiles for crCi-WT, with red guide lines for locations of initial rise, 50% increase and peak ptc-lacZ. The difference between the average Ci-155 intensity for Ci-P(1-3)A and Ci-S849A at each point along the x-axis was subtracted from the maximum Ci-155 intensity for those genotypes (observed in cells anterior to the AP border) to calculate values for Hh-stimulated Ci-155 reduction. These values were added to the Ci-WT Ci-155 profile at each location to produce the blue curve, representing Ci-155 levels in the absence of Hh-stimulated reduction. Blue guide lines show the locations where inferred Ci-155 processing is first inhibited, 50% inhibited, and fully inhibited. See also Figure 2—figure supplement 1 and Figure 2—figure supplement 2.

Figure 2—figure supplement 1
Loss of Hib binding sites does not greatly affect Ci-155 activity or Hh-stimulated proteolysis.

(A–F) ptc-lacZ (red) and Ci-155 (black) intensity profiles for wing discs expressing one copy of the designated ci transgene or alleles from Figure 2A–F. Profiles are from three wing discs for all samples, aligned and measured as described in Materials and methods. (G–K) ptc-lacZ (red) and (G’–K’) Ci-155 (gray-scale) in wing discs with one copy of the indicated crCi alleles (with ci94) and (J, K) homozygous loss of Su(fu) at high (63x objective) magnification, with AP boundary (dashed yellow line at posterior ptc-lacZ boundary). Scale bars are (G–K) 40 μm. (L, M) Intensity profiles for (L) Ci-155 and (M) ptc-lacZ for crCi-WT and crCi-S3-5 in the presence (black and red, respectively) or absence (gray and green, respectively) of Su(fu). Arrows in (L) indicate the locations of ptc-lacZ initial rise (orange), 50% increase (brown) and peak (green) for crCi-WT discs. Note that the green arrow corresponding to maximal ptc-lacZ effectively represents the AP compartment boundary. Thus, the profiles of ptc-lacZ and Ci-155 that report responses to Hh are in the territory anterior to the ptc-lacZ peak; territory posterior to the ptc-lacZ peak has yellow shading to indicate that it does not contain useful information. (N, O) Anterior En (green) induction, revealed by ptc-lacZ (red) marking of the AP boundary (yellow line) extended further anterior for (N) crCi-WT than (O) crCi-S3-5. Scale bars 40 μm. (P) ptc-lacZ intensity profiles (from Figure 2G”–J”) for crCi-P(1-3)A (green), crCi-S849A (red) and crCi-WT (black) were overlapping, while ptc-lacZ induction was much lower for crCi-Δ1270–1370 (gray).

Figure 2—figure supplement 2
Processing-resistant Ci-155 induces ectopic dpp expression and anterior disc expansions are suppressed by adding a constitutive Ci repressor.

(A, B) Wing discs with gCi-S849A and no other source of functional Ci (B) induced ectopic anterior dpp-lacZ (gray-scale) in addition to the AP border stripe but (A) ectopic dpp-lacZ expression is suppressed by a wild-type ci allele (on the Dp[y+] chromosome). (C, D) Wing discs with one copy of (C) gCi-WT or (D) gCi-S849A have similar expression of ptc-lacZ (red) confined to the AP border (at a lower level than in wing discs without ciCe) and normal morphology in the presence of a ci allele that produces constitutive Ci repressor (ciCe). 2A1 antibody staining (gray-scale) is high throughout the anterior in both cases because the ciCe product includes the 2A1 epitope and does not undergo regulated processing. (E) Wing discs with anterior clones (GFP, green, yellow arrows) that have lost a second chromosome gCi transgene, leaving one copy of the crCi allele as a source of Ci. ptc-lacZ was not detected in clones expressing Ci∆1270–1370 (arrows), in contrast to the AP border (arrowheads). (F) Anterior En was much reduced for Ci∆1270–1370 in wing discs expressing one copy of the crCi allele as the only source of Ci. Scale bars (A–D) 100 μm and (E, F) 40 μm.

