Dally is not essential for Dpp spreading or internalization but for Dpp stability by antagonizing Tkv-mediated Dpp internalization
- Growth & Development, Biozentrum, Spitalstrasse, University of Basel, Switzerland
- International Max Planck Research School for Immunobiology, Epigenetics, and Metabolism, Germany
- Institute for Biology I, Faculty of Biology, University of Freiburg, Germany
- CIBSS – Centre for Integrative Biological Signalling Studies, University of Freiburg, Germany
- BIOSS – Centre for Biological Signalling Studies, University of Freiburg, Germany
- Hilde Mangold Haus, University of Freiburg, Germany
Figures
Figure 1
Distinct models on the roles of HSPGs on Dpp/BMP gradient formation.
(A) Schematic view of the wing pouch (future wing tissue) and adult wing tissue. Dpp spreads from the anterior stripe of cells along the A-P compartment boundary to generate pMad gradient and an inverse Brk gradient to specify adult wing veins such as L2 and L5. (B) HSPGs are proposed to transport Dpp via repetitive interaction of HS chains with Dpp (facilitated diffusion model). (C) HSPGs are proposed to hinder Dpp spreading via reversible interaction of HS chains with Dpp (Hindered diffusion model). (D) HSPGs are proposed to stabilize Dpp on the cell surface via reversible interaction of HS chains with Dpp that antagonizes Tkv-mediated Dpp internalization. (E) HSPGs are proposed to internalize and recycle Dpp.
Figure 2 with 2 supplements
Dally, but not Dlp, interacts with Dpp.
(A–D) α-HS (3G10) (A, C), extracellular α-Ollas (B, D), and α-pMad (B’, D’) staining of Ollas-dpp/+, ap >dally disc (A–B) and Ollas-dpp/+, ap >dlp disc (C–D). (E–H) Average fluorescence intensity profile of (B, B’, D, D’) respectively. Data are presented as mean +/- SD. Scale bar: 50 μm.
Figure 2—figure supplement 1
Comparison of endogenous Dpp gradient and signaling between dorsal and ventral compartment.
(A) Extracellular α-Ollas staining of Ollas-dpp/Ollas-dpp wing disc. (B) Average fluorescence intensity profile of (A). (C) α-pMad staining of Ollas-dpp/Ollas-dpp wing disc. (D) Average fluorescence intensity profile of (C).
Figure 2—figure supplement 2
Preferential interaction of Dlp with Wg.
(A) Extracellular α-Wg staining of Ollas-dpp/+ (A), Ollas-dpp/+, ap >dally (B), and Ollas-dpp/+, ap >dlp (C). (D) Average fluorescence intensity profile of (A–C).
Figure 3 with 2 supplements
Dally, but not Dlp, is required for Dpp signaling gradient.
(A–D) α-pMad staining of yw (A), dallyMH32 (B), dallyKO (C), and dlpKO (D) wing disc. (E–H) Adult wings of yw (E), dallyMH32 (F), dallyKO (G), and dlpKO (H). (I) Average fluorescence intensity profile of (A–D). Data are presented as mean +/- SD. (J) Comparison of adult wing size of yw (n=15), dallyMH32 (n=20), dallyKO (n=13), and dlpKO (n=13). Data are presented as mean +/- SD. Two-sided unpaired Student’s t test with unequal variance was used for all the comparison. ****p < 0.0001. n.s; not significant. (K) Comparison of normalized A-P length against D-V length of adult wings of yw (n=15), dallyMH32 (n=20), dallyKO (n=13), and dlpKO (n=13). Data are presented as mean +/- SD. Two-sided unpaired Student’s t test with unequal variance was used for comparison under black lines. Two-sided Mann−Whitney test was used for comparison under red lines. ****p < 0.0001, **p < 0.01. (L–O) α-pMad staining of yw (L), dallyKO (M), YFP-dally (N), and 3xHA-dlp in dallyKO (O) wing disc. (P–U) Adult wings of yw (P), dallyKO (Q), YFP-dally (R), 3xHA-dlp in dallyKO (S), dlpKO (T), and 3xHA-dlp (U). (V) Average fluorescence intensity profile of (L–O). Data are presented as mean +/- SD. (W) Comparison of adult wing size of yw (n=10), dallyKO (n=8), YFP-dally (n=13), 3xHA-dlp in dallyKO (n=13), dlpKO (n=13), and 3xHA-dlp (n=13). Data are presented as mean +/- SD. Two-sided unpaired Student’s t test with unequal variance was used for all the comparison. ****p < 0.0001. Scale bar: 50 μm.
