Volume 20, Issue 18, Pages 1641-1647 (September 2010) Imaging of Transgenic Cricket Embryos Reveals Cell Movements Consistent with a Syncytial Patterning Mechanism  Taro Nakamura, Masato Yoshizaki, Shotaro Ogawa, Haruko Okamoto, Yohei Shinmyo, Tetsuya Bando, Hideyo Ohuchi, Sumihare Noji, Taro Mito  Current Biology  Volume 20, Issue 18, Pages 1641-1647 (September 2010) DOI: 10.1016/j.cub.2010.07.044 Copyright © 2010 Elsevier Ltd Terms and Conditions

Figure 1 Cellular Dynamics during Early G. bimaculatus Embryogenesis (A–H) Time-lapse recording of a transgenic (pBGact-eGFP) embryo. (A) Image at 4 hr and 20 min after egg laying (AEL) (this time point is shown as t0), when no nuclei are seen, probably because they are too deep inside of the yolk to be detected by fluorescence microscopy. (B–D) Cumulative images recapitulating the position of energids at selected time periods during early mitotic cycles. (E–H) Distribution of energids or cells during early to middle blastoderm stages at each time point. (I–K) Distribution of the cortical cytoplasm at the surface of a transgenic (pBGact-eGFP) egg. (I) At 40 min AEL, the cortical cytoplasm is distributed in the egg surfaces in a net-like structure. (J) When energids emerged in the periphery of the egg (11 hr and 40 min AEL), the net-like cortical cytoplasm was dramatically reduced. (K) At midblastoderm stages (19 hr AEL), the cortical cytoplasm was nearly gone, but thin connections of the filament-like cytoplasm remained between cells. (L and M) High-magnification images of cells at the midblastoderm stage. Cells having many filopodia were connected to each other via the thin cortical cytoplasm. (N–Q) Trajectories of the marked cells. White arrows show the trajectories of the marked energids or cells, and the distance of cell migration is indicated by the length of the arrows. t0: 17 hr AEL. (R–U) Determination of the timing of cellularization. The rhodamine conjugated Dextran (10,000 kDa, red color) dye was injected into the eGFP-expressing eggs of the various developmental stages. After the 7 hr incubation, the fluorescence was observed for examination of whether energids around the site of injection incorporated the rhodamine dye. The egg in each panel is shown after the 7 hr incubation subsequent to the dye injection. The injection time and observation time (hour after egg-laying; AEL) are indicated in each panel. (R–T) In eggs injected with the dye before 14 hr AEL, all energids expressing eGFP colocalized the dye (yellow in color) around the site of injection. However, the region of colocalization of the dye and eGFP became smaller, because the injection time was getting late. (U) In eggs injected with the dye at 16 hr AEL, colocalization was not observed. The arrow indicates a mass of dye leaked from the egg during injection. (V and W) Plots of the changes in the relative positions of cells in different regions of the egg are indicated by lines drawn between them at t0 (V) and at 410 min (W). See also Figure S1, Movie S1, Movie S2, and Movie S3. Current Biology 2010 20, 1641-1647DOI: (10.1016/j.cub.2010.07.044) Copyright © 2010 Elsevier Ltd Terms and Conditions

Figure 2 Cellular Dynamics during Germ-Anlage Formation in G. bimaculatus (A–F) Time-lapse recording of a transgenic (pBGact-eGFP) embryo. Ventral views. Anterior toward the left. Time after egg laying (hr AEL) is shown by yellow numbers. (A and B) At late blastoderm stages, blastoderm cells aggregated in both of the posterolateral patch-like regions of the egg to form a germ anlage (third phase). (C and D) The initially diffusely delimited lateral aggregations have become more condensed and gradually shift from their site of origin toward the posterior and ventral side of the egg, where they first fuse by a caudally located bridge of cells (early and mid fourth phase). (E) Fusion is completed by about 31 hr AEL, producing the unitary single-layered germ anlage that consists essentially of head and thoracic primordial regions (late fourth phase). (F) The germ anlage contracts to produce a clearly delineated germ band (fifth phase). The uniformly distributed cells showing more intense fluorescence than other cells (C–F) are vitellophages [3], lying below the layer of the extraembryonic region (presumptive serosa) and germ-anlage cells. (G–H″) The trajectories of marked presumptive germ-anlage cells (G″ and H″) from the start points shown in (G) and (H) to the end points shown in (G′) and (H′). (I and J) Schematic illustrations of cellular dynamics. Lateral view (I) and ventral view (J). Outlines of aggregation of the germ-anlage cells are shown in blue (21 hr AEL), green (28 hr AEL), or red colors (33 hr AEL). The arrows indicate the direction of cell movements. See also Movie S4 and Movie S5. Current Biology 2010 20, 1641-1647DOI: (10.1016/j.cub.2010.07.044) Copyright © 2010 Elsevier Ltd Terms and Conditions

