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. 2021 Mar 5;9:637873. doi: 10.3389/fcell.2021.637873

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Copyright © 2021 Khan, Akhtar, Umer, Shaheen, Shaukat, Munir, Mithani, Anwar and Tariq.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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ballchen mutation exhibits trxG like behavior. (A,B) Ballchen mutant flies (ball²) were crossed to two different alleles of Pc (Pc¹ and Pc^XL5). Pc alleles (Pc¹ and Pc^XL5) crossed to w¹¹¹⁸ flies were used as control crosses. Heterozygous Pc/+males, Pc¹/+ (A) and Pc^XL5/+ (B), from control crosses exhibit strong extra sex comb phenotype. In contrast, ball² strongly suppressed the extra sex comb phenotype in both ball²/Pc¹ (A) and ball²/Pc^XL5 (B) male flies. 200 male flies were analyzed for each cross and data shown represents two independent experiments. Male flies were categorized according to the severity of extra sex comb phenotype. These categories are: –, no extra sex combs; +, 1–2 hairs on 2nd leg; ++, more than three hairs on 2nd leg; +++, more than 3 hairs on 2nd leg and 1–2 hairs on 3rd leg; ++++, strong sex combs on both 2nd and 3rd pairs of legs as described previously (Tariq et al., 2009). (C) ball² mutant flies were crossed to two different alleles of trx (trx¹, trx^E2). A cross between trx mutants and w¹¹¹⁸ served as a control. Males from the resulting progeny were scored for A5 to A4 transformation (loss of pigmentation in A5, marked by asterisk), a known trx mutant phenotype. trx/+heterozygotes from the cross of w¹¹¹⁸ with trx mutants were used as control. Compared to the control, ball²/trx¹ and ball²/trx^E2 showed a higher percentage of flies with A5 to A4 transformation, indicating a strong enhancement of trx mutant phenotype. Representative images of w¹¹¹⁸, ball, and trx heterozygous mutants and ball/trx double mutants are shown. The expressivity of A5 to A4 transformation phenotype of trx*/ball² was comparable to trx*/+. All crosses were carried out in triplicates and independent t-tests were performed for analyzing each category (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, or ****p ≤ 0.0001). (D) Significantly low levels of abd-A, Abd-B and pnt expression was detected through qRT-PCR in homozygous ball² embryos when compared with w¹¹¹⁸ embryos. Independent t-tests were performed for each gene analysis (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, or ****p ≤ 0.0001). (E–H) Immunostaining of stage 15 embryos with Abd-B antibody is shown in w¹¹¹⁸ (E,F) as well as homozygous ball² (G,H) embryos. As compared to w¹¹¹⁸ (F), ball² embryos showed strongly diminished Abd-B (H) expression.