A staging scheme for the development of the scuttle fly Megaselia abdita

Abstract

Model organisms, such as Drosophila melanogaster, provide powerful experimental tools for the study of development. However, approaches using model systems need to be complemented by comparative studies for us to gain a deeper understanding of the functional properties and evolution of developmental processes. New model organisms need to be established to enable such comparative work. The establishment of new model system requires a detailed description of its life cycle and development. The resulting staging scheme is essential for providing morphological context for molecular studies, and allows us to homologise developmental processes between species. In this paper, we provide a staging scheme and morphological characterisation of the life cycle for an emerging non-drosophilid dipteran model system: the scuttle fly Megaselia abdita. We pay particular attention to early embryogenesis (cleavage and blastoderm stages up to gastrulation), the formation and retraction of extraembryonic tissues, and the determination and formation of germ (pole) cells. Despite the large evolutionary distance between the two species (approximately 150 million years), we find that M. abdita development is remarkably similar to D. melanogaster in terms of developmental landmarks and their relative timing.

Figure 1. The life cycle of M. abdita.

Embryonic development is covered in detail in the main text of the paper. After hatching, M. abdita goes through three larval instars before forming a pupa. The whole life cycle takes 18–20 days to complete.

Figure 2. Embryonic staging and developmental events in M. abdita.

Embryos are shown as lateral views: anterior is to the left, dorsal is up. Stage numbers (roughly corresponding to Bownes' stages in D. melanogaster [27]) are shown in red at the top left, and time after egg laying (AEL) in hrs:min in white at the bottom left corner of each panel. White arrows and bars 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 developmental stages in M. abdita and D. melanogaster.

The duration of each stage is shown for M. abdita and D. melanogaster in alternating black and blue bars. The time scale is divided into blocks of 1 hr on the far left hand side. A brief description of each stage is given on the right. Landmarks of extraembryonic tissue formation and retractions are indicated to the left of the M. abdita time scale.

Figure 4. Cleavage cycles of M. abdita.

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 at the surface of the embryo 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.

Figure 5. Comparison of the length of blastoderm cycles in D. melanogaster and M. abdita.

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 laying, AEL) along with duration (in min) are shown for cleavage cycles C10–14. For D. melanogaster, time for the start of C10 is taken from , and times for the duration of the blastoderm cycles from . Corresponding embryonic stages (see Figures 2 and 3) are indicated on grey background.

Figure 6. M. abdita early blastoderm cycles (C10–13).

Captured images from live DIC movies. Images show lateral views, anterior is to the left, dorsal is up. Times in min after egg laying (AEL). Schematic overlays show vitelline membrane (thick black line), nuclei (red circle) and metaphase (pseudo-cleavage) furrow front (thin black line).

Figure 7. Cellularisation and time classification scheme for M. abdita during cleavage cycle 14A.

Images captured from time-lapse movies showing the membrane morphology at mid-dorsal positions are shown on the left-hand side for time classes T1–T8 and for gastrulation. Starting times after egg laying for each time class are shown in the bottom left of each image in hrs:min(:sec). Schematic overlays show vitelline membrane (thick black line), nuclei (red circle, oval or rectangle), and invaginating membrane front (thin black line). Grey nuclei indicate the size of the nuclei at T3 for reference. Descriptions of features used to distinguish each stage are provided on the right (see text for details).

Figure 8. M. abdita eve mRNA expression staged using nuclei number, nuclear density, and membrane morphology.

Our staging method first distinguishes cleavage cycles based on the number or density of nuclei observed. Dorsal membrane morphology is then used to check the assignment of embryos to cleavage cycles C10–14 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 (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 right-hand column shows details of dorsal membrane/nuclear morphology (sagittal views). See text for details.

Figure 9. Extension and retraction of the serosa in M. abdita.

Time is shown in hrs:min after egg laying (AEL) for each image. The serosa is highlighted in yellow. The black arrow in (A) indicates the amniosersal lip, the white arrow in (G) the position where the serosa ruptures. Corresponding embryonic stages (see Figures 2 and 3) are indicated on grey background. See text for details.

Figure 10. Pole cell formation in M. abdita.

(A–D, E, F) Embryos stained with antibodies against Vasa protein at cleavage cycles C3, C5, C10, C14A (stages 1–5), as well as stage 6 and stage 8. Lateral views, anterior is to the left, dorsal is up. Arrow in (F) indicates the position of the pole cells after their inwards migration. (C') scanning electron micrograph (SEM) of C10 embryo; close-up in (C'') shows pole cells.