Morphological staging definitions and values from quantitative time-lapse analyses at 30°C (see also Fig. 1).

a<p>Age is given in hours after egg lay for the minimum age from a 4-hour range, as the mean ± standard deviation. Parenthetical values for percent of development are based on a total embryogenesis period of 72 hours.</p>b<p>As the complete blastoderm stage was not quantified, we do not provide a definitive staging landmark and hence do not plot this stage in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103967#pone-0103967-g001" target="_blank">Figure 1H</a>.</p>c<p>For GBE, data were pooled from movie recordings 1 and 2. The separate values are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103967#pone-0103967-g001" target="_blank">Fig. 1H</a>.</p>d<p>Note that the staging landmark definition corresponds to the “SR” plot point in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103967#pone-0103967-g001" target="_blank">Figure 1H</a>, whereas the designations SR1–SR4 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103967#pone-0103967-g001" target="_blank">Figure 1</a> micrographs subdivide the subsequent morphological progression of serosal tissue contraction and withdrawal from the embryo.</p

Expression in the line G04609: muscle and leg sensory cell anatomical details.

<p>For the retracted germband (<b>A</b>) and post-dorsal closure (<b>B</b>) stages, representative embryos are shown with G04609-mesodermal×G12424-serosal heterozygote EGFP expression (green; white in single-channel images) and a nuclear counterstain (magenta). The serosa and dorsal yolk have been removed in image A. G04609 muscle EGFP persists in lateral segmental blocks (A,B1,B2). Meanwhile, expression in the appendages is dynamic. The distal tips of the labral, antennal, maxillary, and labial segments (but not the mandibulary segment) initially express EGFP, but this is subsequently lost (compare arrowheads in A and B1). In the legs, distal expression is initially broad (A) and later refines to a subset of putative sensory structures (B1,B3): a small, anterior-proximal patch in the trochanter, a circular structure and single anterior-distal cell within the femur, and cells throughout the pretarsus (shown for the T2 leg). The expression in the trochanter and single labeled cell in the femur are superficial and likely represent sensory cells within the ventral epidermis, such as campaniform sensillae <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103967#pone.0103967-Akay1" target="_blank">[55]</a>. The circular structure lies in the center of the femur’s diameter and is anatomically consistent with identification as the chordotonal organ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103967#pone.0103967-Zill1" target="_blank">[56]</a>. The expression in the pretarsus is distally superficial but innervates the leg more proximally and also likely represents sensory structures. Lastly, EGFP expression also occurs weakly throughout the ventral nerve cord at post-dorsal closure stages, due to the 3xP3 enhancer in the transposon (B1′: curly bracket). All views are maximum intensity projections in lateral aspect, with anterior left or proximal left (B3 only). Abbreviations: A(x), abdominal segment (x); Cb, cardioblasts; E, eye; Fe, femur; Pr, pretarsus; Sm, segmental muscle blocks; T(x), thoracic segment (x); Ti, tibiotarsus; Tr, trochanter. Scale bars are 100 µm (A,B1) and 20 µm (B2,B3).</p

Specific expression features for the lines G12424 and KT650.

<p>Views are dorsal (A–C), ventral-lateral (D), or lateral (E), with anterior left. (<b>A</b>–<b>C</b>) In both lines EGFP (green; white in single-channel images) fills serosal cells, showing the nucleus and cytoplasmic protrusions or overall cell shapes (A–B: G12424×G04609-mesodermal heterozygote: EGFP in the cardioblasts (Cb) is from the G04609 line, described below; C: KT650). EGFP expression throughout the serosa (S) clearly demarcates the tissue boundary with the unlabeled amnion (Am). In fixed specimens (A–B) a nuclear counterstain in shown is magenta. In live imaging panels (C1–C3), whole mount views merge the EGFP and transmitted light channels: the opaque yolk appears black and the embryonic tissue is light grey. Time stamps in C1–C3 are relative to serosal rupture (at 24°C). Inset images C1′–C3′ show details of cell shapes: orderly polygons over the embryo (left) and more irregular apical areas over the yolk (right) in the pre-rupture serosa (C1′); at the anterior edge of the serosa during early contraction (C2′); and at the posterior edge as the serosa begins to degenerate, with trailing cytoplasmic extensions (C3′). See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103967#pone.0103967.s001" target="_blank">Movie S1</a>. (<b>D</b>–<b>E</b>) In addition to serosal expression, both lines exhibit late embryonic expression. EGFP in the eyes (E) and ventral nerve cord (curly brackets) is the result of the 3xP3 promoter within the transposon and is not line specific. Line G12424 (panel D) exhibits EGFP in the pleuropodia (Pl) and a small proximal region of the legs (dashed yellow outline encompasses T1–T3), while line KT650 (panel E) has expression in the distal portion of the legs (yellow arrows) and strongly in the central nervous system, including the brain and at heightened levels in the ventral nerve cord compared to 3xP3 expression alone. Scale bars are 100 µm for all whole mount images; for the inset images, scale bars are 10 µm (B) and 20 µm (C1′-C3′).</p