Processing-resistant Ci variants support normal Hh signaling and wing patterning (A–C) Wing discs with one copy of indicated crCi alleles.

ptc-lacZ (red) indicates the AP compartment boundary (yellow line) to reveal induction of the high-level Hh target gene En (green) in anterior cells at the AP border. (D–F) Wing discs with anterior clones (GFP, green, yellow arrows) that have lost a second chromosome gCi transgene, leaving one copy of the indicated crCi alleles as a source of Ci. (D’–F’) Little (E’) or no (D’, F’) ptc-lacZ induction was observed in the clones (arrows) relative to the AP border (arrowheads). Scale bars are (A–F) 40 μm. (G–L) Wings from adult flies with the indicated ci transgenes and alleles (ciCe encodes a constitutive repressor). The spacing between veins 3 and 4 is (J–L) normal for two copies of WT or S849A ci alleles and (G–I) similarly reduced for one copy of WT or S849A alleles. At least five high-quality mounted wings were examined for each genotype. Scale bars are (G–I) 500 μm.

Activation by Fused kinase is enhanced by blocking Ci-155 processing.

(A–I) Wing discs from animals with one copy of the designated ci transgenes and alleles (together with ci94) with clones (GFP, green, arrows) that express UAS-GAP-Fu and (A–E) lack smo activity or (F–I) are heterozygous for smo (arrowheads indicate AP border), showing (A’–I’) ptc-lacZ (red) and (A”–I”) Ci-155 (gray-scale). (A”–I”) Ci-155 levels were much reduced in clones whenever pathway activity was strongly induced (A”, B”, D”, F”, G”). Scale bars are 40 μm. (J, K) Average intensity of ptc-lacZ in clones (red), Ci-155 in clones (green) or neighboring anterior territory (blue), as a fraction of AP border levels for (J) smo GAP-Fu clones and (K) GAP-Fu clones. Mean and SEM shown. Significant differences between values for a given genotype compared to those for crCi-WT, calculated by paired t-tests, are indicated for p<0.001 (*) and p<0.05 (#). Additionally, in (J) ptc-lacZ was significantly increased for gCi-S849A versus gCi-WT (p<0.0001), as was the anterior level of Ci-155 (p<0.0001).

PKA and Cos2 reduce the activity of Ci-155 that is not processed.

(A–K) Wing discs from animals with one copy of the designated ci transgenes and alleles (together with ci94) with clones (GFP, green, arrows) that lack (A–G) pka activity or (H–K) cos2 activity (arrowheads indicate AP border), showing (A’–K’) ptc-lacZ (red) and (A”, C”–K”) Ci-155 (gray-scale). (B”) En (gray-scale) was weakly induced in pka clones from discs expressing Ci-P(1-3)A. Ci-155 levels were (A”, F”, H”) increased relative to neighboring anterior territory for Ci-WT but were (C”–E”, G”, I”–K”) either unchanged or slightly reduced, presumably from full proteolysis, for processing-resistant Ci variants. Scale bars are 40 μm. (L) Average intensity of ptc-lacZ in pka clones (red) or neighboring anterior territory (pink), and in cos2 clones (dark blue) or neighboring anterior territory (light blue), as a fraction of AP border levels. Mean and SEM shown. Significant differences between ptc-lacZ values in pka or cos2 mutant clones and neighboring anterior pka/+ or cos2/+ cells for a given genotype, calculated by paired t-tests, are indicated for p<0.001 (*) and p<0.05 (#).

Cos2 reduces Ci-155 activity by binding to the CORD region.

(A–J) Wing discs from animals with one copy of the designated ci transgenes and alleles (together with ci94) with clones (GFP, green, arrows) that lack (A–F) pka activity or (G–J) cos2 activity (arrowheads indicate AP border), showing (A’–J’) ptc-lacZ (red) and (A”–F”) Ci-155 (gray-scale). (A”–F”) Ci-155 levels were increased relative to neighboring anterior territory for all Ci proteins, but the increase was relatively small for (B”) Ci-ΔCORD, suggesting that processing outside the clones may be inefficient. By contrast, a large change was observed for Ci-ΔCDNΔCORD, suggesting very efficient processing. Scale bars are 40 μm. (K) Average intensity of ptc-lacZ in pka clones (red) and in cos2 clones (blue), as a fraction of AP border levels. Mean and SEM shown. Significant differences between values for a given genotype compared to those for crCi-WT, calculated by paired t-tests, are indicated for p<0.001 (*) and p<0.05 (#). Additionally, ptc-lacZ was significantly increased for gCi-ΔCORD versus gCi-WT in pka mutant clones (p<0.0001).

Figure 7 with 1 supplement
Loss of both Cos2 inhibition and processing combine to activate Ci-155.

