Figure 1.
The life cycle of C. albipunctata.
Embryonic development is covered in detail in the main text of the paper. After hatching, C. albipunctata goes through four larval instars before forming a pupa. The whole life cycle takes 27±5 days to complete.
Figure 2.
Embryonic staging and developmental events in C. albipunctata.
Embryos are shown as lateral views: anterior is to the left, dorsal is up. Stage numbers (roughly corresponding to Bownes' stages in D. melanogaster [13] are shown in red at the bottom left corner of each panel. Times after egg activation from onset to end of each stage are shown in white at the bottom right corner in hrs:min. Arrows, asterisks, and white bar indicate morphological landmarks. See main text for a detailed description, and Figure 3 for comparative timing of stages with reference to D. melanogaster.
Figure 3.
Comparative timing of stages in C. albipunctata and D. melanogaster.
The duration of each stage is shown for C. albipunctata and D. melanogaster in alternating black and blue bars. Purple bars for C. albipunctata show fused stages due to missing landmarks. Bars with graded colours indicate hetrochronic stages. The time scale is divided into blocks of 1 hr on the far left- and right-hand side (the latter applies to C. albipunctata stage 16/17 shown on the right). A brief description of each stage is given. Events exclusive to C. albipunctata are highlighted as red crossbars.
Figure 4.
Cleavage cycles of C. albipunctata.
Fluorescence images of embryos with DAPI-counterstained nuclei are shown as lateral views. Anterior is to the left. C1–14 indicates cleavage cycle number. The focus is on the sagittal plane for embryos at cleavage stage (C1–C9), and lateral views are shown at blastoderm stage (C10–14). As in D. melanogaster, nuclei begin to move towards the periphery from C7 onwards. Corresponding embryonic stages (see Figures 2 and 3) are indicated on grey background.
Table 1.
Observed number of nuclei in C. albipunctata.
Figure 5.
Comparison of the length of blastoderm cycles in D. melanogaster and C. albipunctata.
The duration of each division cycle is shown for both species with alternating black and blue bars. The onset of each cycle corresponds to the reappearance of nuclear envelopes in DIC movies. The time scale on the left is divided into blocks of 10(in hrs:min after egg activation) and duration (in min) are shown for cleavage cycles C10–14. For D. melanogaster, time for the start of C10 is taken from [13], and times for the duration of the blastoderm cycles from [15]. Corresponding embryonic stages (see Figures 2 and 3) are indicated on grey background.
Figure 6.
C.albipunctata early blastoderm cycles (C10–13).
Captured images from live DIC movies. Images show lateral views, anterior is to the left. Images on the right show metaphase (pseudo-cleavage) furrows (membrane progression marked by white arrows). Note that we cannot see metaphase furrows at C10. Times indicate time of cycle start in hrs:min after egg activation.
Figure 7.
Cellularisation and time classification scheme for C. albipunctata during cleavage cycle C14A.
Images captured from time-lapse movies showing the membrane morphology at mid-dorsal (left column) and mid-ventral (right column) positions are shown on the left-hand side of each column for time classes T1–T8. Schematic overlays in the middle of each column show vitelline membrane (thick black line), nuclei (red circle, oval or rectangle), and the edge of the periplasm, where the yolk begins (thin black line). Text on the right-hand side represents descriptions of features used to characterise each stage. See text for details.
Figure 8.
C. albipunctata eve expression staged using nuclei number, nuclear density, and membrane morphology.
Our staging method first distinguishes cleavage cycles based on the number of nuclei observed. Membrane morphology is then used to check the assignment of embryos to cleavage cycles C10–13 based on the size and spacing of the nuclei (see Figure 6). Embryos assigned to cleavage cycle C14A are further classified into time classes T1–8 based on membrane morphology and nuclear shape (see Figure 7). Using this method, we provide a detailed time-series for expression of the pair-rule gene even-skipped (eve) during the blastoderm stage. Lateral views are shown: enzymatic in situ hybridisation stains to the left, and DAPI-counterstain in the middle. The two columns on the right show details of dorsal (left) and ventral (right) membrane/nuclear morphology (sagittal views). See text for details.
Figure 9.
Extension and retraction of extraembryonic tissues in C. albipunctata.
Time is shown in hrs:min after egg activation for each image. Extraembryonic tissues are highlighted in yellow. Corresponding embryonic stages (see Figures 2 and 3) are indicated on grey background. See text for details.
Figure 10.
Germ plasm in C. albipunctata.
(A) Scanning electron micrograph of a stage 4 embryo showing the absence of morphologically distinguishable pole cells. (B) Close up of the posterior pole of the embryo shown in (A). (C–E) Vasa antibody stains of embryos at stage 4 (C), 6 (D), and 8 (E). Lateral views, anterior is to the left, dorsal is up. See text for details.