List of primers used for generating <i>in situ</i> hybridization templates.

a<p>In the <i>Tribolium</i> official gene set, the coding sequence for <i>Tc-H15</i> is incorrectly split between these two gene models: see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103967#pone-0103967-g006" target="_blank">Fig. 6A</a>.</p>b<p>Forward primers also included the 5′ adapter sequence 5′-GGCCGCGG-3′ for subsequent amplification with the T7 promoter universal primer for sense strand transcription (adapter not shown in table).</p>c<p>Reverse primers also included the 3′ adapter sequence 5′-CCCGGGGC-3′ for subsequent amplification with the T7 promoter universal primer for antisense strand transcription (adapter not shown in table).</p

EGFP expression in the G04609 line across embryogenesis.

<p>Micrographs labeled with the same letter are of the same embryo(s). Views are in lateral (A–B) or dorsal-lateral (C) aspect, with anterior left (A–B) or up (C, except left-most embryo, where the head is labeled: H). (<b>A</b>) Onset of expression from blastoderm through germband extension stages (4.8–24.8 hAEL, n = 11), including in the yolk and dorsal-anterior serosa. (<b>B</b>) Increasing EGFP signal through the completion of dorsal closure (17.9–64.2 hAEL, n = 7), in lateral segmental muscle blocks, distal leg regions, and the cardioblast cell row. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103967#pone.0103967.s002" target="_blank">Movie S2</a>. (<b>C</b>) Overview of expression from dorsal closure through hatching of the larvae (n = 11). Images A1–A2 show expression in yolk globules, where the schematic in A2 tracks all globules visible at each of the three ages indicated for the entire duration that a given globule is visible. In all subsequent panels, colored annotations indicate: EGFP expression in the serosa (blue, arcs and arrowheads: A3,B1–B3,C1), in the segmental muscle blocks (yellow arrows: A4,B1–B4,C1–C3), and in the cardioblast cell row (red double-headed arrows: B1–B4,C1–C3). In B1–B4, only the segmental muscle block on abdominal segment 5 is indicated. Percentage values (A–B) are normalized EGFP signal intensity values relative to the maximum intensity achieved during embryogenesis. Time stamps show embryo minimum age (A–B, at 30°C) or elapsed time from start of filming (C, 30°C then room temperature). All scale bars, shown in the first panels only, are 100 µm except for 200 µm in C3.</p

Genomic mapping of the serosal lines G12424 (A) and KT650 (B).

<p>Genome browser views (version Tcas_3.0) show the insertion site (red) within the context of the entire chromosomal linkage group (ChLG, grey background) and enlarged for the region ±50 kb from the insertion site (white background). Predicted genes (Tcas_3.0 official gene set) within this region are numbered and shown in green (dark green: entire gene, light green: exons); see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103967#pone-0103967-t002" target="_blank">Tables 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103967#pone-0103967-t003" target="_blank">3</a> for details of specific genes. Candidate genes inspected by <i>in situ</i> hybridization are labeled in blue.</p

Progression of serosal expression in the lines G12424 and KT650 across embryogenesis.

