Fog signaling has diverse roles in epithelial morphogenesis in insects
- University of Cologne, Germany
- University of Cambridge, United Kingdom
- University of Warwick, United Kingdom
- University of Illinois, United States
Figures
Figure 1 with 4 supplements
Expression of Fog signaling components during early embryogenesis.
(A–D) Schematics showing embryo condensation as described in the text. Serosa is shown in purple, germ rudiment tissue is shown in gray, arrows display tissue movements. aaf: anterior amniotic fold, …
Figure 1—figure supplement 1
Fog and T48 pathway in Drosophila.
In Drosophila the secreted protein Fog activates the two G protein coupled receptors (GPCRs) Mist (Mesoderm-invagination signal transducer, also known as Mthl1 (Methuselah-like1)) (Manning et al., …
Figure 1—figure supplement 2
Insect Fog proteins.
(A) Schematic representation of Fog proteins from Drosophila melanogaster (Dm-Fog), Gryllus bimaculatus (Gb-Fog), Oncopeltus fasciatus (Of-Fog) and Tribolium castaneum (Tc-Fog). The proteins contain …
Figure 1—figure supplement 3
Expression of Tc-cta, Tc-mist and Tc-fog during early embryogenesis.
(A–X) Whole mount ISH for Tc-cta (A–H), Tc-mist (I–P) and Tc-fog (Q–X). (A’–X’) DAPI staining of respective embryos. Anterior is left, all panels show optical sagittal sections, except G, H, L, O, …
Figure 1—figure supplement 4
Tc-fog and Tc-twi are co-expressed only within the posterior presumptive mesoderm.
Double whole mount ISH for Tc-twi (red) and Tc-fog (blue). Anterior is left, all panels show optical sagittal sections.
Figure 2 with 5 supplements
Fog signaling is required for posterior amniotic fold formation.
(A–H) Stills from live fluorescent imaging of LifeAct-eGFP transgenic embryos, ranging from late blastoderm to germband extension stages. (A–D) wildtype control. (E–H) Tc-cta KD. The asterisk marks …
Figure 2—figure supplement 1
KD of Tc-fog, Tc-mist and Tc-cta by RNAi results in similar phenotypes.
(A–D) Nuclear (DAPI in A, C and D; nGFP transgene in B) staining of embryos of similar age. (A) Wildtype embryo at early elongating germ band stage. (B–D) Tc-cta, Tc-fog or Tc-mist KD embryos. aaf: …
Figure 2—figure supplement 2
Fog signaling is required for posterior amniotic fold formation.
(A– C) Tc-iro expression (ISH). (A’– C’) DAPI staining of respective embryos. In wildtype embryos, Tc-iro is expressed at the serosa/germ rudiment border and in a dorsal germ rudiment domain. (C) …
Figure 2—figure supplement 3
Morphogenetic defects in late Tc-cta KD embryos.
(A–D) Stills from fluorescent live imaging of a Tc-cta KD embryo carrying a LifeAct-eGFP transgene. aaf: anterior amniotic fold, blue asterisk: lateral rupture, yellow asterisk: ventral rupture. …
Figure 2—figure supplement 4
Frequencies of phenotypic defects upon Tc-fog KD.
(A) 98% of offspring of beetles injected with H2O show normal development (N = 124). 76% (N = 183) of offspring of beetles injected with Tc-fog dsRNA showed phenotypic defects. (B) The 139 embryos …
Figure 2—figure supplement 5
Late development of Tc-cta KD embryos.
(A, B) Optical cross sections (DIC) of fixed embryos after germband retraction. The elongation of the hindgut has started. (A’, B’) Enlarged views of the areas enframed in A, B. Red arrows indicate …
Figure 3
Schematic representation of the embryonic phenotype produced by Fog pathway disruption.
Schematics showing wildtype development and the effects on embryo formation of RNAi disruption of Tc-fog, Tc-mist or Tc-cta. Anterior is left, ventral is down.
Figure 4
Fog signaling affects the positioning of the primordial germ cells.
Whole mount ISH for the germ cell marker Tc-tapas. (A, B, D, E) Wildtype. (C, F) Tc-fog KD. (A–C) Optical sagittal sections of whole embryos. (D–F) Optical sagittal sections of posterior regions. (A’…
Figure 5 with 4 supplements
Tc-fog RNAi delays mesoderm internalization.
(A–C) Ventral views of whole mount embryos (anterior left) stained for the segmental marker Tc-gsb (yellow), nuclei (DAPI; blue). Embryos are also stained for Tc-twi expression but this is only …
Figure 5—figure supplement 1
Mesoderm internalization varies along the AP axis in Tribolium.
