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Decoding temporal interpretation of the morphogen Bicoid in the early Drosophila embryo

  1. Anqi Huang
  2. Christopher Amourda
  3. Shaobo Zhang
  4. Nicholas S Tolwinski
  5. Timothy E Saunders  Is a corresponding author
  1. National University of Singapore, Singapore
  2. Yale-NUS College, Singapore
  3. Agency for Science Technology and Research, Singapore
Research Article
Cite this article as: eLife 2017;6:e26258 doi: 10.7554/eLife.26258
9 figures and 2 videos

Figures

Figure 1 with 1 supplement
Optogenetic tool to manipulate Bcd-dependent transcription activity.

(A) Schematic illustration of CRY2::mCh::Bcd construct; CRY2 optogenetic cassette tagged with mCherry fluorescent protein is fused to the N-terminal of Bcd coding sequence; expression of the construct is under the regulation of the endogenous bcd regulatory sequence. (B–E) Embryos maternally expressing cry2::mch::bcd having developed in the dark (B–E) or light (B’–E’) as compared to bcd-/- embryos (B’’–E’’) fixed at the end of the blastoderm stage and stained for Hb (B), Gt (C), Kni (D) and Kr (E). Yellow and green arrowheads indicate, respectively, the anterior and posterior Kr boundaries in the dark. White arrowheads point to ectopic posterior Hb and Gt expression in the anterior. (F and G) Embryos in the dark (F and G) or light (F’ and G’) compared to bcd-/- (G’’) stained for mCh (F) and Cad (G) at early n.c. 14. (H) Cuticle patterns of embryos with maternally loaded cry2::mch::bcd having developed in the dark (H) or light (H') in the first 2.5 hr AEL as compared to bcd-/- embryos (H’’). White arrowhead indicates duplicated telson. (J and K) Average nuclear intensity of CRY2::mCh::Bcd (J) or Cad (K) normalized to peak value is plotted vs. AP position (% EL) in cry2::mch::bcd, bcd-/- embryos having developed in dark (blue curve) and light (red curve). (J) Data were fitted to an exponential curve shown by smooth lines with length scales around 80 µm. Shaded error bars are across all nuclei of all embryos at a given position. (B–K) n = 5–7 embryos per condition. Scale bar, 50 µm.

https://doi.org/10.7554/eLife.26258.002
Figure 1—figure supplement 1
Caudal expression in embryos of different bcd dosage.

(A) Average nuclear Caudal intensity normalized to the peak value is plotted vs. AP position (% EL) in embryos deposited by female flies carrying 1, 2 and 4 copies of bcd gene (shown in purple, green and orange, respectively). Shaded error bars are across all nuclei of all embryos at a given position. (B) The corresponding boundary position of Caudal distribution at which the expression profile crosses the half maximum posterior expression value (51.32 ± 4.95 %EL, 45.55 ± 5.05%EL and 38.91 ± 3.66%EL for 1x, 2x and 4xbcd, respectively). Data are presented as Mean ± SD, *p<0.05 and ***p<0.001. n = 9, 13 and 25 embryos for 1x, 2x and 4x bcd, respectively.

https://doi.org/10.7554/eLife.26258.003
Figure 2 with 4 supplements
Inhibitory effect on Bcd-dependent transcription is gene-dosage and light-power dependent.

