1. Developmental Biology
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Embryonic geometry underlies phenotypic variation in decanalized conditions

  1. Anqi Huang
  2. Jean-François Rupprecht
  3. Timothy E Saunders  Is a corresponding author
  1. Mechanobiology Institute, National University of Singapore, Singapore
  2. CNRS and Turing Center for Living Systems, Centre de Physique Théorique, Aix-Marseille Université, France
  3. Department of Biological Sciences, National University of Singapore, Singapore
  4. Institute of Molecular and Cell Biology, Proteos, A*Star, Singapore
Research Article
Cite this article as: eLife 2020;9:e47380 doi: 10.7554/eLife.47380
7 figures, 3 videos and 1 additional file

Figures

Figure 1 with 1 supplement
The Bcd gradient in embryos with differing geometry is consistent with the SDD model.

(A) Distribution of embryonic length in wild type (n = 239) and fat2RNAi (n = 364) embryos. (B) Distribution of embryonic width in wild type (n = 239) and fat2RNAi (n = 364) embryos. (C) Aspect ratio (EL/EW) against embryonic length for each embryo. Black dots denote expected aspect ratio if embryonic volume is conserved (assuming ellipsoidal geometry). (D) Number of nuclei along the AP axis plotted against embryonic length in wild type and fat2RNAi embryos. Line indicates linear regression of all data. (E) En expression in fat2RNAi embryos showing defective dorsal closure (left) or normal morphogenesis (right). Arrows indicate locations of defects. Green dots indicate En stripes. (F) Midsagittal plane of ctrl (top) and fat2RNAi (bottom) embryos expressing eGFP::Bcd in mid n.c. 14. (G) eGFP::Bcd profiles of both ctrl and fat2RNAi embryos plotted as a function of absolute distance from the anterior pole (left) or scaled AP position (right). Each dot represents the average concentration in a single nucleus. Colormap indicates the absolute AP length of each individual. (H) Mean and standard deviation of nuclear intensity within each 2% EL were computed for group of embryos longer (red, n = 27) and shorter (blue, n = 17) than 450 µm. Inset is close-up of profile near embryo midpoint to show the intersection of the two curves. (I) Representative fluorescent in situ hybridization (FISH) against bcd mRNA in wild type (top) and fat2RNAi (bottom) in n.c. 11 embryos. Scale bar, 50 μm. (J) Fluorescent intensity profile of FISH assay along AP axis in n.c. 4 (top) and n.c. 11 (bottom). Normalization to measured fluorescence signal in the region 120 μm from anterior. n = 5, 2 (n.c. 4) and n = 2,2 (n.c. 11) for wild type (red) and fat2RNAi (blue) respectively. Error bars show standard deviation. (K) Fitting of SDD model to experimentally measured Bcd gradient. All parameters, as outlined in Materials and methods, are kept constant, with only change being embryonic geometry. See Materials and methods for details. Lower panel shows intensity difference in experimental measurements and predicted profiles along the AP-axis.

Figure 1—figure supplement 1
Effects of perturbing embryonic geometry by fat2RNAi.

(A) Embryonic width plotted vs. embryonic length in three genotypes. (B) Embryo aspect ratio (length/width) for OreR and fat2RNAi embryos measured from live and fixed embryos (n > 200 in all conditions). Error bars, standard deviation. (C) Comparison of mean embryo length and width for OreR (red) and fat2RNAi (blue) embryos collected from live and fixed embryos. Error bars, standard deviation. n > 200 in all conditions. (D) Rate of embryonic hatching, puparation and adult emergence in wild type, fat2RNAi individuals longer or shorter than 400 µm, 6xbcd and 10xbcd embryos. (E) Max projection of Dapi staining in wild type and fat2RNAi embryos. Scale bar, 100 µm. (F) Representative images of in situ against bcd in wild type and fat2RNAi embryos at n.c. 4, 8 and 12. (G) Effect of protein folding time on gradient profiles in SDD model. Predicted functional Bcd concentration in fat2RNAi (blue) and ctrl (red) embryos with fast folding (assuming immediate folding, dashed line) or slow folding (~50 min, solid line) plotted against absolute distance from anterior (top panel) or relative AP positions (mid panel). Same normalization is used as in Figure 1K. The concentration difference in the fast folding scenario at each relative AP position is plotted in the lower panel. Scale bars, 50 µm.

Embryonic patterning is robust to perturbation of embryonic geometry.

(A) Max projection of ctrl (top) and fat2RNAi (bottom) embryos expressing maternally loaded eGFP and hb >LlamaTag. (B) Scaled Hb boundary positions plotted against EL in two genotypes. (C–F) Comparison of gap gene expression between wild type and fat2RNAi embryos. Profiles normalized to max intensity and the computed mean and s.d. plotted against scaled AP length. Boundary positions of each individual is plotted to show the distribution in two genotypes (WT, red and fat2RNAi, blue). Scale bars, 100 µm.