Twi protein expression within the ventral furrow of a gastrulating Tribolium embryo. Cross section through anterior (A) and posterior (B) region of the same embryo. The sections are modified from Han…
Figure 5—figure supplement 2
Apical constrictions during mesoderm internalization.
Transverse cryosections at posterior positions of wildtype (A) and Tc-fog KD (B) embryos of similar age stained for F-actin. (A’, B’) Magnified insert with mesoderm cells outlined in magenta. (A’’, …
Figure 5—figure supplement 3
Delayed mesoderm internalization upon Tc-fog KD.
(A, B) Transverse section of staged embryos stained for F-actin (Phalloidin; red) and nuclei (Sytox; green). (A) Wildtype. The amnion/dorsal ectoderm covers the ventral side of the embryo. The …
Figure 5—figure supplement 4
The mesoderm is internalized upon Tc-fog KD.
(A, B) Sagittal cryosections of embryos at the early elongating germ band stage showing Tc-twi expression (ISH, dark blue). (A’, B’) DAPI staining of respective embryos. (A) Wildtpype. Tc-twi is …
Figure 6 with 1 supplement
Regulation of Tc-fog and Tc-mist expression.
Whole mount ISH for Tc-fog (A–E) and Tc-mist (H–L) expression in wildtype embryos (A, H) and embryos in which DV and AP genes have been knocked down (B-E, I-L; specific KD shown in panels). All …
Figure 6—figure supplement 1
Regulation of Tc-fog and Tc-mist by DV and AP patterning genes.
Arrowheads indicate activation, the bar-headed line indicates inhibition. Dashed lines indicate that the regulation is indirect.
Figure 7 with 2 supplements
Local Tc-fog expression and posterior folding.
Nuclear (DAPI) staining of wildtype (A) and KD (B, C, D; specific KD shown in panels) embryos at the early posterior amniotic fold stage. Anterior is left, ventral is down (where possible to discern).
Figure 7—figure supplement 1
Tc-fog expression after Tc-zen1 KD.
Whole mount ISH for Tc-fog. Optical sagittal sections. (A, B) anterior Tc-fog expression is lost upon KD of Tc-zen1. Anterior is left, ventral is down.
Figure 7—figure supplement 2
Tc-fog expression after Tc-Toll and Tc-zen1 KD.
Whole mount ISH for Tc-fog. Optical sagittal sections. (A–C) Tc-fog expression is lost (or greatly reduced) upon Tc-Toll and Tc-zen1 double KD by pRNAi. (A’–C’) nuclear (DAPI) staining of respective …
Figure 8
Serosal cells undergo intercalation during serosal expansion.
Stills from confocal live imaging of wildtype embryos with cell membranes marked via transient expression of GAP43-YFP. The tracked cells are colored as rows parallel to the serosa/germ rudiment …
Figure 9
Fog signaling affects serosal expansion.
(A, B) Stills from confocal live imaging of wildtype (A) and Tc-fog eRNAi (B) embryos with cell membranes marked via transient expression of GAP43-YFP. Embryos are undergoing germband elongation. (A’…
Figure 10 with 1 supplement
Fog signaling is required for blastoderm formation in Tribolium, Oncopeltus and Gryllus.
(A–D) Stills from confocal live imaging of wildtype (A), and Tc-fog, Tc-mist, Tc-cta eRNAi (B–D) Tribolium embryos with cell membranes marked via transient expression of GAP43-YFP. Embryos are at …
Figure 10—figure supplement 1
Developmental delay upon KD of Fog pathway components.
Violin plot showing effect of Tc-fog, Tc-cta or Tc-mist KD on timing of early development. All data points are shown in a quasi-random offset. ***p<0.001 (unpaired t-test) comparing control and KD.
Figure 11
Distinct functions of Fog signaling in Tribolium.
Summary schematic showing the different roles of fog signaling during early Tribolium embryogenesis.
Videos
Video 1
Fluorescent live imaging of wildtype and Tc-fog RNAi nGFP transgenic embryos.
Maximum intensity projections of one egg hemisphere are shown with anterior to the left and ventral to the bottom.
Video 2
Fluorescent live imaging of wildtype and Tc-cta RNAi LifeAct-GFP transgenic embryos.
Maximum intensity projections of one egg hemisphere are shown with anterior to the left and ventral to the bottom.
