www.sciencemag.org/cgi/content/full/science.1218256/DC1
Supporting Online Material for A Segmentation Clock with Two-Segment Periodicity in Insects Andres F. Sarrazin, Andrew D. Peel, Michalis Averof* *To whom correspondence should be addressed. E-mail:
[email protected] Published 8 March 2012 on Science Express DOI: 10.1126/science.1218256
This PDF file includes: Materials and Methods Figs. S1 to S6 Movie Captions S1 to S3 References Other Supporting Online Material for this manuscript includes the following: (available at www.sciencemag.org/cgi/content/full/science.1218256/DC1) Movies S1 to S3
Materials and Methods In situ hybridization Alkaline phosphatase and fluorescence in situ hybridization on whole mount embryos were carried out as previously described (24, 25). DIG-labeled RNA probes against Tc-odd and Tc-eve were transcribed using standard protocols (25) from clones isolated by PCR from Tribolium genomic DNA. The 3321 bp Tc-odd probe targets most of the second and third exons (738 bp) and the entire second intron (2583 bp). The 3001 bp Tc-eve probe targets all but the first 11 bp of the coding sequence (778 bp), both introns (2181 bp) and some 3'UTR sequence (42 bp). Primer sequences: Tc-odd-forward: 5’-TGTCCGGCAAGTCAAACAGG A-3’, Tc-odd-reverse: 5’-CGCTTCATAATGTCCTCGATACTGAACCC-3’, Tc-eveforward: 5’-CGACAAGATGTTCAACCAAGAGCA-3’, Tc-eve-reverse: 5’-ACAAAGCAA CCATGCACGAA-3’. The primary and secondary antibodies used in FISH were Sheep antiDIG (Roche, 1:500) and Alexa 555 Donkey anti-Sheep (Molecular Probes, 1:400). Embryos were imaged on a Leica MZ16F epifluorescence stereoscope with a DFC300FX digital camera, and on a Leica SP2 laser scanning confocal microscope. Staging of Tc-odd stripe formation We tentatively subdivide the formation of each Tc-odd stripe in three phases (I, II and III), based on the distribution of Tc-odd transcripts within the growth zone. Each stage is named after the primary Tc-odd stripe that is being formed within the growth zone and the phase of Tc-odd expression; e.g. during stages 4.I, 4.II and 4.III the fourth primary stripe emerges from the growth zone. At phase I (Figure 1 C,F) the growth zone has low levels of Tc-odd, except for two lateral spots of expression which mark the initiation of a new cycle of Tc-odd expression. At phase II (Figure 1 A,D) Tc-odd expression extends throughout the growth zone, including the ectoderm, the overlying amnion and the underlying mesoderm (medially), such that a broad posterior domain of high Tc-odd expression is established. At phase III (Figure 1 B,E) Tc-odd expression in the posterior-most part of the growth zone (amnion, ectoderm and mesoderm) is reduced to low levels while more anteriorly positioned cells still express high levels, establishing a broad primary stripe of Tc-odd expression. Tribolium GFP-expressing transgenic line The GFP-expressing construct, named EFA-nGFP, was generated by combining 3.3 kb of upstream regulatory sequences from Tribolium EF1-alpha gene LOC655495 (cloned using primers 5'-GGAATTCATCAGCGTTACCTTCGGTGT-3' and 5'-CGGGATCCAACCTGC AAAACCAC-3') with GFP-6xMyc-NLS (kindly provided by Gary Struhl), followed by the SV40 early polyadenylation sequence. Cloning details and sequences are available on request. The construct was inserted in the unique AscI site of the piggyBac{3xP3-DsRed} vector (26) and injected into Tribolium embryos (vermillion white strain) to generate stable transgenic lines using standard methods (27). Two transgenic lines were recovered. In both lines, nuclear-localized GFP is expressed ubiquitously during most developmental stages, including oogenesis, embryogenesis and larval life. Tribolium embryo culture Tribolium castaneum embryos from the EFA-nGFP transgenic line were collected from overnight matings at 30°C and dechorionated in hypochlorite solution. Embryos at the stage of amnion closure were selected under an epifluorescence stereoscope and placed in a glass 2
dish supplied with M3+ medium, consisting of Shields and Sang insect medium (Sigma S8393) supplemented with 10% heat-inactivated fetal calf serum (Gibco) and 50 µg/ml gentamycin, adjusted to pH 6.5 and filter-sterilized (0.22 µm PES filter, Corning). Germbands were micro-dissected out of the egg using a pair of fine forceps and an eyebrow hair, under a Leica MZ16F epifluorescence stereoscope, taking special care to preserve the amnion membrane and to remove all the yolk. Dissected germbands were transferred to a new glass dish with fresh medium and photographed in brightfield to record the stage before performing bisections or time-lapse experiments. The embryos were cultured at 30˚C in a covered glass dish containing M3+ medium. Expression analysis in half-embryos Dissected embryos were placed with the ventral side up and were bisected along the midline using an eyebrow hair. One half (left side) was immediately fixed in 5% formaldehyde in PBS and the other half (right side) was cultured in 1 ml of M3+ medium in a 24-well culture plate at 30°C for different time intervals (15-105 minutes). At the required times, the embryos were fixed in 5% formaldehyde in PBS. Fixation and in situ hybridization on bisected germbands was carried out in baskets with a 31 micron nylon mesh (03-31/24, Sefar AG). Each germband half was placed in its own basket and transferred between reagents in 24-well Nunclon plates. The proteinase K treatment was omitted. Live imaging and cell tracking Individual embryos were immobilized on a coverslip using heptane glue, mounted on a microscope slide and surrounded with halocarbon oil 700 (Sigma) to prevent desiccation (28). Dissected germbands were mounted flat in M3+ medium; they were kept flat between a coverslip and a microscope slide while leaving some space to allow respiration. Halocarbon oil was placed at the edge of the coverslip to prevent desiccation. Time-lapse movies were recorded with a 10x or 20x objective on an Olympus BX61 spinning disc confocal microscope, in an air-conditioned room kept at 30°C. Z-stacks were captured over a period of 2-6 hours (for whole embryos) or 2-3 hours (for germbands). Cell tracking was performed in 4D (3D time-lapse) image stacks captured every 1-3 minutes. Using ImageJ we manually tracked the GFP-marked nuclei of individual ectodermal cells through time, across several optical sections. 24. 25. 26. 27. 28.