(A and E) Proportion of the nuclei positive for hb transcription plotted vs. the persistence of transcription activity during n.c. 13 in control (blue), CRY2low (orange) and CRY2high (red) embryos of hb anterior (A) and posterior (E) domain. n = 3 embryos per genotype. (B–D) Snapshots of embryos expressing hb>MS2, MCP::GFP in n.c.13. Embryos are maternally loaded with only endogenous bcd (B), or together with cry2::mch::bcd at low(C) or high (D) level. MCP::GFP signals are tracked and marked with green dots. (F and G) Chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) characterization of CRY2::mCh::Bcd binding to Hb (P2 enhancer), Gt, Otd and Kr in the light for 45 mins (4 mW) as compared to embryos aged in the dark (F) and to 1 kb downstream of the Hb P2 enhancer in the dark and light conditions (G). Data are presented as Mean ± SD, *p<0.001. (H–O) Eve staining in embryos maternally loaded with cry2::mch::bcd in bcdWT (H–K) or bcd null (L–O) background; embryos have developed in dark (H and L) or illuminated with blue light at 0.04 mW (I and M), 0.4 mW (J and N) or 4 mW (K and O). n = 5–7 embryos per condition. Scale bar, 50 µm.

https://doi.org/10.7554/eLife.26258.004
Figure 2—figure supplement 1
Expression profiles in CRY2low and CRY2high embryos.

(A) Snapshots of embryos expressing CRY2::mCh::Bcd at comparatively low (top panel) and high (bottom panel) level in a bcd wild-type background at late n.c. 14. Scale bar, 50 µm. (B) Average nuclear mCherry intensity in CRY2low (orange curve) and CRY2high (red curve) embryos at late n.c. 14 is plotted vs AP position (% EL). Shaded error bars are across all nuclei of all embryos at a given position. n = 8 embryos for CRY2low and n = 5 embryos for CRY2high.

https://doi.org/10.7554/eLife.26258.005
Figure 2—figure supplement 2
Effect of illumination on hb mRNA production along the AP axis.

(A and B) Box plot showing the persistence of hb>MS2 puncta in control (blue), CRY2low (orange) and CRY2high (red) embryos. Anterior hb domain is subdivided into five distinct regions along AP axis, i.e., 100–75, 75–70, 70–65, 65–60 and 60–40% EL. Data is quantified in these regions as well as hb posterior domain, respectively. The cut-off value for statistical analysis is 160 s as shown by the greyed regions. Each box shows the counts for a single embryo.

https://doi.org/10.7554/eLife.26258.006
Figure 2—figure supplement 3
MCP::mCh shows the same reporter kinetics as MCP::GFP.

(A) Schematic demonstration of imaging and illumination configuration. Rectangular image represents a typical field of view of hb transcription along mid 50% EL of an embryo, anterior to the left. (B and C) Segmented fluorescent spots of transcription along the AP axis in the field of view in n.c.12 to 14 shown by MCP::GFP (B) and MCP::mCh(C). Heatmap color represents relative intensity of each spot. (D) Average density of fluorescent spots in the anterior hb domain in n.c.12–14. Shaded error bars are across all embryos at a given time point. (E) Proportion of the nuclei positive for hb transcription plotted vs. the persistence of transcription activity during n.c. 13 in MCP::GFP (green) and MCP::mCh (red) embryos. n = 6 embryos for each line.

https://doi.org/10.7554/eLife.26258.007
Figure 2—figure supplement 4
Rate of light-induced loss and recovery of Bcd-dependent transcription.

(A–C) Segmented fluorescent spots of hb transcription along the AP axis in the field of view in n.c.12 to 14 in embryos from females of nos>MCP::mCh; CRY2::mCh::Bcd crossed to males of hb>MS2-yellow in different illumination conditions. (A) dark control; (B) 1 min illumination after the onset of n.c.13; (C) 5 min illumination during the 12th division. (A’ - C’) Average density of fluorescent spots in the anterior hb domain in n.c.12–14 corresponding to the illumination conditions in the left panels. Shaded error bars are across all embryos at a given time point. Data from dark control (red) is duplicated in all conditions for comparison. Blue shadowed area indicates illumination time window. n = 10, 4, 4 embryos for condition A – C, respectively.

https://doi.org/10.7554/eLife.26258.008
Illumination across different time windows causes embryonic lethality with varied severity.