Video 3
Fluorescent live imaging of the posterior pole of a wildtype embryo transiently expressing GAP43-YFP.
Apical constrictions are visible at the center of the forming fold. Embryo was mounted with the posterior pole towards the objective and the resulting movie was digitally rotated. Maximum intensity …
Video 4
Single section through the subapical region of the cells at the posterior pole of a wildtype embryo transiently expressing GAP43-YFP.
Cells constrict over time and this occurs in a pulsatile manner, and cell intercalation is also visible. Ventral is to the bottom.
Video 5
Fluorescent live imaging of wildtype embryos transiently expressing GAP43-YFP.
Embryos were mounted with their posterior poles towards the objective. Maximum intensity projection of posterior view is shown. Ventral is to the bottom.
Video 6
Fluorescent live imaging of the presumptive serosa and germ rudiment epithelium of a wildtype embryo transiently expressing GAP43-YFP.
The cuboidal-to-squamous transition of the serosa cells during germband formation can be seen. Maximum intensity projection of the epithelium as well as transverse and sagittal sections along …
Video 7
Fluorescent live imaging of aTc-cta RNAi LifeAct-GFP transgenic embryo.
Maximum intensity projection of one egg hemisphere is shown with anterior to the left and ventral to the bottom.
Video 8
Fluorescent live imaging of Tc-Toll1 and Tc-fog double RNAi nGFP transgenic embryo.
Maximum intensity projection of one egg hemisphere is shown with anterior to the left.
Video 9
Fluorescent live imaging of the dorsolateral side of a wildtype embryo transiently expressing GAP43-YFP.
Serosa cells at/near the serosa/germ rudiment boundary were tracked and colored as rows (pink cells closest to the boundary). Only cells that were visible from the beginning of the timelapse are …
Video 10
Fluorescent live imaging of the lateral side of a wildtype embryo transiently expressing GAP43-YFP.
Serosa cells at/near the serosa/germ rudiment boundary were tracked and colored as rows (pink cells closest to the boundary). Only cells that were visible from the beginning of the timelapse are …
Video 11
Fluorescent live imaging of the ventrolateral side of a wildtype embryo transiently expressing GAP43-YFP.
Serosa cells at/near the serosa/germ rudiment boundary were tracked and colored as rows (pink cells closest to the boundary). Roughly half the cells (at the anterior) are tracked from the beginning …
Video 12
Fluorescent live imaging of part of the forming serosa in a wildtype embryo transiently expressing GAP43-YFP.
A group of cells intercalating via rosette formation are tracked. The field of view was manually stabilized to follow this group of cells. Anterior is to the left.
Video 13
Fluorescent live imaging of the wildtype and Tc-fog weak KD embryos transiently expressing GAP43-YFP.
Serosa cells at/near the serosa/germ rudiment boundary were tracked. The top panels show cells coloured as rows (pink cells closest to the boundary). The bottom panels show cells colored randomly …
Video 14
Fluorescent live imaging of a Tc-fog weak KD embryo transiently expressing GAP43-YFP.
Maximum intensity projection of one egg hemisphere with anterior to the left and ventral to the bottom is shown in the center. A transverse section near the anterior pole is to the left, a …
Video 15
Fluorescent live imaging of wildtype and Tc-fog, Tc-T48, and Tc-fog and Tc-T48 double.
RNAi embryos transiently expressing GAP43-YFP. Average intensity projections of one egg hemisphere are shown with anterior to the left and ventral to the bottom.
Video 16
Fluorescent live imaging of wildtype and Tc-fog, Tc-cta, and Tc-mist RNAi embryos transiently expressing GAP43-YFP.
Average intensity projections of one egg hemisphere are shown with anterior to the left and ventral to the bottom (where possible to discern).
Video 17
Fluorescent live imaging of additional Tc-fog, Tc-cta, and Tc-mist RNAi embryos transiently expressing GAP43-YFP.
Further examples of blastoderm formation defects to demonstrate the variability in the phenotypes. Average intensity projections of one egg hemisphere are shown with anterior to the left and ventral …
Video 18
Fluorescent live imaging of wildtype and Gb-mist RNAi histone2B-EGFP transgenic embryos.
Maximum focus projections of one egg hemisphere are shown as ventral views with anterior to left.
Additional files
-
Supplementary file 1
Primer List.
Primers used to produce anti-sense RNA for in-situ hybridization and dsRNA for RNAi-mediated gene knockdown.
- https://doi.org/10.7554/eLife.47346.048
-
Transparent reporting form
- https://doi.org/10.7554/eLife.47346.049