<p>Live imaging detection of GFP in the transgenic <i>Tribolium</i> lines nGFP, G12424, and KT650. Micrographs labeled with the same letter are of a single embryo. Views are ventral (A1<b>–</b>A3), lateral (A4,B,C,E,F), or dorsal (D,G), with anterior left. (<b>A–B</b>) Exemplar nGFP embryos, illustrating the uniform blastoderm (BL), primitive pit (PP, white arrowhead), closing serosal window (SW), maximum germband extension (GBE), and mid serosal rupture (SR1) stages. Dashed lines label the serosal edge (A3,B) and posterior abdomen (A4). The embryo in panels A1–4 naturally rotated to a lateral view. (<b>C–D</b>) G12424 serosal EGFP increases from shortly after serosal window closure through mid germband retraction. (<b>E</b>–<b>F1</b>) KT650 serosal EGFP increases from shortly after maximum germband extension until just before serosal rupture. Both lines exhibit early EGFP expression in yolk globules (C1,E1: dashed outlines show embryo position, SW stage). The onset of serosal EGFP expression shows an anterior-dorsal or anterior bias (C2,E2: blue lines). During germband retraction, expression in both lines becomes dynamic, with streaks of EGFP between serosal nuclei (shown for G12424: compare C3′ and C4′, dots mark selected nuclei). (<b>F2</b>–<b>G3</b>) In both lines, serosal expression persists throughout the lifetime of the tissue (shown for KT650: sequential stages following serosal rupture, SR1-4, through tissue degeneration). Percentage values (C–F1) denote normalized EGFP intensity for each line. Time stamps show embryo minimum age (at 30°C). Anatomical abbreviations: H, head; S, serosa; T(x), thoracic segment (x). Scale bars are 100 µm, except for 50 µm in C3′. (<b>H</b>) Quantification of EGFP expression time courses, showing the mean ± standard deviation, with sample sizes indicated in the legend. Asterisks mark the maximum EGFP signal. Also plotted are the durations of the two films (younger, “y”; older, “o”: grey lines) and the morphological stages defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103967#pone-0103967-t003" target="_blank">Table 3</a> (black plot points). See Methods for details.</p

Candidate target genes in the vicinity (±50 kb) of the G12424 insertion, as numbered in Fig. 3A.

a<p>Genes are identified by protein prediction models from Tcas_3.0 (official gene set (OGS): TC#, shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103967#pone-0103967-g003" target="_blank">Fig. 3A</a>), AUGUSTUS2 (au2, from July 2012), and AUGUSTUS3 (au3, from September 2013).</p>b<p>Indicates a gene with multiple OGS identifiers but a single Augustus model.</p>c<p>Indicates genes for which <i>in situ</i> hybridization was tested.</p>d<p>Based on reciprocal BLAST identification of the probable <i>Drosophila melanogaster</i> orthologue, or non-orthologous best hit, within the <i>Tribolium</i> genome browser.</p

Candidate target genes in the vicinity (±50 kb) of the KT650 insertion, as numbered in Fig. 3B.

a<p>Genes are identified by protein prediction models from Tcas_3.0 (official gene set (OGS): TC#, shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103967#pone-0103967-g003" target="_blank">Fig. 3B</a>), Augustus2 (au2, from July 2012), and Augustus3 (au3, from September 2013).</p>b<p>Indicates a gene with multiple OGS identifiers but a single Augustus model.</p>c<p>Indicates genes for which <i>in situ</i> hybridization was tested.</p>d<p>Based on reciprocal BLAST identification of the probable <i>Drosophila melanogaster</i> orthologue, or non-orthologous best hit, within the <i>Tribolium</i> genome browser.</p

Mesodermal expression in the line G04609: heart anatomical details.

<p>For the retracted germband (<b>A</b>–<b>C</b>), dorsal closure (<b>D</b>), and post-dorsal closure (<b>E</b>–<b>G</b>) stages, representative embryos are shown with G04609-mesodermal×G12424-serosal heterozygote (A–C,E–G) or G04609 homozygote (D) EGFP expression (green; white in single-channel images) and a nuclear counterstain in fixed specimens (magenta). The serosa is visible in panel A only. Throughout these stages, G04609 heart EGFP is stably expressed in a subset of cardioblast cells, near to the dorsal epidermis-amnion boundary (B: dashed magenta line approximation, based on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103967#pone.0103967-Panfilio2" target="_blank">[16]</a>) and directly below the epidermis (C1–C3,G1–G3). As heart formation progresses during dorsal closure, small bends form in the cardioblast cell row (D2–D5: red arrows), reflecting the overall scalloped, or wavy, geometry of dorsal closure (compare with shape of dashed line in B). Note that in D1 the upper cell row is not yet fully in view due to the slightly oblique angle. Views are lateral (A–C) or dorsal (D–G), with anterior left. All views are maximum intensity projections except C1–C3 and G1–G3 provide orthogonal views of a single plane (depth below surface is indicated in the xy panel: C2,G1). Boxed regions in A and F correspond to panels B and G, respectively. The focal point for orthogonal views is indicated by a white asterisk (B–C,F–G). Time stamps in D1–D5 show elapsed time from start of filming (at 24°C). Abbreviations: A(x), abdominal segment (x); Am, Amnion; Cb, cardioblasts; Ep, epidermis; S, serosa; T(x), thoracic segment (x). Scale bars are 100 µm (A,D,E), 20 µm (B,C,G), and 50 µm (F).</p