(A) Schematic demonstration of illumination time windows. Blue bars indicate illumination while the absence of blue bars indicates dark condition. The onset of gastrulation is defined as time 0. Negative values refer to specific time before gastrulation. The start and the end of illumination is indicated by the number on the left and right side of the blue bars, respectively. (B–K) Cuticle preparation of OreR (B), bcd-/- (H) and embryos illuminated in different time windows (C–G and I–M). The illumination time is indicated on the left of each image. (C) Arrow, pharynx wall; (D) Arrow, absence of pharynx wall. Arrowhead, missing structures lying between mouth hooks and cephalo-pharyngeal plates. (J) Arrowhead, denticle belt of thorax segment. n = 10 embryos per condition. Scale bar, 50 µm.

https://doi.org/10.7554/eLife.26258.010
Figure 4 with 1 supplement
Illumination at the end of blastoderm stage causes wrong cell fate determination in anterior embryonic segments.

(A) Schematic demonstration of illumination time windows at the end of blastoderm stage. Illumination starts at certain time before gastrulation and ends at the onset of gastrulation as indicated by blue bars. (B–F, B’–F’ and B’’–F’’) Embryos illuminated in different time windows as indicated at the left side of each panel are fixed by the end of GBE and stained for En (B–F), Deformed (Dfd, white) and Scr (magenta) (B’–F’), Scr (magenta) and Abd-B (cyan)(B’’–F’’); (B–F) Colored dots represent embryonic segments. Red, clypeolabrum; orange, stomodeum; yellow, mandibular lobe; light green, maxillary lobe; dark green, labial lobe; light blue, prothorax; dark blue, mesothorax; dark purple, metathorax; and light purple, abdominal segments. (E’’ and F’’) Arrows, ectopic Abd-B expression. n = 5–10 embryos per condition. Scale bar, 50 µm. (B’’’–F’’’) Schematic representation of hox gene expression maps.

https://doi.org/10.7554/eLife.26258.011
Figure 4—figure supplement 1
Hox genes expression pattern in increasing illumination time window.

(A) Schematic demonstration of illumination time windows at the end of blastoderm stage. Illumination starts at certain time before gastrulation and ends at the onset of gastrulation as indicated by blue bars. (B–F, B’–F’ and B’’–F’’) Embryos illuminated in different time windows as indicated on the left side of each panel are fixed by the end of GBE and stained for Dfd (white) and Scr (magenta) (B–F), Scr (magenta) and Abd-B (B’–F’) and Antp (yellow) and Ubx (green)(B’’–F’’). Panels (B–F) and (B’–F’) show the unprocessed images previously shown in Figure 4. (E’ and F’) Arrows, ectopic Abd-B expression. (F’’) Arrow, ectopic Ubx expression. n = 5–10 embryos per condition. Scale bar, 50 µm.

https://doi.org/10.7554/eLife.26258.012
Figure 5 with 3 supplements
Impact of temporally patterned illumination on downstream gene expression.

(A) Schematic demonstration of eight different illumination time windows. (B–D) Average Hb intensity normalized to posterior peak values plotted vs. AP position (% EL) in embryos having developed in different temporally patterned illumination. Sample numbers correspond to time windows shown in (A). (B–C) Double-headed arrows point out the changed expression level of Hb. (E and F) Embryos having developed in dark (E) or illuminated for 30 min before n.c.11 (F) are stained for Hb. Red dashed line indicates the position of posterior border of anterior Hb domain of embryo in (E). Scale bar, 50 µm. (G and H) Position of Kr anterior border (G, blue dots), Kr posterior border (G, red dots), Kni anterior border (H, yellow dots) and Kni posterior border (H, green dots) under different temporal illumination as indicated by sample numbers. Error bars indicate s.d.; t-test was used for the statistical evaluation with *p<0.05. A total number of 70, 53 and 59 embryos were analyzed for expression of Hb, Kr and Kni, respectively.

https://doi.org/10.7554/eLife.26258.013
Figure 5—figure supplement 1
Impact of temporally patterned illumination on downstream gene expression.

