Figure 1.
Rib is required for cell growth in the embryonic SG. (A) SG tube morphology at distinct embryonic stages. Placode cells (stage 10) internalize to form an incipient tube (stage 11). Elongation and posterior turning (stage 12) positions the tube along the anteroposterior axis as the cells collectively migrate during tube maturation (stages 13 and 14). Scale bar: 10 µm. (B) The bilateral secretory tubes (magenta nuclei) of the late embryonic SG connect with each other and to the digestive tract via the ducts (green nuclei). Scale bar: 5 µm. (C) SG cell volumetry of embryos stained with α-Mipp1 (magenta, cell boundaries) and α-CrebA (green, nuclei) in WT and rib mutants. Scale bars: 10 µm. (D)rib SG cells are smaller than WT at all embryonic stages. Quantitative analysis of stage-wise cell volumetric data (mean with SEM) from a total of 1,200 3D-rendered cells. n = 150 cells from three embryos of each genotype at each stage. WT: stage 11, 42.13 ± 0.93 µm3 (mean ± SEM); stage 12, 53.81 ± 1.46 µm3; stage 13/14, 75.39 ± 1.73 µm3; and stage 15/16, 98.44 ± 1.86 µm3. rib null: stage 11, 24.73 ± 0.65 µm3; stage 12, 48.43 ± 0.95 µm3; stage 13/14, 49.33 ± 1.29 µm3; and stage 15/16, 63.05 ± 1.63 µm3. P < 0.05; two-tailed unpaired t test for each stage-wise comparison. (E) WT and rib mutant SG cell volumes differ during early stages of tubulogenesis (top: stage 11–12), when glands are internalizing (P < 0.05; two-tailed unpaired t test; median and quartiles) and during late stages of tubulogenesis (bottom: stages 13–16), after internalization (P < 0.05; two-tailed unpaired t test; median and quartiles). (F)rib mutant SGs fail to fully elongate. α-SAS staining of the luminal membrane (arrowhead) reveals that rib mutant SGs are shorter than WT. >50 WT and rib null embryos were examined and images were captured for ∼10 samples of each. Scale bars: 50 µm. (G) Rib-overexpressing (Oex) SG cells (fkh-GAL4 > UAS-rib) are larger than WT. Left: 3D rendering of SG cells showed ∼46% volume increase in Rib-Oex. A total of 300 cells were 3D rendered, n = 150 from three late-stage (15–16) embryos of each genotype. Scale bars: 10 µm. Right: Quantitative analysis of cell volume in WT and Rib-Oex glands (two-tailed, unpaired t test; median and quartiles). (H) SG nuclear DNA volume is unaffected by rib loss. Left: 3D rendering of DAPI staining showed no difference in nuclear DNA volume between WT and rib mutant SGs. A total of 300 nuclei were 3D rendered, n = 150 from three late-stage (15–16) embryos of each genotype. Scale bars: 10 µm. Right: Quantitative analysis of DNA volume in WT and rib mutants (two-tailed, unpaired t test; median and quartiles). (I) Whole embryo volume is unchanged in rib mutants. Left: 3D rendering of whole embryos. Scale bar: 50 µm. Right: Quantitative analysis of embryonic volume showed no difference in mean volume of rib mutants versus WT (n = 5 embryos/group; two-tailed, unpaired t test; mean with SEM).

Rib is required for cell growth in the embryonic SG. (A) SG tube morphology at distinct embryonic stages. Placode cells (stage 10) internalize to form an incipient tube (stage 11). Elongation and posterior turning (stage 12) positions the tube along the anteroposterior axis as the cells collectively migrate during tube maturation (stages 13 and 14). Scale bar: 10 µm. (B) The bilateral secretory tubes (magenta nuclei) of the late embryonic SG connect with each other and to the digestive tract via the ducts (green nuclei). Scale bar: 5 µm. (C) SG cell volumetry of embryos stained with α-Mipp1 (magenta, cell boundaries) and α-CrebA (green, nuclei) in WT and rib mutants. Scale bars: 10 µm. (D)rib SG cells are smaller than WT at all embryonic stages. Quantitative analysis of stage-wise cell volumetric data (mean with SEM) from a total of 1,200 3D-rendered cells. n = 150 cells from three embryos of each genotype at each stage. WT: stage 11, 42.13 ± 0.93 µm3 (mean ± SEM); stage 12, 53.81 ± 1.46 µm3; stage 13/14, 75.39 ± 1.73 µm3; and stage 15/16, 98.44 ± 1.86 µm3. rib null: stage 11, 24.73 ± 0.65 µm3; stage 12, 48.43 ± 0.95 µm3; stage 13/14, 49.33 ± 1.29 µm3; and stage 15/16, 63.05 ± 1.63 µm3. P < 0.05; two-tailed unpaired t test for each stage-wise comparison. (E) WT and rib mutant SG cell volumes differ during early stages of tubulogenesis (top: stage 11–12), when glands are internalizing (P < 0.05; two-tailed unpaired t test; median and quartiles) and during late stages of tubulogenesis (bottom: stages 13–16), after internalization (P < 0.05; two-tailed unpaired t test; median and quartiles). (F)rib mutant SGs fail to fully elongate. α-SAS staining of the luminal membrane (arrowhead) reveals that rib mutant SGs are shorter than WT. >50 WT and rib null embryos were examined and images were captured for ∼10 samples of each. Scale bars: 50 µm. (G) Rib-overexpressing (Oex) SG cells (fkh-GAL4 > UAS-rib) are larger than WT. Left: 3D rendering of SG cells showed ∼46% volume increase in Rib-Oex. A total of 300 cells were 3D rendered, n = 150 from three late-stage (15–16) embryos of each genotype. Scale bars: 10 µm. Right: Quantitative analysis of cell volume in WT and Rib-Oex glands (two-tailed, unpaired t test; median and quartiles). (H) SG nuclear DNA volume is unaffected by rib loss. Left: 3D rendering of DAPI staining showed no difference in nuclear DNA volume between WT and rib mutant SGs. A total of 300 nuclei were 3D rendered, n = 150 from three late-stage (15–16) embryos of each genotype. Scale bars: 10 µm. Right: Quantitative analysis of DNA volume in WT and rib mutants (two-tailed, unpaired t test; median and quartiles). (I) Whole embryo volume is unchanged in rib mutants. Left: 3D rendering of whole embryos. Scale bar: 50 µm. Right: Quantitative analysis of embryonic volume showed no difference in mean volume of rib mutants versus WT (n = 5 embryos/group; two-tailed, unpaired t test; mean with SEM).

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