Figure 3—figure supplement 1
Generation of dallyKO and dlpKO (A) Schematic view of manipulation of the glypican genomic loci exemplified with dlp.
(B) The exact sequences at the lesion of dallyKO and dlpKO: color code as in (A) blue: genomic sequences at the inner boundaries of homology arms for HDR, yellow: attP, green: loxP, black: ‘scars’ (remnants of vector sequences including cloning sites).
Figure 3—figure supplement 2
Validation of dlpKO allele.
(A, B) α-Dlp and DAPI staining of dlpKO/+ (A) and dlpKO/ dlpKO (B).
Figure 4
Interaction of core protein of Dally with Dpp.
(A–F) α-HS (3G10) (A, D), extracellular α-Ollas (B, E), GFP (B’, E’), α-pMad (C, F) staining of Ollas-dpp/+, ap >dally disc (A–C) and Ollas-dpp/+, ap >dallyΔHS disc (D–F). (G) Average fluorescence intensity profile of (B). (H) Average fluorescence intensity profile of (E). (I) Relative Dpp accumulation (α-Ollas signal) (B, E) normalized against expression level of Dally or DallyΔHS (GFP fluorescent signal) around the Dpp producing cells (B’, E’). Two-sided unpaired Student’s t test with unequal variance was used for the comparison. ***p< 0.001. (J) Average fluorescence intensity profile of (C). (K) Average fluorescence intensity profile of (F). (G–K) Data are presented as mean +/- SD. (L–N) α-pMad staining of YFP-dally (L), YFP-dallyΔHS (M), and dallyKO (N) wing disc. YFP signal of YFP-dally (L, inset) and YFP-dallyΔHS (M, inset) wing disc. We note that, similar to the overexpression results (B’ and E’), YFP-dallyΔHS wing discs showed higher expression levels than YFP-dallyΔHS (L and M inset). (O) Average fluorescence intensity profile of (L–N). Data are presented as mean +/- SD. (P–R) Extracellular α-HA staining of HA-dpp (P), HA-dpp; YFP-dallyΔHS (Q), and HA-dpp; dallyKO (R) wing disc. (S) Average fluorescence intensity profile of (P–R). Data are presented as mean +/- SD. (T–U) Adult wings of YFP-dally (T) and YFP-dallyΔHS (U). (V) Comparison of adult wing size of YFP-dally (n=12) and YFP-dallyΔHS (n=20). Data are presented as mean +/- SD. Two-sided unpaired Student’s t test with unequal variance was used for the comparison. ****p < 0.0001. Scale bar: 50 μm.
Figure 5 with 1 supplement
HS chains of Dally act largely independent of interaction with Dpp.
(A–B) extracellular α-Ollas (A, B), GFP (A’, B’), and α-pMad (A’’, B’’) staining of JAX;HA-dppΔN, ap >dally disc (A-A’’) and JAX;HA-dppΔN, ap >dallyΔHS disc (B-B’’). (C) Relative Dpp accumulation (α-Ollas signal) (A, B) normalized against expression level of Dally or DallyΔHS (GFP fluorescent signal) around the Dpp producing cells (A’, B’). Two-sided Mann−Whitney test was used for comparison. n.s; not significant. (D) Average fluorescence intensity profile of (A’’). Data are presented as mean +/- SD. (E) Average fluorescence intensity profile of (B’’). Data are presented as mean +/- SD. (F–G) α-pMad (F, G) and extracellular α-HA (F’, G’) staining of JAX;HA-dpp disc (F, F’) and JAX;HA-dppΔN disc (G, G’). (H) Average fluorescence intensity profile of (F, G). Data are presented as mean +/- SD. (I) Average fluorescence intensity profile of (F’, G’). Data are presented as mean +/- SD. (J–K) Adult wings of JAX;HA-dpp (J) and JAX;HA-dppΔN (K). (L) Comparison of adult wing size of JAX;HA-dpp (n=19) and JAX;HA-dppΔN (n=18). Data are presented as mean +/- SD. Two-sided unpaired Student’s t test with unequal variance was used for the comparison. ****p < 0.0001.