J. Schinko, N. Posnien, S. Kittelmann, N. Koniszewski, G. Bucher, Single and double whole-mount in situ hybridization in red flour beetle (Tribolium) embryos. CSH Protoc 2009, pdb prot5258 (2009). D. Kosman et al., Multiplex detection of RNA expression in Drosophila embryos. Science 305, 846 (2004). C. Horn, E.A. Wimmer, A versatile vector set for animal transgenesis. Dev Genes Evol 210, 630 (2000). A.J. Berghammer, M. Weber, J. Trauner, M. Klingler, Red flour beetle (Tribolium) germline transformation and insertional mutagenesis. CSH Protoc 2009, pdb prot5259 (2009). I. Davis, R.M. Parton, in Live Cell Imaging: A Laboratory Manual, R.D. Goldman, D.L. Spector, Eds. (CSHL Press, Cold Spring Harbor, NY, 2005), pp. 385-407.
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Figure S1 Explanation of Tc-odd staining in early germbands. (A) The early Tribolium germbands shown in Figure 1 are multilayered structures consisting of ectoderm, mesoderm and an extra-embryonic membrane called the amnion. The posterior growth zone (area within dotted lines) may be subdivided into a medial sector (M) and two lateral sectors (L). The mesoderm is only found in the medial sector, while the ectoderm extends through the lateral sectors and gradually spreads over the mesoderm medially during early germband elongation (see ref. Handel et al. 2005). The amnion lies over (ventral to) the entire growth zone. White arrowheads point to a stripe of Tc-odd expression in the amnion. (B) The intensity profiles of Tc-odd expression shown in Figure 1 A'-F' were measured within a rectangle placed on the lateral part of the germband (area within dotted line) using ImageJ. (C) The intensity plot profile depicts staining intensity along the anteroposterior axis of the germband, in arbitrary units.
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Figure S2 Expression of Tc-odd prior to germband elongation. (A-D) Lateral views of Tc-odd expression in blastoderm embryos of increasing age (ventral is to the top). (A'-D') Nuclear staining with DAPI on the same embryos. (A) In early blastoderm embryos, Tc-odd is expressed in a broad posterior domain and in a smaller anterior-ventral domain (asterisk). (B) The broad posterior domain resolves into the first (P1) and second (P2) primary stripes, while the anterior-ventral domain fades from the anterior to form a small anterior-ventral spot of expression (asterisk). (C,D) During germdisk condensation and primitive pit formation the third primary stripe appears in the posterior (P3), within the forming pit, and the first secondary stripe (S1) appears anterior to the first primary stripe (P1). The first primary stripe passes through the amnion on the dorsal side of the egg (arrowheads), and migrates over the posterior pole of the embryo during primitive pit formation, as the amnion gradually surrounds the sinking germband (later stages of this process are depicted in Figure 1). The anterior-ventral spot of Tc-odd expression (asterisk) marks extra-embryonic tissue near the forming head lobes, before fading. The embryo in panel D is at a similar stage as the dissected germband shown in Figure 1A. (E-H) Tc-odd expression seen in panels A-D, viewed from the posterior pole of the embryo. The posterior-most cells do not appear to express Tc-odd during the resolution of the first (P1) and second (P2) primary stripes (panels E,F), but show dynamic expression of Tc-odd during the formation of the third (P3) primary stripe (compare panels G and H). The posterior pole of the embryo in panel H has been dissected and mounted on a slide.
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Figure S3 Expression of Tc-odd in relation to Tc-eve. Double fluorescent in situ hybridizations against Tc-odd (red) and Tc-eve (green) on embryos at successive developmental stages. (AC) Surface views of the posterior half of the blastoderm. Tc-odd and Tc-eve are expressed in separate domains during formation of the first two primary Tc-odd stripes (oddP1 and oddP2). (D-G) Ventral views of dissected germbands. Tc-odd and Tc-eve expression are 'outof-phase' but partly overlapping in the growth zone. The intensity of expression in the posterior most ectoderm and amnion (region x) is compared to the intensity in the immediately more anterior ectoderm and amnion (region y) for Tc-odd and Tc-eve: when Tcodd is expressed at higher levels at the posterior (x/y odd >1), the reverse is true for Tc-eve (x/y eve
