Limits...
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.

Show MeSH

Related in: MedlinePlus

Mesodermal expression in the line G04609: heart anatomical details.For the retracted germband (A–C), dorsal closure (D), and post-dorsal closure (E–G) 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 [16]) 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).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4117572&req=5

pone-0103967-g006: Mesodermal expression in the line G04609: heart anatomical details.For the retracted germband (A–C), dorsal closure (D), and post-dorsal closure (E–G) 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 [16]) 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).

Mentions: As development proceeds, EGFP signal arises in multiple domains. Among these, expression is first detected in lateral, segmentally repeated blocks of muscle during germband extension, beginning 16.4±0.9 hAEL (n = 8; embryos with particularly strong overlying serosal EGFP were excluded from this measurement; Fig. 4A4: yellow arrows). Subsequent expression also develops in the cardioblast cell row (presumptive heart), beginning about midway through germband retraction (Fig. 4B1: red arrow). Expression in both of these domains strengthens through the end of embryogenesis and then persists in the hatched larva (Figs. 4B–C, 5, 6). Additionally, EGFP expression occurs in the distal region of the appendages, in a dynamic fashion that refines over time and likely comprises a subset of sensory cell structures (Figs. 4B1–B4, 5A,B1,B3). While the lateral muscle block regions seem to be fully labeled by EGFP, in a segmentally iterated and characteristic shape (Fig. 5B2), heart expression does not occur in all cardioblasts (Fig. 6A–C) although EGFP expression appears to be stably on or off in a given cell (Fig. 6D1–D5).


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

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

Mesodermal expression in the line G04609: heart anatomical details.For the retracted germband (A–C), dorsal closure (D), and post-dorsal closure (E–G) 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 [16]) 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).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0103967-g006: Mesodermal expression in the line G04609: heart anatomical details.For the retracted germband (A–C), dorsal closure (D), and post-dorsal closure (E–G) 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 [16]) 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).
Mentions: As development proceeds, EGFP signal arises in multiple domains. Among these, expression is first detected in lateral, segmentally repeated blocks of muscle during germband extension, beginning 16.4±0.9 hAEL (n = 8; embryos with particularly strong overlying serosal EGFP were excluded from this measurement; Fig. 4A4: yellow arrows). Subsequent expression also develops in the cardioblast cell row (presumptive heart), beginning about midway through germband retraction (Fig. 4B1: red arrow). Expression in both of these domains strengthens through the end of embryogenesis and then persists in the hatched larva (Figs. 4B–C, 5, 6). Additionally, EGFP expression occurs in the distal region of the appendages, in a dynamic fashion that refines over time and likely comprises a subset of sensory cell structures (Figs. 4B1–B4, 5A,B1,B3). While the lateral muscle block regions seem to be fully labeled by EGFP, in a segmentally iterated and characteristic shape (Fig. 5B2), heart expression does not occur in all cardioblasts (Fig. 6A–C) although EGFP expression appears to be stably on or off in a given cell (Fig. 6D1–D5).

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