Figure 3 with 1 supplement
Defects due to decanalization by Bcd over-expression are length-dependent.

(A) Schematic illustration of tandem bcd construct. (B) Embryos expressing 2x (wild type), 6x and 10x of maternal bcd fixed at the onset of gastrulation and stained with Phalloidin. Green arrowheads indicate the positions of cephalic furrow (CF) formation. (C) Distribution of defective cuticular segments in non-hatched 10xbcd (top), fat2RNAi, 6xbcd (mid) and fat2RNAi (bottom) embryos. Colormap indicates the frequency of defects in each segmental region. (D) Bar plots showing the distribution of defective cuticular segments in three genotypes. (E) En expression patterns in 4x (top), 6x (mid) and 10x (bottom) bcd embryos with different EL. Green dots, normal En stripes; red dots, defective En stripes; A, anterior; P, posterior. (F) Representative 6xbcd embryos with different EL expressing en >mCD8::GFP. Numbers indicate En stripe identities and red mask indicate defective segmental regions. Scale bar, 100 µm.

Figure 3—figure supplement 1
Embryonic patterning with maternal bcd overexpression.

(A) Expression level of maternally loaded bcd mRNA in fat2RNAi, 6xbcd and 10xbcd relative to wild type. (B) FISH against bcd in wild type (top) and 10xbcd (bottom) embryos at n.c. 8. Scale bars, 50 µm. (C) Normalized FISH intensity plotted vs. distance from the anterior for wild type, fat2RNAi and 10xbcd embryos at n.c.4 (top) and 11 (bottom). Normalization to measured signal at 120 μm into embryo. (D–E) Representative cuticle patterns of 10xbcd (D) and fat2RNAi, 6xbcd (E) embryos. Red arrowheads indicate defective regions. M, mouthparts and T, thoracic segments.

Embryonic patterning breaks down at A4 segment with bcd overexpression.

(A–B) Embryonic length of 6xbcd individuals plotted against the scaled (A) or absolute (B) AP position of four gap gene boundaries (Hb, blue; Gt, magenta; Kr, red and Kni, green). Data from every 30 µm EL interval were binned to compute mean and s.d. and the colored areas are generated by connecting mean values of different EL ranges. Dashed line indicates posterior boundary of individuals; green and magenta crosses overlapping the dashed line indicate individuals with corresponding EL not expressing Kni and Gt, respectively. (C) Representative segmentation gene expression in 6xbcd embryos with different EL. Asterisk indicates repressed Eve stripe five and arrowhead indicates failed activation of Kni. Scale bar, 100 µm.

Figure 5 with 3 supplements
bcd mutant phenotypes correlate with embryonic length.

(A) Phenotypic frequency showing different number of normal abdominal segments in bcdKO mutant individuals (n = 202). Dashed area indicates proportion of embryos showing fully developed ectopic spiracles (see Figure 5—figure supplement 1D(i)). (B) Phenotypic frequency of different ectopic spiracle morphology as shown in Figure 5—figure supplement 1D (n = 168). (C) Variation of gap gene boundary positions in wild type vs. bcdKO embryos. Error bars are computed by bootstrapping with data shown in Figure 5—figure supplement 1E–H. (D) Representative cuticle patterns of fat2RNAi, bcdKO embryos within different ranges of embryonic length, from left to right, 330–360, 360–390, 390–420, 420–450, and 450–480 µm. Stars indicate normal abdominal segments. (E) Number of normal abdominal segments plotted against EL range of each individual. (F) En expression in wild type embryo. Green dots indicate En stripes. (G) Representative images of fat2RNAi, bcdKO embryos showing different number of En stripes. Magenta dots indicate En stripes. Scale bar, 100 µm. (H) Number of En stripes plotted vs. EL in individuals from three genotypes. Magenta triangles indicate individuals showing defective morphogenesis at the end of dorsal closure; cyan triangles indicate normal morphogenesis.

Figure 5—figure supplement 1
Characterization of bcd mutant phenotypes.

(A) Representative cuticle pattern of wild type individual. Black asterisks highlight normally formed denticle belts. (B) Representative cuticle patterns in bcdKO mutant individuals. Blue asterisks denote aberrant denticle belts. (C) Representative cuticle patterns in bcdE1 mutant individuals. (D) Various phenotypes of ectopic posterior spiracle observed in the anterior region of the bcdKO embryos. Arrows point to ectopic spiracles showing complete morphogenesis (i), partial morphogenesis (ii), only primordial structures (iii), and no ectopic spiracle differentiation (iv). Letter h indicates spiracular hair; fk, filzkörper and st, stigmatophore. (E–H) Gap gene expression by the end of blastoderm stage in wild type (upper panel) and bcdKO (lower panel). Dashed lines mark the boundary of expression domains, and the scaled boundary positions of individuals are plotted in the graphs below (wild type in blue and bcdKO in green). In (H), asterisk indicates weak transcriptional activation of anterior Gt domain.

