In situ hybridisation (ISH) was performed as described in Kainz, 2009.

To analyse BMP pathway activity in control and Gb-Toll KD embryos, pMad antibody staining was performed as described in Pechmann et al., 2017.

For the pMad antibody staining, early cricket embryos (until egg stage 5) were dechorionated using 50% bleach (DanKlorix). Subsequently, embryos were washed several times with water until the embryos floated to the top of the water surface. Water was removed and embryos were fixed for 20 min in heptane/PBS containing 5% formaldehyde. The fixative was removed completely and the vitellin membrane was carefully removed in PBST using fine forceps (Dumont #5). Devitellinised embryos were transferred to PBST containing 5% formaldehyde and were fixed for overnight.

In situ hybridisation on blastoderm and early germ band embryos (until egg stage 5) was performed on heat-fixed embryos. For this, we collected 20–30 eggs in a 1.5 ml Eppendorf-tube. The eggs were washed two times with distilled water. Embryos were heat fixed in 100 µl distilled water at 99°C for 90 s and cooled down on ice for 2–5 min. To crack the eggshell, embryos were washed two times with 100% methanol. After the methanol shock, embryos were transferred to a glass dish and the vitelline membrane was removed using sharp needles and forceps. Peeled embryos were transferred to a fixative (5% formaldehyde in 0.01% PBST) and were fixed for several hours to overnight. Embryos were washed with 0.01% PBST and were gradually transferred to 100% MeOH for long-time storage at −20°C.

For in situ hybridisation on embryos of egg stages > 6, living embryos were dissected out of the vitelline membrane using sharp needles and forceps in PBST. Yolk was removed and embryos were fixed in PBST containing 5% formaldehyde overnight.

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