Formation of the germ-disc in spider embryos by a condensation-like mechanism

Background Determination of the embryonic body axes is a crucial developmental process in all animals. The establishment of the embryonic axes of spiders has been best studied in the common-house-spider Parasteatoda tepidariorum. Here, anteroposterior (AP) polarity arises during germ disc formation; the centre of the germ-disc marks the future posterior pole, and the rim of the disc the future anterior pole of the spider embryo. The centre of the germ disc is also needed for the formation of the cumulus, a group of migratory cells needed to establish dorsoventral (DV) polarity. Thus, both body axes depend on proper germ disc formation and patterning. However, these processes have not been fully analysed at the cellular and molecular level. Results Here I present new techniques to stain the cell membranes/outlines in live and fixed spider embryos. I show that the germ-disc is formed from a regular and contiguous blastoderm and that co-ordinated cell shape changes, rather than migration of single cells, are required to drive germ-disc formation in P. tepidariorum embryos. Furthermore, I show that the rate of cell divisions within the embryonic and extra-embryonic region is not involved in the rapid establishment of the germ-disc. Finally, I show that the process of germ-disc formation is dependent on the initiation of zygotic transcription. Conclusions The presented data provide new insights in to the formation of the germ-disc in spider embryos. The establishment of the germ-disc in Parasteatoda embryos is a highly dynamic process that involves wide scale cell-shape changes. While most of the blastodermal cells become cuboidal to form the dense germ-disc, the remaining blastodermal cells stay squamous and develop into huge extra-embryonic, yolk rich cells. In addition, this study shows that the onset of zygotic transcription is needed to establish the germ-disc itself, and that the mid-blastula transition of Parasteatoda tepidariorum embryos is prior to any overt axis establishment. Electronic supplementary material The online version of this article (doi:10.1186/s12983-016-0166-9) contains supplementary material, which is available to authorized users.

stage 3 embryo has been injected with FITC WGA. Dividing cells have been labelled in orange and blue. Scale bar is 25µm in b, c, d, f and g''.
Fig. S2 Cell shapes at late stage 2 . Living embryos have been injected with FITC WGA and maximum intensity projections of confocal scans have been used to analyse the shape of the cells.

Fig. S3
(a-a'') The same embryo before (a) and after the injection of cytochalasin D (a' and a''). The asterisk in a indicates the centre of the future germ-disc. After the injection of cytochalasin D (CD) no germ-disc forms and cells cluster. In addition, cytokinesis is disrupted and cells show multiple nuclei (compare insets in a and a'). Regions of blastoderm disruption are indicated by the arrowheads in a''. a' and a'' are stills taken from movie 5. Time corresponds to the time points shown in the movie. (b-b''') Stills from movie 4. During germ disc formation a yolk granule (labelled with an asterisk in bb'') is shifted to a more basal position within or is excluded from the germ-disc cells. (cc'') Tracking of an extra-embryonic nuclei with its attached perinuclear cytoplasm. The nuclei with its attached perinuclear cytoplasm first divide (c') but fuse again (c''). This results in an extra-embryonic cell with two nuclei. (d) Confocal scan (maximum intensity projection) of extra-embryonic cells stained with phalloidin and DAPI. Two nuclei are present in one cell (arrowheads). Scale bar is 50 µm in d.

Fig. S4
Two stage 1 embryos of the same cocoon have been injected with water (a) or alpha amanitin (b). While the control embryo developed normal until germ-band stage, the alpha amanitin injected embryo arrested at stage 2 but was still alive when the control embryo reached stage 8. The development from a stage 2 to a stage 8 embryo takes around two days (see movie 2). The outline of the germ-band has been marked by the dotted line in a.

Semi-quantitative-RT-PCR -supplemental Material and Methods
The expression levels of Pt-hh and Pt-PolyUb has been analysed by running 28, 30, 32 and 35 PCR cycles on cDNA of stage 1, stage 2 and stage 3 embryos. The cDNA was prepared from total RNA that has been extracted separately from stage 1, stage 2 and stage 3 embryos. cDNA concentrations of the different embryonic stages have been adjusted to 1ng cDNA/µl PCR reaction. To analyse the effect of alpha-amanitin injections on the maternal to zygotic transition, stage 1 embryos of three different spider females (three biological replicates) have been injected with alpha-amanitin or water. The development of the injected embryos has been monitored and the total RNA of around 50 embryos of the water and alpha-amanitin injected embryos has been extracted. For the alpha-amanitin injected embryos only the embryos that got stuck at stage 2 were used for total RNA extractions after the water-injected embryos had reached the beginning of stage 3. cDNA has been prepared from the three water and the three alphaamanitin RNA samples separately and the absence or presence of Pt-PolyUb and Pt-hh transcripts has been analysed via PCR.
Primer set used for semi-quantitative-RT-PCR Shown is the published coding sequence (CDS) for Pt-hedgehog (Pt-hh) and the partial genome sequence for Pt-hedgehog. Primer sequences are in yellow. The different exons are coloured in green and magenta.