Figure 5—figure supplement 2
Variations of bcd mutant phenotypes.

(A–B) Comparison of bcdKO individuals showing different expression patterns of Kr (A) and Gt (B). Solid triangle indicates anterior Gt domain and dashed triangles indicate failed activation of Kr or anterior Gt. (C–D) Phenotypic frequency of cuticle patterns and ectopic spiracles in individuals generated by a single bcdKO female crossed to a single male and raised in constant environments. C and D show two independent single-cross experiments. n = 23 (C) and n = 42 (D).

Figure 5—figure supplement 3
Bcd mutant phenotypes correlate with embryo length.

(A–E) En expression at the end of head involution stage in ctrl (A) and bcdKO (B–E) embryos. Dots indicate En stripes (ctrl, green; bcdKO, magenta) and the numbers indicate embryonic length. (F) Number of En stripes as function of embryo length for ctrl and bcdKO embryos.

Figure 6 with 2 supplements
Phenotypic discordance can be traced back to variations in gap gene expression in bcdKO individuals.

(A) Representative gap gene expression patterns within different range of EL. Range1, 330–360 µm; range2, 390–420 µm; and range3, 510–540 µm. (B) Embryonic length of bcdKO individual plotted vs. boundary position of four gap genes shown as absolute distance from the anterior pole (Hb, blue; Gt, magenta; Kr, red and Kni, green). Data from every 30 µm EL interval were binned to compute mean and s.d. and the colored areas are generated by connecting mean values of different EL ranges. Dashed boxes indicate ranges of EL corresponding to (A). Dashed line indicates posterior boundary of individual embryos; red and green crosses overlapping the dashed line indicate individuals with corresponding EL not expressing Kr and Kni, respectively. (C) Schematic illustration of positional information transfer from maternal systems to gap gene expression in bcdKO embryos within different range of embryonic length. Scale bar, 100 µm.

Figure 6—figure supplement 1
Positional information transfer in the absence of maternal bcd.

(A) Embryos of wild type (left), bcdKO (mid) and tll (right) fixed at the end of the blastoderm stage and stained for Tll (magenta) and Hb (cyan). The symmetric expression of Hb and Tll in bcdKO and the absence of posterior Hb stripes in tll embryos indicate that Tll is necessary for the activation of this Hb domain. (B) Embryos of wild type, bcdKO, fat2RNAi, and fat2RNAi, bcdKO fixed at presyncytial stages (around n.c. 8) and stained with Dapi and Hb antibody. The Hb intensity at these stages represent maternal Hb expression. (C) Mean and s.d. of normalized Hb profiles from four genotypes, respectively, were plotted vs. scaled AP position. (D) Mean and s.d. of maternal Hb boundary positions in four genotypes. *p<0.01. (E) Representative images of bcdKO embryos within different EL ranges (corresponding to Figure 6A) fixed at the end of blastoderm stage and stained for Gt (magenta) and Eve (cyan). Scale bar, 100 μm.

Figure 6—figure supplement 2
Strong correlation between gap gene boundary positions and embryonic length in the absence of maternal bcd.

(A–D) Relative boundary positions of Hb (A), Gt (B), Kni (C) and Kr (D) plotted vs. embryonic length in bcdKO and bcdKO, fat2RNAi individuals. Lines indicate linear regression of each gap gene boundary.

Author response image 1
Bcd gradient at steady-state using SDD model on surface of a cylinder (blue: L = 500μm, r = 100μm; red: L =350μm, r = 143μm).

Bcd is introduced at rate J at the anterior end of the cylinder. We exclude the ends of the cylinder. (A) Bcd gradient assuming immediate protein folding (total Bcd). (B) Bcd gradient accounting for protein folding time. Dashed lines highlight how the crossover point shifts due to accounting for folding..

Videos

Video 1
Embryogenesis of different geometry.

Wide field movies of wild type (top) and fat2RNAi (bottom) embryos from the onset of gastrulation until hatching.

Video 2
Defective morphogenesis in late embryo development due to extreme embryonic geometry.

Confocal movies of fat2RNAi embryos expressing en >mCD8::GFP from germband retraction to dorsal closure stages. Two embryos in the movie represent individuals with EL shorter (top) and longer (bottom) than 400 µm. The dashed box indicates mismatch between opposing ectodermal tissue during dorsal closure. The numbers indicate the EL of each embryo.

Video 3
Engrailed expression in bcdKO mutant individuals.

Live imaging of bcdKO embryogenesis from onset of gastrulation to the end of dorsal closure. Embryos express H2Av::mCh (red) and en>mCD8::GFP (green). Dots indicate En stripes and the numbers on top of the embryos indicate the AP length of each individual.

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