Figure 5—figure supplement 1
JAX does not rescue dpp disc allele.
(A–D) α-pMad staining (A, C) and α-Brk staining (B, D) of dppd8/dppd12 (A, B) and JAX;dppd8/dppd12 (C, D). (E, F) Adult wings of dppd8/dppd12 (E) and JAX;dppd8/dppd12 (F).
Figure 6
HS chains of Dally stabilize Dpp by antagonizing Tkv-mediated Dpp internalization.
(A, B) Extracellular α-Ollas staining of Ollas-dpp/+, ap >tkv RNAi wing disc (A) and dallyKO, Ollas-dpp/+, ap >tkv RNAi wing disc (B). (C, D) Average fluorescence intensity profile of (A, B) respectively. Data are presented as mean +/- SD. (E) Average fluorescence intensity profile of the dorsal compartment of (A, B). Data are presented as mean +/- SD. (F, G) α-pMad staining of Ollas-dpp/+, ap >tkv RNAi wing disc (F), and Ollas-dpp/+, ap >dally RNAi wing disc (G). (H) Extracellular α-Ollas staining of dallyΔHS, Ollas-dpp/+, ap >tkv RNAi wing disc. (I) Average fluorescence intensity profile of (H). Data are presented as mean +/- SD. Scale bar: 50 μm.
Figure 7
A revised model on the roles of Dally on Dpp/BMP gradient formation and signaling.
(A) A previous model. Dally and Dlp competes with Tkv for Dpp binding to antagonize Tkv-mediated Dpp internalization. (B) A revised model. Dally, but not Dlp, reversibly interacts with Dpp partially through its HS chains and mainly through its core protein. The former interaction is not essential and the latter interaction is not sufficient to antagonize Tkv-mediated Dpp internalization under physiological conditions. Upon interaction of Dpp with the core protein of Dally, HS chains of Dally antagonize Tkv-mediated Dpp internalization through Tkv to stabilize Dpp on the cell surface. HS chains of Dally control Dpp signaling also independent of interaction with Dpp likely through Tkv. It remains unknown whether and how Dpp signal activation by core protein bound Dpp and stability by HS chains are coordinated. (C) In dallyKO mutants, Tkv-mediated Dpp internalization by Tkv is not antagonized. Dpp is irreversibly bound to Tkv and then removed by Tkv from the extracellular space and degraded. (D) In dallyΔHS mutants, the core protein of Dally can interact with Dpp but this interaction is not sufficient to antagonize Tkv-mediated Dpp internalization. Without HS chains, Dpp is irreversibly bound to Tkv and then removed by Tkv from the extracellular space and degraded. (E) In dppΔN mutants, Dpp can interact with core protein but not with HS chains of Dally. Without interaction of HS chains with Dpp, HS chains can antagonize Tkv-mediated Dpp internalization through Tkv to stabilize Dpp on the cell surface.
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/86663/elife-86663-mdarchecklist1-v1.docx
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Supplementary file 1
Raw data and statistics to produce all the graphs in the study.
- https://cdn.elifesciences.org/articles/86663/elife-86663-supp1-v1.xlsx
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Dally is not essential for Dpp spreading or internalization but for Dpp stability by antagonizing Tkv-mediated Dpp internalization
eLife 12:RP86663.
https://doi.org/10.7554/eLife.86663.3
