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Visualizing late insect embryogenesis: extraembryonic and mesodermal enhancer trap expression in the beetle Tribolium castaneum.

Koelzer S, Kölsch Y, Panfilio KA - PLoS ONE (2014)

Bottom Line: We show that EGFP expression is readily detected, including in heterozygote crosses that permit the simultaneous visualization of multiple tissue types.The tissue specificity provides live, endogenous marker gene expression at key developmental stages that are inaccessible for whole mount staining.Furthermore, the nonlocalized EGFP in these lines illuminates both the nucleus and cytoplasm, providing cellular resolution for morphogenesis research on processes such as dorsal closure and heart formation.

View Article: PubMed Central - PubMed

Affiliation: Institute for Developmental Biology, University of Cologne, Cologne, Germany.

ABSTRACT
The beetle Tribolium castaneum has increasingly become a powerful model for comparative research on insect development. One recent resource is a collection of piggyBac transposon-based enhancer trap lines. Here, we provide a detailed analysis of three selected lines and demonstrate their value for investigations in the second half of embryogenesis, which has thus far lagged behind research on early stages. Two lines, G12424 and KT650, show enhanced green fluorescent protein (EGFP) expression throughout the extraembryonic serosal tissue and in a few discrete embryonic domains. Intriguingly, both lines show for the first time a degree of regionalization within the mature serosa. However, their expression profiles illuminate distinct aspects of serosal biology: G12424 tracks the tissue's rapid maturation while KT650 expression likely reflects ongoing physiological processes. The third line, G04609, is stably expressed in mesodermal domains, including segmental muscles and the heart. Genomic mapping followed by in situ hybridization for genes near to the G04609 insertion site suggests that the transposon has trapped enhancer information for the Tribolium orthologue of midline (Tc-mid). Altogether, our analyses provide the first live imaging, long-term characterizations of enhancer traps from this collection. We show that EGFP expression is readily detected, including in heterozygote crosses that permit the simultaneous visualization of multiple tissue types. The tissue specificity provides live, endogenous marker gene expression at key developmental stages that are inaccessible for whole mount staining. Furthermore, the nonlocalized EGFP in these lines illuminates both the nucleus and cytoplasm, providing cellular resolution for morphogenesis research on processes such as dorsal closure and heart formation. In future work, identification of regulatory regions driving these enhancer traps will deepen our understanding of late developmental control, including in the extraembryonic domain, which is a hallmark of insect development but which is not yet well understood.

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Progression of serosal expression in the lines G12424 and KT650 across embryogenesis.Live imaging detection of GFP in the transgenic Tribolium lines nGFP, G12424, and KT650. Micrographs labeled with the same letter are of a single embryo. Views are ventral (A1–A3), lateral (A4,B,C,E,F), or dorsal (D,G), with anterior left. (A–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. (C–D) G12424 serosal EGFP increases from shortly after serosal window closure through mid germband retraction. (E–F1) 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). (F2–G3) 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′. (H) 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 Table 3 (black plot points). See Methods for details.
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pone-0103967-g001: Progression of serosal expression in the lines G12424 and KT650 across embryogenesis.Live imaging detection of GFP in the transgenic Tribolium lines nGFP, G12424, and KT650. Micrographs labeled with the same letter are of a single embryo. Views are ventral (A1–A3), lateral (A4,B,C,E,F), or dorsal (D,G), with anterior left. (A–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. (C–D) G12424 serosal EGFP increases from shortly after serosal window closure through mid germband retraction. (E–F1) 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). (F2–G3) 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′. (H) 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 Table 3 (black plot points). See Methods for details.

Mentions: We took a live imaging approach to characterizing the enhancer trap EGFP expression. To contextualize the enhancer trap lines’ spatially and temporally restricted expression, in parallel we filmed embryos from a line that ubiquitously expresses nuclear-localized GFP (nGFP, [4]). This allowed us to precisely determine the timing of landmark morphological stages (Fig. 1A–B, Table 1). Briefly, gross morphogenesis and development of the external structures in the Tribolium egg proceed as follows. The cellularized epithelium of the blastoderm on the yolk surface differentiates into the serosa and the germ rudiment (embryo and amnion). The first morphological change is a small depression at the posterior pole, the primitive pit, which rapidly enlarges into a posterior fold. Fold outgrowth culminates in the internalization of the embryo relative to both extraembryonic membranes, where the constricting region through which the embryo is visible is termed the serosal window[3], [14], [21]. The embryo then proceeds to add body segments during germband extension, and then to thicken during germband retraction[17]. Subsequently, the serosa ruptures in the ventral-anterior region where it had previously completed closure, leading to its withdrawal and final degeneration during dorsal closure of the embryonic epidermis; during this process the folded, compacted serosa is known as the ‘dorsal organ’ [15]–[17]. After dorsal closure, continued maturation of the embryo includes increasing physical activity as the longitudinal body muscles twitch periodically (Table 1).