(A) Schematic demonstration of eight different illumination time windows. (B–G) Embryos illuminated in different time windows are fixed at the end of blastoderm stage and stained for Hb (B1–B8), Otd (C1–C8), Gt (D1–D8), Kr (E1–E8), Kni (F1–F8), and Eve (G1–G8); row 1–8 corresponds to 8 temporal illumination conditions shown in (A). n = 5–10 embryos per condition. Scale bar, 50 µm.

https://doi.org/10.7554/eLife.26258.014
Figure 5—figure supplement 2
Cephalic furrow position shifts in different illumination conditions.

(A–D) Snapshots from time-lapse movies of embryos with maternally expressed CRY2::mCh::Bcd and Gap43::mCh in dark condition (A), illuminated during n.c.12–14 (B), illuminated during n.c.10–11 (C) and illuminated before n.c.10 (D). Left panels: onset of cephalic furrow (CF) invagination. Mid panels: 3 min after invagination initiates. Right panels: 6 min after invagination initiates. White arrowheads indicate CF positions in the dark condition. Transparent arrowhead indicate abolished CF formation. Yellow arrowhead indicates anteriorly shifted CF. (E) Average CF position in condition A, C and D. Data are presented as Mean ± SD, ***p<0.001. n = 4–9 embryos for each condition. Scale bar, 50 µm.

https://doi.org/10.7554/eLife.26258.015
Figure 5—figure supplement 3
Using optogenetic perturbations to test gap gene models.

(A) Interaction network between gap genes (Hb, Gt, Kr, Kni) and regulatory inputs (Bcd, Cad, Tailless and Huckebein). Green and red arrows correspond to positive and repressive interactions respectively, with color intensity qualitatively representing interaction strength. (B) Hb, Kr, Gt and Kni expression profiles from the optimal parameter set (red), see Materials and methods. Blue curves are resulting profiles if Bcd activity is restricted from the beginning of cycle 12. (C) Top: shift in Kr boundaries at the end of cycle 14 in the simulations under different perturbations (as in Figure 5A) of Bcd activity. Bottom: corresponding shifts in Kni boundaries. (D) Hb, Kr, Gt and Kni expression profiles from the parameter set (#19) that qualitatively agrees best with our optogenetic perturbations. Blue curves are resulting profiles if Bcd activity is restricted from the beginning of cycle 12. Black arrows represent shifts observed experimentally; red arrows highlight differences between simulation prediction and experimental observation. (E) Parameter variation (see Materials and methods) between the nine parameter sets satisfying at least three of the observations from our optogenetic perturbations and all 21 parameter sets identified in Bieler et al.. Each column represents the target gene (kni, gt, kr, hb) and each row is the interacting element (including gap genes and maternal inputs). Hb m and Hb d represent interactions with the monomeric and dimeric forms of Hb. Red (yellow) shading denotes a negative (positive) shift in the interaction between the interacting element and the target gene in the parameter subset compared with the mean of all 21 parameter sets.

https://doi.org/10.7554/eLife.26258.016
Figure 6 with 1 supplement
Temporal requirement of Bcd-dependent transcription for proper cell fate determination.

(A) Schematic representation of required time windows of Bcd-dependent transcription for downstream gene expression; color bars indicate the time windows required for Bcd-dependent transcription to be on for correct expression of Kni1 (red), Gt1 (orange), Otd (yellow) and Gt2 (green); (B–D) embryos exhibiting the correct gene expression when Bcd-dependent transcription is active in required time window; colored boxes point out the corresponding expression domain; (B’–D’ and C’’) embryos showing defects in gene expression when Bcd-dependent transcription is interrupted in required time window; dashed boxes indicate failed expression. Scale bar 50 µm. (E) Schematic diagram of temporal interpretation of Bcd morphogen. Colored bars indicate the time windows required for Bcd-dependent transcription for correct cell fate determination in different embryonic segments.

https://doi.org/10.7554/eLife.26258.017
Figure 6—figure supplement 1
Impeding Bcd-dependent transcription during cycle 14 delays cephalic furrow invagination.