(A–J) Wing discs from animals with one copy of the designated ci alleles (together with ci94), have (A–D) no ectopic anterior ptc-lacZ (red) unless (E) both the CORD domain is removed and processing blocked (by the S849A alteration). (A’–E’) Ci-155 (gray-scale) in the same wing discs. (F–J) Anterior En (green) induction, revealed by marking the AP compartment boundary (yellow lines) with the posterior extent of ptc-lacZ (red), was reduced for Ci variants (G, J) lacking the CORD domain, (J) especially together with the S849A alteration. (K) Wing disc with anterior clones (GFP, green, yellow arrows) that have lost a second chromosome gCi transgene, leaving one copy of crCi-S849AΔCORD as the only source of Ci, showing (K’) ptc-lacZ induction in the clones (arrows) to levels similar to the AP border (arrowheads); (K”) Ci-155 (gray-scale) is uniformly high because of blocked processing. Scale bars are (A–J) 40 μm. See also Figure 7—figure supplement 1.

Figure 7—figure supplement 1
Reduced AP border activity of Ci lacking the CORD domain.

(A–H) Wing discs expressing one copy of crCi-WT or crCi-ΔCORD as the only source of Ci, showing (A, C, E, F) Ci-155 (gray-scale), (B, D, G, H, A’–H’) ptc-lacZ (red) or (B, D, G, H, B”, D”, G”, H”) En (green) and AP compartment boundary (yellow line). Reduced En induction by Ci-ΔCORD was (B, D) not restored to normal by loss of Su(fu). (E–H) Loss of Fu kinase activity in fumH63 wing discs produced (E, F) similarly strong, broad Ci-155 at the AP border and (E’, F’) drastic reduction of ptc-lacZ for Ci-WT and Ci-ΔCORD, while (G, H) additional loss of Su(fu) restored ptc-lacZ without anterior En in both cases. (I–L) induction of ptc-lacZ (red) in smo mutant clones (green, arrows) expressing GAP-Fu was similar for Ci-WT and Ci-ΔCORD encoded by either (I, J) gCi transgenes or (K, L) crCi alleles and was much lower than at the AP border (arrowheads) in all cases, as presented graphically in (N). (M, M’) Much higher ptc-lacZ (red), matching AP border levels (arrowheads) were observed in GAP-Fu clones (green, arrows) expressing processing-resistant Ci-S849AΔCORD, accompanied by (M”) significant Ci-155 (gray-scale) proteolysis. (N) Average intensity of ptc-lacZ in clones as a fraction of AP border levels. Significant differences between values for a given genotype compared to those for crCi-WT, calculated by paired t-tests, are indicated for p<0.001 (*). There was no significant difference (p<0.05) for gCi-ΔCORD versus gCi-WT. Scale bars are (A, C, E, F) 100 μm and (D, G, H, I–M) 40 μm.

Summary of graded processing, activation, and proteolysis of Ci-155 and underlying mechanisms at the AP border.

At the AP border, Hh (emanating from posterior, mustard yellow, territory) inhibits Ci-155 processing stimulated by Cos2, PKA, CK1, and GSK3, and activates Fu protein kinase activity to activate Ci-155, overcoming inhibition by Su(fu) and other factors. Hh also promotes a reduction of Ci-155 levels, most likely by promoting full Ci-155 proteolysis through induction of Rdx/Hib or reducing Su(fu) association. Here we used processing-resistant Ci variants to show that PKA and Cos2 (through binding the CORD domain on Ci-155) limit Ci-155 activity in anterior cells (left) and (right) to deduce the spatial profiles of Hh-stimulated Ci-155 reduction (‘proteolysis’, upward arrows) and inhibition of Ci-155 processing (downward arrows, blue triangle) at the AP border that underlie steady-state Ci-155 levels (brown). Graded target gene (En, Ptc, Dpp) activation is normally elicited by a combination of activated Ci-155 and Ci-75 repressor but was still observed when there was no regulation of Ci-155 processing, indicating that Ci-155 activation must be graded. Although graded Hh signaling was observed when Ci-155 processing is not regulated, Ci-75 repressor must be present in anterior cells to prevent ectopic dpp expression and inhibition of processing was shown to be important for activated Fu to induce high levels of ptc expression. Thus, Hh normally elicits graded inhibition of Ci-155 processing and graded activation of full-length Ci-155 but the activation gradient can suffice provided there is some repressor in anterior cells and Ci-155 is substantially spared from processing at the AP border.

