The inbred lines used in this study and in Wu et al. [31 ] were kind gifts of Dr. Cecelia Miles [77 (link)]: #2.46.4, #2.49.3, #9.17.1 and #9.31.2, representing LΑ, Lλ, SΑ and Sλ, respectively. Embryo collection, FISH and imaging were performed for Lλ and Sλ as previously reported [31 ]. Briefly, 0–4 h embryos were collected from 5 to 10-day-old females under standard conditions at 25°. We performed mRNA FISH using digoxigenin-labelled RNA probes synthesized as before [31 ,78 (link)], and the fluorescence signals were detected by sheep anti-digoxigenin (Roche, 1:400) and goat anti-sheep AlexaFluor 594 (Life technologies, 1:400) as the primary and secondary antibodies, respectively. The nuclei of collected embryos were counterstained with 4,6-diamidino-2-phenylindole (DAPI).
We performed imaging on Zeiss Imager Z1 ApoTome microscope, and the associated software AxioVision 4.8 was applied to capture images in linear settings without normalization as before [31 ]. Imaging for embryos was focused on the mid-sagittal section and taken with 10× objective, capturing embryos at the stage of interest (nc13 or nc14 before gastrulation) and avoiding embryos with severe morphological distortion and deformations. We adjusted the exposure time through using stained embryo with highest intensity to effectively avoid pixel intensity saturation. To make direct comparisons of expression profiles between lines, all experiments and imaging were conducted side by side.
We performed imaging on Zeiss Imager Z1 ApoTome microscope, and the associated software AxioVision 4.8 was applied to capture images in linear settings without normalization as before [31 ]. Imaging for embryos was focused on the mid-sagittal section and taken with 10× objective, capturing embryos at the stage of interest (nc13 or nc14 before gastrulation) and avoiding embryos with severe morphological distortion and deformations. We adjusted the exposure time through using stained embryo with highest intensity to effectively avoid pixel intensity saturation. To make direct comparisons of expression profiles between lines, all experiments and imaging were conducted side by side.
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