Embryos expressing CRY2::mCh::Bcd and Gap43::mCh are imaged on a custom built light-sheet microscope. The embryos are illuminated with a 488 nm laser for 20 min starting 5 min after the establishment of the cellularization furrow. Panels (A-D) and (A’-D’) show, respectively, the lateral and ventral views for dark control embryos while (E-I) and (E’-I’) show the lateral and ventral views for illuminated embryos. The red and blue rectangles indicate the time point marking the onset of cephalic furrow (white arrows) and ventral furrow (yellow arrows) invagination, respectively. n = 7 embryos per condition. Scale bar, 50 µm.

https://doi.org/10.7554/eLife.26258.018
Author response image 1
Exploring the mechanism underlying optogenetic manipulation of Bcd-dependent transcription.

(A) Live imaging of cry2::mch::bcd, bcd-/- embryo in dark (left panels) and illuminated (right panels) conditions. The top panels show the zoom-in of most anterior embryo regions. (B – D) Cross-section of late cycle 14 embryos stained for Phalloidin. Cry2::mch::bcd, bcd-/- embryo in dark (B) and illuminated (C) conditions are compared to bcd null embryo (D). Red bars indicate the depth of membrane ingression in the anterior end of the embryos (shallow cellularization front in dark embryos versus excessive ingression in illuminated and bcd-/- embryos).

https://doi.org/10.7554/eLife.26258.020
Author response image 2
Precise measurements on four gap genes.

(A – D) Max projection images of Drosophila embryos stained for Hb (A), Kr (B), Kni (C) and Gt (D) fixed at around 40 min into cycle 14 (top panels). Average intensity normalized to peak value is plotted vs. AP position (% EL) for each gap gene (bottom panels). Shaded error bars are across all nuclei of all embryos at a given position. n = 7-9 embryos for each gap gene quantification.

https://doi.org/10.7554/eLife.26258.021
Author response image 3
Embryonic regions distinguished by different sensitivity of Bcd activity and duration.

Red, orange and blue segments represent distinct embryonic regions requiring Bcd activation potency at WT, intermediate hypomorphic and weak hypomorphic level for proper differentiation, respectively. Concomitantly, they require Bcd for long, intermediate and short duration in time.

https://doi.org/10.7554/eLife.26258.022

Videos

Video 1
CRY2::mCh::Bcd expression reduces the transcription activity of hb in the anterior domain in blue light

Embryos expressing the hb>MS2, MCP::GFP system were imaged throughout n.c. 13 and 14 with a time resolution of 40 s. Rows 1, 2 and 3 show respectively Control, CRY2low and CRY2high embryos. The left column represents the raw data whilst the right column shows the segmented MS2 dots (in green) as described in the Materials and methods section.

https://doi.org/10.7554/eLife.26258.009
Video 2
Bilateral cephalic furrow formation is uncoupled by single sided illumination

Embryo expressing CRY2::mCh::Bcd and Gap43::mCh mounted on a custom-built Light-Sheet microscope. The embryo was mounted during n.c. 13 and Gap43::mCh was used to follow its developmental stage. Five minutes after the start of cellularization, one lateral side of the embryo was illuminated for 20 min, starting from its most apical section to 70 µm deep (0.7 µm interval). Whole embryo recording (561 nm laser) and hemi embryo illumination (488 nm laser) were done simultaneously with a time resolution of 30 s. The top two panels show the lateral sides of the embryo, the second panel being the illuminated one. The bottom two panels show the ventral and the dorsal, respectively. n = 7 embryos per condition.

https://doi.org/10.7554/eLife.26258.019

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