Visualizing late insect embryogenesis: extraembryonic and mesodermal enhancer trap expression in the beetle Tribolium castaneum.

Koelzer S, Kölsch Y, Panfilio KA - PLoS ONE (2014)

Progression of serosal expression in the lines G12424 and KT650 across embryogenesis.Live imaging detection of GFP in the transgenic Tribolium lines nGFP, G12424, and KT650. Micrographs labeled with the same letter are of a single embryo. Views are ventral (A1–A3), lateral (A4,B,C,E,F), or dorsal (D,G), with anterior left. (A–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. (C–D) G12424 serosal EGFP increases from shortly after serosal window closure through mid germband retraction. (E–F1) 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). (F2–G3) 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′. (H) 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 Table 3 (black plot points). See Methods for details.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4117572&req=5

pone-0103967-g001: Progression of serosal expression in the lines G12424 and KT650 across embryogenesis.Live imaging detection of GFP in the transgenic Tribolium lines nGFP, G12424, and KT650. Micrographs labeled with the same letter are of a single embryo. Views are ventral (A1–A3), lateral (A4,B,C,E,F), or dorsal (D,G), with anterior left. (A–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. (C–D) G12424 serosal EGFP increases from shortly after serosal window closure through mid germband retraction. (E–F1) 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). (F2–G3) 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′. (H) 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 Table 3 (black plot points). See Methods for details.
Mentions: We took a live imaging approach to characterizing the enhancer trap EGFP expression. To contextualize the enhancer trap lines’ spatially and temporally restricted expression, in parallel we filmed embryos from a line that ubiquitously expresses nuclear-localized GFP (nGFP, [4]). This allowed us to precisely determine the timing of landmark morphological stages (Fig. 1A–B, Table 1). Briefly, gross morphogenesis and development of the external structures in the Tribolium egg proceed as follows. The cellularized epithelium of the blastoderm on the yolk surface differentiates into the serosa and the germ rudiment (embryo and amnion). The first morphological change is a small depression at the posterior pole, the primitive pit, which rapidly enlarges into a posterior fold. Fold outgrowth culminates in the internalization of the embryo relative to both extraembryonic membranes, where the constricting region through which the embryo is visible is termed the serosal window[3], [14], [21]. The embryo then proceeds to add body segments during germband extension, and then to thicken during germband retraction[17]. Subsequently, the serosa ruptures in the ventral-anterior region where it had previously completed closure, leading to its withdrawal and final degeneration during dorsal closure of the embryonic epidermis; during this process the folded, compacted serosa is known as the ‘dorsal organ’ [15]–[17]. After dorsal closure, continued maturation of the embryo includes increasing physical activity as the longitudinal body muscles twitch periodically (Table 1).

Bottom Line: We show that EGFP expression is readily detected, including in heterozygote crosses that permit the simultaneous visualization of multiple tissue types.The tissue specificity provides live, endogenous marker gene expression at key developmental stages that are inaccessible for whole mount staining.Furthermore, the nonlocalized EGFP in these lines illuminates both the nucleus and cytoplasm, providing cellular resolution for morphogenesis research on processes such as dorsal closure and heart formation.

View Article: PubMed Central - PubMed

Affiliation: Institute for Developmental Biology, University of Cologne, Cologne, Germany.

ABSTRACT
The beetle Tribolium castaneum has increasingly become a powerful model for comparative research on insect development. One recent resource is a collection of piggyBac transposon-based enhancer trap lines. Here, we provide a detailed analysis of three selected lines and demonstrate their value for investigations in the second half of embryogenesis, which has thus far lagged behind research on early stages. Two lines, G12424 and KT650, show enhanced green fluorescent protein (EGFP) expression throughout the extraembryonic serosal tissue and in a few discrete embryonic domains. Intriguingly, both lines show for the first time a degree of regionalization within the mature serosa. However, their expression profiles illuminate distinct aspects of serosal biology: G12424 tracks the tissue's rapid maturation while KT650 expression likely reflects ongoing physiological processes. The third line, G04609, is stably expressed in mesodermal domains, including segmental muscles and the heart. Genomic mapping followed by in situ hybridization for genes near to the G04609 insertion site suggests that the transposon has trapped enhancer information for the Tribolium orthologue of midline (Tc-mid). Altogether, our analyses provide the first live imaging, long-term characterizations of enhancer traps from this collection. We show that EGFP expression is readily detected, including in heterozygote crosses that permit the simultaneous visualization of multiple tissue types. The tissue specificity provides live, endogenous marker gene expression at key developmental stages that are inaccessible for whole mount staining. Furthermore, the nonlocalized EGFP in these lines illuminates both the nucleus and cytoplasm, providing cellular resolution for morphogenesis research on processes such as dorsal closure and heart formation. In future work, identification of regulatory regions driving these enhancer traps will deepen our understanding of late developmental control, including in the extraembryonic domain, which is a hallmark of insect development but which is not yet well understood.

Show MeSH
Related in: MedlinePlus