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Gene (Drosophila melanogaster)CiFlybase ID:
FBgn0004859
CG2125
Gene (Drosophila melanogaster)Cos2Flybase ID:
FBgn0000352
CG1708
Gene (Drosophila melanogaster)PKAFlybase ID:
FBgn0000273
CG4379
Gene (Drosophila melanogaster)FusedFlybase ID:
FBgn0001079
CG6551
Gene (Drosophila melanogaster)Suppressor of FusedFlybase ID:
FBgn0005355
CG6054
Genetic reagent (Drosophila melanogaster)hs-flpPMID:7867064FBti0002738hsp70-driven Flp recombinase on X
Genetic reagent (Drosophila melanogaster)ci94PMID:7705626
PMID:10102270
FBal00454435 kb deletion removing promoter and first exon
Genetic reagent (Drosophila melanogaster)ciCePMID:10102270ciCe2 FBal00016578 bp deletion that is expected to result in a truncation of the protein at amino acid residue 975
Genetic reagent (Drosophila melanogaster)Dp[y+]PMID:10102270Dp(1;4)1021[y+] svspa-pol FBab0003151
Genetic reagent (Drosophila melanogaster)Su(fu)LPPMID:1468628FBal0016296Amorphic 1.5 kb deletion extending into neighboring kar gene
Genetic reagent (Drosophila melanogaster)pka-C1H2PMID:8391504FBal0033960G203D alteration to key kinase domain residue
Genetic reagent (Drosophila melanogaster)smo2PMID:15592457FBal0015765Behaves as a null
Genetic reagent (Drosophila melanogaster)FRT 42D P[Smo+]PMID:10102270P[Smo+, hsp70-GFP] FBtp0012072Fully rescues loss of smo function
Genetic reagent (Drosophila melanogaster)FRT 42D cos22PMID:11090136FBal0001772To generate loss-of-function cos2 clones
Genetic reagent (Drosophila melanogaster)fumH63PMID:8846897FBal0120493G203D loss of kinase activity
Genetic reagent (Drosophila melanogaster)tub-GAL80 FRT 40ABDSC BL-5192For MARCM clones on 2L
Genetic reagent (Drosophila melanogaster)FRT 42D P[Ci+] tub-GAL80PMID:10102270P[Ci+] 16 kb segment rescues ci null in stock for 2R MARCM clones
Genetic reagent (Drosophila melanogaster)C765 > Gal4Flybase ID:
FBti0002765
Spatially uniform wing disc GAL4 driver
Genetic reagent (Drosophila melanogaster)UAS-GAP-FuPMID:17658259FBal0284373Fu coding sequence with Myristoylation sequence from hGAP43 at N-terminus and CFP at C-terminus
Genetic reagent (Drosophila melanogaster)ptc-lacZPMID:8898207P[ptcA-lacZ] FBal004786410.8 kb ptc promoter driving lacZ
AntibodyAnti-Ci-155 (rat monoclonal)DSHBAB_2109711(1:3)
AntibodyAnti-beta-galactosidase (rabbit polyclonal)MP BiomedicalsAB_2334934(1:10,000)
AntibodyAnti-Engrailed (mouse monoclonal)DSHBAB_528224(1:5)
AntibodyAlexaFluor
488, 546, 594, 647
Thermofisher ScientificAnti-rabbit,
Anti-mouse
Anti-Rat
(1:1000)
Recombinant DNA reagentpCFD4Mann LabAddgene: 83954Gibson cloning of gRNA
Recombinant DNA reagentBluescript genomic Cubitus interruptusBasler Lab
Recombinant DNA reagentatt-Pacman Expression VectorDGRC
Chemical compound, drugNormal Goat SerumJackson Immunoresearch laboratoriesRRID:AB_2336990
Chemical compound, drugAqua PolymountPolySciencesCN: 18606–20
Commercial assay or kitGibson AssemblyNew England BiolabsCN: E5510S
Commercial assay or kitPfuUltraII Fusion HS DNA polymeraseAgilent TechnologiesCN: 600670
Commercial assay or kitZero Blunt Topo cloning vectorInvitrogenCN: K270020
Strain, strain background (Escherichia coli)Transformax EPI 300 Electrocompetent E. coliEpicentre
Now lucigen
CN: EC300110Electro-competent cells
Strain, strain background (Escherichia coli)One Shot TOP10 Chemically Competent E. coliThermofisher ScientificCN: C4040-10Chemically Competent cells
Software, algorithmImage JNIH Bethesda Maryland
Software, algorithmA Plasmid Editor (APE)

Data availability

All data reported in this study are included in the manuscript and supporting files.

Additional files

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)