Figure 3 Expression and Function of G. bimaculatus otd1 in Early Embryos (A) Expression pattern of Gb'otd1 during blastoderm stages. The transcripts of the otd ortholog (Gb'otd1) are distributed in all superficial energids or cells of the syncytial blasdoderm (13 hr AEL) and uniform cellular blastoderm (18 hr AEL). The transcripts are localized around each nucleus of the blastoderm cells (insets in each panel; in situ hybridization result merged with the nuclear staining by DAPI). At 22 hr AEL, the onset of cell aggregation into the posterior region, Gb'otd1 expression is still ubiquitous (this sample lacking expression signals around the posterior poles as a result of damage in the egg surface through the sample preparation). Instead, the right small panels show Gb'otd1 expression around both egg poles (merged with DAPI staining). By 23 hr AEL, intense expression of Gb'otd1 rapidly becomes restricted to the germ-anlage-forming region, exhibiting a broad stripe in 20%–40% in the egg length. As germ-anlage cells become more densely packed toward the ventral region of the egg, the Gb'otd1 expression becomes further restricted to a small patched region in each head lobe of the germ anlage by 32 hr AEL. Expression of Gb'otd1 in the presumptive serosa cells is observed in this stage (bottom panel). (B) Expression pattern of G. bimaculatus hunchback (Gb'hb) during blastoderm stages. Although no transcripts of Gb'hb were detected until 16 hr AEL (data not shown), at 18 hr AEL, faint expression was observed in superficial cells in the middle region of the cellular blastoderm. At 26 hr AEL, the onset of fusion of the bilateral cell aggregations at the ventral surface of the egg, Gb'hb is expressed in superficial cells from the anterior pole to the middle region (bracket). At 34 hr AEL, the germ anlage is just completed but is still not covered with the amnion, and Gb'hb expression becomes localized to the serosal region. Higher-magnification images at 34 hr AEL egg show that Gb'hb expression formed a sharp boundary between the extraembryonic and the embryonic region. On the other hand, in the germ anlage, Gb'hb is expressed as a gap-like pattern in the presumptive gnathal region. (C and D) RNAi analysis of Gb'otd1. (C) Effect of Gb'otd1 RNAi on the morphology of early embryos (38 hr AEL). The left column shows the lateral view, and the right column shows the ventral view in the same embryo. The transgenic (pBGact-eGFP) line was used for the RNAi experiment to visualize morphological defects during early embryogenesis. (D) Control (DsRed RNAi) and Gb'otd1 RNAi embryos (46 hr AEL) stained for expressions of the segment-marker gene G. bimaculatus wingless (Gb'wg). See also Figure S2 and Movie S6. Current Biology 2010 20, 1641-1647DOI: (10.1016/j.cub.2010.07.044) Copyright © 2010 Elsevier Ltd Terms and Conditions

Figure 4 Comparison of the Timing of Developmental Events during Early Embryogenesis among D. melanogaster, T.castaneum, and G. bimaculatus The developmental events plotted against the period since egg laying (log time). The maternal gradient system is simply represented by a red gradient in an egg cartoon of each species. Periods of regional specification by the head gap gene (H; otd) and/or trunk gap genes (T; hb and Kr) are indicated by red dots on each line plot. Expression domains of otd (light blue), hb (blue), and Kr (pink) are shown in cartoons of embryos. The orange dotted line indicates the timing of cellularization. In Drosophila, the head and trunk regions are specified almost simultaneously through the activities of gap genes, which are regulated primarily by Bcd morphogenetic activity, during syncytial blastoderm stages. In Gryllus, the anterior head and gnathal/thoracic regions are specified by activities of otd and hb/kr, respectively, in a temporally differential manner in the cellularized condition. Phases 1–5 indicate the phases of cellular dynamics in Gryllus early development (defined in text). In Tribolium, although the establishment of specific domains of hb is later than otd, the hb expression in the germ-anlage region starts in the H stage (represented by H/(T)). Abbreviations: p.f., pronuclear fusion; a.e., axial expansion; s.b., syncytial blastoderm formation; c.b., cellularization of the blastoderm; GAf., germ-anlage formation; GAe., germ-anlage (or germband) elongation. Current Biology 2010 20, 1641-1647DOI: (10.1016/j.cub.2010.07.044) Copyright © 2010 Elsevier Ltd Terms and Conditions